On the preparation of 80Br-biomolecules—III: The effect of various experimental parameters on radiochemical yields

International Jourmd of Applied Radiation and Isotopes, 1976, Vol. 27, pp. 627-630. Pergamon Preus. Printed in'Northern Ireland

On the Preparation of Br-Biomolecules III: The Effect of Various Experimental Parameters on Radiochemical Yields*

80.

STEVEN H. WONG, JACK F. MUSTAKLEM and HANS J. A C H E t Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. The effect of several experimental parameters on the efficiencyof the 8°Br incorporation via the "CF3S°mBr-KBrO3'' gas exposure resulting in carrier free radiobrominated compounds was investigated by using two model compounds, L-tyrosine and guanosine. The S°Br labelling proceeds very rapidly in the case of the L-tyrosine and is fairly independent of "labelling time" and substrate concentration as long as a certain minimum amount of L-tyrosine is used. This is in contrast to the guanosine system where rapid secondary reactions reduce the initially high yields of 8°Br-8-bromoguanosine at extended "labelling times" and where larger amounts of substrate are needed to produce optimum yields. INTRODUCTION IN Tim tWO preceding papers in this seriesO-2) we have discussed several direct methods of incorporating radiobromine into biomolecules, such as deoxyuridine, L-tyrosine, guanosine, deoxycytidine, phenylalanine and acetic acid, resulting in the formation of the carrier free radiobromine derivatives of these molecules. In these studies the incorporation of radiobromine was achieved via the "direct CF3 S°mBr gas exposure," the modified "CF3 S°mBr-C12 gas exposure", and the "CF3 s°"Br-KBrO3 gas exposure" techniques inducing a S°Br for H exchange in the biomolecules listed above or a S°Br for I exchange in the iodo derivatives of these biomolecules. The most promising methods appeared to be the "CF3 s°"Br-C12" and the "CF3 s°"Br-KBrO3" gas exposure techniques, the latter being similar to the "Xe-KIO3" iodination method first reported by STtCgJ.IN eta/. (3) The labelling procedures were carried out

mostly by employing standard techniques and in the previous work (~-2) no particular effort was made to vary the experimental parameters in order to maximize the radiochemical yields of the radiobromine labelled biomolecules. On the other hand the great differences between the chemical properties of the biomolecules studied in this investigation make it appear unlikely that one standard procedure could be used to obtain optimal yields with all of these compounds. One would expect that experimental parameters such as labelling time, amount of substrate, etc. would greatly affect the observed radiochemical yields. Thus in the following we would like to discuss the effect of these and other experimental conditions on the S°Br incorporation via the "CFaS°"Br-KBrO3 gas exposure" technique into two model molecules, Ltyrosine and guanosine.

EXPERIMENTAL The basic features of the "CF3S°~Br* Work supported by the U.S. Energy Research KBrO3 gas exposure" technique have been previously described, cl-2) and Development Administration. In the first series of experiments, which were t Address correspondence to this author. 627

628

S. H. Wong, Z F. Mustaklem and H. Z Ache

carried out to ascertain the effect of labelling time on the radiochemieal yields, 10mg of KBrO3 were transferred after a 40 min exposure to 60 Torr of CF3 s°"Br to another vessel and dissolved in 0.15ml of 0.1N aq HCI containing 10 mg of guanosine or L-tyrosine, respectively. The solutions were kept at room temperature for a predetermined time and subsequently analyzed. In the second series of experiments where the effect of substrate concentration was studied, 10 mg of KBrO3 crystals, which had been exposed for 40rain to 60Torr of CF3 s°"Br, were dissolved in 0.15 ml of 0.1 N aq HCI containing various amounts of substrate. High pressure liquid chromatographic analysis was started one rain after dissolving the crystals. In the third series of experiments various amounts of guanosine were exposed in 0.15 ml of a 0.1 N aq HC1 solution or slurry (if higher concentrations of guanosine were used) which also contained 10 mg of KBrO3. This solution or slurry was exposed for 40 min to CF3 S°mBr gas. The reaction vessel was in each case a 50 ml Pyrex bulb. The carrier-free radiobrominated molecules were purified by high pressure liquid chromatography (HPLC) utilizing Aminex A25 columns as previously reported, cl) The radioactivity assay of the labelled compounds was achieved by (discontinuous) liquid scintillation spectrometry of the effluent from the HPLC columns. ~1) The absolute amount of S°Br activity incorporated into the labelled substrate molecules depends on the activity of CF3 a°"Br and exposure time and ranged in our experiments to up to 10/xCi of S°Br. Relative radiochemical yields quoted are based on the sum of the activities of all products separated by HPLC including the activity appearing as S°Br-. Guanosine and L-t~,rosine, as well as authentic samples of S°Br-8-bromoguanosine, were obtained from ICN Life Sciences Group, Cleveland, Ohio. The position of the s°13r label in L-tyrosine has not been established.

