UBC Theses and Dissertations

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UBC Theses and Dissertations

I. Synthesis of specifically substituted D-mannitol derivatives. II. A color precursor isolated from… Finlayson, Alexander James 1956

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I . SYNTHESIS OF SPECIFICALLY SUBSTITUTED D-MANNITOL DERIVATIVES. I I . A COLOR PRECURSOR ISOLATED FROM WESTERN HEMLOCK WOOD by ALEXANDER JAMES FINLAYSON A t h e s i s submitted i n p a r t i a l f u l f i l m e n t of the Requirements f o r the Degree of Master of Science i n the Department of Chemistry We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree of MASTER OF SCIENCE Members of the Department of Chemistry The U n i v e r s i t y of B r i t i s h C o l u m b i a A p r i l , 1 9 5 6 . ABSTRACT ^- M a n n i t o l was simultaneously t r i t y l a t e d and a c e t y l a t e d to give l , 6 - d i - 0 - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e i n 70% y i e l d , using a procedure that gave a product completely f r e e from t r i p h e n y l c a r b i n o l . D i r e c t t r i t y l a t i o n of D-mannitol y i e l d e d 1 , 6 - d i - O - t r i t y l - D - m a n n i t o l which was c r y s t a l l i z e d , probably as a s o l v a t e d molecule, from a l c o h o l and water. A c e t y l a t i o n of t h i s compound gave l , 6 - d i - 0 -t r i t y l - D - m a n n i t o l t e t r a a c e t a t e i n good y i e l d . D e t r i t y l a t i o n of l , 6 - d i - 0 - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e to D-mannitol-2,3,^,5*-tetraacetate was e f f e c t e d by hydrogen bromide i n g l a c i a l a c e t i c a c i d , c a t a l y t i c hydrogenolysis over platinum oxide, and r e f l u x i n g w i t h d i l u t e a c e t i c a c i d , and the l a t t e r convenient method was found to give the highest y i e l d of pure product. A airupy di-O-methyl-D-mannitol was prepared from D-raannitol-2,3, 1+,5-tetraacetate by methylation f o l l o w e d by d e a c e t y l a t i o n on an i o n exchange column. Th£« s t r u c t u r e of is th« compound has not been e s t a b l i s h e d . A n e a r l y c o l o r l e s s , amorphous s o l i d was i s o l a t e d i n 2.7% y i e l d by 50% aqueous ethanol e x t r a c t i o n of the f i n e l y - d i v i d e d wood of a Western Hemlock t r e e (Tsuga het e r o p h y l l a ) which had p r e v i o u s l y been e x h a u s t i v e l y e x t r a c t e d w i t h benzene. Treatment of t h i s m a t e r i a l w i t h concentrated h y d r o c h l o r i c a c i d i n methanol y i e l d e d a purple s o l i d whose r e d - v i o l e t methanol s o l u t i o n was s t a b l e to o r d i n a r y l i g h t . The paper chromatographic behaviour and c o l o r r e a c t i o n s compared w i t h a red rose p e t a l e x t r a c t and the methoxyl content i n d i c a t e d that the purple s o l i d was probably an anthocyanidin c o n t a i n i n g methoxyl groups. A c e t y l a t i o n of the o r i g i n a l s o l i d e x t r a c t w i t h p y r i d i n e and a c e t i c anhydride gave a d e x t r o r o t a t o r y yellow s i r u p s o l u b l e i n chloroform. ACKNOWLEDGEMENTS The w r i t e r wishes t o express h i s s i n c e r e thanks t o Dr. L. D. Hayward f o r h i s encouragement and guidance throughout the course of t h i s i n v e s t i g a t i o n * The w r i t e r a l s o wishes t o express h i s thanks t o the Powell R i v e r Company f o r the award of a s c h o l a r s h i p , and t o the Sugar Research Foundation Incorporated f o r f i n a n c i a l a s s i s t a n c e . TABLE OF CONTENTS PART I Page INTRODUCTION ..' 1 HISTORICAL INTRODUCTION.... 4 DISCUSSION OF RESULTS A. 1,6-Di-O-trityl-D-mannitol Tetraacetate 10 B. 1,6-Di-O-trityl-D-mannitol 10 C. D e t r i t y l a t i o n Procedures 11 D. D-Mannitol-2,3,4,5-tetraacetate 13 E. Di-O-methyl-D-mannitol ., 14 EXPERIMENTAL A. Materials..... 18 B. A n a l y t i c a l Methods 20 C. D-Mannitol hexaacetate 21 D. 1,6-Di-O-trityl-D-mannitol Tetraacetate 21 E. 1,6-Di-O-trityl-D-mannitol 23 F. Acetylation of 1,6-di-O-trityl-D-mannitol 25 G. D e t r i t y l a t i o n Procedures 25 H. Acetylation of D-Mannitol-2,3,4,5-tetraacetate 28 I. Methylation of D-Mannitol-2,3,4,5-tetraacetate 29 J, Deacetylation of the Di-O-methyl-D-mannitol tetraacetate 30 K. Di-O-methyl-dimethylene-D-mannitol 31 CLAIMS TO ORIGINAL RESEARCH 32 BIBLIOGRAPHY 33 PART II INTRODUCTION 35 HISTORICAL INTRODUCTION. 38 DISCUSSION OF RESULTS 45 EXPERIMENTAL A. Preparation of the Wood 51 B. Extraction of the Wood with Benzene 52 TABLE OF CONTENTS (cont.) Page EXPERIMENTAL (cont.) C. E x t r a c t i o n of the Wood w i t h Acetone... 52 D. E x t r a c t i o n of the Wood w i t h Aqueous Et h a n o l . . . 53 E. A c i d Treatment of the Ethanol E x t r a c t 54 F. E x t r a c t i o n of the Pigment from Rose P e t a l s . . . . 55 G. Chromatography of the Pigments.. 56 H. Tests of Lin k and Robinson 57 I . A c e t y l a t i o n of the Ethanol E x t r a c t 61 CLAIMS TO ORIGINAL RESEARCH 62 BIBLIOGRAPHY. 63 LIST OF TABLES TABLE I S o l u b i l i t y C h a r a c t e r i s t i c s of the Ethanol E x t r a c t . 54 TABLE I I Chromatography of the Crude Rose E x t r a c t . . . 56 TABLE I I I Chromatography of the P u r i f i e d Rose E x t r a c t 57 TABLE IV Anthocyanidin Tests.... 58" TABLE V Robinson's Tests . 60 1. INTRODUCTION I t has long been known that the s e v e r a l hydroxy}, groups present i n a carbohydrate molecule possess d i f f e r e n t fies r e a c t i v i t i e s ( 2 6 ) . Where these d i f f e r e n t are known i t i s p o s s i b l e by choice of appropriate reagents and c o n d i t i o n s to syn t h e s i z e carbohydrate d e r i v a t i v e s w i t h s u b s t i t u e n t groups l o c a t e d a t s p e c i f i e d p o s i t i o n s on the carbon c h a i n . The purpose of t h i s research was to study the s p e c i f i c i t y of some s e l e c t i v e s u b s t i t u t i o n and e l i m i n a t i o n r e a c t i o n s i n the mannitol molecule and the f o l l o w i n g s e r i e s of s y n t h e t i c steps was c a r r i e d out: D-mannitol (I) l , 6 - d i - 0 - t r i t y l -D-mannitol t e t r a a c e t a t e ( I I ) D - m a n n i t o l - 2 , 3 , ^ , 5 - t e t r a a c e t a t e ( I I I ) 1 ,6-di-O-methyl-D-mannitol t e t r a a c e t a t e (IV) 1 ,6-di-O-methyl-D-mannitol (?) (V) I s o l a t i o n of c r y s t a l l i n e 1 , 6-di-O-methyl-D-mannitol, which has been p r e v i o u s l y prepared by another method (30) would show that D - m a n n i t o l - 2 , 3 , 5 - t e t r a a c e t a t e r e s u l t e d from the d e t r i t y l a t i o n of 1 , 6 - d i - O - t r i t y l - D - m a n n i t o l t e t r a -a c e tate and that the a c e t y l groups i n D-mannitol -2,3,**>5-t e t r a a c e t a t e d i d not undergo m i g r a t i o n i n a methy l a t i o n r e a c t i o n u s i n g the Purdie method ( 2 3 ) . I f a di-O-methyl * T r i t y l i s used as an abbreviated form f o r t r i p h e n y l m e t h y l 2. d e r i v a t i v e other than V was obtained some group m i g r a t i o n would be i n d i c a t e d . 3. CH9OH i * HOCH i HOCH f HCOH t HCOH t CH20H CH-OTr HOCH » HOCH t HCOH t HCOH t CHgOTr CH o0Tr t * AcOCH t AcOCH t HCOAc t HCOAc i CH 20Tr II CHoOTr t * AcOCH t AcOCH t HCOAc i HCOAc t CH 2OTr II CHo0H i * AcOCH t AcOCH t HCOAc t HCOAc t CH20H CH2OAc AcOCH t AcOCH t HCOAc t HCOAc t CH2Ofic CHo0Me t c AcOCH t AcOCH t HCOAc t HCOAc t CH20Me CH2OMe HOCH i HOCH i HCOH i HCOH t CH20Me I I I IV HISTORICAL INTRODUCTION T r i t y l ethers have been v a l u a b l e i n carbohydrate s y n t h e t i c work because of t h e i r s p e c i f i c i t y of s u b s t i t u t i o n and s t a b i l i t y . They are formed by the r e a c t i o n of t r i p h e n y l -e a r b i n o l w i t h an a l c o h o l i n the presence of an a c i d c a t a l y s t (25) * or from t r i t y l c h l o r i d e by r e a c t i o n w i t h an a l c o h o l ( 7 ) . I n compounds c o n t a i n i n g s e v e r a l h ydroxyl groups, both primary and secondary, i t i s o f t e n convenient to s p e c i f i c a l l y s u b s t i t u t e the primary hydroxyls w h i l e another r e a c t i o n i s undertaken with the secondary hydroxyls to s u b s t i t u t e them. Subsequent removal of the groups on the primary hydroxyls leaves these p o s i t i o n s f r e e to undergo f u r t h e r r e a c t i o n . The t r i t y l ethers serve t h i s purpose. Experience has shown tha t i n compounds c o n t a i n i n g both primary and secondary hydroxyl groups, the primary hydroxyls r e a c t p r e f e r e n t i a l l y -w i t h the t r i t y l a t i n g agent ( H e l f e r i c h 1 s Rule) ( 1 0 ) . Although t r i t y l a t i o n was considered to s u b s t i t u t e only the primary hydroxyls i n carbohydrate molecules, t r i t y l a t i o n does occur a t secondary hydroxyls but only a t a much slower r a t e . Hockett and coworkers (15) i n v e s t i g a t e d q u a n t i t a t i v e l y the t r i t y l a t i o n of the f o l l o w i n g compounds: 5. C H , O H V I l j a s S A - d i i s o p r o p y l i d e n e -«-D-galactopyranose ,0. CH2OH V I I 2,3'^,6-diisopropylidene-Lrsorbofuranose. / O K C -No«cu V I I I l , 2 : 5 , 6-diisopropylidene-cx -D-glucofuranose. A l l three compounds gave c r y s t a l l i n e t r i t y l e t h e r s , however a considerable d i f f e r e n c e i n v e l o c i t y of t r i t y l a t i o n was found. Using a f o u r - f o l d excess of t r i t y l c h l o r i d e and f o l l o w i n g the r e a c t i o n p o l a r i m e t r i c a l l y , i t was found that compounds V I and V I I were h a l f - t r i t y l a t e d a f t e r 21.6 and 57.9 hours r e s p e c t i v e l y , w h i l e compound V I I I was h a l f -t r i t y l a t e d only a f t e r 2500 hours. These r e s u l t s show the marked d i f f e r e n c e i n t r i t y l a t i o n r e a c t i o n r a t e s between the primary and secondary hydroxyl groups i n carbohydrates. 6. Hockett and Hudson (16) prepared two d i t r i t y l ethers of c< -methyl-D-xylopyranoside showing that more than one secondary hydroxyl group i n a molecule i s s u b s t i t u t a b l e . The r e l a t i v e r a t e s of t r i t y l a t i o n i n primary hydroxyls and secondary hydroxyls have not been stud i e d i n the h e x i t o l s e r i e s . In t h e i r s t u d i e s of the r e a c t i o n s between benzene and carbon t e t r a c h l o r i d e , F r i e d e l and C r a f t s (7) prepared the f i r s t t r i t y l ethers. H e l f e r i c h (1*+) f i r s t s t u d i e d the t r i t y l a t i o n of o the f r e e monosaccharides and prepared the mon^-trityl d e r i v a t i v e s of D-glucose and D-galactose. He noted t h a t these compounds reduced F e h l i n g ' s s o l u t i o n and when a c e t y l a t e d gave a c r y s t a l l i n e t e t r a a c e t a t e d e r i v a t i v e which, when t r e a t e d w i t h phosphorus pentabromide y i e l d e d l,6-dideoxy-dibromo - 2 , 3, l+-tri - 0-acetyl-D-glucose or D-galactose. He concluded that he had t r i t y l a t e d glucose i n the s i x p o s i t i o n . The 6 - t r i t y l - D - g l u c o s e and galactose were probably glasses s i n c e t h e i r m e l t i n g p o i n t s (13) were not sh a r p l y d e f i n e d . By means of the r e l a t i v e s p e c i f i c i t y of the1 t r i t y l group f o r the primary hydroxyls H e l f e r i c h (12) was able to develop the sy n t h e s i s of some d i s a c c h a r i d e s which p r e v i o u s l y had been very d i f f i c u l t . He synthesized g e n t i o b i o s e , which i s 6-(/3 -D-glucopyranosyl)-D-glucopyranose, by the f o l l o w i n g r e a c t i o n s : 0< -D-glucose ex -D-glucose _ 2 ,3,1+,-6-tetra-O-acetyl-cX- 6 - 0 - t r i t y l - 2 , 3 ^ - t r i - O - b e n z o y l -- D - g l u c o s y l bromide ON-D-glucosyl f l u o r i d e I " i I : . 2 ,3, »+-tri-Obenzoyl-o\-D-glucosyl I f l u o r i d e 6 - ( t e t r a a c e t y l - / 3-glucopyranosyl ) ' - 2 , 3 , 5-tribenzoyl- o<-D-glucosyl f l u o r i d e 6 - ( ( i -D-glucopyranosyl)-QC-D-glucopyranose ( g e n t i o b i o s e ) . 