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The synthesis and properties of some peptides and the specificity of pepsin Harris, Clifford Kaye 1954

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THE SYNTHESIS AND PROPERTIES OF SOME PEPTIDES AND THE SPECIFICITY OF PEPSIN by CLIFFORD KAYE HARRIS A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biochemistry We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE Members of the Department of Biochemistry THE UNIVERSITY OF BRITISH COLUMBIA August, 1954. ABSTRACT A number of peptide intermediates and derivatives have been synthesized from both optically-pure and racemic amino acids. Carbo-benzoxv-DL-alanvl chloride was coupled with J^leucine methyl ester and the mixed, crystalline carbobenzoxy^L-alanyl-L-leucine methyl ester was isolated. Carbobenzoxy-DL phenylalanyl chloride and carbobenzoxy-DL phenylalanyl azide were coupled with ^ -leucine methyl ester and two products were separated by fractional crystallization. One of the products has been identified as carbobenzoxy-L-phenylalanyl-L-leucine methyl ester. A series of synthetic, dipeptide derivatives, containing some of those peptide bonds (present in the phenylalanyl chain of insulin) that Sanger and coworkers (31,32) found to be split by pepsin, were subjected to the action of pepsin at pH 2.0 and at pH 4-.0. Proteolysis was detected by paper chromatography of the enzyme-substrate solutions. Of the compounds tested, carbobenzoxy-DL-phenylalanyl-^."phenylalanine ethyl ester and carbobenzoxy^L-phenylalanyl-LrtyTosine methyl ester were found to be hydrolysed by pepsin at pH 2.0 but not at pH 4-.0. Synthetic compounds containing the peptide bonds - phenylalanyl-valyl, leucyl-valyl, glutamyl-alanyl, alanyl-leucyl, and glycyl-phenylalanyl -were resistant to pepsin even though i t had been found (31,32) that, in insulin, these peptide bonds were pepsin-sensitive. These r e s u l t s i n d i c a t e t h a t , i n the case o f p e p s i n , obse rva t ions on the a c t i o n o f p e p s i n on s y n t h e t i c , d i p e p t i d e - t y p e subs t ra tes shou ld not be used t o p r e d i c t , o r to e x p l a i n , the s p e c i f i c i t y o f p e p s i n on p r o t e i n s o r on h i g h molecu la r weight p e p t i d e s . ACMOWLEDGEMENT The author wishes to express his very sincere thanks to • Dr. W. J. Polglase for the help, advice and encouragement offered throughout the course of this thesis. The author also wishes to thank the National Research Council for personal assistance in the form of two summer research grants. TABLE OF CONTENTS-Page INTRODUCTION 1 HISTORICAL A. Synthesis of peptide derivatives . . . . . . . . 2 B. Specificity of pepsin 5 EXPERIMENTAL A. Synthesis of peptide derivatives I. Carbobenzoxy chloride . . . . . . . . 12 II. Purification of ^ leucine 12 III. Preparation of amino acid esters 13 IV. Carbobenzoxy-DL-alanyl-L-leucine methyl ester . . . 14 V. Carbobenzoxy-DL-alanyl-L-leucine 17 VI. DL-Alanyl^L-leucine 17 VII. Carbobenzoxy-DI^alanyl-Ij-leucinaEU.de . . . 18 VIII. Carbobenzoxy-L-alanyl-L-leucine methyl ester . . . . 18 IX. Carbobenzoxy-If alanyl-L- leucine 19 X. Carbobenzoxy-?-phenylalanyl-L-leucine methyl ester . 19 XI. Carbobenzoxy-L-phenylalanyl-L-leucine methyl ester . 24 XII. Carbobenzoxy-J^pherrylalahyl-DIr-phenylalanine ethyl ester . 26 XIII. Carbobenzoxy-pT^phenylalanyl-DL-phenylalanine . . . 2 7 XIV. DL-Phe nylalanyl-DL-phe nylal anine 28 XV. Carbobenzoxy glyi^l-DL-pheiylalanihe ethyl ester . . 28 Page X V I . Carbobenzoxy g lycy l - J3L-pheny la l an ine 30 X V I I . Ca rbobenzoxy-DL-pheny la l any l -DL-va l ine methy l e s t e r 30 X V I I I . Carbobenzoxy-DL-phe n y l a l a n y l - D L - v a l i n e . 31 X I X . Carbobenzoxy-L-c<-glutamy1-DL-alanine methyl e s t e r . 31 X X . Carbobenzoxy- I^o( -g lu tamyl -DL-a la nine 33 X X I . Ca rbobenzoxy-DL-pheny la l any l -L - ty ros ine methyl e s t e r 33 X X I I . Carb obenzoxy-DL-phenyla l any 1 - L - t y r o s ine . . 34 X X I I I . C a r b o b e n z o x y - L - l e u c y l - D L - v a l i n e methyl e s t e r . . . . 35 B . S p e c i f i c i t y of p e p s i n . 36 DISCUSSION A . Chemical p a r t 41 B . Enzymatic p a r t 45 BIBLIOGRAPHY 49 TABLES Page I.. Preparation of PL-alanine 14 I I . Preparation of L-leucine methyl ester hydrochloride . 15 I I I . Preparation of carbobenzoxy-DL-alanyl-L-leucine methyl ester .~T . . . ~ . . . . . . . . 16 IV. Preparation of carbobenzoxy-DL-phenylalanine 20 V. Preparation of DL-phenylalahine e t h y l ester hydrochloride 20 VI. Preparation of carbobenzoxy-DL-phenylalanine e t h y l ester 21 VII. Preparation of carbobenzoxy-DL-phenylalanyl hydrazide 22 VIII. Pepsin hydrolysis of bovine \ - g l o b u l i n at increasing concentrations of methanol 37 IX. A c t i o n of pepsin on various synthetic substrates 38 X. A c t i o n of pepsin on various synthetic substrates . . . 40 XI. Compounds separatea from the coupling of carbobenzoxy phenylalanine with L-leucine methyl ester 44 XII. A summary of the bonds s p l i t by pepsin . . . . . . . . 47 DIAGRAMS I. A chromatogram of pepsin hydrolysates of synthetic substrates 3 8 INTRODUCTION The work reported In this thesis consists of two parts; f i r s t , a study of peptide synthesis and the resolution of diastereoisomeric peptide derivatives; second, a re-investigation of the specificity of the gastric protease, pepsin. The compounds prepared in the f i r s t part of the work were tested, in the second part of the work, as potential pepsin substrates. HISTORICAL A. Synthesis of Peptide Derivatives With the increasing importance of naturally occuring peptides or peptide derivatives containing r>airr*no acids (23, 36) and with'the expense or unavailability of the "unnatural" _p_-amino acids i t would be of great value i f inexpensive, effective methods of resolution of the racemic amino acids could be obtained. Most early workers when preparing peptides containing a D-amino acid or an L_-amino acid f i r s t resolved a racemic mixture of the amino acid by the use of an. optically active base. This method is both expensive and time consuming. More recently, the stereo specificity of certain enzymes has been used for the preparation of optically pure amino acids. An out-standing example of this is the method used by Greenstein (12, 28, 16) in which an aqueous extract of rat or hog kidney is allowed to act, rapidly and asymmetrically, upon many N-acylated DL-amlno acids. The acylase hydrolyses preferentially the N acylrLj^amino acid bond producing the free Jj-amino acid and the N-acyl _P_-amino acid. From such a digest, the free L_-amino acid is separated by addition of alcohol, leaving the soluble acylrgramino acid in the mother liquor. The acyl-D-amino acid is then hydrolyzed by hot mineral acid to yield the free JJ-amlno acid. - 3 - -This enzymatic method cannot be applied to the resolution of a l l L_-amino acids. In the case of arginine and histidine, i t is difficult to acylate both basic groups with susceptible, and conveniently removed, acyl radicals, and the N-acyl derivatives of cystine and proline are completely resistant to enzymatic hydrolysis by animal enzyme preparations (22). An alternative method of resolution based upon the asymmetric enzymatic hydrolysis of the racemic amino acid amides by a non-specific amidase has, therefore, been developed (17, 22)• Other enzymatic methods of resolution of racemic amino acids include that of asymmetric synthesis with aniline (11). Such a method makes use of the enzymatic synthesis of the insoluble anilide of the L -isomer which subsequently crystallizes. The diasterioisomer can then be recovered from the mother liquor. The anilide form of the _L_-isomer is , however, of no further use in coupling unless subjected to hydrolysis which may result in loss by racemization. Scattered reports of resolution of unsubstituted amino acids have been recorded. However, most of the workers make use of salt formation between an optically active acid or base and a racemic amino acid with subsequent separation of one or both of the diastereoisomers by fractional crystallization (30, 29, 37, 21). In the preparation of a dipeptide by the carbobenzoxy method (4) i t is often possible to obtain one or two crystalline intermediate compounds, as well as the final crystalline dipeptide. The steps of this method are as follows: o p 0 ff—V " / ' R R, I I v i a a z i d e 0 0 0 o r c h l o r i d e r. a || || II . ( _ y CH 2 - 0-C-NH-CH-C-NH-CK-C-C-R 2 R R j I I I 0 0 0 s a p o n i f i c a t i o n |j || || > ( y CH 2 -Q - C - m - C H - C - N H - C H - C - 0 H NaOH I ^ IV 0 0 hyd rogeno lys i s || |l -> NH 2 -CH-C-NH-CH-C-0H Pd H 2 R R x V Thus, i f a carbobenzoxy DL-amino a c i d ( i ) i s coupled t o an Xramino a c i d e s t e r ( I I ) there may be as many as three s y n t h e t i c s teps a t which t o e f f e c t a s e p a r a t i o n o f the r e s u l t i n g d i a s t e r e o i s o m e r s . Fur thermore , the r e s u l t i n g o p t i c a l l y pure" carbobenzoxy d i p e p t i d e s (IV) o r d i p e p t i d e e s t e r s ( I I I ) a re i n such a form t h a t they can be used d i r e c t l y f o r f u r t h e r c o u p l i n g s t o produce l o n g e r c h a i n p e p t i d e s . T h i s method o f r e s o l u t i o n has been used i n a few cases . P o l g l a s e and Smi th (27) c o u p l e d ^ L - l e u c i n e a z i d e w i t h the methyl e s t e r o f D L - a l a n i n e . C a r b o b e n z o x y - L - l e u c y l - D - a l a n i n e methyl e s t e r c r y s t a l l i z e d from the r e a c t i o n m i x t u r e . The d ia s t e reoc i somer cou ld not be c r y s t a l l i z e d from the mother l i q u o r , but a f t e r s a p o n i f i c a t i o n w i t h a l k a l i , the r e s u l t i n g carbobenzoxy-^-leucyl-L-alanine ciystallized. Hunt and du Vigneaud (20) prepared carbo-benzoxy-Lj-alanyl-Lrhistidine from carbobenzoxy-DL^alanine and L-histadine methyl ester. After saponification of the resulting product the carbo-benzoxy- L-alanyl-L-histidine crystallized. The combined mother liquors were hydrogenolysed and the dipeptide D^alariyl-Ir-histidine isolated by-means of i t s copper salt. Behrens, Doherty and Bergmann ( 3 ) , prepared acetyl-Dj-phenylalanyl-L-leucine and acetyl-L-phenylalanyl-L-leucine by reduction of acetyldehydrophenylalanyl-L^-leucine and fractional c r y s t a l l i -zation from f i f t y percent dioxane. Cook, Cox and Farmer (7) coupled L-c<-bromoisovaleryl chloride and DL-N-methyl valine. These products were converted to the hydroxy isomeric lactones and then separated chromate— graphically. In this thesis, the f i r s t part of the experimental section describes the coupling of carbobenzoxy-DL-alanine and of carbobenzoxy-DL-phenylalanine with L-leucine methyl ester, and the separation or attempted separation of the corresponding isomers. Some of the L-leucine used i n the syntheses