Condensation of cytidylic acid in presence of polyphosphoric aci

Fox, S. W.

Schwartz, A. W.

English

NASA Technical Reports Server

. 
I- 
.* 
Condensation of Cytidylic Acid in the Presence of 
Polyphosphoric Acid 
by 
Alan W. Schwartz* and Sidney W. Fox 
GPO PRICE f 
CFSTI PRICEW s 
Department of Chemistry 
/, &B Florida State University 
Tallahassee, Florida 
Hard copy (HC) 
Microfiche (M F) / 6 3  
and 
Institute of Molecular Evolution** 
University of Miami 
Coral Gables, Florida 
In the press, 
Biochimica et Biophysics Acta 
v66 (ACCESSION 37311 NUMBER) (THRU) 
https://ntrs.nasa.gov/search.jsp?R=19660028021 2020-03-16T17:35:26+00:00Z
SUMMARY 
1. Cytidine-2'(3')-phosphate can be condensed in the presence 
of polyphosphoric acid. 
2. The product was found to contain polyphosphate end groups 
which, however, could be removed by treatment with E. coli alkaline 
phosphatase (EC 3 . 1.3 . 1) . - -  
3. A single 2'(3')-P-teminalend groupwas found per chain. 
4. With the exception of the free P-terminal end group, 
there was found to be one internally bound phosphate for every 
cytidine residue. 
5. Although resistant linkages were detected, recovery of 
starting material from mild alkaline hydrolysis indicated that 
the linkages were predominantly 2'(3')-phosphodiester in nature. 
6 .  The presence of 3'-phosphodiester linkages was demon- 
strated by hydrolysis with bovine pancreatic ribonuclease (EC 
2.7.7.16). 
7. The presence of a large proportion of 5'-phosphodiester 
linkages was demonstrated by hydrolysis with venom phosphodi- 
esterase (EC 3.1.4.1). 
c 
- 2.F- 
INTRODUCTION 
Preliminary publications have reported on the synthesis of 
phosphodiester linkages through the simple heating of c tidine- 
2 ' ( 3  ' ) -phosphate in the presence of polyphosphoric acidx and on 
the cocondensation of mixtures of cytidylic acid with other 
mononucleotides2. The present paper reports the partial struc- 
tural characterization of the oligomericmixture obtained from 
cytidine-2'(3')-phosphate and polyphosphoric acid. The presence 
of a high proportion of phosphodiester linkages is demonstrated. 
MATERIALS AND METHODS 
Condensation of Cytidine-2' (3')-Phosphate 
In order to obtain sufficient material for structural charac- 
terization, five condensations were carried out under identical 
conditions, and the purified products were pooled. In each case, 
10 g of polyphosphoric acid*** and 5 g of cytidine-2'(3')- 
phosphate were mixed under nitrogen, and the mixture was heated 
at 65O for 1 h. After cooling, a magnetic stirring bar and a 
small quantity of water were added to the reaction flask, and the 
reaction mixture was dissolved by adding conc. NH40H. The flask 
was cooled during the addition by an ice-salt bath, and the pH 
was not permitted to rise above 7.0. The solution was transferred 
to a dialysis sac and was dialyzed against water in the cold for 
3 days. The dialyzate was then absorbed on a large column 01 
Dowex 1x8 chloride (2-3 lb) and, after washing with water, the 
product was eluted with 4 N formic acid in 0.5 N ammonium formate 
at 4O. The formate effluent was dialyzed against water for an 
additional 3 days in the cold and lyophilized to give the final 
product in 2.6% yield****. 
Ultraviolet Spectra 
Spectra were obtained in 0.01 N HC1 and in 0.1 N NaOH on a 
Beckman DK2A ratio-recording spectrophotometer. 
chromicity was measured in 0.1 N - NaOH after 48 h at 37O. 
Alksline hyper- 
Elemental Analysis 
Four samples of the product were dried over P2O in vacuo at 
room temperature for 24 h. The samples were analyze8 for C, H, 
N and P, and an anhydrous formula weight was calculated for the 
average residue by setting the parts of carbon equal to 9.0. 
