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 anomalous nucleosides as well as those occurring naturally. The dependence
of the reaction course on time, concn. 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 simulating those of the primitive earth. The uv
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