The Synthesis of a Dipeptide from its Component Amino Acids Protecting Groups in the Elementary Organic Laboratory Paul E. Young and Andrew Campbell Department of Natural Sciences, York College, C.U.N.Y., Jamaica, NY 11451 In recent years, the trend in elementary organic lecture classes has been toward greater emphasis on biologicallyrelated topics. Most courses now devote some lecture time to a discussion of amino acids, peptides, and proteins, and yet very few elementary organic laboratory experiments have been designed to illustrate the theory. This lack of experimental procedure is especially noteworthy in the case of peptides. Nonetheless, peptide synthesis is now a well-refined art ( I ) , and many of the procedures, all designed to produce high experimental yields, can be readily adapted to the elementary laboratory. Interest in peptides, always high, has heightened even more with the recent discovery of the importance of the so-called "opiate" peptides (2). The methods used in peptide synthesis illustrate important organic blocking techniques and provide interesting polyfunctional organic products which may be studied in solution by standard physical techniques. We wish to describe a simple three-step procedure for synthesizinga dipeptide from its component amino acids. The particular peptide presented was chosen for several reasons:
(1) The amino acids are inexpensive and have hydrocarbon side-chains, giving rise to fewer side products and simplifying the work-up procedure; (2) Each of the three steps in the synthesis provides white crystalline product in a straightforward manner using standard laboratoryglassware and procedures; and (3) The dipeptide product exhibits conformational multiplicity in solution and may be observed in both its E and Z forms by nuclear magnetic resonance spectrosCOPY. Synthesis
The synthetic sequence is illustrated below:
0
HN-CHCOOH t (CH3C0)20 C6H5$H2 NH2CHCOOH
@
The 270 MHz NMR spectrum of Nacetylr-prolyl-L-phenylalanine methylester in DMSW..
+
+
CH30H
MIXED
0
-
NoOH
S0Cl2
CH3CO-N-
CH3CO-N-CHCOOH
@
7H2C6H5
HCI
. NH2CHC02CH3
@
5H2C6H5 CHCONHCHCOOCH3
In the first step of the sequence, the amino group of Loroline is orotected bv acetvlation and in the second steo, the i nprotected e a s the carhoxyl terminus 0: ~ - ~ k e n ~ l a l a nis methyl ester. This esterification, often accomplished with gaseous HCI, is more satisfactorily carried out in the elementary lab with the liquid, thionyl chloride. The final step in which the two amino acids are coupled to form the dipeptide is accomplished by the mixed anhydridemethod (2). The entire sequence, somewhat modified from standard peptide protocol, can be accomplished in seven hours. The first two reactions require approximately 1%hr each, and the third step can be completed in approximately 3% hr. Thus, the proce-
Volume 59
Number 8
August 1982
701
dure can he completed in two standard 4-hr lab periods if the first two reactions are run the same day. For a more relaxed pace, the procedure may be spread out over three periods. The experimental time may he reduced t o one period by purchasing acetyl-l-proline and L-phenylalanine methyl ester hvdrochloride. thoueh this increases t h e cost of the exneriment cot~&lcral~ly. With thr amin,, acids as starting mnrerinl;, the n s t in chemicals should nor exceed V!5 ner LO students and will decrease significantly with larger scale. Theoretical Aspects T h e mixed anhydride method is so-named because of the intermediacy of a n anhydride of acetyl-L-proline
Experimental
N-Acetyl-L-proline In a 25-ml Erlenmeyer flask, dissolve 2.3 g (0.02 mole) L-prolinein 2.5 ml 1 N NaOH. To this mixture, add 2.0 ml (0.021 mole) acetic anhydride. Stopper and shake the flask vigorously over a 30-min period. The flask will become warm. Allow the flask to return to room temperature and neutralize the mixture with several drops of concentrated hydrochloric acid, swirling gently during the addition. AUow the flask to cool in an ice bath. Crystalsshould begin forming within 15 min, and crystallization should be complete after approximately 1 hr. (If the phenylalanine methyl ester is being made during this laboratorv neriod, this is a eoad time to beein its . nrenaration.) Filter . the crys& by &uum and wash with a small volume of coldwater. Allow the crvstals to drv and wash with n similnr -amnll o~ l~ ~-~ r n. nf ~ ~ -v-. ..e ~ ~ ~ . ether. The yield generally ranges from 60-8090, m.p. 112-115T (lit. ( 5 ) 115-117°C). If desired, the product may be recrystallized from water ~~
It is instructive to discuss the mechanism of the anhydride's formation and t o have the students account for the use of two moles of the weak base, N-methylmorpholine, for each mole of acetylproline a s well a s for the preferential attack of the second amino acid a t the prolyl carhonyl. Another aspect of this project, mentioned earlier, enhances the didactic usefulness of the synthesis. In solution, both N-acetyl-L-proline and the dipeptide, N-acetyl-L-prolyl-Lphenylalanyl methyl ester show conformational heterogeneity about t h e acetyl-proline amide bond.