Molecular Weight 
Weight average molecular weight was determined from equili- 
brium sedimentation in a Spinco Model E analytical ultracentrifuge 
using Rayleigh interference optics4. 
Digestion with Alkaline Phosphatase and Ribonuclease 
in conjunction with an analysis for inorganic orthophosphate5 to 
detect the presence of phosphornonoester end groups (or any 
Incubation with E. - -  coli alkaline phosphatase was employed 
w 
phosphate groups not involved in internucleotide linkage). 
liberation of additional inorganic orthophosphate after addition 
of pancreatic ribonuclease to the incubation mixture was used as 
a measure of the proportion of 3'-phosphodiester linkages in 
the product. 
The 
In duplicate experiments, 5-6 mg of product was dissolved 
in 10-15 ml of 0.20 N sodium acetate (pH 5.3), and 0.5 mg of 
E. coli alkaline Phosphatase was added. A second solution of 
identical concentration of product, but without phosphatase, 
served as a control for autohydrolysis, 
of alkaline phosphatase in 10 ml of buffer were also prepared, 
and the solutions were incubated at 37'. Aliquots were taken 
from all solutions periodically and were analyzed for inorganic 
phosphate. After maximal liberation of phosphate was observed, 
the experimental and enzyme blank solutions were divided into 
halves, 0.5 mg of pancreatic ribonuclease was added to one- 
half of each solution, and incubation was continued. Additional 
aliquots were taken from each pair of solutions and were analyzed 
for inorganic phosphate. 
i 
Enzyme blanks of 0.5 mg 
, Hydrolysis in Alkali 
Duplicate samples of the product were hydrolyzed in 0.10 N 
sodium hydroxide at 37' for 48 h. 
to pH 8.0 with HC1 and were fractionated on a 1 x 28 cm column 
of Dowex 1x4 formate. Cytidine-2'- and cytidine-3'-phosphates 
were obtained by elution with 0.2 N HCOOH. 
prior dephosphorylation with alkalxne phosphatase was also 
studied . 
The hydrolyzates were adjuszed 
Hydrolysis after 
End Group Analysis of a Dephosphorylated,Preparation 
In order to verify the presence of a single cytidine residue 
corresponding to the dephosphorylated P-terminal end group, 
duplicate samples of the oligomer were treated with E. coli 
alkaline phosphatase, and the dephosphorylated produEts were 
isolated on a 1 x 10 cm column of Dowex 1x4 chloride with 0.01 N 
HC1 as the eluent. 
lyzed in 0.1 N NaOH at 37' for 48 h, and the hydro1 zates were 
was eluted with water to remove anionic components, and then with 
1 - N HC1 to remove cytidine. 
The dephosphorylated products were then hydro- 
The column fractionated on a 2 x 12 cm column of Dowex 50x4 (H T 1 .  
Analvsis for Labile Phosohate 
In order to test for the presence of polyphosphate end 
groups, samples of product were heated at 80' in 1.0 N H2SO4, and 
aliquots of the solutions were periodically analyzed For inorganic 
phosphate. 
I 
Hydrolysis with Ribonuclease 
1 
r 
Samples of the product (6-7 mg) were each dissolved in 
.O ml of 0.20 N sodium acetate (pH 5.31, and 0.5 mg of pancreatic 
ibonuclease wag added to each. The solutions were incubated at 
37" for 4-12 h and were then transferred to a 1 x 28 cm column of 
Dowex 1x4 formate. After washing with water, the column was 
eluted with 0.02 N - HCOOH. 
Hvdrolvsis with Ribonuclease and Alkaline Phosphatase 
Duplicate samples of the product (1.9-2.0 mg) were each 
dissolved in 0.5 ml of 0.20 N sodium acetate (pH 5.3) and 0.1 mg 
of E. coli alkaline phosphatase. Each solution was incubated at 
37O-fo- h and then diluted to 2.0 ml and adjusted to 0.03 N 
in potassium chloride and 0.02 N in sodium borate. 
were then fractionated on a 1.2-x 8 cm column of Dowex 1x4 
chloride, which had been prepared by washing with 0.03 N HC1 in 
0.02 N sodium borate. 
same Solvent. 
