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Mechanism and specificity of the phenanthrenequinone test for monosubstituted guanidines
ANALYTICAL
BIOCHEMISTRY
76, 134-141
(1976)
Mechanism and Specificity of the Phenanthrenequinone Test for Monosubstituted Guanidinesl HARVEY
A. ITANO.
KAZUHIRO HIROTA,* ICHIRO AND SHIGEKI YAMADA”
KAWASAKI.”
Department of Puthology. University of Colijbrnic~. Snrl Diego, Ltr Jolla. California 92093 Received
April
12. 1976: accepted
June
18. 1976
The reaction of phenanthrenequinone with arginine yielded 2-amino-IHphenanthro[9.10-djimidazole (API) and a’-pyrroline-5-carboxylic acid. The reaction of phenanthrenequinone with benzylguanidine yielded API and benzaldehyde. The same product . 2-benzylamino1 H-phenanthro[Y, IO+/]imidazole was obtained when either a reaction mixture of phenanthrenequinone and benzylguanidine or a reaction mixture of API and benzaldehyde was reduced with sodium borohydride. Thus the reaction mechanism involves the formation of a Schiff base intermediate. l.l-Disubstituted guanidines. t-butylguanidine. and phenylguanidine produce neither a Schiff base intermediate nor API. On the other hand. guanidine and canavanine. which also do not produce Schiff base intermediates. result in API through mechanisms that require the presence of a reducing compound in the reaction medium.
The formation of a fluorescent compound in the reaction of a guanidino group with phenanthrenequinone in alkaline medium is a sensitive test for the detection of arginine. arginine-containing peptides. and other monosubstituted guanidines ( I-4). Phenanthrenequinone reacts with 1, I-disubstituted guanidines to form stable condensation products; however, these products are not fluorescent. The fluorescent product of the former reaction is 2-amino-IH-phenanthro[9, IO-(llimidazole (API) (5). and the nonfluorescent products of the latter are spirofluoreneimidazolinones (6). The mechanism of the reaction of phenanthrenequinone with certain monosubstituted guanidines has been elucidated in the present study. This mechanism accounts for the absence of API as a product in ’ A part of this investigation was carried out during the tenures of Shigeki Yamada and Ichiro Kawasaki as Visiting Associates of the National Institute of Arthritis and Metabolic Disease and of Harvey A. Itano as Visiting Professor at the University of California. San Francisco. 2 Present address: Institute for Protein Research. Osaka University. Osaka. Japan. :3 Present address: Brewing Science Research Institute. Suita, Japan. ’ Present address: Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd.. Osaka. Japan. 134 Copyl-ighl G 15176 by Academic Press, Inc .4ll rights of reproduction in any fol-m I-esewed.
MECHANISM
OF PHENANTHRENEQUINONE
TEST
the reactions of phenanthrenequinone with r-butylguanidine phenylguanidine as well as with l,l-disubstituted guanidines.
13s and with
METHODS Nuclear magnetic resonance spectra of samples in dimethylsulfoxide-d,i (Me,SO-ci,) solution containing internal tetramethylsilane (TMS) were recorded with a Varian HR-220 spectrometer. Infrared spectra of crystalline samples were recorded as Nujol mulls with a Beckman IR-33 spectrometer. Mass spectra were obtained in a LKB Type 9000 spectrometer at an ionizing energy of 70 eV. Melting points were determined with a Kofler hot-stage microscope and are uncorrected. Elemental analyses were performed by Galbraith Laboratories, Inc.. of Knoxville, Tenn. A’-Pyrroline-5-carboxylic acid hydrochloride was prepared according to Vogel and Davis (7). API hydrochloride was prepared by the reaction of arginine with phenanthrenequinone (I). and benzylguanidine sulfate was prepared by the reaction of benzylamine with 0-methylisourea (8). Other compounds and solvents used in this work were of the best commercially available grades. RESULTS
A reaction mixture of 0.019 M phenanthrenequinone and 0.01 M arginine in 0.2 M NaOH (EtOH:H,O. 4: I) was analyzed periodically by high voltage filter paper electrophoresis (9). API was detected as a diffuse. fluorescent zone near the origin. After 3 min the mixture contained, in addition to arginine and API. a ninhydrin-positive fluorescent product that moved about 0.6 as rapidly as tryptophan. which is the slowest moving of the amino acids in the standard mixture, and a nonfluorescent product that stained pink with ninhydrin and migrated between methionine and threonine. After IS min only a trace of arginine remained; after I .5 hr arginine was not detected. but API. the ninhydrin-positive fluorescent product. and the pink-staining product remained. Only API and the pink-staining product remained 3 hr after the reactants were mised. The published procedure for the preparation of API from phenanthrenequinone and arginine (l! was followed. After API was removed by filtration, the filtrate was passed through a column of Dowex 50 resin (H+ form). and the column was eluted with 1.O hl NH,OH to separate the pink-staining product from unreacted arginine. The eluate was concentrated. and ethanol was added to the concentrate to obtain a precipitate. which was collected and dried to a powder. The powder was refluxed with 6 N HCI and evaporated to dryness. The ir spectrum of this sample
136
ITANO
ET AL.