RESULTS AND DISCUSSION In discussing the S°Br for I exchange reaction via the CF3 S°mBr-KBrO3 gas exposure technique in substrates such as iodo-acetic

acid and 5-iododeoxyuridinet2) we have already drawn the attention to the effect of labelling time on the yields of the radiobrominated product. The labelling time was defined as the time elapsed between the dissolving of the KBrO3 crystals (previously exposed to CF3 S°mBr) in the acidic solution containing the substrate and the start of the HPLC separation. In the "CF3 S°~Br-KBrO3 gas exposure" induced S°Br for H exchange in L-tyrosine and guanosine this effect is even more pronounced. As shown in Fig. 1 the maximum radiochemical yields are reached in both systems within less than I rain. The yield of S°BrL-bromotyrosine remains subsequently almost constant over the total time range of 60 rain investigated in this study, while the yield of S°Br-8-bromoguanosine dramatically drops from initially (after 1 min) 52% to less than 3% after a labelling time of 10 min. The apparent explanation for this drastically different behavior displayed by these two model compounds seems to be that in the former case the radiobromine becomes incorporated into a stable position, whereas in the i

90

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RADIOCHEMIC/~YIELDSVS "L,~ELLING TIME" [KBrO.,j-CF3 B¢ GAS EXPOSURETECHNIQ(.E]

80

~q ~60 ,~

• s°Br-L-TYROSINE o S°Br-GUANOSINE

50 Q

20

\

I0 0

I0

20 30 40 50 60 "LABELLING TIME"(MIN.)

FIG. 1. RadiochemicaI yields vs "labelling

time." (KBrOs-CF3 S°mBrgas exposure technique: 10rag KBrO3 exposed to 60Torr of CF3 s°"Br gas for 40 rain at room temperature and subsequently dissolved in 0.15 ml of 0.1 N aq HCI solution containing 10 mg substrate.)

On the preparation of ~°Br-biomolecules--llI latter case the initially formed S°Br-8bromoguanosine undergoes under these experimental conditions rapid hydrolysis leading to the loss of the radiobromine. This example clearly demonstrates in conneetion with the previously reported results(2) for the S°Br-bromoacetic acid system the need for investigating the effect of "labelling time" on radiochemical yields if one wants to optimize the labelling procedure. If the S°Br-8-bromoguanosine yields can be drastically reduced by rapid secondary processes one might expect that the amount of guanosine present in the solution used to dissolve the KBrO3 crystals is another factor controlling the yields; e.g. one can argue that the presence of a relatively large amount of substrate could suppress, at least to a certain extent, the secondary processes, hydrolysis, etc. and in this way avoid the decomposition of the labelled product. This behavior is indeed borne out in Fig. 2, where the S°Br-8-bromoguanosine yields are plotted as a function of the amount of guanosine present in the 0.1 N aq HCI solution. The yields increase slowly up to 75% at substrate concentrations of 2500/~g guanosine/ml 0.1 N aq HCI. On the other hand the results in Fig. 1 provide very little evidence for secondary decomposition of the S°Br labelled L-tyrosine under these experimental conditions. I

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1

I I°Br-GUANOSINEYIELD~ VS AMOUNTOF /

~

IN 0.=. oq, .cl

80 ~- (KBrO~'CFsBrGAS EXPOSURETECHNIQUE)

0 [

0

I

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500 IOO0 1500 L~O0 p.g GUAt~SINE/ml 0.1N aq. HCI

I

2500

FIG. 2. Radiochemieal yields of S°Br-8bromoguanosine vs amount of guanosine/ml of 0.1 N aq HCI. (0.15 ml of this solution were used to dissolve 10rag of KBrOa crystals, which have been previously exposed to 60 Torr of CF38°'Br gas at room temperature for 40 min; labelling time I min.)

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629 I

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Q= L-/NR(~INE IN 0.1 N oo~HQ SOLUtiON I00