The solutions 
Cytidine was eluted from the c o l h  in the 
Hydrolysis with Venom Phosphodiesterase 
Duplicate samples of dephosphorylated product (2 mg) were 
dissolved in 2.0 ml portions of 0.20 N sodium glycinate (pH 9.3) 
containing 0.025 N MgC12, and 0.1 mg zf venom phosphodiesterase 
(Worthington) was-added to each. The solutions were incubated 
at 37O for 3 and 4 h and were subsequently analyzed on a 1 x 27 cm 
column of Dowex 1x4 formate. After washing with water, cytidine- 
5'-phosphate was eluted in 0.02 - N HCOOH. 
. 
The ultraviolet spectra were completely analogous to spectra 
obtained with previously reported preparations and indicated that 
phosphoram'date linkages did not participate in the internucleo- 
tide bonds', . 
Elemental analysis of the product revealed a ratio of 1.52 
atoms of phosphorus for each nine-carbon cytidine residue (C, 26.2; 
H, 4.0; N, 10.6; P, 11.4). In other words, there is an "excess" 
of 0.52 phosphorus atom per cytidylic acid residue. On the basis 
of the elemental analysis, the anhydrous formula weight of the 
average cytidylic acid residue was taken at 350 (in agreement with 
the molar extinction coefficient). Since the molecular weight as 
determined by ultracentrifugation was 1960, the average chain 
length was 1960/350 = 5.6. An average chain of 5.6 residues will, 
therefore, have a total of 1.52 x 5.6 = 8.5 phosphorus atoms, of 
which 0.52 x 5.6 = 2.9 will be in excess of the single phosphorus 
per residue required for ideal phosphodiester linking. In addi- 
tion, if we assume the presence of one phosphate end group per 
chain, we would expect to find 2.9 + 1.0 = 3.9 "external" 
phosphate groups (i,e., those not involved in the internucleotide 
linking). This would produce a phosphate end group analysis of 
3.9/8.5 = 46% of the total phosphorus of the oligomer. This 
figure agrees with that determined by dephosphorylation of the 
oligomer with alkaline phosphatase (Table I). The conclusion 
drawn from this agreement is that the high proportion of "external" 
phosphate revealed by alkaline phosphatase treatment can be 
accounted for completely as the product of phosphorylation of the 
oligomer by the reagent polyphosphoric acid. Treatment of the 
oligomer with alkaline phosphatase thus removes only one end 
group per chain in addition to the "excess" phosphate, Therefore, 
the linkages of the oligomer must be entirely through phosphate 
groups, since other types of linkages (e,g., C-0-C chain branching) 
would produce more than one P-terminal end group per chain. 
Isolation of an alkaline phosphatase-treated fraction of the 
oligomer and subsequent alkaline hydrolysis produced one cytidine 
end group per 5.6 residues, in agreement with the conclusion of 
one P-terminal end group per chain (Table 11). 
The results of the hydrolysis of oligomer in dilute NaOH are 
tabulated in Table 111. The yield of monomer increases from 58% 
to about 76% after dephosphorylation. This increase of 18% 
represents 0-18 x 5.6 = 1.0 residue. This observation and the fact 
that the "excess" phosphate was found to be entirely "external" 
led to the conclusion that the 2.9 "excess" phosphate groups per 
chain might be in the form of single polyphosphate end groups. 
This conclusion was verified by the analysis for labile phosphate 
(Table I). The 22.4% of the total phosphate which was liberated 
in 3 h corresponds to 1.9 phosphate groups per chain and is 
- 7 -  
e 
precisely what would be expected from the hydrolysis of a statistical 
polyphosphate group of 2.9 units. 
