L-Amino Acid Ethyl Ester Hydrochlorides Derivatives for Qualitative Organic Analysis C. L. Borders, Jr., D i a n n e M. Blech, a n d K a r e n D. McElvanyi College of Wooster, Wooster, OH 44691 As a culmination of t h e laboratory in our second . experience . organic rhrmistry m u r s r , each s t u d m i must idmtify three unknuwnl;. T h r student3 have one "narural product" a s a n unknown, taken from a list of common carbohydrates a n d o-amino acids. W e require t h e students t o determine t h e specific rotation of their natural product and t o make one chemical derivative. While carbohydrates present few prohlems, compilations of derivatives of t h e natural L-amino acids a r e n o t readily available. One of few such lists is t h a t for t h e amino acid ethvl ester hvdrochloride salts ( 1 ) . b u t there is no general procedure repoited for t h e synthksii of these comoounds. W e r e w r t here our results in the attemnted svnthesis bf t h e ethyl ester hydrochlorides for 21 common c-amino acids. Experimental The a-amino acid (1.0 g) was suspended in 15ml of absolute ethanol in a 1M)-mlround bottomed flask. The HCI gas was generated by the controlled addition of concentrated hydrochloric acid to concentrated sulfuric acid. Fifty milliliters of 18M HzSOawas added toa 1-Lsuetion flask containing a Teflon-coated magnetic stirring bar. The side arm of the suction flask was fitted with a oieee of latex tuhine to a 9-in. I k w u r pilet. Twenty milliters of 12 A! ~ I Cwasaddrd I t,, a'iung-stem 125-ml druppind funnel and attached to the topof thcsuct~untladk with a rubber stopper. The suction flask was securely clamped atop a magnetic stirrer and the dropping funnel was also, in turn, securely clamped. The round-bottomed flask containing the amino acid was clamped on a separate ring stand at a 45- angle, and the Pasteur pipet was secured so that the tip was just below the surface of the ethanol. The hydrochloricacid was added drop by drop to the stirred HzS04, and the HCI gas generated was hubhled through the shaken suspension of the amino acid in ethanol. Care must he taken to prevent the accumulation of solid in the tip of the pipet. The dissolution of HCI gas in ethanol is quite exothermic, and the contents of the roundbottomed flask become quite warm in the process. One could generate HCI gas by adding HzSOa to solid NaC1, but we found this to be too cumbersome for routine use. We also found it unnecessary to dry the generated HCI by passing it through a HzSOl trap. After all the hydrochloric acid was added, the contents of the round-bottomed flask were heated under reflux for 0.5 h. In the initial stages of this work, we assumed that the point of dissolution of all the solid amino acid in ethanol marked the endooint of the reaction. For some amino acids, solution was obtained after 5 or 10 ml of hydrochloric acid had been added. We subsequently found that solids isalated after the reaction was stopped a t this point often were not the ethyl esters, but rather the HCI salts of the amino acids or a mixture of the amino acid and ester hydrochloride salts. We, thus, routinely refluxed the reaction mixture for 0.5 h, or untilcom~letesolutionwas obtained. Crystallization of the products was first attempted by placing the stoppered reaction flask in the freezer for one or two days. If no crystals were obtained, the reaction mixture was poured into an evapxating dish and placed in a good fume hood overnight to remove the volatile solvent and excess HCI. After thorough drying, the m.p. of each product was obtained. Proton magnetic resonance (PMR) spectra (Varian T-60 in Dz0) were ohtained for all derivatives to confirm the formation of the desired ethyl ester hydrochlorides. ~
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ethyl ester hydrochloride salts of 15common amino acids. T h e melting points of these products were, in nearly every instance, very close t o literature values, even though the products were not recrystallized. By careful attention t o conditions, we were able t o obtain pure samples of both t h e diester a n d 8-monoester hydrochlorides of L-aspartic acid. W e were unable t o locate t h e melting point of 3,5-dinitro-L-tyrosine ethyl ester hydrochloride i n t h e literature, a n d ours may he t h e first reported synthesis of this compound. L-Amlno Acid Ethyl Ester Hydrochloridesa Amino Acid
m.p.) 1%)
retiux cryst. (h) me*.
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Current address: Mallinckrodt InstitLte of Rad~ology.Washington University School of Medocme. St. LOUIS. MO 6 3 1 10.
