July, 1945
FURTHER STUDIES OF THE ESSENTIAL GROUPSOF PANCREATIC AMYLASE
2-(4'-Nitrophenoxy)-4-amino4ethylaminos-triazine
(VIII).-2-Chloro-4-amino-6-ethylamino-s-triazine was converted into VI11 by the procedure used for I. The
1079
2-(4'-Arsonophenoxy)-4,6diethylamino-s-triazina (XV)
was synthesized from XI11 in 9% yields according t o directions given for I11 (A). compound was crystallized fro? an absolute ethanolAnal. Calcd. for CLIH18NS0,As: As, 19.55. Found: benzene mixture; m. p. 211-213 ; yield, 90%. As, 19.84. Anal. Calcd. for C I I H I ~ N ~ OC, ~ : 47.82; H, 4.37. 2- [4'-Di-( carborymethylene-thio)-arsenosophenoxy1Found: C, 47.64; H, 4.48. 4,6-diethylamino-s-triazine(XV).-The reaction was-car2-(4'-Aminophenoxy)-4-amino-6-ethylamino-s-triazine ried out as previously described for V. The product was rewas prepared by the procedure described for I1 in crystallized from methanol; yield, 45%; m. p. 170-173'. 77% yields; m. p. 204-206 Anal. Calcd. for CirH2zNsOaS As: As, 14.53. Found: Anal. Calcd. for C11H1,NsO: C, 53.64; H, 5.73. As, 14.52. Found: C, 53.60; H, 5.71. 2-Chloro-4,6-dimo~hopholino-s-triazine (XVI) was obtained 2 4 4'-Arsonophenoxy)-4-aminwS-ethylamino-ptriazine in almost quantitative yields in a manner similar to that (X) was synthesized in 25% yields from IX according t o used to produce XI; m. p. 175-176'. the directions gjven in 111. Anal. Calcd. for C;,HlaNsOzC1: C, 46.23; H , 5.65. Anal. Calcd. for CllHlrNsOIAs: As, 21.09; N, 19.72 Found: C, 46.40; H, 5.71. Found: As, 20.97; N, 19.50. 2-(4'-Nitrophenoxy) -4,6-dimorpholino-s-triazine (XVII). 2-Chloro-4,6-diethylao-s-triaz~~ (XI)was prepared -The procedure employed was the same as that described by a procedure similar to that for 2,4-dichloro-6-ethyl- above for XII. Recrystallization from a benzeneabsolute ethanol ( 3 : l ) solution gave a flocculent white amino-s-triazine. 2-(4'-Nitrophenoxy)-4,6-diethylamino-s-triaziae (HI) compound: m. p. 227-229'; yield, 77%. was synthesized by the method of Otto.* A mixture of Anal. CalcdT .for ClrHiNeOs:. C;-52.57; H, 5.19. XI (34 g., 0.17 mole), 4-nitrophenol (80 g., 0.58 mole) and Found: C, 52.55; H, 5.35. 2-( 4'-Aminophenoxy)-4,6-dimorpholino-s-triazine the sodium salt of 4-nitrophenol (28 g., 0.17 mole) was fused in a Casserole a t 130-140" for fifteoen minUteS, f01- (XVIII).-The reduction was carried out as described for bwed by another fifteen minutes a t 150 The reaction VI1 except that benzene served as the solvent and it was mixture was extracted several times with dilute sodium necessary to warm the reaction mixture to 50-60"; yield, hydroxide, and the residue recrystallized twice from almost nl, p , 227-239 0 . ethanol to give a white product; yield, 39 g. (76%); m . p. Anal.- Calcd. for ClrHnNoO,: C, 56.Y7; H, 6.19. 210-211 Found: C, 56.80; H , 6.0'3. Anal. Calcd. for C13HIeN601: C, 51.31; H, 5.30. Summary Found: C. 51.25; H, 5.33. 2-(4'-Aminophenoxy)-4,6-diethylamino-s-triazine (XIII). Several 2- (4'-arsonophenozy)-4,6-aniino- and -The procedure employed for the reduction of XI1 W a s that previously described in VII; yield, 7770'0; m. p. alkylamino-s-triazines are reported for the first 226-228 '. time. Anal. Calcd. for C , 3 H l a ~ e c~ ,: 56.91; H, 6.61. The preparation of certain intermediates, leading to the syntheses of these arsonic acids, is deFound: C, 56.81; H, 6.61.
