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An interaction between aminopurines, aminopyrimidines, and fluorescent thin-layer plates
146 An
NOTES
interaction
thin-layer
between
aminopurines,
aminopyrimidines,
and
fluorescent:
plates”
6-Monomethylamino-g-(3’-amino-3’-deoxy-~-~-~ibofuranosyl)-purine (MMPA) was hydrolyzed with N HCl for I h at 100~. After concentration ivz zlaczta the hydrolysate was placed on a I mm Silica Gel HP thin-layer plate (Merck, Brinkmann) which was subsequently developed with chloroform-methanol-2.5 N NH40H (150 : IOO : 15, v/v) (CMN) solvent. In this system the Rp of MMPA was 0.33 and the Rp of the base resulting from hydrolysis, 6-monomethylaminopurine (GMAP) , was 0~67. However, the only U.V.-absorbing material on the chromatogram was at the origin. An EICl (0.1 N) extract of the origin showed no U.V. maximum, only generalized absorption increasing from 2go to 230 rnp which suggested repression of the mobility and/or destruction of the purine base since chromatography of an aliquot of the same hydrolysate on Whatman I filterpaper with uc-butanol-water-2s y0 ammonia (172 : IS : IO, v/v) showed that 6MAP was present in the hydrolysate. It is the purpose of this communication to describe and alert other investigators to this problem. When a drop of 2 N WC1 was placed on a Silica Gel HE plate a yellow spot appeared which was no longer fluorescent when viewed under U.V. light. The fluorescent background, manganese-activated zinc silicate, apparently reacts with NC1 to give a non-fluorescent compound which remains at the origin in this solvent system, retards the mobility of the purine base, and may destroy the base. TABLE EFFECT
I OF I-ICI
ON
MOljILLTY
OB
6MAP
ON
SILICA
6MAP (25 1~1, I ,ug//A) in I-ICI was applied Solvent : CMN.
wcz W) 0.05
0.10 0.15 0.25 OS.55 1.05
*+
RF
S$ot size*
0.67 0.67 0.62 (20 %) 0.38 (80%) 0.30 0.19 0.08
-t_ -
= discrete,
GEL
HF
TWIN-LAYER
PLATES
twice to a spot z cm long on a I mm thick plate.
”
-1-
= large, diffused.
Table I shows that with 2 applications of solution to the same spot, the RF of 6MAP decreased and the spot size increased as the normality of HCl was increased above 0.1 N. When the acid was held constant at 0.1 N, the Rp of 6MAP decreased and the spot size increased as additional applications of acid were made to the same spot (Table II). To demonstrate that the decreased & was due to reaction with the fluorescent l
Health
This worlc was supported, Service.
J. Gltvomatog.
21
(rgGG)
146-147
in part,
by Grant
HE-04983
from the United
States
Public
I47
NOTES
TABLE EFFECT LAYER
II OF REPEATED PLATRS
APPLICATIONS
N HCl
OF 0.1
ON
MOBILITY
OF 6MAP
ON SILICA
GEL
WF TNLN-
6MAP (25 141, I pg/pl) was applied twice to a spot z cm long on a 1 mm thick plate followed by the indicated number of applications of 0.1 N I-Xl. Solvent: CMN. 6MAP
HCZ applictilions
i 20
=
*-I-
RF
spot size*
o-74 0.45 0.35 0.23 (76%) o-12 (24%)
-I-
discicte,
-
fl
-I-
= large,
diffused.
background and not to another component of the system, parallel experiments were conducted with Silica Gel G and CF. As shown in Table III, there was no repression of RF ,on Silica Gel G but the mobility of 6MAP was repressed on Silica Gel GF under identical conditions. Further experiments showed that the mobility of adenine, 2,6-diamino-purine, and cytosine was affected even when spotted twice in 0.1 N HCl while the.mobilities thymine, uracil, tryptamine, and tyramine were unof 6-dimethylaminopurine, affected by 1.05 N HCl in this system. TABLE EFFECT
III OF FLUORESCENT
BACKGROUND
ON MOBILLTY
OF 6MAP
GMAP (12.5 ~1, 1 pg/pl) was applied twice to a spot and GF. Solvent: CMN. HCZ (N)
G
0.05 0.25 0.55
0.92 0.92 0.92 0.92
‘i-
= discrelc,
cm long and 0.3 mm thick of Silica Gel G
GF
RF
1.05
2
-
S$ot size*
Rp
spot size *
5
0.74 0.63 0.31
-I-
-I-
0.27
_-
= large, diffused.
Thus, if one applies amino- or monomethylaminopurines or pyrimidines to fluorescent background plates the HC1 concentration should be 0.05 N or lower depending on the number of applications to be made to the same spot. Medical Ressarch Laboratories, Veterans *,Administration Hos$ital, iWinnea$oZis, Minn.
(U.S.A.)
