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Non-Enzymatic Synthesis of Polysaccharides Nucleosides and Nucleic Acids and the Origin of Self-Reproducing Systems.

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ANGEWANDTE CHEMIE
VOLUME 1
*
NUMBER 1
JANUARY 27 . 1 9 6 2
PAGE 1-64
Non-Enzymatic Synthesis of Polysaccharides, Nucleosides and Nucleic Acids
and the Origin of Self-Reproducing Systems
BY PROF. DR. G. SCHRAMM [*I, DR. H. GROTSCH, AND D1PL.-CHEM. W. POLLMANN
MAX-PLANCK-INSTITUT F m VIRUSFORSCHUNG, T-INGEN
Compounds containingfree amino, hydroxyl or carbonyl groups can be activated by reaction
with polyphosphate ester. tf the compoundc;contain a second functional group, polycondensations are possible; e.g. amino acids + polypeptides, carbohydrates + polysaccharides,
nucleotides + polynucleotides. In the presence of polyadenylic acid, polymerization of
uridylic acid is speeded up tenfold. This mutual efect of complementary nucleotide strands
constitutes the experimental basis for a hypothesis concerning the terrestrial origin of
systems capable of self reproduction.
without racemization. The polyphosphate ester can be
readily produced by dissolving P205 in ether and other
solvents containing alkoxyl groups. Its precise structure
is not yet well known; it seems likely that it is a mixture
of cyclic and straight-chain esters.
Schramm and Wissmann [l] have described a synthesis
of polypeptides carried out with the help of polyphosphate esters in which the amino group of one component
was made reactive by forming a phosphamide ester,
reacting subsequently with the carboxyl group of the
second component. This procedure is particularly well
suited for the purpose of polymerization. Thus, it has
been possible to prepare in this manner high-molecular
weight peptides containing as many as 24 amino acids
by starting with alanyl-glycyl-glycine [I]. Similarly, a
polyarginine (mol. wt. 4000-5000) was prepared from
arginine and high molecular weight peptides were obtained from various substituted and unsubstituted
aminobenzoic acids.
These experiments will be described in greater detail
elsewhere. The procedure has the advantage that condensation occurs under exceedingly mild conditions and
Activation of Hydroxyl-Containing Compounds
Experiments were carried out in order to discover
whether or not polyphosphate ester can also be used to
activate hydroxyl groups. We were prompted to undertake these studies by the fact that in the vast majority
of ca3es, cellular biosynthetic processes are initiated by
the transformation of hydroxy compounds into reactive
phosphate esters by adenosine triphosphate or similar
polyphosphates; these esters in turn react with either the
Table I . Biochemical Syntheses
(1)
+
(2) glucose-I-phosphate
(3)
+ saccharose+ uridine diphosphate
glucose-1-diphospho-uridine fructose
ribose-I-phosphate
+ uracil
(4) 5-phosphoribosyl-1-pyrophosphate
(5)
nucleoside diphosphate
(6)
deoxynucleoside triphosphate
(7)
amino acid
+ ATP + s-RNd
[*I Preliminary communication: G. Schramm, H. Grotsch, and
W. Pollmann, Angew. Chem. 73, 619 (1961).
[ I ] G . Schramm and H . Wissrnann, Chem. Ber. 91, 1073 (1958).
Angew. Chem. internat. Edit. / Vol. I (1962) / No. 1
phosphorylase
+ phosphate
-+ uridine + phosphate
nucleotide pyrophosphorylase
adenylic acid + pyrophosphate
starch
uridine phosphorylase
+ adenine
polynucleotide phosphorylase
+
R N A + phosphate
>
DNA
+ pyrophosphate
> aminoacyl-s-RNA + protein
nitrogen or oxygen of another compound while giving
up their phosphate group. Examples of such reactions
are listed in Table 1. In the case of reactions (1) to (4)
1
the activation involves an acetal hydroxyl; in the case of
reactions (5) and (6) a secondary phosphate group is
activated, whereas in reaction (7) a carboxyl group
undergoes activation. Our first attempts were therefore
directed towards the activation of acetal hydroxyl
groups and phosphate esters by means of polyphosphate
ester.