(Ix)
.
O.
scribed.
(8) Hofmann, Bcr., 18, 2755 (1885).
LINCOLN, NEBRASKA
(9) Otto, i b i d . , 20, 2236 ( 1 8 8 7 ) .
[CONTRIBUTION FROM
THE
RECEIVED FEBRUARY.27, 1945
DEPARTMENT RF CHEMISTRY, COLUMBIA UNIVERSITY ]
Further Studies of the Essential Groups of Pancreatic Amylase BY M. L.CALDWELL, C. EDWINWEILLAND RUTHS. WEE' Studies of the inactivation of pancreatic malted barley but appear to be of little if any amylase2"Sb and of @-amylasefrom barley and importance to the activity of pancreatic amylfrom malted b a r l e ~ by ~ ~the , ~ use of special re- ase.a,b This latter conclusion regarding free sulfhydryl agents have shown that certain free groups of these proteins are essential to their amylase groups, suggested by the work of Little and Caldactivities and have brought to light additional well,%is confirmed and extended by the observadifferences in the properties of these two types of tions reported here. starch-splitting enzymes. Experimental Free primary amino groups of the protein are essential to the activity of pancreatic amylaseaa*b Highly purified preparations of pancreatic amylase' were used. The influence of each reagent upon the but appear to be of little if any importance to the amylase activity was judged by direct comparisons of the activity of @-amylasefrom barley or from malted activity of the amylase solution under observation with barley.3a On the other hand, free sulfhydryl and that of an aliquot of the same amylase solution which had free tyrosine groups of the protein are essential been treated in an otherwise identical. manner except for the reagent concerned. These aliquots are referred t o as to the activity of @-amylasefrom barley or from controls in Table I. None of the reagents was found to (1) We are greatly indebted t o the Takamine Laboratory, Inc., for a grant in aid of this investigation. (2) (a) J. E. Little and M. L. Caldwell, J . Biol. Cham., 119,588 (1942); (b) J. E. Little and M. L. Caldwell, ibid., 147, 229 (1943). (3) (a) C. E.Weill and M. L. Caldwell, THISJOURNAL, 67, 212 (1946); (b) C. E. Weill dnd M.L. Caldwell, ibid., 67, 214 (1945).
influence the activity meawrements in the concentrations usually employed. Exceptions with larger concentrations were taken care of by the use of suitable blanks.
(4) € C.ISherman, . M. L. Caldwelland M. Adams, J . B i d . Chcm., 68, 295 (1930), and unpublished work.
1080
H. L. SUNDBERG, C. M. MCCLOSKEY, D. E. KEES AND G. H. COLEMAN
The amylase activities were all measured under the same specified condition9 with 1% soluble potato starch adjusted to 0.02 M sodium chloride, 0.01 M phosphate and PH 7.2.6 The saccharogenic activity refers to the increase in the reducing value (calculated as maltose) of the reaction mixture in thirty minutes at 40 brought about by one milligram of the enzyme preparation when the concentrations of the amylase were selected to give approximately the same (20%) hydrolysis of the starch.
VOl. 67
TABLE I A STUDY OF THE INFLUENCE OF SPECIFIC SULFHYDRYL REAGENTS UPON T H E ACTIVITY OF PANCREATIC &dYLASE Treatment Reagent Concn., M
After treatment Activity % of control
Unitsd
Phenylmercuric0 5,200 100 chloride" (satd. s o h ) 4,920 95 Results p-Chloromercuri0 5,400 100 benzoic acidb 0.0005 4,870 90 A number of reagents which have been reported to be specific for free sulfhydryl groupss were Iodoacetamide' 0 6,150 100 studied for their influence upon the activity of 0.05 6,350 103 pancreatic amylase. Typical data are summarAmylase solutions adjusted to 0.1 M phosthate; ized in Table I. These results show that the re- PH 6.8, reacted with reagent for thirty minutes at 0 r, Amylase solutions adjusted to 0.1 M phosphate; PH agents studied had little if any influence upon the reacted with reagent for thirty minutes at 0 . activity of pancreatic amylase and lead to the 7.0,Amylase solutions were adjusted to 0.1 M phosphate; conclusion that free sulfhydryl groups are of little PH 6.8, treated with the reagent and held at 25" for 120 minutes. The control was treated in the same way except if any importance to the activity of this amylase. This finding with pancreatic amylase is in that no iodoacetamide was added. of "maltose" formed in thirty minutes at marked contrast to the results obtained under 40"Milligrams by one milligram of enzyme acting on 1% soluble similar conditions with these reagents with potato starch at PH 7.2 in the presence of 0.01 M phosp-amylase from barley and from malted barley.3b phate and 0.02 M sodium chloride.
