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N- and C-alkylation of peptides and proteins in dimethyl sulfoxide
361
BIOCHIMICA ET BiOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 33213
N- and C-alkylationofpeptides and proteins in dimethyl sulfoxide In previous communications1, 2, it was demonstrated that neutral amino acid residues, such as glycine and alanine in wheat gluten proteins, participate in anionic graft polymerizations with vinyl compounds, such as methyl acrylate, in dimethyl sulfoxide containing Nail. Neutral amino acid residues in proteins have two potential sites for anion formation under aprotic conditions: the nitrogen and the a-carbon of the amide group of the peptide bond. Model reactions have now been carried out in which anions were produced on glycine peptides and polyglycine and then alkylated with methyl iodide, benzyl bromide and iodoacetamide. A lkylation procedure. All reactions were performed under anhydrous conditions. Peptides, proteins and glassware were dried under vacuum at IOO° for 24 h. A solution containing 2 mmoles of methylsulfinylcarbanion per ml dimethyl sulfoxide was prepared as described by COREY AND CHAYKOVSKY3. A typical alkylation procedure was as follows: 22.8 mg of polyglycine (0. 4 mmole of glycyl residues) was suspended in IO ml of dimethyl sulfoxide (anhydrous 1) containing 0. 4 ml (0.8 mmole) of the methylsulfinylcarbanion solution by stirring for 2 h under an atmosphere of nitrogen. After 2 h, a Io-fold molar excess (8.0 mmole) of the alkylating agent was added and the suspension was stirred overnight. The mixture was then concentrated on a rotary evaporator under reduced pressure and hydrolyzed with constant boiling HC1 (4° m l ) at reflux for 24 h. The hydrolysate was then extracted with chloroform to remove unreacted alkylating agent. The chloroform was discarded, the HC1 removed under reduced pressure and the residue dissolved in water for amino acid analysis. When iodoacetamide was utilized as alkylating agent, the reaction was heterogeneous, due to the insolubility of iodoacetamide in dimethylsulfoxide, and, therefore, allowed to react for 7 days. Amino acid content of the products was determined on a Phoenix Amino Acid Analyzer, Model K-8ooo, according to the rapid analysis system of BENSON AND PATTERSON 4 a s automated by CAVINS AND FRIEDMAN 5. TABLE[ CONVERSION OF GLY'CINE RESIDUES TO N-ALKYL DERIVATIVES
Peptide
A lkylating agent
Percent of conversion * Sarcosine
N-Acetylglycine Tetraglycine Polyglycine
CH3I C6HsCH2Br CH3I C6HsCH2Br CHsI C 8H s C H 2 B r
N-Benzylglycine
Trace 20 Io 27 27 7°
" B a s e d on a m i n o acid a n a l y s i s of h y d r o l y z e d p r o d u c t s .
Biochim. Biophys. Acta, 207 (197 o) 361-36.3
362
SHORT COMMUNICATIONS
Three glycine models were chosen for examination: N-acetylglycine, tetraglycine and polyglycine. Glycine peptides were chosen for models for this study because their structure is simple. If alkylation were to occur, there should be no steric inhibition by bulky side-chain groups, and the alkylation would yield known amino acids. Alkylation of anions produced on these peptides with methyl iodide would yield sarcosine with N-alkylation or alanine with C-alkylation. With benzyl bromide as alkylation agent, the N-alkyl product would be N-benzylglycine and the C-alkyl product phenylalanine. Table I illustrate the results obtained after hydrolysis of the products. Running the amino acid analyses concurrently with a standard mixture of amino acids allowed facile identification of the products. Initially it was thought that the product with benzyl bromide was exclusively C-alkylation since only an enhancement of the phenylalanine peak was observed (along with a decrease in glycine). But later it was found that N-benzylglycine elutes exactly with phenylalanine. NMR analysis Of the alkylation product indicated that little, if any, C-alkylation occurred. To quantitate yields of the alkylation reactions it was necessary to establish the ninhydrin color yield of the N-alkyl amino acids. The concentration of N-alkyl derivative in the product was then determined by amino acid analysis. The color yields for N-methyl and N-benzyl products of glycine and a-alanine and related compounds are summarized in Table II. TABLE
II
NINHYDRIN
COLOR YIELDS
R E L A T I V E TO L E U C I N E
AND
FOR N-ALKYL
AMINO ACID AND RELATED
C O M P O U N D S ON M O L A R B A S I S
A~4onm/A570nm
Amino acid
Color yield"
.4 440/xz~ 570nm
Leucine Glycine Phenylglycine N-Methylglycine N-Benzylglycine N-Acetylglycine a-Alanine N-Methyl-a-alanine N-Benzyl-a-alanine
I.OO
O. 1 5
0.99 o.99 o.42 o. 52 o i .oo 0.05 0.05
o. 16 o. 17 o.2o o. 16 o o. 13 --"* ---'"
* See ref. 9. *" T h e N - a l k y l a l a n i n e d e r i v a t i v e s g a v e e s s e n t i a l l y n o c o l o r a t 44 ° n m .
