Modelling of prebiotic synthesis and selection of peptides under isothermal conditions and thermal cycling mode

Article abstract of DOI:10.1007/s11172-012-0060-3

The model peptide synthesis from mixtures of amino acids was carried out under the thermal cycling and isothermal modes. The compositions of the obtained mixtures of products and the primary amino acid sequence of the synthesized peptides were determined by Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry in combination with high-performance liquid chromatography with the application of de novo sequencing of the synthesized products. The processes of abiogenous synthesis of peptides were shown to occur under relatively mild temperature conditions and give a substantially less number of peptides as compared with the possible statistical set. The evolution of the system takes place in the process of the synthesis in solid phase with the disappearance of a series of the most unstable peptides. The selection process with the formation of complementary peptides takes place in peptide synthesis under the thermal cyclic mode.

Full text of DOI:10.1007/s11172-012-0060-3

Russian Chemical Bulletin, International Edition, Vol. 61, No. 2, pp. 422—441, February, 2012
422
Modelling of prebiotic synthesis and selection of peptides
under isothermal conditions and thermal cycling mode
O. V. Demina,a A. S. Kononikhin,a,b A. V. Laptev,a A. A. Khodonov,a E. N. Nikolaev,a,b and S. D. Varfolomeeva
aN. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences,
4 ul. Kosygina, 119334 Moscow, Russian Federation.
Fax: +7 (499) 137 4101. Eꢀmail: ovd@sky.chph.ras.ru
bInstitute for Energy Problems of Chemical Physics, Russian Academy of Sciences,
38/2 Leninsky prosp., 119334 Moscow, Russian Federation.
Fax: +7 (499) 939 7370
The model peptide synthesis from mixtures of amino acids was carried out under the
thermal cycling and isothermal modes. The compositions of the obtained mixtures of products
and the primary amino acid sequence of the synthesized peptides were determined by Fourier
transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry in combiꢀ
nation with highꢀperformance liquid chromatography with the application of de novo sequencꢀ
ing of the synthesized products. The processes of abiogenous synthesis of peptides were shown
to occur under relatively mild temperature conditions and give a substantially less number of
peptides as compared with the possible statistical set. The evolution of the system takes place in
the process of the synthesis in solid phase with the disappearance of a series of the most unstable
peptides. The selection process with the formation of complementary peptides takes place in
peptide synthesis under the thermal cyclic mode.
Key words: amino acids, biopolymers, mass spectrometry, peptide synthesis, peptides, therꢀ
mal synthesis, thermal cycling.
The problem of the appearance of selfꢀreplicating polyꢀ
ly that the process of the system "ordering" had been ocꢀ
curred with the drastic reduction of possible variants of
macromolecules.1
mer molecules is the key question of life origin. At present,
it is assumed that multiple lowꢀmolecular organic comꢀ
pounds, being the starting substances for the synthesis
of proteins, nucleic acids, and sugars, had arisen on
the early Earth due to various chemical and radiation
processes.1—14 This direction of the search for the methods
of biomacromolecules synthesis with the modelling of
its possible prebiotic conditions of the synthesis is develꢀ
oping intensively due to new physicochemical methods of
investigation.
The emergence of information and catalytically funcꢀ
tioning macromolecular structures, such as nucleic acids
and proteins, is fundamentally important. The almost inꢀ
finite variety of molecules different in real structure could
appear due to the spontaneous formation of phosphodiester
(nucleic acids) and peptide (proteins) bonds. The huge
decrease in the variants of protein structures occurred durꢀ
ing the origin of living systems and their consequent
evolution, and a very limited number of catalytic sites of
enzymes has been observed in the modern biological
world.15,16 The proteins and nucleic acids presently existꢀ
ing on the Earth are a very small part of the molecules that
could appear due to the primary processes and evolve to
real biological structures. This means phenomenologicalꢀ
Thus, it is necessary to explain the mechanisms of
molecular convergence of macromolecular structures and
their selection by some properties, as well as the appearꢀ
ance of the mechanism of their proliferation (selfꢀreplicaꢀ
tion) for the understanding the phenomenon of the origin
of life. It is important that the advanced concepts would
be based on the known fundamental laws of development
of natural phenomena and would not violate the known
thermodynamic limitations, in particular, the second law
of thermodynamics.
The great attention is given to the problems of evoluꢀ
tion of macromolecules at the prebiotic level.10,17—22
In particular, outstanding book written by M. Eigen18
gives the kinetic description of selection processes of
macromolecules using the "competitive advantage"
concept. However, beyond the scope of these studies,
the questions remain unanswered about the mechanism
of macromolecule selfꢀreproduction processes and about
the driving force providing ordering, "competitive advanꢀ
tage," and, finally, molecular convergence of polymer
variety to a restricted number of types of biomacroꢀ
molecules.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 419—437, February, 2012.
1066ꢀ5285/12/6102ꢀ422 © 2012 Springer Science+Business Media, Inc.

