determination of the α-glycol grouping in nucleic acid components

4
METHODS OF ANALYSIS AND QUALITY CONTROL DETERMINATION OF THE a-GLYCOL GROUPING NUCLEIC ACID COMPONENTS II. NEW AMPLIFICATION METHOD OF THE TITRIMETRIC DETERMINATION OF RIBONUCLEOSIDES AND NUCLEOSIDE 5' -PHOSPHATES A. I. Busev, V. Ya. Zakharans, and U. Ya. Mikstais IN UDC 615.014o471:547~ ]~ At the present time, ribonucleosides (I) and nucleoside 5'-phosphates (II) are used in medicine (inosine, ATP, etc.), as taste intensifiers (nueleoside 5'-phosphates), and to obtain valuable physiologically active compounds. Chemical methods for their production, including methods for the chemical phosphorylation of I unsubstituted in positions 2' and 3' with the aim of producing II, are being used ever more widely. To determine nucleosides and their phosphates, methods of UV spectroscopy and acid-base titration have been proposed [1 ]. But both the spectral and the acid-base properties of these compounds depend on the nature of the heterocyelic base and are shown similarly in nucleosides ~:ad their phosphates modified in the ribose moiety [1 ]. By these methods it is impossible to determine I in the presence of 2' -deoxyribonucleo- sides (III) and to determine II in the presence of nucleoside 2'(3')-phosphates IV. The same deficiencies are also inherent in the eoulometric method of determining nueleosides and nucleoside phosphates of cytosine, thymine, and uracil [2]. For these purposes methods based ontheMalaprade reaction, consisting in the selec- tive oxidation of the cis-a -glycol groupings in the ribose moieties of I and I[ are promising~ Periodate oxida- tion is widely used in analytical chemistry mad for structural investigations of carbohydrates and their de- rivatives [3, 4]. It has been established that I and II of adenine and guanine and N-ribosyl derivatives of nucleic acids of the pyrimidine series react with periodate in a molar ratio of 1:1, while the corresponding III ~.nd IV do not react with pertodate [5]: 5 F E 0 GHzOH ~I~' 0'( ~t 1 0-4 ~ HGI OHI + Id-) ~- H~.O HO OH HG OH II H o o where R is a heteroeyelic base. Thus, in the oxidation of I and II the periodate is reduced to todate but no formaldehyde or formic acid is produced at the same time, as is observed in the oxidation of r The usual methods with oxidation by periodate reduce to the determination of formaldehyde or formic acid formed [3, 6] or to the determination of the periodate used [3, 6, 7], for which purpose back-titration of the excess of periodate is used. A method has been proposed previously for determining I and II in which after oxidation an excess of arsenite is added to the excess of periodate, and the residuaI arsenite is then back- titrated with a standard iodine solution [8 ]. We have found no information in the literature on methods of analyzing I and II based on the determina- tion of the iodate formed on their oxidation by periodate. To develop such a method we have concentrated on the method of determining iodate in the presence of periodate by binding the latter in the form of a soluble complex compound with hexavalent molybdenum and determining the iodate iodometrically. It is known that at pH 2.0-5.0 molybdenate anions and periodate form a heteropolyacid -6-molybdoperiodate [I(MoO4)G]~-. M. V. Lomonosov Moscow State University. All-Union Scientific-Research Institute of Chemical Reagents and Ultrapure Chemical Substances "IREA," Latvian Branch, Olaine. Translated from Khimiko- Farmatsevtieheskii Zhurnal, Vol. 11, No. 3, pp. 128-132, March, 1977. Original article submitted March 30, 1976. This material is p "oteeted by copyrigtzt registered in the name of Plenu ~ Publishing Corporation ~7 West 17th Street New York ,V y lo~1; ~ ~ ] of this publication r~2a)' be rep "odz~ced stored in a retrieval 5,v~tem" ~r tr~,,r i ~ .~ ~-.---' ~-- ' ~ . ' ~ ". ...... N~ ~.~rt . . . . . ' ~ ..... ~ .................. , *t, .-Z.~' Jtlrrtt Or o)' a~zy t?leat;s electrot~ic,~ niechanteal, photocopying, 1 [mtcrojttming recoraing or otherwise without written permission of the publisher. A copy of this article is available from the publisher for $ 7 50. 417