Preparation of 0-Glycosides
We have found that when polyphosphate ester is allowed
to react with aldoses and ketoses, the acetal hydroxyl
group is attacked and the resulting reactive intermediate
can readily undergo furiher reactions with other hydroxyl-containing compounds. If, for example, glucose
dissolved in dimethylformamide is allowed to react with
methyl glucose in the presence of polyphosphate ester,
the splitting-off of the methyl group is accompanied by
a good yield of a uniform disaccharide which upon
chromatography can be clearly differentiated from
maltose and gentiobiose, and can be identified as cellobiose (see Diagram 1). Thus, there arises in this case,
a ~1+4-glucosidiccompound. The surprisingly sterically
uniform course of the reaction has been confirmed in
many other examples.
oi'+
m WO
-7OHc
CHzOH
H
3
OH
OH
OH
OH
activated glucose
1
OH
methyl glucoside
- P-OH
V
CHz OH
cellobiose
Diagram 1
So far it has not been possible to isolate in pure form
the active intermediate (glucose-1-P) which is formed
from glucose and polyphosphate ester. Undoubtedly,
the first step in the reaction involves a phosphorylation
of the acetal hydroxyl group. Within a few minutes
following the addition of the polyphosphate ester in the
cold, the aldehyde group of the glucose can no longer be
titrated with iodine; moreover, when the mixture was
analyzed, glucose-1-phosphate was found. However,
the active intermediate is not identical with glucose-lphosphate as the latter does not react to form glucosides
under the conditions described. Possibly, the intermediate compound is an ethyl ester of glucose-l-phosphate or glucose-polyphosphate.
Preparation of Polysaccharides
Polymerization of sugars in acid solution has been observed repeatedly. Miclzeel and co-workers [2] were able
to isolate polyglycosides in good yields when they
treated glucose and other sugars in dimethyl sulfoxide
with hydrochloric acid, provided the water formed in
the course of the reaction was distilled off. However,
the polysaccharides that were formed were highly
branched.
If sugars that have a free carbonyl group are allowed to interact in an inert solvent in the presence of
polyphosphate ester, high molecular weight polyglycosides result which are largely linear and unbranched. We obtained a polymer from glucose in dimethylformamide from which we were able to separate
low molecular weight oligosaccharides by means of
dialysis. With a yield of 30-50%, there remained a
polyglucoside with a mean molecular weight of 50000.
Its specific viscosity, [?I = 100, corresponded to that of
cellulose of equal molecular weight. This indicates that
the vast majority of molecules formed are of the longchain, unbranched type. Both optical rotation (-1-38"),
and the infrared spectral data, indicate the presence of
P-glycosidic linkages. However, the polyglucoside still
contained small amounts of phosphate, probably because
a proportion of the non-acetal hydroxyl groups were
also phosphorylated in the course of the reaction. These
non-acetal phosphate esters do not seem to be sufficiently rich in energy to allow them to undergo further
condensations. The phosphorylation can be prevented
with the use of formamide solutions. Polyphosphate
ester dehydrates acid amides to the corresponding nitriles.
When the reaction is carried out in formamide, hydrocyanic acid results, and the acetamide is transformed
quantitatively into acetonitrile. This reaction has also
been described by others [3]. Formamide slows the
phosphorylating action of the polyphosphate ester
sufficiently for a mixture of both phosphate-free and
phosphate-containing glycosides to form. These compounds can easily be separated from one another by
means of a basic ion-exchanger. With an over-all yield
of 1'0-20 %, a phosphate-free polyglucoside with a
molecular weight of approximately 50000 and an optical
rotation of +16 was obtained from glucose. The precise
molecular composition of this glucoside is still being
determined. Upon decomposition with periodate it uses
up one mole of NaI04; this is to be expected if only
1 -+ 4-glycosidic linkages are present.
Ketoses, e.g. fructose and pentoses, will polymerize in
an analogous fashion in the presence of polyphosphate
ester (see Table 2). We have studied the polyriboside in
more detail. As was found for glucose, a phosphatefree polyriboside, which on the basis of viscosity and
sedimentation determinationshad a molecular weight of
40OO0, was obtained. The optical rotation amounted
[2] F. Micheel and A . Bockmann, Angew. Chem. 72, 209 (1960);
F. Micheel, A . Bockmann, and W. Meckstroth, Makromolekulare
Chem. 48, I(1961).