Modelling of prebiotic synthesis of peptides
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
423
a mixture of methanol, chloroform, and 10% aqueous solution of
ammonia in ratio 4 : 4 : 2 as an eluent and a 5% solution of
ninhydrin in methanol as a developer were used for TLC.
1Н NMR spectra were recorded on Bruker ADꢀ600 and
Bruker DPXꢀ300 devices (Germany) with a working frequency
of 600 and 300 MHz, respectively, in D2O and DMSOꢀd6.
Chemical shifts of the signals were determined relative to acetoꢀ
nitrile (MeCN,  1.98) as an external standard. The 2D COSY
1Нꢀ1Н NMR correlation spectra were also recorded for the asꢀ
signment of signals of separate groups.
We suppose that the primary mechanism of selfꢀrepliꢀ
cating and evolution of macromolecules is interconnected
with the thermocycling conditions.23,24 As a result of rotaꢀ
tion, any point of the Earth surface experiences cyclic
temperature fluctuations. These fluctuations had an exꢀ
tremely high amplitude on the primary Earth with a rather
weak atmosphere. These fluctuations could be characterꢀ
ized by a wide spectrum of amplitudes from –100 to 200 C
in dependence of specific conditions (radiation, heat exꢀ
change, thermal conductivity). A variety of chemical reꢀ
actions and phase transitions are possible under these conꢀ
ditions.
Modern biomacromolecules are the products of polyꢀ
condensations which occurred on the early Earth and reꢀ
sulted in the emergence of amide (proteins and peptides),
phosphodiester (nucleic acids), and acetal (polysacchaꢀ
rides) bonds. In the formalized form, monomeric moleꢀ
cules should be trifunctional including two reactive groups
essential for the polymerization and substituents allowing
one to distinguish chemically one molecule from another.
The substituents can interact with each other supramolecꢀ
ular with one or other selectivity determined by the free
energies of complexes formation owing to hydrogen bonds
and ion or hydrophobic interactions.
Mass spectrometry of all samples was carried out on a Finnigan
LTQ FT combined mass spectrometer (Thermo Electron, Gerꢀ
many) consisting from an ion cyclotron resonance mass specꢀ
trometer with Fourier transform (FT ICR) equipped with a suꢀ
perconducting solenoid of 7 T and a highly sensitive linear quaꢀ
drupole ion trap. Mass spectra were recorded in the FT ICR
mode using such ionization methods as the electrospray (ESI)
and the matrixꢀassisted laser desorption/ionization (MALDI).
Mass spectrometric analysis combined with chromatography was
carried using tandem mass spectrometry in combination with
highꢀperformance liquid chromatography (HPLCꢀMS/MS), inꢀ
cluding the preliminary fractionation of samples by the nano
flow highꢀperformance liquid chromatograph Agilent 1100
(Agilent, US) on the selfꢀmade column 75 m×15 cm with the
reversed phase ReproSil Pur C18 (a particle diameter is 3 m,
a pore diameter is 100 Å (Dr. Maisch GmbH, Germany)) using
the mobile phase Н2О—MeCN with the addition of formic acid
to the concentration of 0.1 vol.% (MeCN gradient in the flow
Thus, the following conditions are needed for the synꢀ
thesis of prebiotic macromolecules:
from 5 to 50% within 90 min at a flow rate of 0.3 L min–1
)
followed by the analysis of fractions by means of the measureꢀ
ment of exact masses with FT ICR mass spectrometer and the
acquiring collisionꢀinduced dissociation (CID) spectra in a linear
quadrupole ion trap in the m/z range from 300 to 2000.
Tandem mass spectrometry with CID as a fragmentation
method was used for the confirmation of peptide nature of the
molecular ions and the determination of possible structures of
molecular ion fragments.
(1) trifunctional monomers containing several types of
substituents (for example, for amino acids: amino group,
carboxyl group, and a substituent containing an additional
functional group);
(2) excess of monomers;
(3) thermal cycling, i.e., a cyclic change of temperaꢀ
ture providing the reversible phase transition of water from
the liquid to gaseous state and back.
The kinetic theory of prebiotic evolution under the
thermal cycling conditions was described in detail in pubꢀ
lications.1,23 It is supposed that the process of prebiotic
evolution occurred successfully with very little characterꢀ
istics of "competitive advantage."
The purpose of this work is the experimental study of
the synthesis of peptides from mixtures of oppositely
charged amino acids under two possible synthesis condiꢀ
tions, such as an isothermal and a thermocyclic (thermal
cycling) modes.
The compositions of the samples and the conditions of synꢀ
thesis are given in Tables 1 and 2.
Synthesis of peptides and their derivatives under the thermal
cycling mode from the mixture of amino acids using different pH
values of the medium (general procedure). Aqueous solutions of
mixtures of amino acids LꢀAsp and LꢀArg (see Table 1, runs 1—4),
LꢀAsp and LꢀLys•HCl (see Table 1, runs 5—8) in ratios of 1 : 1
and 1 : 2, respectively, were prepared with the solutions pH
reduced to 4.5 and 8.5 with HCl or NaOH. The volume of the
initial solutions was 15—25 mL for LꢀAsp (0.68 mmol) and
LꢀLys•HCl (0.68 mmol), as well as for LꢀAsp (0.68 mmol) and
LꢀArg•HCl (0.68 mmol), at the 1 : 1 ratio. An aliquot of the
initial solution (2 mL) was placed into a thermoꢀresistant beaker
covered with a perforated lid, and water was evaporated by slow
heating to 120, 130, or 140 C. After water evaporation, the reꢀ
action mixture was heated for 12 or 24 h at 120 or 130 C, cooled
to ambient temperature, and dissolved in a minimal volume of
water (~1 mL). A sample (50 L) was taken, and a new aliquot of
the starting solution of amino acids was added. The cycle
was repeated 7—9 times. After the last sample taking, the reacꢀ
tion mixture was heated for 36 h without the addition of
the initial mixture aliquot for subsequent comparison with
the penultimate probe. The synthesis of peptides from mixtures
of LꢀGlu and LꢀLys•HCl, LꢀAla and LꢀGly was done similarly
Experimental
The following reagents were used in experiments: amino acids
Lꢀlysine hydrochloride (LꢀLys, K), Lꢀaspartic acid (LꢀAsp, D),
Lꢀarginine hydrochloride (LꢀArg, R), and Lꢀglutamic acid (LꢀGlu,
E) (special purity grade, DiaꢀM, Russia); sulfuric acid (special
purity grade), hydrochloric acid (special purity grade), and sodiꢀ
um hydroxide (special purity grade) (produced in Russia).
The monitoring of reactions was performed by thin layer
chromatography (TLC). Plates with silica gel 60 F254 (Merck),

424
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
Demina et al.
Table 1. Reagents, samples, and conditions of the thermal cycling synthesis
Run
Reagents and conditions
Probe 1
Probe 2
Probe 3*
Sample
t/h (N)
Sample
t/h (N)
Sample
t/h (N)
1
2
3
4
5
6
7
8
LꢀArg•HCl—LꢀAsp (1 : 1), 120 C, рН 4.5
LꢀArg•HCl—LꢀAsp (1 : 1), 120 C, рН 8.5
LꢀArg•HCl—LꢀAsp (2 : 1), 120 C, рН 4.5
LꢀArg•HCl—LꢀAsp (2 : 1), 120 C, рН 8.5
LꢀLys•HCl—LꢀAsp (1 : 1), 120 C, рН 4.5
LꢀLys•HCl—LꢀAsp (1 : 1), 120 C, рН 8.5
LꢀLys•HCl—LꢀAsp (2 : 1), 120 C, рН 4.5
LꢀLys•HCl—LꢀAsp (2 : 1), 120 C, рН 8.5
1А
2А
3А
4А
5А
6А
7А
8А
36 (3)
36 (3)
36 (3)
36 (3)
36 (3)
36 (3)
36 (3)
36 (3)
1B
2B
3B
4B
5B
6B
7B
8B
85.5 (7)
85.5 (7)
85.5 (7)
85.5 (7)
108 (9)
108 (9)
108 (9)
108 (9)
1C
2С
3С
4C
5C
6C
7C
8C
121.5 (7)
121.5 (7)
121.5 (7)
121.5 (7)
144 (9)
144 (9)
144 (9)
144 (9)
Note. t is the heating time, and N is the number of cycles.
* After taking probe 2, the sample was heated for 36 h more without addition of an aliquot of the initial amino acid mixture.
(the same amounts of the catalysts and ratios of the mixture
components).
Experiment 12 was carried out as control without any catalyst
but with the addition of distilled water (2 mL). Samples were
taken after 104 h (probe 1) and 278 h (probe 2) of the heating.
After prolonged heating (104 h and more) the reaction mixꢀ
ture of samples 9А—11А and 9B—11B became semitransparent
and glassy with a slight yellowish tint. The reaction mixture of
samples 12А—13А and 12B—13B remained visually almost
unchanged.
Synthesis of peptides and their derivatives in the solid phase
from the mixture of amino acids using various catalysts (see Table 2,
runs 9—13). Amino acids LꢀAsp (3.4 mmol) and LꢀLys•HCl
(3.4 mmol) were put in the thermoꢀresistant beaker, and the
catalysts were added, such as concentrated sulfuric acid (run 9:
2 equiv.; run 10: 3 equiv.), hydrochloric acid (run 11: 3 equiv.),
or sodium hydroxide (run 13: 4 equiv.) (see Table 2). The mixꢀ
ture was mixed and heated in the drying cabinet at 130 C.
Synthesis of peptides and their derivatives in the solid phase
from a mixture of amino acids using sulfuric acid as a catalyst (see
Table 2. Reagents, samples, and conditions of the synthesis in solid phase
Run
Reagents and conditions
Probe 1
Sample
Probe 2
Sample
t/h
t/h
Series I
9
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, 2 equiv. H2SO4
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, 3 equiv. H2SO4
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, 3 equiv. HCl
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, H2O
19А
10А
11А
12А
13А
104
104
104
104
104
19B
10B
11B
12B
13B
278
278
278
278
278
10
11
12
13
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, 4 equiv. NaOH
Series II
14
15
16
17
18
LꢀLys•HCl—LꢀAsp (1 : 1), 130 C, 2 equiv. H2SO4
LꢀLys•HCl—LꢀAsp (1 : 2), 130 C, 3 equiv. H2SO4
LꢀLys•HCl—LꢀAsp (2 : 1), 130 C, 3 equiv. H2SO4
LꢀAsp, 130 C, 2 equiv. H2SO4
14А
15А
16А
17А
18А
138
138
138
138
138
14B
15B
16B
17B
18B
300
300
300
300
300
LꢀAsp, 130 C, 4 equiv. H2SO4
Series III
19
20
21
22
23
24
25
26
27
LꢀArg•HCl—LꢀAsp (1 : 2), 140 C, 3 equiv. H2SO4
LꢀArg•HCl—LꢀAsp (1 : 1), 140 C, 2 equiv. H2SO4
LꢀArg•HCl—LꢀAsp (2 : 1), 140 C, 3 equiv. H2SO4
LꢀLys•HCl—LꢀGlu (1 : 2), 140 C, 3 equiv. H2SO4
LꢀLys•HCl—LꢀGlu (1 : 1), 140 C, 2 equiv. H2SO4
LꢀLys•HCl—LꢀGlu (2 : 1), 140 C, 3 equiv. H2SO4
LꢀArg•HCl—LꢀGlu (1 : 2), 140 C, 3 equiv. H2SO4
LꢀArg•HCl—LꢀGlu (1 : 1), 140 C, 2 equiv. H2SO4
LꢀArg•HCl—LꢀGlu (2 : 1), 140 C, 3 equiv. H2SO4
19А
20А
21А
22А
23А
24А
25А
26А
27А
100
100
100
100
100
100
100
100
100
19B
20B
21B
22B
23B
24B
25B
26B
27B
300
300
300
300
300
300
300
300
300
Note. t is the heating time, and N is the number of cycles.

