The reactions of oxo-osmium ligand complexes with isopentenyl adenine and its nucleoside

HOINORGANIC

CHEMISTR

Y $343352

343

(1976)

The Reactions of Oxo-Osmium Ligand Complexes with Isopentenyl Adenine and Its Nucleoside JOHN A. RAGAZZO and E. J. BEHRMAN* Department

of Biochemistry,

The Ohio State

University,

Columbus,

Ohio 43210

ABSTRACT We report syntheses of oxo-osmium(VI)bis(ligand) estersof N6 -(A* -isopcntenyl) adenine (6ipAde) and its nucleoside (IPA) which result from the addition of 0~0, to the double bond of the isopentenyl group. A study of the kinetics of these reactions shows that under typical conditions the rates of reaction relative to thymidine are as follows: for CkO,-pyridiner thymidine = I; 6-ipAde = 4600: for OsO,-2, 2’bippidyl: thymidine = 380; 6-ipAde = 8600; IPA = 8600. We also report syntheses of osmate esters of IPA in which the osmium is bonded through the 2’- and 3’-hydroxyl residue.

groups of the ribose

We have been interested in the reactions of oxo-osmium compounds with nucleic acid components [l-3] because of their utility in x-ray crystallographic studies [4--61 and in the direct visualization approach to the sequencing problem 17, 81. N6(Az-isopentenyl) adenosine, IPA, is a component of a number of t-RNA species [9] _ We anticipated that its olefinic group would be many times more reactive than the pyrimidine 5,6-double bond system by analogy with our previous work on 3-cyclohexene carboxylic acid [ 101 and thymidine [ 11. If this were true, the IPA residue could be selectively labeled on a kinetic basis. Another problem for single-site labeling with an oxo-osmium reagent is, however, the occurrence of ester interchange (transesterification) reactions of the type shown in Eq. (I)_

Daniel has shown 131 that the use of 2,2’-bipyridyl as ligand results in the formation of osmate esters of greatly increased inertness as compared with the bis( pyridine) analogs. We report data in this paper on the kinetics of formation and exchange behavior of osmate .esters of isopentenyl adenine and its nucleoside, IPA. *Author

to whom correspondence

should be addressed_ @American Elsevier Publishing Company, Inc., 1976

344

J_ A_ RAGAZZO

AND E. J. BEHRMAN

RESULTS Synthesis -amI Characterization

of the Osmate Esters

Syntheses of the osmate esters were carried out as indicated in Chart 1. Both sugar esters (type I) and oieftic esters (type II) were msde_ Spectral properties are summarized in Table 1. Compounds of type I (sugar esters) are easily distinguished from compounds of type II (olefiic esters) by their nmr spectra The Cl’ proton in the sugar esters is shifted downfield by about 0.4 ppm reIative to the parent nucIeoside and the oIefIc ester The allyhc methyl resonances in the parent nucleoside and in the sugar esters appear as a broad singlet whereas those resonances for the olefiic esters appear as a well separated pair of singlets_ The oleftic proton resonance at about 65.4 is, of course, present only in the parent compounds and in the sugar esters. It should be noted that this Iatter resonance is sometimes obscured by the hydroxyl protons in dry DIVLSOand so is more easily observed in DaO-DMSO mixtures_ Other chemical shifts are similar to those that have been observed previously [ 1, 3]_ Robins and Trip have recorded nmr data for a number of IPA analogs [ 1 l] _ The various esters are also easily distinguished chromatographically (Table 1). Transesterification The bis(pyridine) olefinic ester of IPA can be prepared as a solid without complication due to ester exchange if it is made in the presence of excess

I

sP

0=03=0

C’L

WA is N6-(~2-isopcntcnyl)

1.27(s,3);1.63(~,3) 1.33(~,3);1.60(~,3) 1.27(s,3);1.47(~,3) 1,18(s,3);1.50(~,3) 1.7O(s,G) 1.70(s$)

835 820 830 830

&cc Experimental.

hJq~e I, R=ribosyl, Chart I.

NMRb,G

830 810

adcnosine,

GType II, R=ribosyl, Chnrt I.

fType II, R = H, Chart I.