.
In this case, these sulfhydryl reagents caused complete inactivation of the amylase. Moreover, ing is in accord with previous more qualitative it was possible to restore the amylase activity by indication,+ and is again in marked contrast t o treatment of the inactivated enzyme solutions the observations with beta-amylase from barley with hydrogen sulfide or with cysteine, a con- and from malted barley, With this enzyme, the firmation of the conclusion that sulfhydryl groups amylase activities of the solutions were found to be directly proportional to the concentrations of are essential to /?-amylase activity. In addition, it is interesting to note that the highly active solutions of pancreatic amylase used summary here gave no evidence of tlie presence of free sulfThe data presented here confirm and extend hydryl groups when examined by the nitro prus- previous indications that sulfhydryl groups of the side reaction, as modified by A n ~ o n . ~This find- protein are not essential to the activity of pan(5) H. C.Sherman, hl. L. Caldwell and M. Adams, THISJOURNAL, creatic amylase. BO, 2529, 2535, 2538 (1938). They emphasize another difference between the (6) L. Hellerman and M. E.' Perkins, J . B i d . Chem., 107, 241 two typical starch-splitting enzymes, pancreatic (1834); E. D. Schock, J. Jensen and L. Hellerman. ;bid., 111, 553 (1935); L. Hellerman and M. E. Perkins, ibid., 114, 175 (1935-6); amylase and p-amylase from barley and from L. Hellerman, Physiol. Rev., 17, 454 (1937); L. Hellerman, F. P. malted barley. Chinard and V. R. Dietz, J . B i d . Chcm., 147, 443 (1943). (7) M. L. Anson, J. Gcn. Physiol., 4'4, 399 (1940-1941).
[CONTRIBUTION FROM THE
SEW YORK27, NEWYORK
DEPARTMENT OF CHEMISTRY,
RECEIVED APRIL12, 1945
STATE UNIVERSITY OF I O W A ]
A New Synthesis of Methyl 3,4,6-Trimethyl-p-D-glucosideand the Preparation of Crystalline 3,4,6-Trhethyl-D-glucose B Y ROBERTL.
SUNDBERG,
CHESTER M. MCCLOSKEY, DONALD E. REES AND GEORGEH. COLEMAN
In 1934 Haworth, Hirst and Panizzonl reA sample of 3,4,6-trirnethy~-~-glucose which ported the synthesis of 3,4,6-trimethyl-~-glucose.had been prepared in this Laboratory by the This represented the synthesis of the last of the hydrolysis of methyl 3,4,6-trimethyl-p-~-glucofour possible trimethyl-D-glucopyranoses which side crystallized spontaneously on standing. are so important in the determination of the This leaves, therefore, 2,3,4-trimethyl-~-glucose structures of oligo- and polysaccharides. Two of as the only one of the four which has not been obthe four have previously been reported as crystal- tained crystalline. Samples of 3,4,6-trimethyl-~-glucose subseline, 2,3,6-trimethyl-~-glucose~ and 2,4,6-triquently prepared in this Laboratory by the same methyl-D-glucose. method crystallized readily at room temperature. (1) Haworth. Hirst and Panizzon,J. Chcm. Soc., 154 (1934). The crystals were thick plates (m. p. 97-98' (2) Irvine and Hirst, rbid., 1213 (1922). (:31 Haworth and Sedgwick, i b i d . , 2573 (1926) (cor.) after recrystallization) and proved to be the