was prepared by purification of technical leucine as described by DeWitt and Ingersoll (10) . B. Specificity of Pepsin The proteolytic enzymes of vertebrates f a l l into two groups, the f i r s t of which i s mainly concerned with the degradation of the large molecules of the food proteins to yield smaller fragments, the second group completing the process initiated by the f i r s t and leading eventually to the liberation of free amino acids. The f i r s t group includes pepsin - 6 -which arises from the gastric juice, and trypsin and chymotrypsin formed from precursors present in the pancreatic juice. The second group is composed of carboxypeptidase, contributed by the pancreatic juice, amino-peptidase, and dipeptidase, which are present in the intestinal secretions. Pepsin is secreted by the gastric mucosa in the form of pepsinogen, which is activated, in the f i r s t instance, by the hydrochloric acid of the gastric juice to yield pepsin itself. Pepsin, once formed, is capable of activating more pepsinogen so that the activation of pepsinogen is an autocatalytic process. Northrup (24) was the f i r s t to isolate the crystalline enzyme from commercial pepsin preparations. This was the second crystalline enzyme to be reported and the first crystalline proteolytic enzyme. Since 1930, many more enzymes have been isolated in the crystalline state (25). Biologically speaking, i t is possible to draw some sort of distinction between pepsin and the trypsins on one hand, formerly called proteinases, and the group of peptidases, carboxypeptidase, aminopeptidase and dipeptidase on the other. The digestion of the food proteins is begun by the "proteinases" and the fragmentary products thus formed are further degraded by the "peptidases" to yield, finally, free amino acids. It was formerly believed that pepsin and the trypsins were able to attack only large molecules of the same order of size as the protein molecule of the food and that the "peptidases" were only able to deal with molecules of the order of size found among polypeptides and perhaps peptones. More recent work, which became possible only when Bergmann's "Carbobenzoxy method of peptide synthesis (4), had made a wide variety of synthetic peptides available, has shown that pepsin, trypsin and chymotrypsin, as w e l l as the p e p t i d a s e s , are a b l e t o a c t upon compara t ive ly s imple pept ides) p rov ided t h a t the pep t ide l i n k a g e s o f the r i g h t k i n d and the a p p r o p r i a t e c o n f i g u r a t i o n a re p r e s e n t . Whereas the subs t r a t e s p e c i f i c i t i e s o f t r y p s i n and chymotryps in were c l e a r l y d e l i n e a t e d i n Bergmann 1s f i r s t experiments (19, 14-), the s p e c i f i c i t y o f p e p s i n i s s t i l l i n c o m p l e t e l y unders tood . In t h e i r o r i g i n a l work (15) , Bergmann and F r u t o n o u t l i n e d the subs t ra t e s p e c i f i c i t y f o r p e p s i n as f o l l ows? 1) Presence o f a t l e a s t two c a r b o x y l groups i n the p e p t i d e . 2) Absence, o f a p r o x i m a l f ree amino group. 3) Presence o f an aromat ic r e s idue (pheny l a l any l o r t y r o s y l ) on the amino s ide o f the l i n k a g e to be a t t a c k e d . A t y p i c a l "Bergmann s u b s t r a t e 8 was ca rbobenzoxy-L -g lu tamyl>L - ty rog ine o r ca rbobenzoxy-L -g lu tamyl-L -pheny la lan ine . The pH optima however, f o r these two subs t ra tes were pH A.O and pH 4 . 5 , r e s p e c t i v e l y , as compared w i t h an optimum of 1.8 - 2 .0 f o r the p e p t i c h y d r o l y s i s o f p r o t e i n s . Moreover , the r a t e o f h y d r o l y s i s of the s y n t h e t i c subs t r a t e s i s ex t r eme ly s low, compared t o the r a t e o f h y d r o l y s i s o f p r o t e i n s (25) . I n 1944, H a r r i n g t o n and :Pittr- R i v e r s (18) , s t ud i ed the a c t i o n o f c r y s t a l l i n e p e p s i n on the N-carbobenzoxy d e r i v a t i v e s o f c y s t e i h y l - and c y s t i n y l t y r o s i n e and o f t y r o s y l - c y s t e i n e and c y s t i n e , and on the cor responding f ree p e p t i d e s both a t pH A.O and pH 1.8 . They found t h a t both c y s t e i n y l ( c y s t i n y l ) -t y r o s i n e and t y r o s y l - c y s t e i n e ( c y s t i n e ) pep t ides were s p l i t by p e p s i n and a l though the N-carbobenzoxy d e r i v a t i v e s were the most r e a d i l y h y d r o l y s e d , the f ree d i p e p t i d e s were a l s o a t t a c k e d . I n agreement w i t h Bergmann, H a r r i n g t o n and P i t t R i v e r s found t h a t the a c t i o n o f peps in on t h e i r s y n t h e t i c subs t r a t e s was much more marked a t pH 4 . 0 than a t pH 1 .8 . They - 8 -a l s o found t ha t the subs t r a t e s , whether a c y l a t e d o r no t , were more s u s c e p t i -b l e t o the a c t i o n o f peps in when they were i n the reduced form. A l t h o u g h the s y n t h e t i c subs t ra tes t e s t e d by H a r r i n g t o n and - P i t t r R i v e r s do not meet the s p e c i f i c i t y requirements se t f o r t h by Bergmann (15) they were s p l i t , i n some cases , j u s t as r a p i d l y as subs t ra tes which s a t i s f i e d Bergmann's c r i t e r i a . The s p l i t t i n g o f a f ree d i p i p t i d e by p e p s i n was c o n t r a r y t o a l l the p r e v i o u s l y conceived ideas o f p e p t i c a c t i o n . The .observa t ion t h a t the subs t r a t e s are a t t acked by p e p s i n much more r a p i d l y i n the reduced (-SH) form than i n the o x i d i z e d (-SS) was i n t e r e s t i n g i n v iew o f the f a c t t ha t p e p s i n a c t s much more v i g o r o u s l y on denatured than on n a t i v e p r o t e i n s and t h a t dena tu ra t i on o f p r o t e i n s i s accompanied by the appearance o f -SH groups . A few years l a t e r F r u t o n and coworkers (8 ) , t e s t e d the a c t i o n o f p e p s i n on c a r b o b e n z o x y - L - m e t h i o n y l - L - t y r o s i n e and J g r n e t h i o n y l - j i r t y r o s i n e . They found s u b s t a n t i a l h y d r o l y s i s (35% and 25% h y d r o l y s i s i n 2A n r . , r e s p e c t i v e l y ) o f the two compounds, a g a i n w i t h an optimum pH o f A . O . The o b s e r v a t i o n o f Northrup (26) t h a t t y r o s i n e i s l i b e r a t e d from a s e l f -d i g e s t i o n o f p e p s i n and the r e p o r t o f C a l v e r y and Schock (6) t h a t t y r o s i n e i s l i b e r a t e d from a p e p t i c h y d r o l y s i s o f egg albumin can p o s s i b l y be e x p l a i n e d by assuming t h a t a t l e a s t one p o i n t o f a t t a c k o f the p r o t e i n by p e p s i n may be a c y s t e i n y l t y r o s i n e , t y r o s y l - c y s t e i n e o r m e t h i o n y l t y r o s i n e l i n k a g e . Us ing pep t ide d e r i v a t i v e s c o n t a i n i n g two a romat ic amino a c i d s such as a c e t y l ^ - p h e n y l a l a n y l r L _ - t y r o s i n e or a c e t y l ^ - p h e n y l -a l a n y l r L - d i i o d o t y r o s i n e , Baker (2) has shown an optimum pH f o r p e p t i c h y d r o l y s i s o f 1 .8 , the pH optimum f o r h y d r o l y s i s o f p r o t e i n s . The e x -periment was extended to subs t r a t e s which con ta ined b e n z y l , hydroxy b e n z y l o r d i i o d o h y d r o x y b e n z y l group. A l l the compounds were hydro lysed q u i t e " - 9 -rapidly, the rate being faster at pH 2.0 than at pH 3.0 or 4.0. Under the condition of hydrolysis used, Baker also found carbobenzoxy-L-glutamyl-L-tyrosine to be hydrolyzed much more rapidly at pH 2.0 than at pH 4.0. The differences between Baker's and Bprgwrnn*p results are difficult to explain unless i t is assumed that the enzyme preparations used by the two workers were different. In their study of the sequence of amino acids in the insulin molecule, Sanger and Tuppy (31), and Sanger and Thompson (32) employed proteolytic enzymes. They found that the specificities of trypsin and chymotrypsin, as delineated by Bergmann and his associates (with synthetic substrates) were almost completely applicable to the large polypeptide fragments obtained from insulin. Sanger and associates suggested (31, 32) that specificity of action of trypsin and chymotrypsin on an intact protein was probably similar to the specificity of these enzymes for synthetic substrates. On the other hand, pepsin appeared to have a considerable lower degree of specificity than had the other two enzymes. In the glycyl chain of insulin, Sanger and Thompson (32), found the following bonds to be split by pepsin: - glutamyl - glutamyl - ) - valyl - seryl - ; - leucyl - tyrosyl - j tyrosyl - glutamyl - ; -glutamyl - leucyl - j - leucyl - glutamyl - ; glutamyl - aspartyl - j in the phenylalanyl chain (31) the following bonds were split by pepsin: phenylalanyl - valyl - j - glutamyl - histidinyl - ; - leucyl - valyl - j - glutamyl - alanyl - j - alanyl - leucyl - j - leucyl - tyrosyl - ; - tyrosyl - leucyl - glycyl - phenylalanyl - j - phenylalanyl - phenylalanyl - and - phenylalanyl - tyrosyl - . It does not appear that the specificity - 10 -o f p e p s i n i s l i m i t e d ^ t o bonds adjacent to a romat ic r e s idues as was p r e v i o u s l y b e l i e v e d (15, 18 , 2 , 8 ) . P e p s i n would appear t o have a s p e c i f i c i t y f o r the more f a t s o l u b l e p a r t s o f the pep t ide c h a i n . T h i s f i n d i n g o f a r a t h e r wide s p e c i f i c i t y f o r the a c t i o n o f p e p s i n on the i n s u l i n molecule i s i n agreement w i t h the r e s u l t s o f D e s n u e l l e , Rovery and Bonjour (9, 35) who s t u d i e d the N - t e r m i n a l r e s idues l i b e r a t e d on t reatment o f ovalbumin and horse g l o b i n w i t h p e p s i n . G e n e r a l l y speaking i t was found t h a t p e p t i c h y d r o l y s i s proceeded d i f f e r e n t l y w i t h g l o b i n than w i t h a lbumin . I n the case o f g l o b u l i n , h y d r o l y s i s proceeded i n two success ive phases . Dur ing the f i r s t phase, v e r y l a r g e pep t ides and duodeca pep t ides (on the average) are formed, s i m u l t a n e o u s l y , from the p r o t e i n c h a i n s . The l a r g e pep t ides a re themselves r a p i d l y t ransformed i n t o duodecapeptides which can be cons idered as t y p i c a l o f t h i s p a r t i c u l a r s t age . The f i r s t phase o f degrada t ion i s r a p i d . C h a r a c t e r i z a t i o n o f t e r m i n a l amino a c i d s shows t h a t t h i s degrada t ion occurs a c c o r d i n g t o c e r t a i n r u l e s o f s p e c i f i c i t y . P e p s i n , d u r i n g t h i s phase, breaks up p r e f e r e n t i a l l y those bonds i n wh ich a l a n i n e , p h e n y l a l a n i n e , l e u c i n e and s e r i n e are i n v o l v e d through t h e i r amino groups . The c h a r a c t e r i z a t i o