Thust and Koksch
Anal. Calcd for C21H23F3N4O6: C, 52.07; H, 4.79; N, 11.57.
C27H27F3N4O6: C, 57.86; H, 4.86; N, 10.00. Found: C, 57.60;
Found: C, 52.30; H, 4.83; N, 11.70. MS (m/z) 371 (M + H)+.
H, 5.05; N, 9.75. MS (m/z) 447 (M + H)+.
4-Gu a n id in op h en yl-N-(ben zyloxyca r bon yl)-(r-d iflu o-
E n zym a t ic Syn t h eses. Trypsin- and R-chymotrypsin-
catalyzed reactions were performed in a total volume of 60 µL
containing 0.1 M HEPES buffer, pH 8.0, 0.2 M NaCl, and 0.02
M CaCl2 at 25°C or distilled water at -15°C, respectively.
Stock solutions of acyl donor esters (8 mM) were prepared in
water. To guarantee a complete solubility of the ester deriva-
tives 10-40% DMSO was added as cosolvent. Stock solutions
of amino components were prepared in 0.2 M HEPES buffer,
pH 8.0 containing 0.4 M NaCl and 0.04 M CaCl2. Appropriate
equivalents of NaOH were added to the stock solutions of
nucleophiles in order to neutralize hydrochlorides and acetates.
The final concentrations of acyl donors and acyl acceptors were
4 and 20 mM, respectively. The latter was calculated as free,
NR-unprotonated nucleophile concentration [HN]0 according to
the formalism of Henderson-Hasselbalch [HN]0 ) [N]0/(1 +
10pK-pH). The pK values of the R-amino group of the nucleo-
philic amino components were determined by inflection point
titration on a video-titrator. Measurements were carried out
at a nucleophile concentration of 6.7 mM using 0.1 M HCl as
titrant. The pKR values were calculated by linear interpolation
from the slope minimum range. Standard deviation was
estimated to be pKR ( 0.01. After thermal equilibration of the
assay mixtures, the enzymatic coupling reactions were initi-
ated by the addition of trypsin or chymotrypsin, leading to
active enzyme concentrations of 30-120 µM and stirred at
25°C. Reaction times of 1-24 h led to a complete ester
consumption. Reactions in frozen medium were performed
analogous to the following procedure: The tube containing the
appropriate acyl donor ester as well as the nucleophilic
component was cooled to 0°C. Five microliters of the enzyme
stock was added, the reaction mixture was rapidly shaken, and
the tube was stored in liquid nitrogen for 20 s. After the shock
freezing procedure, the tube was transferred into a freezer and
incubated at -15°C. After varying reaction times, aliquots of
50 µL were withdrawn of the stirred and frozen reaction
mixtures, diluted with 70 µL of aqueous stop solution contain-
ing 50% methanol, 25% DMSO and 3% trifluoroacetic acid,
and stored at -20°C until HPLC analyses. For each substrate
and nucleophile an experiment without enzyme was carried
out to determine the extent of spontaneous ester hydrolysis,
which was strictly less than 5%. On the basis of the same
control experiments, nonenzymatic aminolysis of the acyl
donor esters was investigated and could be ruled out. The data
reported here represent the average of at least three indepen-
dent experiments. The identity of the formed peptide products
was established by thermospray mass spectroscopy.
1
r om eth yl)-a la n in ea te, Tr iflu or oa ceta te. H NMR (300.08
MHz, DMSO) δ (ppm) 1.54 (s, 3H), 5.11 (s, 2H), 6.41 (t, J )
55.4 Hz, 1H), 7.09/7.29 (m/m, 4H), 7.36 (m, 5H), 7.42 (m, 3H),
8.57 (s, 1H), 9.68 (s, 1H); 13C NMR (75.46 MHz, DMSO) δ
(ppm) 18.01, 66.17, 122.55, 125.79, 127.98, 128.39, 133.31,
136.40, 148.00, 156.01, 164.21; 19F (282.33 MHz, DMSO) δ
(ppm) -52.68 (d/d, J FF ) 279.4 Hz, J FH ) 55.6 Hz, 1F), -49.61
(d/d, J FF ) 279.4 Hz, J FH ) 55.6 Hz, 1F), 4,70 (s, 3F, TFA).
