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Amide hydrolysis in vitamin B12 Raleigh, James Arthur 1962

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AMIDE HYDROLYSIS IN VITAMIN B12  by  JAMES ARTHUR RALEIGH B.Sc,  of B r i t i s h Columbia, i960.  University  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR 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 t o the r e q u i r e d standard  THE  UNIVERSITY OF BRITISH COLUMBIA J u l y , 1962  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y  of  B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e , f o r r e f e r e n c e and for extensive  study.  I f u r t h e r agree t h a t p e r m i s s i o n  c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may  g r a n t e d by the Head o f my Department o r by h i s  be  representatives.  I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n  Department o f The U n i v e r s i t y o f B r i t i s h Vancouver 8^ Canada.  Columbia,  permission.  ii ABSTRACT  Six a l i c y c l i c amides which simulate the s t e r i c environment of the amide groupings i n vitamin B12 have been prepared and t h e i r rates of alkaline hydrolysis compared on the basis of second order apparent rate constants^ Iii addition, vitamin B12b, a simple derivative of vitamin B12, has been prepared and hydrolyzed under the same conditions as f o r the model compounds.  iii  ACKNOWLEDGMENT  I wish t o thank Dr. R. Bonnett f o r the encouragement he has g i v e n , and I g r a t e f u l l y acknowledge  the many h e l p f u l  suggestions that he o f f e r e d d u r i n g the course of these investigations.  A l s o , I wish to thank Dr. D. McGreer f o r the  i n t e r e s t he has shown i n the problem and f o r the d i s c u s s i o n s which have been so h e l p f u l .  F u r t h e r , I wish t o thank Dr. L. D.  Hayward f o r the use of h i s l a b o r a t o r y ' s p h o t o l y s i s apparatus and Mr. Imre Czismadia f o r the a s s i s t a n c e which he gave i n the use of t h i s and other apparatus.  iv TABLE OF CONTENTS Page T i t l e Page  i  Abstract  .  ....  i i  Acknowledgment  i i i  Table of Contents  iv  L i s t of F i g u r e s  .  vi  L i s t of T a b l e s  viii  INTRODUCTION...  1  DISCUSSION 1.  P r e p a r a t i o n of the Amides  9  2.  H y d r o l y s i s of the Amides  15  3.  C a l c u l a t i o n of the Rate Constants  16  k.  R e s u l t s and D i s c u s s i o n of R e s u l t s .  18  EXPERIMENTAL A.  PREPARATION OF THE AMIDES 1.  C y c l o p e n t y l c a r b o x y l i c a c i d amide  31  2.  C y c l o p e n t y l acetamide  32  3.  C y c l o p e n t y l pr opionamide  33  k.  1-Methyl c y c l o p e n t y l acetamide  33  5.  1-Methyl c y c l o p e n t y l pr opionamide  39  6.  C i s 2 , 2 , 3 - T r i m e t h y l c y c l o p e n t y l acetamide....  kl  (cis 7.  CX-d-campholanic  a c i d amide)  Trans 2 , 2 , 3 - T r i m e t h y l c y c l o p e n t y l acetamide". k6 (trans  (X-d-campholanic a c i d amide)  v  Page 8. Trans 2 , 2 , 3 - T r i m e t h y l c y c l o p e n t y l propionamide 9. Stearamide  52 ,  10. V i t a m i n B12b (Hydroxo-cobalamin) B.  53 55  THE HYDROLYSIS OF THE AMIDES 1. Apparatus  56  2. Procedure  56  3. Data f o r the H y d r o l y s i s of the Amides c o n t a i n e d i n Table I V . LITERATURE CITED  62 68  vi LIST OF FIGURES Page  1. 2. 3. k.  POSTULATED COILED CONFORMATION FOR CARBON CHAIN OF AN AMIDE  3  POSTULATED NEIGHBORING GROUP ASSISTANCE TO AMIDE HYDROLYSIS IN VITAMIN B12, f o l l o w i n g  7  PREPARATION OF 1-METHYL CYCLOPENTYL ACETAMIDE, following ,  9  ATTEMPTED PREPARATION OF 1-METHYL CYCLOPENTYL PROPIONAMIDE, f o l l o w i n g  9  5.  PREPARATION OF 1-METHYL CYCLOPENTYL PROPIONAMIDE......  6.  PREPARATION OF CIS 2,2,3-TRIMETHYL CYCLOPENTYL ACETAMIDE (CIS Oi -d-CAMPHOLANIC ACID AMIDE, following... A SIDE PRODUCT IN THE PREPARATION OF CIS 2,2,3TRIMETHYL CYCLOPENTYL ACETAMIDE, .following PREPARATION OF TRANS 2,2,3-TRIMETHYL CYCLOPENTYL ACETAMIDE (TRANS OC-d-CAMPHOLANIC ACID AMIDE, following  7. 8.  9.  10.  SUGGESTED MECHANISM FOR THE FORMATION,OF TRANS•2,2,3TRIMETHYL CYCLOPENTYL ACETAMIDE FROM CAMPHORIMINE, following PLOT OF  l o g |5|  vs. t  12.  11 11 13  1^  FOR CYCLOPENTYL '  ACETAMIDE, f o l l o w i n g 11.  11  17  PER CENT NH EVOLVED VS. TIME IN VITAMIN B12b HYDROLYSIS, f o l l o w i n g  27  HYDROLYSIS APPARATUS, f o l l o w i n g  56  3  vii  LIST OF TABLES Page. I.  MODEL COMPOUNDS, f o l l o w i n g  II.  APPARENT RATE CONSTANT CALCULATION FOR THE HYDROLYSIS OF CYCLOPENTYL ACETAMIDE...  17  A ROUGH PATTERN FOR AMIDE HYDROLYSIS IN VITAMIN B12  18  IV.  HYDROLYSIS RESULTS FOR MODEL AMIDES, f o l l o w i n g . . .  18  V.  A COMPARISON OF HYDROLYSIS RATES IN THE ALICYCLIC SERIES WITH HYDROLYSIS RATES IN THE ALIPHATIC SERIES, f o l l o w i n g  23  ELECTROPHORETIC ANALYSIS OF THE COBALT CONTAINING HYDROLYTIC PRODUCTS FROM VITAMIN B12b  27  III.  VI.  .  *f  V I I . -XIV.DATA FROM THE HYDROLYSIS OF THE AMIDES CONTAINED IN TABLE IV CYCLOPENTYL ACETAMIDE  62  VIII.  CYCLOPENTYL PROPIONAMIDE.  63  IX.  1-METHYL CYCLOPENTYL ACETAMIDE  X.  1-METHYL CYCLOPENTYL PROPIONAMIDE  XI.  TRANS 2,2,3-TRIMETHYL CYCLOPENTYL ACETAMIDE  XII.  TRANS 2,2,3-TRIMETHYL CYCLOPENTYL PROPIONAMIDE...  66  XIII.  CYCLOPENTYL CARBOXYLIC ACID AMIDE  67  XIV.  STEAR AMIDE  67  VII.  •  6*f 65 ..  66  1  INTRODUCTION By 1955  the structure of vitamin B^  n  a  d  2  been  f u l l y elucidated through a combination of chemical degradation studies and X-ray crystallographic techniques, t h i s achievement being the r e s u l t of seven years of intensive research important compound i n the s t r u c t u r a l elucidation was carboxylic acid (II) obtained hydrolysis of vitamin B 1 2 .  (1).  An  the hexa-  as the end product of a l k a l i n e  (The f a c t that the hexacarboxylie  acid i s formed i n hot a l k a l i n e hydrolysis rather than the heptacarboxylic to be due  acid r e s u l t i n g from acid hydrolysis i s believed  to the formation of the extremely i n e r t lactam r i n g  fused to r i n g B of the c o r r i n nucleus  (2)).  Examination of the stepwise h y d r o l y t i c path leading from vitamin B 1 2 to the polycarboxylic acids has shown (3, that the amide groups of the vitamin divide broadly i n t o sets according  to t h e i r ease of hydrolysis.  h) two  Thus, i n acid  hydrolysis, as i n mild a l k a l i n e hydrolysis, there appears to be one set of four l a b i l e amide groups and a second set of three r e l a t i v e l y stable amide groups.  Of the four l a b i l e amides, one  i s a secondary amide being substituted with 1 - aminopropan-2-ol which i n turn forms an ester linkage with the nucleotide of the molecule (R i n Structure I ) .  (The o v e r a l l hydrolysis of  vitamin B 1 2 i s s l i g h t l y more complex due nucleotide whose l i b e r a t i o n may  portion  to the presence of the  either accompany or precede the  hydrolysis of the l a b i l e amide groups  (3)).  To f o l l o w page 1  C  H  '  ccy-i  C  2  H  2  C  H  3  C  H  3  ' ' C H  2  C H  2  C 0  2  H  2  These r e s u l t s could be interpreted i n the l i g h t of the known structure of vitamin B 1 2 by assuming that the l a b i l e amide groups were those on the propionamide chains and the  three  r e l a t i v e l y stable amide groups were those on the acetamide chains, their resistance to hydrolysis being due to substitution on the  (3 carbon atom (5)«  However, t h i s explanation  takes  into account neither the s t e r i c e f f e c t of substituents at neighbouring positions on the pyrroline rings of the c o r r i n nucleus nor the possible i n a p p l i c a b i l i t y of empirical r u l e s (such as the "Rule of Six") i n c y c l i c systems such as those involved here which have not previously been examined i n t h i s connection  (6). The b e l i e f that the substituents could be  important to the r e a c t i v i t y of the amides was work of Newman and h i s group (7) who hydrolysis of some highly hindered  sterically  supported by the  studied the rates of  branched chain amides.  The  r e s u l t s of these experiments could be r a t i o n a l i z e d quite successfully i n terms of Newman's empirical r u l e of s i x which was  developed mainly from e a r l i e r observations  made by H.  A.  Smith ( 8 - 1 3 ) on the rates of acid catalyzed e s t e r i f i c a t i o n of a large number of a l i p h a t i c acids (see also Ik).  Smith explained  his r e s u l t s by postulating that the carbon chain of the acids tends to have a c o i l e d rather than a straight chain conformation (see Figure 1)  and c l a s s i f i e d the acids according  number of substituents on the acid ( 1 3 ) .  OL and  (3  to the t o t a l  carbons of the  Newman emphasized the r e l a t i v e l y larger e f f e c t of (3  3 substitution  and s i m p l i f i e d Smith's c l a s s i f i c a t i o n i n h i s r u l e  of s i x which i s s t a t e d  (15):  as  In r e a c t i o n s i n v o l v i n g a d d i t i o n to an uns a t u r a t e d f u n c t i o n , the g r e a t e r number of atoms i n the s i x p o s i t i o n , the g r e a t e r w i l l be the s t e r i c e f f e c t . In the amides, as i n the a c i d s , determined by numbering  the s i x p o s i t i o n i s  the c a r b o n y l oxygen as one and counting 1).  back along the c h a i n (see F i g u r e  C6 5,C-H6  c — c  3  2\ NH  FIGURE A simple a p p l i c a t i o n the  2  1.  of the r u l e of s i x i s found i n  e s t e r i f i c a t i o n of normal a l i p h a t i c a c i d s  acetic acid.  Thus p r o p i o n i c  beginning w i t h  a c i d w i t h no atoms i n the s i x  p o s i t i o n e s t e r i f i e s a t approximately the same r a t e as a c e t i c a c i d but e s t e r i f i e s almost twice as f a s t as b u t y r i c  a c i d which  has three atoms i n the s i x p o s i t i o n and, f u r t h e r m o r e , h i g h e r than b u t y r i c butyric  acid  (8).  a c i d e s t e r i f y at n e a r l y  homologues  the same r a t e  as  k  On t h i s basis, then, i t was  of i n t e r e s t to investigate  more c l o s e l y the hydrolytic properties of the amide groups of vitamin B12,  e s p e c i a l l y as the amide groups are thought to have  a s i g n i f i c a n t function i n the biochemical  action of the vitamin.  The r o l e of the amides i n the microbiological a c t i v i t y of vitamin B12 has been suggested to be at l e a s t twofold (16). possible function i s that of a s s i s t i n g i n anchoring the  One  vitamin  to the s p e c i f i c protein with which i t i s so c l o s e l y associated in i t s activity. the anchors.  The three stable amide groups are envisaged as  The three l a b i l e amide groups are postulated  serving d i r e c t l y i n the b i o l o g i c a l a c t i v i t y of vitamin B12  as as,  f o r example, the part that the vitamin i s believed to play i n protein synthesis. N-substituted  This i s suggested by the f a c t that certain  amides (prepared from a monocarboxylic a c i d , i . e . ,  corresponding to a l a b i l e amide group) behave as antagonists vitamin B12  (17, 18).  to  C l e a r l y i t would be of i n t e r e s t to know  i f the amide involved was  an acetamide or a propionamlde and i t  i s t h i s that the present work i s designed to show. The experimental approach taken to learn more of the hydrolytic properties of the amides was model compounds which simulated  that of synthesizing  as c l o s e l y as possible the  s t e r i c environment of each amide group on the c o r r i n nucleus and subjecting each to alkaline hydrolysis.  Table I contains  a l i s t of the model compounds synthesized, their s i x number and the position on the c o r r i n nucleus which they were intended to represent.  To follow page *f Table  S i x number  Amide  C H  2  C O N H  C H  2  C H  C H  C H  ~ ^ ^ C H  - ^ ^ C H  2  2  2  2  C O N H  C H  2  C H  2  18  3 8 13  2  ;  3  9 "7  2  C O N H  2  17  3  18  2  C O N H  ® JTt?nberinr a ^ o ^ t e d ^ f  Model f o r position  2  C O N H  C O N H  2  I  r  2  the I . U . K A . O .  3.8,13  i n  1959-  5 The  c y c l o p e n t y l s t r u c t u r e f o r the model compounds  thought s u i t a b l e s i n c e t h i s s t r u c t u r e , from a s t e r i c p o i n t view, probably r e p r e s e n t s For example, i t has  the a c t u a l s i t u a t i o n f a i r l y  been p o i n t e d  out  (13,  15)  that  was of  accurately.  