HYDROTHERMAL SYNTHESIS OF OLIGOGLYCINESWITH ADIABATIC EXPANSION COOLING
Yasuhiro Futamura and Kenji Yamamoto*Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan andDepartment of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo
162-8640, Japan*Author for correspondence, e-mail: backen@ri.imcj.go.jp,

fax: +81-3-3202-7364 (Research Institute, International Medical Center of Japan)(Received 31 October 2005 Accepted 9 December 2005)

Abstract
There have been several studies on biopolymer synthesis under hydrothermal conditions. Althoughdimer and short oligomers were obtained from a monomer unit, few of longer oligomers were obtained. Thesestudies that were applied with various quenching methods suggested the importance of quenching speed fromhydrothermal conditions. We hypothesized a rapid quenching could avoid hydrolysis of the oligomers that had
already been synthesized under hydrothermal conditions. We designed a novel hydrothermal flow reactor withadiabatic expansion cooling, which was thought to be one of the most rapid quenching methods. This systemsimulates geysers, fumaroles, hot springs and volcanic eruptions. After aqueous solutions of monomers weretreated at high temperature and pressure, they were released into the atmosphere through an orifice to be
depressurized and cooled down simultaneously with the Joule-Thomson effect. Using the flow reactor, we havedemonstrated oligomerization of glycine up to decamer (Gly10), which had never been yielded with any otherquenching methods. This suggests that rapid quenching methods under non-equilibrium conditions such asadiabatic expansion cooling is an efficient way to produce long oligomers connected by covalent bonds via
dehydration condensation.

Key words: prebiotic synthesis, hydrothermal system, dehydration condensation, biopolymer, peptide, adiabatic
expansion, Joule-Thomson effect, quenching, non-equilibrium, subcritical water.

Viva Origino 33 (2005) 269-274© 2005 by SSOEL Japan- 269 -Viva Origino 33 (2005) 269-274

Introduction

process (pattern A) [3]. In other previous works that

All of the biopolymers like proteins,

simulates hydrothermal vents, the hydrothermal fluid

nucleic acids and polysaccharides are composed ofwas kept at high pressure during cooling and thenmonomer units connected by covalent bonds via

was depressurized. The pathway was shown as

dehydration condensation. There have been several

pattern B [1,2,4,5].

studies on biopolymer synthesis under hydrothermal

Shockpreviously

reported

equilibrium

ofthattheconditions using various quenching methods; forthermodynamic

example, pouring the fluid into cold water [1,2] orhydrolysis reactions between glycine (Gly) andpassing the fluid through a cooling tube [4,5]. These

diglycine (Gly2) would shift to the dehydration

studies suggest the importance of quenching speed,condensation as the temperature increased [6]. Thisbecause in the former studies longer peptides werereport suggests that rapid quenching, that is non-obtained than in the latter, in spite of similar reaction

equilibrium thermodynamics system, could avoidconditions at high pressure and temperature.

hydrolysis of the oligomers that had already been

Fig.1 shows the quenching pathway ofcondensation/

synthesized under hydrothermal conditions.

the previous and this studies on phase diagram of

Now we designed a novel hydrothermal

water. In closed batch systems, the pressure and theflow reactor with adiabatic expansion cooling, which

temperature were changed gradually during cooling

was thought to be one of the most rapid quenching

Fig. 1. Schematic quenching pathways on phase diagram of water. (A)Temperature decreases with pressure, and (B) Temperature decreases withkeeping pressure.

- 270 -

Viva Origino 33 (2005) 269-274

methods [7]. After aqueous solutions of monomers

octaglycines were supplied as a custom synthesis

were treated at high temperature and pressure, theyfrom Invitrogen Co.

were released into the atmosphere through an orifice

Theflow

reactorwithadiabatic

to be depressurized and cooled down simultaneously

expansion cooling was composed of two reservoirs,with the Joule-Thomson effect shown as pattern A inpumps, a pre-heater, a 1.5 mL of flow reactor withFig. 1 [8,9]. The Joule-Thomson effect is a process inheaters, and a needle valve as described previously

which the temperature of a fluid is decreased below[7]. Most parts of the hydrothermal reactor werethe Joule-Thomson inversion temperature by letting

made from SUS316. The orifice was formed by thethe fluid expand adiabatically. When a fluid suddenly

needle valve and its valve seat made from nickel-

expands from a high pressure to a low pressure, there

based alloy, Inconel 625 (including chrome with ais often a temperature change following the Joule-slight amount of molybdenum and niobium) andThomson coefficients (µJP = ∆T/∆p), because theHastelloy C-22 (including chrome and molybdenumhydrothermal fluid must do external work against thewith a slight amount of iron and tungsten),ambient air to consume thermal energy. Using therespectively.