’ CH7

,CH3

l.?l(s,G) 1.67@,6)

u,,oso* ,cm-1 >C

‘%IKBr. bin DMSO.d,,TMS. cln DMSO, flat maximaor shoulders430-450 nnt. d6-ipAdeis N6-(Al-isopcntcnyl)adcainc,

6-ipAded IPAC 6.ipAde ester! PY bipY IPA ester (olefinic)b’ PY bipY IPA ester (sugar)” PY bipY

COMPOUND

IRU

&42(d,l ,J=4Hz) 6,48(d,l,J=4Hz)

5.97(d,l ,J=GNz) 6.06(d,l,J=6Hz)

5,88(d,l,J=GHz)

Cl’-H

Spectroscopic and Chromatographic Properties

TABLE 1

0,80 0061

0.85 0,70

11s 190 270 165

0.65 0.45

0.88 0.88

I

120 140

-

E, 430 nmc

Rf

0.24 0,16

0.48 0.39

0.22 0,19

0.28 0.44

II

Solven ti

R

w

346

J. A. RAGAZZO

and E. J. BEHRMAN

pyridine and if it is promptly precipitated from the reaction mixture_ In solution and in the absence of excess ligand, the transesterification reaction takes place rapidly_ The half-time for exchange at 35” in 20% DaO-80% DMSO-de is about 20 min. The bipyridyi ester is less reactive_ The half-time for exchange under the same conditions is about 2 hr. It is increased by the presence of excess 2,2’-bipyridyl. These observations are in qualitative agreement with those of DameI [3] on an intermoIecuIar but otherwise similar exchange reaction. These reactions can be conveniently followed by observation of the Cl’-proton resonance (Fig. 1).

The kinetics of formation of the olefiic ester, type II’(Chart I) were followed using the free base, isopentenyl adenine, as substrate with both ligands. In this way, the influence of pyridine or bipyridyl could be compared without complications due to exchange_ For the nucleoside, IPA, o:ly the kinetics of the bipyridyl system were studied. Typical runs are shown in Fig. 2_ The reactions were fmt order in both 0s04 and in substrate_ The observed rate constant was linear with the square of pyridine activity and with the first-power of bipyridyl concentration (Fig. 3.). These dependencies thus agreed with those determined previously for similar systems [ 1, 10, 121. Table 2 collects the rate constants and also presents rates calculated for a set of typical conditions_ Our results show that the OsOa-pyridine reagent reacts with isopentenyl adenine about 4600 times faster than with thymidine- When 2,2’-bipyridy1 is the ligand, the reactivity ratio drops to a factor of about 23. This is in accord with

I 6.6

*

8

8

I 5.8

FIG. I: Low field nmr of a transesterification reaction at equilibrium The solution initially contained IPA bis(pyridine) oleftic ester in 90% DMSO-d, -10% D,O. The upfield doublet is at the position of the Ci’qroton in IPA olefmic ester; the Iow field doubIet is shifted to the position characteristic of a sugar ester_

0X0-OSMIUM

LIGAND

COMPLEXES

347

6

4

0

I

2

4

3

5

min.

5.5 ‘d

-

a,

+ 8 0

3.5 0

1 2

I 4

I 6

min.

FIG. 2: Typicd kinetic runs, pH 7, 8”. Left: j&ipAde] = 2.67 x 1O-4 M, [OsO,] = 2.78 x l(r4 M [py] = 128 X 1W’ M. t1/2 = 9.5 min. Right: [6-ipAde] = 2.3 X l(T4 hi, [OsO, ] = 2.01 X 10“ M, [bipy] = 1.28 X lO_” M. t1i2 = 10 min_

348

J_ A. RAGAZZO

and E. J. BEHRMAN

400-

FIG. 3: Ligand dependencies for the reaction of 6-ipAde with the OsO,-pyridine system (Ieft) and for the reaction of IPA with the OsO,-bipyridyl

average of three determinations_

system (right)_ Each point is the

PY bipy PY bipy bipy

Tltymidine Thymidine 6.ipAd& 6.ipAde JPAC

q

1.2x 1.1 x 1.5 x 2.5 X 2.4X

IO3 lo4 lo6 lo5 10’

