Determination of the α-glycol group in nucleic acid components. VI. Modified method for the titrimetric determination of guanosine and xanthosine using periodate oxidation

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  • The test sample (about 700-mg) heneicosane (200 mg), 2% sodium ethoxide (0.25 ml), and chloroform (0.3 ml) were put into a round-bottomed flask, refluxed for 40 min, and cooled to 18-20~ The resulting mixture was chromatographed (volume injected 4 ~i). Figure 1 shows a typical chromatogram.

    Analysis of Morpholinophosphorodichloridate. A metal column of length 1 m was packed with 15% silicone DC 550 on Chromaton NAW-DMCS (0.16-0.20 rmn); column oven temperature 166~ injector temperature 220~ detector current 150 mA; carrier gas (helium) flow rate 60 ml/min.

    To an accurately weighed quantity of morpholinophosphorodichloridate (~150 mg) and hexa- decane (~70 mg) was added dry benzene (0.2 ml) to ensure complete dissolution of the standard. The resulting mixture was chromatographed (volume injected 2 ~i) (Fig. 3).

    Thus, we have developed procedures for the determination of (I), (II), and (III), which could be used for the control of the industrial process and the quality control of these com- pounds.


    i. I. Kolodkina, N. N. Kalinina, E. I. Pichuzkhina, N. G. Surikova, and A. M. Yurkevich, Inventor's Certificate No. 594784; Otkrytiya, No. 8, 59 (1976).

    2. P. Brige and H. Muller, Chem. Ber., 72B, 2121 (1938). 3. M, Ikehara and E. Ohtsuka, Chem. Pharm. Bull., ii, 435 (1963). 4. A .N . Nesneyanov and R. A. Sokolik, Methods of Heteroorganic Chemistry [in Russian], Mos-

    cow (1964), p. 243. 5. V .V . Katyshkina and M. Ya. Kraft, Zh. Obshch. Khim., 26, 3060-3066 (1956).




    V. Zakharans and A. I. Busev UDC 543.862.28:547.963.32

    Guanosine (I) and xanthosine (II), which normally crystallize with two molecules of water [i], are difficultly soluble compounds, which complicates analysis. Their low and gradual solubility is due to the existence of strong intermolecular hydrogen bonding. More- over, in solution purine nucleosides tend to form associates whose concentration increases with the concentration of the components; this is particularly so in the case of (I) [2]. Our intention in the work described here was to select the conditions for their titrimetric determination by eliminating this deficiency and by using the amplification method suggested earlier [3] as the most accurate and to examine the stoichiometry of per iodate oxidation under these conditions. We also examined the possibility of carrying out a titrimetric determina~ tion of xanthosine 5'-monophosphate (III) and xanthosine 5'-triphosphate (IV). The litera- ture makes no mention of the periodate oxidation of (II)-(IV). An electrochemical oxidation method has been reported for the detection and determination of (II) [4].

    We were unable to use such solvents as N,N-dimethy!formamide and dimethyl sulfoxide, which have been used successfully in the acid--base titration of (I) and (II) in nonaqueous media [5, 6], because the indicator (sEarch solution) loses sensitivity at the endpoint. Consequently we used formaldehyde and urea to enhance the solubility of (I) and (II); they are used as denaturants to suppress hydrogen bonding in nucleic-acid and amino-acid chemis- try.

    Reaction of formaldehyde with nucleic acid bases converts the amino and imino groups to the N-hydroxymethyl groups, --N-CH=--OH; moreover the resulting compounds have better solubil- ity [7, 8], which has been exploited in the acid--base titration of (I) and (II) in nonaqueous media [6]. Although under normal conditions aldehyde groups are not oxidized by periodate,

    M. V. Lomonosov Moscow State University. Translated from Khimiko-Farmatsevticheskii Zhurnal, VOlo 13, No. i, pp. 108-112, January, 1979. Original article submitted June 7, 1978.