Modelling of prebiotic synthesis of peptides
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
425
Table 2, runs 14—18). The synthesis was carried out according
to the above described procedure from a mixture of LꢀAsp
(3.4 mmol) and LꢀLys•HCl (3.4 mmol) adding various amounts
of concentrated sulfuric acid as the catalyst (see Table 2). Samꢀ
ples were taken after 138 and 300 h the heating. The ratio of
amino acids was also varied, i.e., 6.8 mmoles of LꢀAsp and
3.4 mmoles of LꢀLys•HCl were taken for the preparation of
samples containing LꢀAsp and LꢀLys•HCl with the ratio equal
to 2 : 1; 3.4 mmoles of LꢀAsp and 6.8 mmoles of LꢀLys•HCl were
taken for the samples LꢀAsp : LꢀLys•HCl in a 1 : 2 ration. Control
samples contained only LꢀAsp (7.5 mmol) and various amounts
of sulfuric acid (see Table 2, runs 17 and 18). The syntheꢀ
ses of peptides from LꢀLys•HCl—LꢀGlu, LꢀArg—LꢀGlu,
and LꢀArg—LꢀAsp mixtures were carried out using the same
amounts of sulfuric acid and the same ratios of the components
(see Table 2).
Alkaline hydrolysis of the obtained reaction mixtures. The hyꢀ
drolysis of the reaction mixture was performed according to the
modified procedure.25 The calculated amount of sodium hydrꢀ
oxide solution in water (2.12 М NaOH, 0.425 g of sodium hyꢀ
droxide per 1 g of the reaction mixture) was placed in a 25ꢀmL
roundꢀbottom flask equipped with a magnetic stirrer. The mixꢀ
ture was cooled to 0 C and then the reaction mixture of the
sample was added by several portions. After the sample was comꢀ
pletely dissolved under the stirring, the resulting mixture was
heated to ambient temperature, stirred for 1 h, and then acidiꢀ
fied to pH 6. The obtained mixture was added by portions to
methanol (40 mL) cooled to 0 C. The formed white precipitate
was filtered off and dried under the reduced pressure. The quanꢀ
tities of polymers in the samples of runs 14—18 after the treatꢀ
ment were the following: in sample 14B (2 equiv. H2SO4), 0.18 g
of the polymer mixture was obtained from 0.51 g of the treated
reaction mixture; in sample 15B (3 equiv. H2SO4), 0.22 g of the
polymer mixture was obtained from 0.5 g of the treating reaction
mixture; in the sample 17B (2 equiv. sulfuric acid), 0.3 g of the
polymer mixture was obtained from 0.5 g of the treating reaction
mixture with the total yield >50%; in the sample 18B (4 equiv.
H2SO4), 0.3 g of the polymer mixture from 0.65 g of the treating
reaction mixture was obtained with the total yield about 50%.
Mass spectrometry of the products of amino acids polyconꢀ
densation. The mass spectrometric analysis of mixtures of the
obtained reaction products (see Tables 1 and 2) revealed that the
reaction mixtures contained products of amino acids polyꢀ
condensation (peptides and their derivatives) and unreacted
initial amino acids. FT ICR mass spectrometry was used for the
study of the obtained reaction mixtures compositions containing
minor amounts of peptides (no more than 2—5%, according to
the 1H NMR spectroscopic data (see below).
ent products in the case of the MALDIꢀTOFF mass spectroꢀ
metry usage.
The method of ion identification by tandem mass spectromꢀ
etry with CID as an ionization method (see below) was used for
the confirmation of peptide bonds presence and the identificaꢀ
tion of separate ions structure in the mass spectrum.
The formation of diketopiperazines for each mixture of amiꢀ
no acids during thermal synthesis was indicated at the decoding
of the mass spectra, for example, the peak with m/z 257.18 at
z = 1 is assigned to diketopiperazine formed during heating
from two lysine molecules with the elimination of two water
molecules.
Thus, it must be noted the use of both positive and negative
modes, as well as complementary ionization and fragmentation
methods for sample analysis are necessary in order to obtain
more reliable information about the mixture composition.
The data for samples 9А and 9B are presented as examples of
the results obtained by HPLCꢀMS/MS for the samples of
runs 9—14 (see below).
Results and Discussion
Mixtures of oppositely charged amino acids were used as
the initial reagents in the experimental study of the pepꢀ
tide synthesis. We used Lꢀamino acids bearing a charge in
the side chain, namely, aspartic (LꢀAsp, D) and glutamic
(LꢀGlu, E) acids with the negatively charged carboxyl
group of the side chain and lysine (LꢀLys, K) or arginine
(LꢀArg, R) with the positively charged amino or guanidinꢀ
ium groups). The possibility of the synthesis of peptides in
these model systems was studied under isothermal (therꢀ
mal synthesis mode) and thermal cycling conditions (therꢀ
mal cycling mode). The prepared dry mixtures were heatꢀ
ed for a certain time under the thermal synthesis mode in
solid phase. Under the thermal cycling mode, the initial
cycle was multiply repeated, namely, the heating of an
aliquot of solution of amino acids initial mixture — the
phase transition—the polycondensation of monomers in
the solid phase — the cooling — water condensation — the
interaction of monomers with synthesized polymer.
The polycondensation process in the isothermal mode
was carried out in a wide range of initial conditions using
the catalysts (see Table 2, series I, runs 9—13): (1) conꢀ
centrated sulfuric acid, (2) concentrated hydrochloric acid,
(3) sodium hydroxide, and (4) distilled water as a control.
The certain amount of each catalyst was added to the
thoroughly stirred mixture of amino acids. It was found
that such polymerization of amino acids in the solid phase
at the isothermal synthesis mode can occur in an acidic
and a neutral media as well as in an alkaline medium. This
process can be catalyzed by acids, bases, and metal cations.
The chosen reaction conditions overlapped practically the
whole range of possible acid—alkaline conditions of the
prebiotic Earth26,27 (pH from 4.2 to 8.2). It was shown
that the polymerization was effective under these conditions.
The reaction rate and the degree of conversion depended
The next conditions for mass spectra recording were chosen
in order to determine the composition of polymers of series I and
II (see Table 2): an acetonitrile—water mixture with the addition
of formic acid to the concentration 0.1 vol.% as a solvent, the
use of several ionization methods (electrospray and MALDI),
and the detection of both positive and negative ions for the same
sample. The system was washed for 15 min with 95% MeCN and
then it was cleaned for 5 min with water containing 0.1% TFA. It was
found that the degree of sample dilution influenced very strongly
to the determination of its composition. The electrospray
was the best ionization method for mass spectrometric analyꢀ
sis of complex mixture of polymers and peptides, since we
failed to select the appropriate matrix for mixtures of so differꢀ