Anal. Calcd for C21H21F5N4O6: C, 48.47; H, 4.07; N, 10.77.
Found: C, 48.16; H, 3.83; N, 10.56. MS (m/z) 407 (M + H)+.
4-Gu a n id in op h en yl-N-(ben zyloxyca r bon yl)-(r-d iflu o-
r om eth yl)-p h en yla la n in a te, Tr iflu or oa ceta te. 1H NMR
(300.08 MHz, DMSO) δ (ppm) 3.27-3.37 (m, 2H), 5.14 (s, 2H),
6.24 (t, J ) 54,3 Hz, 1H), 7.00/7.29 (m/m, 4H), 7.32-7.41 (m,
10H), 7.61 (m, 3H), 8.55 (s, 1H), 10.00 (s, 1H); 13C NMR (75.46
MHz, DMSO) δ (ppm) 63.99, 66.14, 122.39, 125.82, 127.45,
128.03, 128.40, 130.37, 133.31, 136.43, 147.94, 155.96, 166.45;
19F (282.33 MHz, DMSO) δ (ppm) -52.13 to -51,83 (m, 2F),
4.35 (s, 3F, TFA). Anal. Calcd for C27H25F5N4O6: C, 54.37; H,
4.22; N, 9.39. Found: C, 54.09; H, 4.19; N, 9.15. MS (m/z) 483
(M + H)+.
4-Gu a n id in op h en yl-N-(ben zyloxyca r bon yl)-(r-tr iflu o-
r om et h yl)-leu cin a t e, Tr iflu or oa cet a t e. 1H NMR (300.08
MHz, DMSO) δ (ppm) 0.94-1.01 (m, 6H), 1.91-2.10 (m, 3H),
5.12 (s, 2H), 7.16/7.32 (m/m, 4H), 7.37 (m, 5H), 7.57 (m, 3H),
8.62 (s, 1H), 9.95 (s, 1H); 13C NMR (75.46 MHz, DMSO) δ
(ppm) 22.61, 23.37, 24.26, 41.15, 66.28, 122.38, 125.97, 127.93,
128.07, 128.39, 133.36, 136.30, 147.80, 155.95, 164.68; 19F
(282.33 MHz, DMSO) δ (ppm) 4.26 (s, 3F, TFA), 5.81 (s, 3F).
Anal. Calcd for C24H26F6N4O6: C, 49.66; H, 4.51; N, 9.65.
Found: C, 49.50; H, 4.42; N, 9.44. MS (m/z) 467 (M + H)+.
4-Gu a n id in op h en yl-N-(ben zyloxyca r bon yl)-(r-d iflu o-
r om et h yl)-leu cin a t e, Tr iflu or oa cet a t e. 1H NMR (300.08
MHz, DMSO) δ (ppm) 0.91-0.97 (m, 6H), 1.86 (m, 3H), 5.09
(s, 2H), 6.49 (t, J ) 53,8 Hz, 1H), 7.11/7.29 (m/m, 4H), 7.33
(m, 5H), 7.59 (m, 3H), 8.29 (s, 1H), 10.00 (s, 1H); 13C NMR
(75.46 MHz, DMSO) δ (ppm) 23.06, 23.31, 24.13, 62.86, 66.03,
122.44, 125.98, 127.93, 128.36, 133.25, 136.49, 148.00, 155.99,
167.52; 19F (282.33 MHz, DMSO) δ (ppm) -51.62 (m, 1F),
-51.42 (m, 1F), 4.25 (s, 3F, TFA). Anal. Calcd for C24H27
-
F5N4O6: C, 51.25; H, 4.84; N, 9.96. Found: C, 51.09; H, 4.90;
N, 9.73. MS (m/z) 449 (M + H)+.