Relative Rat& 1 386 4600 8600 8600

Rate, Mmin”c 1.8 X IUs 1.7 x l@ 2,1 x l(r2 4x l(T2 4x lo-2

spy = pyridine;bipy = 2,2’.bipyridyl Q’hc rate laws are as follows for py, rate k,[OsO,] [S] + k,‘[OsO,,][S) atpy; for bipy, rate = k, JOsO,] [S] + k,‘(OsO,] [S] [bipy], For the data of this paper, the observed second.ordcr rate constants, kobs, arc related to k,’ by the expression:k,’ = (kobs-k,) / aat,,, whcrc spy is pyridine activity, or, for the cast of 2,2’.bipyridyl,by the expression:k,’ = (kobs -k,) / (bipy], k, vaJuesare: thymidinc,0.1; G-ipAdc,145; WA,170 iW min”. Vh~es for k0 were taken from extrapolationsto zero ligand concentration rather than from measurementsof the rate in the absence of ligandsince Griffith 113, 14) has shown that the product in the absenceof ligandis n dimcr and it is possiblethat in this csse dimerizntionis rate limiting.Indeed,k, valuesmeasuredin the absenceof ligandwere smallerby a factor of about two than those obtainedby extrapolation.Comparisonwith the constantsof refs. 1 and 10 may be carriedout as follows:for pyridinc:k,’ = (k,P, [py]l) /a*py;for 2,2’-bipyridyl:k,’ = k, 0,. cThe rates were calculatedfrom the rutc expressionsgivenin footnote b under the followingconditions: [L] =O.l M;[OsO,] =4X lO-” M;(S] =4X 1(r3 M. d6-ipAdcis N6 .(A* - isopentcnyl)ndenine. clPA is N6-(A1- isopcntcnyl)adenosine,

Ligando

Substrate

kz’, M3 min’ (py)” or kl ‘, M2 Inin-’ (bipy)

Rate Data for the Reaction of 0x.0.Osmium (VIII) L&and Systems (pJi 7, go, water)

TABLE 2

350

J. A. RAGAZZO

and E. J. BEHRMAN

the general principle that the more reactive reagent is the less selective [ 15]_ The choice of reagent for single-site labeling will thus depend on whether one wishes to avoid reaction with a competing olefinic or glycol site in the molecule_ The pyridine reagent is better for the first case, as discussed above, and the bipyridyl reagent for the second because of the low rate of transesterification of the bipyridyl esters [ 3 ] . EXPERIMENTAL Isopentenyl adenine and isopentenyl adenosine were obtained from the Aldrich Chemical Co. or Sigma Chemical Co. These products were chromatographically homogenous in the solvent systems A, B, and C reported by Robins et al_ [ 16]_ The uv molar absorptivities agreed with literature values [ 16]_ Pyridine was distilled from KOH and kept over 4A molecular sieves. Other compounds were reagent grade and were used without purification_ Ultraviolet-visible spectra were measured on a Perkin-Elmer model 202 instrument_ Infrared spectra were taken in KBr discs on a Perk&Elmer model 237B grating instrument_ Proton magnetic resonance spectra were recorded on a Varian Associates model T-60 instrument (60 MHz) at 3S”. Kinetic runs were carried out using the Perkin-Elmer model 202 machine, a time-drive attachment, and S-cm water-jacketed cells. All reactions were carried out in phosphate buffer, pH 7-0, y = 0.2 M. The temperature was held at 8 2 0.2O using a Forma circulating water bath equipped for cooling. Cell fogging was prevented by piping a stream of dry air through the cell compartment. Product formation was followed at 360 nm except for the thymidine-pyridine system which was monitored at 430 nm. Infinity values were taken after about ten half-times_ Reactions with 6-ipAde as substrate were run under pseudo-secondorder conditions with substrate and 0~04 approximately equimolar. Slopes of plots of the reciprocal of reactant concentration vs. time divided by the appropriate function 1171 gave &bs_ Reactions with IPA and thymidine as substrates were run under pseudo-first-order conditions with 0~0~ concentration limiting_ Slopes of plots of ln(A, - Ac / A, - At) divided by substrate concentration at ta gave k,bs_ All slopes were evaluated using a standard least-sqtlares anaIysis with simple linear regression. Silica gel tic was used to separate the products of the reactions. Osmium-containing compounds were revealed by spraying with 2% thiourea in 2 N HCI. Solvent I: methanol-pyridine, 9: 1 (v/v); solvent II: aqueous phosphate .buffer, 0.05 M, pH 7-pyridine, 20: 1 (v/v). Carbon, hydrogen, and nitrogen analyses were carried out by Galbraith and by Het-Chem-Co.