426
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
Demina et al.
on the temperature and the duration of the process. The
polymerization occurred under relatively mild conditions at
temperatures from 120 to 140 C and did not achieve the
high degree of polymerization, which, however, could inꢀ
crease under the certain conditions,25,28 for instance, when
polyphosphoric acid was used as a catalyst and the heating
temperature was higher than 160 C. The low degree of
polymerization was the necessary condition for the deꢀ
tailed analysis of the obtained mixtures of polypeptides.
The reaction conditions for the thermal cycling mode were
chosen in such a way that the degree of conversion would
not exceed 5% per one cycle.
prepared earlier using the polyphosphoric acid as a cataꢀ
lyst at temperatures higher than 160 C under nitrogen
and a low vacuum.25,28,29 The alkaline hydrolysis of these
polysuccinimides mixtures followed by the structures deꢀ
termination of the obtained polyaspartic acids by NMR
spectroscopy revealed the presence of various terminal
fragments, some of which were not identified.25,28,29
We determined the structures of polymers obtained in the
mixtures of amino acids and from LꢀAsp only and also idenꢀ
tified the terminal fragments by FT ICR mass spectrometry.
The duration of amino acid polycondensation deterꢀ
mined the composition of the polymerization products.
The system evolved with time towards a decrease in the
variety and the number of possible structures.
The determination of polymers structures was carried
out with the usage of de novo sequencing by means of FT
ICR mass spectrometry and tandem mass spectrometry in
combination with highꢀperformance liquid chromatograꢀ
phy (HPLCꢀMS/MS). Thus, all synthesized peptides in
the obtained complicated mixtures of the products of amino
acids polycondensations and their primary amino acid seꢀ
quences were first defined by the HPLCꢀMS/MS methods.
Synthesis of peptides. Polymerization of amino acids in
the solid phase under isothermal conditions. The conditions
for the formation of polymer (peptide) chains were chosen
for abiogenic thermal synthesis in model systems at the
periodical mode in the presence of catalysts: mineral acids
(concentrated sulfuric or hydrochloric acids) or base (soꢀ
dium hydroxide) for a mixture of LꢀLys and LꢀAsp (see
Table 2, series I). Amino acids LꢀLys and LꢀAsp were used
in the ratio 1 : 1 without a solvent (water) with the addiꢀ
tion of different equivalents of sulfuric and hydrochloric
acids or sodium hydroxide. It should be mentioned
that polyaspartic acid derivatives, polysuccinimides with
a small amount of lysine, were mainly obtained, as well as
small amounts of peptides. After 278 h of heating of
sample 9B (2 equiv. H2SO4), polymers containing from 2
to 12—13 residues were obtained, whereas for sample 10B
(3 equiv. H2SO4) the number of amino acid residues in
the polymers varied from 2 to 8—11. Both polyaspartic
acid derivatives and usual peptides of rather diverse comꢀ
position, which underwent the cyclization under the furꢀ
ther heating to form polyaspartic acid derivatives, were
obtained in the sample 11А (HCl as a catalyst). After 278 h
of heating, predominantly polymers containing from 2 to
7—8 units (the destruction of the terminal fragment is
possible) were obtained in a mixture of sample 11B. The
initial polymerization of aspartic acid was observed from 2
to 6—8 (very weak signals) residues at the noise level, and
clusters of amino acids were observed too in the control
sample 12B (water). For sample 13B (NaOH as a cataꢀ
lyst), the mass spectrum exhibited only peptides (diꢀ, triꢀ,
tetraꢀ, and pentapeptides) in the absence of aspartic acid
cyclization, and the amount of peptides did not exceed
2—5% (according to the 1Н NMR spectroscopic data).
The polymers, polysuccinimides with a high molecuꢀ
lar weight, being polyaspartic acid derivatives, have been
The compositions of products mixtures for 9А and 9B
samples obtained by the FT ICR and HPLCꢀMS/MS mass
spectra interpretation are presented in Table 3 as the exꢀ
amples (see also Table 2, series I, run 9; Schemes 1 and 2).
Probe 1 and probe 2 were different only by the heating
time, i.e., probe 1 was heated for 104 h and probe 2 was
heated for 278 h.
The comparison of the products obtained in samples of
run 9 (see Table 2) in dependence of the heating time at
the same temperature shows that the increase in the reacꢀ
tion time leads to the chain elongation in products of
series 3 and 1. The decomposition processes of unstable
structures and the formation of new structures occur in
the solid phase under the permanent heating. It was obꢀ
served that the variety of products becomes narrower unꢀ
der the increase in heating time, viz., series 5 disappears
(see Table 3, Schemes 1 and 2). The peptides obtained
experimentally in very small and even trace amounts were
revealed and identified by HPLCꢀMS/MS (see Figs 1 and 2,
respectively). This shows the efficiency of the technique
used by us for the identification of obtained products in
complex mixtures. After incubation of the system during
104 h, we unambiguously identified the peptide derivaꢀ
tives KDddddddddK and the series of products: DDKK,
DDKKd, DDKKdd, DDKKddd, and DDKKdddd (d is
the succinimide residue), which were not revealed after
278 h of heating.
The sequences of amino acids residues or their derivaꢀ
tives were determined for the structures of the same series.
The de novo sequencing and the determination of relative
probabilities were performed for all structures presented in
Schemes 1 and 2 by means of the processing programs for
mass spectra. The peptide nature was confirmed for ions
with m/z 501.1 and 271.2, i.e., the molecular ion with m/z
501.1 corresponds to the ion [NH2 – Asp – Asp – Asp –
–Asp – OH + Na]+, the molecular ion with m/z 386.08
corresponds to the ion [NH2 – Asp – Asp – Asp – OH +
+ Na]+, the molecular ion with m/z 271.18 corresponds to
the ion [NH2 – Asp – Asp – OH +Na]+, and the molecuꢀ
lar ion with m/z 156.0 corresponds to the ion [Asp + Na]+
at z = 1 for all molecular ions (Fig. 3).

