Kinetic Characterization of E. coli MurG
J. Am. Chem. Soc., Vol. 121, No. 37, 1999 8425
2.18 (m, 2 H), 1.95 (m, 1 H), 1.80 (m, 1 H), 1.57 (m, 1 H), 1.48 (m,
1 H), 1.36 (m, 2 H), 1.29 (d, J ) 8.5 Hz, 3 H), 1.25 (m, 2 H), 1.20 (d,
J ) 8.5 Hz, 3 H), 1.13 (d, J ) 8.5 Hz, 3 H), 0.92 (m, 6 H), 0.02-0.00
(3s, 27 H); 13C NMR (DMSO, 500 MHz) δ 175.8, 172.3, 172.0, 171.8,
171.5, 171.4, 156.2, 62.6, 62.4, 61.2, 52.9, 51.3, 50.1, 47.7, 47.6, 31.4,
31.2, 29.2, 27.2, 22.6, 21.4, 18.0, 17.4, 16.9, 16.8, 16.7, -1.4, -1.5,
-1.6; HRMS(FAB) calcd for C36H72N6O10Si3Na [M + Na+]: 855.4515,
found: 855.4564.
52.3, 51.9, 51.2, 41.5, 38.8, 34.0, 32.7, 31.0, 30.0, 28.6, 27.2, 27.1,
24.6, 24.4, 21.0, 19.2, 19.1, 18.8; ESI-MS calcd for C41H74O21N7P2
[M + H+]: 1062, found: 1062.
Compound 1b. To a solution of compound 1a (25 mg, 0.022 mmol)
in 1.5 mL of H2O/dioxane(1:1) was added NaHCO3 (23 mg, 0.4 mmol)
followed by 6-((biotinoyl)amino)hexanoic acid succinimide ester (12
mg, 0.027 mmol). The reaction was stirred at room temperature for 2
h. Solvent was removed under vacuum. The residue was loaded on a
diethylaminoethyl cellulose column (14 mm × 80 mm, from Whatman
Labsales, Inc.), eluted with 250 mM NH4HCO3 to give 16 mg (80%)
of 1b as a white powder after lyophilization. Rf 0.40 (CHCl3:MeOH:
Compound 8. To a solution of compound 6 (85 mg, 0.13 mmol)
and NH2-L-Ala-γ-D-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OT-
MSE (7) (153 mg, 0.18 mmol) in 1.5 mL of DMF was added
diisopropylethylamine (116 mL, 0.66 mmol) followed by HOBt (27
mg, 0.20 mmol) and PyBOP (104 mg, 0.20 mmol). After stirring for
30 min at room temperature, the solution was diluted in 10 mL of
EtOAc and washed with 0.01 N aqueous HCl (3 × 10 mL). The organic
layer was concentrated, dried over anhydrous sodium sulfate, and
purified by flash chromatograghy (5% MeOH/CHCl3) to give 168 mg
1
H2O ) 3:3:1); H NMR (CD3OD, 500 MHz) d 5.49 (dd, J ) 3.0, 7.3
Hz, 1 H), 5.11 (t, J ) 7.2 Hz, 1 H), 4.50 (dd, J ) 4.8, 7.8 Hz, 1 H),
4.37 (m, 2 H), 4.31 (dd, J ) 4.3, 7.8 Hz, 1 H), 4.29 (m, 1 H), 4.24 (m,
3 H), 4.16 (d, J ) 10.4 Hz, 1 H), 4.02 (m, 2 H), 3.99 (m, 1 H), 3.90
(d, J ) 11.0 Hz, 1 H), 3.74 (m, 1 H), 3.70 (m, 1 H), 3.49 (dd, J ) 9.5,
9.5 Hz, 1 H), 3.21 (m, 1 H), 3.17 (m, 4 H), 2.94 (dd, J ) 4.8, 12.8 Hz,