was identical with that of authentic 4’-pyrroline-S-carboxylic acid hydrochloride. API was filtered from the reaction mixture of another preparation, and the filtrate was hydrogenated directly with the aid of PtO, as catalyst for 3 hr at 15°C. The catalyst was removed by filtration, and the filtrate was examined by thin-layer chromatography (phenol:H,O. 3: 1). Ninhydrin revealed two spots that were identified as proline (R, 0.50) and arginine (Rf0.20). Quantitative analysis in an amino acid analyzer revealed that the yield of proline was 73% and that the residue of unreacted arginine was 10%. The yield of proline was increased to 90% when the time of reduction was increased to 5-7 hr. Attempts to isolate the ninhydrin-positive fluorescent product were unsuccessful. The nature of the intermediate product in the reaction of phenanthrenequinone and a monosubstituted guanidine was therefore investigated in the reaction between phenanthrenequinone and benzylguanidine. The Reaction
of Phencrnrhrenec~trinotle
uqith Ben~ylgtm~idine
Phenanthrenequinone (312 mg. 1.5 mmol) and benzylguanidine sulfate (297 mg, 0.75 mmol or I .5 mequiv) were dissolved with heating in ethanol (80 ml) and in water (7.5 ml). respectively. After the solutions were cooled to room temperature. the solution of benzylguanidine was made alkaline with 2 N NaOH (12.5 ml) and then added to the solution of phenanthrenequinone. After 4 hr of stirring. the reaction mixture was acidified with 6 N HCl and was allowed to stand overnight at 5°C. The precipitate that formed was collected on a filter. washed with 80% ethanol (20 ml). and recrystallized from ethanol. The product was identifified as API .HCl.H,O by its ir spectrum (5). 2.4-Dinitrophenylhydrazine hydrochloride (200 mg) in hot ethanol (80 ml) and 6 N HCl (IO ml) were added to the filtrate from the reaction mixture. Yellow crystals that formed overnight at 5°C were recrystallized from ethanol. mp 240- 24 1“C. The melting point and ir spectrum of this product were the same as those of the authentic hydrazone prepared from benzaldehyde and 2.4-dinitrophenylhydrazine. Preparation
of 2-Ben~~latt~it~o-IH-p~~et~at~thro[9,IO-d]itnida~ole
(a ) Reduction ben~ylgucmidine.
of fhe reaction
mixture
of phenanthreneyuinot~e
(BAPI) und
Phenanthrenequinone (624 mg. 3.0 mmol) was dissolved by heating (55°C) in a solution of 2.8 g of KOH in 200 ml of methanol. Finely powdered benzylguanidine sulfate (594 mg. I.5 mmol or 3.0 mequiv) was added. The mixture was stirred for 4 hr, after which sodium borohydride (3.0 g) was added in small proportions over a period of 2 hr. The mixture was allowed to stand overnight at room temperature and then was acidified with 6 N HCl and dried under reduced pressure.