7 . By the t r i t y l a t i o n r e a c t i o n i t was p o s s i b l e to synthesize a molecule w i t h the C-6 p o s i t i o n u n s u b s t i t u t e d , thus opening the way t o l - a - 6 ' and 1 - / 3 - 6 ' d i s a c c h a r i d e synthses ( 1 0 ) . The f i r s t preparations of the t r i t y l ethers of the p o l y o l s were made by V a l e n t i n ( 2 8 ) . By r e a c t i n g one mole of h e x i t o l w i t h two moles of t r i t y l c h l o r i d e he prepared the d i t r i t y l de-r i v a t i v e s of the common h e x i t o l s except d u l c i t o l . He presumed the low s o l u b i l i t y of d u l c i t o l i n p y r i d i n e to be the reason f o r h i s f a i l u r e to prepare l , 6 - d i - 0 - t r i t y l - d u l c i t o l . Wolfrom (31) however, found that 1 , 6 - d i t r i t y l - d u l c i t o l c r y s t a l l i z e d as a d i p y r i d i n i u m a d d i t i o n compound from the r e a c t i o n mixture and when t h i s a d d i t i o n compound was r e c r y s t a l l i z e d from eth a n o l , the a c i d i t y of the a d d i t i o n compound was s u f f i c i e n t to de-t r i t y l a t e i t l e a v i n g t r i p h e n y l c a r b i n o l and d u l c i t o l . The a d d i t i o n compound was decomposed by bases without d e t r i t y l a t i o n to give the l , 6 - d i - 0 - t r i t y l - d u l c i t o l . T r i p h e n y l c a r b i n o l as a t e r t i a r y a l c o h o l has the tendency to r e a c t by s p l i t t i n g out the e n t i r e hydroxyl group together w i t h i t s b i n d i n g e l e c t r o n s , l e a v i n g a f a i r l y s t a b l e t r i t y l carbonium i o n ( 8 ) . This carbonium i o n i s sbable i n a c i d s o l u t i o n . The proton provided "by the a c i d adds to the oxygen atom of the t r i p h e n y l c a r b i n o l or t r i t y l e ther. Ph^C-OH + H+ P n 3 C + + \ ° Ph 3C-Cl + H + P h 3 C + + HC1 Ph 3C + + OR" Ph^C-OR This mechanism shows why t r i t y l ethers are e a s i l y hydrolyzed by d i l u t e a c i d s and are q u i t e s t a b l e to a l k a l i n e c o n d i t i o n s , since a l k a l i causes l i t t l e or no formation of 8 the t r i t y l carbonium i o n (10). Cleavage of t r i t y l ethers i s e a s i l y accomplished by d i l u t e a c i d s which s h i f t s the above e q u i l i b r i u m to the l e f t . T his f a c t i s of value i n syntheses s i n c e a molecule s u b s t i t u t e d w i t h both t r i t y l groups and a c e t y l groups upon treatment w i t h d i l u t e a c i d would have the t r i t y l groups s e l e c t i v e l y removed and leave the a c e t y l groups i n t a c t and thus form a s p e c i f i c a l l y s u b s t i t u t e d molecule. ILsomerization of a p o l y o l d e r i v a t i v e by a c y l m i g r a t i o n has been observed many times (26) . F i s c h e r (6) s t u d i e d a c e y l a -t i o n experiments on s u b s t i t u t e d g l y c e r i d e s and found that i n an i <x,P - d i g l y c e r i d e (IX) m i g r a t i o n took place to form an <x ,o< -d i g l y c e r i d e (X) durin g a n a c y l a t i o n C H o 0 R ' I 2 , C H O R + RC1 CHo0R I 2 CHOR CHgOR X CHgOH IX F i s c h e r proposed the formation of a c y c l i c o r t h o e s t e r (XII) as an intermediate i n t h i s m i g r a t i o n CHg-O-C^ CH2-0-c( CH-O-C JHgOH X I 0 CE, CH-0. .OH NC •> /\ CHj-j-O CH3 X I I CH 2-0-C^ CHOH CH 2-0-C^ I H 3 CH, X I I I I n methylations of a c y l a t e d carbohydrates u s i n g the Purdie technique (23) a c y l m i g r a t i o n has f r e q u e n t l y been 9 . observed. Haworth ( 9 ) showed a c e t y l m i g r a t i o n occurred i n the methylation of m e t h y l - 2 , 3 , l + - t r i - 0 - a c e t y l - o t -D-glucopyranoside, y i e l d i n g m e t h y l ^ - O - m e t h y l ^ j ^ t ^ - t r i - O - a c e t y l glucopyranoside. H e l f e r i c h ( 1 1 ) noted t h a t i n the methylation of the correspond-i n g t r i b e n z o y l d e r i v a t i v e no migration.of the benzoate e s t e r group occurred. I n the m a j o r i t y of cases examined, the movement of the e s t e r group was toward the primary h y d r o x y l . H e l f e r i c h and K l e i n ( 1 2 ) found that a l k a l i present i n s o f t g l a s s was s u f f i c i e n t to cause I s o m e r i z a t i o n of l ^ ^ A " ^ ^ 3 ' 0 " 3 0 6 ^ ! " 0 (" D-glucose to the corresponding 1 , 2 , 3 , 6 t e t r a a c e t a t e . The pr o x i m i t y ( 9 ) of the groups i n p o s i t i o n s f o u r and s i x i n the c y c l i c sugar molecule appeared to enhance the formation of a s t r a i n l e s s r i n g , thus making m i g r a t i o n f e a s i b l e . Although F i s c h e r ( 6 ) s t u d i e d a c y l m i g r a t i o n between the adjacent p o s i t i o n s i n g l y c e r o l d e r i v a t i v e s , no work has been done on a c y l m i g r a t i o n i n p a r t i a l l y a c e t y l a t e d h e x i t o l molecules during a methylation r e a c t i o n . I n 19^6 Wiggins ( 3 0 ) prepared 1 ,6-di-O-methyl-D-mannitol using the f o l l o w i n g s e r i e s of r e a c t i o n s : D-mannitol I triacetone-D-mannitol 3,*+-monoacetone-D-mannitol i l , 6-di - 0-tosyl - 3, if-monoacetone-D-mannitol 1 , 6 - d i - 0 - t o s y l - 2 , 5 - d i - 0 - a c e t y l - 3 ^-monoacetone-D-mannitol 1 , 2 ^ ? , 6-dianhydro - 3, if-monoac e tone-D-manni t o l l , 6-di - 0-methyl - 3,*f-monoacetone-D^mannitol 1 , 6-di-O-dimethyl-D-mannitol 10. DISCUSSION A. 1 . 6-Di-O-trity-l-D-mannitol Tetraacetate The simultaneous t r i t y l a t i o n and a c e t y l a t i o n of D-mannitol proceeded smoothly i n t h i s s y n t h e s i s . The m o d i f i c a t i o n of Jeanloz (18) of the a c e t y l a t i o n procedure gave l , 6 - d i - 0 -t r i t y l - D - m a n n i t o l t e t r a a c e t a t e completely f r e e from t r i p h e n y l -c a r b l n o l . The ol d e r a c e t y l a t i o n procedure r e q u i r e d pouring of the r e a c t i o n mixture i n t o a large-volume of i c e and water. In cases where no s o l i d contaminants or only water s o l u b l e s o l i d contaminants were present t h i s method o f f e r r e d no d i f f i c u l t i e s . However, i n the case of simultaneous t r i t y l a t i o n and a c e t y l a -t i o n t h i s procedure r e s u l t e d i n the p r e c i p i t a t i o n of unreacted t r i t y l c h l o r i d e as t r i p h e n y l c a r b i n o l , and since l , 6 - d i - 0 - t r i t y l -D-mannitol t e t r a a c e t a t e and t r i p h e n y l c a r b i n o l have very n e a r l y the same s o l u b i l i t i e s i n r e c r y s t a l l i z a t i o n s o l v e n t s , the separa-t i o n of these two compounds presented a problem. The Jeanloz method (18) avoided t h i s d i f f i c u l t y and 1 , 6 - d i - O - t r i t y l - D -mannitol t e t r a a c e t a t e was recovered almost uncontaminated from the r e a c t i o n mixture. B. 1 , 6 - D i - O - t r i t y l - D - m a n n i t o l 1 , 6 - D i - O - t r i t y l - D - m a n n i t o l has not p r e v i o u s l y been reported c r y s t a l l i n e . I t was obtained here i n s o l i d form, presumably as a c r y s t a l l i n e s o l v a t e s i n c e s o l i d m a t e r i a l was obtainable o n l y from an alcohol»water solvent p a i r . Examina-t i o n under the microscope c l e a r l y revealed a n e e d l e - l i k e c r y s t a l form, although the melting p o i n t range was 73 - 78°C Wolfrom (31) reported t h a t sometimes t r i t y l ethers form complexes 1 1 . w i t h such s o l v e n t s as a l c o h o l or p y r i d i n e . S e v e r a l preparations of 1 , 6 - d i - O - t r i t y l - D - m a n n i t o l were made and the t r i t y l group a n a l y s i s was the same f o r each p r e p a r a t i o n a f t e r a r e c r y s t a l l i z a -t i o n from alcohol-water. Further p u r i f i c a t i o n of t h i s compound was made by chromatographic a d s o r p t i o n on an alumina column ( 3 2 ). Several e x p l o r a t o r y columns were run to check the a d s o r b a b i l i t y of the alumina toward the l , 6 - d i - 0 - t r i t y l - m a n n i t o l , and i t was found t h a t , using concentrated s u l f u r i c a c i d as a s t r e a k reagent, a high c o n c e n t r a t i o n of t r i t y l group-containing m a t e r i a l remained at the top of the column even a f t e r washing w i t h a considerable volume of chloroform. According to Z e i l e and Kruckenberg ( 3 2 ) , chloroform removes t r i p h e n y l c a r b i n o l from an alumina column. Since the compound was p u r i f i e d chromatographically and could be converted i n good y i e l d to 1 , 6 - d i - O - t r i t y l - D -mannitol t e t r a a c e t a t e by a c e t y l a t i o n , i t appeared that 1 , 6 -d i - O - t r i t y l - D - m a n n i t o l had been prepared, p o s s i b l y i n a s o l v a t e d form, and any monosubstitution or t r i s u b s t i t u t i o n products as w e l l as t r i p h e n y l c a r b i n o l were e l i m i n a t e d i n the p u r i f i c a t i o n . V a l e n t i n (28) reported a melting p o i n t of 98 - 1 0 3°C f o r 1 , 6 -d i - O - t r i t y l - D - m a n n i t o l but h i s product was subsequently shown to be amorphous ( 2 2 ) . C. D e t r i t y l a t i o n Procedures T r i t y l ethers of carbohydrates are extremely s e n s i t i v e to a c i d and even s m a l l amounts are s u f f i c i e n t to cause d e t r i t y l a -t i o n ( 1 0 ) . They are cleaved to t r i p h e n y l c a r b i n o l and an a l c o h o l by a c i d s a t room temperature, a r e a c t i o n a p p l i e d to carbohydrate d e t r i t y l a t i o n s by s e v e r a l authors ( 1 0 ) . 12. C. (1) Hydrogen Bromide I n G l a c i a l A c e t i c A c i d T r i t y l ether cleavage by hydrogen bromide was achieved and the i n s o l u b l e t r i t y l bromide removed. Wolfrom (31) reported t h a t , i n some cases, excess hydrogen bromide present i n the r e a c t i o n mixture brominated the d e t r i t y l a t e d product to form the corresponding deoxybromide compound. TrOR + 2HBr *- TrBr + RBr + H^ O In the present case i t appeared p o s s i b l e that the hydrogen bromide brominated the D - m a n n i t o l - 2 , 3 , t e t r a a c e t a t e . The s i r u p from t h i s r e a c t i o n d i d not c r y s t a l l i z e a f t e r many t r i a l s ; a mixture c o n t a i n i n g 1,6-dideoxy-dibromo-D-mannitol t e t r a -acetate and t r i t y l bromide or t r i p h e n y l c a r b i n o l would be d i f f i c u l t to c r y s t a l l i z e and halogen a n a l y s i s would not be c o n c l u s i v e . C. (2) Hydrogenation i n G l a c i a l A c e t i c A c i d over a Platinum  Oxide C a t a l y s t . This method gave c r y s t a l l i n e D - m a n n i t o l - 2 , 3 , ^ ^ - t e t r a -acetate i n low y i e l d . Tricyclohexylmethane was recovered from the r e a c t i o n i n d i c a t i n g that complete r e d u c t i o n of the benzene n u c l e i had taken place. The c o n d i t i o n s i n t h i s r e a c t i o n were d i f f i c u l t to c o n t r o l and a c r y s t a l l i n e product was only once obtained. Two reasons f o r incomplete r e a c t i o n are suggested. (a) S u s c e p t i b i l i t y of the c a t a l y s t to poisoning: Unless extreme care was e x e r c i s e d i n p u r i f y i n g the 1,6-di-O - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e the c a t a l y s t was poisoned and the s t a r t i n g m a t e r i a l was recovered unchanged. 