1 H), 2.71 (d, J ) 12.8 Hz, 1 H), 2.31 (m, 1 H), 2.28 (m, 2 H), 2.25
(m, 1 H), 2.20 (m, 4 H), 2.02 (s, 3 H), 2.00 (m, 2 H), 1.86 (m, 2 H),
1.82 (m, 1 H), 1.73 (m, 4 H), 1.67 (s, 3 H), 1.63 (m, 5 H), 1.61 (s, 3
H), 1.52 (m, 4 H), 1.45 (m, 2 H), 1.44 (d, J ) 7.3 Hz, 3 H), 1.43 (d,
J ) 6.2 Hz, 3 H), 1.41 (m, 2 H), 1.38 (d, J ) 7.3 Hz, 3 H), 1.37 (d,
J ) 7.2 Hz, 3 H), 1.35 (m, 2 H), 1.17 (m, 1 H), 0.93 (d, J ) 6.7 Hz,
3 H); 13C NMR (CD3OD, 500 MHz) δ 177.2, 176.5, 176.2, 176.1, 176.0,
175.6, 174.7, 174.6, 174.5, 174.2, 166.3, 132.1, 126.2, 96.4, 81.3, 78.8,
75.2, 71.0, 65.7, 63.6, 63.0, 61.8, 57.2, 55.7, 55.0, 54.2, 50.9, 50.7,
50.4, 41.2, 40.4, 40.2, 39.1, 39.0, 38.6, 37.2, 37.0, 33.0, 32.5, 30.6,
30.3, 30.2, 30.0, 29.6, 27.7, 27.1, 26.9, 26.7, 26.1, 24.5, 23.5, 20.0,
19.5, 18.4, 18.3, 18.0, 17.9; HRMS(FAB) calcd for C57H95N10O24P2-
SNa2 [M-3H++2Na+]: 1443.5512, found: 1443.5494.
1
(87%) of 8 as a white solid: Rf 0.24 (5% MeOH/CHCl3); H NMR
(CD3OD, 500 MHz) δ 7.52-7.37 (m, 15 H), 5.88 (m, 1 H), 5.65 (s, 1
H), 5.13 (m, 4 H), 4.41 (m, 2 H), 4.35 (m, 3 H), 4.17 (m, 8 H), 4.06
(dd, J ) 9.5, 3.5 Hz, 1 H), 3.84 (m, 3 H), 3.77 (m, 1 H), 3.10 (m, 2
H), 2.29 (t, J ) 14.5 Hz, 2 H), 2.19 (m, 1 H), 1.90 (m, 1 H), 1.88 (s,
3 H), 1.77 (m, 1 H), 1.67 (m, 1 H), 1.51 (m, 2 H), 1.43-1.35 (m, 14
H), 1.01-0.97 (m, 6 H), 0.06-0.04 (3s, 27 H); 13C NMR (CDCl3, 500
MHz) δ 173.9, 172.8, 172.4, 171.8, 171.3, 157.1, 137.1, 135.5, 135.4,
129.2, 129.0, 128.9, 128.7, 128.4, 128.1, 126.1, 101.6, 97.1, 82.5, 81.0,
78.2, 76.7, 70.0, 69.6, 68.4, 64.8, 64.1, 63.8, 63.0, 53.9, 53.3, 51.4,
50.0, 49.1, 48.4, 40.4, 31.6, 31.5, 29.6, 27.9, 23.1, 22.7, 19.6, 18.0,
17.9, 17.8, 17.5, 17.4, -1.3, -1.4, -1.5; HRMS(FAB) calcd for
C68H106N7O20PSi3Na [M + Na+]: 1478.6436, found: 1478.6417.
Compound 9. To a solution of compound 8 (87 mg, 0.06 mmol) in
5 mL of MeOH was added 20 mg of 20% Pd-C. The reaction vessel
was filled with hydrogen and stirred at room temperature. After 30
min 1 mL of pyridine was added. The solution was diluted with 15
mL of MeOH and stirred for 30 min. The catalyst was filtered off. The
filtrate was concentrated to give product 9 which was used in the next
reaction without further purification. Rf 0.28 (CHCl3:MeOH:H2O ) 3:2:
0.5).
