The Synthesis of an Amino Acid Derivative and spectroscopic Monitoring of Dipeptide Formation Richard J. Simmonds Edward Davies Chemical Laboratories, U.C.W., Aberystwyth SY23 INE, U.K. Virtually all undergraduate chemistry courses include a consideration of the synthesis of peptides from amino acids, but, presumably due to the small number of procedures described ( I ) as suitahle for laboratory classes, this is often not illustrated bv- anv-ex~erimental work. Similarlv the use . of visible spectroscopy to measure a rate of reaction is sometimes omitted from undergraduate practical courses. The experiments described below have been used in our laboratories to give students exverience of both areas in a sinele " day of pr&tical time.
T o obtain the rate constant k, A, must therefore be known. This may he found by measuring the absorbance after allowing the reaction to go to completion (about 2 h is adequate). Provided an ultraviolet (UV). ~Dectrometeris avaifahle, one may alternatively, and more conveniently, calculate A. from the e value of D-nitrovhenoxide ion under the described conditions (15.2 x lo3) a i d the molar concentration of starting material [accurately determined from its UV absorbance a t 274 nm c(, = 9.6 X lo3)prior to accurate dilution with buffered glycine solutionl. These two methods give similar accuracies,and, as shown in the figure, excellent agreement with pseudo-first-order kinetics is obtained provided measurements are confined in the first 15 min. Calculation of the rate constant issimple but may he performed by microcomputer if desired; a listing of a program in BBC BASIC to plot a graph of ln[(A, - A)I(AJ] vs. time and calculate the gradient of the least-squares-fitted lines is available from the author. A
Synthesis
One of the most extensively used methods (2) in modern peptide synthesis is that using an activated ester, in particular p-nitrophenyl esters of N-protected amino acids. In the seauence described here L-alanine is converted to its crvstal, linr A'-ben7yloxycarl1~1nyl derivative and then esterifird usinr D-nitro~henoland either dicvclohexvlrarbodiimide 13) (DCC) or ethyl chloroformate (4): ~~~
NaOH
NHzCH(CH8)C02H+ CsHSCHzOCO .C1 C~HSCHZOCO NII - CH(CHdC02H
DCC or ClCOlEmtsN
Caution: Benzyl chloroformateand ethyl chloroformateare toxic and corrosive. Dicycloheaylcarbodiimide produces severe allergic reactions in sensitive individuals.These chemicals should he used in a fume hood,
.
N-Benzyloxycarbonyl-~4anlne
To a magnetically stirred solution of L-alanine (2 g) in water (20 mL) in a fume cupboard is added simultaneouslyaliquots of sodium hydroxide (5 mL, 2 M) and henzyl chloroformate (1.3 mL). Four more such additions are made over 10 min giving a total addition of 25 mL of sodium hydroxide and 6.3 mL of henzyl chloroformate. The mixture is stirred for an additional 20 min, made basic by adding sodium hydroxide (5 mL, 2 M) and washed with diethyl ether (2 X 50 mL). The aqueous portion is acidified with hydrochloric acid (15 mL, 2 M) and extracted with diethyl ether (2 X 50 mL). This organic extract is dried (MgSOJ and evaporated to an oil,
The reaction using DCC is simple to perform and gives higher yields than that using ClCOzEt, hut, since some indiuiduals display severe allergic reactions to DCC,it should not be used unless close superuision is provided. The resulting Z-Ala-PNP is readily crystallized and its optical rotation may be measured to give an estimate of the extent of any racemization (very little in either case). Klneiics The rate of reaction of the Z-Ala-PNP with aqueous glycine giving the dipeptide derivative Z-Ala-Gly-OH is easily followed by spectroscopy since the ester has a maximum absorbance near 275 nm while that of the D-nitronhenmide ~ ~ ion liberated on reaction is in the visihleAregion.Provided that the concentration of elvcine is huee relative to that of ZAla-PNP the reaction Gliows pseuio-first-order kinetics and so ~A~
~
~
~
~
-
~
~
-
where [x] represents [Z-Ala-PNP]. Since 1 mol of Z-AlaP N P (transparent above 400 nm) produces 1mol of p-nitrophenoxide, the absorbance, A, at 420 nm will increase from 0 ~ A, a t infinite time when [x] = 0, and so when 1x1 = [ x ] to F 1~1i)tdelplot 01 the decompos~iond the pnnrophenyl ester of rtbenzyloxycarbony alan ne (2-A a-PhPl n aqreaLo g yc ne at 19 'C.
