A liquid-phase peptide synthesis in cyclohexane-based biphasic thermomorphic systems

Article abstract of DOI:10.1039/b205156g

Combinations of typical organic solvents composed of cyclohexane and qualified aprotic polar organic solvents were found to realize an effecitive, biphasic thermomorphic system in arbitrary ratios of upper and lower phases that enable a practical applicat

Full text of DOI:10.1039/b205156g

A liquid-phase peptide synthesis in cyclohexane-based biphasic
thermomorphic systems†
Kazuhiro Chiba,* Yusuke Kono, Shokaku Kim, Kohsuke Nishimoto, Yoshikazu Kitano and Masahiro
Tada
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: +81-42-360-8830; Tel: +81-42-367-5700
Received (in Cambridge, UK) 29th May 2002, Accepted 3rd July 2002
First published as an Advance Article on the web 15th July 2002
Combinations of typical organic solvents composed of
cyclohexane and qualified aprotic polar organic solvents
were found to realize an effecitive, biphasic thermomorphic
system in arbitrary ratios of upper and lower phases that
enable a practical application of a liquid-phase peptide
synthesis.
phase and a lower NA(main) phase (Fig. 1, colored by
methylene blue, MB). After heating to 45 °C, it was soon
immixed to form a stable, homogeneous phase. Furthermore, by
cooling back to 25 °C, the solution was immediately separated
into the two initial phases, completely recovering the homoge-
neously dissolved MB into the lower NA layer. This clear
partition of a solute suggested the ability of the thermo-
controlled solubilization and the partition of designed solutes in
this solvent system. It was therefore prompted us to seek the
promising property of the biphasic organic liquid system by
expanding the solvent combinations and ratios. Fig. 2(a) shows
the correlations between the miscible temperature and the ratio
of NE and NM. With increasing of the content of NE, the
miscible temperature was linearly lowered. For example, a
biphasic mixture of CH–NE–NM (CH+NA 17+83, NE+NM
70+30) formed a homogeneous phase at 14 °C and higher
Much current research utilizes the solid-phase as a platform for
organic reactions.1 Solid-phase resins offer many advantages
with regard to compound isolation and ease of handling. The
insoluble nature of the resins has, however, complicated the
characterization of compounds attached to them and led to
reagent accessibility problems. One successful merging of
solid- and liquid-phase chemistry is the use of soluble polymer
platforms that selectively remove excess reaction reagents and
byproducts.2 On the other hand, the use of a liquid–liquid
biphasic thermomorphic process, in which a reagent or a
catalyst is designed as a residue in one of the liquid phases and
as a product in the other liquid phase, can be an enabling
approach for a commercial application of chemical reactions
with high selectivity, efficiency, and ease of handling for the
separation of solutes.3 Based on the immiscibility of per-
fluorinated hydrocarbons with both organic and inorganic
solvents, a novel ‘fluorous biphasic system’ for catalysis has
been proposed.4 Fluorous biphasic systems consist of a fluorous
phase containing a preferentially fluorous soluble reagent and a
catalyst with limited solubility in the fluorous phase.5 The
thermomorphic liquid–liquid separation system composed of
organic solvents of different polarity might allow for extremely
efficient reactions, for example, by the association of apolar
products and polar substrates or catalysts in a one-phase
solution, which could be spatially separated after the comple-
tion of reactions. It should further open the door for the
construction of liquid-phase combinatorial chemistry, industrial
and green chemistry by the ease of the separation of products,
catalyses, reagents, electrolytes, and/or soluble platforms for
organic synthesis. This gave us the incentive to explore a novel
liquid-phase peptide synthesis system in a thermomorphic
system using typical organis solvents. Herein is a report of our
findings of the combination of cyclohexane and some typical
organic solvents to realize the aimed miscible biphasic system
in arbitrary ratios of upper and lower phases. By using the
cyclohexane-based biphasic thermomorphic system, an applica-
tion for liquid-phase peptide synthesis was efficiently accom-
plished.
Fig. 1 (a) An organic solvent mixture composed of CH–NE–NM (75+20+5
v/v/v) formed the biphasic system at 25 °C (a lower NA layer was colored
by methylene blue). (b) The solvent mixture was immixed at 45 °C. (c,d)
After cooling back to 25 °C, it immediately began to exclude the CH phase.
(e) Finally, it formed the initial biphasic solution to recover methylene blue
in the lower layer.
We first explored extensively solvent-constructions for
thermomorphic solutions that could be reversibly immixed at
‘desired’ temperatures in ‘arbitrary ratios’ of upper and lower
layer volumes. Among numerous compositions of organic
solvents, an appropriate mixture of cyclohexane (CH) and
nitroalkane (NA) was initially found to show the desired
function, that is, a 75+25 (v/v) mixture of CH and NA [a 20+80
v/v mixture of nitromethane (NM) and nitroethane (NE)]. At 25
°C, the solvent mixture was separated into an upper CH(main)
Fig. 2 (a) Effect of the solvent compositions (CH+NA ratios and NM+NE
ratios) on the miscible temperatures. Each biphasic solvent mixture (10 mL
in an 18 3 200 mm test tube, 10 °C) was gradually heated (ca. 3 °C min21
)
with stirring to form a homogeneous solution at the temperature plotted
above. (b) Effect of solvent composition on the miscible temperature. The
solvent mixtures were composed of a 50+50 (v/v) mixture of CH–nitriles or
CH–amides with varying ratios of AN+PN or DMF+DMA, respectively.
† Electronic supplementary information (ESI) available: general procedure
and spectral data for compounds 2–7. See http://www.rsc.org/suppdata/cc/
b2/b205156g/
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This journal is © The Royal Society of Chemistry 2002