MECHANISMOFPHENANTHRENEQUINONETEST
137
Water (100 ml) was added to the residue to dissolve inorganic salts. and undissolved material was collected on a filter. suspended in 0.5 N NaOH. and extracted with ethyl acetate. The extract was washed with water. dried over anhydrous Na,SO,. and evaporated to dryness. The residue was loaded on a column packed with silica gel. and the column was eluted with dichloroethane. A yellow fraction which chromatographed with a R, of 0.79 by thin-layer chromatography in a solvent system of II-heptane: isopropanol:concentrated NH, (6:4:0.5) was obtained. Dichloroethane was removed. and the residue was mixed with 6 N HCI (20 ml) to produce a salt which was collected on a filter, washed with water, and dried over P,O, in a vacuum desiccator. The dried material was suspended in ether (30 ml) to dissolve the yellow impurity. The suspended solid was collected on a filter and dried to yield 630 mg (56%) of a white product. Recrystallization from 6 N HCI (50 ml) in ethanol (350 ml) yielded 340 mg (309,). mp 220-224°C (dec); ir (Nujol) 3360. 1700. 1660. 1640. 1600. 1570. 760. and 730 cm-‘; NMR aT,, (Me,SO-d,) 5.07 (d,2). 7.30-7.75 (m. 10. one exchanged with D,O). 8.61 (d.2). 8.79 (ti.2). 9.18 (t. 1. exchanged with D,O). Anal. Calcd for C,,H,,N,.HCl.H,O: C. 69.92: H, 5.33: N. 11.12. Found: C. 70.12: H. 5.31: N. 11.18. The product was dried over PzO, if? 1~7~~0 at 110°C for 24 hr to obtain anhydrous hydrochloride. Anal. Calcd for C,,H,;N,*HCl: C. 73.44; H, 5.00; N, 11.68. Found: C, 73.45; H, 4.95; N. 11.73. The hydrated hydrochloride (189 mg, 0.5 mmol) was added to a solution of 4 N NaOH (5.0 ml) and ethanol (5.0 ml). and the resulting suspension of free base was poured into water (100 ml). The yellow precipitate that formed was collected on a filter, washed with water. and dried over P,O, in r’(fcz~o to yield 147 mg (86%). Recrystallization from methanol yielded 107 mg (63%) of hydrate, mp 217-218°C: ir (Nujol) 3400. 1630, 1620. 1580. 1540. 1070. 1050. 980. 760. and 730 cm-r; m/e (intensity) 325 (21, 324 (16). 323 (62). 322 (7), 321 (4). 320 (2). 306 (3). 294 (2). 234 (2). 233 (22). 232 (100). 219 (2), 218 (5). 206 (S). 205 (27). 204 (2). 191 (2). 190 (8), 180 (2). 178 (4). 177 (5). 176 (2). 165 (4). 164 (2). 163 (9-j. 1’ (2). 151 (5). 92 (2), 91 (20). 77 (2). and 65 (5). A&. Calcd for C,,H,,N,.H,O: C, 77.40: H, 5.61; N. 12.31. Found: C, 77.90; H. 5.75; N, 11.94. The hydrated free base was dried over P,O, in rarzlo at 110°C for 24 hr to obtain anhydrous free base. Awl. Calcd for C,,H,,N,: C, 81.71, H, 5.30; N, 12.99. Found: C, 81.63; H, 5.31: N, 12.90. (B ) Reduction of’ the reuction mixture of’ API und henculdrhyde. API.HCI.H,O (288 mg. 1.0 mmol) and freshly distilled benzaldehyde (1.06 g, 10.0 mmol) were added to a solution of 2.8 g of KOH in 200 ml
138
ITANO ET AL.
NH +
YH2
H,N-II-NH-CH2-CH,-CH,-CH-COO-
I
Y
OH-
“,C=N, ,CH-COO-
W, 4
H:/c-NH
\ CH-COOH
W, CH;
“II
FIG. 1. The reaction of phenanthrenequinone with arginine in alkaline solution and the production of proline from reduction of the reaction mixture.
of methanol. After the reaction mixture was stirred for 2 hr under N?, sodium borohydride (1.5 g) was added slowly in small proportions. The mixture was allowed to stand for 1 hr. acidified with 6 N HCI, and evaporated to dryness under reduced pressure. The residue was dissolved in 30 ml of 0.5 N NaOH and extracted with ethyl acetate. The extract was washed with water, dried with anhydrous sodium sulfate, and evaporated to dryness. The residue was dissolved in ethanol (30 ml) and applied to a glass plate precoated with silica gel (Merck 5766). The plate was developed with a solvent system of ether:n-heptane:isopropanol ( 1: 1: l), and the zone of the product was scraped off the plate and extracted with ethyl acetate. The solvent was removed by evaporation. and 6 N HCl (5.0 ml) was added to the residue. The white crystals that formed were collected on a filter and crystallized from 6 N HCI (50 ml) in ethanol (350 ml) and dried over P,O, to yield 155 mg (41%) of BAPI .HCl .H20, mp 222-225°C (dec). This product had the same ir spectrum as the compound prepared from phenanthrenequinone and benzylguanidine. The mixed melting point of the two products was 223-225°C. The Reactiorl of Pherlcrnthrerleqllirlorle trith Other Gunnidines
Reaction mixtures of 0.01 M guanidino compound and 0.012 M phenanthrenequinone in 0.2 M NaOH (EtOH:H,O. 4:l) were analyzed by high voltage electrophoresis. API was produced with a-amino-
MECHANISM
FIG. 2. The formation routes and the production
OF
PHENANTHRENEQUINONE
TEST
of the Schiff base of API and benzaldehyde by two of BAPI from reduction of each reaction mixture.
