Dehydroalanine-Based Inhibition of an Epimerase
23.1 mmol), and 1-hydroxybenzotriazole hydrate (HOBt, 3.54
g, 23.1 mmol) in 60 mL of dry CH2Cl2 was stirred for 10 min
under argon. Ala-OtBu‚HCl (4.00 g, 22.0 mmol) and Et3N (3.05
mL, 22.0 mmol) were added, and the solution was stirred at
room temperature for 20 h. The mixture was diluted to 200
mL with CH2Cl2 and washed with 200 mL of water, resulting
in the precipitation of triethylamine hydrochloride salt. Fol-
lowing filtration, the organic layer was separated and washed
with 200 mL portions of 5% KHSO4, saturated NaHCO3, and
brine. The organic layer was dried over MgSO4, filtered, and
rotary evaporated. Recrystallization from EtOH/H2O gave 8.4
g (90%) of 5 as white crystals. Spectral properties were in
agreement with those in the literature.41
N -(Be n zyloxyca r b on yl)-3-ch lor o-L-a la n yl-L-p h e n yl-
a la n yl-L-a la n in e ter t-Bu tyl Ester (6). A solution of 5 (2.50
g, 5.86 mmol) in 15 mL of CH3OH containing 5% Pd/C (250
mg) was stirred under 1 atm of hydrogen at room temperature
for 20 h. The mixture was filtered through Celite, and the
filtrate was rotary evaporated to give 1.71 g (100%) of the
amine as a colorless oil. In a separate flask, a solution of Z-3-
chloro-L-alanine18 (1.17 g, 4.55 mmol), EDC (0.916 g, 4.78
mmol), and HOBt (0.766 g, 5.00 mmol) in 20 mL of dry CH2-
Cl2 was stirred for 10 min under argon. A solution of the amine
(1.33 g, 4.55 mmol) in 5 mL of dry CH2Cl2 was then added,
and the mixture was stirred at room temperature for 22 h.
The mixture was diluted to 50 mL with CH2Cl2 and washed
with 50 mL portions of water, 5% KHSO4, saturated NaHCO3,
and brine. The organic layer was dried over MgSO4, filtered,
and rotary evaporated. The residue was purified by silica gel
chromatography (40:40:1 petroleum ether/EtOAc/MeOH) to
give 1.31 g (54%) of 6 as a colorless glass: 1H NMR (200 MHz,
CDCl3) δ 7.35 (s, 5H), 7.28-7.13 (m, 5H), 6.76 (d, 1H, J ) 7.8
Hz), 6.25 (d, 1H, J ) 7.1 Hz), 5.45 (d, 1H, J ) 7.3 Hz), 5.11 (s,
2H), 4.63 (dd, 1H, J ) 7.1 Hz, J ) 7.1 Hz), 4.51 (m, 1H), 4.32
(m, 1H, J ) 7.1 Hz), 3.96 (dd, 1H, J ) 3.9 Hz, J ) 11.2 Hz),
3.68 (dd, 1H, J ) 5.1 Hz, J ) 11.2 Hz), 3.08 (m, 2H), 1.43 (s,
9H), 1.29 (d, 3H, J ) 7.1 Hz); +LSIMS (thioglycerol matrix)
532 (M + H+). Anal. Calcd for C27H34ClN3O6: C, 60.95; H, 6.44;
N, 7.90. Found: C, 61.29; H, 6.57; N, 8.20.
promotes an elimination reaction that generates the
dehydroalanine-containing species. The observed inhibi-
tion by 4 can be attributed to the sp2-hybridization of the
R-carbon in the dehydroalanine unit that mimics the
planar geometry of the anionic intermediate shown in
Figure 2a. These results argue strongly against the
dehydration/rehydration mechanism (Figure 2c), since 4
is the putative intermediate in that mechanism, yet no
signs of the hydration of 4 (to give a mixture of 1 and 2)
were observed. No evidence for the formation of covalent
adducts was observed, as might be expected if the
unnatural elimination step occurred at the covalent ester
adduct stage (Figure 2b). Furthermore, if covalent ca-
talysis were employed, one might not expect compound
4 to be a potent inhibitor.
Elimination reactions of â-halides and inhibition by
planar intermediate analogues have long been hallmarks
of the reactions catalyzed by cofactor-independent amino
acid racemases, all of which utlilize deprotonation/
reprotonation mechanisms.9,11,12,21,26,36-40 Notable ex-
amples include the inhibition of proline racemase by the
planar compounds 12 and 13.21,36 In addition, the enzyme-
catalyzed elimination of HCl or HF from â-halo amino
acids, as well as the elimination of water from N-hydroxy
amino acids, has led to the transient formation of the
planar enamine and imine species 14 and 15 (they
ultimately hydrolyze when free in solution). These species
have been shown to serve as potent inhibitors of the
enzymes diaminopimelate epimerase38,39 and glutamate
racemase.9,40 This work strongly suggests that a similar
mechanistic strategy is at play in this newly discovered
class of peptide epimerases.
