220
[19]
ENZYMES, ANTmODIES, AND OTHER PROTEINS EFFECT OF ~NHIBITORS ON ol-CHYMOTRYPSIN ACTIVITY a-Chymo-
Incubation
Compound
trypsin X 10-s M
Inhib./a-Chy
time (min)
(II) (II) (III) (III) (III) (III)
1.3 1.3 2.2 2.3 38.0 38.0
51 26 26 110 8 8
60 60 60 120a 10 60
Percent inhibition 91 84 98 94 75 50
a I n c u b a t e d a t 0 ° i n s t e a d of room t e m p e r a t u r e .
and assayed for enzymic activity. In this case, the N-benzyltyrosine ethyl ester assay ~ was performed. Results from this procedure are shown in the table. We have observed that at low ratios of inhibitor to chymotrypsin some reversible inhibition occurs. This may be due to alkylation of certain enzymes sites to yield groups that are labile to alkaline hydrolysis. Substrates analogous to (I), (II), and (III) should .function with trypsin, pepsin, papain, and other proteolytic enzymes. Furthermore, oxidases could be labeled by this technique with substrates such as hydrazine derivatives that yield diazonium salts on oxidation. Reductases and other enzymes could presumably be derivatized by other suitable substrates and active species. We have recently found that the D form of compound I effectively and fully inhibits chymotrypsin within 15 min when used at a 10/1 ratio to the enzyme.8 ~B. C. W. Hummel, Can. J. Biochem. Physiol. 37, 1393 (1959). This method is described in "Worthington Enzyme Manual," p. 129. Worthington Biochemical Corp., Freehold, New Jersey, 1972. s j . Am. Chem. Soc., submitted.
[19] P e p t i d e A l d e h y d e s : P o t e n t I n h i b i t o r s o f S e r i n e a n d Cysteine Proteases B y ROBERT C. THOMPSON
The peptide acids generated by protease-catalyzed hydrolysis of proteins are generally good inhibitors of the enzymes that produce them. In the past few years it has become clear that derivatives of peptide acids in which the carboxylic acid group has been replaced by an aldehyde group are even more potent inhibitors of certain proteases than are the
[19]
PEPTIDE ALDEHYDES
221
original acids. This phenomenon was first discovered by Umezawa and his colleagues while screening filtrates of actinomycetes for naturally occurring protease inhibitors, l It was independently discovered by Westerick and Wolfenden2 and by Thompson ~ in a search for transition state analogs of cysteine and serine proteases. The latter workers have proposed that the peptide aldehydes bind to these proteases as enzymealdehyde hemithioacetals and hemiacetals, respectively. The enzymealdehyde adduct would then resemble in structure the transition state or high energy tetrahedral intermediate of the proteolytic reaction. It would therefore be stabilized by those forces that stabilize the transition state and confer on the enzyme its catalytic powers. The most important of these forces may be the stability of the covalent bond formed between the enzyme and the aldehyde.4 There is at present no direct evidence that the enzyme-aldehyde adduct has the proposed structure. However, indirect evidence that the adduct resembles the transition state for substrate hydrolysis comes from studies with elastase in which the relative binding energies of different peptide aldehydes were found to reflect koat/Kn, and not the K,,, for analogous substrates2 The proposed structure is also consistent with the finding that the enhanced binding due to the aldehyde group is more marked with the cysteine protease papain 2 than with any serine protease so far studied. This is in accord with the greater stability of hemithioacetals. ~ Peptide aldehydes have been synthesized both by oxidation of peptide alcohols1,3 and by reduction of peptide acids. ~ Both routes are restricted to peptides that do not contain other functional groups sensitive to the reducing and oxidizing reagents, and the oxidative route at least, subjects the whole peptide to a reaction which sometimes gives only poor yields2 ,3 To provide increased flexibility of synthesis of peptide aldehydes, I have explored another synthetic route, which involves the preparation from a-amino alcohols7 of suitably protected a-amino aldehydes, the coupling of the protected aldehydes to preformed peptides, and the subsequent removal of protection from the aldehyde group. This type of synthesis has 1K. Kawamura, S. Kondo, K. Maeda, and H. Umezawa, Chem. Pharm. Bull. (Tokyo) 17, 1902 (1969). J. Westerick and R. Wolfenden,d. Biol. Chem. 247, 8195 (1972). 3R. C. Thompson, Biochemistry 12, 47 (1973). 4R. C. Thompson, Biochemistry 13, 5494 (1974). 5G. E. Lienhard and W. P. Jencks, J. Am. Chem. Soc. 88, 3982 (1966). B. Shimizu, A. Saito, A. Ito, K. Tokawa, K. Maeda, and H. Umezawa, J. A~ttibiot. 25, 515 (1972). ~S. Yamada, K. Koga, and H. Matsuo, Chem. Pharm. Bull. (Tokyo) 11, 1140 (1963).
