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The reaction of S-methylglucosylisothiourea with histidine
ARCHIVES
OF
The
BIOCHEMISTRY
AND
Reaction
BIOPHYSICS
the Department
(1960)
of S-Methylglucosylisothiourea K. MAEKAWA2
From
101-107
91,
of Agricultural
AND
IRVIN
Biochemistry, Received
with E. LIErZER
University June
Histidine’
of Minnesota,
St. Paul,
Minnesota
24, 1960
The treatment of histidine with S-methylglucosylisothiourea at pH 8.4 resulted in the formation of five Pauly-reactive histidine derivatives which could be separated by paper electrophoresis. A resolution of this mixture could also be achieved on Dowex 50 although there was some indication that the sugar residue had been cleaved from at least two of the components during chromatography. Chromatography on cellulose columns permitted the isolation of the two main products of the reaction. From the analytical data obtained on these two compounds, it is suggested that these are derivatives of histidine in which one glucosylamidyl group is attached to the a-amino N and another to carbons 2 or 4 of the imidazole ring.
a-amino group had undergone some modification. Although the ability to couple with diazotized sulfanilic acid (Pauly’s reaction) was only slightly diminished (about lo%), the reactivity toward reagents which are specific for the unsubstituted imidazole ring of histidine4 was almost completely lost. This paper reports the characterization of
INTRODUCTION
The reaction of S-methylglucosylisothiourea3 (1) with various amino acids was first reported by Micheel and Berlenbach in 1952 (2). The following equation shows how an amino group may be replaced by a glucosylguanidyl group as a result of this reaction :
NH-C-S-CH3
+ H2N-R
:F
NH-C-NH-
-+
R
+ CH~-SH
OH
SMG DIAGRAM
Maekawa and Ishimoto (3), upon reinvestigating the interaction of SMG with various amino acids, concluded that the imidazole ring of histidine as well as its
1.
two histidine derivatives isolated from the reaction of SMG with histidine, both of which appear to be substituted on the imidazole ring as well as the cu-aminoposition.
1 Paper No. 4327, Scientific Journal Series, Minnesota Agricultural Experiment Station. This work was supported by grants from the National Institutes of Health (Grant No. RG 4614) and the National Science Foundation (Grant No. G 5830). * Visiting investigator on leave from the Faculty of Agriculture, Ehime University, Matsuyama, Japan. 3 For convenience this reagent will be referred to as SMG.
METHODS REACTION
OF
SMG
WITH
HISTIDINE
To 2.1 g. of L-histidine.HCl.HzO (10 mmole), dissolved in 15 ml. water, was added 5.7 g. (15 mmole) SMG which had been prepared by a 4 Two such specific tests are Knoop’s reaction with bromine (4, 5) and Cowgill’s p-nitrobenzoyl chloride reagent (6). 101
MAEKAWA
102
method previously described (7). The mixture was brought to pH 8.4 with dilute NaOH and allowed to stand at 5’ for 1 week. By the end of this time, the reaction mixture had undergone a 5Oyc decrease in amino groups (8), a 10% decline in reactivity toward Pauly’s reagent (9), and an 89% diminution in its response to Knoop’s test (5).
CHROMATOGRAPHIC REACTION
EXAMIK~TION MIXTURE
OF
Separation of the products of the reaction mixture was first attempted on Dowex 50-4X (Na+ form), 2013409 mesh, equilibrated with 0.1 1M citrate buffer, pH 3.4, as described by Moore and Stein (10) and Cowgill (11). A portion of the reaction mixture equivalent to 50 mg. histidine was adjusted to pH 3.4 and applied to a column measuring 2 X 37 cm. The flow rate was adjusted to 15 ml./hr., and 5-ml. fractions were collected automatically. Unreacted SMG passed through the column without retardation, whereupon the buffer was changed to 0.1 M citrate, pH 5.6, which eluted all Pa&reactive substances except histidine. The latter was removed from the column with 0.1 M phosphate buffer, pH 6.8. The reaction mixture was also chromatographed on a column of cellulose (Whatman No. 1, chromatographic grade) using l-butanol-pyridinewater (8:8:3, v/v) for elution. A portion of the reaction mixture equivalent to 700 mg. histidine was applied to the column (5 X 60 cm.), and the eluate was collected as described above. After 3 1. of the original eluant had passed through the column, it was replaced by methanol-water (l:l, v/v). Rechromatography of fractions which were unresolved by this procedure was conducted on a column of cellulose employing pyridine-water (3:1, v/v) for elution (12). Eluates were examined for the presence of the imidaaole ring by the Pauly reaction (9) using l-ml. samples which had been diluted to 10 ml. with water. The color which finally developed was read at 530 rnN in a Coleman junior spectrophotometer. Free amino groups were determined by the ninhydrin procedure of Troll and Cannan (8) with final readings at 570 nm. The glucose content (free or combined) of the various fractions was measured with orcinol, and the color of the final reaction product was read at 530 mp (13). The various fractions separated by chromatography were prepared for further examination as follows: The contents of the tubes corresponding to each peak were pooled and evaporated to dryness in vacua at room temperature. The residue was dissolved in methanol and decolorized with charcoal. Precipitation was induced by adding ethyl ether, and the precipitated material was
AND LIENER washed several times with ethyl ether petroleum ether and finally dried in
PAPER
followed vucuo.
