PHOTO-ALTERATION OF ISOVALINE AND ITS PRECURSOR
5-ETHYL-5-METHYLHYDANTOIN EXPOSED TO UV AND γ-RAY RADIATION
Palash K. Sarker1*, Jun-ichi Takahashi2, Yumiko Obayashi1, Takeo Kaneko1 and Kensei Kobayashi1
1Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University,
79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
2
NTT Microsystem Integration Laboratories, 3-1 Morinosato Wakamiya, Atsugi 243-0198, Japan
Fax: +81-45-339-3938, sarker-palash-nd@ynu.ac.jp(Received February 6, 2012; Accepted June 22, 2012)(Abstract)

precursor molecules of Ival [17], was identified in the
Yamato-791198 (24 pmol·g-1) and Murchison (47pmol·g-1) meteorites [18]. But the origins of Ival and
EM-Hyd in the chondrites still remain controversial,
because amino acids in chondrites and inexperimental simulations of ice irradiations aremostly detected after a strong acid hydrolysis of theresidues formed. Therefore, it should be clarified
whether amino acids and their precursors incarbonaceous chondrites have remained as organic
monomer or generated from their relating
compounds.
Organic molecules containing amino acidprecursors formed in ice mantles of ISDs in dense
clouds could be altered by cosmic rays and UV light
before their incorporation in parent bodies of
meteorites and comets. They might be altered again
in the Solar System bodies. Further, organic
compounds in interplanetary dusts particles (IDPs)
are fully irradiated with strong solar UV light as wellas high-energy particles near Earth orbit. Therefore,
photo-alteration of organics against space radiations
could be an important issue in prebiotic chemistryprior to the origin of life on Earth.
Accordingly, we analyzed UV- and γ-ray
irradiated Ival and EM-Hyd to see their possible
photolysis products. Here we report our detection ofserine (Ser) and alanine (Ala) from irradiated Ival,
and Hyd and 5-methylhydantoin (M-Hyd) from
irradiated EM-Hyd.

We present laboratory studies examining
the photolysis products of isovaline (Ival) and its
precursor
molecule,
5-ethyl-5-methylhydantoin(EM-Hyd) against continuous ultraviolet (UV),
monochromatic UV, and γ-ray photons. The
irradiation experiments were performed with Ival and
EM-Hyd in aqueous state. In both UV and γ-ray
irradiation experiments, Ival produced serine andalanine as common photolysis products. On the other
hand, hydantoin and 5-methylhydantoin weredetected as photolysis products of irradiated EM-Hyd.
This experimental result may point out the potential
importance of the prebiotic photochemistry of Ival
and EM-Hyd in Solar System materials.
(Keywords)
isovaline, radiation, ultraviolet, γ-ray, photolysis
products
1.

Introduction
Since obtaining the first evidence of
extraterrestrial amino acids in a carbonaceouschondrite (e.g., Murchison meteorite) [1], a great
number of laboratory experiments have been reported
to study possible abiotic formation of bioorganic
compounds including amino acids and theirprecursors from possible astrophysical media by
irradiation with high-energy particles [2,3] orultraviolet (UV) light [4-7]. It was suggested thatamino acids among various other compounds weresynthesized in quite cold environments such as in ice
mantles of interstellar dusts (ISDs) in dense clouds by
UV light, and they were then delivered to theprimitive Earth by comets and meteorites [8,9].
Moreover, amino acids in several meteorites (e.g.,
Murchison, Murray, Orgueil) have been found tohave L-enantiomeric excess, the same handedness asobserved in biological amino acids [10,11]. Therefore,
a possible hypothesis can be advocated that life on
Earth was seeded by the delivery of organic
compounds from outer space during the intense
bombardment period of the primitive Earth [12-14].
Among the non-proteinogenic amino acids inextraterrestrial meteorites, isovaline (Ival), a
structural isomer of valine, was found in the Orgueil
and Murchison meteorite showing a significant
L-enantiomeric excess up to 18.5%. This observationsuggests a possible prebiotic contribution to theorigin of biological homochirality [15-16]. Moreover,many acid-labile amino acid precursors, for example,
hydantoin (Hyd) and 5-substituted hydantoins weredetected in carbonaceous chondrites. Among them,5-ethyl-5-methylhydantoin (EM-Hyd), one of theViva Origino 40 (2012) 4 - 8© 2012 by SSOEL Japan

2.

