3184 J. Agric. Food Chem., Vol. 45, No. 8, 1997
Gautschi et al.
(NMM) followed by 13.2 mL (93 mmol) of ClCO2iBu. After 2
min of stirring, a precooled solution (-15 °C) of 16.9 g (93
mmol) of H-Leu-OCH3‚HCl and 10.2 mL (93 mmol) of NMM
in 160 mL of dimethylformamide (DMF) was added slowly.
The mixture was stirred at -15 °C for 1 h, the cooling bath
was removed, and stirring continued until the reaction was
complete (monitoring by GC). The reaction mixture was
diluted with EtOAc and washed with a 3% citric acid solution,
saturated NaHCO3, and brine. The organic layer was dried
(MgSO4) and concentrated in vacuo to give 31 g (97%) of a
yellowish oil. This material was dissolved in 50 mL of CH2-
Cl2; 50 mL of trifluoroacetic acid was added dropwise, and the
mixture was stirred at room temp. (ca. 22 °C) until complete
conversion was observed by GC. The solvent was evaporated
in vacuo, and the residue was taken up in EtOAc and washed
with saturated NaHCO3 and brine. The aqueous phase was
adjusted to pH 10 with NaOH and extracted twice with EtOAc;
the combined organic layers were dried (MgSO4) and concen-
trated in vacuo to give 21.1 g (96%) of an orange oil. This
product was dissolved in 100 mL of EtOAc, and the solution
was warmed to reflux temperature. After complete conversion
(GC monitoring) the reaction mixture was cooled slowly to
room temperature with slight stirring. The crystalline pre-
cipitate was separated by filtration, washed with cold EtOAc,
and dried to give 7.8 g (40%) of pure diketopiperazine IV. Mp
162-164 °C; [R]D ) -144.0 (c ) 1.0, CHCl3); 1H NMR δ ) 0.95
(d, J ) 6.6 Hz, CH3), 1.00 (d, J ) 6.6 Hz, CH3), 1.49-1.57 (m,
1H, CH2), 1.74-1.85 (m, 1H, CH), 1.86-1.94 (m, 1H, CH2),
1.97-2.19 (m, 3H, CH2), 2.30-2.39 (m, 1H, CH2), 3.50-3.63
(m, 2H, NCH2), 4.02 (dd, J ) 3.2, 9.5 Hz, 1H, CH), 4.12 (dd, J
) 7.8, 8.6 Hz, 1H, CH), 6.41 (br, NH); 13C NMR δ ) 21.18
(CH3), 22.71 (CH2), 23.24 (CH3), 24.57 (CH), 28.04 (CH2), 38.49
(CH2), 46.44 (CH2N), 53.35 (CH), 58.93 (CH), 166.22 (CO),
170.34 (CO); IR (KBr) 3260 m, 2950 m, 2875 m, 1680 s, 1670
s, 1635 s, 1435 m; MS (EI) m/z (%) 154 (96), 125 (12), 86 (26),
70 (100), 55 (12), 43 (17), 41 (23), 30 (13).
egg, white bread, tuna, whole milk, chocolate milk, fish
sauce, and dried shrimp. One of the principal degrada-
tion products of the high-intensity sweetener, aspar-
tame, is 3-carboxymethyl-6-benzyl-2,5-diketopiperazine
(e.g. Tateo et al., 1988; Prodolliet and Bruelhart, 1993);
the latter constituent can often therefore be found in
certain diet foods and beverages. However, in many of
the instances where DKPs have been reported, one of
the constituent amino acids is proline. Rizzi (1989)
carried out model studies of DKP formation in cocoa and
concluded that the mechanism involved intramolecular
cyclization of linear peptide precursors rather than
stepwise cyclic condensation involving individual amino
acids.
DKPs have been shown by several researchers to be
intensely bitter in taste and to contribute, for example,
to the bitter taste of cocoa (e.g. Pickenhagen et al.,
1975a) and aged sake (Takahashi et al., 1974). On the
other hand, DKPs were not found to be a source of
bitterness in the case of a commercial soybean HVP
(Eriksen, 1977, 1980). Cocoa DKPs apparently act
synergistically with theobromine (Pickenhagen et al.,
1975a,b; Ney, 1986). Ney (1986) showed that the
bitterness of DKPs followed the so-called “Q rule”,
developed previously for amino acids, peptides, and
proteins, which relates perceived bitterness to amino
acid composition. Ishibashi et al. (1988) also proposed
a mechanism for the bitter taste sensation elicited by
DKPs and certain other peptides. Gardner (1980)
demonstrated good correlations (R > 0.94; p < 0.01)
between bitterness thresholds of DKPs (and related
compounds) and molecular connectivity. Cyclo(Asp-Phe)
has been used to help simulate the taste and aroma of
cheese (Smith et al., 1977). Esser and Essig (1980)
employed DKPs in a patented method for preparing
beverages with bitter taste. Pickenhagen et al. (1975b)
also used cyclic (as well as open-chain) dipeptides, in
combination with theobromine, to impart desirable
bitter taste to food and beverage formulations.