by
ELECTROPHORESIS
The material obtained from each peak was examined for homogeneity by paper electrophoresis on Whatman No. 4 paper ( 6.5 X 56.5 cm.) employing a commercial apparatus” which had been modified to provide a Durrum-type bridge (14). The buffer vessels contained 1 N acetic acid, pH 2.4, and a voltage of 11 V./cm. was applied for a period of 2 hr. Spots were visualized by applying the diazonium spray of Ames and Mitchell (15) which detects imidazole derivatives which are unsubstituted on the ring nitrogens. Control strips to which had been applied aliquots of the original reaction mixture or histidine were run concurrently.
TITRATION
CURVES
Solutions containing 5-10 mg. of material dissolved in 5 ml. of 0.1 M KC1 were titrated with 0.1 N NaOH or HCl at 25’ using an automatic titrator (Radiometer TTTr) equipped with a recording device (Ole Dich).
INFRARED
ABSORPTION
SPECTRA
These analyses were conducted by the Spectroscopy Laboratory of the School of Chemistry. A Perkin-Elmer model 21 spectrophotometer with a sodium chloride prism was employed. RESULTS
The lower part of Fig. 1 shows the pattern which was obtained when the total reaction mixture of histidine treated with SMG was chromatographed on Dowex 50. Table I summarizes the reaction of each Pauly-reactive peak with orcinol and ninhydrin. A total of 6 Pauly-reactive peaks was obtained of which 3 (A, B, and C) were combined with glucose (orcinol-positive) and 4 (A, D’, E’, and F) contained free amino groups (ninhydrin-positive). The orcinol-positive but Pauly-negative peaks which are eluted from the column with 0.1 M citrate buffer, pH 3.4, most likely represent unreacted SMG and possibly other glucose-containing degradation products.6 6 E-C Apparatus Co., 23 Haven Avenue, New York 32, N. Y. 6 Notably absent from electrophoretic and chromatographic patterns of the total reaction mixture were components which were ninhydrinpositive but Pauly-negative.
REACTION
k
I -*..--.-
OF
SMG
WITH
HISTIDINE
lI
103
-mm
FIG. 1. Chromatography of reaction mixture of SMG and histidine on Dowex 50. Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-) or orcinol (- - -). Roman numerals refer to buffer systems used for elution: I, 0.1 M citrate buffer, pH 3.4; II, 0.1 J4 citrate buffer, pH 5.6; and III, 0.1 M phosphate buffer, pH 6.8. Upper portion of figure shows electrophoretic patterns on paper strips corresponding to each Pauly-reactive peak. Relative distance (Rd) refers to distance traveled toward the cathode as compared with histidine taken as 1.0 (actual distance moved by histidine was 12.4 cm.). Patterns obtained with the total reaction mixture (Rx) and histidine (His) are shown for comparison.
The paper electrophoretic patterns obtained with each of the Pauly-reactive peaks are shown in the upper part of Fig. 1, and the relative distance (Rdj which the spots so obtained migrated are tabulated in Table I. The patterns of the total reaction mixture as well as that of histidine are presented for comparison. From the Rd values obtained for peaks A, B, C, and F, it was possible to establish their identity to corresponding spots of the total reaction mixture. Peak F was thus shown to be excess, unreacted histidine. In the case of peaks D’ and E’, however, the Rd’s were somewhat greater than their apparent counterparts in the original reaction mixture. It may be further noted from the data of Table I that these tlvo peaks were orcinol-negative, raising the possibility that these components may actually be histidine derivatives from which the glucose residue had been cleaved as a result of passagethrough the ion-exchange column.
TABLE
I
REACTIVITY OF PAULY-POSITIVE PEAKS TOWARD ORCINOL AND NINHYDRIN AND THEIR MIGRATION ON PAPER ELECTROPHORESIS Total CornPO-
A B C Db Eb F
Dowex 50
reaction mixture
0.37 0.50 0.66 0.79 0.84 1.00
0.37 0.50 0.66 0.87 0.95 1.00
-
CdlUlOS~
+ + + +
0.37 0.50 0.66 0.79 0.84 1.00
+ -c + +
5 Relative distance of migration as compared to histidine (= 1.00). b In the case of peaks from Dowex 50, these two peaks were not identical to D and E and have therefore been referred to as D’ and E’, respectively, in Fig. 1 and the text. c Based on reaction of rechromatographed material (see Fig. 3).
104
MhEKAWA
VOLUME
AND
LIENER
OF EFFLUENT
(ml.)
FIG. 2. Chromatography of reaction mixture of SMG and histidine on cellulose column. Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-)or orcinol (- - -). Butanolpyridine-water (8:8:3, v/v) replaced by methanol-water (l:l, v/v) as eluant at point indicated by the vertical arrow. Upper portion of figure shows electrophoretic pattern of each peak (see Fig. 1 for details). Double-headed arrow indicates effluent which was rechromatographed (see Fig. 3).