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427

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Modelling of prebiotic synthesis of peptides
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
429
Table 3. Composition of the mixture of products determined by the HPLCꢀMS/MS method for samples 9А and 9B
Number
of residues
Formula*,**
Molecular ion
[M + H]+, m/z
Number
of residues
Formula*,**
Series 3
Molecular ion
[M + H]+, m/z
Series 0
4
5
5
Kddd
Kdddd
KdddK
438.1631
535.1795
566.258
2
2
3
DK
KK
DKD
262.1409
275.2089
377.16783
6
6
7
7
8
8
9
9
Kddddd
632.1958
663.2744
729.2122
760.2908
826.2286
857.3072
923.245
954.3235
1020.261
1117.278
1214.294
Cерия 1
KddddK
Kdddddd#
KdddddK
Kddddddd#
KddddddK
Kdddddddd#
KdddddddK
Kddddddddd#
Kdddddddddd#
Kddddddddddd#
4
5
6
7
8
9
10
11
12
13
DddK
DdddK
DddddK
DdddddK
DddddddK
DdddddddK
DddddddddK
DdddddddddK
DddddddddddK
DdddddddddddK#
456.1736
553.19
650.2064
747.2228
844.2391
941.2555
1038.272
1135.288
1232.305
1329.31
10
11
12
Series 4
Series 2
2
3
4
5
6
7
8
9
DD
DDK
DDdK
DDddK
249.0644403
377.16783
474.18421
571.20058
668.21696
765.23333
862.24971
959.26608
1056.2825
1153.2988
1250.3152
2
3
4
5
6
7
8
9
KD
KDK
KDdK
KDddK
KDdddK
KDddddK
KDdddddK
KDddddddK
KDdddddddK
KDddddddddK##
262.1409
390.23585
487.25223
584.2686
681.28498
778.30135
875.31773
972.3341
DDdddK
DDddddK
DDdddddK
DDddddddK
DDdddddddK
DDddddddddK
DDdddddddddK
10
11
12
10
11
1069.3505
1166.3669
Series 3
Series 5
2
3
3
3
4
Kd
244.1303
372.2253
341.1467
359.1573
469.2417
4
5
6
7
8
DDKK##
505.2628
602.2792
699.2955
796.3119
893.3283
KdK
Kdd
KdD
KddK
DDKKd##
DDKKdd##
DDKKddd##
DDKKdddd##
* d is the succinimide residue.
** Products marked by index # were not identified for the reaction time of 104 h but were identified for the reaction time of 278 h;
products marked by ## were identified for the reaction time of 104 h and were not identified after 278 h.
The peptides NH2—Lys—Asp—COOH (KD) and
NH2—Asp—Asp—COOH (DD) may be initial compounds
for a number of presented series: NH2—Lys—Asp—COOH
for series 2 and 3, and NH2—Asp—Asp—COOH for series
1 and 4. Possibly, the peptides of series 5 are formed from
dipeptides NH2—Asp—Asp—COOH and NH2—Lys—
Lys—COOH (KK), without the exclusion the possibility
of consequent growth of the peptide chain (see Scheme 1
and Table 2, run 9, sample 9А). Probably, the number of
peptides and their derivatives included the Lys—Lys fragꢀ
ment is too insignificant, and we did not observe the strucꢀ
tures with this fragment by means of HPLCꢀMS/MS, alꢀ
though similar structures were revealed by the preliminary
interpretation of the mass spectra without HPLC.
The analysis of the obtained reaction products in exꢀ
periments of series I (see Table 2) indicated that concenꢀ
trated sulfuric acid was the most efficient catalyst of
the process.
Based on the results of studies for the choice of condiꢀ
tions for the formation of polymer structures with masses
more than 1000 Da (see Table 2, series I) in order
to increase the yield of the polycondensation products,
the influence of concentrated sulfuric acid amounts on
the polypeptides and polymers synthesis in the Lys—Asp
system was investigated with the addition of 2, 3, and
4 equiv. H2SO4 under the heating at 130 C without
a solvent for a long time (138—300 h) (see Table 2, series II,
runs 14—18, and Experimental). Dry amino acids, LꢀLys

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Demina et al.
a
I (arb. units)
11.02
14.12
14.22
15.85
16.80
12.94
12.50
80
60
40
17.59
27.73
25.51
24.47
9.75
7.92
9.08
28.97
31.42
20
33.39
4.68
6
10
14
18
22
26
30
t/min
I (arb. units)
b
937.40
Kd7K
80
60
40
20
891.27
826.36
780.32
909.32
615.28
472.30
729.47
954.53
300 350 400 450 500 550 600 650 700 750
800 850 900
m/z
Fig. 1. The HPLCꢀMS/MS data for the sample 9B: a, a peak in the mass chromatogram corresponding to the fraction containing
peptide with a relatively higher molecular weight; b, the mass spectrum of the CID fragmentation products obtained with the linear
quadrupole ion trap. The amino acid sequence of the peptide (Kd7K, where d is the succinimide residue) was calculated manually
using the MS/MS data.
and LꢀAsp, were taken in ratios of 1 : 1 and 1 : 2 and LꢀAsp
only was used for the comparison and control.
tioned that the mass of these polysuccinimide derivatives
increases under the registration of negative ions in comꢀ
parison with that under the resitration of positive ions.
The mass spectrum of a polymer mixture of sample 18B
after the alkaline treatment and acidification with positive
ion registration is shown in Fig. 4, c, and a period of
115 corresponds to the mass of the aspartic acid residue
and unambiguously indicates the presence of Asp residues
as polymer chain units, but we did not determine the ratio
of ꢀ and ꢀisomers. Since the alkaline hydrolysis is
not mild, the racemization of the final product occurs in
these samples.
It was established that under the interpretation of the
mass spectra the polyaspartic acid containing from 20 to
23 units (when recording negative ions) or 17—18 units
(when recording positive ions) was formed for the same
sample after alkaline hydrolysis and acidification of the
dried precipitate from samples 17B and 18B of series II
(see Table 2). This means that the detection of both posiꢀ
tive and negative ions is necessary when recording similar
mass spectra.
According to TLC data, the reaction mixtures of samꢀ
ples 14А, 14B, 15А, and 15B contained Asp, Lys, polysucꢀ
cinimide, and various polymer products of reaction, whereas
Asp and polysuccinimides were observed in the samples of
experiments 17 and 18. According to the mass spectroꢀ
metric analysis data, the reaction mixture obtained from
a mixture of Lys and Asp even after 300 h of heating conꢀ
tained the structures with the aspartic acid residue instead
of the succinimide fragment. This was not observed for the
samples consisting of aspartic acid only. The obtained
experimental data allow one to suggest the spontaneꢀ
ous polymerization of aspartic acid at the beginning folꢀ
lowed by the cyclization of the polyaspartic acid to polyꢀ
succinimide.
Mass spectra of samples 14B and 18B of series II are
presented in Fig. 4 (see Table 2). The period equal to 97
and corresponding to the mass of the succinimide residue
is well seen in the mass spectrum of sample 14B (see
Fig. 4, а). Periods of 97 characteristic of succinimide resiꢀ
dues in the polymer chain were revealed in the mass specꢀ
tra of sample 18B (see Fig. 4, b and c) in the final reaction
mixture (before alkaline hydrolysis). It should be menꢀ
Using the mass spectra of the final reaction mixtures
of samples 14B, 15B, and 16B (before alkaline hydrolysis),
we obtained the data confirming the formation of peptide