139
reaction
@guanidinopropionic acid. a-amino-y-guanidino-butyric acid. and canavanine. Only the reaction mixture with canavanine contained some unreacted guanidino compound after 4 hr; an amino acid with the electrophoretic migration velocity of homoserine was produced in this reaction. Guanidine. methylguanidine. guanidinoacetic acid. p-tosylarginine. p-tosylarginineamide. homoarginine. octopine. the phenylthiohydantoin derivative of arginine. and arginine-containing peptides have been reported to produce a positive fluorescence reaction with phenanthrenequinone (l-4). The procedure described in the reaction of benzylguanidine with phenanthrenequinone was followed with tbutylguanidine and phenylguanidine. t-Butylguanidine did not yield any fluorescent product. Phenylguanidine yielded a fluorescent product that differed by thin-layer chromatography from API. Previously only creatine and 1. I-dimethylguanidine did not produce API when tested with phenanthrenequinone t I .6). DISCUSSION The results are in accord with the schemes of Fig. I and 2, respectively, for the reactions of phenanthrenequinone with arginine and benzylguanidine. The formation of benzaldehyde was demonstrated in the latter reaction, but the corresponding product of the reaction with arginine, namely glutamic y-semialdehyde (V). is known to cyclize to A’-pyrroline-5-
140
ITANO
ET AL
carboxylic acid (VI) (7). The formation of this cyclic amino acid was confirmed by comparison with an authentic sample and by the formation of proline (VII) upon reduction of the reaction mixture. The same product, namely BAPI (Xl). was obtained when a reaction mixture either of phenanthrenequinone (I) and benzylguanidine (VIII) or of API (IV) and benzaldehyde (IX) was reduced with sodium borohydride. Thus the intermediate product in the reaction of phenanthrenequinone with benzylguanidine must be 2-(benzylideneamino)-IH-phenanthro[9. IO-dlimidazole (X), the Schiff base of API and benzaldehyde: and the ninhydrin-positive fluorescent intermediate product of the reaction of phenanthrenequinone with arginine must accordingly be the Schiff base (III) of API and glutamic y-semialdehyde. In the former case the Schiff base is present in reversible equilibrium with the amine and aldehyde. and BAPI results from reduction of the reaction mixture; in the latter case cyclization removes the aldehyde from the equilibrium and leads to complete hydrolysis of the Schiff base. and proline results from reduction of the reaction mixture. The above mechanism accounts for the absence of API formation with the monosubstituted guanidines r-butylguanidine and phenylguanidine as well as with I.l-disubstituted guanidines. In each case rearrangement of the condensation product of the guanidino compound with phenanthrenequinone to a Schiff base is not possible. Intermediate Schiff base products are not possible with guanidine or cananavine; nevertheless these compounds, which give negative or weakly positive Sakaguchi reactions (lo- 12). resulted in the formation of API and gave strongly positive fluorescence tests with phenanthrenequinone. The yield of API from guanidine was 30-40% of the yields from monosubstituted guanidines (5). and cananavine reacted more slowly with phenanthrenequinone than did other monosubstituted guanidines. The formation of API from phenanthrenequinone and guanidine and of API and homoserine from phenanthrenequinone and cananavine requires the incorporation of hydrogen atoms into the respective products from the reaction medium. ACKNOWLEDGMENTS This work was supported in part by Grants AM 14982 and GM Institutes of Health. The LKB Type 9000 mass spectrometer University of California on NSF Grant GP-18245. The expert Leila Kotite and Elaine Norris Verrusio is gratefully acknowledged.
17702 from the National was purchased by the technical assistance of
REFERENCES I. Yamada. 2. Inagami. 3. Johnson. E. W.
S.. and Itano. H. A. (1966) Bioc~kim. Bioplly.s. Ac,ttr 130, T. (1973) Ano/. Biochrm. 52, 318-371. R.. Guderian, R. H.. Eden. F.. Chiiton. M. -D.. Gordon, (1974) Proc. Not. Acd. Sri. USA. 71. 536-539.
538-540.
M. P.. and Nester.
MECHANISM 4. 5. 6. 7. 8. 9. 0. I. 2.
OF
PHENANTHRENEQUINONE
TESI
Doolittle. R. F. (1971) Metl~ods Enz~mol. 25, 23 l-144. ltano. H. A.. and Yamada, S. (1972) Antrl. Biochem. 48. 483-490. Itano. H. A. (1974) .I. Nrter.oc~c./. Cl~rm. 11, 1071- 1073. Vogel. H. J.. and Davis. B. D. (1951) J. Am. Chrm. Ser. 74, 10% I I?. Cramer. F.. Scheiffele. E.. and Vollmar. A. (1962) Chr,n. Bev. 95, l670- 1682. Dreyer. W. J.. and Bynum. E. (1967) Mrfl~ods En:yrno/. 11, 32-39. Sakaguchi. S. (1925) J. Biocl~ern. (Tokyo) 5. 25-31. Kitagawa. M.. and Tsukamoto. J. (1937) J. Bi~~clx~~~~. (7‘0/,?0) 26, 373-385. Watson. D. (1966) Nurrrrc (Lo,&~z) 211, 41 I-417.