In view of the uncertainty regarding the identity of peaks D’ and E’, the total reaction mixture was chromatographed on a column of cellulose using l-butanol-pyridine-water for elution. The resulting chromatogram and the electrophoretic patterns which were obtained to establish the identity of each peak are shown in Fig. 2. The Rd’s and color reactions of each peak with orcinol and ninhydrin are included in Table I. In all cases the Rd’s of the components of each peak coincided with one or more spots in the original reaction mixture. Each peak represented a single component with the notable exception of C which was clearly contaminated with histidine (F). It will be noted from Table I that the color reactions of peaks D and E were quite different from peaks D’ and E’ obtained by chromatography on Dowex 50. Both D and E were now orcinol-positive, and E had become ninhydrin-negative. These results suggest that E’ represents a hi&dine derivative from which a glucose residue had been split from the a-amino position, and, in t#hecase of D’, from someother position.
To effect a separation of histidine from component C, the effluent corresponding to the double-headed arrow shown in Fig. 2 was evaporated to dryness in uacuo. The resulting 50 mg. of material was redissolved in water and rechromatographed on cellulose with pyridine-water as the eluant. As shown in Fig. 3, histidine was completely separated from component C so that the latter was obtained in a pure state suitable for further characterization. Of the various reaction products which were demonstrated to be present, only the two main products of the reaction, C and E, were isolated in quantities sufficient to permit more detailed characterization. Analytical data which were obtained on these two compounds are summarized in Table II. DISCUSSION
The data of Tables I and II permit certain deductions to be made concerning the possible structures of compounds C and E. (a) The elementary analysis of both compounds is consistent wit)h the formula C&o. H3JS7012, which is what would be expected
REACTION
OF
SMG
WITH
105
HISTIDINE
--L F
1
ii; 0
100
200
300
500
VOLUME
600
700
OF EFFLUENT
800
900
(ml.)
FIG. 3. Rechromatography of component C on cellulose column. Effluent corresponding to doubleheaded arrow of Fig. 2 was applied to column and eluted with pyridine-water (3:1, v/v). Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-) or orcinol (- - -). Upper portion of figure shows electrophoretic pattern of each peak (see Fig. 1 for details).
PROPERTIES
OF COMPOUNDS
TABLE II C AND E, THE
MAIN
Compound C
analysis,o/o” C H N 0 Melting point, “C. Optical rotation in water, [cx]“~ Dissociation constants”
REACTION
PRODUCTS
Compound E
Elementary
pK1' p&' p&'
Ultraviolet Infrared
absorption, hm,, (mF) EM absorptiond (cm.?)
40.72 6.02 18.11 35.15 192 -41.7
5.80 17.11 37.81 203b -26.0
3.5 6.9 9.4 235 1.10 x 104 1635, 1085, 1045 1663
3.5 7.2 10.5 232 1.15 x 10’ 1635, 1085, 1045 1575
39.08
a Calculated for C20H33N101z : C, 42.62; H, 5.86; N, 17.40; 0, 34.12. These analyses were conducted by the Microanalytical Laboratory, School of Chemistry. *With browning and decomposition. c Based on curves shown in Fig. 4. d Taken from Fig. 5.
if 1 mole of histidine were combined with 2 moles of the glucosylamidyl residue. (b) The failure of compounds C and E to react with ninhydrin (Table I) points to
the absence of any free a-amino groups. Hence, at least one of the glucosylamidyl groups must be attached to the a-amino nitrogen.
106
MAEKAWA
AND
C&N302
2.
DIAGRAM
2
4
6
0
IO
12
PH
FIG. 4. Titration curves of compounds and E (- - -) at 25”.
C (-)
(c) The observation that compounds C and E are capable of coupling with diazotized sulfanilic acid (Pauly’s reagent) indicates the absence of substitution on the nitrogens of the imidazole ring (15, 16). Substitution is therefore restricted either to C-2 or C-4 of the imidazole ring. Substitution at both positions would give an imidazole derivative which would also be Pauly-negative (4). Substitution at C-2 or C-4 of the imidazole ring is further sup-
0 e-??ubo
,2c+Jc
z
Ike
LIENER
ported by the observation that compounds C and E failed to react with KIIOOP’S bromine solution (4, 5) or p-nitrobenzoyl chloride (6), reagents which do not react with imidazole derivatives substituted in the 2 or 4 position. (d) The titration curves of Fig. 4 show three distinct pK’s: pK1’, in the region of pH 3.5 (carboxyl group), pKz’ in the region of neutrality (imidazole groups), and pK3’ in the neighborhood of pH 10 (guanidine or amidine group). Substitution of the imidazole ring is known to influence the pK’ of such derivatives. Thus, 4(or 5)-methylimidazole has a pK’ of 7.6 whereas 2-methylimidazole has a pK’ of 8.1 (17). By analogy, therefore, it is suggested that compound C, which has a pK2’ of 6.9, is substituted in the 4 position of the imidazole ring, whereas compound E, with a pKz’ of 7.2, is substituted in the 2 position. (e) The infrared absorption spectra of compounds C and E (Fig. 5) were quite similar and showed a strong absorption band in the region of 1635 cm.-’ which is typical of the imidazole ring (18-21). Also characteristic of these spectra are absorption bands at 1086 cm.-l and 1045 cm.-’ indicative of a glycosyl residue in the P-configuration (22-25). Although the absorption of a guanidine residue (substitution at the a-amino position of histidine) could be expected at 1615 cm.-’ (26, 27), this region of the spectrum is dominated by the imidazole ring. The main differences between compounds C and E lie in the regions of 1663 cm.-’ and 1575 cm.-‘, and are probably a reflection of the isomeric relationship that exists between these two compounds.