Figure 1 illustrates the effect of ribonuclease on the frac- 
tionation of the oligomeric mixture. The extent of hydrolysis to 
monomer units is tabulated in Table IV. The formation of cytidine- 
2'-phosphate indicates that the 2' (3')-terminal end group (P-terminal) 
of the oligomer is unsubstituted by "excess" phosphate. The 2'- 
isomer can only originate through the liberation of such end groups 
by hydrolysis of a penultimate 3'-phosphodiester linkage. 
presence of cytidine-2'(3')-cyclic phosphate in the 4 h hydrolysis 
is not unexpected, since cytidine-2'-phosphate is known to inhibit 
the opening of the intermediate cyclic phosphate which is formed 
as the first step in the cleavage of phosphodiester bonds by 
ribonuclease6 . 
similar inhibition7. 
under these conditions results in the conversion of virtually all 
of the cyclic phosphate to cytidine-3'-phosphate, since the yield 
of the 3'-isomer plus cyclic phosphate remains constant. Dephos- 
phorylation with alkaline phosphatase in conjunction to the 
ribonuclease digestion produced a 32% conversion to monomer. The 
simplification in structure which orcurs by removal of the poly- 
phosphate end groups in this manner can be observed in Figure 2 ,  
which illustrates the fractionation of the dephosphorylated 
oligomeric mixture. 
The 
Polyphosphates have also been shown to produce 
Apparently, extending the incubation to 12 h 
The dephosphorylated fraction which was hydrolyzed with venom 
phosphodiesterase amounted to 71% of the original sample. The 
yields of cytidine-5'-phosphate and cytidine from each of two 
hydrolyzates are tabulated in Table V. The presence of a small 
amount of cytidine is expected, since the enzyme preparation is 
known to contain a contaminating 5'-nucleotidase. 
DISCUSSION 
Preliminary studies on the products of the polyphosphoric 
acid-catalyzed polymerization of mononucleotides had suggested that 
these materials were extensively cross-linked because of the high 
proportion of apparent end groups (i.e., pho phate liberated by 
digestion with E. coli alkaline phosphatase) s,2. 
"external" phos-ha-min excess of the expected 2'(3') -end group) 
now is seen to reside in single polyphosphate termini which 
apparently are the legacy of the method of condensation. Since 
the 2'(3')-end of the chain was shown to carry the single, expected, 
phosphomonoester end group, these polyphosphate groups are probably 
located on 5'-hydroxyl ends. The presence of linkages other than 
through phosphate groups, therefore, is not consistent with the 
data. Resistance of some of the linkages to alkaline hydrolysis 
and to the action of venom phosphodiesterase, however, indicates 
that "non-natural" linkages or chain branches through 2' (3')- 
2'(3')-phosphodiester bonds are possible structural features of 
the product. However, 2'-5' and 3'-5' phosphodiester linkages 
must constitute the predominant mode of bonding. Although further 
work is necessary to clarify the total nature of the products 
obtained in this manner, it is clear that substantial quantities 
of phosphodiester bonds can be synthesized through the simple 
heating of cytidine-2' (3')-phosphate in polyphosphoric acid. 
The simplicity inherent in this method, the extensive phylogenetic 
evidence ointing to the role of polyphosphate in primitive 
polyphosphoric acid and its salts attractive materials12t13 in the 
development of a theory of abiogenesis. 