[3] T. Mukaiyama and T. Hata, Bull. chem. SOC.Jap. 34, 99
(1961); quoted from Angew. Chem. 73, 414 (1961).
Angew. Chem. internat. Edit. / Vol. I (1962) / No. 1
Table 2. Synthesis of Polysaccharides with Polyphosphate Ester
Monosaccharide
Polysaccharide
Glucose
Polyglucoside
weight
50000
(PI +4)
Ribose
Fructose
Polyriboside
(al-tS)
Polyfructoside
40000
40000
to +35 ’. On the basis of its optical rotation, as well as
its reaction with periodate, it appears to be a sterically
uniform product with predominantly al+5-glycosidic
linkages. This polyriboside readily decomposes into
ribose in dilute acid and is therefore comparable with
other furanoside compounds.
Under mild conditions, sugars will react with polyphosphate ester to yield oligosaccharides with a low
degree of polymerization. These can be readily separated
by means of chromatography. In addition to yielding
uniform products, this procedure has the advantage
that even the more labile polyglycosides can be produced; furthermore, small amounts of water do not
interfere with the reaction. Glucose, with the usual
water of crystallization, can readily serve as starting
material.
is necessary to carry out a chlorination in order to
loosen the proton at the N(9)-position sufficiently for
salt formation to occur. Following glycoside formation,
the halogen atoms must be removed by means of
catalytic reduction. In the free form, sugars do not react
with salts of the heavy metals, but must first be transformed into the 1-halogenidesby means of acylation of
all hydroxyl groups. However, these halogenides are
frequently quite unstable. At the end of the reaction
the protecting acyl groups must be removed. As a large
number of steps is involved, the yields are frequently
quite low [4].
Because of the selective action of polyphosphate ester
on the aldehyde groups, free sugars can be reacted with
purine and pyrimidine bases in one step.Thus, by allowing
adenine to react with deoxyribose we obtained an
approximately 30 % yield (on the basis of the quantity of
sugar employed) of 2’-deoxyadenosine, which in all of
its characteristics, i.e. UV. absorption and R, values
in various solvent systems, was identical with the natural product. As indicated in Diagram 2, adenosine was
NH2
polyphosphateester
+
+
Example: Preparation of Polyphosphate Ester
OH
Pure P205 (150 g.) is boiled under reflux in the presence
of 150 ml. chloroform and 300 ml. ether until the
solution is clear. This takes about 12 hours. The solvent
is then evaporated and the viscous, colorless residue is
used directly in the subsequent synthesis.
Example: Preparation of Phosphate-Free Polyglucosides
Glucose monohydrate (5 g.) and the polyphosphate
ester (10 g.) are dissolved in 50ml. formamide and
heated with stirring to 50-60°C. for 6 hrs. After the
solution has cooled to room temperature, an equal
volume of water is added and dialysis is carried out
for 48 hrs. The residue inside the dialysis bag is then
reduced to dryness in a rotary evaporator, triturated
with methanol, and washed with ether. The powdery
residue is taken up in a small volume of water and passed
onto a basic ion-exchanger (Dowex 1 X 10) which has
been pretreated with formic acid. After elution with
water, a phosphate-free polyglucoside is obtained in a
yield of about 15 %.
Angew. Chem. internat. Edit. / Vol. I (1962) / No. 1
adenine
OH
Diagram 2
OH
adenosine
prepared in good yield from ribose and adenine; on the
basis of ultraviolet and infrared spectral data, and its
behavior on chromatography, this compound was
identical with the natural product. This means that
substitution occurred only in the 9-position. It is interesting to note that the amino group in the 6-position
was unaffected. If it reacted at all, it was undoubtedly
reformed in the course of the purification. The only
sideproduct was a small quantity of 5‘-adenylic acid
which also was identical with the natural product.