Surprisingly, the glycine derivatives give color yields about half those found for the unsubstituted amino acids, in contrast to the negligible color constants for N-acetylglycine and the alanine derivatives. These unexpected observations cannot be rationalized readily in terms of the previously postulated mechanism for the ninhydrin reaction. 6 Amino acid analysis of the hydrolysate of the product from alkylation of polyglycine with iodoacetamide showed a small amount of the expected C-alkylation product, aspartic acid, along with glycine and two unidentified additional products, Biochi~n. Biophys. Acta, 207 ( i 9 7 o) 3 6 i 363
363
SHORT COMMUNICATIONS
possibly HOOCCH2-NHCH2C00H and HOOC-CH-CH2COOH.
I
NHCH,-C00H Our results indicate that it is possible to N-alkylate peptide amide groups and to C-alkylate groups adjacent to peptide bonds under mild conditions and that the alkylations are best carried out with preformed methylsulfinylcarbanion solutions rather than in solutions of N a i l in dimethyl sulfoxide. Increased yields of the products can be obtained by longer reaction time and higher concentrations of alkylating agents. Since N-alkylation of proteins replaces the N - H protons of peptide bonds by alkyl groups, the derivatized proteins may be useful in studies on the effect of hydrogen-bonding interactions, in which peptide bonds participate, on the conformation and related structural properties of proteins 7. Peralkylation of peptides is also a useful technique for sequence determination of oligopeptides by mass spectroscopys. The observation that C-alkylation may be achieved under certain conditions suggests that it may be possible to transform a nutritionally nonessential amino acid to an essential one in the protein.
Northern Regional Research Laboratory, Northern Utilization Research and Development Division, U S. Department of Agriculture, Agricultural Research Service, Peoria, Ill. 616o 4 (U.S.A.)