Experimental Section

2.1. Chemicals
As starting material for the radiation experiment,
EM-Hyd (equimolar mixture of D- and L-EM-Hyd)
and Ival (equimolar mixture of D- and L-Ival) wereabiotically synthesized in the Mita Laboratory at the
Fukuoka Institute of Technology, Fukuoka, Japan.
Ethylmethylketone was condensed with ammonium
carbonate in the presence of potassium cyanide to
produce EM-Hyd according to the Strecker synthesis.
Then, Ival was synthesized by hydrolyzing EM-Hyd
with barium hydroxide. Purity of the synthesized Ival
and EM-Hyd was over 99%.
2.2. Continuous
Spectral
UV
Irradiation
Experiment
Aqueous solution of Ival and EM-Hyd wereplaced separately in synthesized quartz cells for UV
irradiation with continuous spectra (190 to 400 nm)
using a deuterium lamp (L1835; HAMAMATSU
Photonics), as shown in Figure 1. Every sample (2mL, 10 mM, pH = 7) was irradiated with a power4

Viva Origino 40 (2012) 4 - 8

density of 66 µW/cm2 for 8 and 16 h at a 30-cm
distance from the photon source of the lamp. The
beam spot was 6 cm in diameter.

reversed-phasehigh-performanceliquidchromatography (RP-HPLC). The RP-HPLC system
was composed of two pumps (SHIMADZU
LC-20AD), a column (Capcell Pak C18 UG120 S-5µm, 6.0 mm i.d. × 250 mm), and a detector(SHIMADZU SPD-20AV, 210-nm detection
wavelength). The temperature of the column wasmaintained at 37 °C. Phosphate buffer (25 mM, pH =3.5, flow rate 0.90 mL/min) and acetonitrile (100%,flow rate 0.10 mL/min) were used as the mobile
phase under isocratic conditions. Hydantoin and
5-substituted hydantoin standards were purchasedfrom Tokyo Chemical Industry Co., Ltd. for
RP-HPLC analysis.
Furthermore, the photolysis products (organic
acids) of UV irradiated (monochromatic) Ival wereanalyzed by a capillary electrophoresis system(Photal CAPI-3300, Otsuka Electronics Co., Ltd).
Analyses were performed in a pH 6.1 carrier
electrolyte
containing
20mM
2,3-pyridinedicarboxylic
acid,
0.5
mM
cetyltrimethylammonium bromide. The samples wererun at 30 °C with applied voltage from -15〜30 kV.
The indirect UV detection wavelength was set at 205
nm.

Fig. 1. Schematic diagram of the experimental setup for
UV irradiation (continuous, 190–400 nm) from a
deuterium lamp (L1835; HAMAMATSU Photonics). The
lamp is positioned horizontally and its two nozzles (water
inlet and outlet) are aligned vertically. The target cell(window: quartz) is set up at a 30-cm distance from thephoton source of the lamp.

2.3. Monochromatic UV Irradiation Experiment
Ival and EM-Hyd were taken individually insynthesized quartz cells for UV irradiation withmonochromatic spectra (215 nm). Each sample (2 mL,
10 mM, pH = 7) was irradiated with themonochromatic UV light generated from freeelectron laser (FEL) of UVSOR-II facility (Institutefor Molecular Science, Okazaki, Japan) at the beamline BL5U. The total irradiation doses were 100 and
150 mWh (360 and 540 joule) and the beam spot sizeat the sample surface was 5 mm in diameter.

3.