Oi  (3  and  s u b s t i t u e n t s which are p o s i t i o n e d away from the r e a c t i o n s i t e involvement i n a r i n g are l e s s e f f e c t i v e i n s t e r i c a l l y r e a c t i o n a t the s i t e of u n s a t u r a t i o n .  hindering  This i s i l l u s t r a t e d  the f a c t t h a t cyclohexane c a r b o x y l i c a c i d i s e s t e r i f i e d methanol i n the presence of HCl) acid.  Furthermore, a cyclopentane r i n g has  In the present  would appear to serve  by  (with  2h times as f a s t as d i e t h y l a c e t i c been shown to o f f e r  l e s s s t e r i c hindrance than a cyclohexane r i n g to (15).  by  esterification  case, then, the c y c l o p e n t y l  structure  as a s u i t a b l e model i n c o n t r a s t , f o r  example, to an a l i p h a t i c compound w i t h the same number of atoms i n the s i x p o s i t i o n . 5 and  (A s l i g h t l y u n d e s i r a b l e  f e a t u r e of models  6 i s the methyl group s u b s t i t u t e d at p o s i t i o n 3 of  cyclopentane r i n g . )  T h i s methyl group, however, i s t r a n s  the amide f u n c t i o n (see d i s c u s s i o n of i t s p r e p a r a t i o n , and  i s too f a r removed from the r e a c t i o n center  h i n d r a n c e to the r e a c t i o n . )  (19)  the plan-  non-planarity  p o s t u l a t e d f o r the p y r r o l i n e r i n g s of the c o r r i n nucleus As mentioned, the models are designed t o s t e r i c c o n d i t i o n s alone and no attempt has  )  to o f f e r s t e r i c  Another f a c t o r i n f a v o r of  which would correspond to a s i m i l a r  to  p. 13  c y c l o p e n t y l s t r u c t u r a l u n i t i s i t s s l i g h t d e v i a t i o n from ar i t y  the  (20).  represent  been made to  6 incorporate, a l l the features of the environment of the amides of vitamin B12.  In p a r t i c u l a r no account i s taken of the  unsaturation i n the c o r r i n nucleus nor of the nitrogen  contained  i n the pyrroline rings and i t s co-ordination with the c e n t r a l oobalt ion.  However, the e f f e c t of these elements, which would  be l a r g e l y inductive i n nature, should be quite secondary to the more important s t e r i c factors since the reaction centers  (i.e.,  amide groups) are insulated by at l e a s t two methylene groups from the s i t e s of unsaturation.  It must be noted, though, that  the higher degree of saturation i n rings A and D creates a s i t u a t i o n i n which the acetamide groups of vitamin B12 are not affected equally by the elements of unsaturation and bonding between nitrogen and cobalt.  co-ordinate  Thus, the acetamide groups  i n rings A and D could possibly hydrolyze  at a s l i g h t l y slower  rate than that expected on purely s t e r i c grounds when compared to the rate of a l k a l i n e hydrolysis of the acetamide group at position 8 i n r i n g B.  (Throughout t h i s discussion i t has been  assumed that the rate c o n t r o l l i n g step i n hydrolysis, whether acid or base catalyzed, i s attack at the e l e c t r o p h i l i c carbon atom of the carbonyl f u n c t i o n — b y  a water molecule on the  protonated carbonyl group i n the acid catalyzed hydrolysis and by hydroxyl ion on a free carbonyl group i n the base catalyzed (  hydrolysis ( 2 1 ) ) . The usefulness of the models, then, i s based upon the reasonably well founded assumption (6,  22)  that s t e r i c and polar  7 e f f e c t s are independent variables and upon the a d d i t i o n a l assumption that, i n the present case, the v a r i a t i o n i n inductive e f f e c t s accompanying the s t r u c t u r a l changes are n e g l i g i b l e compared to the r e l a t i v e l y large changes i n s t e r i c e f f e c t s ( s t e r i c e f f e c t s which, on the framework of the cyclopentyl structure, p a r a l l e l those i n vitamin One  B12).  s i t u a t i o n f o r which the models are not  suitable i s found at p o s i t i o n 17 i n r i n g D;  strictly  i . e . , the secondary  amide formed between the propionic acid residue and 1-aminopropan-2-ol.  The e f f e c t of t h i s substitution by l-aminopropan-2-ol  on the rate of a l k a l i n e hydrolysis i s d i f f i c u l t to predict since there are a number of f a c t o r s to be considered which are absent i n the primary amide case and about which there seems to be a shortage of recorded  information.  From a study on the rates of  alkaline hydrolysis of primary a l k y l acetates c a r r i e d out by Newman and h i s group of workers (22,  23) i t might be expected  that the amino portion of the amide would offer some s t e r i c hindrance to hydrolysis i n the present case.  However, compari-  son between the rates of hydrolysis f o r primary and secondary amides cannot properly be made on the basis of sterie e f f e c t s alone because of the d i f f e r i n g b a s i c i t i e s of the leaving amino moieties  (22).  Furthermore, there i s a p o s s i b i l i t y of neigh-  bouring group assistance to hydrolysis by the phosphate ester group (2k).  A possible mechanism f o r t h i s suggested assistance  to hydrolysis i s i l l u s t r a t e d i n Figure  2.  To follow page 7  Figure  2  C H  92 H  OH N H  0  C H  Q  C H C H  2  q  -f  C-^ OH  8 Although the present models are not s u i t a b l e f o r p r e d i c t i n g the r e l a t i v e r a t e of h y d r o l y s i s of t h i s  secondary  amide f u n c t i o n , p r e v i o u s h y d r o l y s i s work ( 3 ) has i n d i c a t e d  that  under some c o n d i t i o n s l - a m i n o p r o p a n - 2 - o l i s f r e e d d u r i n g the h y d r o l y s i s of the l a b i l e primary amide groups of v i t a m i n B 1 2 and i f the proposed working  s t e r i c hindrance and p o l a r e f f e c t s a r e  to slow the r e a c t i o n , they are a t l e a s t balanced by some  other f a c t o r such as the suggested n e i g h b o u r i n g group e f f e c t of the phosphate e s t e r  group.  B e s i d e s the model compounds above, v i t a m i n B 1 2 b , simple d e r i v a t i v e of v i t a m i n B 1 2 , was  a  prepared and h y d r o l y z e d as  were c y c l o p e n t y l carboxamide and stearamide, the former f o r the sake of completeness,  the l a t t e r i n order to c o r r e l a t e  r e s u l t s o b t a i n e d here w i t h those of Cason's ( 5 , h y d r o l y t i c method was  followed.  the  2 5 ) whose  The p r e p a r a t i o n of the model  compounds i n t r o d u c e d some i n t e r e s t i n g c h e m i s t r y which w i l l presented i n the f o l l o w i n g  discussion.  be  9  DISCUSSION 1.  P r e p a r a t i o n of the amides C y c l o p e n t y l c a r b o x y l i c a c i d amide, c y c l o p e n t y l  acetamide, c y c l o p e n t y l propionamide  and stearamide were prepared  from the commercially a v a i l a b l e a c i d s by t r e a t i n g the r e s p e c t i v e a c i d c h l o r i d e s with ammonia i n dry ether*  These amides, l i k e  a l l the amides prepared, were r e c r y s t a l l i z e d  ( u s u a l l y from  benzene) t o a constant m e l t i n g p o i n t . 1-Methyl c y c l o p e n t y l acetamide was t i o n a l methods as i n d i c a t e d i n F i g u r e 3 .  prepared by  The step i n v o l v i n g the  replacement  of c h l o r i d e w i t h malonate ( 2 6 ,  y i e l d (13$)  w i t h a h i g h r e c o v e r y of d i e t h y l malonate.  was  expected s i n c e the competing  conven-  27)  gave a v e r y poor This  e l i m i n a t i o n r e a c t i o n to g i v e  1-methyl cyclopentene c o u l d be p r e d i c t e d under the p r e v a i l i n g b a s i c c o n d i t i o n s t o be much f a s t e r than the replacement with i t s h i g h s t e r i c requirements ( 2 6 ) .  The f i r s t  reaction  s t e p , the  treatment of cyclopentanone w i t h methyl magnesium i o d i d e , gave, besides the 1-methyl c y c l o p e n t a n o l , two by-products one of which was  i d e n t i f i e d as compound V ( 2 8 ,  6=0  29).  *  To follow page 9 0 II  •OH HCI  1. C H | C 0 Figure  3  2  C 0 N H  1.  S 0 C l .62. N h L  2  E t )  2  2. H C I 3.  C H  2  o  ^y  H e a t , - C 0  c H  2  C 0  2  2  H  XH CH OH 2  2  Y E  Figure  CH C0 H 2  4  vCH COCHN  2  2  1. S O C l  2 j  2. C H N 2  2  ^  >  \  Figure  /  5  2  AgNOg . NH  3  \CH CH CONH 2  2  2  10 The  second by-product,  which was  obtained as p a l e 1,3  y e l l o w c u b i c c r y s t a l s , i s almost c e r t a i n l y the compound dicyclopentylidenecyclopentanone  and formed by base c a t a l y z e d condensation  of three molecules  cyclopentanone with the subsequent l o s s of water. spectrum showed a peak at 1685  and 1621  conjugation group. violet  cm"  1  ( s p l i t t i n g of  The  (cyclopentanone two  spectrum of the compound corresponds of 305  f o r VI.  infrared  carbonyl  C = C a d s o r p t i o n peak due  ( 3 0 b ) ) and no peak corresponding  of  s t r o n g peaks a t to  to an hydroxy  A strong peak a t 302 TC\JA (€ = 22,*+00) i n the  value ( 3 1 ) for  -1  with exoring u n s a t u r a t i o n ( 3 0 a ) ) ,  conjugated 16^2  cm  (28)  (VI) d e s c r i b e d by Wallach  ultra-  to a c a l c u l a t e d  A s a t i s f a c t o r y elemental  analysis  the compound has not been obtained, however. An attempt was  made to prepare  1-methyl c y c l o p e n t y l  propionamide ( V I I ) as i n d i c a t e d i n F i g u r e k ( 3 2 , 3 3 ) . r o u t e was  abandoned due  to the extremely  This  low y i e l d s obtained i n  the step i n v o l v i n g r e a c t i o n of the G r i g n a r d reagent w i t h oxide.  ethylene  11  An a l t e r n a t e r o u t e as i n d i c a t e d i n F i g u r e 5 was used which i n v o l v e d A r n d t - E i s t e r t homologation  of the a c i d I I I , the  amide being formed d i r e c t l y from the diazoketone by treatment with s i l v e r n i t r a t e and c o n c e n t r a t e d ammonium hydroxide (3*+)»  C H C 0 H  .  2  2  '•  S  Q  C  I  \ C H  2.  2.CH N 2  2  C O C H N  2  9  ^  ( 1 2  ^  A  '  N  Q  N H  3  \ C H  (  ] \  2  C H  2  C O N H  2  ^ /  FIGURE 5 . S i n c e a c i d I I I was a key i n t e r m e d i a t e here i t s s t r u c t u r e was checked occurred.  to make c e r t a i n no rearrangement had  The n u c l e a r magnetic  resonance  spectrum  agreed i n every r e s p e c t w i t h the g i v e n s t r u c t u r e .  of t h i s  acid  In particular  the l a c k of s p l i t t i n g of the methyl peak a t 9 . 0 2 t and the exo r i n g methylene peak a t 7 . 7 9 t i n d i c a t e d a quaternary carbon linkage. The s y n t h e s i s of 2 , 2 , 3 , - t r i m e t h y l c y c l o p e n t y l acetamide  (0(-d-campholanic a c i d amide) proved t o be q u i t e  i n t e r e s t i n g from a c h e m i c a l p o i n t of view. was  I n i t i a l l y , an attempt  made to prepare t h i s a c i d f o l l o w i n g a procedure g i v e n by  LiPP ( 3 5 , see a l s o 36-ltO) which i s o u t l i n e d i n F i g u r e 6 . The course of the r e a c t i o n , however, was not as s t r a i g h t f o r w a r d as the l i t e r a t u r e suggested and i n p a r t i c u l a r a  To follow page 11  H  ?  S  ^ - ^  Q  ^ C H  2  C N ^ ! i  •NOH  C H  o  C 0 N H  2  J -  Q  S  C  l ?  NH  2.  2  Q  3  XI  R g u r e _ 6  C=N H© *N-OH  = 0  .  X Figure  7  12  g a s - l i q u i d chromatography examination the hydrogenation ratios 3:8:1.  of the methyl e s t e r of  product X y i e l d e d three components i n the  The i n f r a r e d spectra of the carbonyl r e g i o n  showed peaks at 1 7 3 3 , three components.  1736,  and 176^ cm."  1  r e s p e c t i v e l y f o r the  The f i r s t component d e c o l o r i z e d a 5$  s o l u t i o n of bromine i n carbon t e t r a c h l o r i d e while the other components d i d not d e c o l o r i z e the bromine s o l u t i o n .  The  two  center  f r a c t i o n was considered to be the methyl e s t e r of c i s 2 , 2 , 3 t r i m e t h y l c y c l o p e n t y l a c e t i c a c i d (X) ( c i s -Ck -d-campholanic 2 -lactone  a c i d ) and the l a s t f r a c t i o n was considered to be the  X I I formed by the a d d i t i o n of the a c i d f u n c t i o n to the double bond created during the r e a c t i o n ( 3 7 ,  see F i g u r e 7 ) .  