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Page 1: Determination of the α-glycol grouping in nucleic acid components

METHODS OF ANALYSIS AND QUALITY CONTROL

D E T E R M I N A T I O N O F T H E a - G L Y C O L G R O U P I N G

N U C L E I C A C I D C O M P O N E N T S

II. NEW AMPLIFICATION METHOD OF THE TITRIMETRIC

DETERMINATION OF RIBONUCLEOSIDES AND

NUCLEOSIDE 5' -PHOSPHATES

A. I . B u s e v , V. Y a . Z a k h a r a n s , a n d U. Y a . M i k s t a i s

IN

UDC 615.014o471:547~ ]~

At the p r e s e n t t ime , r ibonucleos ides (I) and nucleoside 5 ' -phospha tes (II) are used in medic ine (inosine, ATP, etc.) , as tas te in tens i f ie rs (nueleoside 5 ' -phospha tes ) , and to obtain valuable physiological ly active compounds. Chemical methods for the i r production, including methods for the chemica l phosphoryla t ion of I unsubst i tuted in posi t ions 2' and 3' with the a im of producing II, a re being used eve r m o r e widely.

To de t e rmine nucleosides and the i r phosphates , methods of UV spec t roscopy and a c i d - b a s e t i t rat ion have been p roposed [1 ]. But both the s pec t r a l and the a c i d - b a s e p rope r t i e s of these compounds depend on the nature of the he te rocye l i c base and a re shown s i m i l a r l y in nucleosides ~:ad the i r phosphates modified in the r ibose moie ty [1 ]. By these methods it is imposs ib le to de t e rmine I in the p r e s e n c e of 2' -deoxyr ibonucleo- s ides (III) and to de t e rmine II in the p r e s e n c e of nucleoside 2 ' (3 ' ) -phospha tes IV. The s a m e def ic iencies a re a lso inherent in the eou lomet r i c method of de termining nueleosides and nucleoside phosphates of cytosine , thymine , and urac i l [2]. F o r these purposes methods based o n t h e M a l a p r a d e react ion, consis t ing in the s e l e c - t ive oxidation of the c i s - a -glycol groupings in the r ibose moie t ies of I and I[ a re promis ing~ Per ioda te oxida- tion is widely used in analyt ical c h e m i s t r y mad for s t ruc tu ra l invest igations of ca rbohydra te s and their de - r iva t ives [3, 4]. It has been es tabl i shed that I and II of adenine and guanine and N-r ibosy l de r iva t ives of nucleic acids of the pyr imid ine s e r i e s r e a c t with per iodate in a m o l a r rat io of 1 : 1 , while the cor responding III ~.nd IV do not r e a c t with per toda te [5]:

5 F E 0 G H z O H

~ I ~ ' 0 ' ( ~t 1 0-4 ~ HGI OHI + I d - ) ~- H~.O

HO OH H G O H II H o o

where R is a he te roeye l i c base . Thus, in the oxidation of I and II the per ioda te is reduced to todate but no formaldehyde or fo rmic acid is produced at the s a m e t ime, as is observed in the oxidation of r

The usual methods with oxidation by per ioda te reduce to the de terminat ion of formaldehyde or fo rmic acid fo rmed [3, 6] o r to the de te rmina t ion of the per ioda te used [3, 6, 7], for which purpose back- t i t r a t ion of the excess of pe r ioda te is used. A method has been proposed previous ly for de te rmin ing I and II in which a f te r oxidation an excess of a r sen i t e is added to the excess of per ioda te , and the res iduaI a r sen i te is then back- t i t ra ted with a s tandard iodine solution [8 ].