The preparation of the corresponding nucleosides from
adenine with fructose or acetylglucosamine pioceeded in similar fashion (see Table 3). In order topreTable 3. Synthesis of Nucleotides with Polyphosphate Ester
ribose
+
fructose
Sugars readily react in acid solution with primary or
secondary amines to form the corresponding N-glycosides. These in turn are easily decomposed. It is more
difficult to make sugars react with heterocyclic bases to
form N-glycosides of the type that occur in nature as
nucleosides. In order to synthesize the latter, Hg or Ag
salts of the bases are prepared. Some of these are not
readily available. In the case of adenine, for example, it
H
ribose
adenine
deoxyribose
Preparation of Nucleosides
I
OH
+
-4
adenosine
~
adenine --
+ adenine
N-acetylglucosamine
~
(+2’,3’-adenosine phosphate)
-+deoxyadenosine
__- > fructosyladenine
~
+
adenine
-+
N-acetylglycosaminyl adenine
vent the sugars from polymerizing, the reaction was
always carried out in the ‘presenceof an excess of base.
Yields varied between 20 and 60 % relative to the quantity of sugar used. For the reaction, a certain amount
of water in the solvent is advantageous, since otherwise
the sugars decompose. The mode of formation corre[4] Review article: A . M. Michelson, Annu. Rev. Biochem. 30,
133 (1961).
3
sponds in large measure to the biosynthesis of these
nucleotides. This may be the reason why the isomers
occurring in nature are formed preferentially.
Example : Preparation of Adenosine (9-[ 1‘-p-ribosyI]adenine)
Adenine (1 g.) is dissolved with magnetic stirring in
100 ml. dimethylformamide (water content ca. 0.2 %)
with the addition of 0.5 ml. concentrated HCl. After
the addition of polyphosphate ester (3-4 g.), 200 mg.
D-ribose dissolved in 50 ml. dimethylformamide is
added dropwise with stirring. The solution is kept in a
glyceriiie bath at 50°C. for 20 hours and then the dimethylformamide is distilled off in vucuo. The moist
residue is dissolved in a little water and brought to
pH 7. Part of the unreacted adenine usually precipitates
after about 3 hours in the ice-box. The solution is
filtered, adjusted to pH 10 with ammonia, and passed
onto a Dowex-1 ion-exchange column (previously
prepared in the formate form). Adenosine, adenine, and
adenylic acid are eluted according to the method of
Cohn [5].
same for both types of nucleic acids, but that the glycosidic linkages are broken 650 times faster in the deoxyribonucleic acid than they are in the ribonucleic acids.
Thus the stabilizing effect of the 2’-hydroxyl group in
the ribonucleic acids is surprisingly strong. Therefore in
DNA, a large number of purine residues can be split
off by acid hydrolysis without breaking the chain. In
the case of RNA, on the other hand, the molecule
undergoes rupture in six places before a purine residue
is split off.
In view of the genetic role played by DNA it seemed of
interest to fill the gaps caused by the removal of adenine
and guanine with other purines or pyrimidines. The
free aldehyde groups in the apurinic acid were activated
by means of polyphosphate ester and allowed to react
with the corresponding bases. In the case of DNA from
thymus gland, it proved possible to quantitatively fill
with adenine the gaps which were caused by the removal
of more than 90% of the purine bases (Diagram 3).
DNA
H+
+ apurinic acid + adenine + guanine
apurinic acid
+ polyphosphate ester
(adenine)
-+
DNAadenine
Diagram 3.
Synthesis of Nucleic Acids from Apurinic Acids
It is well known that the N-glycosidic linkages between
deoxyribose and either adenine or guanine can be
broken in deoxyribonucleic acids (DNA) by cautious
treatment with acid, without causing at the same time
many of the phosphate bridges between the nucleotides
to split. These purine-free nucleic acids have been
studied in detail by Chargar and collaborators who
have termed them apurinic acids [6]. In order to develop
a quantitative basis for the synthesis of apurinic acids,
Schramm and Pollmann [7] have compared the rate of
splitting of phosphate bridges between sugar residues
with the rate of splitting of N-glycosidic linkages. A
potentiometric method was developed in order to
measure the rate at which phosphate di-esters are split
to form secondary phosphates. The splitting-off of
purine residues was determined chromatographically.