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Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
431
I (arb. units)
a
39.97
39.40
39.15
40.31
40.61
80
60
41.10
38.54
42.25
42.68
n = 14
37.67
37.29
42.99
1.64
40
8.95
4.14
43.36
19.08
23.05 24.87
36.86
20
36.11
28.77
6.29
20.63
32.73
45.47
5
10
15
20
25
30
35
40
t/min
[M – H2O]+
b
I (arb. units)
808.58
Kd14K
80
60
40
20
799.53
+
b14
+
b10
1505.20
+
+
+
+
b7
b4
b11
b13
1117.48
1054.21
+
b2
786.48
472.12
923.47
1244.44
1408.22
535.22
244.29
300 400
500 600
700 800 900 1000 1100 1200 1300 1400 1500
m/z
Fig. 2. The HPLCꢀMS/MS data for the sample 9B: a, a peak in the mass chromatogram corresponding to the fraction containing
a trace amount of a highꢀmolecularꢀweight peptide; b, the mass spectrum of the CID fragmentation products obtained with a linear
quadrupole ion trap. The amino acid sequence of the peptide was calculated manually using the MS/MS data.
bonds under the heating of amino acids mixtures. The
samples of this series are characterized by the following
components: the succinimide residue with a molecular
weight of 97, the Asp residue with a molecular weight of
115, and the Lys residue with a molecular weight of 128,
water molecules and Na ions. The peak with m/z 469.4 at
z = 1 corresponds to the cation [NH2 – Lys – bis(succinꢀ
imidyl) – Lys – OH + H]+.
0.5 g of the initial mixture. For control samples containꢀ
ing only Asp, the amount of the mixture of obtained polyꢀ
mer products was greater for the use of 2 equiv. H2SO4 as
[Asp—Asp—Asp + Na]+
I (arb. units)
386.08
Under polymerization conditions used by us, a series
of peptide derivatives are formed: polysuccinimides for
LꢀAsp and polysuccinimide fragments in the products of
an LꢀAsp—LꢀLys mixture. The formation of diketopiꢀ
perazines and trace amounts of ethylamine is also observed.
The alkaline hydrolysis of samples 14B—16B gave polyꢀ
aspartic acids of variable composition with different
terminal groups, such as residues of lysine, aspargine,
and aspartic acid and, possibly, ethylamine residue (see
Experimental) whereas before the treatment of these
samples, the succinimide and 3ꢀaminosuccinimide fragꢀ
ments and residues of lysine and aspartic acid are obꢀ
served. It follows from the results of hydrolysis that the
amount of the mixture of obtained polymer products was
greater when using 3 equiv. H2SO4 for a mixture of amino
acids (sample 15B) than that when 2 equiv. H2SO4 were
used (sample 14B): 0.22 g and 0.18 g, respectively, from
[Asp—Asp—Asp—Asp + Na]+
80
501.11
[Asp—Asp + Na]+
60 [Asp + Na]+
156.08
271.05
115
115
115
40
20
432.80
484.08
150 200 250 300 350 400 450 500 550 m/z
Fig. 3. The use of tandem mass spectrometry to confirm the
peptide nature of the peak with m/z 501.11 in the mass spectrum
of sample 18B after 300ꢀh heating and the treatment of the
reaction mixture with detection of positive ions.

432
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Demina et al.
I (arb.units)
a
80
60
40
20
200
400
600
800
1000
1200
1400
1600
m/z
I (arb.units)
b
365.058
596.076
80
60
40
20
400
600
800
1000
1200
1400
1600 m/z
I (arb.units)
c
301.1
80
60
40
20
M = 115
400
600
800
1000
1200
m/z
Fig. 4. Mass spectra of sample 14B (see Table 2, run 14) before the alkaline treatment using FT ICR with MALDI for the detection of
positive ions (а) and the reaction mixture of sample 18B before (b) and after the alkaline treatment of the reaction mixture (c) with
detection of positive ions. The samples were heated for 300 h.

Modelling of prebiotic synthesis of peptides
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
433
a catalyst (sample 17B) than that for the use of 4 equiv.
H2SO4 (sample 18B), i.e., 0.3 g from 0.5 g and 0.3 g from
0.65 g of the initial mixture, respectively.
and samples 19B—27B after 300 h of heating (probe 2)
(see Table 2).
The synthesis of peptides from mixtures of LꢀAsp with
LꢀArg (Scheme 3), LꢀGlu with LꢀLys, or LꢀGlu with LꢀArg
under the same reaction conditions gave mixtures of pepꢀ
tides and biopolymers, i.e., peptide derivatives containing
the pyroglutamic acid fragment. The composition of the
mixture of obtained products for the ratio of amino acids
LꢀArg : LꢀAsp = 1 : 1 is shown in Scheme 3. According to
the obtained experimental data, the oligomerization rate of
LꢀGlu was lower than that of LꢀAsp and, hence, the comꢀ
position of the final products of mixtures LꢀGlu—LꢀLys or
LꢀGlu—LꢀArg is more diverse than that in the case of mixꢀ
tures LꢀAsp—LꢀLys or LꢀAsp—LꢀArg (Schemes 4 and 5).
The glutamic acid residue is transformed into the pyroꢀ
When 2 equiv. H2SO4 were used as a catalyst, the
number of residues in peptides attained 21—23 for Asp
(sample 17B) and 14—20 for a mixture of amino acids
(sample 14B), decreasing to 10—12 residues when 3 equiv.
H2SO4 were used (sample 15B). It should be noted that
a decrease in the amount of amino acid containing an addiꢀ
tional amino group in a mixture of amino acids at the
same amount of sulfuric acid results in polymers with
a smaller number of residues in the chain, which is charꢀ
acteristic of all pairs of amino acids used by us.
We analyzed the samples of experiments 19—27 of
series III: samples 19А—27А after 138 h of heating (probe 1)
Scheme 3
Reagents and conditions: LꢀArg : LꢀAsp = 1 : 1, 2 equiv. H2SO4, 140 C, 300 h (see Table 2, run 20, sample 20B).

434
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
Demina et al.
Scheme 4
Reagents and conditions: LꢀLys : LꢀGlu = 1 : 1, 2 equiv. H2SO4, 140 C, 300 h (see Table 2, run 23, sample 23B).
glutamic acid residue to form peptide derivatives under
the heating.
obtained products. For the ratio Asp : Arg = 2 : 1 in series
1, the maximum number of succinimide residues (nmax) is
18 and trace amounts of polysuccinimides with nmax equal
to 19 and 20 were also observed, whereas nmax = 11 for
Asp : Arg = 1 : 1. In series 2 for the ratio Asp : Arg = 2 : 1,
the number of succinimide residues nmax is 19 (nmax = 15
for Asp : Arg = 1 : 1), in series 3 n = 21 (nmax = 17 for
Asp : Arg = 1 : 1), and in series 4 and 5 nmax = 8 and 9
(nmax = 4 and 9 for Asp : Arg = 1 : 1), respectively. For the
ratio Asp : Arg = 1 : 2, the number of succinimide residues
nmax is 9 in series 1, nmax = 13 in series 2, nmax = 14 in
The data for the exact structures of two series of biopolyꢀ
mers for sample 20B are presented in Table 4 (LꢀAsp : LꢀArg =
= 1 : 1, see Table 2, series III, run 20). Both singleꢀ and
doubleꢀcharged ions correspond to these structures in the
mass spectra. It must be noted that all series of derivatives
shown in Scheme 3 are characteristic for the samples
19B—21B (runs 19—21, probe 2) and different by the ratio of
amino acids only (see Table 2), and the ratio of amino
acids determined the number of amino acid residues in the