814
Journal of Chemical Education
Corn
('c)
ments5
The following p v e produck by the standard Procedure, with any changes in procedure noted Cysteine 125-126 0.5 d 127-128 Glycine 143-145 0.5 d 144 koleucine 88-90 0.5 e 92-93 Leucine 132-133 0.5 e 134 Methionine 81-83 0.5 e 81-82 Phenylalanine 155-156 0.5 d 154-156 Serine 128-130 0.5 e 130-131 Tryptophan 223-225 0.5 d 225-226 Tyrosine 165-168 0.5 e 166 Valine 95-97 0.5 e 102-104. 93-97 Lysine 142-144 4 d 143.5-144.5 Cystine 178d 48 d 188 177-1786 2-Nitrotyrosine 182-183 4 d 179-180 3.5-Dinitrotyrosine 1826 1 d Aspanic Acid 94-96 0.5 e 109-110". 95" Aspanic Acid 199-201 0 d 199-200' The foilowing gave difficulties. Glutamic Acid* 102-104 0.5 e 113-1141 Glutamic Acidk 188-190 0 20Zm Threonine 145-147 0 144-145O Histidine 2436 72 252dQ Alanine 77-78 0.5 e 76
Arginine Proline
t t
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T k standard procedwe for oynmeair is given in me Experimental section. T a ~ m horn ( Ii m PIS nolea o m r w w ' Tho lonnalm 01 em, esters war a n l m v d ov om w c t r a in Dfl 3 5 0 " sor)ro? nr clml eslrr n,arm,or ae was olsso .ain u?OD n D.O 10 PUR m a r s * mr reacton rnwm war keot I O O C lor one or i.one.r 'If cwstallizatiandid not occur by d, me solution was evswrafed to dryness by. .~lacino . it in an evaporating dish in a good fume hoad overnight. 'Crystals dried on a clay plate. 9 Solid did not dissolve until 4s h retlux. The product is apparently the diestw 1PMR)dihydochhMde. "or h e diester hydmchloride. 'Far &monoester hydrodlwae. 1 No reflux. It HCl addition war StoDDadassoon as the solid went into rolution and me solution olaced In the freezer, the 8-ionoesterhydrochloride apparently formed. * Vol. of emanol had I0 ~ treaxed l lo 5 m Dclme 3 so a prod.a MLO 08 o ~ t a n e d For tnc aerler nyaro11 nul ref .rea cnlormr "For glulam c sc a njarorn onor '' ~ n -amp e edrd no! o rro me PMR roecsm of me o r m m s m o e r t a no e m eswr lorrns~on. ra ulvbtmc~audhydrochbride is likely me ~roduct. For L-mreontne hvdrochloride. 0 As HCI was added.
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Od. the90 o pmwdlo be ..mrron ne.hCI IPMR, m D I 11 inereact on war rpl .xed no D ~ L m. F uI oeonta nwilnm w r a p ho !erawem o c o ~ o o e f o m lor .-mrconmr n m ester nvorocn made ' * m ..n,s, d nr 0 n.nrornlol d s ' n a not " I S X I . P anel 72 h reflux, Tk tilted solid is likes LhistidinediHCl IPMR, m.p.).'hl only one m i o n were we able to isalate thedesired product by using t o ml of and evaporating to ~ q r r s one, s rct .A we m e .nao e lo repMt r ~ e e e u mm , r procd.ro, am 0.- omattemptat ~ n t h so ~e OM a mcL lyl[l me I n1. pr~n.c. ' A ~ Qn ne and here voodman, me$ .wer ,ar a s condxonr, am me llna omastr ~ e r e a l w a stnmc* W J O, ~ which could not be crystallized.No literature m.p. for either ester hydrochloride could be 01
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Results and Discussion As shown in t h e table, we were able t o obtain readily t h e
lit. m.pb
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tound.
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The syntheses of solid ethyl ester hydrochlorides of Lglutamic acid, L-alanine, L-threonine, L-histidine, L-arginine, and L-proline gave problems. If one wishes to make a derivative of arginine, histidine, alanine, or glutamic acid, the methyl ester hydrochloride salts of these acids have been reported (12).We synthesized the methyl ester hydrochlorides of arginine and histidine by simply replacing ethanol with methanol in the standard procedure. The methyl derivative of alanine was obtained with more difficulty, while we were unable to obtain a solid product with glutamic acid. Neither threonine nor proline gave solid methyl ester hydrochlorides, consistent with the fact that melting points for these compounds have apparently not been reported. If one is to use the ~rocedurerenorted herein to make the ethyl ester hydrochloiide as a solidderivative of an unknown r (i.e.. L-amino acid. it would be useful to have ~ r e l i i i n a ndata specific rotation, PMR spectrum) to limit the possible alter: natives before runnine the reaction. Not all common L-amino acids give the ethyl r s k hydrochloridesalt by our procedure, hut enough do to make this a valuable chemical derivative technique for the positive identification of L-amino acids in the qualitative organic analysis laboratory.
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Acknowledgment
We thank Ann I?. Mc1.mghlin for data on the methyl rswr hydrochloride salts and Barhara Morrison and David Haueisen for confirmatory syntheses of some of the ethyl ester hydrochlorides. C. L. B. thanks the Henry Luce 111Fund for support of this work. Literature Cited (1) Gmenslein. J. P..and Winitz, M., '"Chemistry ofthe Amino Acids." John Wiley and Sons, New York, 1961, Vol. 2, p. 932. (2) En., W., and Ceahinsfo, M., H d u . Chim Acto, 44.706 (19611. (3) Holler,E..Rainey. P., 0rme.A.. Bennett, E.L.,and Caluin,M.,Bimhambtry. 12,1150
,.".",. "-, " ofOrganicCompaunds."4th ed., Oxford Univ. Pleas. NearYork, 1965. Vol. ,,ST,,
(4) "Dictionary
p. "SQ
16) Reference I51.1966.Vol. 5.0.3235.
R. ~:.~~dHoffm&, W.F.,J. B ~ Othem., I n.433 (19271. is) co7tner, (7) Dall'hfa, L., and Ferrsrio, P., H d u . Chim. Aelo, 45,1065 (19621. (8) Referenee (51,Vol. 1,p. 286 (91 Referanee (51, Vol. 3, p. 1530. (10) Brookes, P., Fuller, A. T., and Walker, J., J Cham. Soc., 669 (19571. (11) Reference (5).Vol. 3, p. 1623. (12) Gematein, J. P., and Winitz. M.,'Chemiatry of the Amino Acids: John Wiley and Sons,NewYork, 1961,Vd.2.p.929.
Volume 61
Number 9
September 1964
815
