viii. researches on pyrimidins : a method of · viii. researches on pyrimidins : a method of...

VIII. RESEARCHES ON PYRIMIDINS : A METHOD OF SEPARATING THYMIN FROM URACIL.

(Twenty-ninth Paper.)

BY TREAT B. JOHNSOX

(From the Sheffield Laboratory of Yale Unkrsity.)

(Received for publication, March 28, 1908)

New data about the cleavage products of nucleic acids con- tribute to our knowledge of the constitution of these acids. Practical analytical methods for the quantitative determination of the products of hydrolysis are of the greatest importance. Kossel and his co-workers have developed excellent methods for determining the purin bases-xanthin, guanin, adenin and hypoxanthin. On the other hand, methods for separating quantitatively the pyrimidins are lacking. A delicate qualitative test for uracil and cytosin, in presence of thymin, has been described in a publication from this laboratory,’ but at present there is no test, or characteristic derivative known, which serves for the identification of thymin in presence of uracil. For its detection we make use of the sublimation, the behavior towards silver nitrate and its elementary analysis.

A careful review of the nucleic acid literature reveals the fact that several investigations, in this field, signify the necessity of a practical method of separating thymin from uracil. Kut- scher2 in an investigation on the autolysis of thymus glands isolated 0.6 gram of a crystalline substance whose properties corresponded with those of thymin, but whose content of nitrogen did not agree with the theoretical value. He found 23.4 and 24.1 per cent of nitrogen while the calculated value for thymin is 22.22 per cent. His views are summarized in his own words : “ Diese Reactionen der Krystalle sowie ihr Verhalten gegen ammoniakalische Silberlosung sprachen fur Thymin, doch

1 Wheeler and Johnson: This Journal, iii, p. 183. 2Zeitschr. f. physiol. Chem., xxxiv, p, 114.

407

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408 Separation of Thymin from Uracil

bestatigte die Analyse diese Voraussetzung nicht vollig. Auch durch vielfache Umkrystallization und andere Reinigungsver- suche liess sich dieser Stickstoffwerth nicht herunterdriicken. Es musste also dem Thymin eine stickstoff reichere Substanz viel- leicht Uracil beigemengt sein.”

Steudell in an investigation on the hydrolysis of thymus nucleic acid with hydriodic acid isolated 5.9330 grams of a substance which gave 23.38 per cent of nitrogen on analysis. He says: “Als die Flussigkeit jetzt eingeengt wurde, schied sich ein Ge- menge von Thymin und Uracil aus, das ich in Ermangelung einer guten Trennungsmethode nicht weiter aufgeteilt, sondern als solches analysiert habe.” He2 also obtained a mixture of thymin and uracil in a later investigation on the oxidation of nucleic acid with nitric acid. In a recent paper, entitled “Die Zusammensetzung der Nucleinsauren aus Thymus und aus Heringssperma,” SteudeP wrote: “ Der nach der Krystallization der alloxurbasen noch bleibende Rest lieferte weiterer Behand- lung Thymin und Uracil. Diese beiden Korper liessen sich durch fraktionierte Krystallization zwar nicht quantitativ aber doch qualitativ gut trennen.”

I shall describe in this paper a new, characteristic derivative of thymin, and a method of separating, practically quantitatively thymin from uracil.

Uracil and thymin do not react, below IOO’, with nitric acid of density 1.41. On the other hand uracil dissolves, at ordinary temperature, in fuming nitric acid of density I. 5 giving practically a quantitative yield of 5-nitrouracil I. Thymin reacts, under the same conditions, giving a quantitative yield of the addition product-oxynitrohydrotJay&n4 II.

NH-CO NH-CO

I I HNO, I I CO CH --4 co CNO,

I II I II NH-CH NH-CH

I Zeitschr. f. physiol Chem., xiii, p. I 69. ZIbid., xlviii, p. 425. J Ibid., liii, p. 14. 4 Johnson: Amer. Chem. /own. (To be published in vol. xl.)


