- Email: [email protected]
A new uracil nucleoside from Penicillium Charlesii
54
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25461 A N E W U R A C I L N U C L E O S I D E FROM P E N I C I L L I U M C H A R L E S I I D. E. M A Y N A R D * AND J. E. G A N D E R * *
Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio (U.S.A.) (Received J u n e 24th , 1965)
SUMMARY
When cultured on a medium containing D-~aC61glucose and DL-tartrate, Penicil, lium charlesii G. Smith synthesizes a polygalactofuranoside containing ~4C. Isotope from DL-[I,4J4C~]tartrate is also incorporated under identical growth conditions. The novel structure of the polyhexose led to an investigation of the acid-extractable nucleotides of the organism to determine whether unique nucleosides or nucleotide diphosphofuranosides might be present. The isolation and partial identification of one such compound is described.
INTRODUCTION
Previous investigations in our laboratory 1 have shown that Penicillium charlesii G. Smith, grown 28 days on the Raulin-Thom medium (0.28 M in D@4C6]glucose and 0.035 M in DL-tartrate), produced in the medium a polygalactofuranoside containing 14C. The specific activity of galactose isolated from the polysaccharide was only one-third that of the initial specific activity of D-glucose. I t was also shown that ~4C from DL-[i,4-~4C~]tartrate was incorporated into the galactose subunits of the polysaccharide. These and other data suggested that glucose was not incorporated intact into the polygalactofuranoside. These investigations led to a search in the fungal pads for nucleosides and nucleotide diphospho-sugar furanosides. This communication describes some of the properties of a unique nucleoside obtained from a cold perchloric acid extract of P. charlesii. METHODS
P. charlesii G. Smith, ATCC strain 1887, was grown at 25 ° on 15o ml of RaulinThorn medium as previously described 1. The mycelial pads were separated from the culture medium IO to 20 days after inoculation, washed thoroughly with distilled water and frozen in liquid nitrogen. The frozen pads were homogenized and extracted with 0.25 M perchloric acid at o-5 °. All Subsequent steps through elution from Dowex-i-formate resin were also performed at 0-5 o. The extract was neutralized with KOH, the KC1Oa and cell debris were removed * P r e s e n t a d d r e s s : D e p a r t m e n t of Clinical P h a r m a c o l o g y , H o f f m a n n - L a Roche, Inc., Nutley, N.J. ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of M i n n e s o t a , St. P a u l , M i n n e s o t a .
Biochim. Biophys. Acta, 115 (1966) 53-58
URACIL NUCLEOSIDE FROM Penicillium charlesii
55
b y centrifugation and the supernatant solution was lyophilized. An aqueous solution of the residue was titrated to pH 9 with N H 4 0 H and chromatographed on a Dowex1-formate column 2. With water as the eluent, Io-ml effluent fractions were collected from the column. The contents of fractions 2-1o, comprising about 8o % of the 26o m/~absorbing materials, were pooled and concentrated to a small volume. Ascending paper chromatography of this concentrated sample was performed in the first of the following solvent systems: (I) isobutyric a c i d - H 2 0 - N H 4 0 H (66 : 33 : I, v/v/v) ; (2) ethanol- I. o M ammonium acetate (7 : 3, v/v) : (3) o. I M K H 2PO4(pH 6.8) (NH~) 2S04- i-propanol (IOO: 6o: 2, v/w/v). The chromatograms were viewed with a Mineralite lamp (2537 A) to locate ultraviolet-absorbing