[26]
AROMATIC THIOETI-IERS OF PURINE NUCLEOTIDES
289
[26] Aromatic Thioethers of Purine Nucleotides
By HuGo FASOLD, FRANZ W. HVLLA, FRANZ ORTANDERL, a n d MICHAEL RACK
Modification of the SH-group of 6-mercaptopurine by suitable substituents results in strong activation of the thioether bonds. A family of substances (Fig. 1) has been used to attach a covalent label to amino acid side chains, preferentially to the - - S H group. The reaction proceeds to a cleavage of the aromatic thioether with liberation of a thiophenol. The carbon-6 of the purine is bound to the amino acid side chain although, in some cases, the nitrophenyl moiety is transferred onto the protein. Preparation of S-(Dinitrophenyl)-6-mercaptopurine
Riboside Triphosphate
~',3'-O-Isopropylidene-S- (dinitrophenyl) -6-mercaptopurine Riboside. Isopropylidated 6-mercaptopurine riboside, 2.88 g (9.6 mmoles), prepared from the riboside with the aid of 2,2-dimethoxypropane, 1 is suspended in 300 ml of ethanol and dissolved by the addition of 10 ml of a 1 N solution of sodium hydroxide in 50% ethanol. A solution of 1.24 ml (10 mmoles) of 2,4-dinitro-l-fluobenzene in 60 ml of ethanol is added in several portions over a period of 30 min, and the mixture is stirred for
: No2
C02H
S/~ NO2 N
C ®1_3 O. H2@
®,_3 HO OH
ttO
~ N
OH
Fie. 1. Substances used to attach a covalent label to amino acid side chains. 1j. A. Zderic, J. G. Moffat, D. Kan, K. Gerson, and W. E. Fitzgibbon, J. Med. Chem. 8, 275 (1965).
290
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[25]
another 30 rain. After evaporation to a turbid syrup, the residue is taken up in 300 ml of methylene chloride, and the mixture is extracted three times with 50 ml of water. After removal of the solvent, the product is sufficiently pure for phosphorylation; it is only slightly contaminated by dinitrophenol. Purity may be checked by thin-layer chromatography on silica gel in methanol or dibutyl ether with RI of 0.6 and 0.5, respectively. The compounds carrying the aromatic thioether bonds can be detected on thin-layer chromatograms or by paper electrophoresis by spraying with alkaline 1% mercaptoethanol. The slight yellow color of the bands deepens perceptibly after a few minutes. The yield at this stage is 95%. The riboside thioether may be completely purified by dissolving a sample of about 100 mg in 4 ml of warm ethanol, adding the same amount of ethyl acetate and 200 mg of dry silica gel powder. The mixture is applied to a silica gel column in ethyl acetate, and the column is developed with the same solvent. ~ S-(Dinitrophenyl)-6-mercaptopurine Riboside Monophosphate. The isopropylidated thioether riboside, 3 g (6.16 mmoles), is dissolved in 12 ml of dry triethyl phosphate, and the solution cooled to --20 °. A solution of 0.85 ml (9.3 mmoles) of freshly distilled POCI3 in 2 ml of triethyl phosphate is added in small portions, and the reaction mixture is maintained below --16 ° with the aid of a cryostat. Moisture should be strictly excluded during the addition. The solution is stirred for 2 hr at room temperature. A mixture of 70 ml of a freshly prepared 10% barium acetate solution, 35 ml of acetone, and 40 g of ice is stirred vigorously, and the phosphorylation mixture is added from a pipette. The pH is brought to 7.0 by addition of 4 N NaOH and kept constant until the excess of POC13 is hydrolyzed. Then ethanol is added to a final concentration of 80% (v/v). The slightly yellow precipitate is centrifuged, and extracted four times with 200 ml each of 80% ethanol and twice with 200 ml each of acetone to remove salts and dinitrophenol. The barium salt of the monophosphate may be extracted from the precipitate with portions of 200 ml of water at pH 6 until the absorbance at 260 nm of the supernatant liquids has decreased to 5% of the value of the first extract (approximately 6 extractions are required). The barium salt may be stored in the cold for several months. To isolate the monophosphate, the barium salt is mixed with an equal weight of Dowex 50 X 2 resin in the H ÷ form. The mixture is applied to a Dowex 50 >( 2 column (2 X 35 cm for 2 g of barium salt) in the H ÷ form in water. Upon elution of the column with water, the monophosphate emerges as the second peak after a small peak of impurities. Fractions may be monitored by electrophoresis at pH 3.5 in 0.1 M amU. Faust, It. Fasold, and F. Ortanderl, Eur. J. Biochem. 43, 273 (1974).