Exp er im en ta l Section
N-Acetylglycyl-L-leu cin e (7). To a stirred solution of
glycyl-L-leucine (5.42 g, 28.8 mmol) in 20 mL of 0.1 M AcOH
was added Et3N to pH 6. Acetic anhydride (15.9 mL, 144 mmol)
was added in small portions, while pH 6 was maintained by
addition of Et3N. Amberlite IR-120 (H) resin was added to pH
< 2 and then filtered off. The filtrate was rotary evaporated
under reduced pressure to give 6.19 g (93%) of 7 as a colorless
glass: 1H NMR (200 MHz, D2O) δ 4.37 (dd, 1H, J ) 5.7 Hz, J
) 8.9 Hz), 3.88 (s, 2H), 2.00 (s, 3H), 1.73-1.50 (m, 3H), 0.89
(d, 3H, J ) 6.1 Hz), 0.84 (d, 3H, J ) 6.1 Hz); +DCI-MS (NH3)
231 (M + H+). Anal. Calcd for C10H18N2O4: C, 52.16; H, 7.88;
N, 12.17. Found: C, 51.99; H, 7.81; N, 12.01.
N -Ace t ylglycyl-L-le u cyl-3-ch lor o-L-a la n yl-L-p h e n yl-
a la n yl-L-a la n in e ter t-Bu tyl Ester (8). A solution of 6 (3.01
g, 5.66 mmol) in 30 mL of distilled CH3OH containing 5% Pd/C
(300 mg) was stirred under 1 atm of hydrogen at room
temperature for 24 h. The mixture was filtered through Celite,
and the filtrate was rotary evaporated to give 2.10 g (93%) of
the amine as a pale yellow foam. In a separate flask, a solution
of 7 (1.22 g, 5.28 mmol), EDC (1.06 g, 5.54 mmol), and HOBt
(0.848 g, 5.54 mmol) in 20 mL of DMF was stirred for 10 min
under argon. A solution of the amine in 20 mL of DMF was
then added, and the solution was stirred at room temperature
for 58 h. The solvent was removed by rotary evaporation under
reduced pressure, and the resulting amber residue was
triturated in 50 mL of CH2Cl2, leaving 8 as a white solid that
was collected by filtration. The filtrate was concentrated and
partitioned between 50 mL of water and 50 mL of EtOAc. The
organic layer was washed with 50 mL portions of 5% KHSO4,
saturated NaHCO3, and brine and then dried over MgSO4. The
mixture was filtered and rotary evaporated, and the resulting
Gen er a l. Et3N, CH2Cl2, and EtOAc were distilled under N2
from CaH2. MeOH was distilled under N2 from magnesium
methoxide. Anhydrous DMF was dried over 4 Å molecular
sieves and stored under argon. The pentapeptide substrates
1 and 2 were purchased from the Nucleic Acids Protein
Services Unit at the University of British Columbia (UBC).
Flash chromatography was performed on 230-400 mesh silica
1
gel. H NMR data were obtained on a 200, 300, or 400 MHz
spectrometer. Mass spectrometry was performed by the Mass
Spectrometry Centre at UBC by liquid secondary ionization
mass spectrometry (LSIMS) or chemical ionization (CI). El-
emental analyses were performed by Mr. Peter Borda in the
Microanalytical Laboratory at UBC and by Canadian Mi-
croanalytical Service, Ltd. Deprotected peptides were purified
by reversed-phase HPLC on a C18 column (19 × 300 mm, 15-
µm particle size, 100-Å pore) with detection at 220 nm. Elution
was achieved in mixtures of 0.1% TFA in water and 0.05%
TFA in CH3CN using linear gradients optimized for each
peptide with a flow rate of 14 mL/min.
N-(Ben zyloxyca r bon yl)-L-p h en yla la n yl-L-a la n in e ter t-
Bu tyl Ester (5). A solution of Cbz-Phe (6.60 g, 22.0 mmol),
3-(3-dimethylaminopropyl)-1-ethylcarbodiimide (EDC, 4.42 g,
(36) Keenan, M. V.; Alworth, W. L. Biochem. Biophys. Res. Commun.
1974, 57, 500-504.
(37) Baumann, R. J .; Bohme, E. H.; Wiseman, J . S.; Vaal, M.;
Nichols, J . S. Antimicrob. Agents Chemother. 1988, 32, 1119-1123.
(38) Lam, L. K. P.; Arnold, L. D.; Kalantar, T. H.; Kelland, J . G.;
Lane-Bell, P. M.; Palcic, M. M.; Pickard, M. A.; Vederas, J . C. J . Biol.
Chem. 1988, 263, 11814-11819.
(39) Gelb, M. H.; Lin, Y.; Pickard, M. A.; Song, Y.; Vederas, J . C. J .
Am. Chem. Soc. 1990, 112, 4932-4942.
(40) Glavas, S.; Tanner, M. E. Bioorg. Med. Chem. Lett. 1997, 7,
2265-2270.
(41) Kaminski, Z, J . Synthesis 1987, 917-920.
J . Org. Chem, Vol. 67, No. 24, 2002 8393