Viva Origino 32 (2004) 53-59

FORMATION OF N-CARBAMOYLALANINE FROM UREA AND PYRUVIC ACID IN AQUEOUS
FORMIC ACID [1]Masanori Terasaki*, Shinya Nomoto, Hajime Mita and Akira Shimoyama**

Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan
Fax: +81-29-853-6503; E-mail: nomoto@chem.tsukuba.ac.jp*Present address: University of Shizuoka, Shizuoka 422-8526, Japan. terasaki@u-shizuoka-ken.ac.jp** Present address: Kochi Gakuen College, Kochi 780-0955, Japan(Received 22 October, 2003 Accepted 20 December, 2003)(Abstract)
When an aqueous solution of urea and pyruvic acid was heated under reflux in the presence of formic acid,
2-ureidopropionic acid, that is, N-carbamoylalanine, was obtained in the yield of about 35%. This compound
was considered to be formed by Leuckart reaction, in which N-alkylation is achieved by formic acid reduction ofprimarily formed Schiff base of a ketone and an amine. It had been believed that Leuckart reaction of amides orurea with formaldehyde does not afford N-alkylated products. Although the intended reaction practically failedto proceed below 100 oC, we found that this reaction can be applied to the alkylation of urea at 100 and 120 oC.
This study proposed a novel pathway leading to formation of alanine from precursors, which are likely present
on the primitive Earth. The formation of N-carbamoylalanine is of another significance, since we found in a
separate experiment that it undergoes thermal polymerization.

(Keyword)chemical evolution, urea, pyruvic acid, Leuckart reaction, N-carbamoylalanine.

Viva Origino 32 (2004) 53-59© 2004 SSOEL Japan- 53 -Viva Origino 32 (2004) 53-59

Introduction

rarely been investigated in the standpoint of chemical

In the course of our studies on the role played

evolution.

by urea in the prebiotic formation of biomolecules on

On the other hand, propionic acid had been

the primitive Earth, we had revealed that molten urea

demonstrated to be formed from methane or graphite

acts as a good nitrogen source and a highly polarin appreciable amounts together with formic andreaction

acetic acids by means of ultraviolet lights or electric

mediumnitrogen-incorporating

thatofdischarges that simulate the energy sources available

maleic and fumaric acids producing polyaspartic acidon the primitive Earth [8, 9]. These energy sources[2]. Under irradiation with UV lights, urea had

are also known to bring about dissociation of a water

proved to add to maleic acid in aqueous solution

molecule to a hydroxyl radical and a hydrogen atom

throughaffordin water phase [10]. Many simulation experiments

2-isoureidosuccinic acid, which underwent thermal

have demonstrated that these reactive species canrearrangement to give 2-ureidosuccinic acid, that is,play a role in modifying non-biomolecules intoN-carbamoylaspartic acid [3]. In the Miller’sbiomolecules, which generally contain many oxidized

experiment (1953) [4], urea was generated amongcarbon atoms in the molecules [11-13]. As one of theother biomolecules from methane, ammonia andmodification, the stepwise oxidation induced bywater by the action of electric discharge followed byhydroxyl radicals has been thoroughly investigatedwater-phase thermal reaction. As pointed out by

and, for instance, propionic acid was transformed into

Miller and Orgel, urea should have been accumulated

pyruvic acid through lactic acid in aqueous solution

to a considerable extent on the primitive Earth, on the[14]. Therefore, formic and pyruvic acids could bebasis of its chemical stability and variety of processes

present in the primitive hydrosphere.

radicalthermal

facilitates

condensation

mechanism

tothat produce urea abiotically, such as hydrolysis of

In this study, we examined abiotic formation of

cyanide polymers or dicyan, UV irradiation to

2-ureidopropionic acid, that is, N-carbamoylalanine

ammonium cyanide in the presence of ferrous ions,from urea and pyruvic acid in aqueous formic acidand so on [5]. Urea and ureido-compounds have beensolution (Equation 1 in Scheme 1). This type of

discovered in the meteorites [6-7]. However, thereaction is known as Leuckart reaction, which usuallysignificance of urea has been little appreciated andemploys ammonia or amines as nitrogen sources [15].

- 54 -Viva Origino 32 (2004) 53-59

OO
H2N

O

NH2

H2N

NHOH

OH

O

(1)

O

HCOOH

N-Carbamoylalanine
OO
H2N

NH2+

H2C=O

H2NONHOH

H2N

HCOOH
NO

CH2

H2N

NH

CH3(2)

Scheme 1In this reaction, formic acid acts as a reducing
agent.

kV.
TheThe reaction of urea or amides withyieldofN-carbamoylalaninewasonlydetermined as that of alanine after hydrolysis of thehydroxymethyl compounds and to fail in obtaining

product. A 5-ml portion of the heated solution was

N-methylated products (Equation 2 in Scheme 1), butconcentrated

Leuckard reaction of urea with ketocarboxylic acidhydrolyzed with 6M HCl (2 ml) for 48 h at 110 oC in

has not been reported [16].

a degassed sealed tube. The hydrolyzate wasformaldehyde

wasreportedtogive

underareducedpressure

andconcentrated under a reduced pressure and analyzed
for amino acids with LC. The amino acid analysis

