Laboratory. The figures listed have been corrected for acid is added to PH 4.6 to the aqueous solution, moisture and ash, the latter amounting to only 0.1%. Ni- ainorphous protein, only slightly soluble in water, trogen was. determined by the micro-Kjeldahl method, direct oxygen analyses were made by the Irnterzaurher is precipitated. It may be that the crystalline method, and sulfur was determined after Carius digestion. protei11 is actually some type of proteinsalt coniOnly trace amounts (0.02%) of phosphorus were found. hiiiation or simply an ammonium salt and that the No carbohydrate could be detected by thc orcitiol n ~ e t h o d . ’ ~ crystalline protein isolated by Kekwick14 was likeTABLE 1
70
PjLEMENTART c:O.?ZIWSh‘IOS (Jii WLACTALBUYIN,
s
I1
15.S6 32 . 32 7.01
S
51.X 1.91
c
o
9!1. nn
Tot a1
Tryptophan AnaIysis.---’l‘ry~)tc,l,ltaii \vas tictcriiiitietI 1)y the Spies and Chambers neth hod'^ 0 1 1 the protein in solitl form. a-Lactdbumin was found t o contain approximately 7% tryptophan. There is some uncertainty about the exact value because the minirnuin trarisiriittancy of the color produced by this protein was a t 620 ms. Readings at 620 m p gave a value of 7.2% tryptophan while a t 590 mp, a value of 6.7% was obtained. S$rensen and S$rensens reported that “crystalline insoluble substance” contained 0.3710.633 mg. tryptophan per mg. protein N (5.9-10.0% tryptophan, calculated on the basis of our determined nitrogen content of 15.86%).
Discussion The method for preparing a-lactalbumin described above, which was worked out from the observations of Sgrensen and S@rensen,has consistently yielded the same crystalline protein. It is of some importance, therefore, to attempt to correlate our findings with some of the earlier studies on the lactalbumin fraction of whey as has already been done with the ultracentrifuge data. We have been able to prepare a-lactalbumin in crystalline form only in the presence of ammonium sulfate a t pH 6.6. I t has been mentioned that the crystals are easily soluble in water and that when (12) M. Serensen and G. IIaugaard, Contpl. rcnc. I r a v . lab. Corlsbarn, Ser. chim., 19, 1 (1933). (13) 5. R . Spies and D. C. Chambers, Anal. Chem , 21, 1219 (1949).
[ C O Y I’RIF3IJlION FROhf TIIB
wise a water-soluble complex and hence called an albumin. The crystalline albumin prepared by Sjiigrcn and SvedbergI5 by the method of Wich111a1113~~ (salting out with ammonium sulfate in the of dilute acid) was also soluble in water. ~~resence However, the protein was not homogeneous and speculation concerning its solubility is unwarranted. In regard to the~electrophoreticcharacterization of a-lactalbumin, it is interesting to compare the o1)served mobility of -4.2 with the data of Smith.I7 In his investigation of the proteins of whey by the electrophoretic inetliod, Smith found that one of the components comprising 12% of the total proteins. had a mobility of -4.5 (protein concentration = 1.23%, veronal buffer PH 8.6, ionic strength = 0.1). It seems reasonable to suggest that this component may now be identified as a-lactalbumin. If the present work has been correlated correctly with Pedersen’s ultracentrifuge data and Smith’s electrophoretic experiments, it may be concluded that a-lactalbumin is a major component of whey which can be prepared in crystalline form without difficulty. Further studies on its properties and amino acid composition are in progress. Acknowledgments.--We wish to thank T. L. 11cMeekin for much helpful advice and for the original suggestion that “crystalline insoluble substance” and a-lactalbumin might be identical. We are also indebted to J. H. Custer for the electrophoretic experiments. (11) R. A. Kckwick, iinpublished; see K . 0. Pederseo, Biochcm. J.. 30, 0 18 (1936). (15) B. Sjiigren and T. Svedberg, THISJ O U R N A L , 62, 3650 (1930). (16) A. Wiehmann, 2. physiol. Chcm., 87, 575 (1899). (17) E. L. Smith, J . B i d . Chcm., 166, 665 (1946).