[26]
AROMATIC THIOETttERS OF PURINE NUCLEOTIDES
291
monium citrate. Yield: 45%. The solution of the purified monophosphate is lyophilized, and the product is used immediately for protein reactions or for the synthesis of the triphosphate. Triphosphate I. The monophosphate, 425 mg (0.8 mmole), is dissolved in 6 ml of dry picoline and 0.19 ml of tributylamine. The solution is evaporated to dryness under reduced pressure, and the tributylammonium salt is taken up in 5 ml of dry dioxane. Tributylamine, 0.36 ml, and 0.24 ml (1.16 mmoles) of freshly distilled diphenyl chlorophosphonate are added, and the mixture is stirred for 2 hr. Evaporation under reduced pressure leaves an oily residue, which is extracted with 10 ml of diethyl ether for 15 rain and usually is transformed into a fluffy yellow precipitate adhering to the glass wall. The ether is decanted, the residue is dissolved in 2 ml of dry dioxane, and the solvent is again evaporated under reduced pressure. The gum is suspended in a few milliliters of picotine and dissolved by the addition of 0.5 ml of dimethylformamide. The solution is added to well-dried tributylammonium pyrophosphate, prepared from 356.8 mg (0.8 mmole) of tetrasodium pyrophosphate after removal of the cations over a small Dowex 50 column in the usual manher2 Pyrophosphorylation is begun by the addition of 0.8 ml of dry pyridine. After 30 min, the solution is concentrated under reduced pressure, the syrup is shaken for a few minutes with 5 ml of diethyl ether, and this solvent is decanted. The syrup is taken up in 10 ml of water, and the turbid solution is extracted four times with 10 ml each of ether. The pH of the water phase is brought to 7.0 and the solution is applied to a 2 X 20 em column of DEAE-Sephadex A-25 in the bicarbonate form that has been well washed with water. The column is developed with a linear gradient of 2000 ml total volume from 0.02 to 0.5 M LiCt or from 4.0 to 1 M triethylammoniumbiearbonate at pH 7.2. The elution diagram shows three major peaks with several shoulders; the triphosphate is found in the third peak. The column fractions are again monitored by eleetrophoresis at pH 3.0 and the fractions containing the triphosphate are lyophilized. The fluffy material is extracted twice with 5 ml of absolute ethanol to remove salts, dried well, and used for labeling experiments within a few days. It may be stored as a concentrated aqueous solution for a few weeks. Yield: 28%. Synthesis of S- (Nitro-4-carboxyphenyl) -6-mercaptopurine Riboside 5'-Monophosphate
S- (2-N itro-4-carboxyphenyl) -6-mercaptopwrine Riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) 3.96 g (10 mmoles) ~J. G. Moffat, Can. J. Chem. 42, 599 (1964).