222
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[19]
been used previously by Shimizu et al., 6 who prepared the aldehyde group by reduction of a carbobenzoxy amino acid imidazolide and protected it as a semicarbazone during the coupling reaction. However, using the acidic conditions quoted by Shimizu et al., I have experienced difficulty in regenerating the aldehyde group from the peptide aldehyde semicarbazone without simultaneously degrading the peptide. I have therefore used the more acid-labile diethyl acetal of the aldehyde to mask the aldehyde function during the coupling reaction. The potential success of this method had been indicated previously by work of Westerick and Wolfenden,~ who used the commercially available a-amino acetaldehyde dimethyl acetal. The procedure described below has been used successfully to synthesize peptides containing phenylalaninal (Pheal), alaninal (Alaal), or glycinal (Glyal). It should be applicable to the preparation of peptides containing other a-amino aldehydes, including those with reactive side chains, provided that suitable acid-labile protecting groups are available for those side chains. For a list of acid-labile protecting groups for amino acids, the reader should consult a text or recent review of the synthesis of peptides, e.g., "Peptide Synthesis. ''s N - Trifluoroacetyl L-P henylalaninol A mixture of 520 mg of L-phenylalaninol (Fluka AG, Switzerland) (3.4 mmoles) and 15 ml of 1 M aqueous sodium bicarbonate is stirred vigorously in an Erlenmeyer flask, and 2 ml of trifluoroacetyl ethanethiol (Pierce Chemical Co.) (2.6 g, 16 mmoles) are added. The flask is closed with a paper plug, and stirring is continued overnight. Nitrogen gas is blown through the reaction mixture to remove unreacted thiolester and thiol, and the solid product is removed by filtration. After drying under reduced pressure over phosphorus pentoxide, 750 mg of product (89% yield), m.p. 134°-136 °, are ob¢ained. The product is homogeneous by thin-layer chromatography (TLC) on silica gel, Rf 0.3 in chloroform: methanol (98:2). It is visualized by exposing the plate to chlorine vapor for 1 min, t~o air for 10 min, and then spraying with a 0.3% solution of tolidine in 1% aqueous potassium iodide 6% acetic acid. N - Trifluoroacetyl L-Phenylalaninal A mixture of 100 mg of N-trifluoracetyl L-phenylalaninol (0.4 mmole), 500 mg of 1-ethyl(3,3-dimethylaminopropyl)carbodiimide hydrochloride 8 M. Bodansky and M. A. Ondetti, "Peptide Synthesis." Wiley (Interscience), New York, 1966. Later work is summarized each year in "Specialist Periodical Reports. Amino Acids, Peptides, and Proteins." The Chemical Society, Letchworth, England.