Compound 11. Compound 9 (58 mg, 0.04 mmol) was coevaporated
with toluene (3 × 1 mL) and dissolved in 1 mL of CH2Cl2. A portion
of the citronellol diphenylpyrophosphate (10) solution (0.4 mL) was
added to the reaction followed by an addition of pyridine (20 µL, 0.24
mmol). The reaction was stirred at room temperature for 18 h. Solvent
was removed under vacuum and the residue was loaded to a C18 reverse
phase column (8 mm × 80 mm, particle size 40 mm, pore size 60 Å,
from J. T. Baker) and eluted with CH3CN/0.1% NH4HCO3 aqueous
solution (0, 5%, 10%, 15%, 20%, 25%, 30%, 35% of 10 mL each).
The fractions containing desired product were combined and concen-
trated to give 34 mg (68%) of 11 as a white powder. Rf 0.21 (CHCl3:
MeOH:H2O ) 4.5:1.5:0.2). This product was used in the next reaction
without further purification. ESI-MS calcd for C57H109N7O23P2Si3Na
[M + Na+]: 1429, found: 1429.
Compound 1a. To a solution of compound 11 (43 mg, 0.023 mmol)
in 0.7 mL of DMF was added tetrabutylammonium fluoride (1 M in
THF, 0.7 mL). The reaction was stirred at room temperature for 24 h.
Solvent was removed under vacuum. The residue was loaded to a C18
reverse phase column (8 mm × 80 mm, particle size 40 µm, pore size
60 Å, from J. T. Baker), and eluted with CH3CN/0.1% NH4HCO3
aqueous solution (0, 5%, 10%, 15%, 20%, 25%, 30% of 10 mL each).
The fractions containing the desired product were combined and
concentrated. The crude product was further purified on a diethylami-
noethyl cellulose column (14 mm × 80 mm, from Whatman Labsales,
Inc.), eluted with 250 mM NH4HCO3, to give 24 mg of 1a (93%) as a
white powder after lyophilization: Rf 0.18 (CHCl3:MeOH: H2O ) 3:3:
1); 1H NMR (CD3OD, 500 MHz) δ 5.58 (m, 1 H), 5.11 (t, J ) 6.5 Hz,
1 H), 4.50-3.56 (m, 12 H), 2.94 (m, 2 H), 2.34 (m, 2 H), 2.10 (s, 3
H), 2.00 (m, 1 H), 1.98 (m, 2 H), 1.92 (m, 1 H), 1.74 (m, 2 H), 1.67
(s, 3 H), 1.62 (m, 1 H), 1.60 (s, 3 H), 1.50-1.39 (m, 12 H), 1.23 (m,
2 H), 0.93 (d, J ) 6.5 Hz, 3 H); 13C NMR (D2O, 500 MHz) δ 178.2,
177.9, 176.7, 176.6, 176.5, 176.4, 176.3, 165.3, 135.5, 127.6, 97.0,
82.2, 80.3, 75.4, 74.1, 72.0, 71.9, 71.8, 70.4, 67.6, 62.7, 56.6, 55.8,
Compound 12a. Compound 9 was deprotected with TBAF using
the same method for making 1a. Rf 0.16 (CHCl3:MeOH:H2O ) 3:4:
1.5); 1H NMR (CD3OD, 500 MHz) δ 5.34 (dd, J ) 3.0, 7.0 Hz, 1 H),
4.24 (m, 3 H), 4.17 (dd, J ) 6.7, 6.7 Hz, 1 H), 4.08 (dd, J ) 4.6, 8.5
Hz, 1 H), 4.03 (q, J ) 7.0 Hz, 1 H), 3.93 (m, 1 H), 3.80 (m, 1 H), 3.75
(m, 1 H), 3.59 (dd, J ) 5.5, 11.6 Hz, 1 H), 3.56 (m, 1 H), 3.38 (dd, J
) 9.7, 9.7 Hz, 1 H), 2.82 (t, J ) 7.3 Hz, 2 H), 2.22 (m, 2 H), 2.15 (m,
1 H), 1.86 (s, 3 H), 1.70 (m, 4 H), 1.58 (m, 2 H), 1.40 (m, 1 H), 1.31
(m, 6 H), 1.25 (m, 6 H); 13C NMR (CD3OD, 500 MHz) δ 179.4, 178.8,
178.0, 176.2, 175.9, 174.7, 174.0, 173.8, 95.3, 81.2, 78.7, 74.9, 71.2,
62.8, 55.5, 55.3, 55.0, 51.9, 51.1, 50.8, 40.5, 33.1, 32.5, 30.4, 28.4,
23.7, 23.4, 19.8, 19.4, 18.4, 18.0; HRMS(FAB) calcd for C31H53N7O18P
[M - H+]: 842.3185, found: 842.3212.
