ACCEPTED MANUSCRIPT
6
Tetrahedron
chromatography (CH
7 (1.81 g, 41%, cis : trans = 70 : 30). White solid. Mp 115–
17 °C. Anal. Calcd for C32 C 63.14, H 6.62, N 9.20.
2
Cl
2
– MeOH (20 : 1) as an eluent) to give
Compound 30 was prepared from 25 analogously to 29. Yield
0.385 g (63%, cis : trans = 2 : 1). White solid. Mp 78–80 °C.
2
1
H
40
N
4
O
8
Anal. Calcd for C27
C 67.39, H 6.93, N 6.15. IR (cm ): 3406, 3337, 1708. MS (CI,
H N O C 67.48, H 6.71, N 5.83. Found
32 2 6
–
1
–1
Found C 63.07, H 6.93, N 8.86. IR (cm ): 3409, 3330, 3288,
+
+
+
+
1
1
727, 1643, 1616. MS (ESI, m/z): 609 (MH ), 553 (MH – C
4
H
8
).
4 8 2 3
m/z): 503 (MNa ), 381 (MH – C H – CO ). Н NMR (CDCl ), δ
1
Н NMR (СDCl
3
), δ 1.50 (18Н, s), 2.20–2.26 (1H, m), 2.37–2.56
1.49 (s, 9H), 1.69 (br s, 2H), 1.96–2.58 (br m, 4H), 2.76 (br s,
1H), 3.06 (br s, 2H), 4.21 (br s, 1H), 4.37 (br s, 2H), 4.51 (br s,
0.4H), 4.64 (br s, 0.6H), 5.78–6.28 (br m, 1H), 7.30 (t,
J = 6.9 Hz, 2H), 7.38 (t, J = 6.9 Hz, 2H), 7.60 (d, J = 6.9 Hz,
(
(
1H, br m), 2.69 (0.7H, br s), 2.76–2.83 (1.3H, br s), 3.34–3.62
2H, m), 2.23 (1Н, m), 4.20–4.25 (1H, m), 4.38–4.44 (2Н, m),
4.62 (1H, br s), 5.71 (0.3H, br s), 6.03 (0.7Н, br s), 7.31 (2Н, t,
13
J = 7.6 Hz), 7.40 (2Н, t, J = 7.3 Hz), 7.61 (2H, m), 7.76 (2Н, d,
J = 7.3 Hz), 8.46 (1H, br s), 11.50 (1Н, br s). C NMR (СDCl
2H), 7.75 (d, J = 6.9 Hz, 2H), 10.00 (br s, 1H). C NMR
(CDCl ), δ 26.0 and 27.0, 28.5, 36.1 and 36.4, 37.2 and 37.4,
1
3
3
),
3
δ 28.2, 28.4, 31.2 and 31.3, 34.6 and 35.0, 46.1, 47.2, 55.4 and
38.6 and 39.5, 47.2, 55.1 and 56.1, 66.8 and 66.9, 79.4 and 80.9,
120.0, 124.9 and 125.2, 127.1, 127.7, 141.4, 143.9, 155.4 and
156.0, 156.2 and 158.0, 176.9 and 177.7.
56.1, 67.0, 79.6, 83.4, 120.0, 125.3, 127.2, 127.8, 141.4, 143.9,
53.3, 155.8, 156.6, 163.4, 177.6.
1
3
.15. 1-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(2-(2,3-
3.18. Synthesis and determination of the minimal inhibitory
concentration (MIC) of the peptides
bis(tert-butoxycarbonyl)guanidino)ethyl)cyclobutanecarboxylic
acid (28).
Peptides were prepared manually or using automatic peptide
synthesizer by solid-phase peptide synthesis (SPPS) using the
standard Fmoc/tert-butyl (t-Bu) protocol. Commercially
Compound 28 was prepared from 25 analogously to 27. Yield
8
1.4 g (47%, cis : trans = 2 : 1). White solid. Mp 124–127 °C.
Anal. Calcd for C H N O C 63.65, H 6.80, N 9.00. Found
avaiable Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH and Fmoc-
Phe-OH were used for incorporation of amino acid residues into
peptides. 2-Chlorotrityl chloride resin was used. The first Fmoc-
protected amino acid (0.3 mmol per 1 g of resin) was introduced
by treatment with ethyl diisopropylamine (0.6 mmol) in dry
CH Cl (10 mL) for 1 h, followed by capping of the resin with
3
3
42
4
8
–
1
C 63.20, H 7.12, N 8.77. IR (cm ): 3405, 3333, 3290, 1721,
+
+
1
640, 1619. MS (ESI, m/z): 623 (MH ), 523 (MH – C H –
4 8
1
2 3
CO ). Н NMR (CDCl ), δ 1.48 (18Н, s), 1.72–1.89 (2Н, br m),
2
3
.28–2.49 (3Н, br m), 2.57 (br s, 0.5H), 2.63–2.75 (1.5H, m),
.20–3.38 (2Н, br m), 4.21 (1Н, br s), 4.34–4.49 (2Н, br m), 4.58
2
2
(
1H, br s), 5.42 (0.3H, br s), 5.81 (0.7H, br s), 7,31 (2Н, t,
excess of ethyl diisopropylamine and MeOH. Further Fmoc-
protected amino acids (4 eq) were activated in DMF by a mixture
of O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluoro-
phosphate (HBTU, 3.9 eq) and ethyl diisopropylamine (DIPEA,
8 eq) before coupling. A solution of piperidine (20% in DMF)
was used for Fmoc-deprotection (2 × 15 min). Cleavage of linear
peptide was done in a TFA/TIS cleavage cocktail (95:5 v/v) at
ambient temperature (3 h). The volatile products were removed
in vacuo and the residue was triturated with ether, and the
precipitate was subjected to centrifugation. The solvent was
removed by decantation; the residues were dried on air and then
lyophilized. Head-to-tail cyclization of linear precusor peptides
(0.1% solution in dry DMF) was performed using PyBOP
(0.3 mmol) and DIEA(0.6 mmol). Cyclic peptides were purified
by reversed-phase high-performance liquid chromatography (RP-
HPLC) and then lyophilized.