13 (b) The p o s s i b i l i t y t h a t the r e d u c t i o n of the benzene n u c l e i had occurred before the ether cleavage thus i n h i b i t i n g the r e a c t i o n (21 ). c» <3) D i l u t e A c e t i c A c i d This method gave the most c o n s i s t e n t r e s u l t s , h i g h e s t y i e l d s , and e a s i e s t p u r i f i c a t i o n of product. The a d d i t i o n of excess water p r e c i p i t a t e d the t r i p h e n y l c a r b i n o l almost qu a n t i -t a t i v e l y and the mannitol t e t r a a c e t a t e remained i n s o l u t i o n . The melting poi n t (158 - 162°C.) of the p r e c i p i t a t e was almost tha t f o r pure t r i p h e n y l c a r b i n o l i n d i c a t i n g that very l i t t l e h e x i t o l m a t e r i a l had c o - p r e c i p i t a t e d . The a c e t i c a c i d method was used almost e x c l u s i v e l y a f t e r s e v e r a l t r i a l s w i t h methods (1) and (2). The use of m i n e r a l a c i d i n chloroform was not considered s i n c e the s o l u -b i l i t y of t r i t y l c h l o r i d e , t r i p h e n y l c a r b i n o l , and D-mannitol-2,3,^,5-tetraacetate i n chloroform was h i g h and an i n i t i a l s e p a r a t i o n of the end products would be d i f f i c u l t . Chloroform petroleum-ether formed a good r e c r y s t a l l i -z a t i o n solvent since any t r i p h e n y l c a r b i n o l , which was not removed by the i n i t i a l water p r e c i p i t a t i o n , remained i n s o l u t i o n and was not p r e c i p i t a t e d by the a d d i t i o n of petroleum-ether. D. D-Mannitol-2 . 3.h.^-Tetraacetate D-Mannitol-2,3,H,5-tetraacetate was reported c r y s t a l l i n e by Micheel (21). He assumed that d e t r i t y l a t i o n of 1,6-di-O-t r i t y l - D - m a n n i t o l t e t r a a c e t a t e would give D-mannitol-2,3,h,5-t e t r a a c e t a t e because d e t r i t y l a t i o n of 6 - t r i t y l - l , 2 , 3 , 1 + - t e t r a -O-acetyl-D-glucopyranose gave the corresponding c r y s t a l l i n e 1,2,3,^-tetraacetate. l h . The p h y s i c a l constants and a c e t y l a n a l y s i s of the compound i n t h i s l a b o r a t o r y agreed w i t h the prepared data given by Micheel (21) and a c e t y l a t i o n of our D-mannitol - 2 , 3 , h,5-tetraacetate gave D-mannitol-hexaacetate i n good y i e l d . An attempted t r i t y l a t i o n of the t e t r a a c e t a t e to give 1 , 6 -d i t r i t y l - D - m a n n i t o l t e t r a a c e t a t e y i e l d e d only t r i p h e n y l c a r b i n o l and a s i r u p . D-Mannitol - 2 , 3 , * + , 5 - t e t r a a c e t a t e was slow to c r y s t a l l i z e , o_ and even when seeded r e q u i r e d one to two weeks at 0 C. f o r complete c r y s t a l l i z a t i o n (m.p. of pure compound 1 2 3 ° c ) • A l i t e r a t u r e search revealed that of the nine p o s s i b l e isomeric D-mannitol-tetraacetates only three have been prepared: D - m a n n i t o l - 1 , 3 6 - t e t r a a c e t a t e , m.p., 1 0 7 ° c « ( 3 ) . D - m a n n i t o l - l ^ ^ ^ - t e t r a a c e t a t e , m.p., 92°C. ( 2 9 ) . D - m a n n i t o l - 2 , 3 , ^ , 5 - t e t r a a c e t a t e , m.p., 1 2 3 ° c . ( 2 1 ) . E. Di-O-methyl-D-mannltol M e t h y l a t i o n of the D - m a n n i t o l - 2 , 3 A > 5 - t e t r a a c e t a t e f o l l o w e d by d e a c e t y l a t i o n gave a syrupy di-O-methyl h e x i t o l d e r i v a t i v e . The s o l u b i l i t y and methoxyl value reported by Wiggins (30) f o r 1 , 6-di-O-methyl-D-mannitol agreed w e l l w i t h the compound prepared here. The dimethyl-mannitol was a s i r u p and i t was i n v e s t i g a t e d by means of paper chromatography to show one or more c o n s t i t u e n t s were present. I t i s known that s e p a r a t i o n of i n d i v i d u a l h e x i t o l s on paper chromatograms i s d i f f i c u l t , s i n c e they a l l have approximately the same R f values ( 1 9 ) . F i n d i n g a s a t i s f a c t o r y spray reagent a l s o proved d i f f i c u l t s i nce sodium metaperiodate i n a c i d i c potassium permanganate; a l k a l i n e potassium permanganate; ammonical 15. s i l v e r n i t r a t e gave no r e a c t i o n and n e u t r a l sodium metaperiodate f o l l o w e d by d i l u t e ethylene g l y c o l and aqueous potassium i o d i d e was so s e n s i t i v e that no d e f i n i t e conclusions could be drawn. In an attempt to get a c r y s t a l l i n e d e r i v a t i v e the dimethyl mannitol was t r e a t e d w i t h formaldehyde to o b t a i n a dimethylene compound. l , 6-DImethyl - 2,*f : 3 , 5-dimethylene-D-mannitol was a l s o reported by Wiggins ( 3 0 ) . Seed c r y s t a l s of t h i s compound and of 1 , 6-di-O-methyl-D-mannitol have^Deen obtained r e c e n t l y from Dr. Wiggins but t h e i r use had not yet y i e l d e d any c r y s t a l l i n e products. A c e t y l m i g r a t i o n has been reported (9) from the f o u r p o s i t i o n to the s i x p o s i t i o n i n p a r t i a l l y a c e t y l a t e d D-gluco-pyranose d e r i v a t i v e s during the course of m e t h y l i o d i d e - s i l v e r -oxide methylation. According to Haworth (9) the methylation i t s e l f was not the determining f a c t o r i n a c e t y l m i g r a t i o n but r a t h e r the a l k a l i n e c o n d i t i o n s produced by the s i l v e r oxide. In the present case, i f m i g r a t i o n occurred even to a small degree, a mixture of dimethyl compounds would r e s u l t which would make c r y s t a l l i z a t i o n d i f f i c u l t . I s o l a t i o n , i n good y i e l d , of c r y s t a l l i n e 1 , 6-di-O-methyl-D-mannitol would prove that the mannitol t e t r a a c e t a t e obtained was a c t u a l l y the 2 , 3 > ^ , 5 - t e t r a a c e t a t e isomer, s i n c e methylation would s u b s t i t u t e only the primary hydroxyls of the molecule. Furthermore, i t would show i n t h i s case a t l e a s t t h a t a c e t y l m i g r a t i o n d i d not occur during a methylation u s i n g the Purdie reagents. This was p a r t i a l l y borne out i n the f i r s t methylation which, a f t e r proceeding f o r three hours f o l l o w e d by the usual treatment of the r e a c t i o n mixture, y i e l d e d c r y s t a l s of the s t a r t i n g m a t e r i a l . Although not a l l the 16. s t a r t i n g m a t e r i a l was recovered i t i n d i c a t e d t h a t , i n t h i s case at l e a s t , m i g r a t i o n d i d not r e a d i l y take p l a c e , since some of the compound must have been methylated. I f a c e t y l m i g r a t i o n had taken place then a f t e r de-a c e t y l a t i o n a mixture of dimethyl mannitols was obtained. Se p a r a t i o n of t h i s mixture i n t o two or more components would show that a c e t y l m i g r a t i o n had occurred. The f o l l o w i n g t a b l e gives a l i s t of the nine isomeric dimethyl-D-mannitols and the r e s u l t s of t h e i r r e a c t i o n w i t h p e r i o d i c a c i d . Except f o r case 5 and case 6 the behavior of each of the dimethyl-D-mannitols toward p e r i o d i c a c i d would be unique. From a c o n s i d e r a t i o n of the s t r u c t u r e s i n v o l v e d the f o u r compounds 1,2,7>9 would be the most probable compon-ents of a mixture r e s u l t i n g from a c e t y l m i g r a t i o n and these are e a s i l y d i s t i n g u i s h a b l e by t h e i r r e a c t i o n towards p e r i o d a t e . I f m i g r a t i o n occurred here i t would be of i n t e r e s t t o prepare other D-mannitol t e t r a a c e t a t e s and study a c y l migra-t i o n under methylation c o n d i t i o n s . Since a c y l m i g r a t i o n during methylation i s known to occur (9) i n p a r t i a l l y a c e t y l a t e d r i n g forms of sugars i t would be of i n t e r e s t to study the r e l a t i v e ease of m i g r a t i o n o c c u r r i n g i n these forms w i t h that o c c u r r i n g i n the s t r a i g h t c h a i n h e x i t o l molecules. Since t r i t y l c h l o r i d e has been shown to r e a c t w i t h both primary hydroxyls and secondary hydroxyls i n a polyhydroxy compound c o n t a i n i n g these groups, the study of the r e l a t i v e t r i t y l a t i o n r a t e s of the primary and secondary hydroxyls i n a h e x i t o l molecule could be made. Theoretical Products of the Periodate Oxidate of D-Mannitol Dimethyl Ethers n n Moles Oxidation Products (moles) Case P o s i t i o n of -OCH, Periodate consumed HCHO HCOOH Other groups -> CHoOMe 1 1,6 3 2 2| ^  CHO 0HC-CH20Me 2 2 > 6 2 1 H0H2C MeOCH-CHO CHO CHO 3 3 , 6 2 1 HCOMe & CH20Me CHO TH 20Me ^ 1 , 2 3 1 2 MeOCH CHO CH20Me 2 1 1 HOCH MeOCH CHO C^OH 2 x x MeO(jH MeOCH CHO CH?OH 7 2,5 1 2MeOCH CHO CHO Q , e T i MeOCH O 3 , 5 1 1 H J 0 H HC0Me-CH20H O i l C H 0 9 3 , H - 2 2 MeO^H HCOMe CHO 1,3 2,3 EXPERIMENTAL 18. A l l m e l t i n g points were determined i n an apparatus p r e v i o u s l y c a l i b r a t e d w i t h a s e r i e s of pure compounds. I n view of the s e n s i t i v e nature of the carbohydrate m a t e r i a l a l l evaporations were c a r r i e d out at reduced pressure and tempera-tures not exceeding 5 0°C A. M a t e r i a l s  Benzene Absolute benzene was prepared by the method of F i e s e r (5) and stored over sodium. P y r i d i n e P y r i d i n e was stored over s o l i d potassium hydroxide f o r s e v e r a l weeks and d i s t i l l e d from barium oxide immediately before use. Methanol Absolute methanol was prepared by two successive d i s t i l l a t i o n s of the commercial absolute grade m a t e r i a l from magnesium t u r n i n g s . A c e t i c Anhydride A n a l y t i c a l reagent grade a c e t i c anhydride s u p p l i e d by B r i t i s h Drug Houses was d i s t i l l e d before use. Toluene Absolute toluene was prepared by the method of F i e s e r (5) and stored over sodium. A c e t y l C h l o r i d e Reagent grade a c e t y l c h l o r i d e as s u p p l i e d by Merck and Company and used without f u r t h e r p u r i f i c a t i o n . 19. Methyl Iodide Methyl i o d i d e was obtained from B r i t i s h Drug Houses and d i s t i l l e d before use. T r i p h e n y l c a r b i n o l A student p r e p a r a t i o n of t r i p h e n y l c a r b i n o l was p u r i f i e d by one r e c r y s t a l l i z a t i o n from carbon t e t r a c h l o r -ide and one from 95% ethanol: m.p., 161.0 - 162.5 ° c . D-Mannitol D-Mannitol as supplie d by the Matheson Chemical Company was r e c r y s t a l l i z e d from aqueous ethanol: m.p., 165 - 166°C [o(]*k - 0.195° (c, 5.359; l , l ; H 2 0 ) . The s p e c i f i c r o t a t i o n was a l s o observed i n 5% aqueous ammonium molybdate s o l u t i o n (27). 1 1 22 * J + 2h.0° (C, 5.093; l , l ; a q . (NHk.)6 M0702u.) Barium Methylate A s o l u t i o n of barium methylate i n absolute methanol was prepared by the method of I s b e l l (17). T i t r a t i o n of a 6.0 ml. a l i q u o t w i t h 0.572 N s u l f u r i c a c i d showed the barium methylate c o n c e n t r a t i o n to be 0.381 normal. . Triphenylmethyl C h l o r i d e (1) Dry, pure t r i p h e n y l c a r b i n o l (100.0 g.) was d i s s o l v e d i n dry, thiophene-free benzene (32 ml.), the s o l u t i o n was heated on a steam bath and, when hot, a c e t y l c h l o r i d e (20.0 ml.) was added w i t h s t i r r i n g . A f t e r f i v e minutes heating a d d i t i o n a l a c e t y l c h l o r i d e (30.0 ml.) was added over a period of one-half hour. The r e a c t i o n mixture was 20. r e f l u x e d a f u r t h e r haIf-hour a f t e r the f i n a l a d d i t i o n of a c e t y l c h l o r i d e . The a d d i t i o n of two volumes of low-b o i l i n g petroleum-ether to the cooled r e a c t i o n mixture caused the formation of a heavy white p r e c i p i t a t e . The f l a s k was placed i n an i c e bath f o r two hours to complete p r e c i p i t a t i o n . The c r y s t a l s were removed by f i l t r a t i o n , washed w i t h 100 ml. petroleum-ether, and d r i e d i n vacuo over c a l c i u m c h l o r i d e , soda lime and p a r a f f i n shavings. Y i e l d : 71.5 g. (68$), m.p., 110 - 111°C. ( 1 ) . The product was c o l o r l e s s but f o r a pale green luminescence and was used without f u r t h e r p u r i f i c a t i o n . T r i t y l c h l o r i d e prepared by t h i s method was much su p e r i o r to that commercially a v a i l a b l e . The commercial product even a f t e r s e v e r a l r e c r y s t a l l i z a t i o n s had a dark y e l l o w c o l o r and melted a t 106 - 108°C. A n a l y t i c a l Methods  A c e t y l a n a l y s i s A c e t y l groups were determined by the method of C l a r k ( i f ) . Methoxyl a n a l y s i s Methoxyl groups were> determined by the modified Z e i s e l method as described by C l a r k (**). T r i t y l a n a l y s i s T r i t y l groups ( t r i p h e n y l m e t h y l groups) were deter-mined by the method of V a l e n t i n (28). I n order to determine the maximum s o l u b i l i t y l o s s e s i n a t r i t y l d etermination by the use of excess reagents a sample of t r i p h e n y l c a r b i n o l was analyzed f o r t r i t y l 21. content. T r i p h e n y l c a r b i n o l (0.10285 g.) was d i s s o l v e d i n concentrated s u l f u r i c a c i d (5.0 ml.) and the r e s u l t i n g orange s o l u t i o n was poured i n t o c o l d water (50.0 ml.). The c h a r a c t e r i s t i c p r e c i p i t a t e was c o l l e c t e d on a s i n t e r e d g l a s s f u n n e l , washed w i t h c o l d water (175 ml.) to remove a l l t r a c e s of s u l f u r i c a c i d and d r i e d i n vacuo over phosphorus pentoxide t o constant weight; (0.10180 g.), m.p., 159 - 161°C. I t was concluded that approximately a one per cent l o s s might be expected i n a t r i t y l a n a l y s i s . D-Mannitol Hexaacetate D-Mannitol hexaacetate was prepared by the method of Baer and F i s c h e r (2) f o r use as a reference compound. The me l t i n g p o i n t a f t e r two r e c r y s t a l l i z a t i o n s from 95$ ethanol was 122 - 123 °C; y i e l d , 89.5$. Found: a c e t y l , 58.7$, 59.h% C a l c u l a t e d f o r C ^ H ^ O ^ ' a c e t y l , 59.