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Treat B. Johnson 409

NH-CO NH-CO

I I HNO, I co CCH, -+ co

I /CH3

b!H----!H I +NO,

NH - CHOH

II

In a paper, entitled “Die Constitution des Thymins,“’ Steudel has described the action of concentrated nitric acid on thymin. He obtained a compound to which he assigned the empirical for- mula, C,H,O,N,. He gave no melting point for the compound, but states that it did not contain water of crystallization; that it was soluble in warm water and ammonia and gave, on reduction, a derivative which responded to Weidel’s alloxan reaction. The data which I have obtained2 seem to indicate that Steu- del’s Yzitrothymin was a secondary decomposition product and not a simple thymin derivative.

The formation of the hydropyrimidin II, involves a direct addition of nitric acid to the double bond between the four and five positions of the pyrimidin ring. Oxynitrohydrothymin II, exists in two modifications which I have designated by the Greek letters a and ,B. The two isomers are formed under practically the same conditions. The a-derivative is the stable modification and melts at 183~ to 185”. The @derivative melts at 230” to 235O and rearranges to the a-form at the ordinary temperature. These isomeric oxynitrohydrothymins are especially characterized by their crystalline habit, and are converted quantitatively into thymin by reduction with tin and hydrochloric acid.

5-Nitrouracil and oxynitrohydrothymin show a remarkable difference in solubility in cold, absolute ethyl alcohol. The hydropyrimidin is extremely soluble in this reagent, while 5-nitrouracil requires approximately 800 to 900 parts of cold alcohol for complete solution. I have devised a simple method of separating thymin from uracil by the use of this difference in solubility in alcohol. The mixture of uracil and thymin is treated with the proper proportions of fuming nitric acid (sp. gr. 1.5) and converted into 5-nitrouracil and oxynitrohydrothymin respectively.

1 Zeitsckr. f. pkysiol. Ckem., xxxii, p. 241. = Lot. cit.


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The conditions of this operation are described in detail in the experimental part of this paper. The hydrothymin is then separated from nitrouracil by trituration with absolute alcohol. The hydrothymin can be identified by its melting point and crystalline habit. Furthermore the thymin can be recovered by reduction of the hydropyrimidin with tin and hydrochloric acid. The nitrouracil is identified by elementary analysis and reduction to 5-aminouracil’ with aluminum amalgam. This base gives a characteristic picrate which melts at ~247’.~

The Action of Nitric Acid on Thymin.

Nitric Acid (sp. gr. 1.415): Five-tenths of a gram of thymin was dissolved in 2 cc. of concentrated nitric acid and the solution evaporated to dryness on the steam bath. There was no evidence of any reaction and the thymin was recovered unaltered. It deposited from hot water in plates that decomposed at about 321’. When mixed with pure thymin this decomposition point was not lowered. Analysis (Kjeldahl) :

Calc111ated for C.,HoOgNz: Found :

N.................................. 22.22 22.33

Nitric Acid (sp. gr. 1.5) : The Forwaatiovt of a-Oxynitrohydro- thymin:

NH-CO

I co

I /NO2

I r\CH, NH - CH.OH

Thymin dissolves in cold, fuming nitric acid with slight evolution of heat. If red fumes are evolved by this treatment it is an indication that the thymin is not pure. The hydropyrimidin was obtained perfectly pure, and the yield was quantitative, when thymin was treated with fuming nitric acid under the following conditions: One gram of thymin was dissolved in 6 cc. of nitric acid and the solution allowed to evaporate at ordinary temperature. The nitropyrimidin deposited in large, well-de-

1 Behrend and Griinwald: Ann. d. Chew, cccix, p. 256. 2 Wheeler and Bristol: Amer. Chem. /own., xxxiii, p. 437.


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Treat B. Johnson

veloped prisms or blocks (see figure) which decrepitated above 130’ and melted at 183’ with violent effervescence. The compound was soluble in hot water, extremely soluble in alcohol, and separated from both these solvents in blocks that melted at 183’ to 185~. It was insoluble in benzene. The pyrimidin did not lose weight after drying for 1.5 hours at 100' to IIO'

and again for one-half hour at I IO' to I I 5’. When heated above I 20' it slowly underwent decomposition. The composition of the compound was not altered by recrystallization from absolute alcohol (Analysis III) :

0.2086 gram substance gave 0.2454 gram CO, and 0.0695 gram H,O. Nitrogen determinations (Kjeldahl) :

Calculated for Calculated for C~H~OSN~: C5HoOzNz (Thymin) : I. F”z.d : III.