It was found that the rate of hydrolysis of phosphate
groups, as well of the glycosidic linkages, was proportional to the hydrogen ion concentration, but that the
ratio of these rates to one another was independent of
the pH. Table 4 lists the rates of hydrolysis uf deoxyribonucleic acid and ribonucleic acid at pH 2.4. The
table shows that the rate of phosphate hydrolysis is the
Table 4. Rate Constants for Hydrolysis at pH 2.4; T
=
37OC.
RNA
6.6
151 W. E. Cohn, J. Amer. chem. SOC.72, 1471 (1950).
[6] Ch. Tumm, M . E. Hodes, and E. Chargaff,J. biol. Chemistry
195, 49 (1952).
[7] W. Pollmann and G . Schramm, Z. Naturforsch. 166 673,
( 1 96 I).
4
The newly synthesized nucleic acid, in contrast to the
apurinic acid, no longer exhibited an aldehyde group;
following hydrolysis it proved possible to demonstrate
chromatographically the amounts of deoxyadenosine
expected from theoretical considerations. A further
argument in favor of the natural configuration of this
synthetic product is the fact that it could be hydrolyzed
both by deoxyribonuclease and by snake venom diesterase.
By activation with polyphosphate ester, it was possible
to make the apurinic acids react with other bases.
In order to measure the incorporation quantitatively we
employed radioactive bases. A reaction in which the
polyphosphate ester was replaced by an equivalent
amount of phosphoric acid served as a control. The
rates of incorporation are shown in Table 5 , which
Table 5. Incorporation of ‘‘C-Bases into Apurinic Acid in the Presence
of Polyphosphate Ester. Reaction in Dimethylformamide at 37OC.
Base
adenine
guanine
orotic acid
thymine
uracil
1
Incorporation
[%I
96
40
40
5
8
Time of reaction
[hrs.]
24
24
24
48
48
indicates that pyrimidines are incorporated more slowly
than are purines. The amounts incorporated can be
increased by permitting the reactions to proceed for
longer periods.
It is of interest that the incorporation of the bases proceeded not only in dimethylformamide but also in
aqueous solution, although the yield was smaller in the
latter instance. This raises the possibility of altering the
DNA in a cell in a controlled manner and, thereby, to
Angew. Chem. internat. Edit. Vol. I (1962) I No. 1
study the effect of such alteration of the sequence of
bases on the gene function. Some experiments are now
in progress. In addition to acid hydrolysis, other
methods are available for selectively removing certain
bases from a DNA or RNA molecule. Schuster [ 8 ] ,
working in our Institute, studied the removal of uracil
from RNA by means of hydroxylamine. Activation of
liberated aldehyde groups with polyphosphate ester and
incorporation of either those bases which occur in
nature, or others that do not, can lead to the production
of a wide spectrum of different nucleic acids.
Synthesis of Nucleic Acids
by Polymerization of Nucleotides
The procedure described above can be considered a
partial synthesis of nucleic acids. We have also achie\;ed
total non-enzymatic synthesis of nucleic acids by starting from nucleotides. Many laboratories have worked
on the general problem of polymerizing nucleotides to
form nucleic acids.
Khorana and co-workers [9] allowed thymidylic acid to react
with dicyclohexyl carbodiimide and they were able to make
the activated phosphates polymerize to form oligonucleotides,
achieving an average degree of polymerization of 3 to 4.
Michelson [lo] was able to transform nucleotides in dry
dioxane into cyclic 2’,3’-phosphates by adding tri-n-butylamine and either tetraphenyl pyrophosphate or diphenyl
phosphorochloridate. If additional tetraphenylpyrophosphate was allowed to act on the ammonium salts of these
cyclic phosphates, oligonucleotides were formed with a
maximum chain length of 12 nucleotides. Cramer 1111 has
described a method of polymerizing nucleotides which consists of sharply drying the latter and then causing them to
react with the enol phosphate of malonic ester in absolute
dimethylformamide and tributylamine. In this manner
Cramer obtained an adenosine diethylpyrophosphate ester
which could be polymerized to form polyadenylic acid. His
yield was only 3 % of a polythymidylic acid with a maximum number of 5 nucleotide units.