Results and Discussion

3.1. Photolysis Products of Irradiated Ival
A recent study investigated that serine (Ser) andalanine (Ala) were detected as common photolysis
products of Ival during continuous UV and γ-ray
irradiation. Moreover, two other amino acids (i.e.,
aspartic acid, glutamic acid) were formed from Ivalby γ-ray irradiation [19]. In the present study, Ival
was additionally subjected to monochromatic UV
irradiation, and we detected the same photolysis
products as Ser and Ala (Figure 2). The
chromatograms for control samples (i.e., trace c, dand e in Figure 2 for Ival without γ-ray irradiation,
Ival without UV-continuous irradiation and Ival
without UV-monochromatic irradiation respectively)
assured that Ser and Ala were formed as photolysis
products of Ival. It is assumed that the formation of
Ser needs an additional OH group, which might comefrom COOH groups during UV and γ-ray irradiation,or the solvent (e.g., H2O) might be another possible
source of OH group as H2O could be attacked by
newly formed ions during γ-ray irradiation. Other
than amino acids, formic acid, acetic acid andisovaleric acid were formed from the monochromatic
UV irradiated Ival (Figure 3). The wholephoto-alteration process by the energetic photons
might involve the release of methyl (-CH3), ethyl(-CH2CH3), carboxyl (-COOH) and amino (-NH2)
groups from Ival in the transition period, which
would lead to the formation of the photolysis
products (Figure 4a and 4b). But extensive sets offuture experiments are required to clarify the precise
formation mechanism of the photolysis products
generated from Ival during UV and γ-ray irradiation.

2.4. γ-Ray Irradiation Experiment
Ival and EM-Hyd were sealed in borosilicate
glass tubes in air. Every sample (2 mL, 10 mM, pH =7) was irradiated with γ-rays from a 60Co source(1.25-MeV-photon energy) at the Japan Atomic
Energy Agency, Takasaki, Japan. The total
irradiation doses were 10 and 20 KGy (20 and 40 J)
as absorbed by the solvent.
2.5. Analysis of Ival and EM-Hyd
The photolysis products of irradiated Ival and
EM-Hydwere
analyzedbyion-exchange
high-performance liquid chromatography (IE-HPLC)
using
post-column
derivatization
witho-phthalaldehyde and N-acetyl-L-cysteine. The
IE-HPLC system was equipped with two HPLC
pumps (Shimadzu LC-10AT), a cation-exchange
column (4 mm i.d. × 150 mm; Shimpak
ISC-07/S1504), a post column derivatization system,
and a Shimadzu RF-535 fluorometric detector(358-nm excitation wavelength; 450-nm emission
wavelength). The temperature of the column wasmaintained at 55 °C. Gradient elution was performed
using eluents A (0.07 M trisodium citrate perchloric
acid with 7% ethanol, pH 3.20) and B (0.2 M
trisodium citrate boric acid with NaOH, pH 10). The
flow rate of the carrier was 0.3 mL/min. A Wako
amino acid mixture (type AN-II and type B) was usedas the amino acid standard for IE-HPLC analysis.
In addition, the photolysis products (other thanamino acids) of irradiated EM-Hyd were analyzed by5

Viva Origino 40 (2012) 4 - 8

(a)

(b)
Fig. 4. The photo-alteration process of Ival by theenergetic photons forms dominantly (a) Ser and Ala, (b)formic acid, acetic acid and isovaleric acid.

Fig. 2. Ion exchange chromatograms of amino acids: (a)Wako amino acid standard, (b) Blank (milli-Q water), (c)Ival without γ-ray irradiation, (d) Ival without UV(continuous) irradiation, (e) Ival without UV(monochromatic) irradiation (f) UV (monochromatic)
irradiated Ival, (g) UV (continuous) irradiated Ival, (h)γ-ray irradiated Ival. Abbreviations: Asp: Aspartic acid,Thr: Threonine, Ser: Serine, Glu: Glutamic acid, α-AAA:
α-Aminoadipic acid, Gly: Glycine, Ala: Alanine, α-ABA:
α-Aminobutyric acid, Val: Valine, Cys: Cystine, Met:
Methionine, Ile: Isoleucine, Leu: Leucine, Tyr: Tyrosine,Phe:
Phenylalanine,β-ala:
β-Alanine,
β-AiBa:β-Aminoisobutyric acid, γ-ABA: γ-Aminobutyric acid,
His: Histidine, Orn: Ornithine, Lys: Lysine. The IE-HPLC
system gives identical retention time for Val and Ival.

(a)

(b)

Fig. 3. Electropherogram of UV (monochromatic)
irradiated Ival.