flow reactor, we have demonstrated oligomerization

High performance liquid chromatographyof glycine up to decamer (Gly10), which have never(HPLC) analysis was performed using an HPLC

been yielded with any other quenching methods. In

apparatus, Waters 600E/2487 system attached with an

this paper, the efficiency of rapid quenching methods

octadecyl bonded silica-based column, X-Terra MS

such as adiabatic expansion will be discussed inC18 (Waters, φ = 2.5 µm; 4.6 mm, inner diameter; 50

prebioticmm length). As oligopeptides consisting of glycine

synthesis

ofhydrolysable

compounds

including biopolymers.

residues, which have no side chain, are toohydrophilic to be analyzed using reversed-phase

Materials & Methods

liquid chromatography (RP-LC), we applied ion-pair

Glycine and diglycine was purchased

RP-LC [10,11]. The mobile phase consisted of 5from Wako Pure Chemical Industries, Ltd. (Osaka,mmol L-1 potassium dihydrogen phosphate (KH2PO4,Japan). Distilled water was purchased from Gibco

Wako Pure Chemical Industries, Ltd.) and 2.5 mmol

BRL and Kozakai Seiyaku Co., Ltd. As authenticL-1 sodium 1-hexanesulfonate (C6H13SO3Na, Sigma-samples for LC, diglycine and glycine anhydride

Aldrich Co.), and the pH was maintained at 2.5 with(diketopiperazine) were purchased from Wako Purephosphoric acid (H3PO4, Sigma-Aldrich Co.). The

Chemical Industries, Ltd., tri-, tetra-, penta- andflow rate of the mobile phase was 0.5 mL min-1.

hexaglycines were from Sigma, and hepta- andBefore loading, the sample was filtrated by a 0.22 µm- 271 -Viva Origino 33 (2005) 269-274

centrifugal filter device (Millipore Ultrafree-MC).

cooling and performed hydrothermal oligomerization

The UV detector monitored the absorbance at 200 nmof glycine. First, the pressure of fluid was maintained

to detect oligoglycine series.

at 10 MPa with a backpressure regulator. Pressurizedmass

aqueous solution of 2 mol L-1 of glycine was mixed

spectrometry (LC/MS) was performed using awith pre-heated water to be 1 mol L-1 of glycine

module of Waters 2695 and Micromass Quattro micro

aqueous solution at 270 °C. The flow rate was 2.6

ZQ2000. As the mobile phase for LC/MS, a volatile

mL min-1 and the residence time at high pressure andion-pairacidtemperature was about 30 seconds. Then, the solution(C5F11COOH, Tokyo Kasei Kogyo Co. Ltd.) was used

was quenched and depressurized through the orifice

at the concentration of 1 mmol L-1 [12]. Mass

into the ambient air with the adiabatic expansion

numbers were scanned with positive and negative ion

method. Almost all the solution was collected in spitemode, but no signals were detected with negative ionof some evaporation. As shown in Fig. 2, LC analysis

mode.

was performed and various oligoglycines wereLiquidreagent,

chromatography/undecafluorohexanoicdetected. Production of olygomers was confirmed byResultscomparing with authentic samples (up to octamer)Tosimulate

rapid

quenchingfromhydrothermalconditions,wedesigned

new

LC/MS with selected ion recording modehydrothermal flow reactor with adiabatic expansion

was also performed and qualitatively detected nona-aand LC/MS analysis.

Fig. 2. Chromatogram of the products in the sample that reacted from 1 mol L-1 glycine aqueous
solution at 270 °C and 10 MPa.

- 272 -Viva Origino 33 (2005) 269-274(Gly9) and decaglycine (Gly10), of which protonatedhydrothermal conditions would be widely useful formass [M+H]+ is 532.2 and 589.2, respectively (datahydrolysable bond formation, for example, not only

not shown), that had never been obtained with any

peptide bond but also phosphoester, glycoside, acyl

other cooling methods.

bond and so on.

Discussion

Acknowledgements
In this paper, we demonstrated that theWe thank Mr. Tomomasa Goto for his

adiabatic expansion cooling method was one of theassistance in performing the experiments reported

most rapid quenching processes to polymerize

here, Prof. Yukio Yamaguchi, Univ. of Tokyo for

peptides from amino acid. The qualitative analysis

helpful comments, Mr. Kazuyuki Ito for preparationshows that longer peptides were synthesized than inof the manuscript and Dr. Etsuko Suzuki, Nihon

the previous works [1-5]. This suggests the rapid

Waters K. K. to help LC-MS analysis using

quenching method is quite effective to synthesize

Waters/Micromass Quattro micro. This work waspolymer from monomer. We cannot, however, lead tosupported in part by a research grant program forthe conclusion whether the rapid quenching would

Learning from Nature for Production 2002-2003

avoid hydrolysis of oligomers synthesized in thefrom Sekisui Chemical Co., LTD., Japan and by thehydrothermal reactor, or whether the rapid quenching

grants-in-aid forwould facilitate dehydration condensation of glycines

Ministry of Education, Culture, Sports, Science andas a result of evaporation of solvents and nucleation

Technology of Japan.

Scientific

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Our hydrothermal flow reactor withReferences

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