Materials and methods
The Leuckart reaction was performed by

was performed by a pre-column derivatization

heating an aqueous formic acid solution (10 ml) ofmethod using o-phthalaldehyde/2-mercaptoethanol

urea and pyruvic acid. Heating at 120 oC was carriedaccording to the method of Jones et al. [17]. The

out in a degassed sealed tube. The reaction mixture

analysis was carried out by an LC (Shimadzu LC-10)

was directly analyzed for N-carbamoylalanine with aequipped with an ODS-80T column (Tosoh Ltd.), andliquid chromatograph (LC) coupled to a mass

thespectrometer (MS) (Hewlett Packard series 1100

buffer-MeCN-MeOH.

elutionwaseffectedbyNaphosphate

The authentic sample of N-carbamoylalanine

MSD). The LC was equipped with an amide-80
normal phase column (Tosoh Ltd.), and the elution

waswas effected by MeOH-H2O (99:1). The MS wasisocyanate in a similar manner to that reported [18].

acquired every 0.07 s over m/z 50 to 300 inIn the isolation of the product, we used aelectrospray ionization method by positive mode at 4

cation-exchange resin (Amberlite IRC 120, H+ form)- 55 -

preparedfromL-alanine

andpotassiumViva Origino 32 (2004) 53-59

to acidify and desalt the reaction mixture. This

Hydrolyzate of thereaction mixture

give pure crystals (82 %, mp 182 oC (dec witheffervesce) (lit. 183 oC [18])) without contaminationby the starting material and inorganic salts.

Optical density ( 334 nm)

method facilitated the purification of the product to(a)Results
Authentic Ala

A mixture of urea (0.4 mol/l) and pyruvic acid(0.2 mol/l) in 10% aqueous formic acid was heated(b)1015Retention time / mino

at 40 to 120 C for 6 h, and the Leuckart reaction
proceeded over 100 oC in the yields of about 35%,
as shown in Fig. 1. The yield of the reaction wasFig. 2. Liquid chromatograms of hydrolyzate of thereaction mixture of urea and pyruvic acid at 100 oC for
6 h in the presence of 10% aqueous formic acid (a) andthe authentic sample of alanine (b).

h, only small amounts of alanine were detected. Sinceurea and ureido-compounds are known to be resistantto acid hydrolysis, we hydrolyzed the products for48h, and larger amounts of alanine were found inhydrolyzates (data not shown). These results show
thattheproducts

ureidocarboxylic

aremainlycomposedofacid.

The

formationofN-carbamoylalanine by this reaction was also
confirmed on the [M + 1]+-mass fragmentogram (Fig.
Fig . 1. Yie ld of N-carbamoylalanine from a mixtu resoluti on of ure a (0.4 M) and pyruvic acid (0.2 M) at
various temperatures for 6 h in the pre sence of 10%
aqueous formic acid.

3) of the reaction mixture. This reaction gave the best
yields by using 2 equivalents of urea to pyruvic acidin 5 to 20% aqueous formic acid for more than 5 h,estimated by determining alanine in the hydrolyzateof the reaction mixture (Fig. 2). When the reaction
mixtures were analyzed after being hydrolyzed for 24- 56 -and the yield did not exceed about 35% under all theconditions tested here (data not shown).

Viva Origino 32 (2004) 53-59

Discussion

mixtures. From these results, the Leuckard reaction

Pyruvic acid is rather unstable due to theof urea may be limited to special ketocarboxylic acid.

reactivity of a polarized carbonyl group in its

This study proposed a novel pathway leading totheamino acids from precursors in water phase on thehemiaminal-type (1, Scheme 2) or the aminal-type

primitive Earth. N-Carbamoylamino acid may have aderivative (2) is considered to be formed at first, as inpotential as an intermediate of polyamino acids.

the reaction of urea with glyoxylic acid, which wasCollet

reported to give a mixture of a hemiaminal and an

N-carbamoylamino acids with a mixture of NO andaminal of the acid at room temperature. In theseO2 affords N-carboxyanhydrides, which are known toderivatives, the polarity should be cancelled, andpolymerize under mild conditions [19]. Furthermore,their lifetimes may be longer than that of thewe have recently found that N-carbamoylalanine is

ketocarboxyliciscapable of undergoing thermal polymerization at its

considered to be formed through dehydration of 1 or

melting point [20]. Further researches are under way

elimination of urea from 2 followed by formic acidin order to find out an abiotic formation pathway to

reduction of a C-N double bond of a Schiff base (3).

other N-carbamoylamino acids and to investigate

We further carried out the reaction of urea withpolymerizing

glyoxylic and oxaloacetic acids at 100 oC, expecting

N-carbamoylamino acids.

molecule.

Onformationaccount

acid.

ofN-carbamoylaspartic

of

its

polarity,

N-Carbamoylalanine

respectively,al.

andshowedthat

copolymerizing

reaction

abilities

ofofandN-carbamoylglycine
acid,

etbut

nodesired products were detected in the reaction

OR

Urea
R'HO

NH-CO-NH2

R

R'UreaH2N-CO-HN

NH-CO-NH2

R

12or- H2O- Urea

N-CO-NH2 HCOOH
RR'

3Scheme 2- 57 -R'

H

NH-CO-NH2RR'

Viva Origino 32 (2004) 53-59

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