14
BIOCHEMISTRY
76, 134-141
(1976)
Mechanism and Specificity of the Phenanthrenequinone Test for Monosubstituted Guanidinesl HARVEY
A. ITANO.
KAZUHIRO HIROTA,* ICHIRO AND SHIGEKI YAMADA”
KAWASAKI.”
Department of Puthology. University of Colijbrnic~. Snrl Diego, Ltr Jolla. California 92093 Received
April
12. 1976: accepted
June
18. 1976
The reaction of phenanthrenequinone with arginine yielded 2-amino-IHphenanthro[9.10-djimidazole (API) and a’-pyrroline-5-carboxylic acid. The reaction of phenanthrenequinone with benzylguanidine yielded API and benzaldehyde. The same product . 2-benzylamino1 H-phenanthro[Y, IO+/]imidazole was obtained when either a reaction mixture of phenanthrenequinone and benzylguanidine or a reaction mixture of API and benzaldehyde was reduced with sodium borohydride. Thus the reaction mechanism involves the formation of a Schiff base intermediate. l.l-Disubstituted guanidines. t-butylguanidine. and phenylguanidine produce neither a Schiff base intermediate nor API. On the other hand. guanidine and canavanine. which also do not produce Schiff base intermediates. result in API through mechanisms that require the presence of a reducing compound in the reaction medium.
The formation of a fluorescent compound in the reaction of a guanidino group with phenanthrenequinone in alkaline medium is a sensitive test for the detection of arginine. arginine-containing peptides. and other monosubstituted guanidines ( I-4). Phenanthrenequinone reacts with 1, I-disubstituted guanidines to form stable condensation products; however, these products are not fluorescent. The fluorescent product of the former reaction is 2-amino-IH-phenanthro[9, IO-(llimidazole (API) (5). and the nonfluorescent products of the latter are spirofluoreneimidazolinones (6). The mechanism of the reaction of phenanthrenequinone with certain monosubstituted guanidines has been elucidated in the present study. This mechanism accounts for the absence of API as a product in ’ A part of this investigation was carried out during the tenures of Shigeki Yamada and Ichiro Kawasaki as Visiting Associates of the National Institute of Arthritis and Metabolic Disease and of Harvey A. Itano as Visiting Professor at the University of California. San Francisco. 2 Present address: Institute for Protein Research. Osaka University. Osaka. Japan. :3 Present address: Brewing Science Research Institute. Suita, Japan. ’ Present address: Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd.. Osaka. Japan. 134 Copyl-ighl G 15176 by Academic Press, Inc .4ll rights of reproduction in any fol-m I-esewed.
MECHANISM
OF PHENANTHRENEQUINONE
TEST
the reactions of phenanthrenequinone with r-butylguanidine phenylguanidine as well as with l,l-disubstituted guanidines.
13s and with
METHODS Nuclear magnetic resonance spectra of samples in dimethylsulfoxide-d,i (Me,SO-ci,) solution containing internal tetramethylsilane (TMS) were recorded with a Varian HR-220 spectrometer. Infrared spectra of crystalline samples were recorded as Nujol mulls with a Beckman IR-33 spectrometer. Mass spectra were obtained in a LKB Type 9000 spectrometer at an ionizing energy of 70 eV. Melting points were determined with a Kofler hot-stage microscope and are uncorrected. Elemental analyses were performed by Galbraith Laboratories, Inc.. of Knoxville, Tenn. A’-Pyrroline-5-carboxylic acid hydrochloride was prepared according to Vogel and Davis (7). API hydrochloride was prepared by the reaction of arginine with phenanthrenequinone (I). and benzylguanidine sulfate was prepared by the reaction of benzylamine with 0-methylisourea (8). Other compounds and solvents used in this work were of the best commercially available grades. RESULTS
A reaction mixture of 0.019 M phenanthrenequinone and 0.01 M arginine in 0.2 M NaOH (EtOH:H,O. 4: I) was analyzed periodically by high voltage filter paper electrophoresis (9). API was detected as a diffuse. fluorescent zone near the origin. After 3 min the mixture contained, in addition to arginine and API. a ninhydrin-positive fluorescent product that moved about 0.6 as rapidly as tryptophan. which is the slowest moving of the amino acids in the standard mixture, and a nonfluorescent product that stained pink with ninhydrin and migrated between methionine and threonine. After IS min only a trace of arginine remained; after I .5 hr arginine was not detected. but API. the ninhydrin-positive fluorescent product. and the pink-staining product remained. Only API and the pink-staining product remained 3 hr after the reactants were mised. The published procedure for the preparation of API from phenanthrenequinone and arginine (l! was followed. After API was removed by filtration, the filtrate was passed through a column of Dowex 50 resin (H+ form). and the column was eluted with 1.O hl NH,OH to separate the pink-staining product from unreacted arginine. The eluate was concentrated. and ethanol was added to the concentrate to obtain a precipitate. which was collected and dried to a powder. The powder was refluxed with 6 N HCI and evaporated to dryness. The ir spectrum of this sample
136
ITANO
ET AL.