292
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[26]
is suspended in 50 ml of water and neutralized with 4 N NaOH. Isopropylidated 6-mercaptopurine riboside (see above) 300 mg (1 mmole) is added, and the pH is adjusted to 8.0. The reaction mixture is stirred for 7 days, the pH being readjusted each day. This somewhat unusual reaction proceeds to the desired thioether with elimination of the SH group of the nucleoside. 4 The reaction is monitored by occasional electrophoresis in 0.1 M pyridine acetate at pH 6.5. The resultant solution is applied to a DEAE-Sephadex A-25 column (3 X 35 cm) in the bicarbonate form, and the product is eluted with a linear gradient of 2000 ml total volume from water to 0.2 M annonium bicarbonate at pH 6.5 (adjusted with CO2). It appears as a second peak after a small amount of unreacted mercaptopurine riboside. Purity is checked by electrophoresis at pH 6.5, and the thioether is detected with a mercaptoethanol spray as described above. The solution of the thioether is lyophilized several times to remove almost all the bicarbonate. Yield:
80%. Monophosphate II. The thioether riboside, 489.5 mg (1 mmole), is dissolved in 8 ml of dry triethyl phosphate, and 1 ml of freshly distilled POC18 is added at temperatures below 0 ° in small portions; the solution is maintained at 0 ° for 20 hr. Thereafter, 20 ml of 1 M barium acetate solution are added with vigorous stirring while the mixture is cooled to below 20 °. After addition of 80 ml of water, the pH is held between 7 and 8.5 by the addition of triethylamine. When all the POC13 is hydrolyzed, the pH is adjusted to 8.5 and the precipitated barium phosphate is removed by centrifugation. The precipitate is redissolved at pH 2.0 and reprecipitated at pH 8.5 several times to wash out the copreeipitated barium salt of the desired nucleotide. The combined supernatant fluids are mixed with 3 volumes of ethanol. After standing at 0 ° for 3 hr, the precipitated barium salt of the monophosphate is isolated by centrifugation and washed 3 times with 80% ethanol and with ether. The free monophosphate may be obtained by passage of the solution of its barium salt over a Dowex 50 X 2 column in the H ÷ form, as described above. The isopropylidene group is also removed during this operation. Fractions containing the compound are monitored by electrophoresis at pH 3.0, as described above, or by paper chromatography in isopropanol-0.5 M ammonium acetate pH 6.0 (5:2), with the aid of alkaline mercaptoethanol spray. The solution is lyophilized twice. The compound may be stored as a concentrated aqueous solution at pH 4 for a few weeks whereas the barium salt is quite stable. Yield: 65%. Triphosphate. The procedure described above for the S-(dinitro4F. W. ttulla and It. Fasold, Biochemistry11, 1056 (1972).
[25]
AROM&TIC THIOETHERS OF PURINE NUCLEOTIDES
293
phenyl)-6-mercaptopurine riboside triphosphate may also be applied to the prophosphorylation of this monophosphate. The yield in this case is about 55%. These compounds show rapid reaction with sulfhydryl groups at pH values above 7.5. Of all other amino acid side-chain functional groups tested, only primary amines and phenols are able to react with the aromatic thioether at elevated temperatures and over incubations of several hours' duration.2, 4
Related Compounds The compounds described above may be regarded as analogs of adenosine nucleotides. The guanosine nucleotide analogs of the same group have been synthesized from 2-amino-6-mercaptopurine nucleoside using very similar procedures. A 3',5'-cyclic monophosphate was synthesized from the 5'-monophosphate (I) after the method of Smith et al2
Examples o] A~nity Labeling o] Proteins Phosphorylase b. Phosphorylase b is dependent in its activity upon the allosteric effector AMP, and thus AMP analog (II) could be used to label the effector binding site. 4 The protein was first freed from AMP by filtration with Sephadex G-50 and charcoal columns. Portions of the enzyme, 60 mg, were incubated in 2 ml of 0.01 glycerophosphate-HC1 at pH 8.0 with 40 mg of (II) for 0.5 to 2.0 hr. The analog in this case had been labeled by 32p. The pH was then lowered to 6.5, thereby terminating the modification reaction. Excess nucleotide was removed by filtration over Sephadex G-25, and specific enzymic activity as well as covalently bound 32P-labeled nucleotide was determined. The activation of phosphorylase b by the attached label is shown in Fig. 2. It was not possible to activate the enzyme to a higher degree since the nonspecific reaction of the nucleotide with superficial SH groups became prevalent. The peptic fingerprint of a preparation covalently activated to 20% (see Fig. 2) gave only a single labeled peptide spot in an autoradiogram. Rabbit Muscle Actin. The monomeric G form of actin carries an ATP firmly bound, which is hydrolyzed in a stoichiometric reaction to ADP during polymerization to the fibrillar F form under the influence of inorganic cations, especially Mg. 2+ By removal of the triggering cations, and displacement of the ADP by fresh ATP, the protein depolymerized again to the G form. 5 M. Smith, G. I. D r u m m o n d , and H. G. Khorana, J. Am. Chem. Soc. 83, 698
(1961).