n o f the c a r b o x y l t e r m i n a l groups l i b e r a t e d by p e p s i n was not a t tempted . Dur ing a second phase o f the h y d r o l y s i s , the pep t ides undergo a f u r t h e r degrada-t i o n down to the t e t r a pep t ide (average) s t age . T h i s phase proceeds v e r y s l o w l y and seems n o n - s p e c i f i c , (at l e a s t as f a r as the amino s ide o f the l i n k a g e i s concerned) . On the o the r hand, i n the case o f a lbumin , there does not seem t o be any gene ra l fo rma t ion o f l a r g e p e p t i d e s . A f i r s t phase proceeds r a p i d l y - 11 -t o the hexa- o r penta-pept ide s tage . No s p e c i f i c i t y can be demonstrated i n the h y d r o l y s i s o f i n d i v i d u a l l i n k a g e s . A f t e r t h i s r a p i d i n i t i a l de -g r a d a t i o n , v e r y l i t t l e f u r t h e r h y d r o l y s i s o c c u r s , a l though e v e n t u a l l y , a g a i n , an average o f f o u r amino a c i d s pe r pep t ide res idue i s r eached . No s p e c i f i c i t y can be demonstrated i n t h i s second phase (9, 35). A n f i n s e n ( l ) i n c o n s i d e r i n g a p e p t i c d i g e s t i o n o f r i b o n u c l e a s e suggests t h a t the i n i t i a l stage o f d i g e s t i o n c o n s i s t s o f a change i n the f i n e s t r u c t u r e o f the mo lecu le , i n v o l v i n g min imal c leavage o f pep t ide bonds and no change i n sed imenta t ion cons t an t . Subsequent d i g e s t i o n by p e p s i n then r e s u l t s i n the rupture o f app rox ima te ly t e n pep t ide bonds, w i t h the r e s u l t i n g pep t ides c o n t a i n i n g , on the average, 7 - 8 amino a c i d r e s idues per m o l e c u l e . From the f o r e g o i n g d i s c u s s i o n , i t i s c l e a r t h a t the subs t ra te i s p e c i f i c i t y o f p e p s i n needs f u r t h e r s tudy . Such a s tudy r e q u i r e s t h a t s u i t a b l e , c h e m i c a l l y - d e f i n e d , p o t e n t i a l subs t r a t e s be p repared . U s u a l l y , i n work on enzyme s p e c i f i c i t y , the syn thes i s and the i s o l a t i o n and c h a r a c t e r i z a t i o n o f subs t r a t e s become the major r e sea rch t a s k s , w h i l e the work o f t e s t i n g enzyme s p e c i f i c i t y (the u l t i m a t e o b j e c t i v e o f the research) may be completed i n a r e l a t i v e l y shor t t i m e . T h i s has been t r u e f o r the r e sea rch repor ted h e r e i n . A l l m e l t i n g p o i n t s are r epor t ed i n degrees cen t igrade and are u n c o r r e c t e d . - 12 EXPERIMENTAL A . Syn thes i s o f Pep t ide D e r i v a t i v e s I . Carbobenzoxy C h l o r i d e (4) One l i t e r o f to luene was coo led i n an i c e ba th and phosgene (200 g . ) i n t roduced s l o w l y (34) . B e n z y l a l c o h o l (160 m l . ) was then added and the s o l u t i o n a l lowed to stand f o r 1 h r . i n an i c e ba th and a f u r t h e r 2 h r . a t room temperature . A t the end o f t h i s t ime the excess phosgene was f l u s h e d out w i t h a stream o f d r y a i r and the s o l u t i o n concent ra ted under reduced pressure t o a volume o f 120 m l . The carbobenzoxy c h l o r i d e was t e s t e d by p r e p a r a t i o n o f c a rbo -benzoxy g l y c i n e , as f o l l o w s : g l y c i n e (7.5 g . ) , was d i s s o l v e d i n 25 m l . o f 4_N sodium h y d r o x i d e . To t h i s s o l u t i o n , carbobenzoxy c h l o r i d e (20 g . ) and 4 N. sodium hydroxide (25 m l . ) were added i n e q u i v a l e n t , s m a l l p o r t i o n s over a p e r i o d o f 35 min . A f t e r 1 h r . a t room temperature , the s o l u t i o n was a c i d i f i e d to congo red w i t h concent ra ted h y d r o c h l o r i c a c i d . The p r e c i p i t a t e was f i l t e r e d and r e c r y s t a l l i z e d from w a t e r - e t h a n o l ; y i e l d : 18 .5 g . , m.p . 1 1 8 - 1 1 9 ° . T h i s i n d i c a t e d a y i e l d o f 88.5$ f o r the carbobenzoxy c h l o r i d e . I I . P u r i f i c a t i o n o f 1,-Leucine (10) T e c h n i c a l ^ - l e u c i n e , 131 g . , ( l mole , assuming pure l e u c i n e ) was suspended i n 350 m l . o f water and t r e a t e d w i t h 3 moles o f - 13 -a c e t i c anhydr ide and a s o l u t i o n o f 7 moles o f sodium hydroxide i n 350 m l . o f wa te r . Throughout the a d d i t i o n , the mix ture was kept s l i g h t l y a l k a l i n e . The reagents were added over a p e r i o d o f 2 n r . , w i t h s t i r r i n g and c o o l i n g i n an i c e - s a l t ba th . Twenty minutes a f t e r the l a s t a d d i t i o n , the mix ture was a c i d i f i e d w i t h 7 moles o f 37% h y d r o c h l o r i c a c i d and p l a c e d i n the r e f r i g e r a t o r . The product was c o l l e c t e d on a Buchner funne l the next day. The p r e c i p i t a t e was washed w i t h 200 m l . o f c o l d water and 200 m l . o f acetone; y i e l d : 110 g . For r e c r y s t a l l i z a t i o n , t h i s compound was d i s s o l v e d i n 9 p a r t s o f 33% methanol and t r e a t e d w i t h c h a r c o a l . A second r e c r y s t a l l i z a t i o n was from a s o l u t i o n o f 2 .5 m l . methanol and 5 m l . hot water pe r gram o f m a t e r i a l ; m.p . 1 7 9 - 1 8 0 ° . Twice r e c r y s t a l l i z e d , N - a c e t y l - L r - l e u c i n e ( 1 7 . 3 / g . ) , was r e f l u x e d w i t h 35 m l . o f 3 N hydrobromic a c i d . A t the end o f 2 n r . , the s o l u t i o n was d i l u t e d w i t h 100 m l . o f hot methanol and brought to pH 6.0 w i t h aqueous ammonia. The ; S O L O T .IO ' M . t a o f - l e u c i n e was coo led ove rn igh t and f i l t e r e d ; y i e l d : 10 .8 g . joC]]25-) +15.6 (c 4, 6N H C l ) . The r epor t ed r o t a t i o n f o r L - l e u c i n e (10) i s : [c * ] 2 5 n +15.3 (c A, 6 N H C l ) . A paper ehromatogram o f the p u r i f i e d i r l e u c i n e showed o n l y one spot c o r r e s -ponding t o a sample of pure _L_-leucine. I I I . P r e p a r a t i o n o f Amino A c i d E s t e r s The cor responding amino a c i d e s t e r h y d r o c h l o r i d e s (see pages 14, 20, 30, 32 ) were d i s s o l v e d i n the l e a s t amount o f water and covered w i t h a l a y e r of e t h e r . The s o l u t i o n was coo led t o 0° and an excess o f 12 N. sodium hydroxide was added s l o w l y w i t h s h a k i n g . - 14. -Anhydrous potass ium carbonate was then added t o absorb a l l the wa te r . The mix tu re was f i l t e r e d and the p r e c i p i t a t e e x t r a c t e d r epea t ed ly w i t h e t h e r . The combined f i l t r a t e s were d r i e d over sodium s u l f a t e . IV. Carbobenzoxy-Di ' -Alf lTiyl - L - L e u c i n e Me thy l E s t e r a) Carboben gmry-nT . - a lan ine D L - A l a n i n e (5 g . ) was d i s s o l v e d i n 15 m l . o f 4 K sodium hydroxide which had been coo led to 0°. Carbobenzoxy c h l o r i d e (10.5 g . , 20% excess) and 4 N. sodium hydroxide (15 m l . ) were added over a p e r i o d o f t h i r t y minutes w i t h s h a k i n g . A f t e r s t and ing a t room temperature f o r 1 h r . , the s o l u t i o n was a c i d i f i e d to congo red w i t h concent ra ted h y d r o c h l o r i c a c i d . The p r e c i p i t a t e was f i l t e r e d and r e c r y s t a l l i z e d from wate r -ace tone . TABLE I P r e p a r a t i o n o f carbobenzoxy-DL-alanine P r e p a r a t i o n No. S t a r t i n g amt, o f D L - a l a n i n e Y i e l d (g .) m.p. <°c> 1 5.0 8.5 109 - 111 2 10 .0 14 .2 109 - 111 3 5.0 8.0 109 - 111 4 2 .5 4 . 2 110 - 112.5 b) L - I e u c i n e methyl e s t e r h y d r o c h l o r i d e J t r l e u c i n e (11.3 g . ) was d i s s o l v e d i n t e n t imes i t s weight o f anhydrous methanol and the s o l u t i o n sa tu ra t ed w i t h d r y hydrogen - 15 -c h l o r i d e gas a t 0 ° . The s o l u t i o n was a l lowed to s tand a t 0 ° o v e r n i g h t . The r e a c t i o n mix ture was then concentra ted under reduced p r e s s u r e , where--upon c r y s t a l l i z a t i o n r e s u l t e d . The c o n c e n t r a t i o n was repeated three o r f o u r t i m e s , a f t e r the a d d i t i o n o f anhydrous methanol . The r e s u l t i n g amino a c i d e s t e r h y d r o c h l o r i d e was suspended i n e t h e r , f i l t e r e d , and r e c r y s t a l -l i z e d from methanol -e ther . TABLE I I P r e p a r a t i o n o f J L - l e u c i n e methyl e s t e r h y d r o c h l o r i d e P r e p a r a t i o n S t a r t i n g amt. Y i e l d m.p. No. o f L - l e u c i n e (g.) (°c) ~ ( g . ) 1 10 .0 2 . 0 148 - 149.5 2 10 .0 4 . 0 145 - 147 3 5.8 4 .5 147 - 148 4 10 .0 6.8 • 147 - 148 5 9.0 3 . 9 147 - 148.5 6 11.3 9.92 1 4 8 . 5 - 149.5 7 20 .0 5.1 147 - 148 8 9 .0 3 .86 147 - 148.5 9 10 .0 4 .31 146 .5 - : 14855 10 10 .0 5.72 146 - 148 c) Ca rbo teng iovy-D^-a l any l -L- l euc ine methyl e s t e r Carbpbenzoxy-DL-alanine (5.45 g . ) , was d i s s o l v e d i n 20 m l . o f anhydrous e the r and the s o l u t i o n coo led t o 0 ° . Phosphorous p e n t a c h l o r i d e (5.45 g.) was added and the solution shaken at 0° until a l l but a trace of the latter had dissolved. Light petroleum ether (previously cooled to -5o°) was added, whereupon the solution turned cloudy. On stirring at i -50° the carbobenzoxy-DL-alanyl chloride crystallized. The supernatant I_IG?I»D was decanted and the chloride washed by decantation, with light petroleum ether (previously cooled to -50°). The product was then dissolved in 30 ml. of chloroform (previously cooled to -50°) and added to a solution of _L^ leucine methyl ester (from 8.9 g. of the hydrochloride) in ether. After 30 min. at 0° and 2 hr. at room temperature, 2.24 g. of L^leucine ester hydrochloride was filtered from the reaction mixture. The filtrate was washed with saturated sodium bicarbonate, N_ hydrochloric acid and fianlly with water. The chloroform-ether solution was dried over sodium sulfate—,, and concentrated to a syrup. The syrup was dissolved in ethyl ether and crystallization occured on slow evaporation. The compound was recrystal-lized from ether-petroleum ether (30-60°) j (p^2 )^ " 2^*° ( ethanol) TABLE III Preparation of carbobenzoxv-DL-alanylrLrleucine methyl ester Preparation Starting amt. Yield m.