We have found no informat ion in the l i t e ra tu re on methods of analyzing I and II based on the d e t e r m i n a - tion of the iodate fo rmed on the i r oxidation by per iodate . To develop such a method we have concentra ted on the method of de te rmin ing iodate in the p r e s e n c e of per iodate by binding the l a t t e r in the f o r m of a soluble complex compound with hexavalent molybdenum and de te rmin ing the iodate iodometr ica l ly . It is known that at pH 2.0-5.0 molybdenate anions and per ioda te fo rm a he teropolyacid - 6 - m o l y b d o p e r i o d a t e [I(MoO4)G] ~-.

M. V. Lomonosov Moscow State Univers i ty . All-Union Sc ien t i f i c -Resea rch Inst i tute of Chemica l Reagents and Ul t rapure Chemica l Substances "IREA," Latvian Branch, Olaine. Trans la ted f r o m Khimiko- F a r m a t s e v t i e h e s k i i Zhurnal , Vol. 11, No. 3, pp. 128-132, March, 1977. Or ig inal a r t ic le submit ted March 30, 1976.

T h i s m a t e r i a l is p "o t ee t ed b y co p yr i g t z t r eg i s t e red in the n a m e o f P l enu ~ P u b l i s h i n g C o r p o r a t i o n ~ 7 West 1 7 t h S t r e e t N e w Y o r k ,V y l o ~ 1 ; ~ ~ ] o f th is p u b l i c a t i o n r~2a)' be rep "odz~ced s t o r e d in a retr ieval 5,v~tem" ~r t r ~ , , r i ~ .~ ~-.---' ~ - - ' ~ . ' ~ ". . . . . . . N ~ ~.~rt

. . . . . ' ~ . . . . . ~ . . . . . . . . . . . . . . . . . . , *t, .-Z.~' Jtlrrtt Or o)' a~zy t?leat;s electrot~ic,~ n iechantea l , p h o t o c o p y i n g , 1 [ m t c r o j t t m i n g r e c o r a i n g or o t h e r w i s e w i t h o u t w r i t t e n p e r m i s s i o n o f t he pub l i sher . A c o p y o f th i s art ic le is avai lable f r o m the p u b l i s h e r for $ 7 50.

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Page 2: Determination of the α-glycol grouping in nucleic acid components

TABLE 1. Results of the Titrimetric Determination of sides and Nucleoside 5'-Phosphates

Mean Compound .demrmined Molecular n result

weight (~-), %

Uridinr Disodium uridine 5'-mono-

phosphate Trisodium uridine 5'-triphos-

phate 5- t~hyluridine 5-Bromouridine Cytidine Cytidine 5'-monophosphate Di$odium cytidine 5'=diphos-

phate Adenosine

Adenosine 5'-monophosphate Trisodium adenosine 5'-diphos-

phate

Disodium adenosine 5'-triphos- pilate tetrahydrate

N, N" Dimetlv)ladenosinr 5'-Tosyladenosine Inosine Disodium inosine 5'-

monophosphate Disodium inosine 5'- diphosphate

C-uanosine

Guanosine 5'-monophosphate Disodium guanosine 5'-diphos-

phate Disodium guanosine 5'-triphos-

phate

Amount of I compound t determined l correspond- / ing to 1 rail of 0.0250 N i sodium thiomlfate

