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Vol. 85
COMMUNICATIONS TO THE EDITOR
cine,? cis- and trans-3-hydroxyproline, serine, allothreonine, and threonine. Basic hydrolysis produces tryptophan and P - m e t h y l t r y p t ~ p h a n . ~Quantitative amino acid determinations by both the Moore-Stein technique and mass spectrometry show that only one of each amino acid is present. None of the amino acids was attacked by D-amino acid oxidase. Potentiometric titration indicated that telomycin has one free carboxyl function and one free amino group; molecular weight determinations averaged 1300. The molecular weight of Telomycin is calculated a t 1273.3 on the basis of the eleven amino acids linked by ten peptide bonds and one lactone, The presence of a lactone is confirmed by the disappearance of the characteristic ester-type infrared band a t 1745 cm.-', accompanied b y a change in optical ~ to [ c u ] ~ * D-32'; ( c 1, rotation [from [ a I z 8 -133" 2 : l methanol-water)] on treatment with 0.32 N barium hydroxide a t ambient temperature. Potentiometric titration of the newly formed "telomycic acid" disclosed an additional carboxylic acid function (pKa 3.6). Chromic acid oxidation5 showed t h a t threonine is protected in Telomycin but not in telomycic acid, thus establishing threonine as the 0-terminus of the lactone. As expected, Telomycin travels to the cathode while telomycic acid migrates to the anode on electrophoresis at pH 6.4. Although attempts to degrade Telomycin or telomycic acid to peptides by means of acidic or enzymatic partial hydrolysis were unpromising, partial basic hydrolysis was successful. Prolonged treatment with aqueous sodium hydroxide, followed by ion-exchange neutralization, gave (in addition to traces of amino acids) five water-soluble peptides and one water-insoluble hexapeptide. The water-soluble peptides were separated b y electrophoresis, yielding a pentapeptide (Asp, Ser, T h r , ulio-Thr, Ala); a tetrapeptide (Asp, Ser, Threo, u l b T h r ) ; two tripeptides (Asp, Ser, Thr) and (Thr, alloT h r , Ala); and a dipeptide (-4sp, Ser). T h e pentapeptide had aspartic acid as the N-terminal amino acid, as did Telomycin itself (Sanger D N F B method). Hydrazinolysis (Akabori) of the pentapeptide revealed alanine as C-terminal. These observations, together with pK studies, establish the N-terminal sequence of teloniycin as P-.~sp-Ser-Thr-ulio-Thr-.~la. T h e purified (thin-layer chromatography) waterinsoluble hexapeptide contains both 3-hydroxyprolines, glycine, tryptophan, P-methyltryptophan, and P-hydroxyleucine; the N-terminal amino acid is glycine (Sanger D N F B ) . Since the C-terminal amino acid of telornycic acid is cis-C-hydroxyproline (Xkabori), this must also be the position occupied in the hexapeptide. The Edrnan technique confirmed glycine as N-terminal and revealed /runs-3-hydroxyproline as the next amino acid in the hexapeptide. Partial hydrolysis (acidic) of the hexapeptide gave a tripeptide containing both tryptophans and P-hydroxyleucine (acidic and basic total hydrolyses). Edman degradation on this ( 2 ) J C . S h e e h a n , K . h l a e d a , A K S e n , a n d J. A . S t o c k , J . A m . Chenr 3 ) T h e i y a i i c c o n f i g u r a t i o n h a s been assigned t o t h e "slow-moving" 3 - h y d r < , x y p r o l i n e on t h e basis of t h e classically rignrous conversion nf i t s prec u r s o r . ,(-meth~,xy-I.-prtrline.t o I - m e t h o x y s u c c i n a m i d e . J C. S h e e h a n a n d J G W h i t n e y . tbid , in press. ( 4 j B y electrophiiresis a n d p a p e r c h r o m a t o g r a p h y t h e B - m e t h y l t r y p t o p h a n f r o m T e l o m y c i n h a s been s h o w n t o correspond t o s y n t h e t i c ".4" r a c e m a t e k i n d l y provided b y Professor H. R S n y d e r of t h e U n i v e r s i t y of Illinois C,f H . R S n y d e r a n d 1) S h l a t t e s o n . J . A m C h e m . S o r . . 1 9 , 2217 (1957). T h e J - m e t h y l t r y p t o p h a n s t r u c t u r e wac suggected b y Professor K l a u s Biem a n n :IfI T ) nn t h e basis o f mas? s p e c t r o m e t r i c m e a w r e m e n t s o n t h e isolated a m i n o acid t u r n i s h e d b y n u r l a b o r a t o r y . C f . K . B i e m a n n , " h l a s s S p e c t r o m e t r y . SIcC,ram-Hill Book C,o I n c , S e w Y o r k . N Y , 1902. 11 2 7 5 . ( 5 ) J C. S h e e h a n , H. G Z a c h a u , a n d W R . L a w w n . . I . A m Cheni S o r . , 8 0 , :3:3-19 (1'258).