-p. No. of DL-alanine (g.) (°C) ~~(g.)  1 3.05 1.5 74.5 - 75.5 2 5.45 4.19 74 - 75 3 5.45 1.23 72 - 73 This compound has previously been reported only as an o i l (20, 27). - 17 -Anal. Calcd. for C 18 H24°5 W 2 J N > 8 # 0 0 ; F o u n d » N» 7- 8 1» 7*9-+* u P o n admixture with an authentic sample of carbobenzoxy-D^lanyl-L-leueine methyl ester," the melting point was depressed to 62-67°. V. Carbobenzoxy-I&-Alflnyl-L-Lftur'1 np I Carbobenzoxy-DL-alanyl-Lrleucine methyl ester (2.18 g.) was dissolved in 20 ml. of acetone and treated with 6.05 ml. of N_ sodium hydroxide. After 4-5 min. at room temperature, the solution was acidified, and the acetone evaporated in a stream of air. The resulting syrup was extracted into ethyl acetate and this solution extracted with a saturated i solution of sodium bicarbonate. The sodium bicarbonate solution was acidified and extracted with ethyl acetate and this extract dried with DRIED sodium sulfate. TheAethyl acetate solution was concentrated to a syrup. Attempts to induce crystallization were unsuccessful. i" } VI. gl-Alanyl-^Leucine Carbobenzoxy-pjj-alanyl^L-leucine from above was dissolved in methanol (15 ml.), water ( 1 ml.) and glacial acetic acid (1 ml.) and hydrogenated over a palladium catalyst. Hydrogenolysis was complete in 28 hrs. The catalyst was filtered off and the filtrate concentrated under reduced pressure to a syrup from which the acetic acid and water were removed by repeated concentration with anhydrous methanol. The syrup could not be induced to crystallize. However, on standing at room ' A sample of carbobenzoxy-D_-alanyl-L-leucine methyl, ester was kindly supplied by Dr. Emil L. Smith of the University of Utah. - 18 -temperature i t d i d s o l i d i f y ; ^ Q ^ 2 ^ ~ 7 - 7 8 (s. 5 w a t e r ) . The recorded r o t a t i o n (27) f o r J ^ - a l a n y l - ^ - l e u c i n e i s j o ^ 2 1 ^ - . 1 7 . 0 (c 5 water) V I I . Carbo ben y.mry -DL-Alanyl -L.-Le uc inamide Ca r bobenzoxy - D L- a l any l -Xr l eu c in e methyl e s t e r (200 mg.) was d i s s o l v e d i n methanol (15 m l . ) , p r e v i o u s l y sa tu ra ted a t 0 ° w i t h ammonia. A f t e r 40 h r s . a t room temperature, the s o l u t i o n was concent ra ted whereupon c r y s t a l l i z a t i o n o c c u r r e d . The produc t was r e c r y s t a l l i z e d from e thano l -wa te r ; m.p. 1 6 3 - 1 6 4 ° ; jf^J^D ~ 2 7 * ° / e t h a n o l ) . A n a l . C a l c d . f o r C ^ H ^ O ^ : N , 12 .54 Found, N 1 2 . 4 3 . The cons tan ts repor ted (27) f o r the two d i a s t e r eo - i somer s are* C a r b o b e n z o x y - ^ - a l a n y l ^ r l e u c i n a m i d e ; m.p . 1 8 7 - 1 8 8 ° ; K 3 0 D " 6 k 1 e t h a n o l ) C a r b o b e n z o x y - L - a l a n y l - L - l e u c i n a m i d e : m.p . 1 8 8 - 1 8 9 ° ; J k ] 3 ° D 1 - 4 1 ( e l e thanol ) V I I I . Carbobenzoxyr^-Alanyl -J^-Leucine M e t h y l E s t e r a) Carbobenzoxy ^ - a l a n i n e i ^ A l a n i n e (2.5 g . ) was t r e a t e d i n the same way as f o r the p r e p a r a t i o n o f carbo benzoxy-DL.-alanine. The o i l y product was d i s s o l v e d i n e ther and e x t r a c t e d w i t h sodium b i c a r b o n a t e . The b icarbona te l a y e r was a c i d i f i e d and r e - e x t r a c t e d w i t h e t h e r . The e ther l a y e r was d r i e d over sodium s u l f a t e and the compound was c r y s t a l l i z e d by the a d d i t i o n o f pe t ro leum e ther ( 3 0 - 6 0 ° ) ; - y i e l d : 3 .2 g ; m.p . 8 1 - 8 4 ° . The r epor ted m e l t i n g p o i n t (4) f o r ca rbobenzoxy-L-a lan ine i s 8 4 ° . b) -19 -Carbobensnvy-L -a lanyl -Li r leuc ine methyl e s t e r Carbo tenzoxy-L-a l an ine (3.2 g . ) was coupled t o ^ - l e u c i n e methyl e s t e r (from 5.56 g . o f the h y d r o c h l o r i d e ) through the a c i d c h l o r i d e by the u s u a l procedure (see page 15 ). Ca rbobenzoxy-L-a l any l c h l o r i d e was induced to c r y s t a l l i z e a t -50° a l though l i k e carbobenzoxy-jLI<-alanyl c h l o r i d e , p rev ious r e p o r t s (27) have d e s c r i b e d t h i s substance as an o i l . C a r b o b e n z o x y - L - a l a n y l - L - l e u c i n e methyl e s t e r c o u l d o n l y be i s o l a t e d as an o i l . I t was d r i e d i n vacuo: we igh t , 3.0 g . I X . Carbobenzoxy qLrA"! anyl=Lj-Leucine C a r b o b e n z o x y r L r a l a n y l r L y l e u c i n e methyl e s t e r (3.0 g . ) was d i s s o l v e d i n acetone (20 m l . ) and t r e a t e d w i t h .N sodium hydroxide (9.37 m l . ) i n the same manner as f o r the p r e p a r a t i o n o f carbobenzoxy-DL—alanyl-L-l e u c i n e (page 17 ). The product c o u l d not be induced to c r y s t a l l i z e and was i s o l a t e d as an o i l . X . Ca rbobenzoxy-^ -Pheny la l anv l -L -Leuc ine M e t h y l E s t e r a) Carbobep7.nyy-pLi-T3henylalanine DL-Pheny la l an ine (6.6 g . ) was d i s s o l v e d i n 2_N sodium hydroxide (20 m l . ) which had been cooled t o 0°. To t h i s s o l u t i o n , carbobenzoxy c h l o r i d e (7.0 g . ) , and 2_N sodium hydroxide (20 m l . ) were added g r a d u a l l y , w i t h s h a k i n g . A f t e r 0.5 h r . a t room temperature the s o l u t i o n was a c i d i f i e d t o congo red w i t h concent ra ted h y d r o c h l o r i c a c i d . A n o i l separated which s o l i d i f i e d on s t i r r i n g . The compound was r e -c r y s t a l l i z e d from t o l u e n e . TABLE IV P r e p a r a t i o n o f ca rbobenzoxy-DL-phenyla lan lne . P r e p a r a t i o n No. S t a r t i n g amt. o f DL-pheny la lan ine _ (g.) Y i e l d (g.) m.p . ( ° 0 1 6.6 5.67 102 - 103 2 6 .6 10 .1 102 - 103 3 6 .6 8.5 100 - 103 4 6 .6 3 .5 9 7 - 9 9 b) DX-Pheny la lan ine e t h y l e s t e r h y d r o c h l o r i d e TABLE V P r e p a r a t i o n o f PL-phenyl al, a n ine e t h y l e s t e r h y d r o c h l o r i d e P r e p a r a t i o n No. S t a r t i n g amt. o f DL-pheny la l an ine ~ (g.) Y i e l d <g-> m.p , (°c) 1 10 .0 6.38 124 -125 .5 2 10 .0 7.85 125 -126 3 25 .0 19 .0 124 -125.5 4 25.0 21 .6 127 .5-129.5 DL-Pheny la l an ine was suspended i n t e n t imes i t s weight o f anhydrous e thano l and sa tu ra ted w i t h d r y hydrogen c h l o r i d e gas a t 0 ° . The r e a c t i o n mix ture was heated under r e f l u x f o r 2 h r . and concent ra ted r epea t ed ly w i t h - 2 1 -anhydrous e thano l under d imin i shed p r e s s u r e . The r e s u l t i n g amino a c i d e s t e r h y d r o c h l o r i d e was c r y s t a l l i z e d from e t h a n o l - e t h e r . c) Carbobengn-xY-pT.-phenylalanlne e t h y l e s t e r (30) DL-Pheny la l an ine e t h y l e s t e r h y d r o c h l o r i d e (3.6 g . ) was d i s s o l v e d i n 50 m l . o f ch lo ro form and t r e a t e d , p o r t i o n - w i s e , under i c e c o o l i n g and V/GO/TOI/S shak ing w i t h carbobenzoxy c h l o r i d e (2.4. g . ) .and .N sodium hydroxide (27 m l . ) . A f t e r 0 .5 h r . a t room temperature , the ch lo ro form l a y e r was washed w i t h N h y d r o c h l o r i c a c i d (30 m l . ) and d i l u t e potass ium carbonate 0-0%) and d r i e d over sodium s u l f a t e . On c o n c e n t r a t i o n • reduced u n d e r p r e s s u r e , an o i l remained which c r y s t a l l i z e d on s t a n d i n g . The product was r e - c r y s t a l l i z e d from e t h y l e the r -pe t ro leum e the r ( 6 5 - 1 1 0 ° ) . TABLE VI P r e p a r a t i o n o f carbobenzoxy-DL-phenyla lanine e t h y l e s t e r P r e p a r a t i o n No. S t a r t i n g amt. o f DL pheny la l an ine ~~ (g.) Y i e l d (g.) m.p. (°c) - 1 3.0-1 " 5.5 79.5 - 80 3 . 6 J 2 5.5 5.0 " 80 - 82 3 6.6 6 .4 77 .5 - 79 .5 4 6 .0 5.5 ' 80 " - "81 " d) . Carbobenzoxv-DL-nhenyla lanvl hydraz ide (30) Carbobenzoxy-DL-phenyla lanine e t h y l e s t e r (4.0 g . ) was -22-suspended in 12 ml. of absolute ethanol and warmed with 0.91 g. of hydrazine hydrate (2.2 moles of 35%), and left at room temperature for 24 hr. At the end of this time, the reaction mixture had crystallized into a solid mass. The crystal 3 were brought into solution by the addition of more absolute ethanol with warming. The solution was treated with a second portion (0.2 g.) of hydrazine hydrate. At the end of 24 hr., the reaction mixture was diluted with water and the crystalline product collected on a Buchner funnel. The compound was re-crystallized from dilute ethanol. TABLE VII Preparation of carbobenzoxy-DL-phenylalanyl hydrazide Preparation No. Starting amt. of carbobenzoxv-DL-phenylalanine ethyl ester (g.) Yield (g.) m.p (°c) 1 4.0 3.0 135 - 135.5 2. 4.0 3.61 134.5 - 135 - . 3 6.0 5.42 133 - 135 4 4.66 4.04. 134.5 - 136 e) Carbobenzoxy^ phenylalanyl - J L - leucine methyl ester 1. Coupling through carbobenzoxy-DJ^r-phenylalanyl chloride Carbobenzoxy-DL-phenylalanine (4.7 g.) was suspended in 15 ml. of anhydrous ether and cooled to 0°. Phosphorous pentachloride (3.3 g.) was added and the mixture shaken until a l l but a trace of the latter had reacted. Light petroleum ether (200 ml.) was then added, whereupon the carbobenzoxy-IJL-phenylalanyl chloride crystallized. This was filtered off (yield: 4.0 g.) and added to an ether solution of L-leucine methyl ester (from 7.5 g. of the hydrochloride). The solution was kept at 0° for 1 nr., then left at room temperature overnight. At the end of the reaction the precipitated L-leucine methyl ester hydrochloride was filtered off and the filtrate washed with saturated sodium bicarbonate, N_ hydrochloric acid and water and dried over sodium sulfate. The dried ether solution was concentrated whereupon crystallization occurred* yield: 2.63 g. By fractional crystallization from ethyl ether and then from ethyl ether - petroleum ether (65 - 110°) two different compounds were separated; "Xa" which had: m.p. 123.2 - 124^  ( f > ^ D - 2 0 ' 8 (Sr1- ethanol)L Anal. Calc'd. for C 2 4H 5 00 5N 2: N, 6.57, Found: N, 6.15; and "Xb" which had: m.p. 108 - 108.5° [oC.] 1 7' 5 D - 21.64 (c-1, ethanol). Anal. Calc'd. for 024^0 N2- N, 6.57, Found: N, 6.17, 6.03. 2. Coupling through carbobenzoxy-DL-phenylalanylazide Carbobe nzoxy-DL-phenylalanyl hydrazide (2.18 g.) was dissolved in a mixture of 17 ml. of glacial acetic acid and 41 ml. of 2N hydrochloric acid and cooled to -3° in an ice-salt bath. The cooled solution was treated, with stirring, with a solution of sodium nitrate (0.59 g.) in 4 ml. of water. The azide, which separated immediately as a thick, stringy precipitate, was extracted into 40 ml. of ice cold ether. The ether solution was washed with water, sodium bicarbonate and again with water (all solutions ice cold) and dried for a short time (10 min.) with sodium sulfate. The dried solution of - 24 -carbobenzoxy-DIi-phenylalanyl azide was then treated with an "absolute ether solution of 0.98 g. of L-leucine methyl ester (prepared from 1.23 g. of the hydrochloride). After standing for 24 nr.