[19]
PEPTIDE ALDEHYDES
223
(2.6 mmoles), and 2.0 ml of redistilled dimethyl sulfoxide is stirred for 10 min, and 200 ~l of a 2 M solution of anhydrous phosphoric acid 9 in dimethyl sulfoxide are added. The mixture is stirred for 2.5 hr, then 5 ml of 1 M potassium phosphate at pH 7.5 and 5 ml of ethyl acetate are added, the mixture is shaken, and the layers are separated. The ethyl acetate solution is washed with an additional 3 ml of the phosphate buffer, dried over sodium sulfate, and evaporated. The residue is dissolved in 5 ml of ethanol and added to a solution of 4 g of sodium bisulfite in 7 ml of water while stirring vigorously. After standing for 10 rain, the ethanol is removed by evaporation of the solution to approximately 7 ml, and the aqueous residue is extracted twice with 5 ml of ether. Solid sodium carbonate is added to the aqueous solution to bring the pH to between 8.5 and 9.0. The mixture is allowed to stand for 5 rain and is then extracted twice with 5 ml of ether. The ether extracts from the basic solution are pooled, dried, and evaporated to yield 68 mg (69%) of product, homogeneous by TLC, Rs 0.5 on silica gel in chloroform:methanol(98:2). The aldehyde is visualized either by spraying the plate with a solution of dinitrophenylhydrazine in phosphoric acid prepared according to Johnson, TM or by the chlorine-tolidine reagent described above. A solution of the product in hexane kept at --20 ° deposited 58 mg of product (m.p. 78°-80 °) with the N M R spectrum anticipated for an N-acyl phenylalaninal. We have found that 1-ethyl(3,3-dimethylaminopropyl)carbodiimide hydrochloride generally gives better yields of product than does another water-soluble carbodiimide, 1-eyclohexyl 3-morpholinoethyl carbodiimide hydroehloride. This is particularly true when the dimethyl sulfoxide is not redistilled before use. The use of dimethyl sulfoxide that has been redistilled under reduced pressure is recommended for all oxidation reactions. Prolongation of the oxidation reaction leads to a decreased yield of aldehyde product. After 24 hr, the yield of TFA Pheal is 36% before, and 27% after, crystallization from hexane.
L-Phenylalaninal Diethylacetal N-Trifluoroacetyl L-phenylalaninal, 50 mg (0.20 mmole), is dissolved in 2 ml of ethanol. Then 100 ~l of 1 M p-toluenesulfonic acid monohydrate in ethanol and 1 ml of triethylorthoformate are added and the mixture is allowed to stand for 30 rain. The solvents are evaporated under high vacuum. The residue is dissolved in ethyl acetate and extracted twice with 5% aqueous sodium bicarbonate. The ethyl acetate solution R. E, Ferrell, H. S. Olcott, and H. Fraenkel-Conrat, J. Am. Chem. Soc. 70, 2101 (1948). lo C. D. Johnson, J. Am. Chem. Soc. 73, 5888 (1951).
224
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[19]
is dried over sodium sulfate and evaporated. The residue is homogeneous by TLC on silica gel, RI 0.8 in hexane:ether(4:l). The product is visualized either by the dinitrophenylhydrazine or chlorine-tolidine reagents described above. Unlike the parent aldehyde, the diethylacetal forms a yellow spot with dinitrophenylhydrazine solution only on heating the plate after spraying. The residue is dissolved in 1.2 ml of methanol, and 1.6 ml of 0.5 M aqueous sodium hydroxide are added. After standing for 45 min, 5 ml of water are added, the solution is partially evaporated to remove the methanol, and is then extracted with ethyl acetate. The ethyl acetate solution is dried and evaporated to give 35 mg of an oil. TLC of the oil on silica gel showed the major product to have R~ 0.5 in chloroform: methanol(8:2). The product is visualized by spraying with a 0.3% solution of ninhydrin in acetone or with the dinitrophenylhydrazine reagent. Occasionally a trace of a second product, RI 0.4, was observed when the product was visualized with the ninhydrin reagent. The amount of this product increased with prolongation of the base hydrolysis. A cetylprolylalanylprolylphenylalaninal