PIUIADELPHIA 18, PENNA.
1)EI’ARI‘MENT OF ORGANIC
CHEMISTRY, T H E F I E B R E W
UNIVERSITY]
Syntheses of DL-Aspartic Acid and DL-Asparagine Vza Their N-Benzyl Derivatives I ( Y AlAX ITRANKEL,
Y . LIWSCIIITZ AND Y . RMIEL
RECEIVED JULY 14, 1952 New syntheses for the preparation of dl-aspartic acid and dl-asparagine through their N-benzyl derivatives have been worked out. a-Benzylamino-N-benzylsuccinamicacid has been prepared much more efficiently than reported previously. A course for the reaction between maleic anhydride and benzylamine is proposed, differing from that postulated by Reppe and Ufer.2
In the course of work on syntheses of poly-aamino acids we were in need of a-amino-N-benzylsuccinamic acid which had been prepared by LIcMillan and Albertson by hydrogenolysis of a-benzylamino-N-benzylsuccinamic acid (11). Their synthesis of this latter compound is based on a procedure developed by Reppe and Ufer2 and consists in the reaction of one mole of maleic (1) F. E1 hIchfllidn a n d N. E’. Albertson, THISJ O U R W A L , 7 0 , 3778 (1948). (2) W. Reppe dod H. Ufer, U. S Patent 2,200,220 ( M a y 7, 1940).
anhydride with two moles of benzylamine in water a t reflux temperature for 16 hours. On repeating the preparation of I1 we invariably found that the yield was only 18--25y0 as against 48y0 obtained by the above authors. On the other hand, from the reaction mixture another compound which proved to be the benzylamine salt of N-benzyldl-aspartic acid (111) was isolated in a yield of about 70%. I11 could be obtained quantitatively by a modified procedure with a reaction time of only 1.5 hours, by first allowing one mole of maleic
Jan. 20, 1953
SYNTHESIS OF DL-ASPARTIC ACIDAND DL-ASPARAGINE
33 1
anhydride to be completely hydrolyzed and then Experimental adding two moles of benzylamine. Microanalyses are by Drs. Weiler and Strauss. Melting I11 yielded almost quantitatively N-benzyl-dl- points were d e t e d n e d in a Fisher- Johns apparatus. a-Benzylamino-N-benzlsuccin~cAcid (11). (A) From aspartic acid (IV) on treatment with sodium hydroxide, extraction with ether of the liberated Maleic Anhydride and Benzylamine.-9.6 grams of maleic was dissolved in 40 ml. of dry dioxane and 21.4 g. benzylamine and acidification with hydrochloric anhydride of benzylamine added portionwise, the reaction mixture acid. Catalytic hydrogenolysis of IV resulted in a becoming very hot. It was boiled under reflux for about 30 minutes, during which time a white voluminous precipitate practically quantitative yield of dl-aspartic acid. We found that a-benzylamino-N-benzylsuccin- settled in the flask. After cooling, the substance was filtered and washed several times with water and acetone. amic acid (11) can be prepared in over 70% yield The yield was 24 g. (72%) of a material (melting at 215' on by carrying out the reaction between maleic recrystallization from ethanol) whose m.p. was not deanhydride and benzylamine in dioxane (thereby pressed when mixed with the substance obtained by the precluding hydrolysis to maleic acid) ; the reaction procedure of McMillan and Albertson.' Anal. Calcd. for C I ( H ~ ~ O * N N,~ 9.0. : Found: N, 8.7. time was reduced to about '/2 hour as against 16 (B) From Benzylmaleamic Acid and Benzylamine.-Two hours reported by McMillan and Albertson. grams of benzylmaleamic acid was dissolved in 15 ml. of We also proved that the reaction between maleic dry dioxane (by warming) and 1 g. of benzylamine added. anhydride and benzylamine proceeds in two stages: The mixture was boiled under reflux and after a few minutes the first consisting in opening of the anhydride and a white crystalline precipitate settled. Reflux was confor five more minutes and after cooling the substance formation of benzylmaleamic acid3 (or its benzyl- tinued was filtered and washed with water and acetone. It was amine salt (I) when using two moles of benzylam- identical with that obtained by the above procedure. ine) a t low temperature, and the second stage in an Benzylmaleamic Acid.