-at 0°, the reaction mixture was washed with _N hydrochloric acid, sodium bicarbonate and water and dried over sodium sulfate. The solvent was removed under reduced pressure; yield (crystals): 1.82 g. By fractional crystalli-zation from ethyl ether and then from ethyl ether - petroleum ether (65 - 110°), the same two compounds were as before (p. 23) when the coupling was performed with carbobenzoxyl - PL - phenylanylchloride; "Za", m.p. 123.5 - 124° (oCf^D " 1 5 , 9 ('-~1> e t h a n o 1 ) - Anal. Calc'd. for C^H^O^: N, 6.57, Found: N, 6.25; "Xb", m.p. 101 - 105° [ ? Q 2 2 , 5 P " 2 3 , 5 e t h a n o 1 ) ' A n a 1 ' C a l c ' d - f°r  C 2 f y o ° 5 \ s N'  6 , 5 7 > Found: N, 5.84. XI. Carbobenzoxy^LrFhenylalanyl-L^Leucine Methyl Ester. a. Carbobenzoxy-L-phenyileal anine Ir-Phenylalanine (6.6 g.) was treated in the same manner as for the preparation of carbobenzoxy-PL-phenylalanine; yield: 5.3 g., [ p ^ ] 2 ^ * ^ + 5*53 (£-1.5 glacial acetic acid). The carbobenzoxy-L-phenylalanine was recrystallized from acetone-petroleum ether (65 - 110°); m.p. 128 - 130°. The constants previously reported for carbobenzoxy-L-phenylalanine (5) are j o ^ 2 " ^ +4.9), m.p. 126 - 128° • b. ^Phenylalanine ethyl ester hydrochloride jr-Phenylalanine (5.0 g.) was treated in the same manner as for the preparation of PL-phenylalanine ethyl ester hydrochloride (p.20); yield: 4.4 g., m.p. 152 - 153° j c ^ ) 2 ^ - 6 - 5 8 water). - 25 -c . Carbobenzoxy-Jc-phenylalanine e t h y l e s t e r . Lr-Phenylalanine e t h y l e s t e r h y d r o c h l o r i d e (3.0 g . ) was t r e a t e d i n the same manner as f o r the p r e p a r a t i o n of carbobenzoxy-DL-pheny la l an ine e t h y l e s t e r . The p roduc t , a f t e r s t and ing about a week at 5 ° c r y s t a l l i z e d i n t o an o i l y mass, and was t ransformed d i r e c t l y i n t o the h y d r a z i d e . d . Carbobenzoxy- j j rphenyla lanyl h y d r a z i d e . The carbobenzoxy-Jrrphenylalanine e t h y l e s t e r from the above p r e p a r a t i o n was d i s s o l v e d i n 15 m l . of abso lu te e thano l and warmed w i t h 1.0 g . of hydraz ine hydrate (38%) u n t i l complete s o l u t i o n r e s u l t e d . At the end of 48 h r . the r e a c t i o n mix ture was t r e a t e d w i t h water and the p r e c i p i t a t e f i l t e r e d ; y i e l d : 3.0 g . On r e c r y s t a l l i z a t i o n from e thano l - wa te r , the carbobenzoxyr-L-phenylalanyl hydraz ide had the f o l l o w i n g cons t an t s : m.p. 138 - 139° {ocQ25£) + 1 9 , 6 e t h a n o l ) . e. Ca rbobenzoxy-L-pheny la l any l - Jg l euc ine methy l e s t e r . 1. Coup l ing through carbobenzoxy-Jc-phenylalanyl  c h l o r i d e • Carbobenzoxy-L-phenyla lanine (2.58 g . ) was conver ted t o the c h l o r i d e i n the same manner d e s c r i b e d i n the p r e p a r a t i o n of c a r b o b e n z o x y - ^ p h e n y l a l a n y l - ^ l e u c i n e methyl e s t e r (p. 22). The c h l o r i d e r e q u i r e d about 1 h r . t o c r y s t a l l i z e ; y i e l d : 1.9 g . The s o l i d c h l o r i d e was t h e n added t o an_ e the r s o l u t i o n of L - l e u c i n e methyl e s t e r (from 2.15 g . of the h y d r o c h l o r i d e ) and l e f t at 0° f o r 0.5 h r . and at room temperature o v e r n i g h t . A f t e r washing and d r y i n g the r e a c t i o n mix tu re i n the u s u a l manner, the so lven t was removed under - 26 -reduced pressure to yield an o i l . This was dissolved in ethyl ether and * the product was crystallized by the addition of petroleum ether (65 - 110°); m.p. 107 - 108°, |o(] 2 5 D - 2 0-7 (c. 1 ethanol). Anal. Calc'd. for Cg^H^QO^^: N, 6.57. Found: N, 6.43. Upon admixture with a sample of carbobenzoxy-^phenyl-alanyl-Jc-leucine methyl ester, m.p. 108 - 108.5°, the melting point was 106 - 107.5°. -Upon admixture with a sample of o carbobenzoxy-^phenylalanyl-L-leucine methyl ester, m.p. 122.8 - 123.5 , o the melting point was depressed to 82 - 88 . 2. Coupling through carbobenzoxy-Jg-phenylalanyl  azide. Carbobenzoxy-j>phenylalanyl hydrazide (2.1 g.) was coupled to Jc-leucine methyl ester (from 2.0 g. of the hydrochloride) in the same manner as described for the preparation of carbobenzoxy-DL-phenylalanyl-L-leucine methyl ester (p. 23); yield: 1.26 g., m.p. 105 - 105.5° [bC] 2 5 - 21.7. Anal. Calc'd. for C0,H, 0 N : N, 6.57, D 24 30 5 2 Found; N, 6.42. Upon admixture with a sample of carbobenzoxy-?-phenyl-alanyl-L-leucine methyl ester, m.p. 101 - 105°, the melting point was 101 - 104°. Upon admixture with a sample of carbobenzoxy-?-phenylalanyl-Lrleucine methyl ester, m.p. 123.2 - 124°, the melting point was depressed to 88 - 91°. XII. Carbobenzoxy-J^Phenylalanyl-£L-Fhenylalanine Ethyl Ester. (38J Carbobenzoxy-DL-phenylalanyl hydrazide (2.4 g.) was dissolved in a mixture of 18 ml. of glacial acetic acid and 42 ml. of 2 _N hydrochloric acid and cooled to -3° in an ice-salt bath. The cooled solution was then treated, under stirring, with a solution of sodium n i t r i t e (0.54 g . ) i n 4 m l . of wa te r . The a z i d e , w h i c h appeared immediate ly as a t h i c k , s t r i n g y p r e c i p i t a t e , was d i s s o l v e d i n 40 m l . of i c e c o l d e ther and the e ther s o l u t i o n washed w i t h wate r , sodium b i c a r b o -nate and aga in w i t h water ( a l l s o l u t i o n s i c e c o l d ) and d r i e d f o r a shor t t ime (10 m i n . ) w i t h sodium s u l f a t e . The d r i e d s o l u t i o n of carbobenzoxy -DL -phenyla lanyl az ide was t h e n t r e a t e d w i t h an abso lu te e ther s o l u t i o n of 2.3 g . o f © ^ p h e n y l a l a n i n e e t h y l e s t e r (from*2.74 g . of the h y d r o c h l o r i d e ) . A f t e r s t and ing 24 h r . a t 0 ° , the r e a c t i o n mix tu re was washed w i t h N h y d r o c h l o r i c a c i d , sodium b i c a r b o n a t e , and water and d r i e d wi th - sod ium s u l f a t e . Upon c o n c e n t r a t i o n o f the d r i e d f i l t r a t e the re remained behind a p a l e y e l l o w g l a s s which c r y s t a l l i z e d on standing.; , y i e l d : 1.74 g . , m.p. 92 - 9 8 ° . A second experiment y i e l d e d 2.36 g . of carbobenzoxy -DL -phenyla lanyl -DL -phenyla lan ine e t h y l e s t e r as a whi te amorphous mass, vtfiich was s a p o n i f i e d w i t h .N sodium h y d r o x i d e , d i r e c t l y . An attempt t o syn the s i ze carbobe nzoxy -DL -phenyl -a lanyl -DJJ -phenylalanine e t h y l e s t e r by c o u p l i n g carbobenzoxy-DTr-p h e n y l a l a n y l c h l o r i d e t o D l fpheny la lan ine e t h y l e s t e r was u n s u c c e s s f u l . X I I I . Carbobenzoxy-pjf P h e n y l a l a n y l - D L - P h e n y l a l a n i n e . Carbobenzoxv -JL -phenyla lanvl -DL -phenvla lan ine e t h y l e s t e r (2.36 g . ) was d i s s o l v e d i n 30 m l . of e thano l and t r e a t e d w i t h -.15 m l . of N sodium hydroxide (3 m o l . ) a t room temperature f o r 12 h r . Upon a c i d i f i c a t i o n w i t h _N h y d r o c h l o r i c a c i d and removal o f the e t h y l a l c o h o l i n vacuo, an o i l remained, which was induced t o c r y s t a l l i z e from methanol - wate r . Re c r y s t a l l i z a t i o n from the same s o l v e n t s gave c r y s t a l s : y i e l d : 1.05 g . , m.p . 107 - 1 3 6 ° . On d r y i n g a t 100° and then at 118° i n vacuo, the m e l t i n g range became 132 - 1 3 6 ° . A n a l . - 28 -C a l c ' d . f o r CggHggOgN : N,. 6.28 Found: N , 6.10, 6.03. C 26 H 26°5 N2 H2° J N> 6 * ° 4 X I V . JJ f -Phenyla lanyl -J^L- Pheny la l an ine Carbobenzoxy-DL-pheny la lany l -^Lrpheny la lan ine (0.5 g . ) was d i s s o l v e d i n 80 m l . of methanol , 6 m l . of g l a c i a l a c e t i c a c i d and 5 m l . o f wa te r . P a l l a d i u m c a t a l y s t was added and hydrogen was i n t roduced i n a s low stream u n t i l no f u r t h e r e v o l u t i o n o f ca rbon-d iox ide occu r r ed , as determined by t e s t i n g the exhaust w i t h a s a t u r a t e d bar ium hydroxide s o l u t i o n . A t the end o f the r e a c t i o n (5 n r . ) , the c a t a l y s t was f i l t e r e d o f f and the s o l u t i o n concent ra ted t o d rynes s . The r e s i d u e was t aken up i n anhydrous methanol , and concent ra ted i n vacuo s e v e r a l t imes t o remove excess water and a c e t i c a c i d ; y i e l d : 0.31 g . R e c r y s t a l l i z a t i o n from hot methy l a l c o h o l gave two f r a c t i o n s . F r a c t i o n 1 was i n s o l u b l e i n hot methanol (91 mg.) and gave o n l y one spot i n a paper chromatogram w i t h R^ .82 ( n - B u t a n o l , A c e t i c a c i d , wa t e r : 4:1:5). A n a l . C a l c ' d . f o r O - J I ^ O N : N . 8 .97 . Found: N , 8 .48 . F r a c t i o n 2 -Lo d\J $ 2 was s o l u b l e i n hot methanol and gave two spots on a paper chromatogram w i t h Rp va lues .82 and . 86 , r e s p e c t i v e l y (same so lven t system as above) . A n a l . C a l c ' d . f o r C ^ g H ^ O ^ : N , 8 .97 , Found: N , 10.19. XV. Carbobenzoxy G l y c y l - . E L - P h e n y l a l a n i n e E t h y l E s t e r , a . Carbobenzoxy g l y c i n e (4) G l y c i n e (7.5 g . ) was d i s s o l v e d i n 25 m l . of 4 IT sodium hydroxide a t 0° and t r e a t e d a l t e r n a t e l y and i n p o r t i o n s over a p e r i o d of about 20 m i n . w i t h 17.0 g . of carbobenzoxy c h l o r i d e and an a d d i t i o n a l 25 m l . o f 4 J L sodium h y d r o x i d e . On a c i d i f i c a t i o n t o congo - 29 -r e d w i t h concent ra ted h y d r o c h l o r i c a c i d the compound c r y s t a l l i z e d ; y i e l d : 18.7 g . R e c r y s t a l l i z a t i o n from ch loroform gave m.p. 115 - 1 1 7 ° . b . Carbobenzoxy g l y c y l c h l o r i d e (4) Carbobenzoxy g l y c i n e (6.3 g . ) was suspended i n 35 m l . of anhydrous e ther and shaken w i t h 6.7 g . of f i n e l y powdered phosphorous p e n t a c h l o r i d e f o r 20 min . under i c e c o o l i n g . At the end o f the r e a c t i o n , the e the r was removed under reduced pressure and the r e s idue washed t w i c e w i t h c o l d pe t ro leum e the r (30 - 6 0 ° ) . Under extreme c o o l i n g (acetone and d ry i c e ) the c h l o r i d e c r y s t a l l i z e d ; y i e l d : 3 .6 g . A second s i m i l a r experiment r e s u l t e d i n an o i l which was d i s s o l v e d i n e the r and coupled t o DL-pheny la l an ine e t h y l - e s t e r . c . Carbobenzoxy g l y c y l - I i l r p h e n y l a l a n i n e e t h y l e s t e r ; Carbobenzoxy g l y c y l c h l o r i d e (syrup from above) • was d i s s o l v e d i n e ther and added t o an e the r s o l u t i o n o f 3 .4 g . o f DL-phenv la l an ine e t h y l e s t e r (from 3 .8 g . o f the h y d r o c h l o r i d e ) . The r e a c t i o n mix ture was kept at 0 ° f o r 0.5 h r . and a t room temperature o v e r n i g h t , washed w i t h N. h y d r o c h l o r i c a c i d , sodium b ica rbona te and water and d r i e d w i t h sodium s u l f a t e . A p r e c i p i t a t e which remained a f t e r the washing was f i l t e r e d o f f and r e c r y s t a l l i z e d from e t h y l ace ta te - pe t ro leum e the r ; m.p . 87.5 - 89. C r y s t a l l i z a t i o n occur red d u r i n g the d r y i n g so t h a t i t was necessary t o e x t r a c t the d r y i n g agent (sodium s u l f a t e ) w i t h hot e t h y l a c e t a t e . The e t h y l ace ta te e x t r a c t s 1 were combined and the so lven t removed under d i m i n i s h e d p r e s s u r e . The r e s u l t i n g r e s idue was r e c r y s t a l l i z e d from - e t h y l ace ta te - pe t ro leum e the r ; y i e l d : 1.45 g . , m.p. 88 - 8 9 . 