B. Dinesh et al. / Tetrahedron 68 (2012) 4374e4380
4379
ꢀ
addition of 10% (v/v) H
silver acetate (1 mmol) in Et
resulting mixture stirred for 3 h. The progress of the reaction was
monitored by TLC (CHCl /MeOH/AcOH 40:2:1 (v/v)). The solvent
was removed under reduced pressure and diluted with H O. The
2
O and then cooled to wꢁ15 C. A solution of
ValeOMe. All the intermediates were characterized by electrospray
ionization mass spectrometry (ESI-MS), 500/700-MHz H NMR and
thin-layer chromatography (TLC) on silica gel (SiO , CHCl /MeOH
2 3
1
3
N (11 mmol) was added, and the
3
9:1 (v/v)) and were used without further purification. The final
peptides were obtained as pure products after washing with hex-
ane/ether mixtures. The peptides were characterized by ESI-MS
2
aqueous phase was extracted with ethyl acetate (AcOEt), and the
resulting colourless aqueous phase was adjusted to pH 2 with 50%
1
and by 700-MHz H NMR spectra. Far-UV circular dichroism (CD)
KHSO
with brine, dried (Na
reduced pressure to obtain Boce
ppm (700 MHz, CDCl
4
and extracted with AcOEt. The AcOEt extracts were washed
spectra (Fig. 6) were recorded for peptides 3 and 4 with methanol
as the solvent. A path length of 1 mm was used. Data were acquired
in the wavelength-scan mode by using a 1 nm bandwidth with
2
SO
4
) and the solvent was removed under
3
1
b
(R)-Val (yield w60%). H NMR,
d
ꢁ1
d
3
): 0.93 (d, 6H, C H
3
), 1.50 (s, 9H, Boc CH
3
),
a step size of 0.2 nm and a scan speed of 10 nm min . Typically,
g
a
b
1.85 (m, 1H, C H), 2.55 (d, 2H, C H
2
), 3.75 (m, 1H, C H), 4.92 (d,
two scans were acquired, and the data were averaged. Solvent
subtraction was carried out by using methanol as a blank, and the
spectra were smoothened. Mass spectral data (m/z): peptides 2,
1
H, NH).
4(R) and
g
4(S)-valine
612.3 [MþH] (Mcal) 611.7 Da, 634.3 [MþNa] , 650.3 [MþK] ; 3
þ
þ
þ
4
.3. Synthesis of
g
þ
þ
and 4, 626.3 [MþH] (Mcal) 625.8 Da, 648.3 [MþNa] , 664.3
A previously described procedure12 was used with minor
[MþK] . Melting points ( C): peptides 2, 154e156 C; 3, 115e117 C
1
þ
ꢀ
ꢀ
ꢀ
ꢀ
modification.
Step 1. Boce
and 4, 172e173 C. H NMR,
d
ppm (700 MHz, CDCl
3
): Boce
D
3
-Pro-
3
L-ValeOH or Boce
D
-ValeOH (20.2 g, 93 mmol) was
Aib-
b
(R)Val-Aib-ValeOMe (2), 0.92/0.95/0.98 (12H, d,
b
(R)Val
12
d
g
b
dissolved with 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s
acid, 14.7 g, 102.3 mmol) and 4-dimethylaminopyridine (DMAP,
C H
3
, Val C H
3
), 1.48 (9H, s, Boc CH
3
), 1.50/1.63 (12H, s, Aib C H
3
),
b
g
3
a
1.87/2.24 (4H, m,
D
-Pro C H
2
/C H
), 3.7 (3H, s, OCH
2
), 2.38 (2H, d,
b
(R)Val C H
2
) 3.43
d
3
b
1
7 g, 139.5 mmol) in 200 ml of CH
2
Cl
2
. The reaction mixture was
(2H, d,
4.11 (1H, t,
(1H, br,
D
-Pro C H
2
3
), 4.05 (1H, br,
b
(R)Val C H),
ꢀ
0
a
a
cooled to 0 C and a solution of N,N -dicyclohexylcarbodiimide
D
-Pro C H), 4.46 (1H, q, Val C H), 6.8 (1H, s, Aib NH), 6.96
b (R)Val NH), 7.28 (1H, s, Aib NH), 7.66 (1H, d, Val NH);
3
(
DCC, 21 g, 102.3 mmol) in 100 ml of CH
2
Cl
2
was added dropwise.
4
The mixture was then stirred for about 2 h at room temperature
and kept in the refrigerator, overnight. The precipitated dicyclo-
Boce -Pro-Aib-g (R)Val-Aib-ValeOMe (3), 0.88/0.9/0.94 (12H, d,
D
4
3
d
4
a
g
(R)Val C H
3
, Val C H
3
), 1.4 (
g
(R)Val C H
3
), 1.46 (9H, s, Boc CH
3
),
b
b
g
hexyl urea was filtered off, washed with 10% KHSO
over anhydrous Na SO . The solution was used for the next step
without purification.
Step 2. To the cooled solution from step 1, 98% acetic acid (61 ml,
023 mmol) was added. The reaction mixture was allowed to cool,
NaBH (8.8 g, 232 mmol) added in small portions with stirring over
.5 h. The reaction mixture was left in the refrigerator overnight.