-Four grams of maleic anhydride addition to the double bond of a second mole of was dissolved in 100 ml. of dry ether and cooled in an icebath. Eight grams of benzylamine was now added dropbenzylamine a t elevated temperature leading to wise with vigorous stirring. A white crystalline mass compound 11. This reaction course does not con- settled immediately. I t was filtered off, washed with ether form to that proposed by Reppe and Ufer2 accord- and dried. It weighed 11.7 g. (97.5%) and represented the ing to whom imides are formed in the reaction be- benzylamine salt of benzylmaleamic acid (I), m.p. 94'. Anal. Calcd. for ClaH2aOlN*: N, 9.0. Found: N, 8.7. tween amines and maleic anhydride. Eight grams of I was dissolved in a dilute sodium hydl-Asparagine was synthesised by treating maledroxide solution and on acidification with hydrochloric acid amic acid (V) with an amount of benzylamine a white crystalline precipitate was obtained which weighed slightly greater than equimolar a t reflux tempera- 4.7 g. (90%) and melted a t 138'. ture in dioxane. The salt (VI) which resulted was Anal. Calcd. for CIIHllOaN: C, 64.4; H , 5.4; N, 6.8. isolated and exposed to a higher temperature by Found: C, 64.4; H, 5.6; N, 6.7. Benzylamine Salt of N-Benzyldl-aspartic Akd (III).reflux in xylene, yielding N-benzyl-dl-asparagine Forty-nine grams of maleic anhydride was boiled under re(VII) from which dl-asparagine was obtained by flux with 150 ml. of water for l/z hour. The reaction mixcatalytic hydrogenolysi~.~ ture was cooled and 107 g. of benzylamine cautiously added
-
while cooling by immersing the flask in cold water. Heating under reflux was then continued for one more hour. After CH-CONHR cooling, acetone has added until the substance was comII II pletely precipitated. The yield was quantitative, m.p. CH-COOH C H C O O H C ~ H ~ C H L N H180' ~ (raised t o 184' on recrystallization from ethanol). V ( R = H) I ( R = Benzyl) Anal. Calcd. for C18H~20dN2: C, 65.5; H , 6.7; N, 8.4. VI(R = H) Found: C, 65.5; H,6.4; N, 8.5. N-Benzyldl-aspartic Acid (IV).-Fifty grams of 111 was dissolved in 60 mi. of a 15% sodium hydroxide solution. The benzylamine was extracted several times with ether RNIly (and subsequently recovered), and the remaining alkaline CHz-CONHR solution was acidified with hydrochloric acid until acid t o CH-CO I congo paper. On cooling and scratching the wall of the CH-COOH vessel, white crystals separated. Thirty-two grams of a I substance melting a t 194' (dec.) was obtained (95%). CH-co Ic'HCHzCeHb Anal. Calcd. for C11Hl8O4N: N, 6.3. Found: N, 6.1. I1 ( R = Benzyl) dCAspartic Acid.-Five grams of IV was suspended ' in VI1 ( R = H ) 110 ml. of glacial acetic acid and 0.3 g. of PdClZ on carbon CHz-COOII catalyst (containing 30% PdC12) added. The reduction was carried out in a Parr low pressure hydrogenation apCH-COOH I 2 CeHsCII2NHz +CHCOOHC~HLCHZNH~paratus (initial pressure 50 p.s.i.) for three hours a t about II 60'. The cold reaction mixture was filtered; the portion CH-COOH I1 NHCH2CeHb of the material which adhered to the catalyst was extracted with cold formic acid. On separate evaporation of both solI11 vents dl-aspartic acid was obtained almost quantitatively. NaOH Anal. Calcd. for C,H,04N: N, 10.5. Found: N, 10.6. HCl Maleamic Acid (V) .-Three grams of maleic anhydride was dissolved in 25 ml. of dry dioxane and. gaseous amCHz-COOkI CHzCOOH monia passed through under stirring. A white crystalline I Hz I precipitate formed immediately. The reaction was conCH-COOH + CH-COOH tinued until the theoretical amount of ammonia was taken I I up. The substance was filtered, washed with dioxane and NHCHzCsHr NHL dried. It melted at 142'. IV Benzylamine Salt of Maleamic Acid (VI).-Three grams of V was sumended in 50 ml. of dioxane and refluxed with (3) G. Piutti, Goss. chim. i l d , 26, I, 438 (1896). (4) Direct treatment of V with benzylamine a t a temperature above 3 g. of benzylamine. Most of the material dissolved after 138' resulted in the decomposition of this substance, whereas a t lower a few minutes, but later on a mass of white crystals of V I temperatures addition to the double bond could not be effected. precipitated. Reflux was continued for 35 minutes alto-
+
TABLE I CsHsCHzNHz CH-CONHR
+
I >.