(3S,6S)-3-Methylhexahydropyrrolo[1,2-a]pyrazine-1,4-di-
one (I). According to the procedure described for the synthesis
of diketopiperazine IV, 50 g (0.23 mol) of Boc-Pro-OH and 32.4
g (0.23 mol) of H-Ala-OCH3‚HCl were condensed to give 3.8 g
(10%) of crystalline diketopiperazine I. Mp 172-174 °C; [R]D
1
) -126.5 (c ) 0.2, EtOH); H NMR δ ) 1.48 (d, J ) 6.8 Hz,
CH3), 1.89-1.96 (m, 1H, CH2), 1.98-2.07 (m, 1H, CH2), 2.09-
2.15 (m, 1H, CH2), 2.32-2.37 (m, 1H, CH2), 3.52-3.65 (m, 2H,
NCH2), 4.09-4.16 (m, 2H, CH), 6.94 (br, NH); 13C NMR δ )
15.83 (CH3), 22.70 (CH2), 28.10 (CH2), 45.38 (CH2N), 51.12
(CH), 59.21 (CH), 166.36 (CO), 170.53 (CO); IR (KBr) 3265 m,
2980 w, 2880 w, 1715 s, 1690 s, 1665 s, 1645 s, 1455 m, 1410
m; MS (EI) m/z (%) 168 (35, M+), 140 (10), 125 (34), 97 (33),
70 (100), 55 (17), 44 (90), 41 (49), 28 (51).
MATERIALS AND METHODS
Ma ter ia ls. Beers were purchased from a local liquor store.
Sodium chloride was ACS/Reagent grade (VWR Scientific,
West Chester, PA). Dichloromethane was OmniSolv HR-GC
grade (EM Science, Gibbstown, NJ ). Methanol and water were
both ACS/HPLC grade (Fisher Scientific, Pittsburgh, PA). GC
retention index standards (straight-chain ethyl esters) were
obtained from various suppliers and were used without further
purification. All reagents used in synthesis of DKPs were
purchased from Fluka (Buchs, Switzerland), except for the
protected amino acids which were purchased from Bachem
(Bubendorf, Switzerland). Small samples of DKPs II and III
were generously provided by Hoffmann-La Roche, Nutley, NJ .
Syn t h esis a n d Ch em ica l Ch a r a ct er iza t ion of DKP s.
Diketopiperazines I and IV-VII were prepared using standard
peptide chemistry (Bodanszky, 1984) in moderate to good
overall yields. Equipment/methods used in their chemical
characterization were as follows. Mp: Bu¨chi apparatus (Dr.
Tottoli), in open capillaries, not corrected. Optical rotation
[R]D: Perkin-Elmer 241 polarimeter, in 10 cm cell. IR: Nicolet
(3S,6S)-3-(2-Methylsulfanylethyl)hexahydropyrrolo[1,2-a]py-
razine-1,4-dione (V). According to the procedure described for
the synthesis of diketopiperazine IV, 26.8 g (0.125 mol) of Boc-
Pro-OH and 25.0 g (0.125 mol) of H-Met-OCH3‚HCl were
condensed to give 0.53 g (1.8%) of diketopiperazine V after
recrystallization of the crude product. Mp 142-146 °C; [R]D
1
) -113.6 (c ) 1.01, CHCl3); H NMR δ ) 1.85-1.97 (m, 1H,
CH2), 1.98-2.12 (m, 3H, CH2), 2.13 (s, 3H, CH3), 2.30-2.43
(m, 2H, CH2), 2.65-2.76 (m, 2H, SCH2), 3.51-3.65 (m, 2H,
NCH2), 4.11 (dd, J ) 6.8, 8.4 Hz, 1H, CH), 4.21 (t, J ) 5.6 Hz,
1H, CH), 7.69 (s, 1H, NH); 13C NMR δ ) 15.06 (CH3), 22.55
(CH2), 28.06 (CH2), 28.78 (CH2), 29.79 (CH2), 45.31 (CH2), 54.20
(CH), 58.91 (CH), 165.46 (CO), 170.74 (CO); IR (KBr) 3270 m,
2930 w, 2820 w, 1715 s, 1690 s, 1645 s, 1635 s, 1430 m, 1305
w; MS (EI) m/z (%) 228 (32, M+), 167 (44), 154 (99), 139 (32),
70 (100), 61 (28), 56 (21), 41 (32), 28 (26).
510 FT-IR spectrometer, absorptions reported in cm-1
.
1H
NMR spectra: Bruker AVANCE DPX-400 (400 MHz), δ in ppm
relative to TMS, J in Hz. 13C NMR spectra: Bruker AVANCE
DPX-400 (100 MHz), δ in ppm relative to TMS. MS: Finnigan
MAT model 212, peak intensities given as percentage of base
peak in parentheses. GC: Carlo Erba GC 6000 Vega Series
2, column DB-1701, 30 m × 0.32 mm i.d.
(3S,6S)-3-Benzylhexa hydropyrrolo[1,2-a ]pyra zine-1,4-di-
one (VI). According to the procedure described for the syn-
thesis of diketopiperazine IV, 50 g (0.23 mol) of Boc-Pro-OH
and 49.6 g (0.23 mol) of H-Phe-OCH3‚HCl were condensed to
give 17.8 g (32%) of crystalline diketopiperazine VI. Mp 132-
1
134 °C, [R]D ) -190.5 (c ) 1.0, CHCl3); H NMR δ ) 1.85-
(3S,6S)-3-(2-Methylpropyl)hexahydropyrrolo[1,2-a]pyrazine-
1,4-dione (IV). To a precooled solution (-15 °C) of 20 g (93
mmol) of Boc-Pro-OH in 400 mL of tetrahydrofuran (THF) was
added dropwise 10.2 mL (93 mmol) of N-methylmorpholine
2.03 (m, 3H, CH2), 2.26-2.36 (m, 1H, CH2), 2.82 (dd, J ) 10.1,
14.5 Hz, 1H, CH2), 3.52-3.68 (m, 2H, NCH2), 3.58 (dd, J )
3.8, 14.5 Hz, 1H, CH2), 4.06 (dd, J ) 7.3, 8.2 Hz, 1H, CH),
4.28 (dd, J ) 3.8, 10.1 Hz, 1H, CH), 5.94 (br, NH), 7.22-7.36