C...... 31.74 47.61 32.08 H...... 3.70 4.76 3.70 N...... 22.22 22.22 22.00 21.95

a-Oxynitrohydrothymin dissolves in water giving an acid reaction. The pyrimidin undergoes decomposition when its aqueous solution is boiled. The addition of barium hydroxide to its aqueous solution produces no precipitate or color. No thymin deposited when an alcoholic solution of the pyrimidin was treated with ammonia. When the nitropyrimidin was dissolved in concentrated sulphuric acid, and a few drops of ferrous sulphate solution added, the characteristic test for nitric acid was obtained.

a-Oxynitrohydrothymin can also be prepared by dissolving thymin in fuming nitric acid (1.5) and evaporating the solution to dryness, at once, on the steam bath. This method of nitration can be recommended for preparing quickly small quantities of the hydropyrimidin. The yields are not quantitative since part of the thymin undergoes oxidation. The best results are obtained by this method, when I gram portions of thymin are nitrated under the following conditions : One gram of thymin is dissolved in 4 cc. of nitric acid (I. 5) and the solution evaporated to dryness, at IOOO, as quickly as possible. The nitrothymin is obtained as a colorless, crystalline residue which crystallizes from water in blocks decomposing at 183~ to 185’. It is possible, in this manner, to prepare several grams of the hydropyrimidin


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in a few minutes. The yields obtained by this method of nitration were very unifrom and are given in the table below:

.-

Nitric acid. sp.gr. 1.5

Weight of crude oxynitrohydro-

thymin.

-- cc. grmn9. grams.

4 1.0 1.1 4 1. 1.1 4 1. 1.1 4 1. 1.1 8 2. 2.15 4 1. 1.2 8 2. 2.1

h a ti

Weight of ydropyrimidin fter crystallisa Km from water

TG 0.82 0.79 0.81 1.60 0.90 1.50

Percentage of theoretical.

per cent.

57.1 58.5 56.4 57.8 55.5 64.4 53.5

Crystallography of Oxynitrohydrothymin by W. E. Ford.

The pyrimidin crystallizes in the triclinic system, showing a combination of b (OIO), c (OOI), a (IOO), M (IIO), d (034) andx (I I I). The crystals were small, averaging about 2 mm. broad by I mm. thick. In habit they present the appearance of dia- mond shaped tables with beveled edges, as is illustrated in the

figure. The face b (010) is always the most prominent, while the prism with the pinacoid a, and the base with the dome d form the beveling faces. Frequently one of these latter faces is much subordinated in size, or may be entirelywanting. The negative pyramid x is always small and often not present. The crystal


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faces, although distinct to the eye, were very poorly adapted for measurement with the reflection goniometer. They were usual1 y quite rough or curved and gave indistinct and broad signals on the goniometer. A series of the crystals were measured on the two circle goniometer and the average of the best readings obtained were taken for the fundamental angles, but the angles as given and the crystallographic constants calculated from them can be considered only as approximate. The measured angles were as follows :

(010) : (110) = 63" 54'. (010) : (100) = 960 15'. (010) : (001) = 73" 38C

Zone (OlO).(llO) : zone (OlO).(OOl) = 79' 35'. Zone (OlO).(llO) : zone (OlO).(lOl) = 50'5'. (010) : (034) = 71" 33'; talc. = 71° 46'.

Using the first five measurements as fundamentals the crystal constants were calculated to be:

a:b:c = 0.578:1.000:0.420.a = 107°35',~=100025',~=80059'

The crystals show a good cleavage parallel to c (001). On account of the nature of the material only a few of the optical facts concerning the compound could be determined. It pos- sesses a strong double refraction. The extinction direction on b (010) is inclined to the edge between b and a at 31~. The crystals when looked at in the polariscope in a direction perpen- dicular to c (OOI), the cleavage face, show the emergence ofan optic axis nearly in the center of the field.