    0091-150X/79/1301- 0099507~50 9 1979 Plenum Publishing Corporation 99

  • TABLE i. Stoichiometry of the Periodate Oxidation of Guanosine and Xanthosine in the Absence of Urea*

    t pound Oxidant pH I t--no,, H.SO, I,,7 II KIO~in0,1N HzSO4 1,7

    I it KIO, in2 N HzSO4 0,2

    Moles periodate per mole compound, n * 0.002, after oxidation mr

    8h [ 16h

    1,000 [ t,000 1,000 1,000 1,000 1,000

    ~4 h [ 48h I 1,000 [ 1,000

    1,000 ] 1,000 1,002 1,0~8


    t,002 1,003

    *Anaiytical conditions: 18~ Cperiodat e = 0.025 M; Cnucleoside = 0.O1 M.

    TABLE 2. Stoichiomatry of (in the Presence of urea),

    Com- pound



    Oxidant pH

    KIO~in0,1 N H2SO.1 1,9 2,0 2,2 2,4

    NalO4in H20 6,2 6,3 6;4 6,7

    K IO~n 2 N. H2SO~. 0,6 KIO4in0,1N H2504 1,9

    2,0 2,1 2,5

    Na IO4 inH~O 6,0 6,6 6,7 6,8

    KIO4in0,1 N H2SO4 1,8

    the Periodate Oxidation of (I), (II) (III), and (IV)*

    Concentration. in oxi- Timing points ] datlon, M I of oxidation [ - - -----] ~n-~a~ [ over which l 1 ratio I n = 1.000 ~= 1 urea I -w I ~erio- 0 .004

    I 0,5 0.0021 6,25 lmin - -24h 1,0 0.0051 2,5 lmin - -8h 2,0 0.005 1 4,0 0.005 i 0,5 0.002 ; 1,0 0.005 1 2,0 0.005 i 4,0 0.005 I 0,5 0.005 I 0,5 0.003 I 1,0 0.005 I 2,0 0.005 1 4,0 0.005 I 0,5 0.005 I 1,0 o.oo5 I 2,0 0.005 I 4,0 0.005 I - - 3 .00251

    - - 3.00251 - - 9.00351

    2;5 3 rain -- 8h 2,5 3 min -- 8h 6,25 2--30 rain 2,5 3--30 rain 2,5 3--20 rain 2,5 3--10 rain 2,5 I-- I0 rain 4,1 1 ra in- - 5 h 2,5 1 min - 8 h 2,5 1 mirr-- 8 h 2,5 1 min - - 8h 2,5 1--20 rain 2,5 1--10 rain 2,5 1--5 rnin 2,5 1--4 rain 5,0 3 min- - 5 h 5,0 2--10 rain 3,6 1--5 min

    Moles periodate per mole compound, n, after 24 h oxi- dation

    Na IO4in H20 7,0 IV KIO4~n0,1 N H2SO~ 1,6

    *Ana ly t i ca l cond i t ions : 18~ Cper iodat e

    1,003 1,005 1,006 1,008 1,071 I , I I0 1,180

    l, l l2 1,012 1,004 1,006 1,01O 1,400 1,410


    = 0.0125 M in all cases.

    the use of formaldehyde in periodate oxidation reactions is precluded by the considerable and variable correction in the blank run. Solutions of formaldehyde in polar solvents (water, alcohols) are known to be equilibrium mixtures of polymeric solvates -- polyoxymethylene glycols HQ(CH20)nH or polyacetals RO(CH20)n H- with a small amount of free formaldehyde. The quan- titative composit ionof the equilibrium mixture depends mainly on the formaldehyde concentra- tion and temperature [9]. The consumption of periodate in the blank run was evidently due to the oxidation of these polymeric solvates.

    Urea proved more suitable for enhancing the solubility of (I) and (II) in periodate oxidation. Urea has been used for the chromatographic separation of oligonucleotides in or- der of chain length [I0]. At 25~ (I) is ten times more soluble in 8 M urea solution than in water [ii]. We found that urea also enhances the solubility of (II), though not to such a great extent, but enough for the determination. In 8 M urea solution 0.01 M solutions of (I) and (II) can be prepared by warming. The resulting solutions can be diluted to i M urea concentration with water without precipitating (I) and (II).