4-Gu a n id in op h en yl-N-(ben zyloxyca r bon yl)-(r-tr iflu o-
1
r om eth yl)-p h en yla la n in ea te, Tr iflu or oa ceta te. H NMR
(300.08 MHz, DMSO) δ (ppm) 3.45-3.47 (m, 2H), 5.14 (s, 2H),
6.98/7.29 (m/m, 4H), 7.33-7.39 (m, 10H), 7.55 (m, 3H), 8.91
(s, 1H), 9.91 (s, 1H); 13C NMR (75.46 MHz, DMSO) δ (ppm)
37.81, 66.40, 122.30, 125.88, 127.61, 128.06, 128.20, 128.41,
130.52, 133.13, 136.22, 147.72, 155.91, 163.85; 19F (282.33
MHz, DMSO) δ (ppm) 4.13 (s, 3F, TFA), 6.88 (s, 3F). Anal.
Calcd for C27H24F6N4O6: C, 52.77; H, 3.94; N, 9.12. Found: C,
52.40; H, 3.83; N, 8.97. MS (m/z) 501 (M + H)+.
4-Gu an idin oph en yl-N-(ben zyloxycar bon yl)-(r-m eth yl)-
D-p h en yla la n in a te, Tr iflu or oa ceta te. 1H NMR (300.08
MHz, DMSO) δ (ppm) 1.62 (s, 3H), 3.35-3.60 (m, 2H), 5.39
(m, 2H), 7.18/7.39 (m/m, 4H), 7.52-7.64 (m, 10H), 7.74 (m,
3H), 8.21 (s, 1H), 10.06 (s, 1H); 13C NMR (75.46 MHz, DMSO)
δ (ppm) 38.11, 59.13, 65.56, 122.69, 125.91, 126.75, 127.97,
128.35, 130.65, 132.82, 135.74, 136.96, 148.89, 155.95, 172.36.
Anal. Calcd for C27H27F3N4O6: C, 57.86; H, 4.86; N, 10.00.
Found: C, 57.52; H, 4.83; N, 9.87. MS (m/z) 447 (M + H)+.
HP LC An a lyses. HPLC measurements were performed by
analytical reversed phase HPLC on a C4 polymer coated
column (10 µm, 250 mm × 4 mm) and a C8 reversed phase
column (5 µm, 250 mm × 4.6 mm). Samples were eluted with
various mixtures of water/acetonitril containing 0.1% trifluo-
roacetic acid under gradient conditions. Detection was carried
out at 254 nm to monitor the aromatic chromophores of the
acyl donors. The reaction rates and product yields were
calculated from the peak areas of the substrate esters and the
hydrolysis and aminolysis products, respectively. The chro-
matograms were analyzed using the software Mc DAcq (ver-
sion 1.39, Bischoff Chromatography, Germany).
Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft (Innovationskolleg
“Chemisches Signal und biologische Antwort”).
4-Gu an idin oph en yl-N-(ben zyloxycar bon yl)-(r-m eth yl)-
L-p h en yla la n in a te, tr iflu or oa ceta te. 1H NMR (300.08 MHz,
DMSO) δ (ppm) 1.34 (s, 3H), 3.02-3.33 (m, 2H), 5.08-5.16
(m, 2H), 7.03/7.21 (m/m, 4H), 7.24-7.40 (m, 10H), 7.52 (m,
3H), 7.91 (s, 1H), 9.88 (s, 1H); 13C NMR (75.46 MHz, DMSO)
δ (ppm) 38.68, 59.11, 65.54, 122.66, 125.85, 127.94, 128.36,
130.62, 132.83, 135.73, 148.84, 155.97, 172.34. Anal. Calcd for
Su p p or tin g In for m a tion Ava ila ble: Characterization of
intermediate chemical products and HPLC retention times and
MS data of enzymatic products. This material is available free
J O020613P
2296 J . Org. Chem., Vol. 68, No. 6, 2003