l&l FRECUENCY
-1400-coo (CM“)
loto
Sk
FIG. 5. Infrared absorbance spectra of compounds C (-) and E (- - -). Absorption bands at 1385, 1470, and 2950 cm.? are due to the mineral oil (Nujol) phase. Absorption at 1025 cm.-’ is an instrumental anomaly.
REACTION
OF
SMG
From the evidence just considered, the following structures are proposed for compounds C and E: CH,OH \NH-C-
Ga=
_
= D GLUCOSYLAMIDYL
Group
NH &
N NH ‘C’
60 Compound
10.
H,C-$-Cl++,-CODH
$-CHz-C,H-COOH
Pi iH “CH’
Compound
C
DIAGRAM
“I” GO E
11. 12.
3.
Concerning the identity of the minor products of the reaction, it can only be surmised that these might include derivatives in which substitution may not have occurred in the a-amino position (such as compounds A and D which are ninhydrinpositive). This would account for the fact that only a 50% decrease in amino groups is observed at the end of the reaction. Substitution on one of the ring nitrogens must also be considered as a possibility inasmuch as there is always a small but measurable decline (10 %) in the color reaction of t’he mixture toward Pauly’s reagent. REFERENCES 1. HELFERICH, B., AND KOSCHE, W., Ber. 69, 71 (1926). 2. MICHEEL, F., AND BERLENBACH, W., Ber. 86, 189 (1952). 3. MAEKAWA, K., AND ISHIMOTO, K., Bull. Chem. Sot. Japan. In press. 4. HOFFMAN, K., in “The Chemistry of Heterocyclic Compounds” A. Weissberger (ed.), part I, p. 184. Interscience Publ. New York, 1953.
13. 14. 15. 16. 17
18. 19. 20. 21.
22. 23.
24. 25. 26. 27.
A., J. Biol. Chem.
216, 391 (1955). R. W., Anal. Chem. 27, 1521 (1955). K., AND ISHIMOTO, K., Bull. Chem. Sot. Japan 77, 999 (1956). TROLL, W., AND CANNAN, P. K., J. Biol. Chem. 269, 803 (1953). MACPHERSON, H. T., Biochem. J. 40, 470 (1945). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 192,663(1951). COWGILL, R. W., J. Am. Chem. Sot. 79, 2249 (1957). LOWY, P. H., AND BORSOOK, H., J. Am. Chem. Sot. 78, 3175 (1956). BRUCKNER, J., Rio&em. J. 60, 200 (1955). DURRUM, E. L., J. Am. Chem. Sot. 73, 4875 (1951). AMES, B. N., AND MITCHELL, H. K., J. Am. Chem. Sot. 74, 252 (1952). COWGILL, R. W., Anal. Chem. 27, 1519 (1955). COHEN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids, and Peptides,” p. 131. Reinhold Publ. Corp., New York, 1943. LARSON, L., Acta Chem. Stand. 4, 29 (1959). SUTHERLAND, G. B. B. M., Advances in Protein Chem. 7, 291 (1952). GORE, R. C., Anal. Chem. 26, 11 (1954). KOEGEL, R. J., GREENSTEIN, J. P., WINITZ, M., BIRNBAUM, S. M., AND MCCALLUM, R. A., J. Am. Chem. Sot. 77, 5708 (1955). WHISTLER, R. L., AND HOUSE, L. R., Anal. Chem. 26, 1463 (1953). Infrared Spectra of BELLAMY, L. J., “The Complex Molecules,” p. 175. Methuen and Co., London, 1954. BARKER, S. A., BOURNE, E. J., STACEY, M., AND WIFFEN, D. H., J. Chem. Sot. 1964,171. TABAHASHI, M., J. Pharm. Sot. Japan 76, 237 (1955). EPP, A., Anal. Chem. 29, 1283 (1957). GOTO, T., NAKANISHI, K., AND OHASKI, M., Bull. Chem. Sot. Japan 30, 723 (1957).
6. COWGILL, 7. MAEKAWA,
9.
107
HISTIDINE
5. HUNTER,
8.
