Total synthesis of elastin peptide using high pressure-liquid phase synthesis assisted by a soluble tag strategy

Article abstract of DOI:10.1021/ol5032798

A highly aggregating elastin peptide was prepared efficiently using a high pressure-liquid phase synthesis approach assisted by a soluble tag strategy. Two standard syringes were connected to each other to construct a reactor. This simple reactor was used

Full text of DOI:10.1021/ol5032798

Letter
pubs.acs.org/OrgLett
Total Synthesis of Elastin Peptide Using High Pressure−Liquid Phase
Synthesis Assisted by a Soluble Tag Strategy
Yohei Okada,‡ Shoichi Hosoya,†,§ Hidenobu Suzuki,§ and Kazuhiro Chiba*,†
†Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo
183-8509, Japan
‡Department of Chemical Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo
184-8588, Japan
§YOSHIDA KIKAI Co., Ltd., 3-13 Sakurada-cho, Nagoya Atsuta, Aichi 456-0004, Japan
S
* Supporting Information
ABSTRACT: A highly aggregating elastin peptide was
prepared efficiently using a high pressure−liquid phase
synthesis approach assisted by a soluble tag strategy. Two
standard syringes were connected to each other to construct a
reactor. This simple reactor was used to apply high pressure to
the highly viscous reaction mixture thereby maintaining its
fluidity. The reactions were completely inhibited due to
aggregation when conducted in a standard flask reactor,
whereas our high pressure approach accelerated the couplings
to realize complete conversion within 5−7 min. All steps were
conducted at 0.10 M concentration, affording grams of the
desired product.
he high modularity and diversity of peptides result in their
peptide is recovered as a precipitate following dilution of the
Tstrong affinity for a wide range of targets, ranging from reaction mixture with excess polar organic solvent (typically
methanol or acetonitrile). Our approach is a promising
alternative for synthesizing aggregating peptides because
intermolecular hydrophobic interactions are minimized in less
polar organic solvents compared with the polar organic solvents
(typically N,N-dimethylformamide or N-methylpyrrolidone)
commonly used in solid phase peptide synthesis.
small metal ions to large biomacromolecules such as proteins
and nucleic acids, depending on the precise sequence of the
peptide. Peptides can recognize and interact specifically with
themselves, called self-assembly, to form supramolecular
networks which cause the gelation of aqueous solvents.1 Such
peptide-based hydrogelators are attractive tools for developing
soft biomaterials because the properties of the gelator can be
tuned by altering the amino acid sequences including both
proteinogenic and nonproteinogenic.2 Strategies that allow the
rational design and control of gelation properties have recently
been proposed to allow novel bioapplications of peptide
gelators.3
However, the unique gelation property of peptides often
makes their chemical synthesis difficult. Although gelation can
seem like a technical issue, it can cause severe problems and
limit the utility of the peptides, especially hydrophobic
peptides. Indeed, peptide aggregation can occur even on solid
phase resin, making the reactive end of the peptide inaccessible
and inhibiting the peptide’s elongation. Microwave-assisted
methods can overcome this obstacle,4 but a synthetic method
applicable to large scale production of aggregating peptides,
regardless of sequence, remains elusive.
Elastin is an extracellular matrix protein that provides
elasticity to several human tissues, including arteries, lungs,
and skin.6 Tropoelastin, a water-soluble precursor, is highly
cross-linked to form native insoluble elastin. The elastic
properties of elastin make it an intriguing scaffold for functional
biomaterials that could have medical, clinical, therapeutic, and
cosmetic applications.7 For example, inadequate elastin in the
skin causes wrinkles. However, these physical properties of
elastin are accompanied by a synthetic challenge: namely, the
gelation and aggregation of the protein. The chemical structure
of elastin comprises a simple repeating sequence, generally of
the nonpolar amino acids glycine (Gly), valine (Val), and
proline (Pro) (Figure 1). Described herein is the highly
efficient large scale production of elastin peptide using high
pressure−liquid phase peptide synthesis assisted by a soluble
tag strategy.8
The tag-assisted liquid phase peptide synthesis approach was
based on our routinely used procedure. The first coupling of
We have developed a soluble tag-assisted liquid phase
peptide synthesis method using hydrophobic benzyl alcohol as
a support.5 Both coupling and deprotection are conducted in
the liquid phase using a less polar organic solvent (typically
dichloromethane or tetrahydrofuran). The desired tagged
Received: November 12, 2014
© XXXX American Chemical Society
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Letter
However, when coupling of Fmoc-Val-OH to H-Gly-Val-O-
TAG was attempted in the biphasic solvent mixture using
COMU and DIPEA, the reaction mixture became a highly
viscous gel within 3 min and stopped the mechanical stirrer,
indicating severe aggregation (Figure S5 in SI). The reaction
was largely inhibited, and most of the starting H-Gly-Val-O-
TAG was recovered. Although the addition of a large excess of
cyclohexane and/or heating could also aid coupling, we
expected that only reestablishing the fluidity of the reaction
mixture was required since the substrate and reagents were both
in “liquid” phase and their collision still potentially took place.
