I
Charles E. Carraher, Jr. WrioM State Universitv Dayton, Ohio 45431
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Synthesis of Poly(fl-Alanine) and
I fl-A1anine
Introductlon-Amino Acids Amino acids, as the name implies, contain both an amino group and an acid group. While it is convenient to write the formula as shown below to the left, a better representation shows it as an internally ionized salt, a zwitterion, as shown to the right.
proteins play critical roles in all life processes acting as enzymes, haptens, metabolic intermediates, etc. Fibrous proteins constitute the structural material of animals in the form of muscle, skin, connective tissue, etc. Wool, casein, and silk are but three of many industrially important protein materials. Proteins are largely polypeptides having the same gross structure as Nylon being composed almost exclusively of namino acids such as phenylalanine, glycine and alanine. (It is interesting to note that the amino acids cystine, methionine, and cysteine contain sulfur which are the major source of the polluting gases SO2 and SO3 formed from burning coal.) H
The most important amino acids are the a-amino variety since they serve as the building blocks of proteins. With the exception of glycine, the naturally occurring amino acids are optically active, thus emphasizingthe complexity in describing proteins derived from them. Most have the same configuration a t the asymmetric n-carbon atom, being of the L form. Alpha amino acids can he chemically synthesized utilizing many routes, the most common including (a) synthesis from halogenated acids
41 R
I I NH,
Whelr X = halide
R+-COOH
(b) malonic ester synthesis exemplified as follows
H
H
I R-4-CWH I Br
I I NH
% R-C-COOH
and the (c). Strecker synthesis illustrated by 0
11 R-C-H
HCN
NH,
H
I R-C-CN
I I
NH2
H:O
H
I
R-C-COOH I I NH.
The synthesis utilized in this exercise employs the hydrolysis of poly(8-alanine) and is discussed below. Proteins are present in all living matter. Soluble or globular 668 / Journal of Chemical Education
O
R
I II I f N-c+€+ I R' Repeating Protein Unit where R and R' are Varied. Molecular weights of proteins range from several thousand (600 for insulin from cattle) to 108 (4 X lo7 for tobacco mosaic virus)-thus they are oligomeric (small number of repeating units) to high polymers with 106repeating units. For a chain where the repeating unit was 6 A this would give a chain that was 6 mm long for the longest proteins. Parameters such as sequence, type and amount of amino acids in the chain, and arrangements in space are critical in describing natural proteins. While the natural synthesis of protein has been conducted for millions to billions of years, the wholly chemical synthesis of proteins is just beginning. The Fisher approach, use of N-carboxyanhydride, solid phase syntheses and automation of the latter serve as a brief reminder of efforts towards this end. (Of interest is the recent large scale synthesis of edible nrotein utilizing bacteria which metabolize wood. etc.. mulkply rapidly an2 which are eventually harvested.) kcrylamide can he polymerized via a Michael type condensation to give poly(&alanine) which upon hydrolysis yields the biologically important 8-alanine in high yield. Beta-alanine is generally hiosynthesized by mammals through transamination of pyruvic acid with glutamic acid. Introductlon-Michael Additions Carbon-carbon double bonds in conjugation with "electron accentors" serve as eood substrates in nucleonhilic additions. S U C ~reactions are inown as Michael additions, a name origi n a l ] annlied ~ in a more restricted sense to rea&ions of malonic este; and acetoacetic ester. Examples of "electron deficient" substrates include
while examples of typical nucleophiles include
Michael reactions are catalyzed by such bases a s sodium ethoxide, sodium, sodium hydroxide and amines such as triethylamine and piperidine. A mechanism for the synthesis of poly(8-alanine) is
Characterlzatlon of Poly(0-Alanlne) (optional) Inf~aredspectra of poly(@-alanine) and acrylamide can be compared noting such items as the absence of the hand about 1600-1680 cm-1 in the polymer-the region characteristic of the C=C moiety. The solubility of poly(@-alanine)and acrylamide in solvents such as glacial acetic acid, benzene, chloroform, dimethylformamide, dimethylsulfoxide, conc. formic acid, conc. sulfuric acid and acetone. The polymer should be soluble in fewer solvents and requires generally several days to go into solution. The question ofwhether degradation (hydrolysis) is occurring in acid solutions and how this can be tested for might be raised. Films can be cast ontoglass plates using 20%solutions of poly(8alanine) in formic acid. The coated plates should he dried for 72 hr under room conditions to give generally taugh-brittle films. (Coherent films are difficult to obtain. Evaporation can he assisted by placing the plates in avacuum oven and heating to 40°C and applying vacuum until bubbles are about to form. These conditionsshould be previously determined using a separate plate with a small portion of polymer solution on it.) These films can be comoared to those obtained from
Hydrolysis of Poly(&Alanine) to &Alanine 0
H:O
B B Poly(@-alanine) T h e poly(8-alanine) is subsequently hydrolized yielding the amino acid alanine. Synthesis of Poly(&Alanine)' Acrylamide2 (10 g, 0.14 mole) in dry dimethyl farmamide3 (10 ml) is added to a two-necked 50-ml flask fitted with a nitrogen inlet tube. Nitrogen is passed through the system. The solution is heated to -100'C and 2 drops of a 50%dispersion of metallic sodium in xylene4 is added.