materials. The major area detected occurred at RF o.49 and will hereafter be designated Ux. A strip (RF o.45-o.5o) containing ultraviolet-absorbing material was cut from each chromatogram and eluted with water. The pooled and concentrated eluates were re-chromatographed in systems 2 and 3. Following each chromatographic procedure, ultraviolet-absorbing materials were eluted and concentrated for further study. A portion of the purified sample was treated with o.ooi M periodic acid in o.I M acetate buffer (pH 4.6) (ref. 3) at 25 ° in a stoppered cuvette. The amount of periodate consumed was estimated by the decrease in absorption at 265 m~. At the end of the oxidation, sodium arsenite was added to destroy any remaining periodate, and formaldehyde was measured by the chromotropic acid method 4. Hydrolysis of another portion of the sample in 6o % perchloric acid for I h at IOO° (ref. 5), followed by purification on charcoal and chromatography in the three previously described solvent systems allowed release and separation of bases. The remainder of the sample was treated with 3 % sodium amalgam at 25 ° for 48 h to reduce the C5-C" double bond of the pyrimidine ring~, rendering the glycosidic linkage labile to acid. Unreacted amalgam and mercury were removed and the solution was made I M with respect to HC1. After 3o min at IOO°, the sample was neutralized and passed through anionic and cationic exchange resins. The deionized and concentrated material was chromatographed in ethyl a c e t a t e - p y r i d i n e H20 (36:1o:15, v/v/v) 7, with the solvent being allowed to ascend the paper three times. Carbohydrate compounds were located by treating strips of the chromatograms with ammoniacal silver nitrate or periodate-benzidine spray reagents 8. The major area of the chromatogram reacting to both spray reagents (Rglueose o.83; glucose = I.OO) was eltlted with water. The eluate was concentrated to dryness, treated with hexamethyldisilazane and trimethylchlorosilane and subjected to gasliquid chromatography on a 3 % SE-52 columng, using a hydrogen flame detector. RESULTS
Preliminary experiments with l~C-labeled purine and pyrimidine bases suggested that the major neutral ultraviolet absorbing material, extracted from P. charlesii pads and isolated as described, was derived from uracil. Table I compares the chromatographic mobility of this material (Ux) in three solvent systems to uracil, uridine, deoxyuridine and UMP. The data show that the ultraviolet-absorbing material migrates similar to, but not identical with, uridine and dissimilar to the other uracil derivatives tested. Hydrolysis released a material that migrated identical Biochim. Biophys. Acta, 115 (1966) 53-58
56
D. E. MAYNARD, J. E. GANDER
TABLE I R E L A T I V E M I G R A T I O N R A T E S OF V A R I O U S N U C L E O S I D E S A N D N U C L E O T I D E S C O M P A R E D TO U x
Material
RF Solvent
z
Solvent 2
Solvent 3
U x (hydrolyzed)
0.62
0.66
0.54
Ux
0.49
0.46
0.57
Uracil
o.62
o.65
o.54
Uridine
o.46
o.49
o.6o
Deoxyuridine
o.54
0.77
....
Uridine- 5 ' - p h o s p h a t e
o.22
o. i I
o.73
].2•
1.o
i
,
•.
/
'..
c ""... .........
\
o 0.6-
/...
//...~ .
. \ .. \
~\...
-]/'
\
\
'....)\ "..\
/
~, ." \-"
°4-4--,\
.'-\
Y,,
//
'~c.-
o2
220
240
260
280
300
Wavelength (rap) Fig. I. U l t r a v i o l e t s p e c t r a of uracil, p H 7 (. . . . a n d t h e base released f r o m U x , p H 6. 5 (. . . . ).
), U x , p H 6. 5 (
), U x , p H II ( . . . . . .