:r
<~-O OH ““4 Go-7 -
WITH
OF
The
BIOCHEMISTRY
AND
Reaction
BIOPHYSICS
the Department
(1960)
of S-Methylglucosylisothiourea K. MAEKAWA2
From
101-107
91,
of Agricultural
AND
IRVIN
Biochemistry, Received
with E. LIErZER
University June
Histidine’
of Minnesota,
St. Paul,
Minnesota
24, 1960
The treatment of histidine with S-methylglucosylisothiourea at pH 8.4 resulted in the formation of five Pauly-reactive histidine derivatives which could be separated by paper electrophoresis. A resolution of this mixture could also be achieved on Dowex 50 although there was some indication that the sugar residue had been cleaved from at least two of the components during chromatography. Chromatography on cellulose columns permitted the isolation of the two main products of the reaction. From the analytical data obtained on these two compounds, it is suggested that these are derivatives of histidine in which one glucosylamidyl group is attached to the a-amino N and another to carbons 2 or 4 of the imidazole ring.
a-amino group had undergone some modification. Although the ability to couple with diazotized sulfanilic acid (Pauly’s reaction) was only slightly diminished (about lo%), the reactivity toward reagents which are specific for the unsubstituted imidazole ring of histidine4 was almost completely lost. This paper reports the characterization of
INTRODUCTION
The reaction of S-methylglucosylisothiourea3 (1) with various amino acids was first reported by Micheel and Berlenbach in 1952 (2). The following equation shows how an amino group may be replaced by a glucosylguanidyl group as a result of this reaction :
NH-C-S-CH3
+ H2N-R
:F
NH-C-NH-
-+
R
+ CH~-SH
OH
SMG DIAGRAM
Maekawa and Ishimoto (3), upon reinvestigating the interaction of SMG with various amino acids, concluded that the imidazole ring of histidine as well as its
1.
two histidine derivatives isolated from the reaction of SMG with histidine, both of which appear to be substituted on the imidazole ring as well as the cu-aminoposition.
1 Paper No. 4327, Scientific Journal Series, Minnesota Agricultural Experiment Station. This work was supported by grants from the National Institutes of Health (Grant No. RG 4614) and the National Science Foundation (Grant No. G 5830). * Visiting investigator on leave from the Faculty of Agriculture, Ehime University, Matsuyama, Japan. 3 For convenience this reagent will be referred to as SMG.
METHODS REACTION
OF
SMG
WITH
HISTIDINE
To 2.1 g. of L-histidine.HCl.HzO (10 mmole), dissolved in 15 ml. water, was added 5.7 g. (15 mmole) SMG which had been prepared by a 4 Two such specific tests are Knoop’s reaction with bromine (4, 5) and Cowgill’s p-nitrobenzoyl chloride reagent (6). 101
MAEKAWA
102
method previously described (7). The mixture was brought to pH 8.4 with dilute NaOH and allowed to stand at 5’ for 1 week. By the end of this time, the reaction mixture had undergone a 5Oyc decrease in amino groups (8), a 10% decline in reactivity toward Pauly’s reagent (9), and an 89% diminution in its response to Knoop’s test (5).
CHROMATOGRAPHIC REACTION
EXAMIK~TION MIXTURE
OF
Separation of the products of the reaction mixture was first attempted on Dowex 50-4X (Na+ form), 2013409 mesh, equilibrated with 0.1 1M citrate buffer, pH 3.4, as described by Moore and Stein (10) and Cowgill (11). A portion of the reaction mixture equivalent to 50 mg. histidine was adjusted to pH 3.4 and applied to a column measuring 2 X 37 cm. The flow rate was adjusted to 15 ml./hr., and 5-ml. fractions were collected automatically. Unreacted SMG passed through the column without retardation, whereupon the buffer was changed to 0.1 M citrate, pH 5.6, which eluted all Pa&reactive substances except histidine. The latter was removed from the column with 0.1 M phosphate buffer, pH 6.8. The reaction mixture was also chromatographed on a column of cellulose (Whatman No. 1, chromatographic grade) using l-butanol-pyridinewater (8:8:3, v/v) for elution. A portion of the reaction mixture equivalent to 700 mg. histidine was applied to the column (5 X 60 cm.), and the eluate was collected as described above. After 3 1. of the original eluant had passed through the column, it was replaced by methanol-water (l:l, v/v). Rechromatography of fractions which were unresolved by this procedure was conducted on a column of cellulose employing pyridine-water (3:1, v/v) for elution (12). Eluates were examined for the presence of the imidaaole ring by the Pauly reaction (9) using l-ml. samples which had been diluted to 10 ml. with water. The color which finally developed was read at 530 rnN in a Coleman junior spectrophotometer. Free amino groups were determined by the ninhydrin procedure of Troll and Cannan (8) with final readings at 570 nm. The glucose content (free or combined) of the various fractions was measured with orcinol, and the color of the final reaction product was read at 530 mp (13). The various fractions separated by chromatography were prepared for further examination as follows: The contents of the tubes corresponding to each peak were pooled and evaporated to dryness in vacua at room temperature. The residue was dissolved in methanol and decolorized with charcoal. Precipitation was induced by adding ethyl ether, and the precipitated material was
AND LIENER washed several times with ethyl ether petroleum ether and finally dried in
PAPER
followed vucuo.