294
ENZY1V[ES, ANTIBODIES, AND OTHER PROTEINS
[26]
30
20
I0
I
!
I
I
I
OI
OZ
03
04
05
Residues of Nucleofide/Enzyme Subuni!
FzG. 2. Activation of phosphorylase b by covalently bound nucleotide after reaction with monophosphate (II). Ordinate: Percentage of maximal activation by AMP.
Prior to labeling by the ATP analog, one easily accessible SH group of the protein was allowed to react with N-ethylmaleimide. It had previously been shown that this modification does not change the polymerization reaction and interaction with myosin in any of its properties. 6,7 The label was attached by incubation of F-actin at a concentration of 10 mg/ml with a 100-fold molar excess of the ATP analog at pH 7.5 for 2 hr in 1 mM Tris chloride containing 0.2 mM ascorbic acid. The depolymerization, as measured by loss of viscosity, proceeded as in the case of ATP. After the removal by centrifugation of nondepolymerized material, approximately 20% of the total, the protein was repolymerized and centrifuged down as modified F-actin. Samples for determinations of bound nucleotide, inorganic 3~Pi, and specific viscosity were taken before and after repolymerization, as well as after the attempt at depolymerization of the modified F-actin. Only 20% of the protein could be brought into the monomeric state again; the major portion was "frozen" in the F form. During repolymerization, the nucleotide was split to the ADP analog and inorganic phosphate. Tryptic fingerprint of this protein revealed only one labeled peptide spot on autoradiography. 2 Other Proteins. Whereas a specific binding site was labeled in the examples cited above, a very different reaction ensued with phosphoribosyl pyrophosphate-ATP-ligase. After incubation of the enzyme in 0.05 M sodium bicarbonate solution with AMP analog (I), label from C. J. Lusty and H. Fasold, Biochemistry 8, 2933 (1969). 7 W. W. Kielley and C. B. Bradley, ]. Biol. Chem. 218, 653 (1956).
[27]
Ne-FLUOROBENZOYL&DENOSINE 5t-TRIPHOSPHATES
295
both 32p and 14C in the dinitrophenyl group 8 was attached to protein. The dinitrophenyl moiety was attached stoichiometrically to its subunits; a tryptic fingerprint revealed only two labeled peptides on autoradio~aphy. With Na÷,K÷-ATPase from nerve tissue, on the other hand, the nucleotide moiety was able to label the enzyme, although extremely long incubation periods of several days were necessary2 s T. Dall-Larsen, H. Fasold, C. Klungs0yr, H. Kryvi, C. Meyer, and F. Ortanderl, Eur. J. Biochem. 60, 103 (1975). P. Patzelt, H. Pauls, E. Erdmann, and W. Schoner, Hoppe-Seyler's Z. Physiol. Chem. 355, 758 (1974).
[27] Ne-o - a n d p - F l u o r o b e n z o y l a d e n o s i n e Y-Triphosphates
By
ALEXANDER HAMPTON a and LEWIS A. SLOTIN
The title compounds (I) and (II) were prepared as potential ATPsite-directed reagents. 1 The fluorobenzoyl substituents were selected because (a) the smallness of fluorine lessens steric hindrance to reaction of (I) and (II) with enzymic groups, (b) fluorine undergoes nucleophilic displacement from aromatic systems under milder conditions than other halogens, (c) o- and p-fluorobenzamides are stable in physiological buffers, and (d) (I) and (II) could be attacked by a nucleophilic group of an enzyme not only at their ortho and para carbons, respectively, but also at their amide carbons, thus approximately doubling the probability that (I) or (II) could act as an affinity label for the ATP site of any given enzyme. RCONH
ooo
II II II
N
(HO)2POPOPOCH2
[
15~°~ I OHOH~ I I
HO
OH
(I): R = o-fluorophenyl (II): R = p-fluorophenyl 1 A. Hampton and L. S. Slotin, Biochemistry 14, 5438 (1975).