Reduction of a-Oxynitrohydrothynzin with Aluminum-Amalgam,

Two and five-tenths grams of the hydropyrimidin were dissolved in cold water and reduced for I. 5 hours with an excess of aluminium-amalgam. The temperature was not allowed to rise above 40~ during the reduction. After filtering from aluminium- amalgam and aluminium hydroxide the filtrate was evaporated to dryness. I obtained a crystalline deposit which was difficultly soluble in cold water, but separated from hot water in plates melting at 320’. When mixed with thymin the melting point


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was not lowered. It dissolved in fuming nitric acid giving the original Lu-oxynitrohydrothymin melting at 183~ to 185’.

Calculated for CbH,j02N~: Found :

N.................................. 22.22 22.01

Reduction of cr-Oxynitrohydrothymin with Tin and Hydrochloric Acid.

Two and two-tenths grams of the pyrimidin were reduced for one hour with an excess of tin and concentrated hydrochloric acid. The acid solution was then evaporated to dryness, the residue dissolved in water, and the tin removed by precipitation with hydrogen sulphide. When the aqueous filtrate was concentrated and cooled, thymin separated in glistening plates which decomposed at 320'. Analysis (Kjeldahl) :

Calculated for CjH,jOzNz: Found:

N.................................. 22.22 22.10

/7-Oxynitrohydrothymin:

NH-CO

NH - CHOH

I have performed fifteen experiments, during this research, to determine the behavior of fuming nitric acid towards thymin under different conditions. I have taken different amounts of thymin (0.5 to 5.0 grams) and have varied the proportions of nitric acid. I have also allowed the acid solutions to evaporate under different conditions-at room temperature, on the steam oven, in a vacuum over sulphuric acid and at Too’-but in only two experiments have I observed the formation of ,B-oxynitrohydrothymin. The conditions, under which I obtained this isomer were as follows: One and six-tenths grams of thymin were dissolved in 8 cc. of cold, fuming nitric acid (I .5) and the solution allowed to evaporate, in the air, over night. The next morning large, transparent blocks had deposited which showed no signs of melting at 183” to 185' but decomposed at 230’ to


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235’ according to the rate of heating. One of the crystals selected for analysis weighed 0.2600 gram. The isomer did not revert to the a-derivative when crystallized from water or absolute alcohol, but separated on cooling in well developed prisms decomposing at 230’ to 236’. In another experiment 0.5 gram of thymin was dissolved in IO cc. of fuming nitric acid and the solution allowed to evaporate over sulphuric acid, in a vacuum desiccator. I obtained practically a quantitative yield of the P-derivative melting at 230’ to 235’. The compound did not lose weight when heated for one-half hour at 90’ to 100’.

Analyses (Kjeldahl) : Calculated for Found :

C.~H,O~NS: I. II.

iv.......................... 22.22 22.00 22.1

Reduction of /?-Oxynitrohydrothymin with Tin and Hydrochloric Acid.

This compound was reduced in the same manner as the a-derivative. The excess of tin was removed with hydrogen sulphide and the filtrate evaporated to dryness. I obtained a crystalline residue which separated from water in plates melting at 3 I 5’ to 320’. The compound sublimed when heated in a test- tube and when mixed with thymin the melting point was not lowered. It dissolved in fuming nitric acid giving a-oxynitrohydrothymin melting at 183’ to 185’. Analysis (Kjeldahl) :

Calculated for C~Hn02Nz: Found:

N . . . . . . . . . . . . . . . . .._..._........... 22.22 22.31

Rearrangement of /l-Oxynitrohydrothykn into a-Oxynitrohydro- thyynin.