was identical with that of authentic 4’-pyrroline-S-carboxylic acid hydrochloride. API was filtered from the reaction mixture of another preparation, and the filtrate was hydrogenated directly with the aid of PtO, as catalyst for 3 hr at 15°C. The catalyst was removed by filtration, and the filtrate was examined by thin-layer chromatography (phenol:H,O. 3: 1). Ninhydrin revealed two spots that were identified as proline (R, 0.50) and arginine (Rf0.20). Quantitative analysis in an amino acid analyzer revealed that the yield of proline was 73% and that the residue of unreacted arginine was 10%. The yield of proline was increased to 90% when the time of reduction was increased to 5-7 hr. Attempts to isolate the ninhydrin-positive fluorescent product were unsuccessful. The nature of the intermediate product in the reaction of phenanthrenequinone and a monosubstituted guanidine was therefore investigated in the reaction between phenanthrenequinone and benzylguanidine. The Reaction
of Phencrnrhrenec~trinotle
uqith Ben~ylgtm~idine
Phenanthrenequinone (312 mg. 1.5 mmol) and benzylguanidine sulfate (297 mg, 0.75 mmol or I .5 mequiv) were dissolved with heating in ethanol (80 ml) and in water (7.5 ml). respectively. After the solutions were cooled to room temperature. the solution of benzylguanidine was made alkaline with 2 N NaOH (12.5 ml) and then added to the solution of phenanthrenequinone. After 4 hr of stirring. the reaction mixture was acidified with 6 N HCl and was allowed to stand overnight at 5°C. The precipitate that formed was collected on a filter. washed with 80% ethanol (20 ml). and recrystallized from ethanol. The product was identifified as API .HCl.H,O by its ir spectrum (5). 2.4-Dinitrophenylhydrazine hydrochloride (200 mg) in hot ethanol (80 ml) and 6 N HCl (IO ml) were added to the filtrate from the reaction mixture. Yellow crystals that formed overnight at 5°C were recrystallized from ethanol. mp 240- 24 1“C. The melting point and ir spectrum of this product were the same as those of the authentic hydrazone prepared from benzaldehyde and 2.4-dinitrophenylhydrazine. Preparation
of 2-Ben~~latt~it~o-IH-p~~et~at~thro[9,IO-d]itnida~ole
(a ) Reduction ben~ylgucmidine.
of fhe reaction
mixture
of phenanthreneyuinot~e
(BAPI) und
Phenanthrenequinone (624 mg. 3.0 mmol) was dissolved by heating (55°C) in a solution of 2.8 g of KOH in 200 ml of methanol. Finely powdered benzylguanidine sulfate (594 mg. I.5 mmol or 3.0 mequiv) was added. The mixture was stirred for 4 hr, after which sodium borohydride (3.0 g) was added in small proportions over a period of 2 hr. The mixture was allowed to stand overnight at room temperature and then was acidified with 6 N HCl and dried under reduced pressure.
MECHANISMOFPHENANTHRENEQUINONETEST
137
Water (100 ml) was added to the residue to dissolve inorganic salts. and undissolved material was collected on a filter. suspended in 0.5 N NaOH. and extracted with ethyl acetate. The extract was washed with water. dried over anhydrous Na,SO,. and evaporated to dryness. The residue was loaded on a column packed with silica gel. and the column was eluted with dichloroethane. A yellow fraction which chromatographed with a R, of 0.79 by thin-layer chromatography in a solvent system of II-heptane: isopropanol:concentrated NH, (6:4:0.5) was obtained. Dichloroethane was removed. and the residue was mixed with 6 N HCI (20 ml) to produce a salt which was collected on a filter, washed with water, and dried over P,O, in a vacuum desiccator. The dried material was suspended in ether (30 ml) to dissolve the yellow impurity. The suspended solid was collected on a filter and dried to yield 630 mg (56%) of a white product. Recrystallization from 6 N HCI (50 ml) in ethanol (350 ml) yielded 340 mg (309,). mp 220-224°C (dec); ir (Nujol) 3360. 