),
to uracil in all three solvent systems. Fig. I shows the similarity of the ultraviolet spectra of Ux and its base to the spectrum of uracil. This evidence, coupled with the lack of phosphate in the 260 m/*-absorbing material and its ability to give the classical furfural reaction in concentrated H2SO 4 suggested U x to be a nucleoside. Calculated on the basis of AM ~ IOOOOfor uracil, the yield of Ux is approx. I / , m o l e per mycelial pad. This figure varies little with the age of the pads at harvest. When Ux was treated with periodate, 2.05 moles of periodate were consumed with the release of I.O mole of formaldehyde per mole of uracil. Hydrolysis of Ux after reduction with sodium amalgam released four materials that could be detected on chromatograms with either benzidine-periodate or ammoniacal silver nitrate reagents. The material migrating at Rote 0.83 was the major constituent and moved at the same rate as galactose. No ribose or deoxyribose was detected on the paper chromatograms. Materials eluted from the Rate 0.80-0.85 area of the chromatogram were trimethylsilylated. Table I I li~ts the relative retention times of the components in the residue compared to the trimethylsflyl derivative of Bioehim. B io phys. Acta, I15 (1966) 53-58
URACIL NUCLEOSIDE FROM P e n i c i l l i u m charlesii
57
TABLE II CHROMATOGRAPHY OF TRIMETHYLSILYL S U G A R DERIVATIVES: A COMPARISON OF RELATIVE RETENTION TIMES OF THE UNKNOWN TO REFERENCE COMPOUNDS
GAS
Material
Relative retention time*
Raze 0.80-0.85
0.58**
0.87
D-Glucose (equi].) D-Galactose (equil.)
0.88
D-Altrose (equil.)
6-Deoxygalactose** * Fructose***
1.o7
1.57
1.93
1.57 I.O 7
0.65 0.68
2-Deoxygalactose*** 2-Deoxyglucose***
I.O I.O
0.42 0.45 0.25
0.46
0.53 0.o 4
0.33 0.38 0-45 0.69
* The values given are retention times c o m p a r e d to a-D-glucose. ** R e p r e s e n t s the m a j o r peak. *** Values t a k e n from SWEELEY et al. 9.
,¢-n-glucose. This table shows that the material in the major peak is not galactose, altrose, 2-deoxygalactose, 2-deoxyglucose, 6-deoxygalactose or fructose. A comparison of the retention time of this peak to those given by SWEELEY et al2 eliminates the other common hexoses, pentoses, sugar alcohols, glucosamine, and galactosamine. However, the relative retention time of the major component compares closely, although not exactly, with 2-deoxygalactose. The retention times of 3- or 5-deoxygalactose have not been reported. Four of the minor peaks (relative retention times of 0.87, i.o, 1.o7 and 1.57) are similar to those observed for galactose and glucose. Glucose was probably derived from the chromatography paper. DISCUSSION
The data suggest the presence of nucleoside material containing uracil. Although a major fraction of the carbohydrate migrated at the same rate as galactose, only a small portion was identified by gas chromatography as galactose. The data also eliminate 5-ketogalactofuranose as the sugar residue since reduction would lead to the formation of L-altrose and/or D-galactose. The periodate oxidation data, although in agreement with the presence of a 1,4-hexofuranoside, could also result from other structures. At present it is not known the extent to which 14C from DL-[i,4-14C~ltartrate is incorporated into the sugar moiety of Ux nor is it known if the nucleoside is metabolically important in the biogenesis of the polygalactofuranoside polymer. These points are currently being pursued. ACKNOWLEDGEMENT
This work was supported in part by Research Grant A-3713 from the National Institutes of Health. Biochim. Biophys. Acta, 115 (1966) 53-58
58
D. E. MAYNARD, J. E. GANDER
REFERENCES
2 3 4 5 6 7 8 9
J. E. GANDER, Arch. Biochem. Biophys., 91 (196o) 307 . E. VOLKIN AND W~. E. COHN, J. Biol. Chem., 205 (1953) 767 . ]3. L. HORECKER, J. HURWlTZ AND A. WEISSBACH, J. Biol. Chem., 218 (1956) 785 . D. A. MACFAYDEN, J. Biol. Chem., 158 (1945) lO7. A. MARSHAK AND H. J. VOGEL, J. Biol. Chem., 189 (1951) 597. D. C. 13URKE, J. Org. Chem., 20 (1955) 643. P- COLOMBO, D. CORBETTA, A. PIROTTA, G. 1RUFFINI AND A. SARTORI, J. Chromalog., (196o) 343, J. A. CIFONELLI AND F. SMITH, Anal. Chem., 26 (1954) 1132. C. C. SWEELEY, R. BENTLEY, M. MARITA AND W. WELLS, J. Am. Chem. Soc., 85 (1963) 2497.