All methods used so far have led to oligonucleotides
with a small degree of polymerization. One of the principal difficultiesis that these oligonucleotideswere cyclized
intramolecularly, a lengthening of the chain thereby
being prevented. It can be readily understood, however,
that high degrees of polymerization can only be achieved
with highly concentrated nucleotide solutions. By
reversing Ziegler’s dilution principle, in termolecular
condensation may take precedence over intramolecular
cyclization. In order to achieve maximum concentration
the nucleotides to be polymerized were mixed directly
with the viscous polyphosphate ester and the resulting
mass was allowed to rotate at 50-60°C. The addition
of pyridine proved particularly favorable; this compound
is known to act as a catalyst in similar reactions because
of its nucleophilic properties. Table 6 lists the properties
of high molecular weight, non-dialyzable polynucleotides obtained from various nucleotides. We also pre[81 H. Schuster, J. mol. Biology 3, 447 (1961).
PI G . M . Tener, H. G. Khorana, R. Markham, and E. H . Pol, J.
Amer. chem. SOC.80, 6223 (1958).
[I01 A. M. Michelson, J. chem. SOC.(London) 1371, 3655 (1959).
1111 F. Cramer, Angew. Chem. 73, 49 (1961).
Angew. Chem. internat. Edit. 1 Vol. I (1962) 1 No. I
Table 6. Properties of the Synthetic Polynucleotides
s20
Poly-A
Poly-C
Poly-G
Poly-U
pO1y-r
1 1
[“V
Molecular
weight
2.5
2.0
25
23
21000
15000
2.9
4.0
2.3
28
28000
50000
18000
35
24
Hyperchromia
pared a number of mixed polymers from various nucleotides; the properties of these polymers resembled those
of the uniform polynucleotides. It is interesting that this
method makes possible the polycondensation of the
unstable 2’-deoxynucleotides. With T-deoxythymidine5’-phosphate as a starting material, the only free group
is the 3’-hydroxyl. There can be no doubt, therefore,
that in the polythymidylic acid (Poly-T) phosphate
bridges are established between the 3‘- and the 5‘hydroxyl groups, thus yielding the same contiguration
as exists in the natural DNA. Further support for this
argument comes from the fact that deoxyribonuclease
attacks the synthetic polydeoxythymidylic acid. The
average molecular weight calculated on the basis of sedimentation and diffusion determinations is 15000 to
50000. It may be possible to increase this by modifying
the procedure further. Polyribonucleotides may be prepared by making use of 2’-, 3‘- or 5‘-nucleoside monophosphates or cyclic 2’,3‘-phosphates. The nature of
the primary active intermediates has not yet been determined. In all likelihood the monophosphates are
first transformed into cyclic phosphates which in turn
react with the polyphosphate ester to form pyrophosphate esters. Inasmuch as two hydroxyl groups are
available for reacting with the phosphate groups of the
adjoining molecule, one cannot be sure of the steric
uniformity of the synthetic polynucleotides. Various
experiments suggest, however, that in the majority of
cases, at least, the configuration corresponds to that
of the natural nucleic acids. Thus synthetic polyribonucleotides that contain pyrimidine nucleotides can be
hydrolyzed by pancreatic ribonuclease; this would be unlikely if the predominant linkage were the unnatural
2’ + 3’- or 2’ -+ 5’-linkage. Pancreatic ribonuclease
acts specifically on the 3‘-phosphate group of the pyrimidine nucleotides.
Examination of the synthetic polyadenylic acid by
electron microscopy reveals long fibers very similar
in appearance to those in natural, high molecular
weight RNA (Figure 1). Branched chains seem, there-
Fig. 1 . Electron Micrograph (x 60000) of Synthetic Polyadenylic Acid
5
fore, to be infrequent. The length of these fibers indicates
that quite high molecular weights appear to have been
achieved at times. As with the natural materials, so the
synthetic polynucleotides exhibit a high degree of
hyperchromia. In high-molecular weight nucleic acids
the ultraviolet extinction coefficient is diminished because of interaction between the heterocyclic bases.
The extinction coefficients rise when the polymers are
hydrolyzed to mononucleotides, the values being equal
to those calculated by summing individual values for
the nucleotides. Michelson [12] has shown that in various
oligonucleotides, hyperchromia is a function of the
molecular weight. The appreciable hyperchromic values
listed in Table 6 are further indication of the high
molecular weight of the synthetic products. Experiments
by Rich 1131 have shown that enzymatically produced
polyadenylic acid will combine with its complementary
polyuridylic acid to form a double helix; at the same
time the extinction value will drop. We were able to
observe a similar effect when we mixed synthetic polyadenylic acid with synthetic polyuridylic acid. This
phenomenon is likely to occur only when both compounds have a helical structure similar to that of the
natural nucleic acids.