Synthesis of the Osmate Esters 6-ipAde bis(pyridine) ester (Type 11, R = H, Chart I): N6-(A2-isopentenyl) adenine ( 102 mg, 0.5 mmoles) was dissolved in about 10 ml 1 M aqueous pyridine. Osmium tetroxide (127 mg, OS mmoles) dissolved in water was added,

OXO-OSMIUM

LIGAND COMPLEXES

351

the mixture stirred, and allowed to stand overnight. The mixture was taken to dryness in vacua below 40°_ The dark brown product was redissolved in 20% aqueous pyridine and purified by chromatography on alumina. The middle fraction from the column showed one spot on silica tic, solvent I. Anal. Calc for C2eH,s04N,0s: C, 39.01; H, 3.77; N, 15.93. Found: C, 38.99; H, 3.99; N, 15.83. Yield, 249 mg (8i%). 6-ipAde 2, 2’-bipyridyl ester (Type II, R = H, Chart I): N64Az-isopentenyl) adenine (0.5 mmoles, 102 mg) and 2,2’-bipyridyl (OS mmoles, 78 mg) were dissolved with warming in about 10 ml water. Osmium tetroxide (OS mmoles, 127 mg) dissolved in water was added with stirring_ A precipitate began to form. This was collected by filtration, washed with water, ethyl acetate, and diethyl ether. The light brown powder was dried at room temperature overnight over PaOs. Anal. Calc for C2eHZ10~N10s~Hs0: C, 38-03; H, 3.64; N, 15.53. Found: C, 37.87; H, 3.80; N, 15.24. Yield: 268 mg (85%). IPA bis(pyridine) olefmic ester (Type II, R = ribosyl, Chart I): N6-(Az-isopentenyl) adenosine (168 mg, 0.5 mmoles) was dissohed in 20 ml of 1 M pyridine in ethyl acetate_ Osmium tetroxide (78 mg, 0.5 mmole) was added in 10 ml ethyl acetate. The precipitate was collected by filtration after 3 hr_ It showed one spot on silica tic, solvent I. Anal. Calc for CssHs r OsN,Os-HsO: C, 39.18; H, 4.35; N, 12.80. Found: C, 39.20, 39.37; H, 4.51, 4.55; N, 12.54. Yield: 345 mg (90%). IPA 2, 2’-bipyridyl olefinic ester (Type II, R = ribosyl, Chart I): N6(A’-isopentenyl)adenosine (250 mg, 0.745 mmoles) and a 25-30% M excess of 2, 2’-bipyridyl were dissolved in 25 ml moist ethyl acetate. Osmium tetroxide (190 mg, 0.745 mmoles) dissolved in 15-25 ml ethyl acetate was added with stirring. The precipitate which formed rapidly was collected by suction filtration, resuspended in ethyl acetate, and stirred for about an hour. The product was collected again by filtration, washed with ethyl acetate and diethyl ether, and dried in VQCUO at 25O. Anal. CaIc for Cz~H,,N,0s0s=2H20: C, 38.38; H, 4.26; N, 12.55. Found: C, 38.18; H, 4.55; N, 12.38. IPA bis(pyridine) sugar ester (Type I, R = ribosyl, Chart I): N6-(A2-isopentenyl)adenosine (250 mg, 0.745 mmoles) was dissolved in 50 ml water_ 0sz06py4 (117 mg, 0.745 mmoles talc as the mOnOm!r) [2] dissolved in 30 ml of 17% aqueous pyridine was added. After standing overnight, the mixture was taken to an oil on the rotary evaporator. The oil was taken up in a pyridine-isopropanol mixture and evaporated to dryness in vacua. The resulting amorphous solid was dissolved in 20% aqueous pyridine, placed on a silica column, and developed with the same solvent. The middle fraction yielded an amorphous solid which was homogenous in Solvents I and II (silica tic)_ This was dried in VQCUOat 2S”. Anal. Calc for Cs sHssN,O6Os: C, 42.04; H, 4.10; N, 13-74. Found: C, 42.2 1; H, 4.26; N, 13.54. IPA 2,2’-bipyridyl sugar ester (Type I, R = ribosyl, Chart I): N6-(A2-isopentenyl)adenosine (270 mg, 0.805 mmoles) and 2,2’-bipyridyl(l25.8 mg, 0.805 mmoles) were dissolved in 150 ml water. Potassium osmate, KsOsOs(OH)4, (297 mg, 0.805 mmoles) [ 181 dissolved in 50 ml water was added, the pH of the solution adjusted to 7 with 0.1 N HCl, and the solution stirred for 30 min. The solution was filtered after standing overnight to remove a small amount of