H-N
I
CH-R
I
CH-R
I
T h e observation of conformational isomerism in compounds containing tertiarv amide bonds is well-known (3) and t h e special importance of what has been called "cis-trans" isomerism in proline-containina compounds is well documented (4). h i h e dipeptide illus&ated-above, small amounts (less than 20%) of the E (cis) isomer can be observed in the N M H spectrum in rhloroform and considerably greater percentages of the E isomer are m.irlrnt in dimethybulfoxide wet. fieurer. l - . The doublinr of the NMR sirnul for the a c ~ t v lnethvl group a t 1.5-2.06 a n 2 for the methyrester a t 3.76 is lspecialiy diagnostic even on a 60 MHz NMR instrument. With instrumerits offering temperature control, more advanced classes can investigate the coalescence of these peaks and thus t h e activation energy for this interconversion in dimethylsulfoxide. T h e infrared spectrum of t h e dipeptide is also of interest since the carhonyl region shows three distinct peaks a t high resolution (KBr pellet). T h e student should be urged to identify the origin of all three peaks and, in particular, to assign the two amide hands.
702
Journal of Chemical Education
~~
.
~~~
~~
~~~~~
~~~~
~~
L-Phenylalanine methyl ester hydrochloride Add 5 ml methanol to a 250-ml round-bottomed flask and note the level with tape or marking pencil. Add another 95 ml methanol and 3.0 g (0.018 mole) L-phenylalanine. Cool the mixture in an ice bath for 10 min and, while still cold, add 2.6 ml(0.036 mole) thionyl chloride droowise. All the solid should dissolve durine the addition. Reflwr the mixture for 20 min and vacuum distil fnsnirntnr nressure) until no leas
N-acety14-prolyl-~-phenylalaninemethyl ester Dissolve 2.0 g (0.013 mole) N-aeetyl-L-proline in 30 ml chloroform in a 1M)-mlround-bottomed flask. Cool to -20'C in a dry icelearbon tetrachloride cold bath.' Add 1.5 ml(0.013 hole) N-methylmorpholine and 1.8 ml(0.014 male) isobutylchloroformate. Allow this mixture to stir at -20'C for 10 min and then add 2.8 g (0.013 mole) L-phenylalanine methyl ester hydrochloride as a solid followed quickly by another 1.5 ml N-methylmarpholine.Swirl the mixture at -20°C for at least 3/4 hr. Allow it to warm to room temperature and pour it into a 125-mlseparatoryfunnel. Wash the mixture with two 20-ml portions of 0.2 N HCI and two 20-ml portions of 1%NaHC08 solution. Dry the organic layer over anhydrous magnesium sulfate and filter it into a 10O-ml mund-bottomed flask. Distil until approximately3 ml of liquid remains and remove the final traces of solvent on asteam bath. Cool the resultant oil to room temperature and crystallize it from ether. Collect the white crystals by vacuum filtration. The yield should be 80-90% m.p. 1 0 6 1 0 8 T (lit. (7) 106.5-108°C).
Acknowledsment This work was supported in part by grant #RR-08153-05 from the National Institutes of Health. We wish t o acknowledge the use of the Northeast Regional N M R facility a t Yale University which is funded bv erant number CAGE-7916210 from the Chemistry ~ivision-ofNSF. Literature Cited
14, Deber, C. M., b e y , F. A,, Carver, J. P.. and Blouf E
R,J. Am,.
C h . Sm., 92(21),
m m ,