Biochim. Biophys. Acta, 115 (1966) 53-58
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25461 A N E W U R A C I L N U C L E O S I D E FROM P E N I C I L L I U M C H A R L E S I I D. E. M A Y N A R D * AND J. E. G A N D E R * *
Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio (U.S.A.) (Received J u n e 24th , 1965)
SUMMARY
When cultured on a medium containing D-~aC61glucose and DL-tartrate, Penicil, lium charlesii G. Smith synthesizes a polygalactofuranoside containing ~4C. Isotope from DL-[I,4J4C~]tartrate is also incorporated under identical growth conditions. The novel structure of the polyhexose led to an investigation of the acid-extractable nucleotides of the organism to determine whether unique nucleosides or nucleotide diphosphofuranosides might be present. The isolation and partial identification of one such compound is described.
INTRODUCTION
Previous investigations in our laboratory 1 have shown that Penicillium charlesii G. Smith, grown 28 days on the Raulin-Thom medium (0.28 M in D@4C6]glucose and 0.035 M in DL-tartrate), produced in the medium a polygalactofuranoside containing 14C. The specific activity of galactose isolated from the polysaccharide was only one-third that of the initial specific activity of D-glucose. I t was also shown that ~4C from DL-[i,4-~4C~]tartrate was incorporated into the galactose subunits of the polysaccharide. These and other data suggested that glucose was not incorporated intact into the polygalactofuranoside. These investigations led to a search in the fungal pads for nucleosides and nucleotide diphospho-sugar furanosides. This communication describes some of the properties of a unique nucleoside obtained from a cold perchloric acid extract of P. charlesii. METHODS
P. charlesii G. Smith, ATCC strain 1887, was grown at 25 ° on 15o ml of RaulinThorn medium as previously described 1. The mycelial pads were separated from the culture medium IO to 20 days after inoculation, washed thoroughly with distilled water and frozen in liquid nitrogen. The frozen pads were homogenized and extracted with 0.25 M perchloric acid at o-5 °. All Subsequent steps through elution from Dowex-i-formate resin were also performed at 0-5 o. The extract was neutralized with KOH, the KC1Oa and cell debris were removed * P r e s e n t a d d r e s s : D e p a r t m e n t of Clinical P h a r m a c o l o g y , H o f f m a n n - L a Roche, Inc., Nutley, N.J. ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of M i n n e s o t a , St. P a u l , M i n n e s o t a .
Biochim. Biophys. Acta, 115 (1966) 53-58
URACIL NUCLEOSIDE FROM Penicillium charlesii
55
b y centrifugation and the supernatant solution was lyophilized. An aqueous solution of the residue was titrated to pH 9 with N H 4 0 H and chromatographed on a Dowex1-formate column 2. With water as the eluent, Io-ml effluent fractions were collected from the column. The contents of fractions 2-1o, comprising about 8o % of the 26o m/~absorbing materials, were pooled and concentrated to a small volume. Ascending paper chromatography of this concentrated sample was performed in the first of the following solvent systems: (I) isobutyric a c i d - H 2 0 - N H 4 0 H (66 : 33 : I, v/v/v) ; (2) ethanol- I. o M ammonium acetate (7 : 3, v/v) : (3) o. I M K H 2PO4(pH 6.8) (NH~) 2S04- i-propanol (IOO: 6o: 2, v/w/v). The chromatograms were viewed with a Mineralite lamp (2537 A) to locate ultraviolet-absorbing materials. The major area detected occurred at RF o.49 and will hereafter be designated Ux. A strip (RF o.45-o.5o) containing ultraviolet-absorbing material was cut from each chromatogram and eluted with water. The pooled and concentrated eluates were re-chromatographed in systems 2 and 3. Following each chromatographic procedure, ultraviolet-absorbing materials were eluted and concentrated for further study. A portion of the purified sample was treated with o.ooi M periodic acid in o.I M acetate buffer (pH 4.6) (ref. 3) at 25 ° in a stoppered cuvette. The amount of periodate consumed was estimated by the decrease in absorption at 265 m~. At the end of the oxidation, sodium arsenite was added to destroy any remaining periodate, and formaldehyde was measured by the chromotropic acid method 