The following notes were sent to me by John H Chalmers. He was looking for experiments connected
with the thermal cycle and nucleotide polymerization. There are more than you might think. I asked
if I could post it on the newsgroup for others and he said yes.
1.Preparation of nucleotides and oligonucleotides by thermal reaction. Moravek, Josef; Skoda, Jan.
Czech. (1969), Abstract
Labeled title compds. are obtained by thermal phosphorylation of nucleosides which are heated at 155-
65° with an alk. metal phosphate or nucleoside 2'(3'😉-phosphate in the ratio 1:5. The mixt. is
chromatographed and the products isolated from the eluate. Treating labeled nucleosides (14C, 3H,
35S) with nonradioactive phosphates gives compds. contg. soft b-emitters in the nucleoside moiety,
reaction of unlabeled nucleosides with 32P-phosphates gives compds. contg. a hard b-emitter in the
phosphate residue. The following nucleosides were phosphorylated: cytidine, uridine, pseudouridine,
orotidine, deoxycytidine, deoxyuridine, thymidine; (with a change in the base) 6-azauridine, 6-
azacytidine; (with a substitution on the base) 5-hydroxyuridine; (with a change in the sugar) N-
xylosyluracil; (antibiotics) tubercidine.
2.Preparation of nucleotides and oligonucleotides labeled with either carbon-14, tritium, or phosphorus-
32 or both, by thermal reaction of nucleosides with inorganic phosphate. Moravek, Josef; Skoda,
Jan. Inst. Org. Chem. Biochem., Prague, Czech. Abstract
The method is suitable for the prepn. of small quantities of radioactive nucleotides and
oligonucleotides. Heating an equimolar mixt. of 14C- or 3H-labeled uridine and inactive or
radioactive inorg. phosphate yields 60% mixts. of labeled nucleotides and oligonucleotides which are
sepd. by paper chromatog. and electrophoresis. The thermal phosphorylation is applicable to many
anomals nucleosides as well as those occurring naturally. The dependence of the reaction course on
time, concn
Z. of inorg. phosphate and the presence of MgSO4 is estd.
3.Thermal phosphorylations. VI. Formation of oligonucleotides from uridine 2'(3'😉-monophosphate.
Moravek, Josef; Kopecky, Jan; Skoda, Jan. Ceskoslov. Akad. Ved, Prague, Czech. Abstract
Heating 2'(3'😉-UMP 5 min. at 160° afforded a mixt. of uridine, dinucleoside diphosphates, and
trinucleoside triphosphates, which were identified by paper chromatog. and electrophoresis and by
treatment with alk. phosphatase. The oligonucleotides contained predominantly the 3' ® 5'
internucleotidic bonds and a lesser amt. of the 2' ® 5' bonds. In comparison with earlier results
(M., K. and S., 1968), the ratio between the di- and trinucleotides changed in favor of the
trinucleotides, which were obtained in 12% yield. In combination with the ion exchange sepn. of bond
isomers, the above method is useful in the prepn. of labeled tri- and dinucleotides contg. solely
the 3' ® 5' or 2' ® 5' bonds. Heating 2'(3'😉-UMP in HCONMe2 at 150° yielded uridine and uridine 2',3'-
cyclic phosphate. Likewise the 2'(3'😉-phosphates of cytidine, adenosine, and guanosine cyclized
under these conditions.
4.Formation of oligonucleotides during heating of a mixture of uridine 2'(3'😉-phosphate and
uridine. Moravek, Josef. Ceskoslov. Akad. Ved., Prague, Czech. Abstract
At 160° the thermal phosphorylation of nucleosides by inorg. phosphate showed high thermal stability
of nucleotides and dinucleotides. Of the 6 theoretically possible internucleotide bonds, only (3' ®
5'😉 and (2' ® 5'😉 bonds were formed.
The pK values of nucleoside monophosphates and inorg. phosphate are similar and internucleotide bond
formation between nucleotide and nucleoside under conditions of thermal phosphorylation seemed
feasible. Dry uridine 2'(3'😉-phosphate and uridine-14C (molar ratio 5:1) was heated 5 min. at 160°
in vacuo and the mixt. resolved by repeated chromatog. on paper in 7:1:2 iso-PrOH-NH4OH-H2O.