244,20 1,0175

368,15 1,5340

550,09 2,2920 272,20 1,1342 323,11 .1,3463 243,20 1,0133

323,20 1,3467

447,20 1.8633 267,24 1,1135

347,23 1,4468

493,15 2,0548

623,23 2,5968 295,30 1,2304 421,43 t,7560 268,23 1,1176

392,19 1,6341

472,15 1,9673 283,25 1,1802

363,23 1,5134

486,16 2,0257

567,15 2,3631

10

8 7 7 8

10

i

5 10

6

10

?o lo

5

5

7

97,9

80,8

81,6 80,6 99.8 99,7

80,2

88,4 99,4

95,0

91,4

99,7 98,8 94,4 97,5

72,7

82,1 87,6

84:5

87,4

90,4

Ribonueleo-

Relative standard deviation (so, %

0.3

0.8

0.4 0.51 0,4 0.3

;).(,

Q6 0.3

0.5

0.4

0,9 , 0,5

0.9 U.4

I

0.6

0,5 0.5

0.7

0,6

0.6

At pH 3.0 in the p r e s e n c e of an e x c e s s of mo lybda t e , p e r i o d a t e no l o n g e r oxid izes iodide, while iodate oxid izes i t to iodine [9]. The o p t i m u m acid i ty f o r this is pH 3.0; at h ighe r pH va lues the r e a c t i o n between iodate and iodide takes p lace m o r e s lowly , and at l o w e r pH va lues the 6 - m o l y b d o p e r i o d a t e d i s s o c i a t e s and, in addit ion, iodide is ox id ized a p p r e c i a b l y by a t m o s p h e r i c oxygen [9]. At tempts have been m a d e to use this s i tua t ion fo r d e t e r m i n i n g g lyco l and g l y c e r o l [10]o It was then c o n f i r m e d that a 40- fo ld e x c e s s of mo lybda t e a t pH 3.0 c o m - p l e t e ly p r e v e n t s the r e a c t i o n of pe r ioda t e with iodide and that the mask ing of the pe r ioda t e takes p lace in - s t an t aneous ly , whi le its d e m a s k i n g can be b rough t about by oxa l ic acid, which was used fo r the s u c c e s s i v e d e t e r m i n a t i o n of iodate and p e r i o d a t e in a m i x t u r e [11 ]. The method has been used fo r the de tec t ion of t a r - t r a t e and D - g l u c o s e [12 ], f o r the s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of v i c - d i o l s - manni to l , so rb i t e l , D - m a n - n o s e , D - g l u c o s e , e tc . [13] - f o r d e t e r m i n a t i o n of t a r t r a t e in the p r e s e n c e o f c i t r a t e [14], and f o r the i o d o m e t r i c d e t e r m i n a t i o n of a - a m i n o a lcohols conta in ing p r i m a r y , s e c o n d a r y , o r t e r t i a r y amino g roups - m o n o - , d i - , and t r i e t hano l amine s - and amino h y d r o x y acids - s e r i n e and threonine [15].

F o r the t i t r i m e t r i c m i c r o d e t e r m i n a t i o n of I and II a method is p r o p o s e d which is based on the d e t e r - ru inat ion of the iodate f o r m e d on the i r oxidat ion by p e r i o d a t e in an amount equ iva len t to the amount of c o m - pound oxidized. Af te r the end of the oxidat ion r eac t i on , the exces s of pe r ioda t e is bound as 6 - m o l y b d o p e r i o - da te by an exces s of mo lybda te at pH 3.0. Under these condi t ions only the iodate r e a c t s with iodide:

IOn- -+- 5 I - "+ 6H + "-+ 3Iz + 3H20,

l i be r a t i ng an amoun t of iodine equiva len t to the iodate , which is found by t i t r a t ing the solut ion with sod ium th iosul fa te . The fac t tha t the va lue of the c o r r e c t i o n in a blank e x p e r i m e n t is v e r y sma l l and is cons t an t f o r a g iven pe r ioda t e so lu t ion shows that pe r ioda t e is c o n s u m e d only in the oxidat ion of the I o r II, and the actual s i z e of the c o r r e c t i o n in the b lank e x p e r i m e n t shows the amount of iodate p r e s e n t in the pe r ioda t e p r e p a r a t i o n as an impur i ty .

418

Page 3: Determination of the α-glycol grouping in nucleic acid components

The resul ts of the t i t r ime t r i c determinat ion of I and II t reated s ta t is t ical ly in accordance with the IUPAC recommendat ions are given in Table 1o The method is applicable to the determinat ion of natural r i b o n u c l e o s i d e s - uridine, cytidine, adenosine, inosine, g u a n o s i n e - and their 5 ' -mono-~ --di-, and - t r iphos - phates , and also of some der ivat ives of I obtained synthetically. The proposed method of determining I and II is s imple in per formance . To obtain accura te and sa t i s fac tor i ly reproducible resul ts the condition pH = 3.0 m u s t be observed. The method is charac te r i zed by selectivi ty, high sensi t ivi ty, and accuracy. The de te r - mination of I and II is not affected by Ig and IV, respect ively , or by adenine or guanine~ The end point of the t i t rat ion is sharp and no rapid turning blue is observed after the equivalence point has been reached. A blue co lor appears slowly only severa l minutes after the end of t i tration and is apparently due to the oxidation of the iodide ions by a tmospher ic oxygen. The relat ive standard deviation does not exceed 0.5-1%.