1700. 1660. 1640. 1600. 1570. 760. and 730 cm-‘; NMR aT,, (Me,SO-d,) 5.07 (d,2). 7.30-7.75 (m. 10. one exchanged with D,O). 8.61 (d.2). 8.79 (ti.2). 9.18 (t. 1. exchanged with D,O). Anal. Calcd for C,,H,,N,.HCl.H,O: C. 69.92: H, 5.33: N. 11.12. Found: C. 70.12: H. 5.31: N. 11.18. The product was dried over PzO, if? 1~7~~0 at 110°C for 24 hr to obtain anhydrous hydrochloride. Anal. Calcd for C,,H,;N,*HCl: C. 73.44; H, 5.00; N, 11.68. Found: C, 73.45; H, 4.95; N. 11.73. The hydrated hydrochloride (189 mg, 0.5 mmol) was added to a solution of 4 N NaOH (5.0 ml) and ethanol (5.0 ml). and the resulting suspension of free base was poured into water (100 ml). The yellow precipitate that formed was collected on a filter, washed with water. and dried over P,O, in r’(fcz~o to yield 147 mg (86%). Recrystallization from methanol yielded 107 mg (63%) of hydrate, mp 217-218°C: ir (Nujol) 3400. 1630, 1620. 1580. 1540. 1070. 1050. 980. 760. and 730 cm-r; m/e (intensity) 325 (21, 324 (16). 323 (62). 322 (7), 321 (4). 320 (2). 306 (3). 294 (2). 234 (2). 233 (22). 232 (100). 219 (2), 218 (5). 206 (S). 205 (27). 204 (2). 191 (2). 190 (8), 180 (2). 178 (4). 177 (5). 176 (2). 165 (4). 164 (2). 163 (9-j. 1’ (2). 151 (5). 92 (2), 91 (20). 77 (2). and 65 (5). A&. Calcd for C,,H,,N,.H,O: C, 77.40: H, 5.61; N. 12.31. Found: C, 77.90; H. 5.75; N, 11.94. The hydrated free base was dried over P,O, in rarzlo at 110°C for 24 hr to obtain anhydrous free base. Awl. Calcd for C,,H,,N,: C, 81.71, H, 5.30; N, 12.99. Found: C, 81.63; H, 5.31: N, 12.90. (B ) Reduction of’ the reuction mixture of’ API und henculdrhyde. API.HCI.H,O (288 mg. 1.0 mmol) and freshly distilled benzaldehyde (1.06 g, 10.0 mmol) were added to a solution of 2.8 g of KOH in 200 ml
138
ITANO ET AL.
NH +
YH2
H,N-II-NH-CH2-CH,-CH,-CH-COO-
I
Y
OH-
“,C=N, ,CH-COO-
W, 4
H:/c-NH
\ CH-COOH
W, CH;
“II
FIG. 1. The reaction of phenanthrenequinone with arginine in alkaline solution and the production of proline from reduction of the reaction mixture.
of methanol. After the reaction mixture was stirred for 2 hr under N?, sodium borohydride (1.5 g) was added slowly in small proportions. The mixture was allowed to stand for 1 hr. acidified with 6 N HCI, and evaporated to dryness under reduced pressure. The residue was dissolved in 30 ml of 0.5 N NaOH and extracted with ethyl acetate. The extract was washed with water, dried with anhydrous sodium sulfate, and evaporated to dryness. The residue was dissolved in ethanol (30 ml) and applied to a glass plate precoated with silica gel (Merck 5766). The plate was developed with a solvent system of ether:n-heptane:isopropanol ( 1: 1: l), and the zone of the product was scraped off the plate and extracted with ethyl acetate. The solvent was removed by evaporation. and 6 N HCl (5.0 ml) was added to the residue. The white crystals that formed were collected on a filter and crystallized from 6 N HCI (50 ml) in ethanol (350 ml) and dried over P,O, to yield 155 mg (41%) of BAPI .HCl .H20, mp 222-225°C (dec). This product had the same ir spectrum as the compound prepared from phenanthrenequinone and benzylguanidine. The mixed melting point of the two products was 223-225°C. The Reactiorl of Pherlcrnthrerleqllirlorle trith Other Gunnidines
Reaction mixtures of 0.01 M guanidino compound and 0.012 M phenanthrenequinone in 0.2 M NaOH (EtOH:H,O. 4:l) were analyzed by high voltage electrophoresis. API was produced with a-amino-
MECHANISM
FIG. 2. The formation routes and the production
OF
PHENANTHRENEQUINONE
TEST
of the Schiff base of API and benzaldehyde by two of BAPI from reduction of each reaction mixture.