    Compounds (I) and (II) are very stable to overoxidation by a solution of KIO, in 0.i N H2SO, at pH 1.7 (Table i); overconsumption of the oxidant occurs only after 72 h oxidation. During oxidation of (II) with Kl04 solution in a more acidic solution -- in 2 N H2SO, at pH 0.2 -- overconsumption of the oxidant occurs earlier, after 24 h.

    Experiments showed that the error due to the presence of large quantities of urea (per- iodate oxidation in 0.5-4 M urea solution) is comparable with the error in the end-point de- termination; in total they do not exceed 0.4-0.8%. When adequately pure preparations of urea are used and the oxidation time does not exceed 30 min-i h, the blank run can be carried out


  • TABLE 3. Titrimetric Determination of (I)-(IV) KI04 solution in 0.i N HaSO,)

    Test corn- Molecular pound weight

    I II

    III tV

    319,28 320,26 408,t7 568,13

    Amount of test iUre a con- compound ] centration equi~ralent to in oxida- 1 ml of 0.025 tton, M N Na2S20~

    1,3303 2,0 4,0 1.3344 2,0--4,0 1,7007 2,3672

    (oxidant 0.025 M

    Mean te- n suit, X, %

    10 98,4 98,3 10 75,7

    g 86,0

    Relative [ standard de- viation, s~:, ~

    0,4 0,5 0,8 1,0

    without urea. When oxidation extends over several hours the correction in the blank run due to the presence of urea becomes appreciable but does not exceed 0.1-0.2 ml.

    Table 2 summarizes our results for the stoichiometry of periodate oxidation of (I)-(IV). The presence of 0.5-4 M urea solution during the oxidation of (I) and (II) with a 2.5-fold excess of KI04 in 0.i N H2SO4 at a pH of about 2.0 causes overconsumption of the oxidant only after 8 h, though it becomes appreciable after 24 h and increases slightly with urea concentra- tion. When (I) and (II) are oxidized with a solution of NaI04 in water at pH 6.0-7.0 over- consumption of the oxidant occurs very rapidly and increases rapidly with urea concentration. Consequently we do not recommend oxidation with aqueous NaI04 for the determination of (I) and (II) in the presence of urea. Thus the periodate oxidation of (I) and (II) in the pres- ence of urea shows, albeit to a greater extent, the same general feature that we have en- countered in the periodate oxidaton of other ribonucleosides: The reaction is more specific when carried with a solution of KIO4 in 0.i N H2S04 at a pH of about 2.0 and change to neu- tral pH causes overconsumption of the oxidant to occur sooner [12]. When (I) and (II) are to be determined in the presence of urea they should be oxidized with a solution of KIO4 in O.i N H2S04 for 10-15 min.

    The solubility of (III) and (IV) in water is enough to obviate the use of agents to enhance their solubility in periodate oxidation. While (III) when oxidized with a solution of KIO4 in 0.i N HeSO4 at pH 1.7 is stable to overoxidation, like other nucleoside 5'-mono- phosphates [12], the oxidation of (IV) lacks the lengthy linear section on the plot of oxi- dant consumption against reaction time (Fig. i). Consequently, in the determination of (IV) the oxidation time should not exceed 3-5 min. The results of the titrimetric determination of (I)-(IV) after statistical treatment according to the IUPAC recommendations are summarized in Table 3.


    Preparation of Solutions. We have described the preparation of the 0.025 M periodate solution from KI04 (pure for analysis grade) in 0.i N HAS04 in an earlier publication [3]. The 0.025 M solution of KIO4 in 2 N HaSO4 was prepared in the same way by changing the con- centration of the acid being diluted. The 0.025 M NaIO~ solution was prepared by dissolving a weighed quantity of the preparation (pure for analysis grade) in distilled water.

    The 8 M urea solution was prepared by dissolving a weighed quantity of the preparation (pure for analysis, chemically pure, and ultrapure grades) in distilled water. The prepara- tion of the other reagents was described in [3].

    Guanosine, Xanthosine, Xanthosine 5'-Monophosphate, and Xanthosine 5'-Triphosphate. We used chromatographically homogeneous preparations of guanosine and xanthosine dihydrates (Olaine Plant of Chemical Reagents). Their purity and content were checked before use by UV spectroscopy [13] and potentiometric titration in dimethyl sulfoxide [5]. We used chromato- graphically homogeneous preparations of the disodium salts of (III) and (IV), whose purity and content were checked before use by ~ spectroscopy.