~8o

o

20

0

l I l 1 I I00 200 400 600 800 /zg L-TYROSlNE/rnI O,I N aq HCI

Fxo. 3. Radiochemical yields of S°Br-Ltyrosine vs amount of L-tyrosine per ml of 0.1 N aq HCI. (0.15 ml of this solution were used to dissolve 10rag of KBrO3 crystals, which have been previously exposed to 60 Torr of CF3S°'~Br gas at room temperature for 40 rain, labelling time 1 min.) Thus one would expect in the latter case a much less pronounced effect of the amount of substrate present in the solution on the obtained yields. As shown in Fig. 3 where the yields of S°BrL-tyrosine are plotted as a function of substrate concentration this seems to be indeed the case. The yields increase drastically at substrate concentrations of 50 tzg Ltyrosine/ml 0.1 N aq HC1 solution from about 2% to their maximum of 90%. The fact that below a threshold concentration of about 50 tzg/ml very little labelling occurs seems to indicate that a certain minimum amount of substrate is required in order to suppress competing reactions between the reactive bromine species and impurities unavoidably present in the solution. It should be emphasized in this context that the use of larger amounts of substrate does not interfere in the present case with the carrier free formation of the radiobrominated molecules, which being chemically different from the substrate, e.g. bromoguanosine vs guanosine, can be easily separated and purified by HPLC. It might, however, be an important consideration if macromolecules are to be radiobrominated by this method where no simple

630

S. 1-1. Wong, J. F. Mustaklem and H. Z Ache

separation between labelled and non-labelled molecule is possible. In this case it would be most desirable to reduce the amount of substrate in the solution to a minimum to obtain high specific activities. Thus we are presently engaged in the search for the exact nature of the reacting bromine species and the assessment of the reactions it undergoes with the impurities in order to refine this technique to allow the labelling of microamounts of substrate. Figure 4 shows the results of an attempt to combine the two steps required in the CF3S°mBr-KBrOa technique, exposure of KBrO3 to CF3 s°"Br and subsequent dissolution of the KBrOa in acidic substrate solution, to a one step procedure, namely to the simultaneous exposure of a solution or slurry of KBrO3 and substrate, in this case guanosine, in 0.1 N aq HC1, to CF3 S°mBr. By comparing the results plotted in Figs. 2 and 4 it can be seen that the "solution~slurry" labelling requires a greater amount of substrate in order to achieve the same yields as in the previously used two step procedure if all the other experimental conditions are kept constant. While in the solution/slurry labelling the maximum yield is reached at about 40,000 # g substrate/ml solution (Fig. 4) the optimum I

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e°Br-GUANOSINE YIELDS VS AMOUNTOF GUANOSINE IN S(~L.UTION (CF-sm'8r gas-Q/N aq. HCI SOLUTION CONTAINING IOm(J KBrO~ AND VARI(~S AMOUNTS OF GUANOSINE)

1(30

~80

Q

•

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0 0

I I 5 I0 rng GUANOSINE/O.15ml 0.1N aq. HCl

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TABLE 1. Radiochemical yields of S°Br-8bromoguanosine vs amount of KBrO3 crystals dissolved in 0.15 ml of 0.1 N aq HCI solution containing 10 mg of guanosine (room temp; labelling time 1 min). % Radiochemical yield of S°Br-8-bromoguanosine

Amount of KBrO3 exposed to CF3 S°mBr (mg)

43±5 38+5 50+3 54±3 504-3

1 5 10 20 30

Experimental conditions: Various amounts of KBrO3 were exposed to 60 Tort of CF3 s°"Br in a 50 cm3 Pyrex bulb and subsequently dissolved in 0.15 ml of 0.1 N aq HCI containing 10mg of guanosine, labelling time 1 min. yields in the two step process are obtained at considerably smaller substrate concentration, i.e. 2500/~g substrate/ml solution (Fig. 2). From these results one might want to conclude that the solution/slurry technique does not offer any significant advantage, except a slightly simpler handling procedure, over the original CF3 S°mBr-KBrO3 method. Another parameter which we considered in this study was the amount of KBrOa crystals used in the labelling process. Here again the possibility existed that if small amounts of KBrO3 were used competing reactions might interfere with the labelling process. Table 1 shows the results obtained under standard conditions, 40 min CF3 S°mBr gas exposure of KBrO3, "labelling time" 1 min (guanosine) dissolved in 0.15ml 0.1 N aq HCI solution containing 10 mg guanosine. Only slight variations can be observed which indicate that the amount of KBrO3 used is not very critical: We would, however, like to emphasize that the presence of KBrO3 is essential for the labelling process which does not proceed if KBrO3 is replaced by KIO3, KCIO3, KBr or KC1.

15

Fzo. 4. Radioehemical yields of S°Br-8bromoguanosine vs amount of guanosine in solution (slurry) of 0.15 ml 0.1 N aq HCI containing 10mg KBrO3. (Solution exposed for 40rain at room temperature to 60Torr of CF3 S°mBrgas.)

REFERENCES

1. WoNo S. and AcHE H. J. Int. J. appl. Radiat. Isotopes 27, 19 (1976). 2. WONO S. H., MUSTAr~EMJ. F. and Acrm H. J. Int. J. appl. Radiat. Isotopes 27, 379 (1976). 3. GARHV M. E. and S'rOci