All of this 
organisms t; , and the interpretation of geochemical evidenceg-11 make 
I -  - 9 -  
TABLE I 
TYPES OF PHOSPHATE IN OLIGOCYTIDYLIC ACID 
P 
total 
a 
p1 P a  2 
a 
p3 P labile 
11.4 46-47 20-21 38-39 22.4 
a 
= phosphate liberated by alkaline phosphatase, % of total p1 
P2 = phosphate liberated with RNAse, % of total 
P3 = % of phosphate linkages cleaved by RNAse ( 2 I P x 100 
100 - P1 
TABLE I1 
END GROUP ANALYSIS OF DEPHOSPHORYLATED 
OLIGOCYTIDYLIC ACID 
Sample Cytidine Yield End Groups 
( % I  per 5.6 
Residues 
A 16.8 0.94 
B 19.4 1.09 
- 1 0 -  ~ 
TABLE I11 
YIELD OF MONOMER AFTER ALKALINE HYDROLYSIS 
OF OLIGOCYTIDYLIC ACID 
BEFORE AND AFTER DEPHOSPHORYLATION 
Sample 
Before 
Dephosphorylation 
( %  yield) 
After 
Dephosphorylation 
( %  yield) 
A 
B 
C 
D 
E 
57 
59 -- 79 
73 
76 
TABLE IV 
YIELDS OF CYTIDYLIC ACID FROM RIBONUCLEASE DIGESTION 
OF OLIGOCYTIDYLIC ACID 
Relative Yields of Isomers 
Incubation Total Yield of 3 ' -CMP 2 '-CMP 2 ' (3 ' ) -cyclic CMP 
(h) Monomer ( % ) ( % I  ( % I  ( % I  
-- 12 19 81 19 
12 19 78 22 -- 
4 21 56 20 24 
TABLE V 
HYDROLYSIS OF DEPHOSPHORYLATED OLIGOCYTIDYLIC 
ACID WITH VENOM PHOSPHODIESTERASE 
Hydrolysis 
(h) 
5'-cMP 
% Yield 
Cytidine 
% Yield 
Total 
% Yield 
3 45 13 58 
4 45 18 63 
A 
I 
- 1 1 -  * 
I 
B 
E f f l u e n t  (ml) 
0 loo 200 so0 400 500 
Effl u e n  t (ml) 
3 
.- z 
U 
0 
v) 
- 
0 
0 
Fig. 1. Effect of ribonuclease on oligocytidylic acid 
(Dowex 1x4 chloride, 1.2 x 8 an). A = before incubation, and 
B = after incubation with ribonuclease. 
c 
0 
B 
8 
0 0 
- 1 2 -  - 
O p t i c a l  d e n s i t y  ( 2 6 5 m ~ ~ )  
. 
\ 
H y d r o c h l o r i c  a c i d  (MI 
Fig. 2. Fractionation of alkaline phosphatase-treated 
oligocytidylic acid (Dowex 1x4 chloride, 1.2 x 8 cm). 
& 
- 13- - 
FOOTNOTES 
*Present address: Biomedical Research Group, Los Alamos 
Scientific Laboratory, University of California, L o s  Alamos, 
New Mexico. From the Ph.D. dissertation of Alan Schwartz, 
Florida State University, 1965. 
**Contribution no. 047 of the Institute of Molecular 
Evolution. Research aided by Grant no. NsG-689 of the U.S. 
National Aeronautics and Space Administration. 
***Polyphosphoric acid was a gift of the Victor Chemical 
Works and was found to correspond to 84-85% P205 on the basis 
of specific gravity and refractive index3. 
****Weight yields were based on the starting weight of 
nucleotide. 
L 
c 
* 
- 
- 14- 
A 
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696. 
2. A. Schwartz, E. Bradley and S. W. Fox, The Origins of Pre- - -- 
biological and of their Molecular Matrices, Academic 
Press, New 9 m , p . m  
3. R, G ,  Downing and D. E, Pearson, J. Am. Chem. SOC., 83 -
(1961) ,. 1718. 
4; E. G. Richards and H. K. Schachman, J. Phys. Chem., 63 (19591, -
1578. 
5. E. J. King, Biochem. J., 26 (1932), 292. 
6. J. P. Hummel, M. Flores and G. Nelson, J. Biol. Chem., 233 -
(1958), 717. 
7. L. Vandendriessche, Arch. Biochem. Biophys., 65 (19561, 347. -
8 .  M. E. Jones and F. Lipmann, Proc. Natl. Acad. Sci. U . S . ,  46 -
(1960), 1194. 
9. G. Schramm, The Origins of Prebiological Systems and of their 
Molecular Matrices, Academic Press, New York (196% E 134. 
10. C. Ponnamperuma, The Origins of Prebiological Systems and of - --
their Molecular Matrices, Academic Press, New York (19651, 
p. 234. 
11. K. Harada and S. W. Fox, The Origins of Prebiological S stems 
and of their Molecular Matrices, Academic Press, New Yor + 
(196FF,m89. 
12. S. W. Fox and K. Harada, Science, 133 (19611, 1923. 
13. T. V. Waehneldt and S. W. FOX, Biochim. Biophys. Acta, 00 
-
-
(1966), 000. 


Condensation of Cytidylic Acid in the Presence of Polyphosphoric Acid

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