+
1
332 gether and the subst,iiice filtered after coolitig. I t I\ eighetl .i 5 g., aiid still gdvc redctioris for double bond%.. I t melted a t 222O. .4nal. Cdkd. lor CI,~II+UJI: N , 12.6. Found: S , 12.5. N-Benzyldksparagine (VI&).-Five grams of V I was heated under reflux in 50 ml. of xylene for orle hour. The resulting material did no longer show reactions for double bonds. It melted at 216' on recrystallization from water. The over-all yield from V was about 70%. .Id. Calcd. for C11HldOaS\Tz: C , 5Q.5; H , 6.3; S , 13.6. Found: C, 59.6; H, 6.4; N, 12.0.
[CONTRIBUTIOK FROM THE &PA&TMENT
dE-Asparagine.-Four grains of V I 1 was dissolved in a iiiisturc of 50 nil. of glacial acctic acid aiid 20 rnl. of water a n d 0.3 g. of I'dCl:! on carbon catalyst (30yo)addcd. The reductio11 was carried out siniilarly to that of N-benzyl-dlzis;lt.artic acid (see above). After about three hours the reduction was complete and on filtration and evaporation of the solvent the practically theoretical amount of dl-asparagine was recovered. A 4 m l . Calcd. for C4HbOaSt 4- € 1 2 0 : N, 18.7. Found:
S ,18.4. JERUSALEM,
ISRAEL
OF CHEMISTRY OF TNh r&IVERSITY OF C.4LIFORNIA AT L O S AhGELES]
Studies in Stereochemistry. XVI. Ionic Intermediates in the Decomposition of Certain Alkyl Chlorosulfite s BY DONALD J. ORAX 21, 1!152
R E C E I V E U hfA1'
The reactiori of thianyl chloride with the atereolsoniers of 3-pheiiyl-~-butdiiol, %-phenyl-a-peiitaiiol diid 3-phenyl2 pentanol has been studigd Decomposition of the initially fornied chlorosulfite derivdtiyes of these secondary alcohols in thioayl chbride, dioxang or formic acid produced phenylalkyl chloride products which were subsequently reduced t o the carrespending akylhenqenes The structwgs and configyrations of the individual components as well as the distributions of products j.n these alkylbmzene ~ x t u r e were s determined through polarimetric and spectral techniques. From the stereochemical aad structural rd%tionships between the starting alcohols and the final hydrocarbon prodgcts, the following cqnelusions regarding the mechanism of the reaction are reached ( 1) Bridged ion-pairs (phenonium chlorosulfite, phenonium chloride or both) occur as discrete intermediates in these reactions (2.) These ion-pairs collapse t o give both rearranged and "arranged products (3) The reactions are highly stereospecific In the 3-phenyl-2-butyl and tLso-2-phenyl-a-pentyl and threa-3-phenyI-2-pentyl systems, but in the erytR~o-2-phenyl-3-pentylarid erytltra-3-phenyl-2-pentyl systems abaut one third of the product arises through a simple substitution rc;tctioii The general mechdnism of the Shi reaction is discussed in the light of these results
The reactions of thionyl chloride,' phosgeneI2 phosphorus pentwhlaride in liquid SOa3 or dry hywith certain secondary drogen bromide (at alcohols to give halides whose contipations are the sqme as thase of the stwting materials hqve been classified as exwples a€ the Sfai reactionas Hughes, Ingold and co-workers5suggested th&tthe reactions involving thionyl chloride occurred by the internal dwomposition of an intermediate chlorosulfite, and although not stated explicitly, these authors imply that only one transition state intervenes bgtween the alkyl chlorosulfite and the alkyl chloride product. This reaction has been studied kinetically by Lewis and Boozer1' who state that if the decoinposition of the chlorosulfite intervediate does occur by the concerted mechapism, then structures -4-D should all be considered as contributing to the 0