966
Journal of Chemical Education
which cwstallizes on trituration with oetroleum ether (10 mL) and is fi~terehoff. Typical yields 2.0-3.8 (40-75%). Recrystallization from diethyl etherlpetroleum ether gives white needles mp 83-84 'C (lit. (5)84 "C).
g
p-Nihophenyl N-benzyloxycarbonyl-L-alanine Using DCC. Benzyloxycarhouyl-L-alanine(1.0 g, crude product from above) is dissolved in ethyl acetate (10 mL) and p-nitrophenol (0.7 g) added with stirring at 0 OC. Dicyclohexylcarbodiimide (5 mL, 20% in EtOAC) is added and stirring continued at 0 'C for 45 min. Dicyelohexylurea is filtered off and the filtrate evaporated to dryness.The solid is taken up in diehloromethane (10 mL) and remaining impurity is filtered off. The solution is evaporated to dryness, dissolved in warm ethanol (15 mL) and recrystallized by addition of water. Typical yields 0.91.1g (5&71%) mp 13-74 "C, [ a ] = ~ -33- (EtOAc), (lit. (6)mp 7% 79.5 "C, [n]o = -38'). Using Ethyl Chloroformate. Benzyloxycarbonyl-L-alanine(1.1g, crude product) and triethylamine (0.68 mL) are dissolved in dry toluene (25 mL) and cooled at 0 "C. Ethvl chloroformate (0.48 mL) is added'and the mixture stirred for 15 Gin, protected from ingress of moisture. A solution of 4-nitrophenol (0.7 g) in dry dimethylformamide (5 mL) and dry toluene (10 mL) is added and the mixture stirred and heated under reflux for 1 h, protected from moisture. After cooling triethylamine hydrochloride is filtered off, and the solution is washed sequentially with hydrochloric acid (2 X 25 mL, 0.1 M), saturated sodium hydrogen carbonate solution (4 X 25 mL), and water (2 X 10 mL) before evaporating a t 60 T and 20mm to an oil (1.2 g). The oil is washed with petroleum ether and then crystallized from ethanollwater. White crystals (0.4 g, 24%) mp 71-72 "C,
[a]:: -31' (e. 1.15, EtOAc), lit. mp 79-79.5 "C, [a]o -3W) were obtained after recrystallization.
Reaction of Benzyloxycarbonyl-~-alaninep-Nitrophenyl Ester with Glycine One small crystal (about 0.1 mg) of henzyloxycarbonyl-L-alanine is dissolved in spectroscopic grade ethanol (10 mL). If a UV spectrometer is available, the maximum absorbance (A,.) in the 200500-nm range should he recorded after ensuring that it lies between 0.5 and 2.0. T o an aliquot (1.8 mL) of this solution in a euvette is added pH 8.45 glycine buffer solution (1.8 mL) (containing glycine 7.15 g/L, NaC15.56 g L , and NaOH 0.2 g/L) and the mixture vigorously shaken. The absorbance at 420 nm is recorded at I-min intervals for 15 min and then (if A., has not been determined) after a t least 2 h. A graph of -In[(A. - A)I(A,)] against time is plotted, where A. is the final absorbance a t 420 nm and may be calculated from A. =
A,,,
X
15.2 X
lo3
2 x 9.6 x 103
or measured directly. The gradient gives the rate constant (typically 0.1 min-I at room temperature).
Literature Cited 1. Young,P.E.;Campbell,A. J. ChemEduc. 1982,59,701. 2. 6 h e ~ a r d . RC. . In C o m p r e h e ~ i wOrmnicCh~misfr)i.H~lcn,E..Ed.:Peqamon: New York. 1979;Vol. 5 . 0 345. 8. Bodanazky, M.; Du Vigneaud, V . J. Am. Chem. Sor. 1959.81,5688. 4. Farrington,J.A.:Heitall,P. J.;Kenner.G. W.;Turner,J.M.J. Chem. Sac. 195'7.1407. 5. Bergmann, M.;Zervss,L. Chem.Rer. 1932,ffi. 1193. 6. Goodman,M.;Stueben,K.C. J,Am.Chem. Sor. 1959,81,3980.
Volume 64
Number 11
November 1987
967