The  first  component was not i d e n t i f i e d . Lipp assigned the c i s c o n f i g u r a t i o n to the 2 , 2 , 3 t r i m e t h y l c y c l o p e n t y l a c e t i c a c i d (X) on the basis of S k i t a ' s r u l e (*+l) which suggested that hydrogenation  i n the presence of  a c i d should give the c i s isomer while hydrogenation or basic c o n d i t i o n s should give the trans isomer.  under n e u t r a l As a confirma-  t i o n Lipp c a r r i e d out the r e d u c t i o n of (X-d-campholenic  a c i d amide  (amide of IX) using the F o l k i n - W i l s t a t t e r method ( 3 5 , i . e . , r e d u c t i o n i n an absolute ether s o l u t i o n with platinum black catalyst.  The c o n d i t i o n s i n the case of the n e u t r a l or very  s l i g h t l y basic amide s o l u t i o n would be expected to give the trans isomer i f S k i t a ' s r u l e h e l d ) .  The amide obtained i n t h i s  way d i f f e r e d from that prepared by r e d u c t i o n under a c i d c o n d i t i o n s  13 but was (lf2)  i d e n t i c a l to the amide prepared by Mahla and  which was The  considered  to have a t r a n s  method used by Mahla and  configuration.  Tiemann (*4-2, see a l s o *+3  to prepare t r a n s 2 , 2 , 3 - t r i m e t h y l  and Mf)  cyclopentyl acetic  a c i d ( X V I I I ) ( t r a n s OC-D-campholanic a c i d ) was as i t turned Lipp's  i n t h i s way  unusual  out, gave a much c l e a n e r p r e p a r a t i o n  (see F i g u r e 8 ) . was  Tiemann  and,  than t h a t  Moreover the t r a n s c o n f i g u r a t i o n  of  obtained  p r e f e r r e d f o r the proposed k i n e t i c s t u d i e s on  the  amides s i n c e i t removed the p o s s i b i l i t y , though s l i g h t even i n the c i s c o n f i g u r a t i o n , of the methyl group i n p o s i t i o n 3 of cyclopentane r i n g c o n t r i b u t i n g to the s t e r i c hindrance of  the  the  molecule t o h y d r o l y s i s . The  products from the treatment of molten camphorimine  w i t h oxygen ( F i g u r e 8 ) were i n i t i a l l y separated i n t o two  fractions.  by  distillation  G a s - l i q u i d chromatography showed that both  these f r a c t i o n s were mixtures and the lower b o i l i n g f r a c t i o n was  f u r t h e r r e s o l v e d i n t o f o u r components by p r e p a r a t i v e  l i q u i d chromatography to y i e l d camphor, cyclopent-3-enyl  acetic acid n i t r i l e  2,2,3-trimethyl  (XVII) ( (X-d-camphblenic  a c i d n i t r i l e ) whose i n f r a r e d spectrum was  i d e n t i c a l to t h a t  compound V I I I prepared by L i p p ' s procedure ( F i g u r e 6 ) , 2,2,3-trimethyl  cyclopentyl acetic acid n i t r i l e  campholanic a c i d n i t r i l e ) and  gas-  (XVI)  of  trans (transcX-d-  an u n i d e n t i f i e d f o u r t h component  which i s probably a s m a l l amount of the h i g h e r  boiling  fraction.  To follow page 13  2. N H  3  F i g u r e  8  This last component showed n i t r i l e (22^-2 cm." ) and carbonyl 1  (1715 cm. ) absorption i n the infrared and could possibly be -1  the 5-isopropylheptanon-2-nitrile-7 described by Mania and Tiemann (**2) but a positive identification of this compound must await an examination of the higher boiling fraction. It seems reasonable especially when the ketonic analogy i s considered (*+5) that this decomposition of camphorimine proceeds by a free radical process and a mechanism which accounts for some of the products i s suggested i n Figure 9« Preliminary results from experiments designed to test this hypothesis have not been conclusive however.  Thus, irradiation 6f  a 0 . 2 M solution of pure camphorimine i n spectroscopically pure cyclohexane with a broad range of U.V. light at room temperature produced no detectable change i n the camphorimine.  Similar  irradiation of a 0.0** M solution of camphorimine i n the presence of a few crystals of benzoyl peroxide did show a decrease i n imine concentration (U.V. spectra and I.R. spectra of samples taken at intervals) and but only a slow development of a n i t r i l e function.  The control for this irradiation (i.e., no irradiation  but benzoyl peroxide present) appeared to follow a different course from that of the irradiated sample.  Lastly, i t was noted  that a sample of pure camphorimine i n a stoppered flask which had been flushed with nitrogen decomposed much less quickly than a sample of camphorimine i n a flask not so treated.  (Cf. *t2,  To follow page 14  2873C I956).)  *NH  Figure 9  15  supporting the autoxidation mechanism.) Further experiments are planned which should make the mechanism of this transformation clearer. Since vitamin B 1 2 i t s e l f contains a labile cyanide group i t was desirable to remove this i f possible before hydrolyzing the vitamin.  The cyanide group i s removed rather  easily by photolysis i n very dilute acid solution (M-6).  The  course of the photolysis was followed by changes in the ultraviolet spectrum of the aqueous solution with vitamin B 1 2 having absorption peaks at 278, 361, 550 Tf[j{ which are unaltered at various pH's.  Vitamin B 1 2 or hydroxocobalamin, the product B  of photolysis, differs from vitamin B 1 2 only i n the replacement of cyanide by hydroxy group on the central cobalt ion and i n i t s U.V.  spectrum which has absorption peaks at 27*+, 351, *t05, 522 ^\pv  at pH 2 that shift to 278, 358, *fl8, 5 3 5 ^ a t pH 10 (1+7). 2.  Hydrolysis of the amides The hydrolysis procedure and apparatus used was that  of Cason and Wolfhagen ( 2 5 ; 12).  see Experimental  section, Figure  Its simplicity was attractive and i t has been found to give  f a i r l y reproducible results. about %  Gason states that a precision of  i n the determination of the rate constants i s permitted  using 0 . 5 N KOH i n boiling 1- propanol except for the faster rates of hydrolysis where the precision i s about 10%,  These  16  limits of precision are adequate for the distinctions desired i n the present hydrolysis  series.  For finer distinctions  (e.g., the steric effect on the rate of hydrolysis of the substituent s size i n the six position) the accuracy of the 1  method may be increased by using more dilute alkaline  solutions  and the lower temperature of boiling ethanol ( 2 5 ) . 3»  Calculation of the rate constants The amides were compared on the basis of apparent rate  constants (see Table IV) expressed i n l i t e r s moles'^hours"^.  The  second order apparent rate constant, k, for the temperature of the boiling solution (95.0°) was calculated from the equation \ t  2.303  ~ kfa^bT  (•, ( l G g  b  a  , , „ a-x% +  l o g  b^x ' 0  where a i s the i n i t i a l molarity of a l k a l i , b i s the i n i t i a l molarity of amide and x i s the moles per l i t e r of amide reacted in time t hours.  By plotting log | ~ against t, the equation  was reduced to the form k  *  = 2*303 , 1 , a-b slope'* v  The plot gave a satisfactory straight line for points taken up to the half time of the reaction.  Selection of the best  straight line for the fastest hydrolysis was somewhat arbitrary however due to the slight curve of the plot and i n this case did not give s t r i c t l y reproducible results (see Table IV).  A  17 sample p l o t i s shown i n F i g u r e 10 and i n Table I I a r e the c a l c u l a t e d v a l u e s from which the p l o t was made.  Data f o r the  other amides was t r e a t e d i n a s i m i l a r manner (see E x p e r i m e n t a l ) . The complicated k i n e t i c s i n v o l v e d i n the h y d r o l y s i s of v i t a m i n B12b prevents t h i s type of treatment  and the r e s u l t s i n t h i s  case were r e c o r d e d as per cent ammonia evolved i n time t hours (see Table V I and F i g u r e  11). TABLE I I  Apparent r a t e constant c a l c u l a t i o n f o r the h y d r o l y s i s of c y c l o p e n t y l acetamide. (hrs.)  x (M)  a-x (M)  0.00960  0.1+782  0.06975  0.8361  k.k8  0.01795  .*t698  .0611+0  .8837  6.58  .02359  . 1+61+2  .05576  .9201+  9.50  .0297*+  .1+581  .01+961  .965^  11.58  .03390  A539  .oi+5!+5  .999 +  13.53  .037^6  A503  .01+189  1.031!+  15.53  .0^071".  .1+1+71  .03861+  1.0631+  slope =  =  •log a  1.97  a = O.I+878 m b l e s / l i t e r .  k  b-x (M)  i^o|-o.iy^  (a-b] (  s l o p  e  1  b = 0.07935 m o l e s / l i t e r ,  hours ( F i g u r e 1 0 ) .  >  =  °-°96 l i t e r s  moles" hours" . 1  1  18 k.  R e s u l t s and d i s c u s s i o n of r e s u l t s The  first  c l a s s e s according  s i x amides of Table  IV f a l l i n t o  two  to t h e i r r a t e s of a l k a l i n e h y d r o l y s i s .  As  can be seen, the three propionamide d e r i v a t i v e s c o n s t i t u t e the f a s t e r hydrolyzing c l a s s while  the acetamide d e r i v a t i v e s form  the more s l o w l y h y d r o l y z i n g c l a s s . experimental  support  These r e s u l t s provide  f o r the previous  assumption t h a t  the  propionamide groups are the l a b i l e ones i n v i t a m i n B 1 2 . the r e s u l t s obtained  i t would, however, be d i f f i c u l t to p r e d i c t  which of the l a b i l e amide f u n c t i o n s i n v i t a m i n B 1 2 the f a s t e s t .  hydrolyzed  I t i s i n t e r e s t i n g t o note t h a t , of the  h y d r o l y z i n g acetamide d e r i v a t i v e s , 3 h y d r o l y z e s than e i t h e r 1 or 5«  From  On t h i s b a s i s i t may  amide group at p o s i t i o n 18  slowly  much more s l o w l y  be expected t h a t  of the c o r r i n nucleus w i l l  the  hydrolyze  c o n s i d e r a b l y f a s t e r than the amide groups at p o s i t i o n s 2 and Thus, i f the s t e r i c e f f e c t s present r e s i d u e s have a dominant e f f e c t i n determining  7.  i n the amide the r a t e s of  h y d r o l y s i s , a rough p a t t e r n of amide h y d r o l y s i s under a l k a l i n e c o n d i t i o n s may  be expected f o r v i t a m i n B 1 2 as g i v e n i n Table I I I . TABLE I I I  Number of amides in class  P o s i t i o n s on c o r r i n nucleus  Relative rates of h y d r o l y s i s  k  3,8,13,17*  27  1  18  9  2  2,7  1  The propionamide a t p o s i t i o n 17 suggested i n the I n t r o d u c t i o n .  may  be anomolous f o r reasons  TABLE IV Apparent rate constants f o r the hydrolysis of the anides. Amide  CH CONH  1. ; .  2:.  Six number  2  0.096  2  /NcH CH CONH 2  r:\ ^ ^ C H  2  2  C O N H  2  2  4.. ^ W c H C H C O N H 2  2  CH CONH 2  2  0  7.  8.  2  / ^ C O N H  CH (CH )  2  2  )CH CH CONH  6 —T  2  ,-.0.094  3  0.31 ,,0..29  9  0*011  3  0.26  6  o 090  3  0.27 , 0.27  (4)  2  CONH  -1 k l i t e r s moles hours" .  ,  ,0.27  0.090  0  0.24  0.011  , 0.23  0.25 , 0.26 ( O 38 ) • * Value found by Cason and Wolfhagen ( 26 ) f o r stearamide. 3  2  ) 6  2  3  0  , . to follow page 18.  19 In terms of the e m p i r i c a l r u l e s d i s c u s s e d  e a r l i e r , the  r e s u l t s of a l k a l i n e h y d r o l y s i s of the amides s t u d i e d here can be i n t e r p r e t e d q u i t e s u c c e s s f u l l y by e i t h e r Newman's r u l e o f s i x or by c o n s i d e r i n g carbon.  the v a r y i n g  degree of s u b s t i t u t i o n on the (3  Thus, the d i f f e r e n c e between the r a t e c o n s t a n t s f o r  h y d r o l y s i s of c y c l o p e n t y l acetamide (amide 1 ) and c y c l o p e n t y l propionamide (amide 2 ) can be s a i d t o be due e i t h e r t o the g r e a t e r number of groups i n the s i x p o s i t i o n ( 6 : 3 ) or t o the increased  s u b s t i t u t i o n on the (2> carbon o f the former amide.  S i m i l a r l y i n the s e r i e s of acetamide d e r i v a t i v e s 1 ,  3 , and 5 of  Table IV, the much slower r a t e of h y d r o l y s i s of 3 as compared t o 1 and 5 can be r a t i o n a l i z e d i n terms of i n c r e a s e d (3 s u b s t i t u t i o n or i n c r e a s e d  s i x number.  These two e m p i r i c a l l y d e r i v e d  concepts  a r e , of course, c l o s e l y r e l a t e d s i n c e they both d e s c r i b e the r e s u l t s o f a more fundamental property sumably the p o s t u l a t e d  of the m o l e c u l e — p r e -  c o i l e d conformations of the carbon  chain  which i s capable of p l a c i n g atoms a t the s i x p o s i t i o n i n c l o s e proximity  t o the c a r b o n y l  f u n c t i o n (see F i g u r e  1).  