The a-amino aldehyde diethylacetal can be coupled to peptides using the mixed anhydride procedure of Anderson et al. 11 In the example given below there is no danger of racemization of the peptide during this process but, even were this possible, the procedure used would reduce t h e risk of racemization to an acceptable level. 11 Acetylprolylalanylproline, 65 mg (0.2 mmole), and 22 ill of N-methylmorpholine (0.2 mmole) are dissolved in 2 ml of acetonitrile and the mixture is cooled to --20 ° in a bath of Dry Ice and carbon tetraehloride. Isobutyl chloroformate, 26 ~l (0.2 mmole), is added with stirring, and 5 min later, the L-phenylalaninal diethylacetal prepared above is added as a solution in 0.5 ml of ethyl acetate. The stirred reaction mixture is allowed to warm to room temperature over a period of about 4 hr. T h e solvents are evaporated, and the residue is dissolved in 5 ml of water. The solution is then cooled to 0 ° and treated with R E X Y N 1-300 ion exchange resin for 10 min and filtered. This procedure gives an aqueous solution of the peptide aldehyde diethylacetal which shows a single spot, RI 0.9 on TLC on silica gel in chloroform: methanol (9:1). The product is visualized using the dinitrophenylhydrazine or chlorine-tolidine reagents. In the former case, it is necessary to heat the chromatography plate. ,1 G. W. Anderson, J. E. Zimmerman, and F. M. Callahan, J. Am. Chem. Soc. 89, 5012 (1967).
[20]
CARBOXYPEPTIDASES K AND B
225
Dowex AG50W resin (H + form), 100 nag, is added, and the solution is stirred for 2.5 hr at 25 °. This results in complete conversion of the diethylacetal to peptide aldehyde. Tile product, 45 mg (47%), is isolated as an amorphous solid by evaporation of tile aqueous solution and trituration under ether. It was homogeneous by TLC on silica gel, Rs 0.7 in chloroform:methanol (8:2). The product is visualized using the dinitrophenylhydrazine or chlorine-tolidine reagents.
Modifications Necessary to Prepare Aldehydes ]rom Water-Soluble Alcohols A similar series of reactions has been used to prepare the peptide aldehyde AeProAlaProAlaal. However, in this case tile intermediate, N-trifluroacetyl alaninal (TFA Alaal), could not be separated from unreacted N-trifluoroacetyl alaninol (TFA Alaol) by purification of a water-soluble bisulfite addition compound since TFA Alaol is itself fairly soluble in water. Instead, the aldehyde is purified by direct conversion to the ether-soluble diethylacetal. Potassium phosphate, 1 M at pH 7.5, 60 ml, is added to the carbodiimide-dimethyl sulfoxide reaction mixture, and the resulting solution is extracted first with 30 mI of ether and subsequently thrice with 60 ml each of ethyl acetate. The pooled ethyl acetate extracts are dried, evaporated, and treated with triethylorthoformate as described for the preparation of TFA Pheal diethylaeetal. Any contaminating TFA Alaol is removed by extraction of an ethereal solution of the product with aqueous sodium bicarbonate. The procedure yields 48 mg (52%) of TFA Alaal diethylacetal which has the correct proton magnetic resonance spectrum and is homogeneous by TLC on silica gel, R~ 0.8 in chloroform. Crystallization from hexane at --20 ° gave 33 mg (36%) of white crystals. The TFA Alaal diethylacetal is deprotected and coupled to acetylprolylalanylproline by the same method with approximately the same yields as those described from the synthesis of aeetylprolylalanylprolylphenylalaninal.