II + CCH-CH,-C-NHt
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0 HtN-CHI-CHJ-C--OH
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8-Alanine is prepared by hydrolysis in an acid solution. One gram of poly(8-alanine) is added t o 5 m l of aqueous 4 M HzS04 and refluxed for 2 hr. The solution is neutralized to p H 7 by addition of hot aqueous 4 M Ba (OH)z. Barium sulfate precipitates and is filtered and washed twice with 30-ml ~ o r t i o n of s water. The combined water filtrates are evaporated t o dryness in a vacuum oven a t a temperature h e l o w ~ 1 0 0 ~ ~ . T h e residual syrup crystallizes on cooling to give 7&90% yield of p-alanine. The product may be recrystallized from hot methanol. 8-Alanine melts a t 195-196°C. A number of tests can he conducted on t h e p-alanine including comparison of solubilities with those of acrylamide and oolv(R-alanine). standard amino acid tests. etc. he ib&e exerci'se requires about 4 h r (excluding optional oortions) b u t reauires t h a t this time he solit between two jaboratoh to permit drying of the poiymer. ~ y d r o ~ y s i s of the poly(@-alanine)can he executed on a portion of t h e polymer directly after synthesis. Additional tests can be uerformed on the amino acid a s desired. Theexercise can h r aimed at thegeneral chemistry or bioc h e m i s t ~studenl* to illustrare the swlhesis of an aminoacid. or can hiaimed a t students of organic chemistry emphasizing the several routes to amino acid synthesis and also emphasizing Michael type condensations. ~~
~~
'
Polymerization begins almost instantly and is completed in 3-5 min after which 50 ml of water is added to quench the reaction. The polymer solution is filtered and solid polymer washed several times. The polymer is dried between 60-80°C for 24 hr in a vacuum
".u...5.6 An alternative preparation callsfor theaddition of 15ml of acrylamide and 100 ml of dioxane to a 500-ml beaker. Then 1ml of a Grignard reagent (about 0.1 M )is added drouwise to the acrvlamide solution. F& best results the acrvlamide sol&ion is heated tonear reflux before addition of the Grignarh reagent. The product is recovered and treated as noted above.
This is n particularly itriking synthesis since it wcurs rapidly in high yirlrl. Polytj-alaninel is also r ~ l l r d Nylm.3 ' Acrvlamide ~ h < ~ uhel dhandled with rnrc iinre it is toxic! Acrvlamide Ean be purified by recrystallization from ethyl acetate ehd sublimation at pressures below 1 mm of Hg; its melting point is
--
R.VC -. 3 Dimethylformamide can he dried by distillation from a small quantity of phosphorus pentoxide at reduced pressure. This amounts to about 0.018 g of sodium. Caution: Always employ - . adequate safety precautions wh& utilizing sodium. "more easily handled product is obtained if the product is broken uo into small oarticles utilizine a hieh meed blender before drvine. ' 6 Generallya yield of SO-%% is ogtained. ~
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Volume 55, Number 10. October 1978 1 669