5 ° . . A n a l . C a l c ' d . f o r - 30 -C21 H24°5 W2 ! N* 1 ' 2 3 ' F o u n d : N, 7.30, 7.21. ' " XVI. Carbobenzoxy glycvl-DT^phenylalanine. Carbobenzoxy glvcvl-D3>phenvlalanine ethyl ester (0.73 g.) was dissolved i n acetone and treated with 57 ml. of N sodium hydroxide at room temperature for 8 hr. On acidification to congo red with N hydrochloric acid and removal of the 'acetone under reduced pressure an o i l remained, which was extracted into ethyl acetate. The ethyl acetate solution was extracted with sodium bicarbonate, the bicarbonate layer acidified and re-extracted with ethyl acetate. After drying, the f i n a l ethyl acetate extract with sodium sulfate and concentrating i t to dryness, a white crystalline precipitate was obtained; y i e l d : 0.63 g. The product was recrystallized from ethyl acetate - ether - methanol; needles, m.p. 159 - 160°. . Anal. Calc'd. for C 2 0H 2 20 5N 2 : N, 7.86. Found: N, 7.92, 7.95. XVII. Carbobenzoxy-JDLrPhenylalanvl-DL-Valine Methyl Ester a) JL-Valihe methyl ester P i -Valine (10 g.) was suspended i n 125 ml. of anhydrous methanol. The suspension was saturated with dry hydrogen chloride gas and then heated under reflux for 3 hr. Repeated concentration, under reduced pressure with methanol, yielded a syrup which was treated twice more by-the- same procedure. The f i n a l syrupy product was taken up i n methanol and crystallization was effected by careful addition of ethyl ether; y i e l d : 8.1 g., m.p. 107 - 108°. Smith, Spackman and Polglase (33) report a melting point of 120— 122° for a preparation of this compound which gave correct nitrogen and - 31 -methoxyl analyses.' Fox and Minard (13) report a m e l t i n g p o i n t of 90 - 97° • f o r the same compound. b) Carboben2oxy-DL-phenylalanyl- gDL-valine methyl e s t e r . Carbobenzoxy-DL-phenylalanyl azide (from 2.65 g. of the hydrazide) was t r e a t e d w i t h an absolute ether s o l u t i o n of 1.98 g. of PL - v a l i n e methyl e s t e r (from 2.7 g. of the h y d r o c h l o r i d e ) . The r e a c t i o n mixture was kept at 0° overnight and 1.04 g. of carbobenzoxy-DL-phenyl-a l a n y l -DL-valine methyl e s t e r which had c r y s t a l l i z e d was f i l t e r e d o f f . The f i l t r a t e was washed and d r i e d i n the usual manner. Concentration of the f i l t r a t e y i e l d e d another 0.41 g. R e c r y s t a l l i z a t i o n from e t h y l ether -ethanol - petroleum ether (65 - 110) gave c r y s t a l s which were d r i e d at 100° i n vacuo, m.p. 132 - 133.5°. A n a l . Calc'd. f o r C,,H n n0J„ : N, 6.68. ' 25 28 5 2 ' Found: N, 6.56, 6.64. X V I I I . Carbobenzoxv-DL-Fhenylalanyl-DL-Valine. Carbobenzoxy-DLrphenylalanyl-DL-valine methyl e s t e r (1.22 g.) was d i s s o l v e d i n acetone and t r e a t e d w i t h 2.96 ml. of W-sodium hydroxide at room temperature f o r 40 min. On a c i d i f i c a t i o n of the mixture and evaporation of the acetone i n a stream of a i r , a c r y s t a l l i n e p r e c i p i t a t e was l e f t , which was taken up i n e t h y l acetate and e x t r a c t e d w i t h sodium bicarbonate. The sodium bicarbonate e x t r a c t was a c i d i f i e d and the r e s u l t i n g c r y s t a l l i n e p r e c i p i t a t e f i l t e r e d under s u c t i o n ; y i e l d : 0.1 g. R e c r y s t a l l i z a t i o n from methanol - water gave a m e l t i n g range of 142 - 149°. An a l . Calc'd. f o r C 2 2 H 2 g 0 5 N 2 : N, 7.04. Found: M, 7.41. XIX. Carbobenzoxy-L-c*-Glutamvl-DL-Alanine Methyl E s t e r . aa) Carbobenzoxy-J^-glutamic a c i d . ^ - g l u t a m i c a c i d (8.8g.) and magnesium oxide (7.4 g.) - 32 -were added to 100 ml. of water that had previously been covered with a layer of 30 ml. of ether. To this mixture there was added, over a period of 0.5 hr., under ice cooling and with shaking, 20.4 g. of carbobenzoxy chloride. The mixture was then shaken at room temperature until almost a l l of the magnesium oxide had gone into solution. The mixture was then acidified to congo red with concentrated hydrochloric acid and extracted four times with ethyl acetate. The ethyl acetate extract was washed with 1 N hydrochloric acid, filtered through a dry f i l t e r and concentrated to a syrup in vacuo. The syrup was dissolved in a l i t t l e ethyl acetate and crystallized by careful addition of petroleum-ether (30 - 60°); yield: 11.0 g., m.p. 114 - 116°. A second experiment gave 11.13 g., m.p. 113 - 116°. It was found in later experiments that a large excess of carbobenzoxy chloride was not necessary and that 1 mole of carbobenzoxy chloride to 1 mole of glutamic acid was satisfactory. b) Carbobenzoxy-Jr-glutamic acid anhydride (4) Carbobenzoxy-L-glutamic acid (10.0 g.) was warmed with 29 ml. of acetic anhydride' for 5 min. at 100°. The solution was then concentrated to dryness under reduced pressure. The resulting syrup was dissolved in a l i t t l e anhydrous chloroform and crystallized by the addition of petroleum-ether (30 - 60°) followed by cooling; yield: 5.5 g., o m.p. 90 - 93 . c) JDL-Alanine methyl ester hydrochloride DJj-Ala nine (10 g.) was treated in the same manner as for Ir-leucine methyl ester hydrochloride (p. 14); yield: 7.0 g. The product obtained upon recrystallization from methanol - ethyl acetate - 33 -o gave m.p. 155 - 156 . d) Carbobenzoxy -^K^g lu tamyl - J JL-a lan ine methy l e s t e r Carbobenzoxy-lr-glutamic anhydride (5 .0 g . ) was added, at room temperature , t o an anhydrous e ther s o l u t i o n o f^a l an ine methyl e s t e r (from 5.26 g . of the h y d r o c h l o r i d e ) . A f t e r 0.5 h r . , sodium b ica rbona te was added u n t i l the s o l u t i o n was s l i g h t l y a l k a l i n e . A f t e r 3 h r . s t i r r i n g and 2 days at room temperature , the sodium b ica rbona te l a y e r was a c i d i f i e d and the r e s u l t i n g o i l e x t r a c t e d i n t o e t h y l a c e t a t e . The e t h y l ace ta te e x t r a c t was d r i e d w i t h sodium s u l f a t e and concent ra ted t o dryness under reduced p r e s s u r e . Attempts t o c r y s t a l l i z e the compound were u n s u c c e s s f u l ' weight of d r i e d o i l : 5.10 g . X X . Carbobenzoxy-tLroC - G l u t amyl-JDIj-Alanine. C a r b o b e n z o x y - L - o l - g l u t a m y l - D L - a l a n i n e methy l e s t e r (3 .1 g . ) was t r e a t e d w i t h 17 .0 m l . (2 moles) of _N sodium hydrox ide f o r 40 m i n . a t room temperature . The acetone was t h e n evaporated i n a stream of a i r and the r e s u l t i n g o i l e x t r a c t e d i n t o e t h y l a ce t a t e . The e t h y l ace ta te l a y e r was e x t r a c t e d w i t h sodium b i c a r b o n a t e . The b ica rbona te s o l u t i o n was a c i d i f i e d and r e - e x t r a c t e d w i t h e t h y l a c e t a t e . The f i n a l e t h y l ace ta te e x t r a c t was d r i e d w i t h sodium s u l f a t e and the so lven t removed under reduced p r e s s u r e . Attempts t o c r y s t a l l i z e the compound were unsucces s fu l ; weight of s y r u p : 2 .18 g . X X I . Carbobenzoxv-DL-Phenyla lany l - JgTyros ine Me thy l E s t e r , a) L r T y r o s i n e methyl e s t e r h y d r o c h l o r i d e L - T y r o s i n e (5 g . ) was suspended i n 35 m l . of anhydrous * Should use a l a r g e r excess o f the e s t e r . - 34 -methanol and s a tu ra t ed w i t h d ry hydrogen c h l o r i d e gas. An a d d i t i o n a l 35 m l . of methanol was t h e n added and the s o l u t i o n r e f l u x e d f o r 2 h r . Repeated c o n c e n t r a t i o n under d i m i n i s h e d pressure gave needlesj y i e l d : 6.0 g . , m.p. 187 - 1 8 8 ° . A second s i m i l a r experiment y i e l d e d 4 .1 g . , m.p. 184 - 1 8 6 ° . b ) Jg-Tyrosine methyl e s t e r . • Tyros ine methyl e s t e r h y d r o c h l o r i d e (4 .1 g . ) was d i s s o l v e d i n water and t r e a t e d w i t h 2 .0 g . o f c a l c i u m carbonate . The s o l u t i o n was e x t r a c t e d s e v e r a l t imes w i t h e t h y l a c e t a t e . E v a p o r a t i o n of t he e t h y l ace ta te s o l u t i o n under reduced pressure y i e l d e d c r y s t a l s which were r e c r y s t a l l i z e d from e t h y l ace t a t e ; y i e l d : 0.5 g . , m.p. 132 - 133.5?-c) Carbobenzoxy-DIj-phenylalanyl-Is- tyrosine methyl e s t e r Carbobenzoxy-DL-phenyla lanyl hydraz ide (1.7 g . ) was conver ted t o the az ide (p. 23) and t r e a t e d w i t h an abso lu te e t h y l ace ta te s o l u t i o n of Ir-tyrosine methyl e s t e r (from 1.3 g . of the h y d r o c h l o r i d e ) . • A f t e r 24 h r . at 0 ° , the s o l u t i o n was washed w i t h _N h y d r o c h l o r i c a c i d , ; sodium b ica rbona te and water and d r i e d w i t h sodium s u l f a t e . C o n c e n t r a t i o n of the s o l u t i o n under d i m i n i s h e d pressure y i e l d e d a g l a s s which would not c r y s t a l l i z e ; w e i g h t : 1.96 g . X X I I . Carbobenzoxy-DJ>Phenylalanyl-Jg-Tyrosine. C a r b o b e n z o x y - J L - p h e n y l a l a n y l - L - t y r o s i ne methy l e s t e r (1.5 g . ) was d i s s o l v e d i n 20 m l . o f e thano l and t r e a t e d w i t h 9.45 m l . o f N sodium hydroxide (3 m o l . ) f o r 1 h r . at room temperature . E v a p o r a t i o n - 35 -of the e thano l i n a stream o f a i r y i e l d e d a syrup which was d i s s o l v e d i n methanol . On evapora t ion of the methanol a brown c r y s t a l l i n e p r e c i p i t a t e remained. X X I I I . Carbobenzoxy-Jf-J^ucvl-^Tr-Valine Methy l E s t e r . a) Carbobenzoxy-Ic- leucyl h y d r a z i d e . i - L e u c i n e methyl e s t e r h y d r o c h l o r i d e ( 5 g . ) was d i s s o l v e d i n 30 m l . of water and covered w i t h 55 m l . o f ch lo ro fo rm and coo led t o 0 ° i n an i c e - s a l t b a t h . Magnesium oxide (1 .8 g . ) and carboben-zoxy c h l o r i d e (7.2 g . ) were added a l t e r n a t e l y , and i n th ree p o r t i o n s , over 0.5 h r . P y r i d i n e was t h e n added t o decompose the excess carbobenzoxy c h l o r i d e and the mix tu re was a c i d i f i e d w i t h 5 N. h y d r o c h l o r i c a c i d . The ch loroform l a y e r was washed t w i c e w i t h wate r , sodium b ica rbona te and _N h y d r o c h l o r i c a c i d , d r i e d w i t h sodium s u l f a t e and evaporated under reduced p r e s s u r e . The r e s idue was d r i e d by repea ted c o n c e n t r a t i o n w i t h anhydrous methanol , d i s s o l v e d i n 30 m l . of abso lu te methanol and t r e a t e d w i t h 2 .0 g . of hydraz ine hydrate (98$) f o r 24 h r . at room temperature . The mix ture was t h e n r e f l u x e d f o r 1 h r . and concent ra ted s e v e r a l t imes w i t h anhydrous e the r . A whi te c r y s t a l l i n e mass r e s u l t e d v h i c h was o r e c r y s t a l l i z e d from e thano l - water ; y i e l d : 3 .0 g . , m.p. 117 - 119 . b ) Carbobenzoxy-J j - l eucy l -DL-va l ine me thy l e s t e r . Ckrbobenzoxy-L-- leucyl hydraz ide (2,25 g . ) was d i s s o l v e d i n a mix ture of 30 m l . of water , 8.0 m l . of g l a c i a l a c e t i c a c i d and 20 m l . o f 2 IJ h y d r o c h l o r i c a c i d , coo led t o - 3 ° and t r e a t e d w i t h sodium n i t r i t e (1 g . ) i n 5 m l . of wa te r . The r e s u l t i n g c a r b o b e n z o x y - L r l e u c y l az ide was e x t r a c t e d i n t o e ther ( p r e v i o u s l y coo led t o 0 ° ) , washed w i t h water , - 36 -sodium b ica rbona te and aga in w i t h water ( a l l s o l u t i o n s at 0 ° ) , and d r i e d w i t h sodium s u l f a t e . The az ide was t h e n t r e a t e d w i t h an abso lu te e ther s o l u t i o n o f D L - v a l i n e methyl e s t e r (from 1.35 g.