3.2. Photolysis Products of Irradiated EM-Hyd
As the photolysis products of EM-Hyd during
UV (continuous) irradiation, Hyd was only detected(Figure 5a), whereas Hyd and M-Hyd were formed
during UV (monochromatic) irradiation, as shown in
Figure 5b. On the other hand, M-Hyd and trace
amount of 5,5-dimethylhydantoin (DM-Hyd) and5-ethylhydantoin (E-Hyd) were formed during γ-ray
irradiation (Figure 5c). Additionally, an aliquot of
UV (monochromatic) irradiated EM-Hyd was
subjected to acid hydrolysis to convert its photolysis
products into amino acids. The hydrolyzed fraction(c)Fig. 5. RP-HPLC chromatograms of (a) UV (continuous)
irradiated EM-Hyd, (b) UV (monochromatic) irradiated
EM-Hyd and (c) γ-ray irradiated EM-Hyd

6

Viva Origino 40 (2012) 4 - 8

Fig. 6. Ion exchange chromatogram of hydrolyzed UV(monochromatic) irradiated EM-Hyd.

Fig. 7. The photo-alteration process of EM-Hyd by theenergetic photons forms dominantly Hyd, M-Hyd,
DM-Hyd and E-Hyd. Hydrolysis of EM-Hyd, Hyd,
M-Hyd, DM-Hyd and E-Hyd involves the cleavage ofN1-C2 and N3-C4 bonds inside the heterocyclic ring andproduces Ival, Gly, Ala, AIB and AABA respectively.

was analyzed using IE-HPLC; as a result, glycine(Gly) and Ala were identified, which were produced
from Hyd and M-Hyd respectively, and Ival was
detected as a hydrolysis product of EM-Hyd (Figure6). These results may carry significant implications
for the photo-alteration process of irradiated EM-Hyd
against UV and γ-ray radiation. Firstly, EM-Hyd inmeteoritic parent bodies might play as a possible
precursor of Hyd and 5-substituted hydantoins (e.g.,
M-Hyd, DM-Hyd and E-Hyd) and secondly, thephoto-altered products (e.g., Hyd, M-Hyd) and
EM-Hyd might produce Gly, Ala and Ival
respectively in the early stage of their hydrolysis(Figure 7). The photo-alteration process by theenergetic photons might involve the release of methyl(-CH3) and ethyl (-CH2CH3) groups from EM-Hyd in
the transition period, which would led to theformation of Hyd and 5-substituted hydantoins (e.g.,
M-Hyd, DM-Hyd and E-Hyd). On the other hand,hydrolysis of Hyd, M-Hyd and E-Hyd involved thecleavage of N1-C2 and N3-C4 bonds inside theheterocyclic ring and produced Gly, Ala and Ival
respectively. Therefore, it is assumed that hydrolysis
of DM-Hyd and E-Hyd may predominantly produce
α-aminoisobutyric acid (AIB) and α-aminobutyricacid (AABA) respectively.