[20] Carboxypeptidases A and B By M. SOKOLOVSKY Carboxypeptidases A (EC 3.4.12.2) and B (EC 3.4.12.3) are pancreatic exopeptidases. CarboxypePtidase A preferentially hydrolyzes terminal peptide bonds in which the amino donor is a hydrophobic amino acid whereas the specificity of carboxypeptidase B is directed toward the
[19]
ENZYMES, ANTmODIES, AND OTHER PROTEINS EFFECT OF ~NHIBITORS ON ol-CHYMOTRYPSIN ACTIVITY a-Chymo-
Incubation
Compound
trypsin X 10-s M
Inhib./a-Chy
time (min)
(II) (II) (III) (III) (III) (III)
1.3 1.3 2.2 2.3 38.0 38.0
51 26 26 110 8 8
60 60 60 120a 10 60
Percent inhibition 91 84 98 94 75 50
a I n c u b a t e d a t 0 ° i n s t e a d of room t e m p e r a t u r e .
and assayed for enzymic activity. In this case, the N-benzyltyrosine ethyl ester assay ~ was performed. Results from this procedure are shown in the table. We have observed that at low ratios of inhibitor to chymotrypsin some reversible inhibition occurs. This may be due to alkylation of certain enzymes sites to yield groups that are labile to alkaline hydrolysis. Substrates analogous to (I), (II), and (III) should .function with trypsin, pepsin, papain, and other proteolytic enzymes. Furthermore, oxidases could be labeled by this technique with substrates such as hydrazine derivatives that yield diazonium salts on oxidation. Reductases and other enzymes could presumably be derivatized by other suitable substrates and active species. We have recently found that the D form of compound I effectively and fully inhibits chymotrypsin within 15 min when used at a 10/1 ratio to the enzyme.8 ~B. C. W. Hummel, Can. J. Biochem. Physiol. 37, 1393 (1959). This method is described in "Worthington Enzyme Manual," p. 129. Worthington Biochemical Corp., Freehold, New Jersey, 1972. s j . Am. Chem. Soc., submitted.
[19] P e p t i d e A l d e h y d e s : P o t e n t I n h i b i t o r s o f S e r i n e a n d Cysteine Proteases B y ROBERT C. THOMPSON
The peptide acids generated by protease-catalyzed hydrolysis of proteins are generally good inhibitors of the enzymes that produce them. In the past few years it has become clear that derivatives of peptide acids in which the carboxylic acid group has been replaced by an aldehyde group are even more potent inhibitors of certain proteases than are the
[19]
PEPTIDE ALDEHYDES
221
original acids. This phenomenon was first discovered by Umezawa and his colleagues while screening filtrates of actinomycetes for naturally occurring protease inhibitors, l It was independently discovered by Westerick and Wolfenden2 and by Thompson ~ in a search for transition state analogs of cysteine and serine proteases. The latter workers have proposed that the peptide aldehydes bind to these proteases as enzymealdehyde hemithioacetals and hemiacetals, respectively. The enzymealdehyde adduct would then resemble in structure the transition state or high energy tetrahedral intermediate of the proteolytic reaction. It would therefore be stabilized by those forces that stabilize the transition state and confer on the enzyme its catalytic powers. The most important of these forces may be the stability of the covalent bond formed between the enzyme and the aldehyde.4 There is at present no direct evidence that the enzyme-aldehyde adduct has the proposed structure. However, indirect evidence that the adduct resembles the transition state for substrate hydrolysis comes from studies with elastase in which the relative binding energies of different peptide aldehydes were found to reflect koat/Kn, and not the K,,, for analogous substrates2 The proposed structure is also consistent with the finding that the enhanced binding due to the aldehyde group is more marked with the cysteine protease papain 2 than with any serine protease so far studied. This is in accord with the greater stability of hemithioacetals. ~ Peptide aldehydes have been synthesized both by oxidation of peptide alcohols1,3 and by reduction of peptide acids. ~ Both routes are restricted to peptides that do not contain other functional groups sensitive to the reducing and oxidizing reagents, and the oxidative route at least, subjects the whole peptide to a reaction which sometimes gives only poor yields2 ,3 To provide increased flexibility of synthesis of peptide aldehydes, I have explored another synthetic route, which involves the preparation from a-amino alcohols7 of suitably protected a-amino aldehydes, the coupling of the protected aldehydes to preformed peptides, and the subsequent removal of protection from the aldehyde group. This type of synthesis has 1K. Kawamura, S. Kondo, K. Maeda, and H. Umezawa, Chem. Pharm. Bull. (Tokyo) 17, 1902 (1969). J. Westerick and R. Wolfenden,d. Biol. Chem. 247, 8195 (1972). 3R. C. Thompson, Biochemistry 12, 47 (1973). 4R. C. Thompson, Biochemistry 13, 5494 (1974). 5G. E. Lienhard and W. P. Jencks, J. Am. Chem. Soc. 88, 3982 (1966). B. Shimizu, A. Saito, A. Ito, K. Tokawa, K. Maeda, and H. Umezawa, J. A~ttibiot. 25, 515 (1972). ~S. Yamada, K. Koga, and H. Matsuo, Chem. Pharm. Bull. (Tokyo) 11, 1140 (1963).