The r u l e of  s i x , perhaps, i s the more convenient of the two concepts s i n c e it  i s more d e s c r i p t i v e and focuses a t t e n t i o n on the fundamental  property  of the molecule which i t i s designed t o r e f l e c t .  (Iti s  w e l l t o emphasize a t t h i s p o i n t t h a t t h e r u l e of s i x i s e m p i r i c a l and Newman c a u t i o n s  t h a t i t i s t o be used only as a s u b s t i t u t e  f o r molecular models s i n c e the examination o f models i s the best way o f e s t i m a t i n g  steric factors (22)0  20  Considering the acetamide derivatives, i . e . , 1, 3 and 5 of Table IV, further, i t i s interesting to observe the relatively small effect on the hydrolysis rate of a gem dimethyl group adjacent to the acetamide chain as shown i n the only slightly decreased rate of hydrolysis of amide 5 compared with that of amide 1.  This result i s i n agreement with Cason and  Wolfhagen's conclusion that methyl substitution i n the  #  position of normal fatty acid amides has l i t t l e or no effect on the rate of hydrolysis ( 2 5 ) .  Their conclusion receives  additional experimental support from the results for the hydrolysis of the propionamide derivatives of Table IV (i.e., amides 2, *t, and 6) which show that these amides with branching at the 6 carbon have approximately the same rate of hydrolysis as stearamide (amide 8) which has no branching at the 6 carbon. This result then, assuming that the model compounds from which i t was obtained represent the actual situation f a i r l y accurately, would indicate that the substituents at neighbouring positions of the pyrroline rings of the corrin nucleus affect the rate of hydrolysis of the amides very l i t t l e . Although, as pointed out, the increased steric hindrance to hydrolysis accompanying the replacement of a hydrogen i n the six position by a methyl group i s small (e.g., the small effect of the gem dimethyl group mentioned above) and although the accuracy of the hydrolysis procedure prevents a  21  rigorous treatment, a trend can be seen for the steric effect of substituting a hydrogen atom i n the six position with a larger group.  This trend i s illustrated by the following  comparisons in which cyclopentyl propionamide (amide 2) hydrolyzes slightly faster than 1-methyl cyclopentyl propionamide (amide k) and cyclopentyl acetamide (amide 1)  hydrolyzes  faster than 2,2,3-trimethyl cyclopentyl acetamide (amide 5 ) . The empirical rules as stated do not account for this and although the rule of six could possibly be adapted to include both size and type of substituent i n the six position rather than just number, a serious limitation i s placed on the rule as i t stands—a limitation which among others recognized by Newman ( 1 5 ) .  was  The effect of substituting a methyl  group for a hydrogen atom in the six position i s , as mentioned, relatively small, but i f instead a highly polar group was substituted in this position, the rule of six could not be expected to predict the relative effect on, for example, the rate of alkaline hydrolysis of an amide since polar effects under these conditions may be more important than steric effects (see the suggested neighbouring group effect of the phosphate ester group on the hydrolysis of the secondary amide at position 17 of the corrin nucleus, Figure 2 ) . A further modification of the rule of six i s required as a result of the fact that i t was derived from reactions i n  22  the aliphatic series and does not necessarily apply to cyclic systems.  Thus, although the results recorded in Table IV (with  the exception of that for cyclopentyl carboxylic acid amide (amide 7 ) ) have been successfully interpreted in terms of the rule of six, up until now no differentiation has been made between atoms in the six position which are ring members and those which are exocyclic.  It has already been pointed out (see Introduction  regarding the suitability of the model compounds) that the atoms in the six position which are also ring members are not as effective sterically as their counterparts i n the aliphatic series.  Newman ( 1 5 ) noted this fact and defined a so-called  "effective" six number by which was meant the number of atoms in the six position capable of yielding a coiled structure (e.g., mesitoic acid was assigned an effective six number of 6).  But, apart from observing that increasing steric hindrance  to acid catalyzed esterification accompanied increasing ring size i n the series cyclobutane carboxylic acid through cyeloheptyl carboxylic acid, Newman did not assign effective six numbers in this series and has subsequently concluded ( 2 2 ) from comparing the rates of acid catalyzed esterification of aliphatic acids branched at the CX. carbon with the rates for the corresponding cycloalkane carboxylic acids that atoms in the six position that are also in a cyclic structure or are directly attached to i t are relatively ineffective from a steric point of view and should be disregarded.  As an example, the esterifica-  tion rate for isobutyric acid (six number 0 ) approximates the  23  rate for cycloalkane carboxylic acids whereas the esterification rate for diethyl acetic acid (six number 6) i s one-twenty sixth that for cyclohexane carboxylic acid (six number 5> i f ring atoms and hydrogens attached to them are counted). Assuming a rough correlation between the results obtained i n the present work and those obtained by Cason and Wolfhagen (25) for branched chain fatty acid amides (the reason for the difference i n the results for stearamide (see Table IV) has not been determined), the decreased steric effect caused by ring formation involving two groups having atoms i n the six position can be illustrated by comparing apparent rate constants in the two series of Table V. It i s apparent from Table V that Newman's conclusion concerning the relatively minor steric effect of atoms i n the six positions which are also members of a ring or directly attached thereto does not seem to apply i n cases other than those i n which the carboxyl group i s directly joined to the ring.  Thus i t seems to be valid i n the case where branching at  the GX. carbon (by groups containing atoms i n the six position) i s modified by the formation of a cycloalkane structure as can be seen by comparing the apparent rates of hydrolysis of cyclopentyl carboxylic acid amide and 2-ethyl octadecanoamide, but i s not valid for the situation where branching at the ^ carbon i s modified by ring formation. This i s evident when the rates of  To follow page 23  TABLE V k  95°  k  (liters moles hours ) 1  2  <  0,095  1  CH (CH ) 3  2  <  6  6  | 4  CHCH CONH 2  )  2  (  9  CONH  2  0.011  3  2  | 2 l  '<9 )  )  2  CH (CH ) 3  0.24  ( 4 )  2  ( 5  3  2  0.008  3  CHCONH  0.008  2  A M  CH ^CH ) ( 3)  0.05  5  2  ( 6 )  3  2  C-CH CONH  CH  CH (CH ^ 3  2  CH (orC H )  CH /^yCH CONH  °  (liters., moles""' hours" )  1  CH CONH^  9 5  2  •  ) 4  CHCONH '  CH  • Valmes i n t h i s column are rounded o f f . Numbers i n brackets are s i x numbers.  3  2  0.06  2k  hydrolysis for cyclopentyl acetamide and 1-methyl cyclopentyl acetamide are compared with the corresponding branched chain aliphatic amides i n Table V.  In these cases the consequence of  modifying the steric effect of atoms i n the six position by involving them i n ring formation i s seen to be a slight increase i n the rate of hydrolysis which, then, corresponds to only a slight decrease i n steric hindrance.  Therefore i t would appear  that i n the case where branching at the Oi. carbon i s modified by involvement of the branching groups i n a ring structure, the steric effect of atoms i n the six position becomes negligible when those atoms are either part of the cyclic structure or are directly attached to i t (the fact that the cyclopentyl radical has electron donating properties intermediate to the isopropyl and the diethyl carbinyl radicals (6) rules out the possibility that the greatly accelerated rate of hydrolysis for cyclopentyl carboxylic acid amide i s due to polar effects), and that the effect of involving branching groups at the (3 carbon i n ring formation slightly decreases the steric effect of atoms i n the six position but does not eliminate their effect.  From the  foregoing considerations i t may be concluded that the rule of six does not properly apply to cyclic systems but requires modification when cyclic systems are compared with acyclic systems. If i t i s true that the steric effect of the atoms i n the six position of cyclopentyl carboxylic acid amide can be  25  disregarded an explanation must be given for the fact that this amide hydrolyzes at approximately the same rate as the propionamide derivatives of Table IV and herein l i e s another limitation of the rule of six. When f i r s t proposing the rule, Newman ( 1 5 ) recognized the importance of CX substitution but emphasized the greater importance of (3 substitution by concluding that really large steric effects were observed only when the number of atoms in the six position was large and for the most part the effect of CX substitution could be ignored (except when this substitution involved atoms i n the six position).  The results  contained i n Table IV provide more experimental support for this view since a comparison of apparent rate constants for amides 1 , 3 , and 7 shows that large steric effects accompany large six number with £X substitution causing relatively minor effects, the comparison being made against stearamide.  However  in those cases where there i s a low degree of substitution i n the Oi and (3 positions, substituents i n these positions may be given almost equal weight from a steric point of view (9> 1 3 ) . This may be seen by comparing the rate constants for the alkaline hydrolysis of 3-methyl octadecanoamide (six number 6 ) and 2 methyl octadecanoamide (six number 3 ) contained i n Table V. These observations lead to the conclusion that substitution at the OX position i s of secondary importance sterically when there i s multiple (3. substitution but becomes proportionately more important as (3 substitution (and consequently six number)  26 decreases.  This i s , therefore, a significant limitation on the  use of the rule of six and caution must be exercised i n those cases where OX substitution i s present i n a compound of low six number as i n the cyclopentyl carboxylic acid amide case. The f i n a l experiment performed was that of hydrolyzing vitamin B 1 2 b under conditions nearly identical to those for the model compounds (see Experimental) and the results of this experiment are contained i n Table VI.  In column 1 i s recorded  the time from zero time at which aliquots of the hydrolysis mixture were taken and the results of the electrophoretic resolution of the aliquots are recorded across Table VI (comparisons of spot intensities should be made horizontally and not vertically).  The column on the far right gives the ammonia  evolved during the time interval indicated i n column 1.  The  percentage i s calculated on the basis of five rather than six primary amide groups since the electrophoresis results clearly show that one of these primary amides i s inert to alkaline hydrolysis under the conditions of the present experiment ( i t has been assumed that 1-amino pprppan-2-ol i s not volatile i n the present experiment).  This result was anticipated on the  basis of earlier work by Bonnett, et a l . ( 2 ) who, as mentioned above, suggested the inertness was due to lactam formation by the acetamide grouping on ring B of the corrin nucleus (see compound II).  Figure 11 represents the rate of ammonia evolution graphi-  cally and shows the rapid decrease i n the rate after 50$ of the  27  TABLE VI Electrophoretic analysis of the cobalt containing hydrolytic products from vitamin B12b. % NH evolve  Effective negative charges on the pigment spots  Time (hrs.) 0  1  2  +  3  h  5  6  7  0.80  ++++  * + +++  1.22  +++  +++  +  17.7  1.78  ++  +++  ++  21.6  2A7  ++  +++  ++  3.80  +  +  +  G  +++++++  5.07  +  +  25.6 31.9  +++++ ++++++ ++++ +++  +  17.10  +  ++++++  +++++  56.7  20.30  +  ++++++  ++++++  58.9  11.06  +  36.8 51.0  2*+.6l  +++  +++++  +  60.7  1+1.15  +  ++++  +  65.2  65.21  +  ++++  ++  68.5  165.87  +++  +++  76.8  219.55  +++  ++•++  79.5  Residue i n 30$ NaOH for 1 hr. at ll+0°  +++++++ (+) * A l l the pigment spots with the exception of this one, which was orange, were purple in contact with the electrolyte solution.  %  NH  80  -  70  --  60  --  Q) 5 0  --  CM CO  CX  >  o  r-t  rH O <H O EH  AO  -  30  20  -  10 0  25  50  75  100  125  150  175  200  225  hrs.  28  labile ammonia had been evolved.  This result, as well as the  electrophoresis results, i s consistent with the presence of three labile primary amide groups i n vitamin B 1 2 , a fact discovered i n earlier hydrolysis experiments ( 3 ) and one expected on the basis of present experiments (see Table III).  These three labile primary  amides are undoubtedly the propionamide substituents at positions 3 , 8 , and 13 of the corrin nucleus, the fourth labile amide being, on the basis of previous hydrolysis experiments ( 3 ) , the secondary amide at position 17 of the corrin nucleus. Table VI shows that of the two relatively stable amides, apart from the amide involved i n lactam formation, one hydrolyzes faster than the other.  This can be seen by comparing  the rate of development of the pigment spots corresponding to the penta- and hexaearboxylic acids (i.e., spots of effective negative charge five and six respectively).  Thus the color intensity  of the spot corresponding to the pentacarboxylie acids equals that of the spot corresponding to the tetracarboxylic acids within about 20 hours whereas the color intensity of the spot corresponding to the hexaearboxylic aeid equals that of the spot corresponding to the pentacarboxylie acids only after about 165 hours (this second estimate i s , of course, quite approximate since few aliquots of the hydrolysis mixture were taken beyond the f i r s t 2h hours of the reaction).  This result was predicted on the basis  of the present hydrolysis experiments i n the a l i c y c l i c series (see Table III) and in the absence of any complicating factors such as  29  lactam formation of the acetamide derivative on ring A, i t would appear that the acetamide substituent at position 18 of the corrin nucleus of vitamin B12 does i n fact hydrolyze at a faster rate than the acetamide substituent at position 2 (and position 7 barring lactam formation). Most of the conclusions above, of course, depend on the validity of the model compounds.  The results from the hydrolysis  of vitamin B12b indicate that the models represent the actual conditions reasonably well, since the rates of hydrolysis for the amides of the vitamin are of the order expected on the basis of the rates for the model amides.  Perhaps this can be best  seen by noting that the half l i f e time for the hydrolysis of 1methyl cyclopentyl propionamide, the slowest hydrolyzing of the propionamide derivatives, i s  hours which f i t s i n well with  the fact that the labile amide groups of vitamin B12b are completely hydrolyzed after approximately 20 hours under the same conditions (Table VI). for  Also, the half l i f e time of reaction  the slowest hydrolyzing acetamide model, 1-methyl cyclopentyl  acetamide (amide 3 of Table IV), i s calculated to be 1 3 0 hours which can be seen to correlate roughly with the results for vitamin B12b since the color intensities of the pigment spots corresponding to the penta- and hexaearboxylic acids are equal ( i . e . , the remaining hydrolyzable amide i s half hydrolyzed) within 165 hours. Therefore, with the validity of the model compounds being established, the results of the present experiments provide  30 strong support for the view that the amide groups of vitamin B12 which are relatively labile to alkaline hydrolysis are the propionamide substituents and that the acetamide substituents constitute the group of amides i n vitamin B12 which are relatively stable to alkaline hydrolysis.  Further, the results  suggest that of the acetamides i n vitamin B12, the substituent at position 18 of the corrin nucleus hydrolyzes in alkaline conditions significantly faster than the acetamide substituents at position 2 (and 7 barring lactam formation) (see Table III). It w i l l be interesting now to carry out the acid hydrolysis of the model compounds and of the vitamin.  In this  case there i s no lactam formation and the predictions contained in Table III can be checked more closely.  31 EXPERIMENTAL A l l melting points were determined on a Fisher-Johns apparatus and are uncorrected. The ultraviolet spectra were recorded on a Cary Ik recording spectrophotometer.  Infrared  spectra were recorded as films or mulls on a Perkin-Elmer Infracord.  Analytical gas-liquid chromatography was carried  out on the Aerograph gas chromatographic instrument (Wilkens Instrument and Research Inc.) and on the preparative scale the Megachrom instrument was used (Beckman Instruments, Inc.).  The  elemental analyses were performed by Dr. A. Bernhardt and his associates at Mulheim (Ruhr), Germany and by Mrs. Aldridge at the University of British Columbia. A.  Preparation of the amides 1.  Cyclopentyl carboxylic acid amide The preparation of this amide from the corresponding  acid (kindly supplied by Dr. R. Bonnett;  b.p. 110°/l5mm.)  followed a procedure given by A.I. Vogel (*f8a) and, since i t was used frequently, an account of the method i s given here. l l A gms. (O.'IO moles) of cyclopentyl carboxylic acid were added slowly with stirring over a period of twenty minutes to ih.k  gms. (0.12 moles) of thionyl chloride (purified after  Vogel (M-8b)) in a flask warmed i n an o i l bath and attached via a condenser to a water trap.  After the addition was complete,  32  the mixture was refluxed slowly until the evolution of gas ceased (ca. k5 minutes). The undistilled acid chloride was added to a saturated, dry ethereal solution of ammonia which was cooled i n an ice-saltwater bath.  Ammonia gas was bubbled i n during the addition of  the acid chloride. The ammonium chloride formed was f i l t e r e d off and extracted three times with boiling ethyl acetate.  This  extract was added to the ethereal f i l t r a t e and the whole concentrated to give two crops of crude amide. Yield:  7 . 9 6 gms.  (70.3$).  The amide was recrystallized twice from benzene-ethanol and once from benzene alone; (lit.  (1*9) m.p.  m.p. 1 7 9 - 1 8 0 ° (sinters at ca. 1 6 5 ° )  179°).  Found: C 6 3 . 7 5 ,  H 9-89,  N 12.37.  C H 6  1 ; L  0K  requires  C 63.68, H 9 . 8 0 ,N 12.39$.  2.  Cyclopentyl acetamide This derivative of acetamide was prepared from  commercially available cyclopentyl acetic acid by the procedure given for cyclopentyl carboxylic acid amide above. Yield: 6 5 $ . The amide was recrystallized twice from benzene to give white plates; m.p. 1 5 0 - 1 5 1 ° (crystal change ca. 1 3 5 ° ) (lit.  ( 5 0 ) m.p.  11*3-1^5°).  33  Pound C 66.2*f, H 1 0 . 3 0 , W 1 1 . 0 9 .  C^H^ON requires  C 66.10, H 10.30, N 11.01$.  3.  Cvclopentvl propionamide This amide was prepared from commercially available  cyclopentyl propionic acid by adding the acid chloride (prepared as described above for cyclopentyl carboxyl chloride) dropwise with stirring to a flask containing concentrated ammonium hydroxide and which was cooled in an ice-salt-water bath. The reaction mixture was stirred for a further hour and the water removed in vacuo on a steam bath and the residue extracted with boiling ethyl acetate.  This extract was chilled and four  crops of white, flaky crystals collected. Yield:  79.9$.  The amide was recrystallized twice from benzene; m.p. 1 2 ^ - 1 2 5 . 5 ° ( l i t . ( 5 D m.p. 122-3° (from acetone)). Found C 6 8 . 0 6 , H 1 0 . 6 3 , N 9 - 9 0 .  CgH^ON requires  C 68.0*t, H 1 0 . 7 1 , N 9 . 9 2 $ .  k.  1-Methvl cvclopentvl acetamide (a) i .  The following i s a typical preparation of  1-methvl cvclopentanol. 220 gms. ( 1 . 5 7 moles) of methyl iodide was added with stirring to 3 3 . 2 gms. ( 1 . 5 7 gm. at.) of Mg in 1*00 ml. of  3k anhydrous ethyl ether and the mixture refluxed for 1 hour. 12k gms.  ( l A 8 moles) of cyclopentanone  (b.p. h0°/2h  mm.)  in 50$ anhydrous ethyl ether solution was added and the mixture again refluxed for 1 hour.  The reaction mixture was then poured  into ice-water, made acidic (dilute sulfuric acid) and extracted with 1200 ml. of ethyl ether.  The extract was dried over  anhydrous sodium sulfate, the ether removed in vacuo and the residue d i s t i l l e d at reduced pressure. 1-Methyl cyclopentanol was collected as a pale yellow liquid;  b.p. 5 0 - 5 5 ° / l 8 mm.,  crystals on cooling; m.p. Yield:  8 8 . 1 gms.  which solidified-to white needle 21-25°. (59.5$ with a 7$ recovery of  cyclopentanone). (a) i i .  The ca 20 gms. of,dark residue from the  vacuum d i s t i l l a t i o n of 1-methyl cyclopentanol was d i s t i l l e d at greatly reduced pressure, a pale yellow liquid being collected; b.p. 88-98°/0.3 mm.,  n|° 1.5120 ( l i t . (28) n ,  dinitrophenylhydrazone m.p.  2  23*+-235° ( 6 0 ) .  0  1.5210)  2,k-  This compound was  shown to be V (see discussion)(60)). The residue from this d i s t i l l a t i o n deposited cubic crystals on standing a week. The crystals were soluble i n ethanol, ethyl acetate, methylene chloride, ethyl ether and benzene but insoluble i n water and were recrystallized three  35  times from ethanol-water solvent pair; ( 2 9 ) m.p. 8 2 ° ) ; y  m  Q  V  m.p. 8 1 - 8 2 . 5 °  (lit.  (nujol mull) 168500, l6l+2(S), l 6 2 1 ( S ) ,  126l(M), 1226(W), 1166(M) em. ; A -1  m a x #  3 0 2 , shoulders 29k,  (e=22,^00) [ 9 5 $ ethanol],  309  :' ou.:cl;G ^H 0 requires: G 8 3 . 2 6 , H 9 . 2 8 and a molecular ,  20  1  i  weight of 216 a.w.u. Found: C 8 2 . 2 2 , H 9.O8 and a M.W. 2 2 ^ a.w.u. k, (b) The following i s a typical preparation of 1-methyl cyclopentyl chloride. 208 gms. ( 2 . 0 8 moles) of 1-methyl eyclopentanol was shaken with 5 2 0 ml. of cold, concentrated HCI for twenty minutes during which time a color change from red to green occurred i n the reaction mixture.  The organic layer was drawn  off, treated with calcium chloride for one half hour and then washed with 5$ sodium bicarbonate followed by water.  The neutral  organic layer was dried over calcium chloride and d i s t i l l e d at reduced pressure. 1-Methyl cyclopentyl chloride was collected as a colorless l i q u i d :  Yield: *f.  b.p. 7 0 - 7 1 ° / 1 3 8  mm.;  n^° l.M+68.  17*+.2 gms. ( 7 0 . 7 $ ) .  (c) Diethyl l-methvl cyclopentyl malonate 1 5 2 . 5 gms. ( 1 . 2 8 moles) of 1-methyl cyclopentyl  chloride mixed with 2 0 6 . 0 gms. ( 1 . 2 9 moles) of diethyl malonate was added to 3 3 * 6 gms. (I.*f6 gm. atoms) of sodium dissolved i n  36  600 ml. of dry ethanol (prepared after Vogel t * t 8 c ) ) .  The  resulting solid mass was broken up by warming on a water bath and the reaction mixture then stirred for five and one half days at room temperature.  The volume of the mixture was then reduced  from 800 to 250 ml. i n vacuo and kOO ml. of water added. organic layer was drawn off and washed with salt water. aqueous layer was extracted with k$0 ml. of ethyl ether.  The The The  extract was reduced i n volume i n vacuo and combined with the organic layer, the whole then being dried over anhydrous sodium sulfate and d i s t i l l e d at reduced pressure. Diethyl 1-methyl cyclopentyl malonate was collected as a colorless l i q u i d :  1757  b.p. 8 8 . 5 - 8 9 . 5 / 1 . 3 mm.;  (S), 1736 (S) cm."  1  Yield:  n^° l.M-500;  V  m  a  (malonate interaction).  ^ 0 . 8 gms.  ( 1 3 . 2 $ with a 6 6 $ recovery of  diethyl malonate). Calculated for C^H^O^ Found k.  : C  6k.k$,  H 9-16$.  : C 6^.67, H 9-10$.  (d)  1-Methyl cyclopentyl malonic acid  9.17  gms. ( 0 . 0 3 7 8 moles) of diethyl 1-methyl cyclo-  pentyl malonate was refluxed with kO ml. of 10$ ethanolic KOH for thirteen hours on a steam bath.  Upon cooling, the reflux  mixture deposited f l a t , hygroscopic crystals of the potassium salt of 1-methyl cyclopentyl malonic acid.  These crystals were  x  37  separated from the hydrolysate and dissolved i n 5 0 ml. of water.  This solution was made strongly acidic (dilute sulfuric  acid) and then continuously extracted with ethyl ether for three and one half hours.  The extract was evaporated to dryness  and the residual solid recrystallized from benzene to give h.kO gms. of 1-methyl cyclopentyl malonic acid.  A further O.67 gms.  of the desired product was obtained by making the alcoholic hydrolysate 30$ i n KOH and refluxing for another ten hours. 