Workup was by successive washing with 5% KHSO
(2ꢂ), brine (2ꢂ)
SO
4
, brine and dried
1.49/1.55 (12H, s, Aib C H
3
), 1.88e2.05 (4H, m,
(R)Val C H
D
-Pro C H
), 3.43 (2H, br, -Pro C H
-Pro
(R)Val NH), 6.79 (1H, br,
Aib NH), 7.03 (1H, s, Aib NH), 7.88 (1H, br, Val NH); Boce -Pro-Aib-
2 2
/C H
, 1H,
4
d
4
b
d
2
4
g
(R)Val C H), 2.1 (2H, m,
g
2
D
2
),
4
g
3.69 (3H, s, OCH
3
), 3.84 (1H, m,
g
(R)Val C H), 4.1 (1H, br,
D
a
a
4
C H), 4.48 (1H, q, Val C H), 6.26 (1H, br,
g
1
D
4
4
3
4
3
g (S)Val-Aib-ValeOMe (4), 0.85/0.89/0.97 (12H, d, g (R)Val C H ,
d
b
1
Val C H
3 3 3
), 1.49 (9H, s, Boc CH ), 1.49/1.55 (12H, s, Aib C H ),
b
g
4
d
4
1.89e2.08 (4H, m,
D
-Pro C H
), 3.49e3.58 (2H, m,
3.72 (3H, s, OCH ), 4.08 (1H, br,
6.36 (1H, s, Aib NH), 6.72 (1H, d,
2
/C H
2
, 1H,
D
g
(S)Val C H), 2.1 (2H, m,
4
b
d
4
g
and water (2ꢂ). The organic layer was dried over anhydrous Na
2
4
g
(S)Val C H
2
-Pro C H
2
, 1H,
g
(R)Val C H),
a
a
and evaporated to dryness. The obtained product was passed
through a 150 g pad of silica gel (60e120 mesh) with 1:1 AcOEt/
petroleum ether mobile phase. The recovered solid was stirred with
petroleum ether for about 20 min. and filtered through sintered
glass funnel to obtain a fine powder.
3
D
-Pro C H), 4.45 (1H, q, Val C H),
(S)Val NH), 7.17 (1H, s, Aib
4
2
g
4
NH),
7.96 (1H, d, Val NH).
4.5. X-ray diffraction
Step 3. The product from the previous step was refluxed in
1
50 ml of toluene for about 3.5 h. The solvent was removed under
Single crystals of the pentapeptides 2, 3 and 4 were obtained by
slow evaporation using a range of solvent conditions. Colourless
single crystals were grown by dissolving w8 mg of peptides 2, 3a,
reduced pressure to obtain a cyclized pyrrolidinone product.
Step 4. The pyrrolidinone (12e14 g, w50 mmol) dissolved in
2
5:35 acetone/water was cooled in an ice bath, NaOH (4e5 g)
added and stirred for 30 min. Acetone was removed under reduced
pressure and the aqueous solution was acidified with 25% KHSO to
pH 2. The precipitated solid was filtered and washed with water to
3b and 4 in acetone/water mixture (200
petroleum ether mixture (100 l/100 l), in ethanol (300
ether (500 l), respectively. Peptide 2 crystallized in the monoclinic
space group P21, with four peptide molecules and two co-
crystallized water molecules in the asymmetric unit. For peptide
3, polymorphic forms (ethyl acetate/petroleum ether and ethanol)
were obtained. Form 3a crystallized with one peptide molecule and
one water molecule in the asymmetric unit. Form 3b crystallized
with one peptide molecule and one ethanol molecule in the
asymmetric unit. Forms 3a and 3b both crystallized in the same
m
l/ 10
m
l), in ethyl acetate/
m
m
ml) and in
4
m
4
4
get pure corresponding Boce
g
(R)-ValeOH or Boce
g (S)-ValeOH
1
(
yields w75%). H NMR
d
ppm (700 MHz, CDCl ): 0.89e0.92 (d, 6H,
), 1.55e1.88 (m, 3H, C H
), 3.4 (m, 1H, C H), 4.36, 5.81 (d, 1H, NH).
3
3
d
b
C H
C H
3
), 1.45 (s, 9H, Boc CH
3
2
, C H), 2.4 (m, 2H,
a
g
2
4
.4. Peptide synthesis
Peptides 2, 3 and 4 were synthesized by conventional solution-
1 1
orthorhombic space group P2 2 21, whereas peptide 4 crystallized
in the triclinic space group P1, without any co-crystallizing solvent.
phase methods, by means of a fragment condensation strategy. The
Boc-group was used for N-terminal protection, and the C-terminus
was protected as a methyl ester. Deprotections (monitored by TLC)
were performed with 98e100% formic acid (HCOOH) and saponi-
fication for the N- and C-terminal protecting groups, respectively.
Couplings were mediated by 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-1H-benzo-
For peptides 2 and 4, X-ray data were collected on Bruker AXS
ꢀ
KAPPA APEXII CCD with MoK
a
(
l
¼0.71073 A) radiation and for
peptides 3a and 3b X-ray data were collected on Bruker AXS ULTRA
APEXII CCD (rotating anode X-ray generator) with CuK
a
ꢀ
(l
¼1.54178 A) radiation. These data sets were collected in phi and
omega scan type mode. For peptides 2 and 4, the structures were
13
solved by using iterative dual space direct methods in SHELXD
triazole (HOBt) (1.01 equiv.). The final peptide Boce
D
-Pro-Aib-Xxx-
(S)Val) was achieved by
-Pro-AibeOH with H NeXxx-Aib-
and for peptides 3a and 3b, the structures were solved by direct
3
4
4
14
Aib-ValeOMe (Xxx¼
b
(R)Val,
g
D
(R)Val,
g
methods in SHELXS. After the initial solution methods all the
2
fragment condensation of Boce
2
structures were refined against F isotropically followed by full