- of the h y d r o c h l o r i d e ) . A f t e r 24 h r . a t 0 ° , the s o l u t i o n was washed and d r i e d i n the u s u a l manner, and concent ra ted i n vacuo. The r e s u l t i n g o i l c o u l d not be c r y s t a l l i z e d r e a d i l y ; we igh t : 1.05 g . B . S p e c i f i c i t y of P e p s i n . The seven carbobenzoxy d i p e p t i d e e s t e r s , one carbobenzoxy d i p e p t i d e amide, and numerous other pep t ide d e r i v a t i v e s , t ha t were syn thes i zed as d e s c r i b e d i n Pa r t A , above, were t e s t e d f o r t h e i r suscep-t i b i l i t y - t o p e p t i c h y d r o l y s i s . These compounds conta ined pep t ide bonds which Sanger and Tuppy (31) found t o be s u s c e p t i b l e t o p e p s i n i n the p h e n y l a l a n y l c h a i n of i n s u l i n . I n the experiments t o be d e s c r i b e d below, p r o t e o l y s i s was de tec ted by paper chromatography of the enzyme-substrate s o l u t i o n s . The amino a c i d ( s ) l i b e r a t e d by p e p s i n a c t i o n were de tec ted by means of the n i n h y d r i n spray reagent , and the i d e n t i t y of the l i b e r a t e d amino a c i d was e s t a b l i s h e d by a p a r a l l e l ehromatogram of the a u t h e n t i c amino a c i d . The main d i f f i c u l t y i n the enzymatic work was the i n s o l u b i l i t y of many of the subs t ra tes at the pH of the h y d r o l y s i s . T h i s d i f f i c u l t y was overcome, i n most cases , by d i s s o l v i n g the subs t r a t e f i r s t i n methanol and adding b u f f e r s o l u t i o n u n t i l the d e s i r e d pH, subs t ra te c o n c e n t r a t i o n and methanol-buffer r a t i o were reached . I n most cases the subs t r a t e remained d i s s o l v e d or as a f i n e suspens ion . A c o n t r o l experiment was - 37 -done with boyine -globulin as substrate. The hydrolysis of bovine "6 -globulin by pepsin in the presence of increasing concentration of methanol is given in Table VIII. TABLE VIII Pepsin hydrolysis of bovine^ -globulin at Increasing concentrations of methanol. Ratio of methanol - 0 .01N HCl 0 - 5 2 - 5 3 - 5 4 - 5 Enzyme action. ++++ +++ +++ + "ft -Globulin concentration, 2 per cent (1 .0 ml. used) Enzyme concentration, 2 mg. per ml., (0.5 ml. used) pH, 2 .0 (0 .011 HCl) Temperature 3 6 . 7 ° In the case of the carbobenzoxy dipeptide esters, the substrate was dissolved in 1.43 ml. of methanol and diluted to 5 ml. with 0 .01N HCl (pH 2 .0 ) or with 0 .1 M acetate buffer, pH 3.5 (pH 4 . 0 ) . It was found that when 3.57 mis. of 0 .1 M acetate buffer (pH 3 .5 ) was diluted to 5 mis. with methanol, the pH changed to 4 . 0 . In a l l the enzymatic experiments 75 -globulin was run as a control substrate. Substrate and enzyme blanks were also run and were negative in a l l cases. Table IX shows the results of the action of pepsin on a series of carbobenzoxy dipeptide esters and on one carbobenzoxy dipeptide amide. - 38 -TABLE IX Act i o n of pepsin on various synthetic substrates Hydrolysis Substrate pH 2.0 pH 4.0 1. Carbobenzoxy-DL-phenylalanyl-jDL-valine methyl ester t - -2. Carbobenzoxy-^L-phenylalanyl-L^-leucine methyl ester - -3. Carbobenz oxy-DL-phenylalanyl-L^ tyros ine methyl ester + i -4. Carbobenzoxy-DL-phenylalanyl -DJr-phenylalanyl e t h y l ester + -5. Carbobenzoxy-DL-alanyl-Lrleucine methyl ester - -6. Carbobenzoxy glycyl-DL-phenylalanine e t h y l ester - -i 7. Carbobenzoxy-DI^alanyl-Jr-leucinamide - . -8. Carbobenzoxy-I^leucyl-pL-valine methyl e s t e r * 9. Enzyme blank (pepsin only) - -10. - G l o b u l i n control + • Synthetic substrate concentration = 4 mM per ml. (1.0 ml. used) $ - G l o b u l i n concentration = 2 per cent (1.0 ml. used) Enzyme concentration = 2 mg. per ml. (0.5 ml. used) Temperature =36.7" Time of h y d r o l y s i s : pH 2.0, 10 hr.; pH 4.0, 18 hr. * As an o i l , therefore exact concentration not known. -J- Some substrate c r y s t a l l i z e d out during the reactions Another chromatogram was set up which contained the substrates carbobenzoxy-DLj-phenylalanyl-L-tyrosine methyl ester, carbobenzoxv-DL-phenylalanyl-DLr-phenylalanine e t h y l ester, plus pepsin at pH 2.0, as well as L-tyrosine, _]>tyrosine methyl ester hydrochloride, and DL-phenyl-alanine e t h y l ester hydrochloride. The r e s u l t s are shown drawn to scale i n diagram I. - 39 -DIAGRAM I ehromatogram of pepsin hydrolysis of synthetic substrates ^ Rf . 4 2 . ' % .85 A - Tyrosine B - Carbobenzoxy-DL-phenylalanyl-L-tyrosine methyl ester plus pepsin C - l£-Tyrosine methyl ester hydrochloride D - TJarbobenzoxy-DL-phenylalanyl-DL-phenylalanine ethyl ester plus pepsin E - DL-Phenylalanine ethyl ester hydrochloride Table X shows the r e s u l t s o f p e p s i n a c t i o n on a s e r i e s o f carbobenzoxy d i p e p t i d e s and on one d i p e p t i d e . TABLE X A c t i o n o f p e p s i n on v a r i o u s s y n t h e t i c subs t r a t e s Subs t ra te H y d r o l y s i s pH 2 .0 pH 4 .0 + 1. Carbobenzoxy-DL-phenyla lany l -^ I r -va l ine ' * 2 . Carbobe n z o x y - D L - p h e n y l a l a n y l - L - t y r o s i n e - -3 . Carbobenzoxy g lycy l -DL-phenyla lan ine 4 . Carb obe nzoxy-L-o(- g l u t amy 1 - D L - a l anine - -5 . C a r b o b e n z o x y-DIr - p h e n y l a l a n y l - D L - p h e n y l a l a n i n e - -6. P L - F h e n y l a l a n y l - D L - p h e n y l a l a n i n e -7 . - G l o b u l i n + S y n t h e t i c subs t ra te c o n c e n t r a t i o n - 4 mM per m l . (1 .0 m l . used) i5-Globul in c o n c e n t r a t i o n , 2 per cent (1.0 m l . used) Enzyme c o n c e n t r a t i o n - 2 mg. per m l . (0.5 m l . used) Temperature - 3 6 . 7 ° Time of h y d r o l y s i s - pH 2 . 0 , 24 h r . ; pH 4 . 0 , 40 h r . * As an o i l , t he re fo re exact c o n c e n t r a t i o n not known. + Some subs t r a t e c r y s t a l l i z e d out d u r i n g h y d r o l y s i s - 41 -DISCUSSION A. Chemical Fart. One of the objectives of this research was to determine i f peptides (or peptide derivatives) formed by reaction between an L-amino acid (or derivative) and a PL-ami no acid • . • (or derivative) can be separated readily to yield an _L.,Dj-peptide and an _L,Lr-peptide (or suitable derivatives thereof). If this type of resolution of optical isomers could be widely used, synthetic peptides could be obtained more readily, at reasonable cost. In one case, described below, the method was applied unsuccessfully while, in a second example, resolution was, apparently, achieved. A crystalline product (I) was obtai ned when carbobenzoxy-PL-alanyl.chloride was coupled with L-leucine methyl ester. The melting point of this product was 74 - 75°. Carbobenzoxy-D-alanyl-L-leucine methyl ester (II) had been reported previously (27) to have a melting point of 72 - 73°. When (I) and (II) were mixed, a melting point of 62 - 67° was observed. This depression of the melting point proves that the crystalline compound (I) obtained from the coupling with racemic carbobenzoxy^alanine was not carbobenzoxy-P-alanyl-L-leucine methyl ester. Carbobenzoxy-L-alanyl-L-leucine methyl ester (III) has been prepared previously but only as a syrup (27). Therefore, i t was not possible to compare (I) with (III) by the mixed melting point technique. - 4 2 -On the other hand, both carbobenzoxy-^-alanyl-L^-leucinamide (D7) and carbobenzoxy-_L-alanyl-Lj-leucinami de (V) have been characterized previously (27) . It was therefore decided that the amide of (I) would allow a decision to be made regarding i t s identity. The results are summarized: from ref. 27: m.p. [o(j B (ethanol) Carbobenzoxy-^ alanyl-J^-leucinamide (IV) 1 8 7 - 1 8 8 ° - 6 ° Carbobenzoxy-^L-alanyl-]>leuc inamide (V) 1 8 8 - 1 8 9 ° - 4 1 ° from this work: The amide obtained (VI) from product (I) from the coupling of carbobenzoxy-DL-alanyl chloride with ^ l e u c i n e methyl ester 1 6 3 - 1 6 4 ° - 2 7 . 9 o The rotation of an equimolar mixture of carbobenzoxy-^ al anvi-l s leucinamide and carbobenzoxy-j>al any 1-L-leuc inamide would be expected to be about - 2 4 ° . This suggests that the crystalline amide (VI) ( joCJp ~ 2 7 . 9 ° ) obtained i n this work was an equimolar mixture of carbobenzoxy-J^alanyl-i-leucinamide and carbobenzoxy-L-aianyl-L-leucinamide. which may be called carbobenzoxy-DL-alanyl-Ir-leucinamide. The original crystalline product from the coupling of carbobenzoxy-DL-alanyl chloride and L-leucine methyl ester i s therefore believed to be carbobenzoxy-DL-alanyl-L-leucine methyl ester. If these deductions are correct, i t indicates that diastereo-isomers of this type may readily crystallize together to give a ].:'..._ • f a i X T o W - and that separation of pure diastereoisomers (at this stage of - 43 -the s y n t h e s i s at l e a s t ) i n the pep t ide s e r i e s f r e q u e n t l y may be d i f f i c u l t . F u r t h e r work would have t o be c a r r i e d out t o determine i f s e p a r a t i o n o f d ias te reoisomers may be p r a c t i c a l at a l a t e r stage i n pep t ide s y n t h e s i s . When carbobenzoxy-DL-phenyla lanyl c h l o r i d e was coupled t o Ir - l euc ine methy l e s t e r , two c r y s t a l l i n e products were separated by f r a c t i o n c r y s t a l l i z a t i o n . One of t he products (VII) had a m e l t i n g p o i n t o f 123.2 - 124° w h i l e the o ther (VIII) had a m e l t i n g p o i n t o f 108 - 108.5°. Carbobenzoxy-_Ir-phenyl a l a n y l ? j > l e u c i n e methy l e s t e r (IX) was prepared (exper imenta l ) and found t o have a m e l t i n g p o i n t o f 107 - 108°. When (VII) and (IX) were mixed a m e l t i n g range of 82 - 88° was observed. When (VTII) and (IX) were mixed a m e l t i n g p o i n t of 106 - 107.5° was ob t a ined . When equa l p o r t i o n s o f (VII) and (VIII) were mixed a m e l t i n g range of 90 - 92° was observed. Mixed m e l t i n g p o i n t da ta suggest t h a t (VIII) i s p robab ly ca rbobenzoxy- j fpher ry la la iy 1-J>leucine methyl e s t e r . Carbobenzoxy-Dj-phenylr.alanyl-j j- leucine methyl e s t e r (X) was not prepared and no r e p o r t o f i t s s y n t h e s i s has been found i n the l i t e r a t u r e , t h e r e f o r e i t was not p o s s i b l e t o compare (X) w i t h e i t h e r (VTI) o r (VTII) by the mixed m e l t i n g p o i n t t e c h n i q u e . E s s e n t i a l l y the same compounds were ob ta ined when carb obe nzoxy-DL-phe n y l a l a n y l az ide was coupled t o L - l e u c i n e methyl e s t e r , a l though the cons tan ts were s l i g h t l y d i f f e r e n t . A l though the r o t a t i o n s of (VIII) and (IX) were q u i t e s i m i l a r (-21.6 and -20.7, r e s p e c t i v e l y ) , the r o t a t i o n s o f t he cor responding compounds prepared from c o u p l i n g ca rbobenzoxy-DL-phenyla lany l az ide w i t h L - l e u c i n e methy l e s t e r were q u i t e d i f f e r e n t (-15.9 and -23.5, r e s p e c t i v e l y ) . The r o t a t i o n of (VII) (-20.8) was a l s o the same as (VIII) and (IX). T h i s s i m i l a r i t y i n - 44 -r o t a t i o n s of (VI I ) and ( V I I I ) i s d i f f i c u l t t o understand i f (VI I ) i s assumed t o be e i t h e r c a r b o b e n z o x y - ^ - p h e n y l a l a n y l - ^ l e u c i n e methy l e s t e r or c a r b o b e n z o x y - D L - p h e n y l a l a r y l - L r l e u c i n e methyl e s t e r ( X I ) . The r e s u l t s are summarized i n Table X I . TABLE X I ' Compounds separa ted from the c o u p l i n g o f carbobenzoxy- pheny la l an ine w i t h J g l e u c i n e methyl e s t e r . m.p. (e thanol ) A B A B . Ca rbobenzoxy-? -pheny la l any l -L-l e u c i n e methyl e s t e r 123.2 - 124° 123.2 - 124° - 2 0 . 