AROMATIC THIOETI-IERS OF PURINE NUCLEOTIDES
289
[26] Aromatic Thioethers of Purine Nucleotides
By HuGo FASOLD, FRANZ W. HVLLA, FRANZ ORTANDERL, a n d MICHAEL RACK
Modification of the SH-group of 6-mercaptopurine by suitable substituents results in strong activation of the thioether bonds. A family of substances (Fig. 1) has been used to attach a covalent label to amino acid side chains, preferentially to the - - S H group. The reaction proceeds to a cleavage of the aromatic thioether with liberation of a thiophenol. The carbon-6 of the purine is bound to the amino acid side chain although, in some cases, the nitrophenyl moiety is transferred onto the protein. Preparation of S-(Dinitrophenyl)-6-mercaptopurine
Riboside Triphosphate
~',3'-O-Isopropylidene-S- (dinitrophenyl) -6-mercaptopurine Riboside. Isopropylidated 6-mercaptopurine riboside, 2.88 g (9.6 mmoles), prepared from the riboside with the aid of 2,2-dimethoxypropane, 1 is suspended in 300 ml of ethanol and dissolved by the addition of 10 ml of a 1 N solution of sodium hydroxide in 50% ethanol. A solution of 1.24 ml (10 mmoles) of 2,4-dinitro-l-fluobenzene in 60 ml of ethanol is added in several portions over a period of 30 min, and the mixture is stirred for
: No2
C02H
S/~ NO2 N
C ®1_3 O. H2@
®,_3 HO OH
ttO
~ N
OH
Fie. 1. Substances used to attach a covalent label to amino acid side chains. 1j. A. Zderic, J. G. Moffat, D. Kan, K. Gerson, and W. E. Fitzgibbon, J. Med. Chem. 8, 275 (1965).
290
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[25]
another 30 rain. After evaporation to a turbid syrup, the residue is taken up in 300 ml of methylene chloride, and the mixture is extracted three times with 50 ml of water. After removal of the solvent, the product is sufficiently pure for phosphorylation; it is only slightly contaminated by dinitrophenol. Purity may be checked by thin-layer chromatography on silica gel in methanol or dibutyl ether with RI of 0.6 and 0.5, respectively. The compounds carrying the aromatic thioether bonds can be detected on thin-layer chromatograms or by paper electrophoresis by spraying with alkaline 1% mercaptoethanol. The slight yellow color of the bands deepens perceptibly after a few minutes. The yield at this stage is 95%. The riboside thioether may be completely purified by dissolving a sample of about 100 mg in 4 ml of warm ethanol, adding the same amount of ethyl acetate and 200 mg of dry silica gel powder. The mixture is applied to a silica gel column in ethyl acetate, and the column is developed with the same solvent. ~ S-(Dinitrophenyl)-6-mercaptopurine Riboside Monophosphate. The isopropylidated thioether riboside, 3 g (6.16 mmoles), is dissolved in 12 ml of dry triethyl phosphate, and the solution cooled to --20 °. A solution of 0.85 ml (9.3 mmoles) of freshly distilled POCI3 in 2 ml of triethyl phosphate is added in small portions, and the reaction mixture is maintained below --16 ° with the aid of a cryostat. Moisture should be strictly excluded during the addition. The solution is stirred for 2 hr at room temperature. A mixture of 70 ml of a freshly prepared 10% barium acetate solution, 35 ml of acetone, and 40 g of ice is stirred vigorously, and the phosphorylation mixture is added from a pipette. The pH is brought to 7.0 by addition of 4 N NaOH and kept constant until the excess of POC13 is hydrolyzed. Then ethanol is added to a final concentration of 80% (v/v). The slightly yellow precipitate is centrifuged, and extracted four times with 200 ml each of 80% ethanol and twice with 200 ml each of acetone to remove salts and dinitrophenol. The barium salt of the monophosphate may be extracted from the precipitate with portions of 200 ml of water at pH 6 until the absorbance at 260 nm of the supernatant liquids has decreased to 5% of the value of the first extract (approximately 6 extractions are required). The barium salt may be stored in the cold for several months. To isolate the monophosphate, the barium salt is mixed with an equal weight of Dowex 50 X 2 resin in the H ÷ form. The mixture is applied to a Dowex 50 >( 2 column (2 X 35 cm for 2 g of barium salt) in the H ÷ form in water. Upon elution of the column with water, the monophosphate emerges as the second peak after a small peak of impurities. Fractions may be monitored by electrophoresis at pH 3.5 in 0.1 M amU. Faust, It. Fasold, and F. Ortanderl, Eur. J. Biochem. 43, 273 (1974).