Compound 12b. Compound 12b was made from 12a and 6-((bi-
otinoyl)amino)hexanoic acid succinimide ester using the same chemistry
described in step i (Scheme 2). Rf 0.27 (CHCl3:MeOH:H2O ) 3:4:
1.5); 1H NMR (CD3OD, 500 MHz) δ 5.45 (dd, J ) 7.0, 3.0 Hz, 1 H),
4.51 (dd, J ) 5.0, 7.5 Hz, 1 H), 4.39 (m, 2 H), 4.32 (m, 2 H), 4.26 (m,
3 H), 4.12 (m, 1 H), 3.91 (m, 1 H), 3.86 (d, J ) 11.6 Hz, 1 H), 3.73
(dd, J ) 5.5, 11.6 Hz, 1 H), 3.69 (m, 1 H), 3.53 (m, 1 H), 3.22 (m, 1
H), 3.17 (m, 4 H), 2.94 (dd, J ) 5.0, 12.8 Hz, 1 H), 2.72 (d, J ) 12.8
Hz, 1 H), 2.30 (m, 4 H), 2.21 (m, 4 H), 1.99 (s, 3 H), 1.89 (m, 1 H),
1.82 (m, 1 H), 1.74 (m, 2 H), 1.63 (m, 4 H), 1.53 (m, 4 H), 1.46 (m,
2 H), 1.44 (m, 6 H), 1.39 (m, 6 H), 1.35 (m, 4 H); 13C NMR (CD3OD,
500 MHz) δ 177.5, 177.4, 177.3, 176.5, 176.3, 174.8, 174.7, 174.6,
174.3, 174.2, 166.0, 94.3, 80.6, 78.6, 73.2, 68.9, 62.8, 61.1, 60.9, 56.1,
55.0, 54.3, 54.2, 51.6, 50.4, 50.1, 40.4, 39.8, 39.6, 36.4, 36.2, 32.5,
31.4, 28.8, 28.7, 28.6, 28.5, 28.4, 26.2, 25.9, 25.8, 23.2, 22.7; HRMS-
(FAB) calcd for C47H78N10O21PS [M - H+]: 1181.4801, found:
1181.4769.
Compound 13. Compound 13 was made following the same scheme
as 1a except that in step e, intermediate 6 was coupled to dipeptide
CH3NH-D-γ-Glu(O-TMSE)-L-Ala-NH2 instead of to 7. Rf 0.41 (CHCl3:
1
MeOH:H2O ) 3:3:1); H NMR (CD3OD, 500 MHz) δ 5.49 (dd, J )
3.0, 7.0 Hz, 1 H), 5.11 (t, J ) 6.6 Hz, 1 H), 4.33 (q, J ) 7.0 Hz, 1 H),
4.27 (q, J ) 7.0 Hz, 1 H), 4.24 (dd, J ) 3.8, 7.6 Hz, 1 H), 4.16 (m, 1
H), 4.04 (m, 2 H), 4.00 (m, 1 H), 3.90 (dd, J ) 1.8, 11.8 Hz, 1 H),
3.75 (dd, J ) 9.6, 9.6 Hz, 1 H), 3.70 (dd, J ) 5.7, 11.8 Hz, 1 H), 3.48
(dd, J ) 9.6, 9.6 Hz, 1 H), 2.64 (s, 3 H), 2.18 (m, 2 H), 2.16 (m, 1 H),
2.02 (s, 3 H), 1.98 (m, 2 H), 1.92 (m, 1 H), 1.72 (m, 1 H), 1.67 (s, 3
H), 1.62 (m, 1 H), 1.61 (s, 3 H), 1.47 (m, 1 H), 1.43 (d, d, J ) 7.0 Hz,