352

J. A RAGAZZO

precipitate_

and E_ J. BEHRMAN

of the solution was reduced in vacua until a precipitate was redissolved by heating and the clear solution placed at 4” overnight The precipitate which forrned was collected by filtration, washed with water, ethyl acetate, diethy ether, and finally dried in vacua over P20s at room temperature overnight. Yield: 3 15 mg (55%). Anal. Calc for CtsH2,N1060s-3H20r C, 39.19; H, 4.35; N, 12.81. Found: C, 38.58, 38.65; H, 4.13,4.40: N, 12.92. Table I gives some of the characteristic spectral data for these compounds_ Reproductions of the spectra can be found in Ref. 19. A preliminary report of some of this work has been published [20]_ began

The volume

to form.

This

material

We thank Prof_ R. L. Clark for many helpful discussions_ This work supported by an NIff grant, Gfif-20375.

was

REFERENCES 1. L. R_ Subbaraman, J. Subbaraman, and E. J. Behrman, Bioinorg. C7ze.w.1, 35 (1971). 2_ L. R. Subbaraman, J. Subbaramzn, and E. J. Beluman,I_ Org_ Chem. 38,1499 (1973). 3. F. B_ Daniel and E. J. Behrman, JI Am Chem Sec. 97,7X52 (1975). 4_ J. J_ Rosa and P_ B_ Sigler, Biochemisrrq- 13,s 102 (1974). 5. F. L Suddath, G. J. Quigley, A. McPherson, D. Sneden, J. J. Km, and A. Rich, Nature 248,20 <1974)_

6. J. D. Robertns, J. E. Ladner, J. T. Finch, D. Rhodes, R. S. Brown, B. F. C. Clark, and A_ Klw. Nature 250.546 (1974). 7. W_ A. Salser,Annu_ Rev. Riochem 43,993 (1974)_ 8_ R F_ Whiting and F. P. Ottensmeyer, J. hfoL Biol. 67,173 (1972). 9_ R H. Hall. 77te kfodified Nucteosides in Nucleic Acids, Columbia Univ. Press, N. Y. 1971; T. V. Venkstem, The primary Shvcture of Transfer RNA. Plenum, N. Y_, 1973_ 10. L. R_ Subbaraman, J_ Subbaraman, and E. J. Bebrman, fnorg. Chem. II, 2621 (1972). ll_ 12

M. J_ Robbins and E. hi. Trip,Biochemistry 12,2179 (1973). R. L. Clark and E. J. Behrman, Inorg. Chem 14, 1425 (1975).

13. R J. Collin, J. Jones, and W_ P_ Grifflth,J. Chem Sot. Dalton 1094 (1974). 14. R. CoIlin, W_ P. Griffith, F_ L. Phillips, and A. C. Skapski, Biochim Biophys Acfa 320, 745 (1973). 15. L. hi_ Stock and H_ C. Brown, Adv. Phys. Org. Chem. 1, 44-49 (1963); J. E. Leffler and E. Grunwald, Rates QnC! Equilibriaof OrganicReactions, John Wiley and Sons, N. Y-, 1963, p 162 ff- See also C. D. Johnson, Chem Rev. 75,755 (1975). 16. M. J. Robins, R. H. Hall, and R. Thedford, Biochemistry 6.1837 (1967). 17. S. LV_ Benson. The Foundations of ChemicalKinetics, McGraw-Hill, N. Y., 1960, pp. 20-x_ 1% K-k K_ Lott and M_ C_ R. Symons,J. Chem Sot_, 973 (1960). 19. J. A. Ragazzo. M. S_ Thesis, The Ohio State University, 1975. 20. J. A. Rzauo and E. J. Behrman, Abstracts, 170th National hieeting, American Chemical Society, Chicago, August, 1975, BIOL 121. ReceivedlOJdy

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