temperatures. On the other hand, the miscible temperature
increased with an increase in the NM ratio. The miscible
temperature was also affected by the ratio of CH, requiring
higher temperature with an increase in the CH ratio to complete
the one-phase formation. The miscible temperature, however,
was not affected when the ratio of CH+NA (v/v) was more than
50+50. This shows that the miscible temperature and the ratio of
CH+NA can be arbitrarily chosen by controlling the ratio of
NE+NM. Furthermore, a similar property was also found in the
mixture of CH–dimethylacetamide (DMA)–dimethylforma-
mide (DMF) and of CH–acetonitrile (AN)–propionitrile (PN)
(Fig. 2(b)). In the mixture of CH–DMF–DMA, the miscible
temperature varied between 18 and 47 °C by changing the
DMF+DMA ratio. A clear phase separation was also observed
for the mixture of CH–AN–PN between 33 and 61 °C.
In the thermomorphic system, less-polar chemicals or
designed less-polar platforms were selectively partitioned in the
CH-layer, and a platform, (3,4,5-trioctadecyloxyphenyl)-
methan-1-ol 16 effectively worked in the sequential peptide
synthesis (Fig. 3). The platform was selectively7 dissolved in
the CH-layer of the biphasic solution composed of CH–DMF–
DMA, which allowed the sequential peptide synthesis in the
liquid-phase. In the homogenized reaction mixture at 35 °C,
peptide-chain elongation was accomplished by treating with
only 3 mol equiv. of activated Fmoc-amino acids. After
deprotection of the Fmoc moiety, the deprotected products (e.g.
3, 5 and 7) were immediately ‘fished out’ from the unreacted
reagents just by cooling to form the biphasic solution. The
deprotected amino group was then repeatedly treated with
another activated Fmoc-amino acid. In these sequential reac-
tions, highly excessive amounts of activated Fmoc-amino acid
was not required, and the peptide intermediates (e.g. 3–7) were
easily isolated in excellent yield and characterized by NMR
and/or TOF-MS.
In conclusion, biphasic, thermomorphic liquid–liquid separa-
tion systems have been constructed for liquid-phase peptide
synthesis by using a CH-soluble platform in CH and typical
organic solvents. It is noteworthy that the regulation of the
separation and the immixing of solutes can be achieved by a
moderate thermo-control in the practical range of 15–65 °C.
Furthermore, products can be easily isolated from the CH-layer
to complement existing solid-phase peptide synthesis, offering
advantages with regard to compound isolation, characterization,
and reagent accessibility in reaction mixtures. Since those polar
media, NM, DMF and AN, have been widely used for organic
reactions with varied catalysts and reagents,8 the extensive
chemical applications can be also assured with the flexibly
controllable temperatures and upper/lower layer ratios.
This work was partially supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology.
Notes and references
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7 For example, 99% of 1 (130 mM) was recovered from upper CH-layer
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Fig. 3 An example of the liquid-phase peptide synthesis using the biphasic
organic solvent mixture. a, (Fmoc-Val-)2O, DMAP in CH–DMF–DMA
50+25+25 (35 °C), 95%; b, 10% Et2NH in DMF–DMA 50+50 (35 °C), 99%;
c, Fmoc-Gly-OBt, DIPCD in DMF–DMA 50+50 (5 to 35 °C), 99%; d, 10%
Et2NH in DMF–DMA 50+50 (35 °C), 99%; e, Fmoc-Phe-OBt, DIPCD (5 to
35 °C), 97%; f, 10% Et2NH in DMF–DMA 50+50 (35 °C), 99%.
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