It is also possible to prepare polynucleotides in one
step directly from nucleosides by means of phosphorylation and subsequent condensation. However, the yield is
lower than when starting from nucleotides. The direct
one-step preparation is of interest in connection with
our subsequent discussion of the possible origin of
polynucleotides in the course of terrestrial evolution.
Example: Preparation of Polyadenylic Acid from 2'- and
3'-Monophosphates
Adenylic acid (350 mg.) was mixed with polyphosphate
ester (8 g.) and heated for 18 hours in a rotating flask
which was submerged in an oil bath of 55°C. After
cooling, the mixture was dissolved in water (50 ml.) and
dialyzed against water containing NaHC03 for a period
of four days in order to remove the orthophosphate and
the low molecular weight oligonucleotides. Polyadenylic
acid was obtained in the form of a white powder after
freeze-drying the solution. Yield: approxymately 20 "/,.
The Origin of Systems Capable
of Self-Reproduction
Taken together, our experiments have shown that the
most important biological macromolecules can be piepared in simple fashion with the aid of polyphosphate
ester. Protein-likepolypeptides are obtained from amino
acids, polysaccharides from sugars, nucleosides from
sugars and heterocyclic bases ; the nucleosides in turn
can be converted to nucleotides and ultimately into
nucleic acid; by continued reaction with polyphosphate
ester. The simple way in which these compounds are
[12] A. M. Michelson, J. chem. SOC.(London) 1371 (1959).
[I31 G. Fetsenfeid and A. Rich, Biochim. biophysica Acta 26, 457
(1957).
6
formed suggests the possibility that polyphosphate
esters may have had a role in the formation of macromolecules in the course of terrestial evolution. This
suggestion derives support from the fact that polyphosphate esters continue to be of fundamental significance in relation to the metabolism of organisms living
today. A number of piimitive micro-organisms contain
large quantities of inorganic polyphosphates which can
be incorporated into organic compounds as needed.
At the beginning of this paper we alluded to the role
played by ATP and similar polyphosphates in plant
and animal metabolism. For this reason Schrarnm [14]
suggested some time ago that polyphosphates or their
derivatives may have played a role when living organisms
or their precursors originated, and that their role
continued to become more specific as life continued to
evolve.
This hypothesis is in accord with geological fact. It is
likely that in the sterile period, i.e. before the origin of
life, the surface of the earth was rich in all kinds of
organic materials which may have originated as a result
of electrical discharges or ultraviolet irradiation [15, 161.
This organic material may have contained heterocyclic
bases. Bredereck [17] has observed that adenine forms
in surprisingly large quantities when certain simple
nitriles are heated. Or0 [ 181 has also noted that adenine
is formed when aqueous solutions of ammonium cyanide
are heated. At temperatures above 300 "C. phosphoric
acid occurs only in the forms of polyphosphate; it can
therefore be assumed that when the crust of the earth
began to cool down a supply of polyphosphates, and
reactive phosphorus oxides, was available which may
have reacted with aIkoxy compounds to form phosphate
esters. The latter in turn may have reacted with amino
acids, sugars and heterocyclic bases, causing them to
polymerize. The model experiments have made it clear
that the pyranose hexoses polymerize preferably to form
stable polysaccharides, compounds which even in
present-day organisms constitute important structural
elements, as for example cellulose .The furanose pentoses
form unstable polysaccharides, but react readily with
heterocyclic bases to form nucleosides.
However, these polymerizations lead to only random
arrangements of the monomers in the macromolecules,
which consequently are incapable of specific functions.
It is extremely unlikely that monomers arrange by
chance to form even the most primitive organism
imaginable. Such an event can hardly have occurred
within the finite time that the earth has existed. It must
be emphasized that the formation of randomly arranged
proteins and nucleic acids can in no way be considered
an explanation of the origin of life.