The amplification t i t r ime t r i c method was f i r s t used to determine I and IL When 1 mole of I o r of II is oxidized by per iodate , 1 g-ion of iodate is formed and in react ion with iodide this gives 6 g-eq of iodine, the t i trat ion of which consumes 6 g-eq of sodium thiosulfate (IQ - 6I - 6 S 2 0 3 2 - }o This fact shows the accu- r acy of the proposed method for determining I and II is determined by the accuracy of standardization of the thiosulfate solution.

In the s tandardizat ion of a 0.025 N solution of sodium thiosulfate, an e r r o r of one unit in the fourth decimal place gives a relat ive e r r o r of 0.4%.

The proposed method em] be used for determining I and iI in their prepara t ions , for analyzing fractions of nucleoside phosphates obtained in the chemical phosphorylation of i unsubstituted in positions 2' and 3' , and for monitoring cer ta in stages of the chemical synthesis of III f rom I. In a number of cases , its use makes it possible to replace labor ious methods of enzymatic determinat ion of II in the presence of IV and to eliminate the chromatographic separat ion of mixtures of I and III for per forming the analysis.

E X P E R I M E N T A L

The oxidation react ion was per formed at room temperature in the optimum sulfuric acid medium with pH 1.0-2.0. A 0.025 M (0.05 N) solution of periodate was prepared by dissolving the appropriate weight of po tass ium metaper iodate KIO 4 ("ch.d.ao" ["analytic"] grade) in 600 ml of distil led water and then 100 ml of 1 N sulfuric acid was added and the mixture was heated until dissolution was complete. The result ing solu- tion was t r ans fe r red quantitatively to a l - l i t e r measur ing flask and, af ter cooling to room tempera ture , was made up to the m a r k with disti l led water .

A 2 M solution of sodium molybdate was prepared by dissolving Na2MoO ~ �9 2H20 ("ch.d.a") in distilled water . Where neces sa ry , the resul t ing solution was f i l tered through a paper f i l ter .

A buffer solution with pH 2.9-3.0 was prepared f rom a 2 M solution of monochloroacet ic acid to which a concentrated sol,ation of caust ic soda was added to give the required pH vahle. The pH was checked by means of a pH-340 pH mete r . Since the aqueous solution of sodium molybdate had a weakly alkaline reaction, to maintain the optimum pH it is desirable to combine the buffer solution with the molybdate solution. Thus, to four par t s of buffer solution was added one pa r t of molybdate solution, and the result ing solution was brought to pH 2.9-3.0 by the addition of a concentrated solution of monochloroacet ic acid.

A 0.025 M (0.025 N) solution of sodium thiosulfate was prepared f rom N ~ ' ) 3- 5H~O ("ch.d.a"). The t i ter of the solution was determined before use with a standard solution of potass ium dichromate or potassium iodate [I 6 ].

A 20% solution of potassium iodide was prepared by dissolving the salt ("ch.d~a. ") in distilled water, and it was stored in a dark-glass flask.

A I% aqueous solution of starch was used as indicator,

Ribonueleosides and Nueleoside 5'-Phosphates. Commercial chromatographically homogeneous prep- arations of I and II produced by varioas firms and containing various amounts of water were used. Before use, their purity and concentration of active material in them were determined by UV spectroscopy [i ]~

Method of Determination. A sample of the compound to be determined containing approximately 0.I meq of I or II weighed with an accuracy of + 0.02 mg was placed in a 250-ml flask with a ground-in stopper~ and i0 ml of 0.05 N periodate solution, measured accurately with a pipette, was added. The flask was closed with the stopper, set in a place protected from sunlight and left for about 15 rain (in the case of the sparingly soluble guanosine for about 2 h) with occasional stirring of its contents. After the end of the oxidation re-

419

Page 4: Determination of the α-glycol grouping in nucleic acid components

action, 25 ml of buffer solution containing molybdate was added to the flask, its contents were well s t i r red , and then 5 ml of 20% potassium iodide solution was added. After a few minutes, the liberated iodine was t i trated with the standard sodium thiosulfate solution in lhe presence of 2-4 drops of starch solution until the bluish-pink color had disappeared. A blank experiment was performed similarly.

