Table Of Content69-22,252
ALBRO, Lewis Pearson, 1922-
RADIOACTIVELY TAGGED 2,6 - DIAMINO PURINE
AND RELATED STUDIES.
University of Delaware, Ph.D., 1952
Chemistry, organic
University Microfilms, Inc., Ann Arbor, Michigan
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University of Delaware
RA£>IOACTIVELY TAGGED 2, 6-DlAMINOPURINE
AND RELATED STUDIES
fey
Lewis Pearson Albro
Submitted to the faculty of the University of Delaware
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
Newark, Delaware September 19 52
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R A D IO A C TIVELY TAGGED 2, G -D IA M IN O PU RINE
A N D RELATED STUDIES
L
Lewis Pearson Albro, Chemistry
The preparation of adenine tagged in the eight position with C14
by a new variation of the method of Clark and Ivalakar has been
performed and the desired product was obtained in 12>c/o yield
based on the radioactive carbon used.
The preparation of 2. ti-diaminopurine tagged in the eight position
with C1* has been performed and the desired product was obtained
in 79r/t yield based on the radioactive carbon used.
A series of biological experiments was performed to determine
whether or not any interaction existed between these two.com-
pounds when they were administered simultaneously to rats. Un
fortunately the methods used for the determination of thfe results
of these experiments led to completely anomalous results anti no
conclusions could be reached.
i i ii ii ii "" i m-n— inn riimuijiiiiiiiijujl
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ACKNOWLEDGMENT
The author wishes to thank Dr. William A. Mosher for
his help in the choice of this project and for the supervision
during its course, Dr. Dean C. Fletcher for his untiring
assistance particularly with the biological phases of this
work, Dr. George Hitchings of Burroughs Wellcome and Co. for
his assistance in some of the chemical steps, end The Bio
chemical Research Foundation for its financial assistance
which made this work possible.
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INDEX
ABSTRACT................................ ii
ACKNOWLEDGMENT.............................. .................. iii
HISTORICAL.............................................. ........ 1
EXPERIMENTAL................ . . 8
Synthesis of Adenine 8
Preparation of Radioactively Tagged Adenine 17
Synthesis of 2,6-Diaminopurine 23
Preparation of Radioactively Tagged 2,6-Diaminopurine27
Paper Chromatography 31
RESULTS AND CONCLUSIONS........................................35
APPENDIX......................................................... 41
CURVES......... ..................................................50
TABLE 1...........................................................54
TABLE II......................................................... 55
BIBLIOGRAPHY. . .............................................. 60
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1
HISTORICAL
General;
Nucleic acid is found in nearly every type of cell in
botH the nucleus and in the cytoplasm, though tne nucleic
acids found in these two sites are generally different* The
two types of nucleic acid which have been studied most
thoroughly are the most common in these two sites. The
nucleic acid of the nucleus is known as desoxyribonucleic
acid (DNA) and is known to be associated with the chromosomes*
It is believed that the DNA may be the part of the nucleopro-
tein which is responsible for the specificity of the genes
and because of this hypothesis a great deal of interest has
been centered on the elucidation of the properties, synthesis,
and metabolism of the nucleic acids* The cytoplasmic nucleic
acid is known as ribonucleic acid (RNA) and differs slightly
from DNA as will be described later*
The nucleic acids are highly polymerized molecules con
sisting of smaller units each of which is made up of a nitro
genous base, a sugar and phosphoric acid* These nucleotides
have long been thought to be joined together by means of phos
phate ester linkages; however, a recent paper by Ronwinl in
dicates that the polymerized unit of the nucleic acid may be
a polymeric phosphoric anhydride with the nucleosides (base
plus sugar) attached to this central core by single
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phosphoester linkages rather than the two phosphoester
linkages per nucleotide. This hypothesis explains some of
the data, particularly from x-ray diffraction studies which
could not be explained on the basis of the older hypotheses.
DNA is known to contain desoxyribose, adenine, guanine
cytosine, thymine and phosphoric acid and the structure, ac
cording to one of the earlier hypotheses, is given in Fig. 1
tVrC^vHv
l »
,i i---- °
H
U - N — CH C H - C H - C H i 0
o P
OH
N - c - |V/-— CH -C I4U-CH - CH- CH L
0
- C — N 14
O
a - ^ 7
OH
/
o —P.
Figure 1
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3
This earlier hypothesis proposed that the nucleic acid con
sisted of polymerized tetranucleotides, i.e. that the building
block of the polymer was the tetranucleotide illustrated in
Fig. 1. This hypothesis was invalidated when more subtle
analytical methods were developed which proved that the various
nucleotides were not present in al:l:l:l ratio. Also the
relatively small number of permutations of the four nucleo
tides in the tetranucleotide would not explain the believed
specificity of the nucleic acids. To account for these anoma
lies a new hypothesis proposed that the various nucleotides
were individually polymerized into the chain in an essentially
random order, with no necessity for a : : ratio of the
1 1 1 : 1
individual nucleotides. This hypothesis allowed an almost
infinite number of permutations of the structure which would
explain the specificity of the genes if it could be proved
that individual genes related to a single characteristic had
the same structure. This has not been proved, and may n«ver
be proved, but the hypothesis persists.
The structure of RNA is very similar to that of DNA ex
cept that uracil is substituted for thymine and ribose for
de soxyribo se.
The molecular weight of the polymerized nucleic acids
vary considerably depending upon the source, the type and the
method of purification. Molecular weights of 500,000 to
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4
3,000,000 have been reported2'3»4»5*6.
Analytical Determinations;
Since the only difference between the various nucleotides
from any one nucleic acid type (DNA, RNA or other) lies in the
nitrogenous bases, these have been used as the criteria for
most of the analytical studies* In general, the nitrogenous
bases have been hydrolyzed off the nucleic acid, separated and
purified In some manner which would make It possible to deter
mine either the total or relative quantities of the various
bases which were present in the original sample of nucleic
acid* The earliest work along this line, which gave rise to
the : : ratio idea and the tetranucleotide hypothesis, was
1 1 1 : 1
based on simple chemical separations of the various bases and,
due to the relative inaccuracies of fractional crystallization,
etc. It Is not surprising that these gave rise to erroneous re
sults* Later more accurate work of the same type led to bette?
results and disproved the 1:1:1:1 ratio theory* This work by
Gulland et al showed that for every four atoms of phosphorous
7
there is one radical of guanine, one of thymine, about of
1 * 2
citosine, and about 0*8 of adenine for DNA.
These results, while they were a considerable improve
ment on the older data, were shown to be considerably in error
by the work of Chargaff and his co-workers using paper chro
matography to separate the various bases. Most of Chargaff*s
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