Comparison of the chromatographic and electrophoretic mobilities of the individual fractions showed
the presence of uridine 2',3'-cyclic phosphate (I) and uridylyluridine (II). The fraction of lower
chromatographic mobility than that of UMP was split completely by snake venom enzymes and contains
only (2' ® 5'😉 or (3' ® 5'😉 internucleotide bonds. Cleavage of II with snake venom enzymes and
bovine pancreatic ribonuclease and electrophoresis in borate buffer showed the presence of a 1:1
mixt. of uridylyl (2' ® 5'😉-uridine and uridylyl-(3' ® 5'😉-uridine. Cleavage of selected fractions
by snake venom phosphodiesterase or 1.0N NaOH gave uridine-14C 5'-phosphate or uridine-14C 2'(3'😉-
phosphate. The formation of radioactive uridine 2'(3'😉-phosphate and detection of radioactivity in
both uridine moieties of II indicated that the overall reaction is not a simple esterification. The
thermal reaction is specific in the sense that the primary phosphate attacks only the 5'-OH group of
the sugar. II was also obtained by heating a mixt. of uridine 2'(3'😉-phosphate and uridine in
HCONMe2 to give mainly I.
5.Process for preparing polynucleotides. (Farbwerke Hoechst A.-G.). Patent Family
Information Abstract
Polynucleotides are obtained by reaction of nucleotides or nucleosides with polyphosphoric acid
alkyl esters in the presence of a tertiary cyclic nitrogenous base. Thus, to prepare polyadenylic
acid (I), adenylic acid (350 mg.) was mixed with 8 g. polyphosphoric acid ethyl ester, heated 18
hrs. in a rotating flask at 55°. dissolved in 75 ml. H2O, dialyzed 4 days against H2O to remove
orthophosphate and low mol. wt. oligonucleotides, and lyophilized to give 15% yield of I. If 2 cc.
pyridine was present, yield was 20%. In 1% soln., I had sedimentation const. S28 = 2.5, diffusion
const. D20 = 12, for calcd. mol. wt. of 20,000. The polynucleotides reacted like natural nucleic
acid with regard to alkaline hyperchromia and helix formation.
6.Thermal synthesis of internucleotide phosphodiester linkages. Schwartz, Alan; Fox, Sidney W.
Florida State Univ., Tallahassee, Abstract
An oligonucleotide (I) was prepd. from cytidine 2',3'-phosphate (II) by thermal synthesis (Schwartz,
et al., Origin of Prebiological Systems, New York: Academic Press, 1964). II (5 g.) and 10 g.
polyphosphoric acid was heated at 65° 1-2 hrs., the product dissolved in NH4OH, and the soln.
dialyzed 3 days, fractionated on Dowex-1 with a mixt. of 4M HCO2H and 0.5M HCO2NH4, dialyzed again
for 3 days, and lyophilized to yield 1% I. I showed alk. hyperchromicity of .apprx.18%. Digestion by
alk. phosphatase (III) showed 40% of the total P in form of terminal phosphates. Joint hydrolysis by
III and pancreatic ribonuclease liberated addnl. 29% of the total P as compared with hydrolysis by
III alone. It was concluded that 50-60% of the phosphate in I was present in the form of 2' or 3'
phosphodiester linkages.
7.
Polymerization of unprotected 2'-deoxyribonucleoside 5'-phosphates at elevated temperature. Pongs,
Olaf; Ts'o, Paul O. P. Abstract
Polymn. of unprotected thymidine 5'-phosphate and 2'-deoxyribonucleoside 5'-phosphate (disodium
salts) in DMF at reflux 30 min was obsd. and studied. This reaction is catalyzed by proton(s) or a
proton donor and is involved with P1,P2-dinucleosidyl 5'-pyrophosphate as a key intermediate. The
main products are two series of oligomers with structural formulas of (pN)n and (pN)np; 5-10% of the
oligonucleotides consist of at least one 5'-5' phosphodiester linkage (or less likely pyrophosphate
linkage) as indicated by their resistance to spleen phosphodiesterase; no 3'-3' phosphodiester
linkage was found since all materials are hydrolyzable by venom diesterase. With the best catalysts
[b-imidazolyl-4(5)-propanoic acid or triethylamine hydrochloride], the yield of dimer to hexamer of
thymidine oligonucleotides ranges from 12 to 5% of each species. The mechanisms of the synthetic and
degradative processes are discussed. The study of this polymn. process may provide addnl.
understanding about the prebiotic synthesis of polynucleotides.