Modelling of prebiotic synthesis of peptides
Russ.Chem.Bull., Int.Ed., Vol. 61, No. 2, February, 2012
435
Scheme 5
Reagents and conditions: LꢀArg : LꢀGlu = 1 : 1, 2 equiv. H2SO4, 140 C, 300 h (see Table 2, run 26, sample 26B).
series 3, and nmax = 2 and 8 in series 4 and 5, respectively.
Thus, the increase of the initial amount of Asp in sample
19B results in the formation of peptide derivatives with
a higher weight in series 1—4 compared to samples 20B
and 21B, and a decrease of the initial amount of Asp in
sample 21B reduces the amount and weight of polymers in
all series of the obtained reaction products. Both singleꢀ
and doubleꢀcharged ions (series 1—4) and also fourꢀ
charged ions (series 5) corresponded to these products in
the mass spectra. The formation of diketopiperazines was
characteristic of all mixtures of series III studied by us.
The general scheme of peptide fragmentation when
the collisionꢀinduced dissociation (CID) method is used
and the mass spectra confirmed the structures of biopolyꢀ
mers and peptides in samples 20B, 23B, and 26B are shown
in Fig. 5 (see Table 2, runs 20, 23, and 26). The mass
spectra of CID fragmentation products of molecules Asꢀ
pArgddddddArg, Glu—Lys—Glu—Lys, and Arg—Glu—
Glu—Arg were recorded in a linear ion trap (see Fig. 5).
Thus, we established that both peptides and their deꢀ
rivatives containing the succinimide or pyroglutamic fragꢀ
ments can be obtained from a mixture of two oppositely
charged amino acids by means of the isothermal peptide
synthesis. The composition of products depends on the
amount of sulfuric acid used as a catalyst, the properties of
the initial amino acids, and their ratio. It was mentioned

436
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Demina et al.
Table 4. Compositions of several series of the products for the sample 20B (Arg—Asp mixture) according to the HPLCꢀMS/MS data
Number
Formula*
Molecular ion
Number
Formula*
Molecular ion
of residues
in peptide or its
derivative
of residues
in peptide or its
derivative
[M + H]+, [M + 2 H]2+
,
[M + H]+, [M + 2 H]2+
,
m/z
m/z
m/z
m/z
(z = +1)
(z = +2)
(z = +1)
(z = +2)
Series1
Series 2
2
3
4
5
6
7
8
9
10
11
12
13
Rd
Rdd
Rddd
Rdddd
Rddddd
Rdddddd
Rddddddd
Rdddddddd
Rd9
272.1365
369.1529
466.1692
563.1856
660.202
757.2184
854.2347
951.2511
1048.267
1145.284
1242.3
0—
0—
0—
0—
0—
0—
0—
0—
0—
0—
0—
0—
5
6
7
8
RdddR
RddddR
RdddddR
RddddddR
Rd7R
622.2703
719.2867
816.3031
913.3195
0—
360.1474
408.6555
457.1637
505.6719
554.1801
602.6883
651.1965
699.7047
748.2129
796.721
9
1010.336
10
11
12
13
14
15
16
17
18
Rd8R
Rd9R
—
—
—
—
—
—
—
—
—
Rd10R
Rd11R
Rd12R
Rd13R
Rd14R
Rd15R
Rd16R
Rd10
Rd11
Rd12
1339.317
845.2292
893.7374
942.2456
Series 2
3
4
RdR
RddR
428.2376
525.254
0—
0—
* d is the succinimide residue.
that the number of polysuccinimides and peptide derivaꢀ
tives containing the pyroglutamic acid fragments increasꢀ
es for the excess of amino acid with the additional carbꢀ
oxyl group (Asp, Glu). It was shown that a decrease in the
polymerization rate of glutamic acid compared to asparꢀ
tic acid results in the greater variety of the mixture of
obtained products. It was found that the presence of
diketopiperazines and amines in the final mixtures of prodꢀ
ucts is characteristic of all mixtures of amino acids
used by us.
Synthesis of peptides under the thermal cycling mode.
Complementarity. The investigation of the abiogenic
thermal synthesis in model systems under the cyclic
mode was done by us. The process includes the following
stages:
(1) the preparation of aqueous solutions of the initial
mixtures of amino acids (LꢀLys—LꢀAsp and LꢀArg—LꢀAsp)
with pH 4.5 and 8.5;
(2) the sampling of an aliquot of the solution into a glass
beaker with the heating in a thermostat to 120—130 C, the
evaporation of water, and the formation of the solid phase;
(3) the heating of the solid phase during 12—24 h and
the polycondensation in the solid phase;
to pentapeptides in the solidꢀphase polymerization mode
at relatively low temperatures and short incubation times.
The byꢀproducts formed under the thermal synthesis conꢀ
ditions in the solid phase, such as diketopiperazines, sucꢀ
cinimide fragments, amines, and others (see above), were
practically absent in this case. The compositions of mixꢀ
tures of peptides obtained under the thermal cycling conꢀ
ditions and the amino acid sequences of individual pepꢀ
tides were determined by chromatography—mass spectroꢀ
metric analysis (Figs 6—8). The determination of the amino
acids primary sequence for tetrapeptide NH2—Lys—Asp—
Lys—Lys—COOH (KDKK) by tandem mass spectroꢀ
metry in combination with HPLC using CID fragmentaꢀ
tion is shown in Fig. 7.
The reproducibility of the obtained results is of note:
several identical experiments were carried out for mixꢀ
tures LꢀAsp—LꢀLys and LꢀAsp—LꢀArg with a time difꢀ
ference of 1 year. A comparison of the compositions of
obtained samples presented in Table 5 shows that the numꢀ
ber of peptides does not almost increase in time. The reꢀ
producibility of the results was confirmed by the compariꢀ
son of the sample 3А (see Table 1, run 3) with the sample
28 (see Table 5). Sample 28 was obtained and analyzed
1 year earlier than sample 3А. The composition of the
both samples is the same; i.e., the final mixtures of prodꢀ
ucts with the same composition were obtained experimenꢀ
tally with the difference of 1 year (see Table 5).
(4) the cooling to ambient temperature, the addition
of an aliquot of the initial solution of amino acids.
Then this cycle is multiply repeated to the complete
consumption of the initial mixture of amino acids.
The polycondensation of amino acids occurred with
the formation of a series of polypeptides from dipeptides
It must be noted that 4 dipeptides, 8 tripeptides,
16 tetrapeptides, and 32 pentapeptides, i.e., 60 variants of

Modelling of prebiotic synthesis of peptides
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437