[26]
AROMATIC THIOETttERS OF PURINE NUCLEOTIDES
291
monium citrate. Yield: 45%. The solution of the purified monophosphate is lyophilized, and the product is used immediately for protein reactions or for the synthesis of the triphosphate. Triphosphate I. The monophosphate, 425 mg (0.8 mmole), is dissolved in 6 ml of dry picoline and 0.19 ml of tributylamine. The solution is evaporated to dryness under reduced pressure, and the tributylammonium salt is taken up in 5 ml of dry dioxane. Tributylamine, 0.36 ml, and 0.24 ml (1.16 mmoles) of freshly distilled diphenyl chlorophosphonate are added, and the mixture is stirred for 2 hr. Evaporation under reduced pressure leaves an oily residue, which is extracted with 10 ml of diethyl ether for 15 rain and usually is transformed into a fluffy yellow precipitate adhering to the glass wall. The ether is decanted, the residue is dissolved in 2 ml of dry dioxane, and the solvent is again evaporated under reduced pressure. The gum is suspended in a few milliliters of picotine and dissolved by the addition of 0.5 ml of dimethylformamide. The solution is added to well-dried tributylammonium pyrophosphate, prepared from 356.8 mg (0.8 mmole) of tetrasodium pyrophosphate after removal of the cations over a small Dowex 50 column in the usual manher2 Pyrophosphorylation is begun by the addition of 0.8 ml of dry pyridine. After 30 min, the solution is concentrated under reduced pressure, the syrup is shaken for a few minutes with 5 ml of diethyl ether, and this solvent is decanted. The syrup is taken up in 10 ml of water, and the turbid solution is extracted four times with 10 ml each of ether. The pH of the water phase is brought to 7.0 and the solution is applied to a 2 X 20 em column of DEAE-Sephadex A-25 in the bicarbonate form that has been well washed with water. The column is developed with a linear gradient of 2000 ml total volume from 0.02 to 0.5 M LiCt or from 4.0 to 1 M triethylammoniumbiearbonate at pH 7.2. The elution diagram shows three major peaks with several shoulders; the triphosphate is found in the third peak. The column fractions are again monitored by eleetrophoresis at pH 3.0 and the fractions containing the triphosphate are lyophilized. The fluffy material is extracted twice with 5 ml of absolute ethanol to remove salts, dried well, and used for labeling experiments within a few days. It may be stored as a concentrated aqueous solution for a few weeks. Yield: 28%. Synthesis of S- (Nitro-4-carboxyphenyl) -6-mercaptopurine Riboside 5'-Monophosphate
S- (2-N itro-4-carboxyphenyl) -6-mercaptopwrine Riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) 3.96 g (10 mmoles) ~J. G. Moffat, Can. J. Chem. 42, 599 (1964).