8.Abiogenic synthesis of oligonucleotides on kaolinite under the action of ultraviolet radiation.
Strigunkova, T. F.; Lavrentiev, G. A.; Otroshchenko,
V. A. A. N. Bakh Inst. Biochem., Moscow, USSR. Abstract
Processes involved in the abiogenic polycondensation of nucleotides adsorbed on the clay mineral
kaolinite under the action of UV radiation are considered. The dependence of the yield of synthesis
products on irradn. dose was studied. The max. yield corresponds to a 6-h exposure. The newly
synthesized substances were analyzed by ion-exchange chromatog. Some fractions were studied for the
type of bonds they contained by venom phosphodiesterase and RNase T2 enzyme hydrolysis.
Some of the products synthesized by exposure of AMP adsorbed on the surface of clay particles to UV
radiation may be looked upon as oligonucleotides in which some fragments have 2'-5'-bonded and
others 3'-5'-bonded nucleotides.
9.Cyanamide mediated syntheses under plausible primitive earth conditions. II. The polymerization
of deoxythymidine 5'-triphosphate. Sherwood E; Joshi A; Oro Abstract
When an aqueous solution (pH 7.0) of 3H deoxythymidine 5'-triphosphate, deoxythymidine 5'-phosphate,
4-amino-5-imidazolecarboxamide, cyanamide and ammonium chloride was dried and heated at 60 degrees C
for 18 h, oligomers were obtained in a yield of approximately 80%. After the chemical degradation of
any pyrophosphate bonds present in these oligomers, linear polynucleotides of up to 7-8 units in
length were isolated by DEAE cellulose column chromatography and identified by enzymatic digestion
procedures. The di- and trinucleotide fractions were degraded 87% and 100% by snake venom
phosphodiesterase and 39% and 9% by spleen phosphodiesterase. This synthesis of deoxythymidine
oligonucleotides was conducted under potentially prebiotic conditions and may offer a possible
method for the synthesis of deoxyoligonucleotides on the primitive Earth.
10.Early chemical evolution of nucleic acids: a theoretical model. Usher D A
SCIENCE (1977 Apr 15), Abstract
Recent experimental work suggests a possible cyclical pathway for early prebiotic oligonucleotide
formation that involves (i) dry-state (nontemplate) synthesis of random copolymers with mixed 2',5'
and 3',5' bonds, (ii) passage of these oligomers into solution at low temperatures, and (iii) a
preferential hydrolysis of the 2',5' bond in any short helices that have formed. This early system
could have selected for complementary sequences that were largely 3',5'-linked, but may not have
selected efficiently for a single enantiomer of ribose.
11.Synthesis of purine and pyrimidine nucleosides under the influence of high temperature and uv
irradiation. Kuzicheva, E. A.; Khenokh, M. A.; Tsupkina,
N. V. Inst. Cytol., Leningrad, USSR. Abstract
The formation of nucleosides from nucleic acid bases and 2-deoxy-D-ribose (I) was studied under
conditions simulatinghose of the primi irradn. of a dry mixt. of adenine and I at 170° for 30-240
min in a N, CO2, or O atm. yielded 3 different deoxyribonucleoside products. Two had higher mol.
wts. than deoxyadenosine and were not further characterized. The other was identified as
deoxyadenosine. The highest yield of this compd. was obtained in the CO2 atm. and the lowest in the
O atm. The deoxyadenosine underwent thermal decompn. when exposed to 170° for >30 min in the O atm.
Similar expts. with thymine only yielded a product with a mol. wt. greater than that of thymidine.
Tom Hendricks