tripeptide showed P-hydroxyleucine to be N-terminal and hydrazinolysis produced only tryptophan. Alkaline hydrolysis under nitrogen of either the hexapeptide or Telomycin gave indole-$aldehyde and a yellow crystalline product, m.p. 280-2X3' dec. Anal. Calcd. for C16H1302?j:3: C, 68.80; H , 4.69; N, 13.05; mol. wt., 279.29. Found: C , GX.71; H , 4 . S l ; N,15.02; mol. wt., 279.1 (mass number). The ultraviolet spectrum shows a band a t 380 mp (6 l G , O O O ) . Hydrogenation over rhodium-charcoal followed by acid hydrolysis gave proline and hydrotryptophan. These data are in good accord with the formulation of the yellow, crystalline product I.
H
I
The ultraviolet spectra of Telomycin, telomycic acid, and the hexapeptide all have, in addition to tryptophantype absorption, a band a t 339 mp ( e 22,000) This ultraviolet chromophore can be accounted for by a dehydrotryptophan system For example, methyl /3-(3-indolyl)-cr-benzamidoacrylate[prepared by the Erlenmeyer condensation of D-indolealdehyde and hippuric acid followed b y methanol] has an absorption band a t 341 mp ( e 21, 000) All of the foregoing observations are compatible with the representation of Telomycin as structure I I. H02CCHCH~CO-Ser-Thr-u~~~-Thr-.~la-Gly-lru~s-3-HOPro
I
KH*
I
I
0 I
C-czs-3-HOPro-~-Try-8-Me-Tr\d-HOLeu I1
0 I1
The formation of tryptophan from the dehydrotryptophan system on alkaline hydrolysis is surprising, and this type of reaction is being investigated further. Acknowledgment.-This work was aided by a Grant from the Public Health Service, National Institutes of Health. i r e are also indebted to Bristol Laboratories, Syrazuse, New York, for our supply of Telomycin, and to Prof. George Biichi for helpful discussions concerning the ultraviolet chromophore. DEPARTMEST O F CHEMISTRY INSTITUTE OF MASSACHUSETTS TECHSOLOGE' 39, MASSACHUSETTS CAMBRIDGE
J O H N C. SHEEHAN PAULE . DRUMMOXD J O H NN.GARDNER RESJI MAEDA D. MASIA SHOSHIRO SAKAMURA .A. K . SES J O H N .A. STiIC'K RECEIVED AUGUST5 , 1963
The Separation of Ketimine Isomers Sir: Earlier claims of the separation of the geometric isomers of aldimines and ketiminesl have been tlisputed.2 We have now succeeded in preparing and separating both forms of a number of ketimines derived from 2-amino-5-chlorobenzophenone. These compounds are typified by the morpholinoethylimines, obtained by heating 2-amino-3-chloro(1) 0 Anselminc B r v . . 40, 8 4 6 5 (14071. W h l a n c h o t a n d J R F:url