ll
-S I
R-o
t ) R
I
+
o=s ++
Cl A
0
0
C1 l3
0
11
R-n-s
c
+
c1-
f-f
R
o=s!I
Cl' D
( 1 ) ('1) P. A. Levene and L .'I Mikeska, . J . A i d . C ' h s t f i . , 76, 587 (1927); ( h ) J . Kenyon, A. G. Lipscomh and H . Phillips. J . C h c m .So< , 413 ( 1 W O ) ; (c) J. Kenyon, H. Phillips and F. Taylor, i b d . , 382 11932); (d) E. S. Lewis and C. E. Boozer, THISJ O U R K A L , 74, :3OX Il952). (2) A. €3.J. Housa and H.PhilJips, J. CPem. Soc., 108, 1232 (1932). (3) E. D. Hughes, C. Y.fagold and I . C. Whitfield, Nalure, 141, 206 (1941). See a180 reference la. (4) P . A. Levene aqd A. R ~ t h w J, . Bioi. Chcm., 19T, 237 11939). ( 5 ) This term was introdufed by W. A. Cowdrey, E . D. Hughes, C . K . Ingold, S. Mnsterman and A . D. Scott [ J . C h s q . S u r . , 1267 (1837) 1 t o designate those nuckophilic di$pl&cement r e w t b n s which occur wit& retention of mnfiguration. This designation is limited t o those reactions that Q not appear to involve the participetion of some neighboring group [se.e S. Winstein i i t i r l R . li Bricklei, Tms Jckr'nvAI, 64, 2780 /l!J12)1
transition state. In a study more similar to that at hand, Lucas and Could6 demonstrated that optically active evythro-3-chloro-2-butanplreacts with thionyl chloride to give a 16% yield of mesa%,3-dichlorobutane,whereas optically active threo3-chloro-2-butanol with the same reagent produces a 20% yield of racemic d,l-2,3-dichlorobutane. The authors interpreted these steric results in terms of ethylene chloronium ion intermediates. The present paper reports the results of a study of the inechanisin of the decomposition of the chlorosulfites of the X-pheny1-2-buty1, 2-phenyl-8pentyl aiid 3-phenyl-2-pentyl systems. The symmetry properties associated with the first of these systems and the structural relationships between the two latter systems have already proved of value in studies of the mechanisms of the solvolytic if-agner-LIeerwein rearrangement reaction,' the E , reaction,8 and the reaction of the p-toluenesulfonate esters with lithium aluminum hydride.g .4 similar approach has been used in the current study in which the stereochemical relationships between the product- and reactants are used to define tlie mechanistic requirements for the rcaction. Methods The reactioiis w r c carried out utilizing the optically pure diastereomers of 3-phe1iyl-2-butanol~~~~ ( I ) , g-phenyl-3pentano17h,8b(111) arid .?-phenyl-2-pentan01'~.~~ ( I Y ) and thionyl chloride as starting materials, and excess thionyl chloride, dioxane or formic acid (the chlorosulfite was preformed) as solvents. The ~~heriylalkyl chloride products ( 6 ) II J . Liica, an11 C' \V. (;auld J r , i b i d , 63, 2.i41 (19.11). (7) (a; 1) T Crain, t h i , ! , 7 1 , :3W3 119&!11, 4,) 71, RS75 119491. (CJ
74, 21? 2 ) ; ( 1 ) ) 74, 2139 : ! ) ) 1 ~ 1 1 I ) I C'rniii, i h i d , 74, 21 I!)ll!l.>2J; i l v 74, (1
!Xj