*+$ 1 . 6 V D i - 0 - t r i t y l - D - M a n n i t o l Tetraacetate A f t e r s e v e r a l p r e l i m i n a r y experiments the f o l l o w i n g procedure adapted from Reynolds and 3rans (2h) and Jeanloz (18) was found to be s a t i s f a c t o r y f o r the p r e p a r a t i o n of 1,6-di-O - t r i t y l - D i - m a n n i t o l - t e t r a a c e t a t e . Dry D-mannitol (0.083 mole) and t r i t y l c h l o r i d e (0.166 mole) were d i s s o l v e d i n d r i e d p y r i d i n e (150 ml.) c o n t a i n i n g D r i e r i t e (3.5 g.) as an i n t e r n a l d e s i c c a n t . The r e s u l t i n g pale yellow mixture was heated to MO°C. and 22. s t i r r e d mechanically f o r 2\ hr. F r e s h l y d i s t i l l e d a c e t i c anhydride (0.8? mole) was then added and s t i r r i n g and heating were continued f o r an a d d i t i o n a l 1+8 hr. At t h i s time the r e a c t i o n mixture was dark yellow i n c o l o r . The d r i e r i t e was removed by f i l t r a t i o n and excess absolute methanol was added s l o w l y to the i c e c o l d f i l t r a t e to decompose the excess a c e t i c anhydride. N o r i t e (1.0 g.) was then added and the mixture was allowed to stand f o r s e v e r a l hours a t room temperature. Removal of the n o r i t e l e f t a yellow s o l u t i o n . The methyl a c e t a t e , methanol, a c e t i c a c i d were removed by d i s t i l l a t i o n under reduced pressure and the p y r i d i n e was removed by a z e o t r o p i c d i s t i l l a t i o n w i t h dry toluene a l s o under reduced pressure. S e v e r a l d i s t i l l a t i o n s w i t h toluene were necessary f o r complete removal o f a l l t r a c e s of p y r i d i n e . When about one-half of the volume of the solvent had been removed a heavy white p r e c i p i t a t e formed which was f i l t e r e d , washed w i t h absolute methanol, and d r i e d i n vacuo over phosphorus pentoxide. Weight of d r i e d product, 21.0 g.; m.p., iQh -186°C. Upon f u r t h e r evaporation two more crops of c r y s t a l s were c o l l e c t e d weighing 2*+.0 g. and 3.0 g. r e s p e c t i v e l y ; m.p., 180 - lSV^C. The mother l i q u o r , being dark red and g i v i n g no f u r t h e r p r e c i p i t a t i o n upon the a d d i t i o n of absolute methanol, was discarded. T o t a l y i e l d , h8.0 g. (70%). The c r y s t a l s were very s o l u b l e i n chloroform and i n s o l u b l e i n methanol, e t h a n o l , and petroleum-ether. Two r e c r y s t a l l i z a t i o n s from chlorofrom-methanol gave a c o l o r l e s s 2 3 -product melting a t 18H.5 - 186.0°C. W ^ = (C, h.878; 1, 1; C H C 1 3 ) . R e c r y s t a l l i z a t i o n from chloroform-methanol gave almost q u a n t i t a t i v e recovery of the compound. Use of t h i s s o l v e n t p a i r gave more s a t i s f a c t o r y r e s u l t s than n-butanol which was used by Micheel (21). Wolfrom ( 3 D reported a melting p o i n t of 183 - 18*+°C. and a s p e c i f i c r o t a t i o n of +l+6.1*0 i n chloroform f o r 1,6-d i t r i t y l - D - m a n n i t o l - t e t r a a c e t a t e . Found: t r i t y l , 57.2%; a c e t y l , 20.25$. C a l c u l a t e d f o r C t ^ H ^ l O : t r i t y l , 57.5$; a c e t y l , 20.6$. From the observed constants i t was concluded th a t the compound was 1 , 6 - d i t r i t y l - D - m a n n i t o l t e t r a a c e t a t e as reported by Micheel (21) and Wolfrom (31). E. 1.6-Di-O-tri tvl-D-Manni t o l Dry D-mannitol (0.028 mole) and t r i t y l c h l o r i d e (0.056 mole) were d i s s o l v e d i n d r i e d p y r i d i n e (75 ml.) con t a i n i n g D r i e r i t e (1.0 g.) as an i n t e r n a l d esiceant. The r e a c t i o n mixture was heated to hO°C. and s t i r r e d mechanically f o r 17 to 18 h r . , a f t e r which the d r i e r i t e was removed by f i l t r a t i o n and the f i l t r a t e added dropwise to three l i t e r s of i c e and water. A v i s c o u s , c o l o r l e s s s i r u p formed which a f t e r standing f o r two days at 0°C. hardened to a s e m i c r y s t a l l i n e mass. This m a t e r i a l was d r i e d i n vacuo over concentrated s u l f u r i c a c i d and s o l i d potassium hydroxide. Y i e l d , 13.28g.; (72$). According to V a l e n t i n (28), d i t r i t y l h e x i t o l s are so l u b l e i n h y d r o x y l i c s o l v e n t s as w e l l as chloroform and benzene. Hence, the 2h. d r i e d product was e x t r a c t e d three times a t room temperature w i t h an equal weight of absolute methanol, and the e x t r a c t s were combined, concentrated, warmed and f i l t e r e d through n o r i t e . Hot water was added to the f i l t r a t e to t u r b i d i t y . A f t e r standing s e v e r a l days a t room temperature, the s o l u t i o n deposited a s e m i c r y s t a l l i n e product, m.p., 75 - 85°C. Two f u r t h e r p r e c i p i t a t i o n s from methanol-water y i e l d e d a c o l o r -l e s s product which softened t o a c l e a r l i q u i d between 73 and 78°C. Observation of t h i s product under a microscope showed no d e f i n i t e c r y s t a l s t r u c t u r e and i t was assumed to be a g l a s s . Three hundred mg. of the l , 6 - d i - 0 - t r i t y l - m a n n i t o l were d i s s o l v e d i n 20 ml. chloroform and placed on a 3*5 x 1 0 . 0 cm. alumina column which was then washed w i t h 300 ml. chloroform to remove any t r i p h e n y l c a r b i n o l . The l a s t two m i l l i l i t e r s of chloroform e l u a t e gave a negative t r i t y l t e s t . Evaporation of the chloroform eluate y i e l d e d o n l y a small amount of s o l i d m a t e r i a l which had a m e l t i n g p o i n t of 151 - 155°C- This was assumed to be t r i p h e n y l c a r b i n o l ( 2 1 ) . The column was extruded and e x t r a c t e d i n a Sohxlet e x t r a c t o r w i t h 95% ethanol c o n t a i n i n g 1 . 0 $ chloroform f o r h hr. Evaporation of the ethanol e x t r a c t y i e l d e d 200 mg. of s o l i d m a t e r i a l which o b s e r v a t i o n under a microscope proved crystarllihe.:.. The product had the same melting p o i n t as before (73 - 78°C.) and was not a l t e r e d by a r e c r y s t a l l i -z a t i o n from methanol-water. Attempted r e c r y s t a l l i z a t i o n s from ethanol petroleum-ether and chloroform petroleum-ether 2 5 . gave s i r u p s i n each case. Found: t r i t y l , , 6 5 . 0 $ C a l c u l a t e d f o r C]+ify.2°6 : t r i t y l , 7 2 . 8 $ C a l c u l a t e d f o r C i f i f ^ f 2 ° 6 * l f H 2 0 1 t r i t v l > 6 5 . 8 $ . A c e t y l a t i o n of 1 . 6 - d i - O - t r i t v l - D - M a n n l t o l l , 6 ~ D i - 0 - t r i t y l - D - m a n n i t o l (100 mg.) was d i s s o l v e d i n d r i e d p y r i d i n e ( 2 . 0 ml.) and f r e s h l y d i s t i l l e d a c e t i c anhydride ( 1 . 5 ml.) was added. The r e s u l t i n g s o l u t i o n was heated to 60 C. on a water hath and then allowed to stand at room temperature f o r 2h hr. when i t waspoured i n t o an excess of i c e and water. The c r y s t a l l i n e product which separated was f i l t e r e d , d r i e d i n vacuo over phosphorus pentoxide and r e c r y s t a l l i z e d from chloroform-methanol s o l u t i o n . Y i e l d , 0 . 0 8 2 g. ( 6 6 $ ) , m.p., 18M-.5 - 186°C. A mixed melting point taken w i t h an authentic sample o f 1 , 6 - d i - O - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e showed no depression. D e t r i t y l a t i o n Procedures A p p l i e d to 1 . 6 - D i t r i t y l - D - M a n n i t o l  Tetraacetate (1) Hydrogen Bromide i n G l a c i a l A c e t i c A c i d (21) 1 , 6 - D i - O - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e ( 2 . 0 g.) was d i s s o l v e d i n warm g l a c i a l a c e t i c a c i d ( 2 5 ml.) ( d i s t i l l e d from chromium t r i o x i d e ) . The s o l u t i o n was cooled to 5 ° c » a n d 3 - 0 ml. of a 30$ s o l u t i o n of hydrogen bromide i n g l a c i a l a c e t i c a c i d was added, One minute l a t e r a p r e c i p i t a t e formed which was Immediately removed by s u c t i o n f i l t r a t i o n , washed w i t h petroleum-ether and d r i e d i n vacuo over phosphorus pentoxide and p a r a f f i n shavings. The f i l t r a t e was immediately 26 evaporated to dryness under reduced pressure i n order to i n s u r e complete removal of any excess hydrogen bromide. The p r e c i p i t a t e weighed 0 . 9 5 g. (62$), melted a t 151 - 152°C. and gave a p o s i t i v e t e s t f o r the t r i t y l group. I t was assumed to be t r i t y l bromide ( 2 1 ) . The f i l t r a t e , a f t e r evaporation, y i e l d e d a l i g h t y e l l o w s i r u p which was d r i e d and then d i s s o l v e d i n 95$ ethanol c o n t a i n i n g a tr a c e of a c e t i c a c i d . F i l -t r a t i o n through n o r i t e y i e l d e d a c o l o r l e s s s o l u t i o n . The solvent was again removed and the s i r u p d r i e d i n vacuo. The s i r u p was very s o l u b l e i n methanol, ethanol; r e l a t i v e l y s o l u b l e i n chloroform and i n s o l u b l e i n ether and petroleum-ether. S e v e r a l attempts to c r y s t a l l i z e t h i s s i r u p from v a r i o u s s o l v e n t s were u n s u c c e s s f u l . ( 2 ) C a t a l y t i c D e t r i t y l a t i o n u s i n g Hydrogen and Platinum  Oxide C a t a l y s t (21) 1 , 6 - D i - O - t r i t y l - D - m a n n i t o l t e t r a a c e t a t e ( 2 . 0 g.) was d i s s o l v e d i n warm g l a c i a l a c e t i c a c i d (*+0 ml.) ( d i s t i l l e d from chromium t r i o x i d e ) , ' p l a t i n u m oxide ( 0 . 2 0 g.) was added and the mixture was hydrogenated i n a low pressure Parr Hydrogenator. A f t e r 28 hr. heating and shaking, the pressure became constant, the used c a t a l y s t was f i l t e r e d o f f and the f i l t r a t e evaporated under reduced pressure. When one-half the volume of the solvent had been removed, an o i l separated which c r y s t a l l i z e d when cooled to 10°C. and seeded w i t h a c r y s t a l of tricyclohexylmethane. Y i e l d , 0 . 3 0 g.; 27. m.p., 60°C.. Fur t h e r evaporation of the mother l i q u o r y i e l d e d a c o l o r l e s s s i r u p which was d i s s o l v e d i n ethan o l , c o n t a i n i n g a t r a c e of a c e t i c a c i d , and petroleum-ether added to t u r b i d i t y . A f t e r standing s e v e r a l days at 0°C., c r y s t a l s appeared which were removed on a s i n t e r e d g l a s s d i s c , washed thoroughly w i t h petroleum-ether, and d r i e d . Two more r e c r y s t a l -l i z a t i o n s from ethanol petroleum-ether gave c r y s t a l s ; m.p., 122 - 123°C. Another r e c r y s t a l l i z a t i o n f a i l e d to a l t e r the melting p o i n t . Y i e l d , 0.055 g. (.7.0%). D e t r i t y l a t i o n using D i l u t e A c e t i c A c i d (31) 1 , 6 - D i - O - t r i t y l - D - M a n n i t o l t e t r a a c e t a t e (h.l g.) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d (25 ml.) and the s o l u t i o n was heated to r e f l u x temperature. D i s t i l l e d water (^.25 ml.) was then added and r e f l u x i n g was continued f o r one hour when, a f t e r c o o l i n g the r e a c t i o n mixture a f u r t h e r 12.5 ml. of water was added. The p r e c i p i t a t e of t r i p h e n y l c a r b i n o l which appeared immed-i a t e l y , was f i l t e r e d o f f , d r i e d i n vacuo over phosphor-us pentoxide, and weighed. Y i e l d , 2. lf0 g. (9*+$); m.p., 158 - 162°C. A f t e r a r e c r y s t a l l i z a t i o n from 95$ ethanol the c a r b i n o l had a melting poi n t of 161 - 162°C and no depression was observed when a mixed mel t i n g poin t was taken w i t h an authentic sample. The mother l i q u o r was evaporated to dryness under reduced pressure and the r e s u l t i n g s i r u p was taken up i n 95$ ethanol and petroleum-ether was added to t u r b i d -i t y . A f t e r standing a t Ooc. f o r one week, c r y s t a l s 28. separated which a f t e r two f u r t h e r r e c r y s t a l l i z a t i o n s from the above solvent p a i r had a melting p o i n t of 122 - 123°C. Y i e l d , 0.35 g. P a r t i a l evaporation of the solvent from the mother l i q u o r and f u r t h e r a d d i t i o n of petroleum-ether y i e l d e d a second c r o p of c r y s t a l s a f t e r f o u r t e e n days a t 0°C. These c r y s t a l s melted a t 122 - 123°C. a f t e r two r e c r y s t a l l i -z a t i o n s from ethanol petroleum-ether. Y i e l d , 0.10 g. The t o t a l y i e l d of D-mannitol-tetraacetate was 0.*+5 g. (26$). F u r t h e r evaporation of the mother l i q u o r s and subsequent treatment w i t h petroleum-ether f a i l e d to y i e l d f u r t h e r amounts of c r y s t a l l i n e m a t e r i a l . Found: a c e t y l , ^-5.8$ C a l c u l a t e d f o r C ^ B ^ O I Q : a c e t y l , ^6.2$. A mixed melting p o i n t taken w i t h a sample of D-mannitol hexaacetate showed a considerable depression (m.p., 105 - 115°C.) A c e t y l a t i o n of D-Mannitol -2.3.*f. 5 - t e t r a a c e t a t e Dry D - m a n n i t o l - 2 , 3 , ^ ^ - t e t r a a c e t a t e (0.10 g.) was d i s s o l v e d i n d r i e d p y r i d i n e (*+.0 ml.) and f r e s h l y d i s t i l l e d a c e t i c anhydride (U-.O ml.) was added. The c o l o r l e s s s o l u t i o n was allowed to stand at room temperature f o r two days and then poured i n t o 100 ml. of i c e and water. The heavy, c o l o r l e s s c r y s t a l l i n e p r e c i p i t a t e which separated was f i l t e r e d , a i r d r i e d , and r e c r y s t a l l i z e d from 95$ ethanol. Y i e l d , 0.106 g. (85$), m.p., 122 - 12^°C. A second r e c r y s t a l l i z a t i o n d i d not a l t e r the melting p o i n t . 2 9 . When mixed w i t h a sample of D-mannitol hexaacetate, no depression i n melting p o i n t was observed. I . M e t h y l a t i o n of D - M a n n i t o l - 2 , 3 A . 5 - t e t r a a c e t a t e D - M a n n i t o l - 2 , 3 , 5 - t e t r a a c e t a t e ( 0 . 3 5 0 g.) was added to dry acetone ( 5 . 0 ml.) c o n t a i n i n g methyl i o d i d e ( 7 . 0 m l . ) , dry s i l v e r oxide ( * + . 0 g.), and d r i e r i t e ( ^ . 0 g.), and the mixture was r e f l u x e d f o r 2 0 hr. The r e a c t i o n mixture was then cooled to room temperature and the s o l i d s removed by f i l t r a t i o n . The s o l i d m a t e r i a l was e x h a u s t i v e l y e x t r a c t e d w i t h acetone and the chloroform and the combined e x t r a c t s added to the f i r s t f i l t r a t e . Evaporation of the c o l o r l e s s s o l u t i o n under reduced pressure l e f t a c l e a r , c o l o r l e s s s i r u p which, when d r i e d i n vacuo, gave a low methoxyl a n a l y s i s . Found: 0 G H " 3 , 1 2 . 2 $ , 1 1 . 8 $ ; C a l c u l a t e d f o r C ^ H ^ ^ Q : 0 C H 3 , 1 6 . 3 $ . The methylation was repeated using the above method except that the acetone was omitted, the methyl i o d i d e acted as the s o l v e n t . From t h i s methylation a c l e a r , c o l o r l e s s s i r u p was obtained; y i e l d , 0 . 2 9 0 g. Found: OCH3 , 15 M, 1 5 . 5 $ ; C a l c u l a t e d f o r C ^ H ^ O ^ : OCH^ 1 6 . 3 $ S e v e r a l attempts to c r y s t a l l i z e t h i s s i r u p were u n s u c c e s s f u l . This compound has not been p r e v i o u s l y reported i n the l i t e r a t u r e . 3 0 . J . D e a c e t y l a t i o n of the Di-O-methyl-D-mannltol t e t r a a c e t a t e (1) Method of I s h e l l (17) The s i r u p ( 0 . 2 7 V g.) from the above methy l a t i o n was d i s s o l v e d i n absolute methanol ( 1 0 . 0 ml.) c o n t a i n -i n g O .38N barium methylate ( 1 . 0 ml.) and the s o l u t i o n was kept at 0°C. f o r 2h hr. The excess barium methy-l a t e was decomposed by the a d d i t i o n of the s t o i c h i o -metric amount of 2.00N s u l f u r i c a c i d which a t the same time p r e c i p i t a t e d the excess barium i o n as the s u l f a t e . The p r e c i p i t a t e was removed by f i l t r a t i o n and the f i l t r a t e was evaporated to dryness l e a v i n g a c o l o r l e s s s i r u p , y i e l d , 0 . 1 5 0 g. The d r i e d s i r u p was e x t r a c t e d s e v e r a l times w i t h hot e t h y l acetate and the combined e x t r a c t s were again evaporated to dryness l e a v i n g a c o l o r l e s s s i r u p . ( 2 ) Method of McKeown and Hayward (2 0 ) This method made use of an anion exchange r e s i n , Dowex 1 , f o r d e a c e t y l a t i o n . The s i r u p ( 0 . 2 8 5 g.) from the methy l a t i o n d i s s o l v e d i n absolute methanol ( 5 . 0 ml.) was run on the r e s i n i n the column and washed on w i t h a f u r t h e r 6 . 0 ml. of absolute methanol. The compound was l e f t i n contact w i t h the r e s i n overnight and then e l u t e d w i t h 2 0 0 ml. 90$ methanol. The eluate when evaporated, l e f t a c l e a r c o l o r l e s s s i r u p . The d r i e d s i r u p was e x t r a c t e d w i t h hot e t h y l acetate ( 1 5 0 ml.). Evaporation of an a l i q u o t of the 31. e t h y l acetate e x t r a c t showed i t contained 0 . 1 2 5 g. of a dimethyl-D-tnannitol, y i e l d , 78$. Found: -OCB3, 29.2$ C a l c u l a t e d f o r C Q H I 8 0 6 : -OCH^, 29.5$. ^Di-O-methyl-dimethylene-D-mannitol The di-O-methyl-mannitol s i r u p ( 0 . 0 6 g.) was mixed w i t h paraformaldehyde ( 0 . 2 g.) and the mixture was s t i r r e d w i t h concentrated s u l f u r i c a c i d ( 0 . 5 ml.) u n t i l i t had cooled to room temperature. The pale y e l l o w s i r u p was shaken w i t h chloroform (25 ml.) overnight. One more e x t r a c t i o n was made and the chloroform e x t r a c t s were combined, n e u t r a l i z e d w i t h d i l u t e ammonium hydroxide, washed w i t h water, and d r i e d over anhydrous magnesium s u l f a t e . Evaporation of the chloroform e x t r a c t l e f t a c o l o r l e s s s i r u p ; weight, 0 . 0 ^ + 3 g., y i e l d , 6h%. 32. CLAIMS TO ORIGINAL RESEARCH 1. A simultaneous t r i t y l a t i o n ana a c e t y l a t i o n procedure • for D-m^v\vuAo\ has been developed Awhich gives 1 , 6 - d i - O - t r i t y l - D -mannitol t e t r a a c e t a t e i n good y i e l d and almost completely f r e e from t r i p h e n y l c a r b i n o l . 2. D i r e c t t r i t y l a t i o n of D-mannitol y i e l d e d 1,6-di-O-t r i t y l - D - m a n n i t o l which was obtained i n c r y s t a l l i n e form f o r the f i r s t time. 3. The aqueous-acetic a c i d d e t r i t y l a t i o n technique has been extended to the h e x i t o l s e r i e s of compounds. h. A s i r u p y dimethyl-D-mannitol has been prepared using a new s y n t h e t i c r oute. C r y s t a l l i z a t i o n of t h i s compound would show the s t a b i l i t y of a c y l groups towards m i g r a t i o n i n p a r t i a l l y a c e t y l a t e d h e x i t o l molecules during a methylation r e a c t i o n A using s i l v e r oxide and methyl i o d i d e . I f 1,6-dimethyl-D-mannitol was i s o l a t e d i t would show that t r i t y l a t i o n Is s p e c i f i c f o r the primary hydroxyls i n D-mannitol i n t h i s r e a c t i o n . 33. BIBLIOGRAPHY (1) Bachmann, W.E. I n Organic Syntheses. V o l . 2 3 , J . Wiley and. Sons, New York. 19hQ. p.100. (2) Baer, E. and F i s c h e r , H.O.L. J . Am. Chem. S o c , 61 : 7 6 1 . 1 9 3 9 . (3) Bourne, E.J., Bruce, G.T. and Wiggins, L.F. J . Chem. Soc. 2 7 0 8 . 1 9 5 1 . (*+) C l a r k , E.P. I n Semimicro q u a n t i t a t i v e Organic A n a l y s i s . Academic Press Inc., New York. 19^3. v.7h. (5) F i e s e r , L.F. Experiments i n Organic Chemistry. 2nd Ed. D.C. Heath and Co. New York. 19I+I. P. 3 5 8 . (6) F i s c h e r , E. Ber. 53 ' 1 6 2 1 . 1 9 2 0 . (7) F r i e d e l , C. and C r a f t s , J.M. Ann. Chim. Phys. 1 5 5 0 3 . 1881+. (8) Hammett, L.P. P h y s i c a l Organic Chemistry, 1 s t Ed. McGraw-Hill Book Co., New York. 19*+0. p. 5 9 . (9) Haworth, W.N., H i r s t , E.L. and Teece, E.G. J . Chem. Soc. 2 8 5 8 . 1 9 3 1 . (10) H e l f e r i c h , B. In Advances i n Carbohydrate Chemistry. V o l . 3 . E d i t e d by W.W. Pigman and M. L. Wolfrom. Academic Press, Inc., New York. 19*+9. p. 7 9 . (11) H e l f e r i c h , B. and Grunther, E. Ber. 6k : 1272. 1 9 3 1 . (12) H e l f e r i c h , B. and K l e i n , W . Ann. *+50 : 2 1 9 . 1 9 2 6 . (13) H e l f e r i c h , B., Moog, L. and Junger, A. Ber. 58 * 8 7 2 . 1 9 2 5 . (11+) H e l f e r i c h , B., S p e i d e l , P.E. and Toeldte, W. Ber. 56 : 7 6 6 . 1 9 2 3 . 3 V . (15) Hockett, R.C., F l e t c h e r , H.G. and Ames, J.B. J . Am. Chem. Soc. 63 J 2 5 1 6 . 1 9 V I . • (16) Hockett, R.C. and Hudson, C.S. J . Am. Chem. Soc. 56 : 9 V 5 . 1 9 3 V . (17) I s b e l l , H.G. Bur. Stan. J . of Res. 5 s 1 1 8 5 . 1 9 3 0 . (18) J e a n l o z , R.W. J . Am. Chem. Soc. 76 : 568V. 195V. (19) Kramer, F. In Paper Chromatography. M c M i l l a n and Co., New York. 195V. ( 2 0 ) McKeown, G.G. and Hayward, L.D. P r i v a t e communica-t i o n . (21) Micheel, F. Ber. 62B : 262. 1 9 3 2 . (22) M u l l e r , A. Ber. 65 * 1 0 5 1 . 1 9 3 2 . (23) Purdie, T. and I r v i n e , J.C. J . Chem. Soc. 1 0 2 1 . 1 9 0 3 . (2*f) Reynolds, D.D. and Evans, W.L. Organic Syntheses. V o l . 2 2 . J . Wiley and Sons, New York. 19V7. p. 5 7 . (25) Salmi, E.J. and Renkonen, E. Ber. 72 : 1 1 0 7 . 1 9 3 9 . (26) Sugihara, J.M. I n Advances i n Carbohydrate Chemistry. V o l . 8 . E d i t e d by W. W. Pigman and M. L. Wolfrom. Academic Press, Inc., New York. 1 9 5 3 . p-.l. (27) Tanret, G. Compt. Rend. 172 : 1 3 6 3 , 1 5 0 0 . 1 9 2 1 . (28) V a l e n t i n , F. C o l l . Czechoslov. Chem. Comm. 3 : V 9 9 . 1 9 3 1 . (29) von Vargha, L. Ber. 66 : 139V. 1 9 3 3 . (30) Wiggins, L.F. J . Chem. Soc. 3 8 V . 1 9 ^ 6 . (31) Wolfrom, M.L., Burke, W.J. and Waisbrot, S.W. J . Am. Chem. Soc. 61 : 1 8 2 7 . 1 9 3 9 . (32) Z e i l e , K. and Kruekenberg, W. Ber. 69 » 15V6. 