by
ELECTROPHORESIS
The material obtained from each peak was examined for homogeneity by paper electrophoresis on Whatman No. 4 paper ( 6.5 X 56.5 cm.) employing a commercial apparatus” which had been modified to provide a Durrum-type bridge (14). The buffer vessels contained 1 N acetic acid, pH 2.4, and a voltage of 11 V./cm. was applied for a period of 2 hr. Spots were visualized by applying the diazonium spray of Ames and Mitchell (15) which detects imidazole derivatives which are unsubstituted on the ring nitrogens. Control strips to which had been applied aliquots of the original reaction mixture or histidine were run concurrently.
TITRATION
CURVES
Solutions containing 5-10 mg. of material dissolved in 5 ml. of 0.1 M KC1 were titrated with 0.1 N NaOH or HCl at 25’ using an automatic titrator (Radiometer TTTr) equipped with a recording device (Ole Dich).
INFRARED
ABSORPTION
SPECTRA
These analyses were conducted by the Spectroscopy Laboratory of the School of Chemistry. A Perkin-Elmer model 21 spectrophotometer with a sodium chloride prism was employed. RESULTS
The lower part of Fig. 1 shows the pattern which was obtained when the total reaction mixture of histidine treated with SMG was chromatographed on Dowex 50. Table I summarizes the reaction of each Pauly-reactive peak with orcinol and ninhydrin. A total of 6 Pauly-reactive peaks was obtained of which 3 (A, B, and C) were combined with glucose (orcinol-positive) and 4 (A, D’, E’, and F) contained free amino groups (ninhydrin-positive). The orcinol-positive but Pauly-negative peaks which are eluted from the column with 0.1 M citrate buffer, pH 3.4, most likely represent unreacted SMG and possibly other glucose-containing degradation products.6 6 E-C Apparatus Co., 23 Haven Avenue, New York 32, N. Y. 6 Notably absent from electrophoretic and chromatographic patterns of the total reaction mixture were components which were ninhydrinpositive but Pauly-negative.
REACTION
k
I -*..--.-
OF
SMG
WITH
HISTIDINE
lI
103
-mm
FIG. 1. Chromatography of reaction mixture of SMG and histidine on Dowex 50. Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-) or orcinol (- - -). Roman numerals refer to buffer systems used for elution: I, 0.1 M citrate buffer, pH 3.4; II, 0.1 J4 citrate buffer, pH 5.6; and III, 0.1 M phosphate buffer, pH 6.8. Upper portion of figure shows electrophoretic patterns on paper strips corresponding to each Pauly-reactive peak. Relative distance (Rd) refers to distance traveled toward the cathode as compared with histidine taken as 1.0 (actual distance moved by histidine was 12.4 cm.). Patterns obtained with the total reaction mixture (Rx) and histidine (His) are shown for comparison.
The paper electrophoretic patterns obtained with each of the Pauly-reactive peaks are shown in the upper part of Fig. 1, and the relative distance (Rdj which the spots so obtained migrated are tabulated in Table I. The patterns of the total reaction mixture as well as that of histidine are presented for comparison. From the Rd values obtained for peaks A, B, C, and F, it was possible to establish their identity to corresponding spots of the total reaction mixture. Peak F was thus shown to be excess, unreacted histidine. In the case of peaks D’ and E’, however, the Rd’s were somewhat greater than their apparent counterparts in the original reaction mixture. It may be further noted from the data of Table I that these tlvo peaks were orcinol-negative, raising the possibility that these components may actually be histidine derivatives from which the glucose residue had been cleaved as a result of passagethrough the ion-exchange column.
TABLE
I
REACTIVITY OF PAULY-POSITIVE PEAKS TOWARD ORCINOL AND NINHYDRIN AND THEIR MIGRATION ON PAPER ELECTROPHORESIS Total CornPO-
A B C Db Eb F
Dowex 50
reaction mixture
0.37 0.50 0.66 0.79 0.84 1.00
0.37 0.50 0.66 0.87 0.95 1.00
-
CdlUlOS~
+ + + +
0.37 0.50 0.66 0.79 0.84 1.00
+ -c + +
5 Relative distance of migration as compared to histidine (= 1.00). b In the case of peaks from Dowex 50, these two peaks were not identical to D and E and have therefore been referred to as D’ and E’, respectively, in Fig. 1 and the text. c Based on reaction of rechromatographed material (see Fig. 3).
104
MhEKAWA
VOLUME
AND
LIENER
OF EFFLUENT
(ml.)
FIG. 2. Chromatography of reaction mixture of SMG and histidine on cellulose column. Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-)or orcinol (- - -). Butanolpyridine-water (8:8:3, v/v) replaced by methanol-water (l:l, v/v) as eluant at point indicated by the vertical arrow. Upper portion of figure shows electrophoretic pattern of each peak (see Fig. 1 for details). Double-headed arrow indicates effluent which was rechromatographed (see Fig. 3).