222
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[19]
been used previously by Shimizu et al., 6 who prepared the aldehyde group by reduction of a carbobenzoxy amino acid imidazolide and protected it as a semicarbazone during the coupling reaction. However, using the acidic conditions quoted by Shimizu et al., I have experienced difficulty in regenerating the aldehyde group from the peptide aldehyde semicarbazone without simultaneously degrading the peptide. I have therefore used the more acid-labile diethyl acetal of the aldehyde to mask the aldehyde function during the coupling reaction. The potential success of this method had been indicated previously by work of Westerick and Wolfenden,~ who used the commercially available a-amino acetaldehyde dimethyl acetal. The procedure described below has been used successfully to synthesize peptides containing phenylalaninal (Pheal), alaninal (Alaal), or glycinal (Glyal). It should be applicable to the preparation of peptides containing other a-amino aldehydes, including those with reactive side chains, provided that suitable acid-labile protecting groups are available for those side chains. For a list of acid-labile protecting groups for amino acids, the reader should consult a text or recent review of the synthesis of peptides, e.g., "Peptide Synthesis. ''s N - Trifluoroacetyl L-P henylalaninol A mixture of 520 mg of L-phenylalaninol (Fluka AG, Switzerland) (3.4 mmoles) and 15 ml of 1 M aqueous sodium bicarbonate is stirred vigorously in an Erlenmeyer flask, and 2 ml of trifluoroacetyl ethanethiol (Pierce Chemical Co.) (2.6 g, 16 mmoles) are added. The flask is closed with a paper plug, and stirring is continued overnight. Nitrogen gas is blown through the reaction mixture to remove unreacted thiolester and thiol, and the solid product is removed by filtration. After drying under reduced pressure over phosphorus pentoxide, 750 mg of product (89% yield), m.p. 134°-136 °, are ob¢ained. The product is homogeneous by thin-layer chromatography (TLC) on silica gel, Rf 0.3 in chloroform: methanol (98:2). It is visualized by exposing the plate to chlorine vapor for 1 min, t~o air for 10 min, and then spraying with a 0.3% solution of tolidine in 1% aqueous potassium iodide 6% acetic acid. N - Trifluoroacetyl L-Phenylalaninal A mixture of 100 mg of N-trifluoracetyl L-phenylalaninol (0.4 mmole), 500 mg of 1-ethyl(3,3-dimethylaminopropyl)carbodiimide hydrochloride 8 M. Bodansky and M. A. Ondetti, "Peptide Synthesis." Wiley (Interscience), New York, 1966. Later work is summarized each year in "Specialist Periodical Reports. Amino Acids, Peptides, and Proteins." The Chemical Society, Letchworth, England.