1 9 3 6 . PART I I A COLOR PRECURSOR ISOLATED FROM WESTERN HEMLOCK WOOD INTRODUCTION In the groundwood pu l p i n g of Western Hemlock (Tsuga h e t e r o p h y l l a ) a pink c o l o r develops d u r i n g the g r i n d i n g operation which must be removed before the pulp i s of s a t i s -f a c t o r y b r i g h t n e s s f o r commercial use. This i s u s u a l l y done w i t h a bleaching agent. The c o l o r has been a t t r i b u t e d , w i t h very l i t t l e experimental evidence, t o the f o r m a t i o n of an anthocyanin s a l t , long known t o be r e s p o n s i b l e f o r some of the c o l o r s appearing i n f l o w e r s and f r u i t s (11), In many p l a n t s these compounds have a c o l o r l e s s precursor, r a t h e r l o o s e l y defined as a leucoanthocyanin (17). T h i s t h e s i s d e s c r i b e s f o r the f i r s t time the i s o l a t i o n and steps toward the i d e n t i f i c a t i o n of a c o l o r p r e c u r s o r from Western Hemlock wood* For purposes of i d e n t i f i c a t i o n leucoanthocyanins a r e u s u a l l y converted t o t h e i r corresponding anthocyanidin s a l t s by treatment w i t h a l c o h o l i c mineral a c i d ( S ) . With the i s o l a t i o n and i d e n t i f i c a t i o n of the anthocyanidin s a l t and the nature of i t s a u b s t i t u e n t groups the s t r u c t u r e of the leuco-compound may be e l u c i d a t e d . Anthocyanidins are f l a v y l i u m s a l t s and have the funda-mental s t r u c t u r e ( I ) . I They may be considered t o represent a chemical c l a s s of n a t u r a l products (11). They are r e s p o n s i b l e f o r many of the c o l o r s appearing i n the f l o w e r s , f r u i t s , and leaves of p l a n t s and may e x i s t f r e e , e s t e r i f i e d -bo an hydroxy a c i d , or as g l y c o s i d e s . The l a t t e r c l a s s i f i c a t i o n comprises the l a r g e s t group of the anthocyanidins. Most of the antho-c y a n i d i n s found i n nature have the 3, 5, 7 p o s i t i o n s hydroxy l a t e d and a sugar r e s i d u e , i f present, attached t o the 3-hydroxyl or at the 3 and 5 p o s i t i o n s i f i t i s an antho-c y a n i d i n d i g l y c o s i d e . The hyd r o x y l or methoxyl content of r i n g C v a r i e s w i t h the i n d i v i d u a l members of the group and hence the c h a r a c t e r i s t i c s of the i n d i v i d u a l members d i f f e r : : d e l p h i n i d i n (3 ,,4',5 t,3,5,7-hexahydroxy f l a v y l i u m c h l o r i d e ) i s very s o l u b l e i n water but m a l v i d i n (3',5'-dimethoxy-4 1 , 3 , 5 , 7 -tetrahydroxyflavylium c h l o r i d e ) i s almost i n s o l u b l e i n water. F l a v y l i u m s a l t s e x i s t as i o n s and d i f f e r from t r u e oxonium s a l t s formed by ethers and ~Y~-pyrones (18). They are considered by Sh r i n e r (19) t o be carbonium s a l t s and carbons 2,3, and 4 represent the members of an a l l y l i c system. O O Flavones form oxonium s a l t s i n acids but these are very-unstable i n the presence of water, unlike the anthocyanidins, whose acid s a l t s are very stable. In f a c t , anthocyanins and anthocyanidins frequently occur as acid or basic s a l t s i n plants (11). Anthocyanins are sensitive to pH changes: the acid s a l t s are red, the neutral s a l t s are purple, and the me t a l l i c s a l t s with bases are blue (11). The i d e n t i f i c a t i o n of the i n d i v i d u a l anthocyanidins before 1948 rested upon color reactions outlined by the Robinsons (15) and a l k a l i cleavage to a polyhyroxy phenol and a substituted benzoic acid using methods outlined by Karrer (9). In 1949, Bate-Smith (1) developed an i d e n t i -f i c a t i o n technique for anthocyanidin glycosides using paper chromatography with n-butanol, a c e t i c acid, water as the solvent. In 1954 (2), he applied the Forestal solvent (acetic acid, hydrochloric acid, water) to the i d e n t i f i c a -t i o n of the anthocyanidin s a l t s obtained by acid hydrolysis of the anthocyanidin glycosides. Those s a l t s f a i r l y soluble i n water gave excellent r e s u l t s and show strong adsorption when viewed under u l t r a v i o l e t l i g h t (2). HISTORICAL INTRODUCTION The term leucoanthocyanin was proposed by Rosenheim (17) who, i n v e s t i g a t i n g an anthocyanin present i n a species of grape, succeeded i n i s o l a t i n g a water s o l u b l e c o l o r l e s s compound which he b e l i e v e d t o be a precursor t o the antho-cyanin. This c o l o r l e s s compound was converted t o the c r y s t a l l i n e anthocyanidin by treatment w i t h h y d r o c h l o r i c a c i d . He obtained the leucoanthocyanidin i n c r y s t a l l i n e c o n d i t i o n . Rosenheim postula t e d t h a t the leucoanthocyanin ( I I I a)tf) was an intermediate i n the formation, by r e d u c t i o n , of an anthocyanidin from the corresponding f l a v o n o l ( I I ) . Rosenheim suggested t h a t the carbohydrate was attached t o the 4-hydroxyl group i n the pseudo base ( I l i a ) and must be removed by a c i d h y d r o l y s i s before anthocyanidin formation i s p o s s i b l e and the presence of a c i d causes the l i b e r a t e d pseudo base ( I l i a ) to isomerize t o the c o l o r base ( I l l b ) . I s o m e r i z a t i o n was assumed t o take place by the m i g r a t i o n of the hydroxyl from p o s i t i o n 4 t o p o s i t i o n 1. The c o l o r l e s s pseudo-base and the pigment were present i n the p l a n t i n equal q u a n t i t i e s . In 1933, the Robinsons (15) took up the work of Rosen-heim and found c o l o r l e s s precursors i n almost every type of p l a n t m a t e r i a l examined. They found t h a t these leuco-anthocyanins were s t a b l e t o aqueous h y d r o c h l o r i c a c i d but methanolic h y d r o c h l o r i c a c i d brought about gradual formation of the anthocyanidin c h l o r i d e . Upon t h i s p o i n t the Robin-sons (15) disagreed w i t h Rosenheim's p o s t u l a t i o n of a c o l o r -l e s s intermediate unstable t o aqueous mineral a c i d . They found t h a t a l k y l s u b s t i t u t i o n of the 4 - p o s i t i o n d i d not pro t e c t the leucoanthocyanin against the a c t i o n of even weak mi n e r a l a c i d . To e x p l a i n t h i s discrepancy the Robin-sons (15) suggested t h a t the group (-CHOH:CHOH-) was present i n the 3,4 p o s i t i o n of the pyran r i n g and postulat e d the s t r u c t u r e (IV) t o be the c o l o r l e s s precursor of c y a n i d i n c h l o r i d e (V). OH HO K T OH is v: Most of the leucoanthocyanins i n v e s t i g a t e d (16) y i e l d e d c y a n i d i n c h l o r i d e (V) a f t e r treatment with methanolic h y d r o c h l o r i c a c i d . ow 40. In a l a t e r p u b l i c a t i o n , the Robinsons (16) d i v i d e d the leucoanthocyanins i n t o t h r e e classes": (1) Those i n s o l u b l e i n water and the us u a l organic s o l v e n t s and g i v i n g only c o l l o i d a l suspension. (2) Those r e a d i l y s o l u b l e i n water and not ext r a c t e d from water by means of e t h y l a c e t a t e . (3) Those capable of e x t r a c t i o n from aqueous s o l u t i o n by means of e t h y l a c e t a t e . C l a s s (3) compounds were sugar-free and would be more c u r r e n t l y termed leucoanthocyanidins. The Robinsons (16) i s o l a t e d from Peltogyne porphvro-c a r d i a a compound p e l t o g y n o l f o r which they p o s t u l a t e d the s t r u c t u r e ( V I ) . O x i d a t i o n of p e l t o g y n o l gave a compound which had the anthocyanidin c h a r a c t e r i s t i c s , but was not one of the known n a t u r a l l y o c c u r r i n g anthocyanidins; hence they d i d not con-s i d e r p e l t o g y n o l t o be a t r u e leucoanthocyanin. Noting t h a t Rosenheim (1$)showed t h a t c o l o r formation was independent of the presence or absence of oxygen, the Robinsons (16) proposed the hypothesis t h a t anthocyanidin formation from the corresponding leucoanthocyanin was an o x i d a t i v e process although they were unable t o e x p l a i n the formation of the pigment when oxygen was excluded from the system. G. M. Robinson i n a l a t e r paper (14) i s o l a t e d c y a n i d i n c h l o r i d e from the gum of Butea frondosa and found t h a t a p r e l i m i n a r y o x i d a t i o n of the leucoanthocyanin was necessary i n order t o obtain the anthocyanidin. I f the gum was t r e a t e d w i t h a l c o h o l i c h y d r o c h l o r i c a c i d i n the absence of oxygen, a red c o l o r developed which was apparently caused by a m o d i f i c a t i o n of c y a n i d i n c h l o r i d e . I t was necessary t o b o i l the s o l u t i o n of the gum with aqueous p i c r i c a c i d before pure c y a n i d i n c h l o r i d e could be obtained. The c o l o r i n g m a t e r i a l obtained without previous o x i d a t i o n was not di s c u s s e d . In 1953, King and Bottomley (6) i s o l a t e d a leucoantho-cyanin, m e l a c a c i d i n , from A c a c i a melanoxylon which gave a l l the c h a r a c t e r i s t i c r e a c t i o n s when t r e a t e d w i t h mineral a c i d . Previous t o t h i s year, most of the work i n the d e t e c t i o n of leucoanthocyanins had been accomplished by t h e i r con-v e r s i o n t o the corresponding anthocyanin or anthocyanidin w i t h the i d e n t i f i c a t i o n of the l a t t e r by the c o l o r r e a c t i o n s and paper chromatography. Although melacacidin i t s e l f i s amorphous, King and coworkers (7) obtained a c r y s t a l l i n e t e t r a m e t h y l ether of mel a c a c i d i n which was i d e n t i f i e d as 7*3:3 1:4'-tetramethoxy-f l a v a n - c i s - 3 : 4 - d i o l ( V I I ) . m S t a r t i n g from 7:8:3' ^ ' - t e t r a m e t h o x y - f l a v o n o l they synthe-s i z e d the compound (VII) by hydrogenation over Raney n i c k e l o b t a i n i n g a racemic tetramethoxy f l a v a n - c i s - 3 : 4 - d i o l . King was able t o i d e n t i f y the s y n t h e t i c d i o l w i t h the n a t u r a l l y o c c u r r i n g l e v o r o t a t o r y d i o l by comparison of t h e i r i n f r a - r e d s p e c t r a , chromatographic behaviour a f t e r treatment w i t h a l c o h o l i c h y d r o c h l o r i c a c i d , and by compari-son of some of t h e i r d e r i v a t i v e s ( 8 ) . M e l a c a c i d i n i s the only f u l l y c h a r a c t e r i z e d f l a v a n -3".4-diol i s o l a t e d from n a t u r a l sources and the anthocyani-d i n d e r i v e d from i t has not been found i n nature, Bate-Smith (3) f e l t t h a t s i n c e anthocyanidins have not been detected as a r e s u l t of a c i d treatment of cate-chins ( f l a v a n - 3 - o l ) the 3 « 4-diol s t r u c t u r e was necessary f o r f l a v y l i u m s a l t formation. Since anthocyanidin forma-t i o n i n t h i s case was an o x i d a t i o n , i t may have occurred because of the presence of atmospheric oxygen or by a d i s -p r o p o r t i o n a t i o n i n which an equivalent of the f l a v a n d i o l was reduced t o a lower o x i d a t i o n s t a t e (6). 43. O 4> O H 4> O H 2 H + H + OH V T T T OH K 4) The formation of the f l a v a n - 3 - o l s t r u c t u r e (X) has been proposed by King and Bottomley (6) since i t has been de-t e c t e d by paper chromatography among the products from the a c i d treatment of the cacao bean leucoanthocyar4din (4). Furthermore, t h i s anthocyanidin formation proceeded i n the absence of oxygen (17). In 1953, Pigman and coworkers (13) i s o l a t e d a leuco-anthocyanin from black spruce i n n e r bark. I t was a water s o l u b l e s o l i d which gave the c h a r a c t e r i s t i c anthocyanidin r e a c t i o n s when t r e a t e d w i t h a l c o h o l i c m i n e r a l a c i d . Hydro-l y s i s of the leucoanthocyanin w i t h mineral a c i d f o l l o w e d by treatment of the hydrolyzate w i t h phenylhydrazine l e d t o the recovery of glucose phenylosazone. They prepared the methyl and a c e t y l d e r i v a t i v e s of the leucoanthocyanin and proposed the f o l l o w i n g s t r u c t u r e ( X I ) . o H The conversion t o the anthocyanidin could r e s u l t from h y d r o l y s i s of the g l y c o s i d e , and r i n g c l o s u r e , w i t h simultaneous dehydration. A c o l o r precursor was found i n a l l p a r t s of Western Hemlock wood by Pigman et a l . Several d i f f e r e n t e x t r a c t i o n methods were t e s t e d , but the m a t e r i a l was not i n v e s t i g a t e d f u r t h e r . DISCUSSION OF RESULTS Wood from a 68 y r . o l d Western Heplock t r e e (Tsuga h e t e r o p h y l l a ) , f e l l e d i n the U n i v e r s i t y f o r e s t , was used i n t h i s work. The wood sample was f r e e of decay and the t r e e was described as suppressed.