Because of the meat imnortance of neotides in hiochemistry and organic sykhesis,*an undergridcate laboratory exercise devoted to synthesis and characterization is clearly desirable (I). We describe the synthesis of a dipeptide from its comnonent amino acids and its analvsis bv chiral-phase thin:layer chromatography.The svnthesis and analvsis of peptides has lone provided a challenge, because of t h e -c h e m A diversity of ihe possible molecules (2). However, thanks to the enormous effort in the last two or three decades, peptide synthesis is efficient and relatively easy (3, 4). The synthesis of a simple dipeptide therefore illustrates a wealth of synthetic techniques. Of further interest, all reactions are performed on microscale, usine technioues similar to those nronosed hv Mavo et al.
. -(5).l-~he meihod of analysis of thk dipeptide exposes students to a novel chromatoeranhic techuiaue. chiral TLC (6).We chose the peptide l&;l.phenylalanine to provide an easilv handled peptide with aromatic functionalitv, and one that-gave reas6nihle separation on the chiral KC plate.The L-&isomer had been synthesized by hoth carbodiimide (7) and mixed anhydride (8) methods. Our synthetic plan, shown in Figure 1, centers on a coupling reaction promoted by the coupling reagent, 2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 3) hecause it is convenient, clean, and eives eood vields in modest reaction times (9). I t is also repGted give less than 0.2% racemization (10)'. The proposedmechanism for the formation of mixed anhydrides has been published (11). While there is a relatively simple procedure for its svnthesis listed (12). the authors have not tried . . . t o duplicate the procedure.A desire to remove both the amino terminal and the carboxy terminal protecting group under the same conditions led us to the t-hutyl protectinggroups, removable withshort treatment in a strong acid like trifluoroacetic arid. Henre, the amino terminal is protected as a t-BOC group, and the carhoxy terminal is protected as a t-butyl ester. While these protected amino acids are commercially available, we give synthetic procedures for hoth.
ExperimentalAll NMR ramplea were dissolved in CI)CI, unless otherwire noted. Spectra wereobtained on an IRM WI'-200SY NMH spectrometer. IR spectra were obtamed on a Perkin-Elmer 7IOH IR spertnmm t-BOCleucine (2) In a 5-mL reaction vial equipped with a reflux condenser, 0.5 mmole (MW 131.2, 66 mg) of L-leucine was suspended in 1 mL of chloroform. To this was added 0.75 mL of an aqueous solution2 containing 1.24 mmol(130 mg) of sodium carbonate and 125 mg of NaCl followed hy 0.5 mmol (MW 218.2.109 mg) of di-tert-butyldicarbonate3 dissolved in a small amount of chloroform. The mixture was refluxed for 60 min, then cooled to room temperature. If two distinct phases were not present, up to 1 mL of water was added. The CHC13 layer was removed, and the aqueous phase was washed ' In our experience, this lab, run in microscale for a class of 75. Is less expensive than an alternative "cheaper" lab run on larger s...