LITERATURE CITED

1. Specifications and Cri ter ia for Biochemical Compounds, Washington (1972), pp. 149-183. 2. J . E . O'Reilly, Anal. Chem., 4_~7, 1077 (1975). 3, G. Dryhurst , Periodate Oxidation of Diol and Other Functional Groups. Analytical and Structural

Applications, Pergamon, P ress , Oxford-New York (1970). 4. A . J . Fatiadi, Synthesis, No. 4 ,229 (1974). 5. G. Schmidt, Meth. Enzymol., 1..~2, Par t B, 230 (1968). 6. A.B. Zanlungo, Anal. Assoc. Quire. Argent,, 6.~1,287 (1973). 7. A. Berka, Ya. Vulterin, and Ya. Zyka, New Redox Methods in Analytical Chemistry [in Russian],

Moscow (1968) pp. 114-127. 8. V. Ya. Zakharans and U. Ya. Mikstais, Abstracts of Lectures at a Scientific Conference on Methods

of Obtaining and Analyzing Biochemical Preparations [in Russian], Riga (1975), pp. 51-520 9. D. Burnel, C R. Acad. Sci., 261, 1982 (1965).

10. D. Burnel, ]3. Gournail, and L. Malaprade, C. R. Acad. Sci., 261, 2117 (1965). 11. R. Belcher and A. Townshend, Anal. Chim., 41,395 (1968). 12. G. Nisli and A. Townshend, Talanta, 1..~5,411 (1968). 13. G. Nisli and A. Townshend, Talanta, 1.~5, 1377 (1968). 14. G. Nisli and A. Townshend, Talanta, 1..~5, 1480 (1968). 15. A. Besada and Y. A. Gawargious, Talanta, 2_1, 1247 (1974). 16. L M. Kolthoff, R. Belcher, V. A. Stenger, et al., Volumetric Analysis. Vol. 3, Wiley-Interscience,

New York (1957) ~uss ian translation: Moscow (1961), pp. 279-285].

G A S - C H R O M A T O G R A P H I C D E T E R M I N A T I O N OF M E T H Y L A N D R O S T E N E D I O L ,

M E T H Y L T E S T O S T E R O N E , AND M E T H A N D R O S T E N O L O N E P R E S E N T

T O G E T H E R IN AN AIR M E D I U M

V. K. E r m a k o v a , I. I. D o z o r o v a , UDC613.632.4:615.357.631]-074 and L. F . S h a s h k i n a

Methandrostenolene (MAS) is a pronounced anabolytic. Methylandrostenediol (MAD), possessing ana- bolic propert ies , is considered in this case as an intermediate of the synthesis of the androgenic preparation methyltestosterone (MT). In the production of the indicated preparations, they may have an undesirable in- fluence on the bodies of the workers [1 ].

Fo r a hygienic evaluation of the conditions of work and a study of the toxic properties of the substances, it is of great importance to analyze the air of the working zone of work rooms. Recently, modern physic . - chemical methods, including gas- l iquid chromatography, permitting a rapid, accurate, and highly sensitive analysis of complex multicomponent mixtures, have been increasingly often used for these purposes.

Branch of the S. Ordzhonikidze All-Union Scientific-Research Institute of Pharmaceutical Chemistry, Moscow Region. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 11, No. 3, pp. 132-134, March, 1977. Original art icle submitted June 25, 1976.

I This material is protected by copyright registered in the name o f Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission o f the publisher. A copy o f this article is available from the publisher for $ 7.50.

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