139
reaction
@guanidinopropionic acid. a-amino-y-guanidino-butyric acid. and canavanine. Only the reaction mixture with canavanine contained some unreacted guanidino compound after 4 hr; an amino acid with the electrophoretic migration velocity of homoserine was produced in this reaction. Guanidine. methylguanidine. guanidinoacetic acid. p-tosylarginine. p-tosylarginineamide. homoarginine. octopine. the phenylthiohydantoin derivative of arginine. and arginine-containing peptides have been reported to produce a positive fluorescence reaction with phenanthrenequinone (l-4). The procedure described in the reaction of benzylguanidine with phenanthrenequinone was followed with tbutylguanidine and phenylguanidine. t-Butylguanidine did not yield any fluorescent product. Phenylguanidine yielded a fluorescent product that differed by thin-layer chromatography from API. Previously only creatine and 1. I-dimethylguanidine did not produce API when tested with phenanthrenequinone t I .6). DISCUSSION The results are in accord with the schemes of Fig. I and 2, respectively, for the reactions of phenanthrenequinone with arginine and benzylguanidine. The formation of benzaldehyde was demonstrated in the latter reaction, but the corresponding product of the reaction with arginine, namely glutamic y-semialdehyde (V). is known to cyclize to A’-pyrroline-5-
140
ITANO
ET AL
carboxylic acid (VI) (7). The formation of this cyclic amino acid was confirmed by comparison with an authentic sample and by the formation of proline (VII) upon reduction of the reaction mixture. The same product, namely BAPI (Xl). was obtained when a reaction mixture either of phenanthrenequinone (I) and benzylguanidine (VIII) or of API (IV) and benzaldehyde (IX) was reduced with sodium borohydride. Thus the intermediate product in the reaction of phenanthrenequinone with benzylguanidine must be 2-(benzylideneamino)-IH-phenanthro[9. IO-dlimidazole (X), the Schiff base of API and benzaldehyde: and the ninhydrin-positive fluorescent intermediate product of the reaction of phenanthrenequinone with arginine must accordingly be the Schiff base (III) of API and glutamic y-semialdehyde. In the former case the Schiff base is present in reversible equilibrium with the amine and aldehyde. and BAPI results from reduction of the reaction mixture; in the latter case cyclization removes the aldehyde from the equilibrium and leads to complete hydrolysis of the Schiff base. and proline results from reduction of the reaction mixture. The above mechanism accounts for the absence of API formation with the monosubstituted guanidines r-butylguanidine and phenylguanidine as well as with I.l-disubstituted guanidines. In each case rearrangement of the condensation product of the guanidino compound with phenanthrenequinone to a Schiff base is not possible. Intermediate Schiff base products are not possible with guanidine or cananavine; nevertheless these compounds, which give negative or weakly positive Sakaguchi reactions (lo- 12). resulted in the formation of API and gave strongly positive fluorescence tests with phenanthrenequinone. The yield of API from guanidine was 30-40% of the yields from monosubstituted guanidines (5). and cananavine reacted more slowly with phenanthrenequinone than did other monosubstituted guanidines. The formation of API from phenanthrenequinone and guanidine and of API and homoserine from phenanthrenequinone and cananavine requires the incorporation of hydrogen atoms into the respective products from the reaction medium. ACKNOWLEDGMENTS This work was supported in part by Grants AM 14982 and GM Institutes of Health. The LKB Type 9000 mass spectrometer University of California on NSF Grant GP-18245. The expert Leila Kotite and Elaine Norris Verrusio is gratefully acknowledged.
17702 from the National was purchased by the technical assistance of
REFERENCES I. Yamada. 2. Inagami. 3. Johnson. E. W.
S.. and Itano. H. A. (1966) Bioc~kim. Bioplly.s. Ac,ttr 130, T. (1973) Ano/. Biochrm. 52, 318-371. R.. Guderian, R. H.. Eden. F.. Chiiton. M. -D.. Gordon, (1974) Proc. Not. Acd. Sri. USA. 71. 536-539.
538-540.
M. P.. and Nester.
MECHANISM 4. 5. 6. 7. 8. 9. 0. I. 2.
OF
PHENANTHRENEQUINONE
TESI
Doolittle. R. F. (1971) Metl~ods Enz~mol. 25, 23 l-144. ltano. H. A.. and Yamada, S. (1972) Antrl. Biochem. 48. 483-490. Itano. H. A. (1974) .I. Nrter.oc~c./. Cl~rm. 11, 1071- 1073. Vogel. H. J.. and Davis. B. D. (1951) J. Am. Chrm. Ser. 74, 10% I I?. Cramer. F.. Scheiffele. E.. and Vollmar. A. (1962) Chr,n. Bev. 95, l670- 1682. Dreyer. W. J.. and Bynum. E. (1967) Mrfl~ods En:yrno/. 11, 32-39. Sakaguchi. S. (1925) J. Biocl~ern. (Tokyo) 5. 25-31. Kitagawa. M.. and Tsukamoto. J. (1937) J. Bi~~clx~~~~. (7‘0/,?0) 26, 373-385. Watson. D. (1966) Nurrrrc (Lo,&~z) 211, 41 I-417.
14