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Demina et al.
Fig. 6. The composition of a peptide mixture obtained from an LꢀLys—LꢀAsp (1 : 1) mixture in the thermal cycling mode (see Table 1,
run 6, sample 6C). The mass spectrum of the CID fragmentation products was detected in the linear ion trap. The reaction conditions:
the heating temperature is 120 C, the heating time is 144 h, 9 cycles, and pH 8.5. The experimentally determined masses of peptides
are given in parentheses.
Table 5. Composition of products for samples 6А and 6C (LꢀAsp—LꢀLys), 3А and 3C (LꢀArg—LꢀAsp), and 28 (LꢀArg—LꢀAsp) obtained
in the thermal cycling mode at different numbers of cycles and heating times (the peptide structure was determined by HPLCꢀMS/MS)
Number
of residues
in peptide
Primary
amino acid
sequence
Molecular ion
Number
of residues
in peptide
Primary
amino acid
sequence
Molecular ion
[Mcalc + H]+ [Mexp + H]+
[Mcalc + H]+ [Mexp + H]+
Sample 6А
Sample 3А
2
2
3
3
3
4
4
4
5
5
5
KK
DK
DDK
KDK
275.2089
262.1409
377.1678
390.2358
403.3039
505.2628
518.3308
492.1948
620.2897
633.3577
646.4258
275.2076
262.1396
377.1663
390.2342
403.3024
505.2607
518.3293
492.2012
620.2425
633.2874
646.3554
2
2
3
3
4
4
RR
DR
DDR
RDR
DRDR
DDDR
331.2212
290.147
405.174
446.2481
561.2751
520.2009
331.2201
290.1459
405.1728
446.2470
561.2892
520.2151
KKK*
DKDK*
KDKK*
DKDD
DKDDK*
DKKDK*
KDKKK*
Sample 3C
2
2
3
3
4
4
5
RR
DR
DDR
RDR
DRDR
DDDR
DDDDR*
331.2212
290.147
405.174
446.2481
561.2751
520.2009
635.2279
331.2201
290.1459
405.1728
446.2470
561.2892
520.2151
635.2422
Sample 6C
2
2
3
3
3
4
4
4
5
5
5
KK
DK
DDK
KDK
275.2089
262.1409
377.1678
390.2358
403.3039
505.2628
518.3308
492.1948
620.2897
633.3577
646.4258
275.2076
262.1396
377.1663
390.2342
403.3024
505.2607
518.3293
492.2012
620.2425
633.2874
646.3554
Sample 28**
2
2
3
3
4
4
RR*
DR
DDR*
RDR*
DRDR*
DDDR*
331.2212
290.147
405.174
446.2481
561.2751
520.2009
331.2201
290.1459
405.1728
446.2470
561.2892
520.2151
KKK
DKDK
KDKK
DKDD
DKDDK
DKKDK
KDKKK
* Trace amounts.
** LꢀArg—LꢀAsp (1 : 1), рН 4.5, 120 С, 52 h, 4 cycles.

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439
I (arb. units)
501.4
[b3]+
372.3
[b2]+
244.2
80
[M]+
K
K
D
[y2]+
275.3
60
[y3]+
390.3
K
D
K
40
[y1]+
147.2
20
[b1]+
129.2
510.6
200
250
300
350
400
450
500
m/z
Fig. 7. The determination of the amino acid sequence of tetrapeptide KDKK synthesized from a LꢀLys—LꢀAsp (1 : 1) mixture in the
thermal cycling mode (see Table 1, run 6, sample 6C) by HPLCꢀMS/MS. The mass spectrum of the CID fragmentation products
was detected in the linear ion trap. The reaction conditions: the heating temperature is 120 C, the heating is time 144 h, 9 cycles,
and pH 8.5.
Fig. 8. The composition of the peptide mixture obtained from a LꢀArg—LꢀAsp (1 : 1) mixture in the thermal cycling mode (see Table 1,
run 3, sample 3C). The reaction conditions: the heating temperature is 120 C, the heating time is 121.5 h, 7 cycles, and pH 4.5. The
experimentally determined masses of peptides are given in parentheses.
structures, can be formed under the statistically equiꢀ
propable synthesis modes from two amino acids. In
the case of the LꢀLys—LꢀAsp mixture, 11 peptides were
identified, whereas 7 peptides were observed in the case of
the LꢀArg—LꢀAsp mixture. The products obtained from
the LꢀLys—LꢀAsp mixture contain no "independent" pepꢀ
tides, since 10 peptides of 11 have complementary the
affinity to each other.

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Demina et al.
Complementary affinity for the "triples"
isothermal and thermal cycling modes. The number of
different synthesized chains is much smaller than the staꢀ
tistically possible one. If to accept that the number of
possible variants of the primary structures is defined by the
proportion , where  is the number of the code letters,
and  is the chain length (some peptides are composed of
the twoꢀletter code, for example, K and D or K and d,
while some others consist of the threeꢀletter code, nameꢀ
ly, K, D, d, where d is the succinimide), then the total
number of potentially possible variants of structures for
the samples presented in Table 3 is 3 472 980. In fact, only
58 structures were determined after the synthesis termination.
5. The evolution of the system takes place with the
disappearance of some structures through the process proꢀ
longation even when the reaction is running in isothermal
mode. For instance, after the incubation of the system for
104 h, the product KDddddddddK and the whole series of
products DDKK, DDKKd, DDKKdd, DDKKddd, and
DDKKdddd were unambiguously identified and disꢀ
appeared after 278 h of incubation.
Complementary affinity for the "quadruples"
We assume that the above mentioned peptides can form
complexes with each other due to the complementary salt
bridges.
6. The synthesis of peptides with the complementary
sequence of amino acids is observed under the thermal
cycling mode. The synthesis of peptides through the therꢀ
mal cycling mode showed the selection process with the
formation of complementary chain, which agrees with the
notions of the kinetic theory of the prebiotic evolution of
macromolecules advanced by us.23
Thus, only peptides were prepared from the mixture of
two oppositely charged amino acids in the peptide syntheꢀ
sis under the thermal cycling mode, and any polysuccinꢀ
imides or peptide derivatives containing succinimide resiꢀ
dues were not formed.
The experimental study of the peptide synthesis based
on the polycondensation of trifunctional monomers under
the thermal cycling and isothermal synthesis modes made
it possible to draw several conclusions.
This work was partially supported by the Presidium of the
Russian Academy of Sciences (Programs Nos 18 and 25).
1. The processes of polycondensation with the peptide
bonds formation in the studied mixtures of amino acids
forming occur under relatively mild conditions at
120—140 C and a normal pressure.
2. The use of FT ICR mass spectrometry and HPLCꢀ
MS/MS allowed us to identify almost all components of
the complicated mixtures without a preliminary sample
preparation.
3. The set of obtained products depends considerably
on the conditions of the process, namely, when using sulꢀ
furic acid as a catalyst in the isothermal synthesis, aspartic
acid undergoes a cyclization into the polymer chain to form
succinimide fragments or polysuccinimide regions of the
chain upon the heating time prolongation. The process of
peptide bond formation under the action of strong acids,
such as H2SO4, runs mainly on the ꢀcarboxyl group with
the appearance of cyclic imide after the formation of the
"regular" peptide bond, which has been described for the
conditions of classical peptide synthesis.30 Under alkaline
conditions, the proceses occur with the formation of norꢀ
mal peptides containing aspartic acid with the free ꢀcarbꢀ
oxyl group. The final set of synthesis products is more
diverse when glutamic acid is used instead of aspartic acid.
4. No statistic inclusion of amino acids into polypeptide
chains is observed for the peptide synthesis under both
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Received June 22, 2011;
in revised form January 27, 2011