In view of the uncertainty regarding the identity of peaks D’ and E’, the total reaction mixture was chromatographed on a column of cellulose using l-butanol-pyridine-water for elution. The resulting chromatogram and the electrophoretic patterns which were obtained to establish the identity of each peak are shown in Fig. 2. The Rd’s and color reactions of each peak with orcinol and ninhydrin are included in Table I. In all cases the Rd’s of the components of each peak coincided with one or more spots in the original reaction mixture. Each peak represented a single component with the notable exception of C which was clearly contaminated with histidine (F). It will be noted from Table I that the color reactions of peaks D and E were quite different from peaks D’ and E’ obtained by chromatography on Dowex 50. Both D and E were now orcinol-positive, and E had become ninhydrin-negative. These results suggest that E’ represents a hi&dine derivative from which a glucose residue had been split from the a-amino position, and, in t#hecase of D’, from someother position.
To effect a separation of histidine from component C, the effluent corresponding to the double-headed arrow shown in Fig. 2 was evaporated to dryness in uacuo. The resulting 50 mg. of material was redissolved in water and rechromatographed on cellulose with pyridine-water as the eluant. As shown in Fig. 3, histidine was completely separated from component C so that the latter was obtained in a pure state suitable for further characterization. Of the various reaction products which were demonstrated to be present, only the two main products of the reaction, C and E, were isolated in quantities sufficient to permit more detailed characterization. Analytical data which were obtained on these two compounds are summarized in Table II. DISCUSSION
The data of Tables I and II permit certain deductions to be made concerning the possible structures of compounds C and E. (a) The elementary analysis of both compounds is consistent wit)h the formula C&o. H3JS7012, which is what would be expected
REACTION
OF
SMG
WITH
105
HISTIDINE
--L F
1
ii; 0
100
200
300
500
VOLUME
600
700
OF EFFLUENT
800
900
(ml.)
FIG. 3. Rechromatography of component C on cellulose column. Effluent corresponding to doubleheaded arrow of Fig. 2 was applied to column and eluted with pyridine-water (3:1, v/v). Absorbance refers to color obtained on l-ml. aliquots of each tube with Pauly’s reagent (-) or orcinol (- - -). Upper portion of figure shows electrophoretic pattern of each peak (see Fig. 1 for details).
PROPERTIES
OF COMPOUNDS
TABLE II C AND E, THE
MAIN
Compound C
analysis,o/o” C H N 0 Melting point, “C. Optical rotation in water, [cx]“~ Dissociation constants”
REACTION
PRODUCTS
Compound E
Elementary
pK1' p&' p&'
Ultraviolet Infrared
absorption, hm,, (mF) EM absorptiond (cm.?)
40.72 6.02 18.11 35.15 192 -41.7
5.80 17.11 37.81 203b -26.0
3.5 6.9 9.4 235 1.10 x 104 1635, 1085, 1045 1663
3.5 7.2 10.5 232 1.15 x 10’ 1635, 1085, 1045 1575
39.08
a Calculated for C20H33N101z : C, 42.62; H, 5.86; N, 17.40; 0, 34.12. These analyses were conducted by the Microanalytical Laboratory, School of Chemistry. *With browning and decomposition. c Based on curves shown in Fig. 4. d Taken from Fig. 5.
if 1 mole of histidine were combined with 2 moles of the glucosylamidyl residue. (b) The failure of compounds C and E to react with ninhydrin (Table I) points to
the absence of any free a-amino groups. Hence, at least one of the glucosylamidyl groups must be attached to the a-amino nitrogen.
106
MAEKAWA
AND
C&N302
2.
DIAGRAM
2
4
6
0
IO
12
PH
FIG. 4. Titration curves of compounds and E (- - -) at 25”.
C (-)
(c) The observation that compounds C and E are capable of coupling with diazotized sulfanilic acid (Pauly’s reagent) indicates the absence of substitution on the nitrogens of the imidazole ring (15, 16). Substitution is therefore restricted either to C-2 or C-4 of the imidazole ring. Substitution at both positions would give an imidazole derivative which would also be Pauly-negative (4). Substitution at C-2 or C-4 of the imidazole ring is further sup-
0 e-??ubo
,2c+Jc
z
Ike
LIENER
ported by the observation that compounds C and E failed to react with KIIOOP’S bromine solution (4, 5) or p-nitrobenzoyl chloride (6), reagents which do not react with imidazole derivatives substituted in the 2 or 4 position. (d) The titration curves of Fig. 4 show three distinct pK’s: pK1’, in the region of pH 3.5 (carboxyl group), pKz’ in the region of neutrality (imidazole groups), and pK3’ in the neighborhood of pH 10 (guanidine or amidine group). Substitution of the imidazole ring is known to influence the pK’ of such derivatives. Thus, 4(or 5)-methylimidazole has a pK’ of 7.6 whereas 2-methylimidazole has a pK’ of 8.1 (17). By analogy, therefore, it is suggested that compound C, which has a pK2’ of 6.9, is substituted in the 4 position of the imidazole ring, whereas compound E, with a pKz’ of 7.2, is substituted in the 2 position. (e) The infrared absorption spectra of compounds C and E (Fig. 5) were quite similar and showed a strong absorption band in the region of 1635 cm.-’ which is typical of the imidazole ring (18-21). Also characteristic of these spectra are absorption bands at 1086 cm.-l and 1045 cm.-’ indicative of a glycosyl residue in the P-configuration (22-25). Although the absorption of a guanidine residue (substitution at the a-amino position of histidine) could be expected at 1615 cm.-’ (26, 27), this region of the spectrum is dominated by the imidazole ring. The main differences between compounds C and E lie in the regions of 1663 cm.-’ and 1575 cm.-‘, and are probably a reflection of the isomeric relationship that exists between these two compounds.