1-Methyl cyclopentyl malonic acid had a sickly sweet odor;  m.p. I 3 6 - 1 3 7 ; 0  v  (nujol mull) 3007 (S, broad),  1 7 1 8 , 1689 (S, malonic acid interaction), 9 3 0 (M, broad) cm."*  1  Yield: \.  5 . 0 7 gms. ( 7 1 . 8 $ ) .  (e) 1-Methyl cyclopentyl acetic acid (III) 5 . 0 gms. ( 0 . 0 5 8 moles) of 1-methyl cyclopentyl malonic  acid was placed i n a two bulb hot box d i s t i l l a t i o n apparatus and heated at 1 6 0 ° ( o i l bath) for six hours.  The evolution of gas  was rapid and decarboxylation appeared to be complete within two and one half hours.  The dark liquid formed was d i s t i l l e d . i n the  hot box apparatus. 1-Methyl cyclopentyl acetic acid was obtained as a sour smelling liquid:  b.p. 7*+°/0.22 mm.;  2978 (S, shoulder at 2 6 ? 5 ) ,  n| 1.1+589; G  V  m  a  x  1 6 9 7 (S), 9 3 9 (M, broad) cm."1; the  38  nuclear magnetic resonance spectrum (recorded on a 1+0 Mc/s Varian spectrophotometer with f i e l d stabilizer VK 3506 and an external standard of hexamethyl disiloxane) showed peaks at 0.1+0  singlet), 7 . 7 9 ? (singlet), 8.1+7 t (singlet), and  9 . 0 2 t (singlet) with relative areas of 0 . 1 6 , 0.21+, 1 . 0 0 , 0.1+7 respectively.  These peaks correspond i n order to one carboxylic  acid proton, two exoring methylene protons attached to a quaternary carbon, eight ring methylene protons, and three protons of a methyl group attached to a quaternary carbon atom. Yield:  3 . 3 1 gms.  (86$).  A sample of the acid was further purified by gas-liquid chromatography and an elemental analysis made. Calculated for G g H 0 ; C 6 7 . 5 7 , H 9 - 9 2 . llf  2  Found: G 6 7 . 2 9 ,  H 9.79$.  1+.  ( f ) 1-Methvl cvclonentvl acetamide (IV) This amide was prepared from the corresponding acid by  the procedure used for cyclopentyl carboxylic acid amide above. The amide formed white platelets from benzene; Calculated for CgH^ON: Found: C 68.1k, H 1 0 . 8 7 ,  N  10.1$.  m.p. 9 6 . 0 - 9 7 . 5 ° .  C 68.0*+, H 1 0 . 7 1 , N 9 . 9 2 .  39  5»  1-Methyl cyclopentyl propionamide (VII) (a) Attempted preparation via ethylene oxide and the  Grignard reagent of 1-methyl cyclopentyl chloride* To 3-9 gms. (0.1*+ gm. at.) of magnesium under 50 ml. of anhydrous ethyl ether was added a total of ±8.0 gms.  (0.15  moles) of 1-methyl cyclopentyl chloride (10 gms. neat followed by 8 gms. i n anhydrous ethyl ether solution), the reaction being initiated by the addition of an iodine crystal.  The mixture was  refluxed for one half hour and then 20 gms. of ethylene oxide (dried by passage through a soda lime tube) was added via an acetone-dry ice condenser over one half hour, the temperature of the reaction mixture being kept below 3° during the addition.  The  reaction mixture was refluxed for one hour after which ether was d i s t i l l e d off with simultaneous addition of benzene until the vapor temperature reached 6 7 ° . refluxed for another hour.  The reaction mixture was  25 ml. of ice cold water were added  to the cooled reaction mixture and then 150 ml. of 3Q$ sulfuric acid.  The organic layer was drawn off and the aqueous layer  extracted with ether.  The organie layer and ether extract were  combined and dried for 2*+ hours on anhydrous sodium sulfate mixed with sodium carbonate.  The solvent was removed i n vacuo  and the residue d i s t i l l e d at reduced pressure. Two fractions were collected:  1*0  i.  colorless liquid:  b.p. *f6-52°/l8 mm.;  V  3**25(S),  m Q V  1080 (S) em." . 1  Yield: ii. 106^  (M)  l.*t0  gms.  colorless liquid:  b.p. 53-6M-°/l8 mm.]  V  m  Q  3^9  V  (S)  cm."  1  Yield:  1.50  Approximately 1 gm.  gms.  of an orange colored liquid, b.p.  85°/l8  mm.,  remained after d i s t i l l a t i o n . 5.  (b).  A sample of 1-methyl cyclopentyl acetic acid  (prepared as above in section •+) was converted to i t s acid chloride i n 91$ yield. 5.78 gms.  (O.O36 moles) of 1-methyl cyclopentyl acetic  acid chloride (purified by distillation:  b.p. 70-72°/12 mm.)  in  70 ml. of ethyl ether was added to 500 ml. of a cold (ca 0°) ether solution of diazomethane (prepared from "diazald" following a procedure supplied by the manufacturer ( 5 2 ) ) , in a d i s t i l l a t i o n flask which had no ground glass joints.  The solution was  allowed to warm to room temperature, during which time evolution of N  2  was  seen.  Evaporation of the ether l e f t ca 5 gms.  of the  diazoketone as a yellow o i l . 5 gms.  of the undistilled diazoketone was  dissolved  in 150 ml. of purified dioxane, 50 ml. of concentrated ammonia  kl and 10 ml. of 10$  s i l v e r n i t r a t e added and the mixture heated  f o r one hour a t 6 0 - 7 0 ° .  2 0 0 ml. of water were added and the  h e a t i n g continued f o r another hour on a steam bath. r e s u l t i n g brown suspension was The f i l t r a t e was  chilled,  The  t r e a t e d w i t h c h a r c o a l and  s a t u r a t e d w i t h sodium c h l o r i d e  extracted with ethyl ether.  The ether was  filtered. and  evaporated, the  r e s i d u e taken up i n b o i l i n g e t h y l a c e t a t e which, a f t e r  treatment  w i t h c h a r c o a l , d e p o s i t e d t h r e e crops of the amide as white platelets:  109-110°.  m.p.  F u r t h e r c o n c e n t r a t i o n of the mother  liquor yielded colored c r y s t a l s : T o t a l crude y i e l d : The amide was m.p.  2.27  m.p.  95-105°.  gms.  r e c r y s t a l l i z e d three times from benzene:  109.5-110.5°.  6.  C a l c u l a t e d f o r C^H^ON:  C 6 9 . 6 3 , H 11.04, N  9.02$,  Found:  C 69.60, H 10.97, N  9-23$.  c i s 2 , 2 , ^ - T r i m e t h y l c y c l o p e n t y l acetamide  campholanic  acid  (a)  amide)(35-38).  Camphoroxime  160 gms.  ( 2 . 3 moles) of hydroxylamine h y d r o c h l o r i d e  d i s s o l v e d i n a minimum amount of water and 270 gms. •of sodium hydroxide p e l l e t s were added to 230 gms. of d-camphor d i s s o l v e d i n 2 l i t e r s of 95$ e t h a n o l . was  ( X l H c i s cx -d-  (6.8 (1.5 The  moles) moles) mixture  r e f l u x e d on a steam bath with c o n t i n u a l s t i r r i n g f o r two  k2  hours after which time an aliquot of the reaction mixture remained clear on the addition of water.  The reaction mixture was then  diluted with 1.5 l i t e r s of water, neutralized with 6N HCI and chilled. Camphoroxime settled out as white, needle-like crystals:  m.p. 122-3° ( l i t . (53) m.p.  118°);  \ J  M  O  V  (nujol mull)  3333(S), 1669(W), 923(S) cm."  1  (b) a-d-Campholenic acid n i t r i l e (VIII) 167 gms. ( 1 . 0 0 moles) of camphoroxime was heated with 600 ml. of 6 N sulfuric acid with continuous stirring. vigorous reaction set i n when the oxime melted.  A  The reaction  mixture was refluxed for forty minutes, cooled and extracted with 1 l i t e r of ethyl ether i n portions.  The extract was  neutralized with sodium bicarbonate and dried over sodium sulfate.  The ether was removed i n vacuo and the yellow o i l l e f t  was d i s t i l l e d at reduced pressure. CX-d-Campholenic acid n i t r i l e was collected as a colorless liquid: n|° 1.1*665);  V  n|° 1.1*675 ( l i t . (**9)  . 2950(S), 2227(W), 1 7 2 7 O O , 797(M) cm" . 1  m a x  Yield: (c)  b.p. 96-106°/25 mm.;  116 gms. ( 7 7 . 8 $ ) .  a-d-Campholenic acid (IX)  36.O gms. (0.2**1 moles) of Ct-d-campholenic acid n i t r i l e was refluxed with 225 ml. of 30$ ethanolic (95$) KOH on  a steam bath for twenty-four hours.  Most of the ethyl alcohol  was then removed i n vacuo, and 200 ml. of water added to the reaction mixture.  This aqueous solution was acidified with 6N  sulfuric acid and extracted with ethyl ether, the extract was dried oyer anhydrous sodium sulfate, the ethyl ether removed and the residual brown liquid heated on a steam bath for one and one-half hours (to lactonize any hydroxy acid present). After cooling, this brown liquid was taken up i n 350 ml. of anhydrous ethyl ether and dry ammonia gas bubbled i n . The ammonium salt was obtained as a white solid which was unstable i n the atmosphere (stable i n a sealed tube); m.p. 65-75° ( l i t . (5^) m.p. 125-126°);  V  m a x  .  (rm^ol  mull)  2959 (S, symmetrical, broad), 1701(M), l5k3(S), 1377(S) cm."  1  Yield:  31-3 (70.2$).  The ammonium salt of CX-d-campholenic acid was dissolved in water, the solution acidified with dilute HCl and the organic acid extracted into ethyl ether.  The ether extract was washed  with water and dried over anhydrous sodium sulfate.  The ether  was removed i n vacuo and the residual yellow o i l d i s t i l l e d at reduced pressure. OC-d-Campholenic  acid was a colorless l i q u i d : b.p.  Ik9-I5l°/17 mm. ( l i t . (1+9) b.p. 157°/15 mm.); (k9) n cm.  1 9  1.1+713);  V  m  a  x  >  n|° 1.1+700 ( l i t .  2950(S), 17Ql+(S), 9kl(M, broad), 800(M)  Yield:  2 5 . 2 gms.  (88.5$).  The a-d-campholenic acid obtained i n this way decolorized a 5$ bromine solution i n carbon tetrachloride. The amide of oc-d-campholenic acid was prepared by heating 0 . 2 gms. of the ammonium salt i n a sealed tube at 200° for four hours: 6.  m.p. 125-127.5? ( l i t . (*+9) m.p. 1 3 1 - 1 3 2 ° ) .  (d) cis 2.2.3-Trimethyl cyclopentyl acetic acid (X)  (cis a-d-Campholanic acid) 17.6 gms. ( 0 . 1 0 5 moles) of cx-d-campholenie acid i n 150 ml. of anhydrous ethyl ether was hydrogenated at room temperature over 0 . 8 gms. of prereduced platinum oxide i n an autoclave for four hours at  ikOO  psi. of hydrogen.  At the end  of four hours the infrared spectrum of a sample of the hydrogenation mixture showed the absence of a peak at 800 cm.  -1  (C=C i n five membered ring) but the bromine i n carbon tetrachloride was s t i l l positive for unsaturation.  The hydrogenation  mixture was f i l t e r e d , the ether removed i n vacuo and the residue d i s t i l l e d at reduced pressure. The cis 2,2,3-trimethyl cyclopentyl acetic acid obtained was a colorless l i q u i d : b.p. lk3-lM+°/lk mm.);  b.p. 1 5 6 ° / 1 2 mm.  n|° 1A6M+ ( l i t . (35)  2967(S), 170*f(S), 9*+0(M, broad) W.CL JL •  cm."  1  ( l i t . (35) i.J+597);  ^5 Yield:  17.3  gms.  This acid s t i l l gave a positive test with the bromine solution. The following derivatives of the acid were prepared: i.  m.p. 1^9-151° (from benzene) ( l i t . (35)  amide:  m.p. 1 5 0 ° ) . ii.  anilide:  m.p. 123-126° (from ethanol-water)  ( l i t . (35) m.p. 128-129° (from benzene-pet ether), iii.  methyl ester:  b.p. 100-102°/19 mm.  Gas-liquid chromatography (Apiezon J column, column temp. 150°, helium flow 50 cm.^/min.) on the methyl ester showed i t to be a mixture of three components.  This mixture was  separated on a preparative scale by gas-liquid chromatography (ApJ column, column temp. 165°, Helium press.- back 1 psi., head 10 psi.) Fraction  Relative weight  V. * cm. max. c m  -1  1  3  2959(S), 1733(VS), 1190(S), 1166(S).  2  8  2967(S), 1736(VS), 1159(S).  3  1  3 5 W W ) , 2985(S), 176*+(VS), 1126(S), 1065(S), 9^2(S).  Fraction 1 decolorized a 5$ bromine solution i n carbon tetrachloride whereas fractions 2 and 3 did not.  k6  k.08 gms. of fraction 2 obtained in this way  was  hydrolyzed by refluxing for 36 hours with 15% ethanolic (95%) KOH.  The reflux mixture was acidified with dilute sulfuric  acid and extracted with ethyl ether, the ether removed i n vacuo and the residual orange o i l d i s t i l l e d at reduced pressure. A slightly yellow liquid was obtained: 15 mm.;  n|° 1 A 6 0 1 ( l i t . (35) n f ^ 1.1*597);  1712(S), 9^1 (broad) 7.  v  b.p. 151-15V/ m a x >  2959(S),  em."  1  trans 2 . 2 . Vtrimethyl cyclopentyl acetamide^0X;EXv)3-,  (trans QC-d-campholanic (a)  acid amide) (k2.  kl  Mt).  r  Camphorimine nitrate (XIE)  7 2 . 0 gms. of concentrated sulfuric acid i n 3 6 G ml. of water were added to 120 gms. oxime i n 1.5 funnel.  ( 0 . 7 2 moles) of camphor  l i t e r s of ethyl ether contained in a separatory  120 gms.  (1.7k moles) of sodium n i t r i t e was shaken and  within three minutes the ether layer changed from brown to deep violet.  The ether layer was immediately drawn off and allowed  to stand at room temperature.  Within three hours the color of  the solution had changed to a pale blue;  overnight the color  completely disappeared from the ether solution and white crystals of imine nitrate had precipitated. The imine nitrate was collected as white, needle-like crystals:  m.p.  159-165° (d.) ( l i t . (k2) m.p.  158-159°);  h7 (nujol mull) 2933 (VS, symmetrical, broad), 1 7 0 9 ( 3 ) , max. , 1577(W), 825(M) cm. . v  Yield:  2 2 . 