292
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[26]
is suspended in 50 ml of water and neutralized with 4 N NaOH. Isopropylidated 6-mercaptopurine riboside (see above) 300 mg (1 mmole) is added, and the pH is adjusted to 8.0. The reaction mixture is stirred for 7 days, the pH being readjusted each day. This somewhat unusual reaction proceeds to the desired thioether with elimination of the SH group of the nucleoside. 4 The reaction is monitored by occasional electrophoresis in 0.1 M pyridine acetate at pH 6.5. The resultant solution is applied to a DEAE-Sephadex A-25 column (3 X 35 cm) in the bicarbonate form, and the product is eluted with a linear gradient of 2000 ml total volume from water to 0.2 M annonium bicarbonate at pH 6.5 (adjusted with CO2). It appears as a second peak after a small amount of unreacted mercaptopurine riboside. Purity is checked by electrophoresis at pH 6.5, and the thioether is detected with a mercaptoethanol spray as described above. The solution of the thioether is lyophilized several times to remove almost all the bicarbonate. Yield:
80%. Monophosphate II. The thioether riboside, 489.5 mg (1 mmole), is dissolved in 8 ml of dry triethyl phosphate, and 1 ml of freshly distilled POC18 is added at temperatures below 0 ° in small portions; the solution is maintained at 0 ° for 20 hr. Thereafter, 20 ml of 1 M barium acetate solution are added with vigorous stirring while the mixture is cooled to below 20 °. After addition of 80 ml of water, the pH is held between 7 and 8.5 by the addition of triethylamine. When all the POC13 is hydrolyzed, the pH is adjusted to 8.5 and the precipitated barium phosphate is removed by centrifugation. The precipitate is redissolved at pH 2.0 and reprecipitated at pH 8.5 several times to wash out the copreeipitated barium salt of the desired nucleotide. The combined supernatant fluids are mixed with 3 volumes of ethanol. After standing at 0 ° for 3 hr, the precipitated barium salt of the monophosphate is isolated by centrifugation and washed 3 times with 80% ethanol and with ether. The free monophosphate may be obtained by passage of the solution of its barium salt over a Dowex 50 X 2 column in the H ÷ form, as described above. The isopropylidene group is also removed during this operation. Fractions containing the compound are monitored by electrophoresis at pH 3.0, as described above, or by paper chromatography in isopropanol-0.5 M ammonium acetate pH 6.0 (5:2), with the aid of alkaline mercaptoethanol spray. The solution is lyophilized twice. The compound may be stored as a concentrated aqueous solution at pH 4 for a few weeks whereas the barium salt is quite stable. Yield: 65%. Triphosphate. The procedure described above for the S-(dinitro4F. W. ttulla and It. Fasold, Biochemistry11, 1056 (1972).
[25]
AROM&TIC THIOETHERS OF PURINE NUCLEOTIDES
293
phenyl)-6-mercaptopurine riboside triphosphate may also be applied to the prophosphorylation of this monophosphate. The yield in this case is about 55%. These compounds show rapid reaction with sulfhydryl groups at pH values above 7.5. Of all other amino acid side-chain functional groups tested, only primary amines and phenols are able to react with the aromatic thioether at elevated temperatures and over incubations of several hours' duration.2, 4
Related Compounds The compounds described above may be regarded as analogs of adenosine nucleotides. The guanosine nucleotide analogs of the same group have been synthesized from 2-amino-6-mercaptopurine nucleoside using very similar procedures. A 3',5'-cyclic monophosphate was synthesized from the 5'-monophosphate (I) after the method of Smith et al2
Examples o] A~nity Labeling o] Proteins Phosphorylase b. Phosphorylase b is dependent in its activity upon the allosteric effector AMP, and thus AMP analog (II) could be used to label the effector binding site. 4 The protein was first freed from AMP by filtration with Sephadex G-50 and charcoal columns. Portions of the enzyme, 60 mg, were incubated in 2 ml of 0.01 glycerophosphate-HC1 at pH 8.0 with 40 mg of (II) for 0.5 to 2.0 hr. The analog in this case had been labeled by 32p. The pH was then lowered to 6.5, thereby terminating the modification reaction. Excess nucleotide was removed by filtration over Sephadex G-25, and specific enzymic activity as well as covalently bound 32P-labeled nucleotide was determined. The activation of phosphorylase b by the attached label is shown in Fig. 2. It was not possible to activate the enzyme to a higher degree since the nonspecific reaction of the nucleotide with superficial SH groups became prevalent. The peptic fingerprint of a preparation covalently activated to 20% (see Fig. 2) gave only a single labeled peptide spot in an autoradiogram. Rabbit Muscle Actin. The monomeric G form of actin carries an ATP firmly bound, which is hydrolyzed in a stoichiometric reaction to ADP during polymerization to the fibrillar F form under the influence of inorganic cations, especially Mg. 2+ By removal of the triggering cations, and displacement of the ADP by fresh ATP, the protein depolymerized again to the G form. 5 M. Smith, G. I. D r u m m o n d , and H. G. Khorana, J. Am. Chem. Soc. 83, 698
(1961).