4. Hydrolysis of another portion of the sample in 6o % perchloric acid for I h at IOO° (ref. 5), followed by purification on charcoal and chromatography in the three previously described solvent systems allowed release and separation of bases. The remainder of the sample was treated with 3 % sodium amalgam at 25 ° for 48 h to reduce the C5-C" double bond of the pyrimidine ring~, rendering the glycosidic linkage labile to acid. Unreacted amalgam and mercury were removed and the solution was made I M with respect to HC1. After 3o min at IOO°, the sample was neutralized and passed through anionic and cationic exchange resins. The deionized and concentrated material was chromatographed in ethyl a c e t a t e - p y r i d i n e H20 (36:1o:15, v/v/v) 7, with the solvent being allowed to ascend the paper three times. Carbohydrate compounds were located by treating strips of the chromatograms with ammoniacal silver nitrate or periodate-benzidine spray reagents 8. The major area of the chromatogram reacting to both spray reagents (Rglueose o.83; glucose = I.OO) was eltlted with water. The eluate was concentrated to dryness, treated with hexamethyldisilazane and trimethylchlorosilane and subjected to gasliquid chromatography on a 3 % SE-52 columng, using a hydrogen flame detector. RESULTS
Preliminary experiments with l~C-labeled purine and pyrimidine bases suggested that the major neutral ultraviolet absorbing material, extracted from P. charlesii pads and isolated as described, was derived from uracil. Table I compares the chromatographic mobility of this material (Ux) in three solvent systems to uracil, uridine, deoxyuridine and UMP. The data show that the ultraviolet-absorbing material migrates similar to, but not identical with, uridine and dissimilar to the other uracil derivatives tested. Hydrolysis released a material that migrated identical Biochim. Biophys. Acta, 115 (1966) 53-58
56
D. E. MAYNARD, J. E. GANDER
TABLE I R E L A T I V E M I G R A T I O N R A T E S OF V A R I O U S N U C L E O S I D E S A N D N U C L E O T I D E S C O M P A R E D TO U x
Material
RF Solvent
z
Solvent 2
Solvent 3
U x (hydrolyzed)
0.62
0.66
0.54
Ux
0.49
0.46
0.57
Uracil
o.62
o.65
o.54
Uridine
o.46
o.49
o.6o
Deoxyuridine
o.54
0.77
....
Uridine- 5 ' - p h o s p h a t e
o.22
o. i I
o.73
].2•
1.o
i
,
•.
/
'..
c ""... .........
\
o 0.6-
/...
//...~ .
. \ .. \
~\...
-]/'
\
\
'....)\ "..\
/
~, ." \-"
°4-4--,\
.'-\
Y,,
//
'~c.-
o2
220
240
260
280
300
Wavelength (rap) Fig. I. U l t r a v i o l e t s p e c t r a of uracil, p H 7 (. . . . a n d t h e base released f r o m U x , p H 6. 5 (. . . . ).
), U x , p H 6. 5 (
), U x , p H II ( . . . . . .
),
to uracil in all three solvent systems. Fig. I shows the similarity of the ultraviolet spectra of Ux and its base to the spectrum of uracil. This evidence, coupled with the lack of phosphate in the 260 m/*-absorbing material and its ability to give the classical furfural reaction in concentrated H2SO 4 suggested U x to be a nucleoside. Calculated on the basis of AM ~ IOOOOfor uracil, the yield of Ux is approx. I / , m o l e per mycelial pad. This figure varies little with the age of the pads at harvest. When Ux was treated with periodate, 2.05 moles of periodate were consumed with the release of I.O mole of formaldehyde per mole of uracil. Hydrolysis of Ux after reduction with sodium amalgam released four materials that could be detected on chromatograms with either benzidine-periodate or ammoniacal silver nitrate reagents. The material migrating at Rote 0.83 was the major constituent and moved at the same rate as galactose. No ribose or deoxyribose was detected on the paper chromatograms. Materials eluted from the Rate 0.80-0.85 area of the chromatogram were trimethylsilylated. Table I I li~ts the relative retention times of the components in the residue compared to the trimethylsflyl derivative of Bioehim. B io phys. Acta, I15 (1966) 53-58
URACIL NUCLEOSIDE FROM P e n i c i l l i u m charlesii
57
TABLE II CHROMATOGRAPHY OF TRIMETHYLSILYL S U G A R DERIVATIVES: A COMPARISON OF RELATIVE RETENTION TIMES OF THE UNKNOWN TO REFERENCE COMPOUNDS
GAS
Material
Relative retention time*
Raze 0.80-0.85
0.58**
0.87
D-Glucose (equi].) D-Galactose (equil.)