Therefore, we connected two syringes to each other to fluidize
the reaction mixture under high pressure (Figure 3).
Figure 1. Chemical structure of the typical repeat sequence of elastin
peptide, and of the soluble tag.
Fmoc-Val-OH to the tag 1 (HO-TAG) was carried out in
toluene using N,N′-diisopropylcarbodiimide (DIPCI) and 4-
dimethylaminopyridine (DMAP), followed by dilution of the
reaction mixture with an excess of acetonitrile to recover the
desired Fmoc-Val-O-TAG as a precipitate. Following depro-
tection of the Fmoc group in tetrahydrofuran using 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) and piperidine, the
reaction mixture was diluted with an excess of acetonitrile to
obtain H-Val-O-TAG as a precipitate.
However, although coupling of Fmoc-Gly-OH to H-Val-O-
TAG proceeded smoothly in tetrahydrofuran using N-[1-
(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-
(morpholino)]uronium hexafluorophosphate (COMU) and
N,N-diisopropylethylamine (DIPEA), during workup the
addition of an excess of acetonitrile caused the immediate
formation of a heterogeneous sticky precipitate containing a
significant amount of COMU which was difficult to remove
(Figure S1 in Supporting Information (SI)).
After investigating numerous conditions, a 1:1 (v/v) biphasic
mixture of cyclohexane and acetonitrile used as the reaction
solvent instead of tetrahydrofuran was found to dramatically
affect the properties of the precipitate (Figure S2 in SI). The
addition of an excess of acetonitrile caused gradual formation of
a homogeneous tight precipitate readily recovered by vacuum
filtration from which COMU could be effectively rinsed away
(Figure S3 in SI).
The precipitate was a white powder, in contrast to the pale
yellow amorphous solid obtained when tetrahydrofuran was
used as the reaction solvent (Figure 2). Particle size analysis
clearly showed a narrower distribution when a 1:1 (v/v)
biphasic mixture of cyclohexane and acetonitrile was used
compared to tetrahydrofuran as the solvent (Figure S4 in SI).
The biphasic solvent mixture was effective in the deprotection
of the Fmoc group using DBU and piperidine, affording H-Gly-
Val-O-TAG in high yield.
Figure 3. Schematic illustration of the connected syringes used as a
reactor.
Coupling of Fmoc-Val-OH to H-Gly-Val-O-TAG in the
connected syringes proceeded smoothly in 5 min during 50
passes to provide Fmoc-Val-Gly-Val-O-TAG (Figure S6 in SI).
The plungers were simply pushed manually, requiring ca. 0.10−
0.20 MPa to induce sufficient fluidity at each pass. The
obtained highly viscous gel was poured into an excess of
acetonitrile, causing gradual formation of a homogeneous tight
precipitate readily recovered by vacuum filtration. The
recovered precipitate was pure Fmoc-Val-Gly-Val-O-TAG and
did not contain Fmoc-Val-OH, COMU, or DIPEA. The Fmoc
group was readily deprotected using DBU and piperidine by
stirring in a standard flask, providing H-Val-Gly-Val-O-TAG
effectively. Although further couplings caused similar highly
viscous gels, use of the syringe reactor accelerated the reactions
to give the elastin repeat sequence, Fmoc-Pro-Gly-Val-Gly-Val-
O-TAG 2, in 68% yield over 9 steps from the tag 1 (Scheme 1).