l&l FRECUENCY
-1400-coo (CM“)
loto
Sk
FIG. 5. Infrared absorbance spectra of compounds C (-) and E (- - -). Absorption bands at 1385, 1470, and 2950 cm.? are due to the mineral oil (Nujol) phase. Absorption at 1025 cm.-’ is an instrumental anomaly.
REACTION
OF
SMG
From the evidence just considered, the following structures are proposed for compounds C and E: CH,OH \NH-C-
Ga=
_
= D GLUCOSYLAMIDYL
Group
NH &
N NH ‘C’
60 Compound
10.
H,C-$-Cl++,-CODH
$-CHz-C,H-COOH
Pi iH “CH’
Compound
C
DIAGRAM
“I” GO E
11. 12.
3.
Concerning the identity of the minor products of the reaction, it can only be surmised that these might include derivatives in which substitution may not have occurred in the a-amino position (such as compounds A and D which are ninhydrinpositive). This would account for the fact that only a 50% decrease in amino groups is observed at the end of the reaction. Substitution on one of the ring nitrogens must also be considered as a possibility inasmuch as there is always a small but measurable decline (10 %) in the color reaction of t’he mixture toward Pauly’s reagent. REFERENCES 1. HELFERICH, B., AND KOSCHE, W., Ber. 69, 71 (1926). 2. MICHEEL, F., AND BERLENBACH, W., Ber. 86, 189 (1952). 3. MAEKAWA, K., AND ISHIMOTO, K., Bull. Chem. Sot. Japan. In press. 4. HOFFMAN, K., in “The Chemistry of Heterocyclic Compounds” A. Weissberger (ed.), part I, p. 184. Interscience Publ. New York, 1953.
13. 14. 15. 16. 17
18. 19. 20. 21.
22. 23.
24. 25. 26. 27.
A., J. Biol. Chem.
216, 391 (1955). R. W., Anal. Chem. 27, 1521 (1955). K., AND ISHIMOTO, K., Bull. Chem. Sot. Japan 77, 999 (1956). TROLL, W., AND CANNAN, P. K., J. Biol. Chem. 269, 803 (1953). MACPHERSON, H. T., Biochem. J. 40, 470 (1945). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 192,663(1951). COWGILL, R. W., J. Am. Chem. Sot. 79, 2249 (1957). LOWY, P. H., AND BORSOOK, H., J. Am. Chem. Sot. 78, 3175 (1956). BRUCKNER, J., Rio&em. J. 60, 200 (1955). DURRUM, E. L., J. Am. Chem. Sot. 73, 4875 (1951). AMES, B. N., AND MITCHELL, H. K., J. Am. Chem. Sot. 74, 252 (1952). COWGILL, R. W., Anal. Chem. 27, 1519 (1955). COHEN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids, and Peptides,” p. 131. Reinhold Publ. Corp., New York, 1943. LARSON, L., Acta Chem. Stand. 4, 29 (1959). SUTHERLAND, G. B. B. M., Advances in Protein Chem. 7, 291 (1952). GORE, R. C., Anal. Chem. 26, 11 (1954). KOEGEL, R. J., GREENSTEIN, J. P., WINITZ, M., BIRNBAUM, S. M., AND MCCALLUM, R. A., J. Am. Chem. Sot. 77, 5708 (1955). WHISTLER, R. L., AND HOUSE, L. R., Anal. Chem. 26, 1463 (1953). Infrared Spectra of BELLAMY, L. J., “The Complex Molecules,” p. 175. Methuen and Co., London, 1954. BARKER, S. A., BOURNE, E. J., STACEY, M., AND WIFFEN, D. H., J. Chem. Sot. 1964,171. TABAHASHI, M., J. Pharm. Sot. Japan 76, 237 (1955). EPP, A., Anal. Chem. 29, 1283 (1957). GOTO, T., NAKANISHI, K., AND OHASKI, M., Bull. Chem. Sot. Japan 30, 723 (1957).
6. COWGILL, 7. MAEKAWA,
9.
107
HISTIDINE
5. HUNTER,
8.
:r
<~-O OH ““4 Go-7 -
WITH