0.88
D-Altrose (equil.)
6-Deoxygalactose** * Fructose***
1.o7
1.57
1.93
1.57 I.O 7
0.65 0.68
2-Deoxygalactose*** 2-Deoxyglucose***
I.O I.O
0.42 0.45 0.25
0.46
0.53 0.o 4
0.33 0.38 0-45 0.69
* The values given are retention times c o m p a r e d to a-D-glucose. ** R e p r e s e n t s the m a j o r peak. *** Values t a k e n from SWEELEY et al. 9.
,¢-n-glucose. This table shows that the material in the major peak is not galactose, altrose, 2-deoxygalactose, 2-deoxyglucose, 6-deoxygalactose or fructose. A comparison of the retention time of this peak to those given by SWEELEY et al2 eliminates the other common hexoses, pentoses, sugar alcohols, glucosamine, and galactosamine. However, the relative retention time of the major component compares closely, although not exactly, with 2-deoxygalactose. The retention times of 3- or 5-deoxygalactose have not been reported. Four of the minor peaks (relative retention times of 0.87, i.o, 1.o7 and 1.57) are similar to those observed for galactose and glucose. Glucose was probably derived from the chromatography paper. DISCUSSION
The data suggest the presence of nucleoside material containing uracil. Although a major fraction of the carbohydrate migrated at the same rate as galactose, only a small portion was identified by gas chromatography as galactose. The data also eliminate 5-ketogalactofuranose as the sugar residue since reduction would lead to the formation of L-altrose and/or D-galactose. The periodate oxidation data, although in agreement with the presence of a 1,4-hexofuranoside, could also result from other structures. At present it is not known the extent to which 14C from DL-[i,4-14C~ltartrate is incorporated into the sugar moiety of Ux nor is it known if the nucleoside is metabolically important in the biogenesis of the polygalactofuranoside polymer. These points are currently being pursued. ACKNOWLEDGEMENT
This work was supported in part by Research Grant A-3713 from the National Institutes of Health. Biochim. Biophys. Acta, 115 (1966) 53-58
58
D. E. MAYNARD, J. E. GANDER
REFERENCES
2 3 4 5 6 7 8 9
J. E. GANDER, Arch. Biochem. Biophys., 91 (196o) 307 . E. VOLKIN AND W~. E. COHN, J. Biol. Chem., 205 (1953) 767 . ]3. L. HORECKER, J. HURWlTZ AND A. WEISSBACH, J. Biol. Chem., 218 (1956) 785 . D. A. MACFAYDEN, J. Biol. Chem., 158 (1945) lO7. A. MARSHAK AND H. J. VOGEL, J. Biol. Chem., 189 (1951) 597. D. C. 13URKE, J. Org. Chem., 20 (1955) 643. P- COLOMBO, D. CORBETTA, A. PIROTTA, G. 1RUFFINI AND A. SARTORI, J. Chromalog., (196o) 343, J. A. CIFONELLI AND F. SMITH, Anal. Chem., 26 (1954) 1132. C. C. SWEELEY, R. BENTLEY, M. MARITA AND W. WELLS, J. Am. Chem. Soc., 85 (1963) 2497.
Biochim. Biophys. Acta, 115 (1966) 53-58