294
ENZY1V[ES, ANTIBODIES, AND OTHER PROTEINS
[26]
30
20
I0
I
!
I
I
I
OI
OZ
03
04
05
Residues of Nucleofide/Enzyme Subuni!
FzG. 2. Activation of phosphorylase b by covalently bound nucleotide after reaction with monophosphate (II). Ordinate: Percentage of maximal activation by AMP.
Prior to labeling by the ATP analog, one easily accessible SH group of the protein was allowed to react with N-ethylmaleimide. It had previously been shown that this modification does not change the polymerization reaction and interaction with myosin in any of its properties. 6,7 The label was attached by incubation of F-actin at a concentration of 10 mg/ml with a 100-fold molar excess of the ATP analog at pH 7.5 for 2 hr in 1 mM Tris chloride containing 0.2 mM ascorbic acid. The depolymerization, as measured by loss of viscosity, proceeded as in the case of ATP. After the removal by centrifugation of nondepolymerized material, approximately 20% of the total, the protein was repolymerized and centrifuged down as modified F-actin. Samples for determinations of bound nucleotide, inorganic 3~Pi, and specific viscosity were taken before and after repolymerization, as well as after the attempt at depolymerization of the modified F-actin. Only 20% of the protein could be brought into the monomeric state again; the major portion was "frozen" in the F form. During repolymerization, the nucleotide was split to the ADP analog and inorganic phosphate. Tryptic fingerprint of this protein revealed only one labeled peptide spot on autoradiography. 2 Other Proteins. Whereas a specific binding site was labeled in the examples cited above, a very different reaction ensued with phosphoribosyl pyrophosphate-ATP-ligase. After incubation of the enzyme in 0.05 M sodium bicarbonate solution with AMP analog (I), label from C. J. Lusty and H. Fasold, Biochemistry 8, 2933 (1969). 7 W. W. Kielley and C. B. Bradley, ]. Biol. Chem. 218, 653 (1956).
[27]
Ne-FLUOROBENZOYL&DENOSINE 5t-TRIPHOSPHATES
295
both 32p and 14C in the dinitrophenyl group 8 was attached to protein. The dinitrophenyl moiety was attached stoichiometrically to its subunits; a tryptic fingerprint revealed only two labeled peptides on autoradio~aphy. With Na÷,K÷-ATPase from nerve tissue, on the other hand, the nucleotide moiety was able to label the enzyme, although extremely long incubation periods of several days were necessary2 s T. Dall-Larsen, H. Fasold, C. Klungs0yr, H. Kryvi, C. Meyer, and F. Ortanderl, Eur. J. Biochem. 60, 103 (1975). P. Patzelt, H. Pauls, E. Erdmann, and W. Schoner, Hoppe-Seyler's Z. Physiol. Chem. 355, 758 (1974).
[27] Ne-o - a n d p - F l u o r o b e n z o y l a d e n o s i n e Y-Triphosphates
By
ALEXANDER HAMPTON a and LEWIS A. SLOTIN
The title compounds (I) and (II) were prepared as potential ATPsite-directed reagents. 1 The fluorobenzoyl substituents were selected because (a) the smallness of fluorine lessens steric hindrance to reaction of (I) and (II) with enzymic groups, (b) fluorine undergoes nucleophilic displacement from aromatic systems under milder conditions than other halogens, (c) o- and p-fluorobenzamides are stable in physiological buffers, and (d) (I) and (II) could be attacked by a nucleophilic group of an enzyme not only at their ortho and para carbons, respectively, but also at their amide carbons, thus approximately doubling the probability that (I) or (II) could act as an affinity label for the ATP site of any given enzyme. RCONH
ooo
II II II
N
(HO)2POPOPOCH2
[
15~°~ I OHOH~ I I
HO
OH
(I): R = o-fluorophenyl (II): R = p-fluorophenyl 1 A. Hampton and L. S. Slotin, Biochemistry 14, 5438 (1975).