    Analytical Procedure. We used the titrimetric version of the amplification method [3] to examine the stoichiometry of periodate oxidation of (I)-(IV) and to determine these com- pounds.

    The results summarized in Table 1 were derived by the following procedure. The 0.01 M solutions of (I) and (II) were prepared by putting weighed quantities into graduated flasks and diluting almost to the mark with a solution of KI04 in 0.i or 2 N H2SO4. The content~


  • t 2 3 4~ 5 6 7 8 9 10

    Fig. i. Stoichiometry of the oxidation of the disodium salt of (IV) with a 3.6- fold excess of KIO~ in 0.i N H2SO4 at 18~ The abscissa denotes oxidation time (min) and the ordinate oxidant consumption (n) [moles of KI04 per mole of (IV)].

    of the flasks were stirred from time to time. When the preparations had dissolved, the solu- tions were diluted to the ma~rk with the same periodate solution, their contents were care- fully stirred, and i0 ml aliquots containing 0.i mg-eq of (I) or (II) were removed for the determination. For the oxidation of (II) with KIO~ solution in 2 N H2SO4, 2 N NaOH solution (2 ml) was first added to the aliquot to neutralize the acid.

    Solutions of (I) and (II) (normally 0.01 M) were prepared by putting weighed quantities into graduated flasks. The preparations were dissolved in a small quantity of 8 M urea solu- tion by warming and stirring. After cooling the solutions were diluted to the mark with the urea solution and distilled water, and i0 ml aliquots of the solutions, normally containing 0.i mg-eq of (I) and (II), Were removed for the determination. The timing points were 30 sec, I, 2, 3, 4, 5, i0, and 30 min, and i, 2, 3, 4, 5, 6, 7, 8, and 24 h.

    The analytical for procedure (III) and (IV) followed that of our earlier work [3, 12].


    i. M. Herceg, J. Fischer, and R. WEiss, Izv. Jugoslav. Centra Kristalogr., 2, Suppl., 130- 132 (1976).

    2. A. V. Borodavkin, E. I. Budovskii, Yu. V. Morozov, et al., in: Electronic Structure, UV Absorption Spectra, and Reactivity of Nucleic Acid Components [in Russian], Moscow (1977), p. 112.

    3. A. I. Busev, V. Zaharans, and U. Mikstais, Khim.-Farm. Zh., No. 3, 128-132 (1977). 4. J. L. Owens and G. Dryhurst, Anal. Chim. Acta, 89, 93-99 (1977). 5. U. Mikstais, A. P. Kreshkov, N. T. Smolova, et al., Izv. Vyssh. Uchebn. Zaved., Khim.

    Khim. Tekhnol., No. i, 146,148 (1978). 6. A. Veveris, "A study of the acid--base properties of pyrimidine and purine derivatives in

    nonaqueous solutions and their quantitative determination by potentiometric titration," Candidate's Dissertation, Moscow (1978).

    7. M. Ya. Feldman, Progr. Nucleic Acids Res. Mol. Biol., 13, 1-49 (1973). 8. J. D. McGhee and P. H. yon Hippel, Biochemistry (Washington), 14, 1281-1296, 1297-1303

    (1975); 16, 3267-3276, 3276-3293 (1977). 9. A. V. Rudnev, G. V. Kovalev, K. S. Kalugin, and E. P. Kalyazin, Zh. Fiz. Khim., 51, 2031-

    2033 (1977). i0. G. N. Tener, in: Methods for the Study of Nucleic Acids [Russian translation], Moscow

    (1970), pp. 85-90. ii. T. T. Herskovits and J. J. Bowen, Biochemistry (Washington), 13, 5474-5483 (1974). 12. A. I. Busev and V. Zaharans, Khim.-Farm. Zh., NO. 5, 137-140 (1978). 13. Specifications and Criteria for Biochemical Compounds, Washington (1972), 3rd ed., pp.

    168, 182.



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