MENDEL FRIEDMAN L. H. KRULL
L. H. KRULL AND M. FRIEDMAN,J. Polymer Sci., Part A, 5 (1967) 2535. L. H. ]~[RULL AND M. FRIEDMAN, Polymer Preprints, 9 (1968) 129o. E. J. COREY AND M. CHAYKOVSKY,J. Am. Chem. Soc., 87 (1965) 1345. J. U. BENSON AND J. A. PATTERSON, Anal. Chem., 37 (1965) 11o8. J. F. CAVINS AND M. FRIEDMAN, Cereal Chem., 45 (1968) 172. M. FRIEDMAN AND C. W. SIGEL, Biochemistry, 5 (1966) 478. M. GOODMAN AND M. FRIED, J. Am, Chem. Soc., 89 (1967) 1264. D. W. THOMAS, g. C. DAS, S. D. GERO AND E. LEDERER, Biochem. Biophys. Res. Commun., 32 (1968) 519. 9 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907I 2 3 4 5 6 7 8
Received February I9th, 197o Biochim. Biophys. Acta, 2o 7 (197 o) 361-363
BIOCHIMICA ET BiOPHYSICA ACTA
SHORT
COMMUNICATIONS
BBA 33213
N- and C-alkylationofpeptides and proteins in dimethyl sulfoxide In previous communications1, 2, it was demonstrated that neutral amino acid residues, such as glycine and alanine in wheat gluten proteins, participate in anionic graft polymerizations with vinyl compounds, such as methyl acrylate, in dimethyl sulfoxide containing Nail. Neutral amino acid residues in proteins have two potential sites for anion formation under aprotic conditions: the nitrogen and the a-carbon of the amide group of the peptide bond. Model reactions have now been carried out in which anions were produced on glycine peptides and polyglycine and then alkylated with methyl iodide, benzyl bromide and iodoacetamide. A lkylation procedure. All reactions were performed under anhydrous conditions. Peptides, proteins and glassware were dried under vacuum at IOO° for 24 h. A solution containing 2 mmoles of methylsulfinylcarbanion per ml dimethyl sulfoxide was prepared as described by COREY AND CHAYKOVSKY3. A typical alkylation procedure was as follows: 22.8 mg of polyglycine (0. 4 mmole of glycyl residues) was suspended in IO ml of dimethyl sulfoxide (anhydrous 1) containing 0. 4 ml (0.8 mmole) of the methylsulfinylcarbanion solution by stirring for 2 h under an atmosphere of nitrogen. After 2 h, a Io-fold molar excess (8.0 mmole) of the alkylating agent was added and the suspension was stirred overnight. The mixture was then concentrated on a rotary evaporator under reduced pressure and hydrolyzed with constant boiling HC1 (4° m l ) at reflux for 24 h. The hydrolysate was then extracted with chloroform to remove unreacted alkylating agent. The chloroform was discarded, the HC1 removed under reduced pressure and the residue dissolved in water for amino acid analysis. When iodoacetamide was utilized as alkylating agent, the reaction was heterogeneous, due to the insolubility of iodoacetamide in dimethylsulfoxide, and, therefore, allowed to react for 7 days. Amino acid content of the products was determined on a Phoenix Amino Acid Analyzer, Model K-8ooo, according to the rapid analysis system of BENSON AND PATTERSON 4 a s automated by CAVINS AND FRIEDMAN 5. TABLE[ CONVERSION OF GLY'CINE RESIDUES TO N-ALKYL DERIVATIVES
Peptide
A lkylating agent
Percent of conversion * Sarcosine
N-Acetylglycine Tetraglycine Polyglycine
CH3I C6HsCH2Br CH3I C6HsCH2Br CHsI C 8H s C H 2 B r
N-Benzylglycine
Trace 20 Io 27 27 7°
" B a s e d on a m i n o acid a n a l y s i s of h y d r o l y z e d p r o d u c t s .
Biochim. Biophys. Acta, 207 (197 o) 361-36.3
362
SHORT COMMUNICATIONS
Three glycine models were chosen for examination: N-acetylglycine, tetraglycine and polyglycine. Glycine peptides were chosen for models for this study because their structure is simple. If alkylation were to occur, there should be no steric inhibition by bulky side-chain groups, and the alkylation would yield known amino acids. Alkylation of anions produced on these peptides with methyl iodide would yield sarcosine with N-alkylation or alanine with C-alkylation. With benzyl bromide as alkylation agent, the N-alkyl product would be N-benzylglycine and the C-alkyl product phenylalanine. Table I illustrate the results obtained after hydrolysis of the products. Running the amino acid analyses concurrently with a standard mixture of amino acids allowed facile identification of the products. Initially it was thought that the product with benzyl bromide was exclusively C-alkylation since only an enhancement of the phenylalanine peak was observed (along with a decrease in glycine). But later it was found that N-benzylglycine elutes exactly with phenylalanine. NMR analysis Of the alkylation product indicated that little, if any, C-alkylation occurred. To quantitate yields of the alkylation reactions it was necessary to establish the ninhydrin color yield of the N-alkyl amino acids. The concentration of N-alkyl derivative in the product was then determined by amino acid analysis. The color yields for N-methyl and N-benzyl products of glycine and a-alanine and related compounds are summarized in Table II. TABLE
II
NINHYDRIN
COLOR YIELDS
R E L A T I V E TO L E U C I N E
AND
FOR N-ALKYL
AMINO ACID AND RELATED
C O M P O U N D S ON M O L A R B A S I S
A~4onm/A570nm
Amino acid
Color yield"
.4 440/xz~ 570nm
Leucine Glycine Phenylglycine N-Methylglycine N-Benzylglycine N-Acetylglycine a-Alanine N-Methyl-a-alanine N-Benzyl-a-alanine
I.OO
O. 1 5
0.99 o.99 o.42 o. 52 o i .oo 0.05 0.05
o. 16 o. 17 o.2o o. 16 o o. 13 --"* ---'"
* See ref. 9. *" T h e N - a l k y l a l a n i n e d e r i v a t i v e s g a v e e s s e n t i a l l y n o c o l o r a t 44 ° n m .