3 gms. (Ik.5% calculated on camphor oxime).  A sample of the imine nitrate was recrystallized twice from ethanol: m.p. 16*+.5-165-5° (d.) ( l i t . Found:  G 56.26, H 8.76,  N  13.07.  0*2) m.p. 1 5 8 - 1 5 9 ° ) . G  io l8 2°3 H  W  r  e  l  (  u  i  r  e  s  s  G 5 6 . 0 5 , H 8.1*7, N 1 3 . 0 8 $ . (b)  Camphor nitramine (XIV)  The ethereal f i l t r a t e from the imine nitrate was evaporated i n vacuo to give the nitramine as a yellow o i l . Yield:  ca 101 gms.  A sample of the crude nitramine was crystallized from an ethanol-water solvent pair and recrystallized twice from ethanol to give white, needle-like crystals: (lit. X  m a x <  (1*2) m.p. 1*3°);  V  m.p. 39.5-^0°  (nujol mull) l637(S), 1563(VS) cm."  1  m Q Y  max. (95$ ethanol) 270 . Found: C 6 1 . 1 9 , H 8 A 3 , H lk.36.  ^ ^i^ 2 0  1Q  2  r  e  (  l  u  C 6 1 . 2 0 , H 8 . 2 2 , N l*+.28$. (c)  Camphorimine (XV)  i« 2 2 . 3 gms. (O.IO * moles) of camphorimine nitrate 1  was added to 9 3 . 0 ml. of concentrated ammonium hydroxide. A  i  r  e  s  l i t t l e ethyl ether was added to dissolve any solid present. The fishy odor of camphorimine was evident immediately after combining the reactants.  The mixture was shaken, set for one  hour and extracted with ethyl ether. The ether was removed i n vacuo to give the imine as an unstable white amorphous solid having a fishy odor. Yield: ii.  8.32 gms.  (53.0$).  The 101 gms, of impure camphor nitramine prepared  above was added to 500 ml. of concentrated ammonium hydroxide and enough ether to complete the solution of the nitramine. The mixture was shaken well and set for twelve hours, after which time i t was extracted with ethyl ether, the extract dried over anhydrous calcium sulfate and the ether removed in vacuo to yield the crude imine as a white amorphous solid. Yield:  7 6 . 8 gms.  A sample of the imine was purified through i t s hydrochloride as follows.  A few grams of impure imine were dissolved  in anhydrous ethyl ether and dry hydrogen chloride gas was bubbled through, the hydrochloride of the imine precipitating as a white crystalline solid:  m.p.  250° (sublimes ca 1 7 5 ° ) .  The hydrochloride salt was recrystallized three times from 100$ ethanol:  y  m Q V  (nujol mull) 2915(VS), 2877(VS)  (shoulders 2770, 2667, 2 5 2 5 ) , l686(S) cm."  1  k9  Found:  C6*K 17,  H 9 . 8 8 , N 7 . 5 6 , G H N C l requires 1G  l8  C 63.98, H 9 . 6 6 , N 7.^6$.  1 gm. of camphorimine hydrochloride was powdered, suspended i n sodium dried benzene and treated with dry ammonia gas for ten minutes.  The mixture was f i l t e r e d , the benzene  removed in vacuo and the free imine further purified by sublimation:  y  m  (nujol mull) 3268(W), 299*KS), l669(S) cm.' ; 1  Q  V  ^max. (cyclohexane) 238 Y T ^ C £ = 5 3 2 ) 7»  (^) trans 2.2.3-Trimethyl cyclopentyl acetic acid  n i t r i l e (XVI) i . 8 5 . 1 gms. ( 0 . 5 6 2 moles) of camphorimine was placed in a two-necked flask which was connected via a condenser and an ethanol trap to a water aspirator and which was f i t t e d with an air "bleed". The imine was melted, the heat source being an o i l bath i n which the flask was immersed, and air bubbled through the molten imine by means of the aspirator and the "bleed". The temperature of the bath was maintained at 1 1 0 - 1 2 0 ° .  Sublimation  of a solid occurred soon after the heating had begun.  This  solid had a melting point of 8 8 - 1 0 0 ° . The reaction was continued for ten hours after which time the ethanol from the trap was combined with the reddish o i l in the two-necked reaction flask and this solution washed successively with 25 ml. of 6 N HCI, 1% NaOH (neutralize the solution) and water.  The resultant o i l was steam d i s t i l l e d , the  50  d i s t i l l a t e (ca 2 . 5 l i t e r s ) salted (NaCl) and then extracted with ethyl ether.  The extract was dried overnight on calcium sulfate.  The ether was removed i n vacuo and the resulting yellow o i l d i s t i l l e d at reduced pressure to give two fractions: 1.  Slightly yellow liquid: b.p. 104«ll8°/20 mm.;  max. 2 9 6 7 ( S ) ,  V  Yield:  2232(W), 1 7 3 9 ( S ) ,  801(VW)  cm."  1  2 1 . 7 gms.  Gas-liquid chromatography showed this fraction to be a mixture of components (ApJ column,column temp. 1 7 7 ° , Helium flow 60 ml./min. gave three components whereas Ucon Polar column under the same conditions gave two components). 2. 17 mm.;  v  m a x  .  Colorless l i q u i d : 2  b.p. 1 2 * + - l 6 0 ° / 2 0 mm.,  9 7 6 ( S ) , 2232(W), 1 7 0 9 ( 8 ) , 1155(M)  Yield:  120-l*f5°/  cm."  1  6 . 0 2 gms.  Gas-liquid chromatography indicated that this second fraction was also a mixture (ApJ column, column temp. 1 8 0 ° , Helium flow 5 0 ml./min. showed two major components). The above procedure was repeated with a total of 9 2 gms. of fraction 1 . , b.p. 1 0 k - l l 8 ° / 2 0 mm., being collected and resolved by preparative gas-liquid chromatography (ApJ column, column temp. 2 1 5 ° , Helium press. - back l p s i . , head 8 p s i . )  were collected:  Four fractions  51 a.  White solid:  m.p.  ca 9 6 ° ;  2,l*-dinitrophenyl  hydrazone m.p.  175.5-177.5° (2,l*-dinitrophenyl hydrazone of  d-camphor m.p.  177° ( 5 5 ) ) ;  semicarbazone m.p.  with d-camphor semicarbazone 2 2 9 -  (ethanol-water), mixed m.p. 233°;  oxime m.p.  226-228°  1 1 6 ° (ethanol-water), mixed m.p.  with d-  camphoroxime 1 1 6 - 1 1 8 ° . b. 800(m) cm.  -1  Liquid:  v  m  a  x  .  2959(S), 2237(W), 1733<W),  (cf. infrared spectrum for  2,2,3-trimethyl  cyclopent-3-enyl acetic acid n i t r i l e ( (X -d-campholenic acid n i t r i l e ) section 6(b) of the Experimental). trans 2.2.3-Trimethyl cyclopentyl acetic acid  c.  b.p. 1 1 0 . 5 ° / 6 m m . ;  n i t r i l e (XVI): 1A611;  V  m a x #  2976(S), 2237(W)  Yield:  3 9 - 8 gms.  Found: N 9.1*0. d. cm.  Liquid:  y  m  v  cm."  1  (distilled). C^H^N  a  n|° 1.1+570 ( l i t . (1*2)  requires:  N 9.26$.  2985(S), 22l*2(W), 1 7 l 5 ( S ) ,  ll63(M)  -1  The relative weights of a:b:c:d were 1 8 : 9 : 6 9 : 1 . 7.  (e)  trans 2,2,3-Trimethyl cyclopentyl acetic acid  (XVIII) 3 7 . 8 gms.  ( 0 . 2 5 moles) of trans  2,2,3-trimethyl  cyclopentyl acetic acid n i t r i l e were refluxed with 30$ ethanolic  52  KOH for twenty-four hours. The ethanol was removed in vacuo and the resulting solid taken up i n 150 ml. of water.  This aqueous solution was  made strongly acidic (dilute sulfuric acid) and extracted with ethyl ether.  The extract was dried on anhydrous sodium sulfate,  the ether removed in vacuo and the residual yellow o i l d i s t i l l e d at reduced pressure. trans 2,2,3-Trimethyl cyclopentyl acetic acid was obtained as a faintly yellow liquid: n|° 1 . 4 6 2 8 ; V „  m a x  b.p. l 4 9 - l 5 4 ° / 6  mm.;  2976(S), 1 7 l 5 ( S ) , 1297(M), 939(M, broad)  .  cm. - 1 m  Yield:  3 5 * 5 gms.  ( 8 3 . 5 $ based on the corresponding  nitrile). 7.  (f) The amide was prepared via the acid chloride as  outlined for cyclopentyl carboxylic acid amide (see section 1 . ) : m.p.  l45.5-lk6.5°  (from benzene) ( l i t . ( 4 2 ) m.p.  143° (from  acetic acid)). Found:  C 70.76, H 11.26, N 8.32.  C 70.96, H 11.32, N  8.  c  i o i 9 requires: H  8.28$.  trans 2.2,3-Trimethy1 cyclopentyl propionamide (amide  6 of Table IV). This amide was prepared from trans 2,2,3-trimethyl  53  cyclopentyl acetic acid chloride (purified by d i s t i l l a t i o n : b.p. 98-lGG°/l5 mm.)  by way of the diazoketone intermediate  using a procedure identical to that used i n the preparation of 1-methyl cyclopentyl propionamide (VII) (see section 5(b)). trans 2,2,3-Trimethyl  cyclopentyl propionamide was  obtained as white, shiny platelets from benzene-petroleum ether solvent pair: Yield:  m.p.  82.5-84°.  24$ based on trans 2,2,3-Trimethyl  cyclopentyl  acetic.'.aicid. The amide was recrystallized three times from benzene-petroleum ether: Calculated for  m.p.  83.5-84°.  C ^ H ^ N O J  C 72.08, H 11.55,  N  7-64$.  Found: C 7 2 . 0 9 , H 1 1 . 5 1 , N 7 . 8 9 $ . 9.  Stearamide (a)  Methyl stearate  25 gms.  of crude stearic acid in dry, absolute methanol  was treated with dry HCl gas for five minutes and the reaction mixture set overnight.  The whole was then made slightly basic  and extracted with ethyl ether.  The extract was washed with  water, dried over anhydrous sodium sulfate, the ether removed in vacuo and the residual liquid d i s t i l l e d :  b.p. l42-l48°/G.l  mm.  5k  The methyl stearate was r e d i s t i l l e d : m.p. 3 7 ° ( l i t . ( 5 3 ) m.p.  b.p. I f 0 - l f 2 / 0 . 2 mm.; l  l  0  35-7°).  Gas-liquid chromatography showed the ester to be 99$ pure. (b)  Stearic acid  1 2 . 6 gms. of methyl stearate was refluxed i n 1 0 0 ml. of k0% ethanolic KOH for thirty-six hours, after which time the reaction mixture was acidified (dilute sulfuric acid) and extracted with ethyl ether. The ether was removed i n vacuo from the extract and the residual solid recrystallized from 95$ ethanol:  Cc)  m.p. 6 8 - 7 0 °  (lit. (53) 69. * ). 1  0  Stearamide  5 . 2 6 gms. of stearyl chloride (prepared i n the usual manner and purified by d i s t i l l a t i o n :  b.p. 8 6 - 9 0 ° / 1 . 5 mm.  was dissolved i n 20 ml. of dry, purified dioxane and added dropwise with stirring to 15 ml. of ice-cold concentrated ammonium hydroxide.  The precipitate of stearamide was re-  crystallized several times from methanol: (53)  m.p. 1 0 8 - 1 1 0 °  (lit.  109°).  Yield:  3 . 1 2 gms.  Found: C 7 6 . 0 3 , H 1 3 . 1 0 , N *t.76. C 7 6 . 2 6 , H 1 3 . 1 5 , N k.9k$,  G^R^ON requires:  55  10.  Vitamin B12b (Hvdroxocobalamin) (46) 100 mg. of vitamin B12 (undried) i n 190 ml. of 0.004  N HG1 (optimum concentration of vitamin B12 for the apparatus used) was irradiated with ultraviolet radiation (100 watt Hanovia Hg arc lamp f i t t e d with corex f i l t e r ) for two and three quarters of an hour, the HCN gas generated being swept out of the system into alkali traps by a stream of helium. At the end of this time a shift in the ultraviolet absorption spectrum of the solution from A occurred.  m  a  x  36l  n\jJt, to A .  m a x  35l>ythad  The photolysis solution was then extracted with 110  ml. of aqueous phenol (freshly d i s t i l l e d phenol plus 10$ water) i n portions.  The red extract was washed with some water,  diluted with an excess of ethyl ether and reextracted into water.  The aqueous extract (washed free of phenol with ether)  was reduced i n volume i n vacuo at room temperature to approximately 10 ml. (in subsequent preparations the volume was reduced by freeze drying) and acetone added until a faint cloudiness appeared i n the solution.  This solution was then kept at 0°  until the precipitation of deep red crystals appeared complete. The ultraviolet spectra of aqueous solutions of the solid (0.03 mg/ml) showed 525,  276  (P > H 2  a  n  d  ^  m  A. ^  279-*yA (pHIO).  Yield:  4 5 - 7 mg.  m o v  (aqueous HCl) 3 5 2 , 4 1 0 ,  (aqueous NaOH) 36O, 4 2 2 , 5^0,  56  B.  The hydrolysis of the amides 1.  Apparatus The hydrolysis of the amides was performed following  a procedure outlined by Cason and Wolfhagen ( 2 5 ) . The apparatus which was designed by these workers and which was used i n the present hydrolysis studies i s shown i n Figure 12. 2. Procedure Approximately 0 . 2 5 gms. of a given amide were weighed accurately (to the nearest ten thousandth of a gram) into the 50 ml. flask (A) and 25 ml. of standardized 0 . 5 N KOH i n 1-propanol (Note 1.) was pipetted into the flask.  In the 25  ml. graduated cyclinder was placed 10 ml. of 2$ aqueous boric acid solution (Note 2 . ) .  The hydrolysis mixture i n the  flask was then heated under reflux (Note 3«) on a hot plate with water circulating i n the upright condenser (G). When a determination was to be made, water was drained from the upright condenser and 10 ml. of 1-propanol was d i s t i l l e d rapidly (within four to six minutes) i n order to ca:rry over a l l the ammonia.  The time (Note k.) was taken at the point  when the d i s t i l l a t i o n was stopped by running water through the upright condenser and 10 ml. of 1-propanol was immediately added through the ground glass joint at the top.  The graduated  cyclinder was lowered for the last 2 ml. of d i s t i l l a t e and the outside of the delivery tube was rinsed with d i s t i l l e d water.  A fresh sample of boric acid was then arranged to  receive ammonia.  To follow page 56  F i g u r e  12  9 m m .  