R. F. DERR C. S. ALEXANDER H. T. NAGASAWA
Received
July 5th, 1965 J. Ckvonzatog.,
21
(1~66)
14G-~47
NOTES
interaction
thin-layer
between
aminopurines,
aminopyrimidines,
and
fluorescent:
plates”
6-Monomethylamino-g-(3’-amino-3’-deoxy-~-~-~ibofuranosyl)-purine (MMPA) was hydrolyzed with N HCl for I h at 100~. After concentration ivz zlaczta the hydrolysate was placed on a I mm Silica Gel HP thin-layer plate (Merck, Brinkmann) which was subsequently developed with chloroform-methanol-2.5 N NH40H (150 : IOO : 15, v/v) (CMN) solvent. In this system the Rp of MMPA was 0.33 and the Rp of the base resulting from hydrolysis, 6-monomethylaminopurine (GMAP) , was 0~67. However, the only U.V.-absorbing material on the chromatogram was at the origin. An EICl (0.1 N) extract of the origin showed no U.V. maximum, only generalized absorption increasing from 2go to 230 rnp which suggested repression of the mobility and/or destruction of the purine base since chromatography of an aliquot of the same hydrolysate on Whatman I filterpaper with uc-butanol-water-2s y0 ammonia (172 : IS : IO, v/v) showed that 6MAP was present in the hydrolysate. It is the purpose of this communication to describe and alert other investigators to this problem. When a drop of 2 N WC1 was placed on a Silica Gel HE plate a yellow spot appeared which was no longer fluorescent when viewed under U.V. light. The fluorescent background, manganese-activated zinc silicate, apparently reacts with NC1 to give a non-fluorescent compound which remains at the origin in this solvent system, retards the mobility of the purine base, and may destroy the base. TABLE EFFECT
I OF I-ICI
ON
MOljILLTY
OB
6MAP
ON
SILICA
6MAP (25 1~1, I ,ug//A) in I-ICI was applied Solvent : CMN.
wcz W) 0.05
0.10 0.15 0.25 OS.55 1.05
*+
RF
S$ot size*
0.67 0.67 0.62 (20 %) 0.38 (80%) 0.30 0.19 0.08
-t_ -
= discrete,
GEL
HF
TWIN-LAYER
PLATES
twice to a spot z cm long on a I mm thick plate.
”
-1-
= large, diffused.
Table I shows that with 2 applications of solution to the same spot, the RF of 6MAP decreased and the spot size increased as the normality of HCl was increased above 0.1 N. When the acid was held constant at 0.1 N, the Rp of 6MAP decreased and the spot size increased as additional applications of acid were made to the same spot (Table II). To demonstrate that the decreased & was due to reaction with the fluorescent l
Health
This worlc was supported, Service.
J. Gltvomatog.
21
(rgGG)
146-147
in part,
by Grant
HE-04983
from the United
States
Public
I47
NOTES
TABLE EFFECT LAYER
II OF REPEATED PLATRS
APPLICATIONS
N HCl
OF 0.1
ON
MOBILITY
OF 6MAP
ON SILICA
GEL
WF TNLN-
6MAP (25 141, I pg/pl) was applied twice to a spot z cm long on a 1 mm thick plate followed by the indicated number of applications of 0.1 N I-Xl. Solvent: CMN. 6MAP
HCZ applictilions
i 20
=
*-I-
RF
spot size*
o-74 0.45 0.35 0.23 (76%) o-12 (24%)
-I-
discicte,
-
fl
-I-
= large,
diffused.
background and not to another component of the system, parallel experiments were conducted with Silica Gel G and CF. As shown in Table III, there was no repression of RF ,on Silica Gel G but the mobility of 6MAP was repressed on Silica Gel GF under identical conditions. Further experiments showed that the mobility of adenine, 2,6-diamino-purine, and cytosine was affected even when spotted twice in 0.1 N HCl while the.mobilities thymine, uracil, tryptamine, and tyramine were unof 6-dimethylaminopurine, affected by 1.05 N HCl in this system. TABLE EFFECT
III OF FLUORESCENT
BACKGROUND
ON MOBILLTY
OF 6MAP
GMAP (12.5 ~1, 1 pg/pl) was applied twice to a spot and GF. Solvent: CMN. HCZ (N)
G
0.05 0.25 0.55
0.92 0.92 0.92 0.92
‘i-
= discrelc,
cm long and 0.3 mm thick of Silica Gel G
GF
RF
1.05
2
-
S$ot size*
Rp
spot size *
5
0.74 0.63 0.31
-I-
-I-
0.27
_-
= large, diffused.
Thus, if one applies amino- or monomethylaminopurines or pyrimidines to fluorescent background plates the HC1 concentration should be 0.05 N or lower depending on the number of applications to be made to the same spot. Medical Ressarch Laboratories, Veterans *,Administration Hos$ital, iWinnea$oZis, Minn.
(U.S.A.)
R. F. DERR C. S. ALEXANDER H. T. NAGASAWA
Received
July 5th, 1965 J. Ckvonzatog.,
21
(1~66)
14G-~47