Surprisingly, the glycine derivatives give color yields about half those found for the unsubstituted amino acids, in contrast to the negligible color constants for N-acetylglycine and the alanine derivatives. These unexpected observations cannot be rationalized readily in terms of the previously postulated mechanism for the ninhydrin reaction. 6 Amino acid analysis of the hydrolysate of the product from alkylation of polyglycine with iodoacetamide showed a small amount of the expected C-alkylation product, aspartic acid, along with glycine and two unidentified additional products, Biochi~n. Biophys. Acta, 207 ( i 9 7 o) 3 6 i 363
363
SHORT COMMUNICATIONS
possibly HOOCCH2-NHCH2C00H and HOOC-CH-CH2COOH.
I
NHCH,-C00H Our results indicate that it is possible to N-alkylate peptide amide groups and to C-alkylate groups adjacent to peptide bonds under mild conditions and that the alkylations are best carried out with preformed methylsulfinylcarbanion solutions rather than in solutions of N a i l in dimethyl sulfoxide. Increased yields of the products can be obtained by longer reaction time and higher concentrations of alkylating agents. Since N-alkylation of proteins replaces the N - H protons of peptide bonds by alkyl groups, the derivatized proteins may be useful in studies on the effect of hydrogen-bonding interactions, in which peptide bonds participate, on the conformation and related structural properties of proteins 7. Peralkylation of peptides is also a useful technique for sequence determination of oligopeptides by mass spectroscopys. The observation that C-alkylation may be achieved under certain conditions suggests that it may be possible to transform a nutritionally nonessential amino acid to an essential one in the protein.
Northern Regional Research Laboratory, Northern Utilization Research and Development Division, U S. Department of Agriculture, Agricultural Research Service, Peoria, Ill. 616o 4 (U.S.A.)
MENDEL FRIEDMAN L. H. KRULL
L. H. KRULL AND M. FRIEDMAN,J. Polymer Sci., Part A, 5 (1967) 2535. L. H. ]~[RULL AND M. FRIEDMAN, Polymer Preprints, 9 (1968) 129o. E. J. COREY AND M. CHAYKOVSKY,J. Am. Chem. Soc., 87 (1965) 1345. J. U. BENSON AND J. A. PATTERSON, Anal. Chem., 37 (1965) 11o8. J. F. CAVINS AND M. FRIEDMAN, Cereal Chem., 45 (1968) 172. M. FRIEDMAN AND C. W. SIGEL, Biochemistry, 5 (1966) 478. M. GOODMAN AND M. FRIED, J. Am, Chem. Soc., 89 (1967) 1264. D. W. THOMAS, g. C. DAS, S. D. GERO AND E. LEDERER, Biochem. Biophys. Res. Commun., 32 (1968) 519. 9 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907I 2 3 4 5 6 7 8
Received February I9th, 197o Biochim. Biophys. Acta, 2o 7 (197 o) 361-363