[14] G. Schramm in: Proceedings of the International Symposium
on the Origin of Life on Earth, held in Moscow. Pergamon Press,
London, New York 1960, p. 216.
[15] L . Roka: Vermutungen uber die Entstehung des Lebens.
Mosbacher Kolloquium iiber Vergleichend Biochemische Fragen. Springer-Verlag, Heidelberg 1956.
[16] A. J. Oparin: Die Entstehung des Lebens auf der Erde. Verlag Volk und Wissen, Berlin/Leipzig 1947.
[17] H. Bredereck, F. Effenberger,and G . Rainer, Angew. Chem.
73, 63 (1961).
1181 J. Oro, Fed. Proc. 20, 352 (1961).
Angew. Chem. intcrnat. Edif. 1 VoI. I (1962) 1 No. I
The formation of biological macromolecules with a
specific sequence of building units can be imagined if it
is assumed that simple molecules underwent a stepwise development that gradually made them capable of
increasingly complex performance. Biological development presupposes, however, the existence of mechanisms
capable of self-duplication, i.e. systems that give rise to
offspring. According to Darwin's theory of evolution by
mutation and selection, those molecular systems capable
of self-duplication would predominate that can multiply
most readily and most precisely. Small changes that the
system undergoes in the course of multiplication could
then lead to its continued improvement.
What are the minimum conditions for the development
of a self-reproducing system? This question can be
answered best by considering the molecular processes
taking place in organisms living at the present time.
Nucleic acids are known to play a commanding role.
With the utmost simplification, the self-reproduction
of organisms can be reduced to the following principle:
Using chemical and steric arguments Watson and Crick
1191 have shown that in molecules consisting of a chain
of nucleotides, pairing can occur only if the nucleotide
chain is complementary. Subsequent research has demonstrated that one strand of nucleotides serves as a
matrix for a complementary strand, which in turn is the
matrix for the formation of the original strand. In other
words, matrix M catalyzes the formation of matrix Mc,
which in turn catalyzes the formation of M (Diagram 4).
matrix M
c-
complementary
matrix M,
protein
However, these matrices undoubtedly also servepurposes
other than self-duplication; indeed they determine in a
quite complicated fashion which will not be discussed
here, the structure of proteins, which in turn influence
and control the self-duplication of the nucleotide chains
by acting as enzymes.
We have found that we can demonstrate the existence
of the principle of mutually and catalytically interacting
matrices, even under the conditions of non-enzymatic
synthesis of nucleic acids. We have studied the polymerization of uridylic acid in the presence, and absence,
of its complementary polyadenylic acid. Figure 2 shows
[I91 J. D . Watson and F. C. C. Crick, Nature (London)171, 737,
964 (1953).
Angew. Chem. internat. Edit. / Vol. I (1962) / No. 1
that the polymerization of uridylic acid was accelerated
more than tenfold by polyadenylic acid, whereas polyuridylic acid had no such effect. This means that
polymerization to polynucleotide chains is favored
Fig. 2. Polymerization ot Uridine Monophosphate in the Presence (A)
and in the Absence (B), of Polyadenylic acid. (The decrease of free
uridine monophosphate was msasured chromatographically).
Abscissa: Time [hours]
Ordinate: % Free Uridine Monophosphate (referred to ths amount of
starting material)
by complementary strands. If, therefore, in the course
of the evolution of the earth the formation of a nucleotide strand carried certain selective advantages, then
these in turn favored the formation of the complementary strand, which again speeded up the formation
of the original strand. This then indicates how such a
system can reach increasing perfection. We have also
observed that the addition of a synthetic polypeptide,
i.e. of polyarginine, favors the formation of a polynucleotide chain. Further study is needed in order to
determine whether nucleotide matrices can speed up the
non-enzymatic synthesis of such polypeptides. If this
is the case, the cycle of self-duplicating and mutually
stimulating catalysts would be closed; this would then
make it possible to postulate a cycle which would continue to improve as a result of small, chance alterations.
The basic idea is, therefore, that small chance alterations
in one catalyst determine the formation of a second,
dependent catalyst which in turn affects the formation
of the first, now improved catalyst. What path could
lead from these molecular systems to the origin of life
shall not be discussed here.
Received July 20, 1961
[A 165/7 IE]
7
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