[19]
PEPTIDE ALDEHYDES
223
(2.6 mmoles), and 2.0 ml of redistilled dimethyl sulfoxide is stirred for 10 min, and 200 ~l of a 2 M solution of anhydrous phosphoric acid 9 in dimethyl sulfoxide are added. The mixture is stirred for 2.5 hr, then 5 ml of 1 M potassium phosphate at pH 7.5 and 5 ml of ethyl acetate are added, the mixture is shaken, and the layers are separated. The ethyl acetate solution is washed with an additional 3 ml of the phosphate buffer, dried over sodium sulfate, and evaporated. The residue is dissolved in 5 ml of ethanol and added to a solution of 4 g of sodium bisulfite in 7 ml of water while stirring vigorously. After standing for 10 rain, the ethanol is removed by evaporation of the solution to approximately 7 ml, and the aqueous residue is extracted twice with 5 ml of ether. Solid sodium carbonate is added to the aqueous solution to bring the pH to between 8.5 and 9.0. The mixture is allowed to stand for 5 rain and is then extracted twice with 5 ml of ether. The ether extracts from the basic solution are pooled, dried, and evaporated to yield 68 mg (69%) of product, homogeneous by TLC, Rs 0.5 on silica gel in chloroform:methanol(98:2). The aldehyde is visualized either by spraying the plate with a solution of dinitrophenylhydrazine in phosphoric acid prepared according to Johnson, TM or by the chlorine-tolidine reagent described above. A solution of the product in hexane kept at --20 ° deposited 58 mg of product (m.p. 78°-80 °) with the N M R spectrum anticipated for an N-acyl phenylalaninal. We have found that 1-ethyl(3,3-dimethylaminopropyl)carbodiimide hydrochloride generally gives better yields of product than does another water-soluble carbodiimide, 1-eyclohexyl 3-morpholinoethyl carbodiimide hydroehloride. This is particularly true when the dimethyl sulfoxide is not redistilled before use. The use of dimethyl sulfoxide that has been redistilled under reduced pressure is recommended for all oxidation reactions. Prolongation of the oxidation reaction leads to a decreased yield of aldehyde product. After 24 hr, the yield of TFA Pheal is 36% before, and 27% after, crystallization from hexane.
L-Phenylalaninal Diethylacetal N-Trifluoroacetyl L-phenylalaninal, 50 mg (0.20 mmole), is dissolved in 2 ml of ethanol. Then 100 ~l of 1 M p-toluenesulfonic acid monohydrate in ethanol and 1 ml of triethylorthoformate are added and the mixture is allowed to stand for 30 rain. The solvents are evaporated under high vacuum. The residue is dissolved in ethyl acetate and extracted twice with 5% aqueous sodium bicarbonate. The ethyl acetate solution R. E, Ferrell, H. S. Olcott, and H. Fraenkel-Conrat, J. Am. Chem. Soc. 70, 2101 (1948). lo C. D. Johnson, J. Am. Chem. Soc. 73, 5888 (1951).
224
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[19]
is dried over sodium sulfate and evaporated. The residue is homogeneous by TLC on silica gel, RI 0.8 in hexane:ether(4:l). The product is visualized either by the dinitrophenylhydrazine or chlorine-tolidine reagents described above. Unlike the parent aldehyde, the diethylacetal forms a yellow spot with dinitrophenylhydrazine solution only on heating the plate after spraying. The residue is dissolved in 1.2 ml of methanol, and 1.6 ml of 0.5 M aqueous sodium hydroxide are added. After standing for 45 min, 5 ml of water are added, the solution is partially evaporated to remove the methanol, and is then extracted with ethyl acetate. The ethyl acetate solution is dried and evaporated to give 35 mg of an oil. TLC of the oil on silica gel showed the major product to have R~ 0.5 in chloroform: methanol(8:2). The product is visualized by spraying with a 0.3% solution of ninhydrin in acetone or with the dinitrophenylhydrazine reagent. Occasionally a trace of a second product, RI 0.4, was observed when the product was visualized with the ninhydrin reagent. The amount of this product increased with prolongation of the base hydrolysis. A cetylprolylalanylprolylphenylalaninal