O.D.  2 5 m l . G R A D U A T E D C Y L I N D E R  HOT  P L A T E  57  The d i s t i l l a t e was rinsed into a 1 2 5 ml. Erlenmeyer w i t h ' 2 5 ml. of d i s t i l l e d water and the ammonia  titrated  directly with standard 0 . 0 2 N HCl (Note 5 . ) using a methyl red indicator (Note 6 . ) and a blank solution (Note 7 . ) to determine the end point. Determinations were made at intervals of one to two hours for the more rapid hydrolysis with intervals ranging up to twelve hours for the slowest hydrolysis (and up to twentyfour hours i n vitamin B12b hydrolysis).  Five to six determina-  tions were commonly made and, except for the slowest hydrolysis, the reaction was followed until half complete. The procedure for the hydrolysis of vitamin B12b (hydroxocobalamin) was essentially the same as above with a few notable exceptions which are elaborated below. 122.9 mg. of vitamin B12b (dried for f i f t y hours at 56°  i n vacuo over  3?2°5*  This  m a  y have caused very slight  decomposition ( 5 6 ) ) was weighed into flask A and 2 5 ml. of standardized 0 . 5 K KOH i n 1-propanol was pipetted into the flask.  This weight of vitamin B12b gave a O . O O 3 6 5 M concentra-  tion of each amide grouping i n the vitamin (molecular weight of the vitamin was taken to be 13k6,k  a.w.u. ( 1 ) ) i n contrast  to a ca 0 . 0 7 M concentration of amide i n the a l i c y c l i c series. Ammonia determinations were made as above (ammonia evolved was titrated with 0 . 0 1 N HCl) with the addition that an  58 aliquot  of  t h e h y d r o l y s i s m i x t u r e was t a k e n a t  (The'hydrolysis mixture refluxing  determination  upon  r e d c o l o r on e x p o s u r e  This a l i q u o t was r e m o v e d i m m e d i a t e l y  o f 1G m l .  the a d d i t i o n  of f r e s h 1 - p r o p a n o l f o l l o w i n g  to  after a n ammonia  and b e f o r e t h e h y d r o l y s i s m i x t u r e had a g a i n  Flask A was a d a p t e d t o accommodate t h e r e m o v a l  begun t o b o i l . of  o f v i t a m i n B 1 2 b t u r n e d brown  and r e t u r n e d t o t h e i n i t i a l  the atmosphereO  t h e same t i m e .  t h e a l i q u o t s by t h e a d d i t i o n o f a s i d e arm w h i c h was c l o s e d  w i t h a B-10  ground g l a s s s t o p p e r .  The  v o l u m e ( 0 . 3 7 m l . a t room t e m p e r a t u r e )  a l i q u o t was o f m e a s u r e d a n d a c o r r e c t i o n was  made t o t h e m e a s u r e d amount o f ammonia e v o l v e d t o a c c o u n t  for  t h i s d e c r e a s e i n t h e amount o f v i t a m i n B 1 2 b p r e s e n t . The  b a s i c a l i q u o t s were a c i d i f i e d w i t h 5 m l .  of  c a 0 . 0 5 N H C 1 and e x t r a c t e d w i t h 5 m l . o f aqueous p h e n o l distilled  phenol plus 10$ w a t e r ) .  was w a s h e d w i t h a l i t t l e w a t e r and an e x c e s s of e t h y l e t h e r  extract  t o remove any a c i d a n d s a l t s  then added.  s o l u t i o n was e x t r a c t e d w i t h w a t e r extract  The r e d p h e n o l i c  (freshly  washed w e l l w i t h e t h e r .  This e t h e r e a l  and t h e orange aqueous The  phenol-free  aqueous  e x t r a c t was t h e n s u b j e c t e d t o f r e e z e - d r y i n g a n d t h e  resulting  r e d s o l i d a n a l y z e d by e l e c t r o p h o r e s i s . The 3 filter cm. f o r  e l e c t r o p h o r e s i s was c a r r i e d o u t on Whatman number  strips  of 1.5  seven hours.  c m . w i d t h i n a potential o f k v o l t s The  0.05 M phosphate b u f f e r  electrolyte  (pH 6 . 5 )  s o l u t i o n c o n s i s t e d of  c o n t a i n i n g 0 . 0 1 $ KGN  (3).  per a The  59  distances travelled by the developing purple spots from the origin were measured and their relative intensities estimated visually and the results recorded in a scheme used by Armitage, et a l . ( 3 ) . The hydrolysis of vitamin B12b was followed for a considerably longer time than for the a l i c y c l i c amides (see Table VI) and during this time the normality of the a l k a l i solution changed by 3 * 8 $ .  This change would lead to approximately  a 2% error i n the rate of the slowest hydrolyzing amide of vitamin B12b and was ignored i n the present experiment (the error was calculated by assuming that the rate of hydrolysis for 1-methyl cyelopentyl acetamide was the same as the rate for the slowest hydrolyzing amide of vitamin B12b).  The residue  from the hydrolysis of vitamin B12b i n 1-propanolie KOH  was  isolated after the manner followed for the aliquots above and heated at  lhO°  i n 30$ aqueous NaOH for 1 hour and a sample  subjected to resolution by electrophoresis as i n the case of the aliqupts. The results of the complete hydrolysis of vitamin B12b are contained i n Table VI and Figure 11 (see Discussion). In addition to the purple spots developing i n the electrophoresis, a fluorescent band was observed i n every case at a position between the tri-and tetra-carboxylic acids of the pigment acid series.  By electrophoretic comparison with an  authentic sample, this band was shown to be "nucleotide b" (i.e., 3« - ribonucleotide  (57)).  60  General notes on the procedure 1.  The 0 . 5 N KOH i n 1-propanol solution was prepared as  described by Vogel  (*+8d),  the propanol being purified by  d i s t i l l a t i o n after drying over calcium sulfate (b.p. 93-96°/ 758.3 mm.).  The KOH solution was standardized just before use  by titrating with standard 0 . 5 N HG1 (Note 5 ) . 2.  The 2$ aqueous boric acid trap for ammonia evolved during  the hydrolysis was prepared after Wagner ( 5 8 ) by dissolving 20 gms. of boric acid i n 1 0 0 0 ml. of d i s t i l l e d water, boiling the solution for some time and allowing i t to cool.  To the  boric acid solution was added enough methyl red indicator solution (Note 6 ) so as to give a good indicator color intensity at the end point of the HCI titration of ammonia trapped i n the boric acid solution during the amide hydrolyses, i . e . , a color intensity which could easily be compared with the blank solution used for the titration (usually 3 ml. of indicator per l i t e r of boric acid sufficed).  This stock solution of boric acid plus  indicator was stored i n a stoppered flask i n the dark. The solution tended to deteriorate on standing and the original color was restored by adding a few drops of 0 . 1 N HCI.  The  amount of boric acid used for each determination would retain about 1 8 mg. of Nitrogen quantitatively ( 5 8 ) . 3.  The temperature of the boiling 1-propanolic KOH solution  was 95.0°C.  61  k.  The zero time for the hydrolysis was consistently taken at  the point when the 1-propanol solution boiled vigorously.  This  occurred at approximately one and one-half minutes after the flask was placed on the hot plate. 5.  A i l the standard HCl solutions used i n these kinetic  studies were prepared from commercially available standard HCl solutions (sold under the trade name of "Aceulute" and produced by Anachemia Chemicals Limited) diluted with d i s t i l l e d water.  A check was made on the specified concentrations of  these solutions by titration against borax ( 5 9 ) and the 0 . 2 $ maximum, positive deviation found, assuming that i t i s real, i s insignificant i n the present hydrolysis studies. 6.  The methyl red indicator was prepared by dissolving 0 . 0 5 gms.  of methyl red i n a mixture of 60 ml. of 95$ ethanol and hQ ml. of water. 7.  The blank solution was prepared by mixing 10 ml. of the  2$ boric acid stock solution containing methyl red indicator, 10 ml. of 1-propanol and 25 ml. of d i s t i l l e d water.  An  extra amount of water was then added to the blank so that i t s volume and the volume of a titrated sample were of approximately the same magnitude.  62  3»  Data for the hydrolysis of the amides contained i n Table I.V. For each case two hydrolysis runs were made. In the  f i r s t column of each table below i s indicated the concentration of KOH i n 1-propanol (a) and the concentration of the amide (b) for each experiment.  (See Discussion section for descrip-  tions of how this data was utilized.) TABLE VII Cyclopentyl acetamide Experiment  t(hrs.)  x(moles) liter  log a-x b-x  1.  1.95  O.OO987  0.839^  a=0.1*878 M.  **.55  .01866  .890^  b=0.0790lf  5.62  . 9103  6.60  .0217^ .021*22  7.63  .02665  .9^6  9.13  .02985  .9680  10.13  .0319^  .9859  1.97  0.00960  0.8361  if.1*8  .01795  6.58  .02359  .8837 .9201*  9.50  .0297*+  .965"+  11.58  .03390  • 999 *  13.53  .037^6  1.0311*  15.53  .01*071  I.063I+  M,  2. 3=0.1*878 b=0.07935  M. M.  .9272  1  63  TABLE VIII Cyclopentyl propionamide Experiment  t (hrs.)  x(moles) liter  Log a-x b-x  1. a=OA920 b=0.07122  M. M.  2. a=0.4902  M.  b=0.07113 M.  0.95  0.01382  0.9207  1.93  .02250  .9839  2.92  .02842  1.0347  4.03  .03398  1.1099  5.13 6.10  .03862  1.1433  .04216  1.1897  8.23  .04814  1.2841  1.03  0.9161  2.21  0.01327 .02228  3.10  .02751  1.0256  5.18  .03655 .04068  1.1179  6.31  .9813  I.I691  6k  TABLE IX 1-methvl cvclopentvl acetamide Experiment  t (hrs.)  x(moles) liter  Log a-x b-x  1.  2.07  O.OOlllf  a=0.5G«+0 M.  5.00  b=0.07161 M.  9.00  .00239 .00398  13.83  .00553  .8776  16.00  .00665  .881+1  2-+.17  .00950  .9010  32.20  .01215  .9176  38.00  .01398  .9296  J+9.17  • 9521  57.51  .01727 .01963  .969k  72.85  .0235-+  .9998  5.02  0.00263  0.8681+  a=0.5073 M. •  15.63  .8921  0=0.07096  26.83  .00679 .01068  39.00  . oi«+M+  • 9-+06  51.02  .01788  .961+7  63.58  .02122  .9900  75.08  .02-+13  I.OI36  • 2. M.  0.8535 .8601 .8688  .9158  65  TABLE X 1-methyl cyclopentyl propionamide Experiment  t (hrs.)  x(moles) liter  Log a-x b-x  2.00  0.01345  0.9756  4.00  .02666  1.0919  6.18  .03510  1.1919  7.15  .03798  1.2330  2. a=0.4808 M.  1.05 2.08  0.01216  0.9505  .01976  b=0.06468 M.  2.95  .02465  1.0113 1.0568  4.20  .02993  1.1131  5.68  .03513 .03881  1.1785 1.2326  1. a=0.50l6  M.  b=0.06509 M.  7.05  TABLE XI trans 2.2.3-trimethyl cyclopentyl acetamide Experiment  t (nrs.)  Log a-x b-x  0.00336  0.9^68  3.07 5.08  .00840  .9832  .01261  1.0168  7.01  .01635  1.0495  9.02  1.0845  13.37  .01999 .02686  1.1611  15.38  .02960  1.1967  19.30  .03400  I.2617  1.  1.03  a=0.5002 M. b = 0 . 0 5 9 5 2 M.  x(moles) liter  66 TABLE XI (continued) trans 2.2.3-trimethyl cyclopentyl acetamide Experiment  t (hrs.)  2. a=0.5l40 M . b=O.G5965 M .  x  2.10 5.70 9.92 11.93 13.75 15.71  (moles) liter  T n  „ a-x  L o g  b^E  O.OO632 .01422 .02142 .02462  0.9786 1.0415 1.1101 1.1452  .02715 .02950  1.1755 1.2060  TABLE XII trans 2.2.3-trimethyl cyclopentyl propionamide Experiment 1. a=0.5l2G M. b=0.05478 M.  2. a = 0 . 5 l l 8 M. b=0.05502 M.  t (hrs.)  .  (moles) liter  T- „ a-x °S P x L  1.92 3.27 4.22 5.28 6.30  0.01732  1.1208  .02440  1.2055  .02786 i03102 .03366  1.2549  0.93 1.95 3.05 3.93 4.95  0.01023 .01715 .02219 .0255k .02894  1.0492  1.3063 1.3550  1.1160 I.I736 1.2174 1,2676  67  TABLE XIII CvcloDentvl carboxylic acid amide Experiment  t (hrs.)  x(moles) liter  T- „ a-x  b-x  L o g  1.  1.00  0.01116  0.8566  a=0.1+8l2 M.  1.98  .OI836  .9005  3.02  .©2M-50  .9«+32  h.55  .03181  1.0019  6.00  .03768  1.0573  0.01102  0.9271  a=0.5228 M.  0.95 2.00  .01922  0.9833  b=0.07155 M.  3.21  .02581*  I.036I+  •+.1+5  .03190  1.0928  6.23  .03566  1.1327  b=0.07655 M.  2.  TABLE XIV Stearamide Experiment  t (hrs.)  x(moles) liter  r  nrr  L o g  §^x  b^i  I.63  0.00922  1.2-+05  3.25  0.011+57  1.3339  tf.77 6.62  0.01783  1A01+0  0.0208-+  1.1+817  2.  1.50  0.00872  1.2297  a=0.-+659 M. b=0.03566 M.  2.80  .01309  1.3021+  3.90  .01585  1.356-+  5.02  .01821+  1.1+099  6.25  .0201+3  1.1+662  1. b=0.03559 M.  68  LITERATURE CITED 1.  E. Lester Smith. I960.  2.  Methuen and Co. Ltd. London.  R. Bonnett, J. R. Cannon, V.M. Clark, A. W. Johnson, L. F. J. Parker, E. Lester Smith, and Sir Alexander Todd. J. Chem. Soc.  3.  Vitamin B 1 2 .  1158 ( 1 9 5 7 ) .  J. B. Armitage, J. R. Cannon, A. W. Johnson, L. F. J. Parker, E . Lester Smith, W. H. Stafford, and A. R. Todd. J. Chem. Soc. 3849 ( 1 9 5 3 > .  4. J. M. Brierly, R. R. Sealock. and H. Diehl. Coll. J. S c i . 2 £ , l 4 l ( 1 9 5 k ) .  Iowa State  5.  J. Cason, G. Castaldo, D. L. Glusker, J. Allinger, and L. B. Ash. J. Ore. Chem. 1 8 , 1129 ( 1 9 5 3 ) .  6.  Cf. R. W. Taft, J r . , i n M. S. 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