* P r e l i m i n a r y treatment of the f i n e l y ground wood w i t h methanol c o n t a i n i n g h y d r o c h l o r i c a c i d produced a r e d - v i o l e t c o l o r , i n s o l u t i o n and on the surface of the wood i t s e l f , c h a r a c t e r i s t i c of an anthocyanin s a l t . Using the standard methanol-hydrochloric a c i d s o l u t i o n of Pigman (13), t h i s r e a c t i o n was used t o f o l l o w the course of e x t r a c t i o n of the c o l o r precursor from the sawdust. The procedures of i s o l a t i o n and i d e n t i f i c a t i o n employed i n the i n v e s t i g a t i o n are summarized i n Fi g u r e 1. Two methods of e x t r a c t i o n were compared c o l o r i m e t r i c a l l y u s i n g the methanol-hydrochloric a c i d r e a c t i o n . P r e l i m i n a r y e x t r a c t i o n w i t h benzene f o l l o w e d by 50$ aqueous ethanol proved t o be the most e f f i c i e n t method, although the acetone e x t r a c t i o n gave a much l a r g e r t o t a l q u a n t i t y of e x t r a c t . The 50$ aqueous ethanol e x t r a c t ( h e r e a f t e r c a l l e d the extract;)' was a n e a r l y c o l o r l e s s amorphous s o l i d , i n c l a s s (1) of Robinson's c l a s s i f i c a t i o n f o r leucoanthocyanins, and gave ft We are indebted t o the l a t e Dr. D.N. Buckland of the Forest Pathology Laboratory, U.B.C, f o r the examination of the d i s c sample. ii 4 6 . an intense r e d - v i o l e t c o l o r i n methanol c o n t a i n i n g hydro-c h l o r i c a c i d . Using the standard methanol-hydrochloric a c i d s o l u t i o n of Pigman (13) the c o l o r produced was s t a b l e t o o r d i n a r y l i g h t f o r s e v e r a l weeks. Since many f l a v o n o i d type compounds e x i s t as g l y c o s i d e s i n the pl a n t (11), the ethanol e x t r a c t was t r e a t e d w i t h a l c o h o l i c mineral a c i d t o e f f e c t h y d r o l y s i s of the glyeo-s i d i c bond. R e f l u x i n g the e x t r a c t i n methanol, 3N i n h y d r o c h l o r i c a c i d , produced a r e d - v i o l e t s o l u t i o n which l e f t a purple s o l i d upon evaporation of the s o l v e n t . I t was i n s o l u b l e i n water and gave a f a i n t pink c o l o r w i t h aqueous 1% h y d r o c h l o r i c a c i d s o l u t i o n which was completely e x t r a c t a b l e by n-amyl a l c o h o l . Robinson (10) reported t h a t the water, n-amyl a l c o h o l d i s t r i b u t i o n c o e f f i c i e n t f o r h i r s u t i d i n A b e c a u s e of i t s low water s o l u b i l i t y . I f an anthocyanidin s a l t was produced by the a c t i o n of methanolic h y d r o c h l o r i c a c i d on the e x t r a c t , the t e s t s o u t l i n e d by Lin k (11) and those of the Robinsons (15) i n d i c a t e d a m a l v i d i n or h i r s u t i d i n type of anthocyanidin. The methoxyl va l u e was s u f f i c i e n t l y high t o i n d i c a t e the presence of ether groups. The t e s t w i t h f e r r i c c h l o r i d e was negative, i n d i c a t i n g t h a t the 1,2-dihydroxy grouping was absent (11). Cyanidin c h l o r i d e (3' , 4 f,3,5,7-pentahydroxy f l a v y l i u m c h l o r i d e ) gave a p o s i t i v e f e r r i c c h l o r i d e t e s t because of the 3',4' -dihydroxy grouping but m a l v i d i n (3',5 ,-dimethoxy - 4 ',3,5,7-tetrahydroxy f l a v y l i u m c h l o r i d e ) gave a negative f e r r i c c h l o r i d e t e s t because the 1,2-dihydroxy grouping was absent. Paper chromatographic behaviour of the purple substance u s i n g F o r e s t a l s o l v e n t gave a s i n g l e spot of h i g h Rf value, whose c o l o r s i n the v i s i b l e and i n the u l t r a v i o l e t i n d i c a t e d the presence of an anthocyanidin. The high Rf value may be explained by the low s o l u b i l i t y of the purple m a t e r i a l i n water. An Rf value f o r h i r s u t i d i n or m a l v i d i n i n F o r e s t a l solvent has not been r e p o r t e d . Flavonoid type compounds are y e l l o w i n both a c i d and a l k a l i and give y e l l o w spots on a chromatogram i n F o r e s t a l s o l v e n t , whether viewed i n the v i s i b l e or i n the u l t r a -v i o l e t . In t h i s solvent i s o f l a v o n e s e x h i b i t e d a pale-blue f l u o r e s c e n c e under u l t r a - v i o l e t l i g h t , which darkened when exposed t o ammonia vapors ( 2 ) . R e f l u x i n g the e x t r a c t w i t h a more concentrated s o l u -t i o n of h y d r o c h l o r i c a c i d i n methanol, approximately 6N, y i e l d e d a purple s o l i d which gave s i m i l a r r e a c t i o n s t o those already described above. A p u r i f i c a t i o n procedure y i e l d e d a product of lowered methoxyl content, which could r e s u l t from e i t h e r the p u r i f i c a t i o n or from p a r t i a l de-methylation a r i s i n g from the strong a c i d treatment. Bate-Smith (2) used c y a n i d i n c h l o r i d e (Rf, 0.5) as a chromatographic standard i n F o r e s t a l s o l v e n t . To ob-t a i n some of t h i s compound, dark red rose p e t a l s were ex-43. t r a c t e d by the method of W i l l s t a t t e r . Treatment of the rose e x t r a c t w i t h a l c o h o l i c mineral a c i d , t o hydrolyze any g l y c o -s i d e s present, f o l l o w e d by paper chromatography of the ex-t r a c t i n F o r e s t a l s o l v e n t , gave f o u r spots on the chromato-gram, which by t h e i r behaviour i n v i s i b l e and u l t r a - v i o l e t l i g h t i n d i c a t e d t h a t two were anthocyanidins, one was a fl a v o n e and one was an i s o f l a v o n e . The p u r i f i c a t i o n procedure of Geissmann (5) removed two of the f l a v o n o i d components since rechromatography of the p u r i f i e d rose e x t r a c t showed two spots c h a r a c t e r i s t i c of anthocyanidins. A spot w i t h R f, 0.47 gave the cha r a c t e r -i s t i c c o l o r r e a c t i o n s of c y a n i d i n c h l o r i d e , both i n the v i s i b l e and u l t r a - v i o l e t . The c o l o r t e s t s of Robinson on the rose e x t r a c t gave evidence f o r t he presence of c y a n i d i n c h l o r i d e . Robinson (16) r e p o r t s t h a t when two anthocyanidins are present i n a p l a n t , one was u s u a l l y a methoxylated de-r i v a t i v e of the other, hence i n t h i s case i t was p o s s i b l e t h a t the anthocyanidin spot w i t h higher Rf value was a methoxyl d e r i v a t i v e of c y a n i d i n c h l o r i d e . I t would be of i n t e r e s t t o r e i n v e s t i g a t e the rose e x t r a c t i n view of the chromatographic evidence t h a t s e v e r a l components were present. In one specie of rose the r e l a t i v e s t r u c t u r e s of the flav o n e s and anthocyanidins could be com-pared and p o s s i b l y the n a t u r a l r e l a t i o n s h i p between these s t r u c t u r e s could be e l u c i d a t e d . A c e t y l a t i o n of the 50% ethanol e x t r a c t i n p y r i d i n e u s i n g a c e t i c anhydride gave a 50% increase i n weight. The a c e t y l a t e d product was completely s o l u b l e i n c h l o r o -form, whereas the o r i g i n a l product was not completely s o l u b l e i n any s o l v e n t . I t had a p o s i t i v e r o t a t i o n i n chloroform i n d i c a t i n g the presence of asymmetic cent e r s , e i t h e r as sugar residues or as a 3>>4-diol s t r u c t u r e i n the case of me l a c a c i d i n . The a c e t y l a t i o n procedure gave three p o s s i b l e modes of a t t a c k on the problem: a method by which a pure c r y s t a l l i n e d e r i v a t i v e may by obtained without the development of the f l a v y l i u m s a l t , a method t o i s o l a t e the pure f l a v y l i u m s a l t a f t e r d e a c e t y l a t i o n , and a method t o i s o l a t e the c o l o r l e s s precursor i t s e l f . SAWDUST benzene extraction sawdust & resinous extract (1.017g.) 50$ aq. ethanol .fractionation acetone extraction sawdust & sirupy extract (5.048g.) sawdust & extract (1.28g.) HC1 i n methanol 6N purple s o l i d (B) p u r i f i e d by pp'tion from acetic acid - O C H 3 : 11.0$ Rf: 0.95 color t e s t s HC1 i n methanol 3N G5H5N, acetic anhydride purple s o l i d (A) yellow sirup -OGH.: 13.9$ fed =+3.78* Rf: 0.95 1 > color t e s t s resinous material & white amorph, powder pos. Fehling's a f t e r t r e a t -ment by acid. Neg. antho-cyanin t e s t s . Figure 1.. EXPERIMENTAL A. P r e p a r a t i o n o f the Wood A f t e r f e l l i n g , the t r e e was cut i n t o t h r e e - f o o t lengths from which the l e n g t h next t o the butt l o g was used. The bark was removed manually and the l o g s p l i t i n t o segments which were sawed i n t o 1.5 i n c h lengths w i t h a t a b l e waw, then s p l i t i n t o s t i c k s approximately one square cm. i n c r o s s - s e c t i o n , d r i e d i n a i r , and ground t o a f i n e sawdust i n a model no. 2 Wiley M i l l (screen s i z e , 2 mm.). The sawdust was stored out of d i r e c t contact with l i g h t i n a 1 5 - l i t e r b o t t l e . A sample of f r e s h l y ground sawdust was d r i e d i n vacuo over phosphorus pentoxide t o constant weight and contained 22.35$ moisture by weight. When sawdust (5.0g.) was r e f l u x e d f o r f i f t e e n minutes with methanol c o n t a i n i n g concentrated hydro-c h l o r i c a c i d (4:1), the methanol turned r e d - v i o l e t and the wood i t s e l f a t t a i n e d a r e d - v i o l e t c o l o r a t i o n . Re-moval of the sawdust by f i l t r a t i o n and evaporation of the f i l t r a t e l e f t a red-brown powder, i n s o l u b l e i n water but s o l u b l e i n n-amyl a l c o h o l . The course of e x t r a c t i o n of the c o l o r precursor from the sawdust was f o l l o w e d us-in g the standard methanol-hydrochloric a c i d s o l u t i o n of Pigman (13). B. E x t r a c t i o n of the Sawdust w i t h Benzene In order t o remove resinous m a t e r i a l the sawdust was e x h a u s t i v e l y e x t r a c t e d w i t h benzene. Sawdust (251.Og.) was d r i e d i n vacuo over phosphorus pentoxide t o 240.Og. and e x t r a c t e d i n a Soxhlet e x t r a c t o r f o r 24 hi*, w i t h dry benzene. A f t e r e x t r a c t i o n the sawdust was a i r d r i e d u n t i l the odor of benzene was no longer apparent and then stored out of contact w i t h l i g h t . Evaporation of the lemon yellow benzene e x t r a c t l e f t a brown resinous m a t e r i a l (1.017g.). I t was r e d i s s o l v e d i n benzene and petroleum-ether was added causing the p r e c i p i t a t i o n of an amorphous white s o l i d which was removed by c e n t r i f u g a t i o n , washed wi t h petroleum-ether, and d r i e d i n vacuo over soda lime and p a r a f f i n shavings* This m a t e r i a l reduced b o i l i n g F e h l i n g ' s s o l u t i o n , de-c o l o r i z e d n e u t r a l potawsium permanganate, and gi v e negative anthocyanin t e s t s . Evaporation o f the mother l i q u o r s gave a dark brown resinous m a t e r i a l which a l s o gave a negative anthocyanin t e s t * The benzene e x t r a c t was not i n v e s t i g a t e d f u r t h e r * 6. E x t r a c t i o n of the Sawdust w i t h Acetone A i r - d r i e d sawdust (80.0g.) was packed i n t o a g l a s s tube (48.5 x 3.3 cm.) closed by a s i n t e r e d g l a s s d i s c and e x h a u s t i v e l y e x t r a c t e d by a l l o w i n g acetone t o p e r c o l a t e through the column at a very slow r a t e . The The amount of m a t e r i a l e x t r a c t e d was determined by evapo-r a t i o n of an a l i q u o t of the el u a t e at i n t e r v a l s . When the amount of m a t e r i a l i n the eluate became n e g l i g i b l e the e x t r a c t i o n w i t h acetone was stopped although a sample of the sawdust from the column s t i l l gave a p o s i t i v e anthocyanin t e s t . Evaporation o f the acetone e x t r a c t (1390 ml,) y i e l d e d a l i g h t brown s i r u p y m a t e r i -a l (5,408 g.) which gave a p o s i t i v e anthocyanin t e s t . D. E x t r a c t i o n of the Benzene-Extracted Sawdust w i t h 50$ Aqueous Ethanol. ;  Benzene-extracted sawdust (47,6 g.) was packed i n t o a column (28 .5 x 3*3 cm.) and e x h a u s t i v e l y e x t r a c t e d w i t h 50$ by volume aqueous ethanol by the above procedure. When a l l the m a t e