Notably, all steps were conducted at 0.10 M concentration to
afford grams of the desired product (Figure S7 in SI).
With the repeat sequence of the elastin peptide in hand, we
focused on fragment coupling to prepare a longer variant
(Scheme 2). Thus, Fmoc-Pro-Gly-Val-Gly-Val-O-TAG 2 was
divided into two aliquots and the tag was removed from one
aliquot in a 5:1 (v/v) mixture of dichloromethane and
trifluoroethanol using trifluoroacetic acid to give Fmoc-Pro-
Gly-Val-Gly-Val-OH 3 in 89% yield (see SI for experimental
details). The reaction mixture was diluted with an excess of
methanol to precipitate the removed tag, and then the crude
peptide was reprecipitated from tetrahydrofuran using isoproyl
ether to give pure Fmoc-Pro-Gly-Val-Gly-Val-OH 3. Fmoc
group deprotection of Fmoc-Pro-Gly-Val-Gly-Val-O-TAG 2
was achieved using a standard 1:1 (v/v) mixture of cyclohexane
and acetonitrile to give H-Pro-Gly-Val-Gly-Val-O-TAG 4.
However, fragment coupling of Fmoc-Pro-Gly-Val-Gly-Val-
Figure 2. Morphology of Fmoc-Gly-Val-O-TAG prepared in (a) a 1:1
(v/v) biphasic mixture of cyclohexane and acetonitrile and (b)
tetrahydrofuran.
B
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peptide H-(-Pro-Gly-Val-Gly-Val-)2-OH 6 in 88% yield over 2
steps (see SI for experimental details). After precipitating the
released tag by adding an excess of methanol, the crude peptide
was washed with acetonitrile to give pure H-(-Pro-Gly-Val-Gly-
Val-)2-OH 6 (Figure S10 in SI).
Scheme 1. Preparation of the Repeat Sequence of Elastin
Peptide
In conclusion, we have demonstrated the efficient prepara-
tion of a highly aggregating elastin peptide using a high
pressure−liquid phase synthesis approach assisted by a soluble
tag strategy. Two syringes were connected to construct a
reactor that could pressurize the highly viscous reaction mixture
and maintain its fluidity. Using this approach, each coupling
was completed during 50 passes within 5−7 min, even at 0.10
M concentration to afford grams of the desired product. After
coupling, the obtained highly viscous reaction mixture was
simply poured into excess acetonitrile, resulting in the gradual
formation of a homogeneous tight precipitate which was easily
recovered by vacuum filtration and showed excellent purity.
Although the reaction mixtures were highly viscous gels, all
substrates and reagents were maintained in a “liquid” phase in
the syringe reactor which provided sufficient fluidity to realize
effective collisions between the reactant molecules. It should be
noted that the procedure required only standard equipment
found in any laboratory. We believe that the approach
described herein could be applied to an automated pressurized
peptide synthesizer to realize the preparation of aggregating
peptides. A description of such a synthesizer will be presented
in due course.
OH 3 and H-Pro-Gly-Val-Gly-Val-O-TAG 4 in a 1:1 (v/v)
biphasic mixture of cyclohexane and acetonitrile was
unsuccessful, as Fmoc-Pro-Gly-Val-Gly-Val-OH 3 was not
soluble in either cyclohexane or acetonitrile. This problem
was solved by use of acetone instead of acetonitrile (Figure S8
in SI); the coupling proceeded smoothly in a 1:1 (v/v) mixture
of cyclohexane and acetone to give Fmoc-(-Pro-Gly-Val-Gly-
Val-)2-O-TAG 5 in 77% yield over 2 steps (Figure S9 in SI).
Finally, the Fmoc group was deprotected and the tag was
removed using standard conditions to give the desired elastin
Scheme 2. Preparation of Longer Elastin Peptide through Fragment Coupling
C
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ASSOCIATED CONTENT
* Supporting Information
Additional figures, general, and experimental information. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: chiba@cc.tuat.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partially supported by Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science.
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