References
1.
Kvenvolden, K. A., Lawless, J. G., Pering, K., Peterson, E.,
Flores, J., Ponnamperuma, C., Kaplan, I. R. and Moore, C. B.
Evidence for Extraterrestrial Amino Acids and Hydrocarbons
in the Murchison Meteorite, Nature 228, 923-926 (1970).
2.
Kobayashi, K., Kasamatsu, T., Kaneko, T., Koike, J., Oshima,T., Saito, T., Yamamoto, T. and Yanagawa, H. Formation ofamino acid precursors in cometary ice environments bycosmic radiation, Adv. Space Res. 16, 21-26 (1995).
3.
Takano, Y., Takahashi, J., Kaneko, T., Marumo, K. andKobayashi, K. Asymmetric synthesis of amino acidprecursors in interstellar complex organics by circularly
polarized light, Earth Planet. Sci. Lett. 254, 106-114 (2007).
4.
Bernstein, M. P., Dworkin, J. P., Sandford, S. A., Cooper, G.
W. and Allamandola, L. J. Racemic amino acids from theultraviolet photolysis of interstellar ice analogues, Nature 416,
401-403 (2002).
5.
Muñoz Caro, G. M., Meierhenrich, U. J., Schutte, W. A.,
Barbier, B., Arcones, S. A., Rosenbauer, H., Thiemann, W. H.
-P., Brack, A. and Greenberg, J. M. Amino acids fromultraviolet irradiation of interstellar ice analogues, Nature
416, 403-406 (2002).
6.
Nuevo, M., Bredehöft, J. H., Meierhenrich, U. J.,d’Hendecourt, L., and Thiemann, W. H. Urea, glycolic acidand glycerol in an organic residue produced by ultraviolet
irradiation of interstellar/pre-cometary ice analogs,
Astrobiology10, 245-256 (2010).
7.
de Marcellus, P., Bertrand, M., Nuevo, M., Westall, F. andd’Hendecourt, L. Prebiotic significance of extraterrestrial ice
photochemistry: Detection of hydantoin in organic residues,
Astrobiology 11, 847-854 (2011).
8.
Nakamura-Messenger, K., Messenger, S., Keller, L. P.,
Clemett, S. J. and Zolensky, M. E. Organic Globules in the
Targish Lake Meteorite: Remnants of Protosolar Disk,
Science 314, 1439-1442 (2006).
9.
Greenberg, J. M., Kouchi, A., Niessen, W., Irth, H., Paradijs,J., van de Groot, M. and Hermsen, W. Interstellar Dust,chirality, Comets and the Origins of life: Life from Dead
Stars? J. Biol. Phys. 20, 61-70 (1994).
10. Cronin, J. R. and Pizzarello, S. Amino acid enantiomer
excesses in meteorites: Origin and significance, Adv. SpaceRes. 23, 293-299 (1999).
11. Sephton, M. A. Organic compounds in carbonaceous
meteorites, Nat. Prod. Rep. 19, 292-311 (2002).
12. Bailey, J., Chrysostomou, A., Hough, J. H., Gledhill, T. M.,
McCall, A., Clark, S., Menard, F. and Tamura, M. Circularpolarization in star-formation regions: Implications forbiomolecular Homochirality, Science 281, 672-674 (1998).
13. Bailey, J. Astronomical sources of circularly polarized lightand the origin of Homochirality, Orig. Life Evol. Biosph. 31,
167-183 (2001).
14. Buschermohle, M., Whittet, D. C. B., Chrysostomou, A.,
Hough, J. H., Lucas, P. W., Adamson, A. J., Whitney, B. A.
and Wolff, M. J. An extended search for circularly polarized
infrared radiation from the OMC-1 region of Orion,
Astrophys. J. 624, 821-826 (2005).
15. Pizzarello, S., Zolensky, M. and Turk, K. A. Nonracemic

4.

Conclusions
We have described here the ways in whichphotoreactions initiated by UV-photons and γ-rays
are capable of generating amino acids and amino acidprecursors from their relating compounds. The
detection of Ser and Ala produced from Ival, and Hyd,
M-Hyd, DM-Hyd and E-Hyd produced from
EM-Hyd by UV and γ-ray photo-alteration process
may facilitate to point out the chemical evolution ofthese prebiotic compounds on the chondrite parent
bodies.
Acknowledgements
The authors convey their gratitude to Masahiro
Katoh, UVSOR-II, Institute for Molecular Science(IMS), Okazaki, Japan, for his kind help andguidance in UV (monochromatic) irradiation
experiments; Issay Narumi, Japan Atomic Energy
Agency, Takasaki, Japan, for his kind help in 60Co
experiments; and Hajime Mita, Fukuoka Institute ofTechnology, Japan, for providing racemic Ival and
EM-Hyd.

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Viva Origino 40 (2012) 4 - 8

16.

17.

isovaline in the Murchison meteorite: Chiral distribution andmineral association, Geochim. Cosmochim. Acta 67,
1589-1595 (2003).
Glavin, D. P. and Dworkin, J. P. Enrichment of the aminoacid L-isovaline by aqueous alteration on CI and CM
meteorite parent bodies, Proc. Natl. Acad. Sci. USA 106,5487-5492 (2009).
Olson, E. S. Amino acids from coal gasification? Nature 357,
202 (1992).

18.

Shimoyama, A. and Ogasawara, R. Dipeptides anddiketopiperazines in the Yamato-791198 and Murchison
carbonaceous chondrites, Orig. Life Evol. Biosph. 32,
165-179 (2002).
19. Sarker, P. K., Takahashi, J. -I., Kawamoto, Y., Obayashi, Y.,
Kaneko, T. and Kobayashi, K. Photostability of Isovaline and
its Precursor 5-Ethyl-5-methylhydantoin Exposed to
Simulated Space Radiations, Int. J. Mol. Sci. 13, 1006-1017(2012).

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