r i a l s o l u b l e i n 50$ ethanol had been removed from the column the r e s i d u a l sawdust gave a very weak anthocyanin t e s t . The t o t a l p e r c o l a t e (1025 ml.) was evaporated under reduced pressure and when i t s volume was halved a heavy, n e a r l y c o l o r l e s s p r e c i p i t a t e formed; the evaporation was continued and the l a s t 100 ml, of solvent was evaporated u s i n g a l y o p h i l i z i n g u n i t l e a v i n g an almost c o l o r l e s s amorphous powder (1.282 g.). The d r i e d 50$ aqueous ethanol e x t r a c t was used i n a l l f u r t h e r t e s t s ; Table I g i v e s the s o l u b i l i t y c h a r a c t e r i s t i c s of the d r i e d m a t e r i a l , A c l e a r s o l u t i o n could not be obtained i n any solvent hence an o p t i c a l r o t a t i o n was not f e a s i b l e at t h i s p o i n t . TABLE I S o l u b i l i t y C h a r a c t e r i s t i c s of the 50$ Aqueous Ethanol E x t r a c t of Western Hemlock Wood, Reagent R e s u l t water methanol ethanol (95$) benzene chloroform 3N aq. HC1 3N HC1 i n methanol very s l i g h t l y s o l u b l e s o l u b l e g i v i n g a c o l l o i d a l suspension s o l u b l e g i v i n g a c o l l o i d a l suspension i n s o l u b l e i n s o l u b l e i n s o l u b l e and gave no c o l o r r e a c t i o n when warmed. immediate s o l u t i o n g i v i n g a r e d - v i o l e t c o l o r . T h i s t e s t p o s i t i v e even i f the a c i d was present i n low concentra-t i o n s . The ethanol e x t r a c t , at a c o n c e n t r a t i o n of 2 mg. per ml. i n methanol c o n t a i n i n g concentrated h y d r o c h l o r i c a c i d (4:1) gave a c o l o r described as 7,5 RP /XQ *>y the Munsell Book of Colo r s (12), approximately r e d - v i o l e t . E. Treatment of the 50$ Ethanol E x t r a c t w i t h A l c o h o l i c M i n e r a l A c i d  The e x t r a c t (0.100 g.) was r e f l u x e d w i t h methanol c o n t a i n i n g concentrated h y d r o c h l o r i c a c i d (4:1) f o r 15 min. then cooled and the so l v e n t s removed under reduced pressure l e a v i n g a purple amorphous s o l i d (A) which was d r i e d i n vacuo over phosphorus pentoxide. I t was com-p l e t e l y i n s o l u b l e i n water but gave a f a i n t pink s o l u -t i o n w i t h 1$ aqueous h y d r o c h l o r i c a c i d . I t was s o l u b l e i n methanol, ethanol, n-amyl a l c o h o l , and g l a c i a l a c e t i c a c i d . Found: -OGR3 j 13.9$. Calc'd f o r C 1 5 H 1 0 0 5 C 1 ( O C H ^ ( m a l v i d i n c h l o r i d e ) } 16.8$. Calc'd f o r C^ H o G ^ C l ( O C H ^ ( m a l v i d i n c h l o r i d e monohexoside) G6 H10°5 i H.72$. A sample of the e x t r a c t (0.100 g.) was r e f l u x e d w i t h methanol c o n t a i n i n g concentrated h y d r o c h l o r i c a c i d (2:1) f o r 30 min. then cooled and the solvent removed under r e -duced pressure l e a v i n g a purple amorphous powder (B) which, when d r i e d , was s i m i l a r t o ( A ) . (B) was d i s s o l v e d i n g l a c i a l a c e t i c a c i d g i v i n g a red-purple s o l u t i o n and was completely p r e c i p i t a t e d by the a d d i t i o n of excess water. The p r e c i p i -t a t e was recoved a t c e n t r i f u g e , washed w i t h water, and d r i e d i n vacuo over phosphorus pentoxide. Found: -0CH 3 ; 11.0$. Galc'd f o r C ,H 0 C l (0CH,) o ( m a l v i d i n c h l o r i d e ) } 16.8$. l p 10 p > F. E x t r a c t i o n o f Pigment from Rose P e t a l s Dark red rose p e t a l s (5.5.0 g») p r e v i o u s l y d r i e d i n a vacuum d e s i c c a t o r over phosphorus pentoxide and magnesium pe r c h l o r a t e was e x t r a c t e d with 2$ methanolic h y d r o c h l o r i c a c i d u s i n g the method of W i l l s t a t t e r (20). Part of t h i s e x t r a c t was evaporated t o dryness under reduced pressure and d r i e d i n vacuo over phosphorus pentoxide l e a v i n g a dark red powder. This m a t e r i a l was t r e a t e d i n the usual-manner t o hydrolyze any g l y c o s i d e s present and the r e -s u l t i n g red powder was d r i e d i n vacuo. G. Chromatography of the Compounds R e s u l t i n g from the A l c o h o l i c A c i d Treatment of the Ethanol E x t r a c t and the Rose E x t r a c t .  Chromatograms were run on No. 1 Whatman paper usi n g F o r e s t a l solvent ( a c e t i c acid:concentrated hyd-r o c h l o r i c acid:water)(10:1:3) (2). Chromatography of e i t h e r (A) or (B) showed one spot on the chromatogram w i t h Rf v a l u e , 0.95. The spot was red-brown i n v i s i b l e l i g h t , b l u e - v i o l e t under the u l t r a -v i o l e t lamp, and became darker blue when exposed t o con-centrated ammonia vapors. A chromatograph of the crude rose p e t a l e x t r a c t under s i m i l a r c o n d i t i o n s gave f o u r spots on the chromatogram as described i n Table I I . TABLE I I Paper Chromatography of the Crude Rose E x t r a c t s % C o l o r , v i s i b l e 1 C o l o r , U . V . u?y?r N H ^ ' 0.63 red-brown s c a r l e t blue 0.48 red-brown s c a r l e t blue 0.38 y e l l o w y e l l o w golden-yellow 0.32 not v i s i b l e p ale blue darker blue The rose p e t a l e x t r a c t was p u r i f i e d u s i n g a method o u t l i n e d by Geissmann (5). A 1% aqueous h y d r o c h l o r i c a c i d s o l u t i o n of the rose e x t r a c t was shaken w i t h one quarteter of i t s volume of n-amyl alcohol containing 30$ by volume aceto-phenone. The pigment was quantitatively extracted by the alcohol layer and the aqueous layer was discarded. Addition of six volumes of benzene to the amyl alcohol layer caused the precipitation of the pigment which was redissolved in 1$ aqueous hydrochloric acid giving a violet-red solution. Evaporation of the aqueous acid solution gave a dark red powder which was chromatographed in Forestal solvent giving two brown-red spots (Table III). TABLE III Paper Chromatography of the Purified Rose Extract Rf Color under U.V. Color under; U.V. NH3 0.63 scarlet blue 0.47 scarlet blue H. Tests Outlined by Link and Robinson Applied to the Acid-treated Ethanol Extract and Rose Extract  The tests outlined by Link (11) are shown in Table IV: other members of the anthocyanidin family are included for comparison. TABLE IV Anthocyanidin Tests. Reaction P e l a r g o n i d i n C yanidin D e l p h i n i d i n Peonidin M a l v i d i n and H i r s u t i d i n (A) or (B) Rose E x t . c o l o r of v i o l e t v i o l e t v i o l e t v i o l e t aq. s o l * red red blue-red red red(?) pink red s o l ' t y i n r e a d i l y only s l . very s l . moderately water r e a d i l y s l i g h t l y very s o l . s o l . s o l u b l e s o l . s o l . F e C l ^ r x . not def. i n t . blue i n t . blue not def. no r x . no r x . i n t . blue F e h l i n g ' s r e d . i f reduces i n reduces i n red . i f re d . i f red. i f Test warmed c o l d c o l d b o i l e d b o i l e d b o i l e d c o l o r i n v i o l e t v i o l e t v i o l e t v i o l e t v i o l e t then v i o l e t Na2GG3 then blue then blue then blue then blue green-blue then blue blue Behavior c o l o r fades c o l o r c o l o r fades c o l o r c o l o r fades c o l o r fades c o l o r i n aq. s o l . on fades on on fades on when d i l u t e when d i l u t e fades standing h e a t i n g standing h e a t i n g s o l . b o i l e d s o l . b o i l e d on heating Behavior blue then t o NaOH green 59. Robinson (15), using the following tests, was able to distinguish between the main classes of anthocyanidins. 1. A portion of an aqueous hydrochloric acid solution was extracted with n-amyl alcohol and then sodium acetate was added followed by a drop of f e r r i c chloride solution. 2. A portion was extracted by cyanidin reagent (1 part cyclohexanol, 5 parts toluene) and the upper layer was observed in a narrow tube. 3. A portion was shaken in a i r with one-half i t s volume of 10$ aqueous sodium hydroxide then immediately acidified with concentrated hydrochloric acid and extracted with n-amyl alcohol. 4. k portion was shaken with a 5$ solution of p i c r i c acid in a mixture of methyl amyl ether (1 part) and anisole (4 parts). Using solutions of (A) or (B) and the rose extract in 1$ aqueous hydrochloric acid the color reactions with the various reagents were compared with Robinson's results as shown i n Table V. TABLE V Robinson's Tests Test P e l a r g o n i d i n Cyanidin M a l v i d i n D e l p h i n i d i n (A) or (B) Rose E x t . l a NaOAc v i o l e t - r e d l b FeGl^ no-change ex t r a c t e d 3 k r e d - v i o l e t blue-red blue intense blue e x t r a c t e d s t a b l e no change in t e n s e blue not not ex t r a c t e d e x t r a c t e d blue-red r e d - v i o l e t no change intense blue s l i g h t l y e x t r a c t e d e x t r a c t e d s t a b l e destroyed s t a b l e not ex t r a c t e d s t a b l e ON o I. Acetylation of the 50$ Aqueous Ethanol Extract A sample of the 50$ ethanol extract (0.10g.) was dissolved with d i f f i c u l t y in dried pyridine (5.0ml.) ani acetic anhydride (5.0ml.). After the dark red solution had stood for two days at room temperature, i t was poured into 100ml. crushed ice and water whereupon a heavy cream colored precipitate formed which did not crystallize after standing two weeks at 0 C. The supernatant liquid was poured off and the sirup was taken up in chloroform. The solution was washed with water and dried over anhydrous magnesium sulfate. Evaporation of the dried chloroform solution l e f t a sirup (0.156g.), insoluble in water and petroleum-ether and soluble in chloroform and benzene. [c<]D + 3.7$* (c, 1.560: 1, 0.5: CHC1 3). CLAIMS TO ORIGINAL RESEARCH 1. The presence of an e x t r a c t a b l e c o l o r p r e c u r s o r i n Western Hemlock wood was confirmed and an e f f i c i e n t pro-cedure was developed f o r i t s i s o l a t i o n . 2. The n e a r l e w c o l o r l e s s , s o l i d , 50% ethanol e x t r a c t of Western Hemlock wood was shown t o g i v e , a f t e r treatment w i t h a l c o h o l i c m i n e r a l a c i d , a purple s o l i d which had the c h a r a c t e r i s t i c r e a c t i o n s of an anthocyanidin of the m a l v i d i n or h i r s u t i d i n type. 3. A c e t y l a t i o n of the d r i e d e x t r a c t y i e l d e d a dextro-r o t a t o r y yellow s i r u p s o l u b l e i n chloroform and s u i t a b l e f o r f u r t h e r p u r i f i c a t i o n . 63. BIBLIOGRAPHY 1. Bate-Smith, E.G. Nature 1 6 4 : 2 5 . 1 9 4 9 . 2. Bate-Smith, E.C. Symposium on "Recent Advances i n the Chemistry of N a t u r a l l y Occurring Pyrones and Related Compounds". U n i v e r s i t y C o l l e g e , D u b l i n . 1955. 3. Bate-Smith, E.C. and Swain, T. Chem. and Ind. 377. 1953. 4. Forsythe, W.G.C. Nature 172:726. 1953. 5. Geissmann, T.A. In Moderne Methoden der Pf l a n z e - a n a l y z e . V o l . 3. E d i t e d by K. Paech and M.V. Tracey. Spri n g e r - V e r l a g , B e r l i n . 1955. p. 450. 6 . King, F.E. and Bottomley, W. Chem. and Ind. 1368. 1953. 7. King, F.E. and Clark-Lewis, J.W. Chem. and Ind. 757. 1954. 8. King, F.E. and Clark-Lewis, J.W. J . Chem. Soc. 3384. 1955. 9. K a r r e r , P. and de Meuron, G. Helv. Chim. Acta 15:507. 1932. 10. Levy, L.F. and Robinson, R. J . Chem. Soc. 2738. 1931. 11. L i n k , K.P. In Organic Chemistry. V o l . 2, 2nd ed. E d i t e d by H . Gilman. John Wiley and Sons, New York. 1943. p. 1316. 12. Munsell Book of C o l o r s . Muhsell Color Co. Inc. Baltimore. 1942. 13. Pigman, W.W., Anderson, E., F i s c h e r , R., Buchanan, M.A., and Browning, B.L. TAPPI. 3 6 : 4 . 1953. 14. Robinson, G.M. J . Chem. Soc. 1157. 1937. 15. Robinson, G. M. and Robinson, R., Biochem. J . 27:206. 1933. 16. Robinson, R. and Robinson, G.M., J . Chem. Soc. 744. 1935. 17. Rosenheim, 0. Biochem. J . 14:178. 1920. 18. S h r i n e r , R.L. and M o f f e t t , R.B. J . Am. Chem. Soc. 61:1474. 1939. 19. S h r i n e r , R.L. and M o f f e t t , R.B. J . A m. Chem. Soe. 62:2711. 1940. 20. W i l l s t a t t e r , R. and Nolan, T.JJAnn. 408>1. 1915. 

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