8 ° - 1 5 . 9 ° Carbobenzoxy-? -pheny la l any l -L-l e u c i n e methyl e s t e r 108 - 108.5° 101 - 105° ' - 2 1 . 6 4 ° - 2 3 . 5 ° C ' D C D Ca rbobenzoxy-L-phehy la l any l -L -l e u c i n e methyl e s t e r 107 - 108° 105 - 105 .5° - 2 0 . f - 2 1 . 7 " A - • Coup l ing performed w i t h ca rbobenzoxy-DL-phenyla lany l c h l o r i d e B - Coup l ing performed w i t h ca rbobenzoxy-DL-phenyla lany l a z i d e C - Coup l ing performed w i t h ca rbobenzoxy-L-pheny la l any l c h l o r i d e D - Coup l ing performed w i t h c a r b o b e n z o x y - ^ p h e n y l a l a n y l a z i d e . I n the syn thes i s of ca rbobenzoxy-DL-phenyla lany l-t lr - l euc ine methyl e s t e r , t h e r e f o r e , i t has been p o s s i b l e t o separate a t l e a s t one of d i a s t e reo i somers by f r a c t i o n a l c r y s t a l l i z a t i o n . I t was observed, upon paper chromatography of D L - p h e n y l a l a n y l -JOL-phenyla lanine , t ha t two spots appeared. S ince f o u r isomers are formed - 45 -from the coupling, J^phenylalany 1-Lrphenylalanine (XII), P^-phenylalanyl-I>r phe nyl alanine (XIII), L-phenylalanyl-It-phenylalanine (XIV) and L-phenylalanyl-Jc-phenylalanine (XV), i t is probable that one spot:'is composed of (XII) and (XIV) while the other spot is made up of (XIII) and (XV). B. Enzymatic Part. From the results of the enzymatic work i t can be seen that the specificity of pepsin is much higher in the hydrolysis of simple dipeptide: derivatives than i t is in the splitting of larger polypeptide or protein molecules. The splitting of carboberi20xy-DIr-phenylalaJiyl-D]>phenylalanine ethyl ester (XVI) and carbobenzoxy-DL-phenylalanyl-L-tyrosine methyl ester (XVII) by pepsin is unique in that no cases of peptic hydrolysis of -a carbobenzoxy dipeptide ester has been reported. The phenylalanyl-tyrosine bond has also not previously been reported as being split by pepsin. Baker (2) treated carbobenzoxy-L-phenylalany1-L-phenylalanine amide with pepsin at pH 2.0 for 6 days without getting any hydrolysis. Baker's substrate, however, was not in solution but was present as a fine suspension. The fact that both (XVI) and (XVII) contain two aromatic residues and are hydrolysed at pH 2.0 is in agreement with Baker's results. The resistance to pepsin of carbobenzoxy-DL-phenylalanyl-DL-phenylalanine i was unexpected since Baker (2) found that acetyl-DL-phenylalanyl-DL-phenyl-alanine1 was hydrolysed quite rapidly at pH 2.0. Greenstein and co-workers (12), however, have observed that in the treatment of certain N-carbo-benzoxy racemic amino acids (aspartic, asparagine, serine, glutamic, glutamine, alanine and arginine) by a crude aqueous extract of hog kidney, - 46 -the amide bond was e i t h e r r e s i s t a n t t o h y d r o l y s i s , or h y d r o l y s i s was much s lower than w i t h the cor responding N - a c e t y l racemic amino a c i d s . F o r example, the r a t e of h y d r o l y s i s of N-carbobenzoxy-DL-glutamis a c i d by the above enzyme p r e p a r a t i o n was 0 . 8 micromoles pe r n r . pe r mg. p r o t e i n n i t r o g e n , as compared t o 140 f o r the cor responding a c e t y l d e r i v a t i v e . I t appears , t h e r e f o r e , t ha t i n u s i n g s y n t h e t i c subs t r a t e f o r the d e t e r m i n a t i o n of p e p s i n s p e c i f i c i t y , the type o f N - a c y l b l o c k i n g group i s c r i t i c a l , . A more p r e c i s e d e l i n e a t i o n of enzyme s p e c i f i c i t y might be ob ta ined i f l o n g e r c h a i n pep t ides were used as s u b s t r a t e s . I n t h i s type of s u b s t r a t e , the b l o c k i n g group would be a pep t ide and the s t r u c t u r e of a p r o t e i n molecule would be more c l o s e l y approximated. I t i s known tha t a pep t ide bond between two amino a c i d s i n a p r o t e i n molecule i s weaker t h a n a bond i n v o l v i n g the same two amino a c i d s i n a s imple d i p e p t i d e . Th i s f a c t may e x p l a i n why c e r t a i n bonds tha t Sanger found t o be s p l i t i n the pheny la l an ine c h a i n o f t h e i n s u l i n molecule were not s p l i t when present as the carbobenzoxy d i p e p t i d e e s t e r . I t may a l s o be p o s s i b l e tha t the f o l d i n g of the p r o t e i n molecule may determine whether h y d r o l y s i s w i l l t ake p l a c e or not and i f t h i s i s the case , each p r o t e i n may have t o be cons ide red s e p a r a t e l y as f a r as p e p t i c h y d r o l y s i s i s concerned. I t t he re fo re appears t h a t , whereas p e p s i n has a low s p e c i f i c i t y toward the h y d r o l y s i s of p r o t e i n m o l e c u l e s , i t does have a f a i r l y h i g h degree of s p e c i f i c i t y towards s imple s y n t h e t i c s u b s t r a t e s . I n the l a t t e r , at l e a s t one of the c o n s t i t u e n t amino ac ids must be a romat i c . A comparison between the a c t i o n of p e p s i n on a l a r g e po lypep t i de and on s imple s y n t h e t i c subs t r a t e s i s g i v e n i n the f o l l o w i n g T a b l e . TABLE X I I A summary o f the bonds s p l i t by p e p s i n Pep t ide bond I n the i n s u l i n I n s y n t h e t i c Reference molecule (from subs t r a t e s Sanger)  Glutamy1-glutamyl V a l y l - c y s t e i c a c i d Tyro s y l - g l u t amy 1 G l u t a m y l - l e u c y l L e u c y l - g l u t a m y l G l u t a m y l - a s p a r t y l P h e n y l a l a n y l - v a l y l G l u t a m y 1 - h i s t i d i n y l L e u c y l - v a l y l G l u t a m y l - a l a n y l A l a n y l - l e u c y l L e u c y l - t y r o s y l T y r o s y l - l e u c y l G l y cy l -phe n y l a l a n y l P h e n y l a l a n y l - p h e n y l a l a n y l Phenyla lany 1 - t y r o s y l G l u t a m y l - t y r o s y l G l u t a m y l - p h e n y l a l a n y l ++ ++ : +++ bond not present Negat ive not t e s t e d nega t ive not t e s t e d Negat ive not t e s t e d it n negat ive p o s i t i v e 15 •5H5-15 - 48 -TABLE X I I ( c o n t . ) Pep t ide bond I n the i n s u l i n molecule (from Sanger)  I n s y n t h e t i c Reference subs t r a t e s G l u t a m y l - g l y c y l G l y c y l - t y r o s y l T y r o s y l - t y r o s y l C y s t e i n y l - t y r o s y 1 C y s t i h y l ^ t y r o s y l T y r o s y l - c y s t e i n y l T y r o s y l - c y s t i n y l M e t h i o n y l - t y r o s y l T y r o s y l - p h e n y l a l a n y l bond not present tt tt tt tt tt tt it tt P o s i t i v e S p l i t weakly P o s i t i v e 15 tt 15 , 2 18 8 2 +++ i n d i c a t e s major s i t e o f a c t i o n by p e p s i n ++ i n d i c a t e s weak a c t i o n by p e p s i n * Th i s bond was s p l i t s t r o n g l y by p e p s i n i n t he g l y c y l c h a i n o f i n s u l i n but much more weakly i n the p h e n y l a l a n y l c h a i n . #*- Table IX of t h i s t h e s i s . - 49 -BIBLIOGRAPHY 1. Anfinson, C.B., J. Biol. Chem., 196, 201 (1952). 2. Baker, L.E., Ibid, 195, 809. (1951). 3. Behrens, O.K., Doherty, D.G., and Bergmann, M., Ibid, 136, 61 (1940). 4. Bergmann, M., and Zervas, L., Ber., 65, 1192 (1932). 5. Bergmann, M., Zervas, L.. Rinke, H., and Schleich, H., Z. physiol. Chem., 224, 33 (1934). 6. Calvery, H.O., and Schock, E.D., J. Biol. Chem., 112, 171 (1935). 7. Cook, A.H., Cox, S.F., and Farmer, T.H., Nature, 162, 61 (1948). 8. Dekker, C.A., Taylor, S.P. Jr., and Fruton, J.S., J. Biol. Chem., 180, 155 (1949). 9. Desnuelle, P., Rovery, M., and Bonjour, G., Biochim. Biophys. Acta., 5j 116 (1950). 10. De Witt, H.D..,. and Ingersoll, A.W., J. Amer. Chem, Soc, T5j 3359 (1951). 11. Doherty, D.G., and Popenoe, A.E. Jr., J. Biol. Chem,, 189, 447 (1951). 12. Fodor, P.J., Price, V.E., and Greenstein, J.P., Ibid 178. 503 (1949). 13. Fox, S.W., and Minardr F.N., J. Amer. Chem, Soc, 74, 2085 (1952). 14. Fruton, J.S., and Bergmann, M., J. Biol. Chem., 117, 189 (1937). 15. Fruton, J.S., and Bergmann, Ii,, Ibid, 127, 627 (1939). 16. Greenstein, J.P., Gilbert, J.B., and Fodor, P.J., Ibid, 182., 451 (1950). 17. Hammer, D., and Greenstein, J.P., Ibid, 195, 81 (1951). 18. Harrington;, C.R., and Pitt-Rivers, R.V., Biochem. J., 38, 417 (1944). 19. Hofmann, K., and Bergmann, H., J. Biol. Chem., 130, 81 (1939). 20. Hunt, M., and du Vigneaud, V., Ibid,12£, 699 (1938). 21. Ingersol, A.W., J. Amer;-Ghem. Soc., 47, 1169 (1925). - 50 -22. Levintow, L., Price, V.E, and Greenstein, J.P., J. Biol. Chem., 184, 55 (1950). 23. Neuberger, A., Adv. in Protein Chem., £, 297 (1948). 24. Northrup, J.H., J. Gen. Physiol., 13, 739 (1930). 25. Northrup, J.H., Kunitz, M., and Herriot, R.M., Crystalline Enzymes, Columbia University Press, New Tork, N.Y. (1948). 26. Northrup, J.H., J. Gen. Physiol., 13, 739 (1930). 27. Polglase, W.J., and Smith, E.L., J. Amer. Chem. Soc, 71, 3081 (1949). 28. Price, V.E., Gilbert J.P., and Greenstein, J.P., J. Biol. Chem., 179, 1169 (1949). 29. Pyman, F.L., J. Chem. Soc, 99, 1386 (1911). 30. Radke, F.H., Fearing, R.B., and Fox, S.W., J. Amer. Chem. Soc, 76, 2801 (1954). 31. Sanger, F., and Tuppy, H., Biochem. J., 49, 481 (1951). 32. Sanger, F., and Thompson, E.O.P., Ibid, 53, 366 (1951). 33. Smith, E.L., Sparkman, D.H., and Polglase, W.J., J. Biol. Chem., 199, 801 (1952). 34. Smith, L.I., Organic Syntheses, 23, 13 (1943). 35. Stare, F.J., Nutritional Reviews, 9, 55 (1951). 36. Stevens, CM., Halpern, P.E., and Gigger, R.P.J., J. Biol. Chem., 190, 705 (1951). 37. Triem; G., Ber., 71, 1522 (1938). 38. Wessely, F., Schlogl, K., and Korger, G., Monatshefte fur Chemie, 82, 4 (1951). 

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