J = 7.6 Hz), 7.40 (2Н, t, J = 7.3 Hz), 7.61 (2H, m), 7.76 (2Н, d,
J =7.3 Hz), 8.29 (0.3H, br s), 8.41 (0.7Н, br s), 11.52 (1Н, br s).
13
3
C NMR (CDCl ), δ 25.9, 28.2, 28.3 and 28.4, 35.2 and 36.2,
3
7
1
6.5 and 37.0, 39.1 and 39.5, 47.3, 55.2 and 56.2, 66.7 and 67.0,
9.5 and 79.8, 83.3 and 83.4, 120.0, 125.3, 127.2, 127.8, 141.4,
44.0, 153.3 and 153.4, 155.4, 156.3, 163.0 and 163.4, 177.0.
3
.16. 1-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-((tert-
butoxycarbonylamino)methyl)cyclobutanecarboxylic acid (29).
To a suspension of 24 (0.640 g, 1.59 mmol) in CH Cl2
2
(
13 mL), ethyl diisopropylamine (0.830 mL, 4.77 mmol) was
added dropwise, followed by Boc O (0.523 g, 2.40 mmol). The
resulting mixture was stirred for 12 h. The solvent was removed
in vacuo, and saturated aq NaHCO (50 mL) was added to the
2
residue. The mixture was washed with Et O (3 × 50 mL). The
2
3
organic phases were washed with H O (60 mL). The combined
aqueous phases were acidified to pH = 5 with 1 M HCl and
extracted with EtOAc (3 × 30 mL). The combined organic
2
Chromatographic characterization was performed on a Jasco
HPLC system (Japan) using a diode array detector operating at
2
(
20 nm. Runs were carried out on a PolyEncap A 300 column
250 mm × 4.0 mm; Bischoff Analysentechnik, Germany). The
sample concentration was 1 mg of peptide/ml in eluent A. The
mobile phase A was 0.1% trifluoroacetic acid (TFA) in H O, and
phase B was 0.1% TFA in 80% MeCN – 20% H O (v/v). The
retention time (t ) of the peptides was determined at rt using a
4
extracts were dried over MgSO and evaporated in vacuo. The
residue was purified by column chromatography (CH Cl2
–
2
MeOH (15 : 1) as an eluent) to give 29. Yield 0.411 g (55%,
cis : trans = 70 : 30). White solid. Mp 80–84 °C. Anal. Calcd for
2
2
C H N O C 66.94, H 6.48, N 6.00. Found C 66.73, H 6.71,
26 30 2 6
–1
R
N 6.28. IR (cm ): 3403, 3335, 1709. MS (ESI, m/z): 489
linear gradient of 5 to 95% phase B during 40 min.
+
+
(
MNa ), 367 (MH – C
4
H
8
– CO
2
). MS (CI, m/z, negative ion
+
1
scan): 465 (M–H ). Н NMR (CDCl ), δ 1.48 (s, 9H), 2.22–2.55
The MIC against Gram-positive B. subtilis (strain DSM 347)
and Gram-negative E. coli (strain DH 5α) was determined in
3
(
br m, 3H), 2.60–2.75 (br m, 2H), 3.04–3.32 (br m, 2H), 4.12–
2
1
4
0
7
.25 (br m, 1H), 4.38 (br s, 2H), 4.54 (br s, 0.5H), 4.76 (br s,
.1H), 4.95 (br s, 0.1H), 5.15 (br s, 0.3H), 5.98–6.60 (br m, 1H),
.30 (t, J = 6.5 Hz, 2H), 7.38 (t, J = 6.5 Hz, 2H), 7.53–7.65 (br
96-well microtiter plates. Cells were cultivated in lysogeny
broth (LB) (Sigma-Aldrich, Germany). The inoculum was
prepared from mid-logarithmic-phase cultures (OD600 = 0.4). The
OD600 = 1.0 for B. subtilis and E. coli corresponds to
13
m, 2H), 7.74 (d, J = 6.5 Hz, 2H), 10.46 (br s, 1H). C NMR
CDCl ), δ 28.0, 28.4, 34.1 and 34.7 and 35.6, 44.9 and 45.4 and
7
8
(
8.8 × 10 cells/mL and 2.2 × 10 cells/mL, respectively. Aliquots
of the cell suspensions were added to the peptide-containing
wells of a microtiter plate. The final peptide concentrations
ranged between 100 µM and 0.1 µM in 2-fold dilution. The final
3
46.2, 47.1, 54.6 and 55.4, 66.8, 79.4 and 81.0, 119.9, 124.8 and
125.2, 127.1, 127.7, 141.2, 143.8, 155.4 and 155.9 and 156.4,
156.5 and 158.1, 177.0 and 177.6.
5
number of bacterial cells per well was 5 × 10 cfu / 200 µL.
3
.17. 1-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(2-(tert-
Peptide concentrations were tested in triplicate. The test plates
were incubated at 37 °C for 17 h whilst shaking at 180 rpm. The
absorbance was read at 600 nm (Safire Microplate Reader;
butoxycarbonylamino)ethyl)cyclobutanecarboxylic acid (30).