Some of the a-pyrimidin, melting at 183’ to 185’, was preserved in a desiccator from May 29, 1907, until January I 7, 1908. It apparently underwent no change and melted at 180~ to 183” with effervescence. A sample of the ,Bpyrimidin, melting at 230’ to 235’ was preserved from June 19, 1907, to October 7,1907. It then decomposed at 227’ to 235’, and a nitrogen determination (Kjeldahl) gave 22 per cent nitrogen; calculated 22.22

per cent. This material was not examined again until January


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16, 1908. It then had completely rearranged to the a-pyrimidin and melted at 183’ to 188’ with effervescence. A mixture of this material and pure a-oxynitrohydrothymin melted 183~ to 186”. Analysis (Kjeldahl) :

Calculated for CbH,OhNs: Found :

N................................... 22.22 21.9

Method of Analysis.

The mixture of thymin and uracil, which is to be analyzed, is dried at IOOO, then pulverized finely, and dissolved, at ordinary temperature (zoo), in fuming nitric acid. It is essential for the success of this separation that the nitric acid have a density of 1.5 and that enough be taken to react with all the uracil and thymin present. It has been my experience that the best proportions are about IO cc. of fuming nitric acid for I gram of the pyrimidin mixture. The operation is performed best in a glass, crystallizing dish of about 5.5 cm. diameter, when working with I gram portions.

When the pyrimidins have completely dissolved the solution is evaporated to complete dryness at a temperature of 50” to 60’. If impurities are present, which are oxidized by nitric acid at this temperature, the excess of acid can be removed by drying in a vacuum over sulphuric acid and potash. The mixture of nitrouracil and oxynitrohydrothymin is then pulverized in a mortar and triturated thoroughly with cold, absolute alcohol, using 15 cc. of alcohol for each gram of the original mixture. The insoluble nitrouracil is filtered off with suction, washed with 5 cc. of cold alcohol and purified by recrystallization from hot alcohol or water. It is identified in the manner described above.

In order to obtain the oxynitrohydrothymin the alcohol wash- ings are allowed to evaporate spontaneously in a good draught. It is not advisable to remove the alcohol by heating on the steam bath. After removal of the alcohol the oxynitrodrothy- min is recrystallized from the least possible quantity of hot water and identified by its characteristic melting point and crystalline habit.

The results of the analyses of three different mixtures of uracil and thymin are given in the following table:


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Pyrim

idins

. ?E

y .

.

gram

.

I. 0.

5 ur

acil.

0.5

thym

in.

II.

0.2

urac

il 0

2 th

ymin.

III.

1.0

urac

il 0.

2 th

ymin

5:

2.0 10

Nitro

urac

il ins

oluble

in

alcoh

ol.

grm

n.

0.61

(

No

test

for

ur

acil)

0.25

(N

o te

st

for

urac

il)

1.32

(N

o te

st

for

urac

il)

Cal

cula

ted

Oxyn

itroh

yd;&

i~op

so

luble

in

gram

.

0.70

0.28

1.40

gram

.

0.83

m

. p.

af

ter

first

cr

ysta

l- liz

ation

, 18

0°-1

820.

0.30

m

. p.

af

ter

first

cr

ysta

l- liz

ation

, 18

0°-1

840.

0.24

m

. p.

af

ter

first

cr

ystd

- liz

ation

, 18

3’.

Nitro

gen

(Kjel

dahl)

=

21.9

. Th

eror

etica

l =

22

.22.

Tot$

we

ight

,-J

&&te

d O

f n’t

ro”fa

cl an

d ox

ymtrp

hy

dfT;

;n%

ymlr

gram

. /

gra?

n

0.75

1.

44

0.30

0.

55

0.30

1.

56

Theo

retic

al we

ight

.

0.58

4 i

1.70

g ‘j


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I take pleasure in thanking Professor Ford for the crystallographic description of oxynitrohydrothymin.


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Treat B. JohnsonFROM URACIL: Twenty-ninth Paper

METHOD OF SEPARATING THYMIN VIII. RESEARCHES ON PYRIMIDINS: A

1908, 4:407-418.J. Biol. Chem.

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viii. researches on pyrimidins : a method of · viii. researches on pyrimidins : a method of...

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