The a-amino aldehyde diethylacetal can be coupled to peptides using the mixed anhydride procedure of Anderson et al. 11 In the example given below there is no danger of racemization of the peptide during this process but, even were this possible, the procedure used would reduce t h e risk of racemization to an acceptable level. 11 Acetylprolylalanylproline, 65 mg (0.2 mmole), and 22 ill of N-methylmorpholine (0.2 mmole) are dissolved in 2 ml of acetonitrile and the mixture is cooled to --20 ° in a bath of Dry Ice and carbon tetraehloride. Isobutyl chloroformate, 26 ~l (0.2 mmole), is added with stirring, and 5 min later, the L-phenylalaninal diethylacetal prepared above is added as a solution in 0.5 ml of ethyl acetate. The stirred reaction mixture is allowed to warm to room temperature over a period of about 4 hr. T h e solvents are evaporated, and the residue is dissolved in 5 ml of water. The solution is then cooled to 0 ° and treated with R E X Y N 1-300 ion exchange resin for 10 min and filtered. This procedure gives an aqueous solution of the peptide aldehyde diethylacetal which shows a single spot, RI 0.9 on TLC on silica gel in chloroform: methanol (9:1). The product is visualized using the dinitrophenylhydrazine or chlorine-tolidine reagents. In the former case, it is necessary to heat the chromatography plate. ,1 G. W. Anderson, J. E. Zimmerman, and F. M. Callahan, J. Am. Chem. Soc. 89, 5012 (1967).
[20]
CARBOXYPEPTIDASES K AND B
225
Dowex AG50W resin (H + form), 100 nag, is added, and the solution is stirred for 2.5 hr at 25 °. This results in complete conversion of the diethylacetal to peptide aldehyde. Tile product, 45 mg (47%), is isolated as an amorphous solid by evaporation of tile aqueous solution and trituration under ether. It was homogeneous by TLC on silica gel, Rs 0.7 in chloroform:methanol (8:2). The product is visualized using the dinitrophenylhydrazine or chlorine-tolidine reagents.
Modifications Necessary to Prepare Aldehydes ]rom Water-Soluble Alcohols A similar series of reactions has been used to prepare the peptide aldehyde AeProAlaProAlaal. However, in this case tile intermediate, N-trifluroacetyl alaninal (TFA Alaal), could not be separated from unreacted N-trifluoroacetyl alaninol (TFA Alaol) by purification of a water-soluble bisulfite addition compound since TFA Alaol is itself fairly soluble in water. Instead, the aldehyde is purified by direct conversion to the ether-soluble diethylacetal. Potassium phosphate, 1 M at pH 7.5, 60 ml, is added to the carbodiimide-dimethyl sulfoxide reaction mixture, and the resulting solution is extracted first with 30 mI of ether and subsequently thrice with 60 ml each of ethyl acetate. The pooled ethyl acetate extracts are dried, evaporated, and treated with triethylorthoformate as described for the preparation of TFA Pheal diethylaeetal. Any contaminating TFA Alaol is removed by extraction of an ethereal solution of the product with aqueous sodium bicarbonate. The procedure yields 48 mg (52%) of TFA Alaal diethylacetal which has the correct proton magnetic resonance spectrum and is homogeneous by TLC on silica gel, R~ 0.8 in chloroform. Crystallization from hexane at --20 ° gave 33 mg (36%) of white crystals. The TFA Alaal diethylacetal is deprotected and coupled to acetylprolylalanylproline by the same method with approximately the same yields as those described from the synthesis of aeetylprolylalanylprolylphenylalaninal.
[20] Carboxypeptidases A and B By M. SOKOLOVSKY Carboxypeptidases A (EC 3.4.12.2) and B (EC 3.4.12.3) are pancreatic exopeptidases. CarboxypePtidase A preferentially hydrolyzes terminal peptide bonds in which the amino donor is a hydrophobic amino acid whereas the specificity of carboxypeptidase B is directed toward the
