The Journal of Organic Chemistry
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2H), 2.14ꢀ1.57 (m, 18H), 1.52ꢀ1.39 (m, 4H), 1.28ꢀ1.13 (m, 2H),
0.98ꢀ0.83 (m, 16H); 13C NMR (75 MHz, H2O/D2O: 9/1 by volume,
20 °C) δ 203.2, 180.4, 179.8, 176.8, 176.6, 176.3, 176, 175.4, 174.7, 174.6,
174.1, 172, 136.4, 131.2, 120.2, 103.8, 60.41, 6.12, 55.3, 54.5, 53.6, 51.8, 45.5,
45.2, 44, 42.5, 42.1, 39.2, 36.9, 32.8, 32.2, 32.2, 29.1, 29.1, 28.7, 27.4, 27, 26.7,
26.5, 24.8, 24.6, 23.9, 21, 20.6, 16.8, 13.2.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3b. The synthesis of peptide 3b was
performed similarly on a 13.9 μmol scale. Purification was done by
RP-HPLC on a C18 nucleosil 120 Å, 5 μm column (230 nm, 6 mL/min,
rt, buffer A water containing 0.05% TFA, buffer B CH3CN/water 4/1 by
volume containing 0.05% TFA, linear gradient of 0ꢀ30% B in 40 min) to
give 13.8 mg of peptide 3b (71% yield).
Chiral GC-MS analysis: 0.1% of D-Val.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C49H82-
N12O13S [M þ H]þ1079.6, found 1079.7.
1H and 13C NMR analysis of 3d (selected NMR data): 1H NMR (300
MHz, H2Oþ D2O) δ8.62 (t, J= 4.1 Hz, 2H), 8.57 (d, J= 6.7 Hz, 1H), 8.46
(m, 3H), 8.34 (d, J = 7.0 Hz, 1H), 8.24 (d, J = 7.2 Hz, 1H), 7.32 (s, 1H),
4.49ꢀ4.25 (m, 3H), 4.11ꢀ3.92 (m, 3H), 3.82 (m, 2H), 3.75ꢀ3.63 (m,
1H), 3.36ꢀ3.17 (m, 2H), 3.12 (t, J = 6.8 Hz, 2H), 2.98 (s, 2H), 2.65 (t, J =
6.8 Hz, 2H), 2.47 (q, J = 6.7 Hz, 2H), 2.34ꢀ2.18 (m, 2H), 2.15ꢀ1.54 (m,
14H), 1.53ꢀ1.31 (m, 3H), 1.28ꢀ1.10 (m, 1H), 1.02ꢀ0.81 (m, 23H); 13C
NMR (75 MHz, H2O þ D2O) δ 205.9, 180.4, 179.8, 176.7, 176.6, 176.2,
176, 174.7, 174.2, 174.1, 172, 136.4, 131.2, 120.2, 68, 62.9, 62.5, 60.4, 56.1,
55.2, 53.6, 42.5, 42.1, 39.2, 37, 33, 32.8, 32.2, 29.1, 27.4, 27, 26.7, 24.8, 24.6,
23.9, 21.2, 21.1, 20.7, 19.7, 16.8, 13.2.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 5. Peptide elongation was performed on
Rink-PEG-PS resin (NovaSyn TGR, 0.25 mmol/g, 500 μmol scale) by
using standard Fmoc/tert-butyl chemistry on an automated peptide
synthesizer with HBTU/DIEA activation in DMF. A capping step was
performed after each coupling with Ac2O/DIEA. At the end of the
synthesis, the Fmoc protecting group of the last amino acid was removed
with 20% piperidine in DMF.
Final deprotection and cleavage from the solid support were per-
formed with TFA/water/1,2-ethanedithiol/TIS 94.5/2.5/2.5/1 by
volume for 2 h (50 mL). The crude peptide was precipitated in diethyl
ether/heptane 1/1 by volume (500 mL), solubilized in 10 mL of
deionized water, and lyophilized.
Chiral GC-MS analysis: 0.37% of D-Ala.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C47H78-
N12O13S [M þ H]þ1051.6, found 1051.5; [M þ Na]þ 1073.5, found
1073.5.
1H and 13C NMR for peptide 3b (selected NMR data): 1H NMR (600
MHz, H2O þ D2O, 20 °C) δ 8.66 (d, J = 7.4 Hz, 1H), 8.61 (s, 1H), 8.58
(m, 2H), 8.51 (d, J = 6.1 Hz, 1H), 8.36 (d, J = 7.0 Hz, 1H), 8.27 (d, J = 7.1
Hz, 1H), 7.32 (s, 1H), 4.52 (m, 1H), 4.46ꢀ4.39 (m, 2H), 4.31 (q, J = 7.2
Hz, 1H), 4.01 (m, 1H), 3.97 (t, J = 5.9 Hz, 2H), 3.86ꢀ3.79 (m, 2H),
3.73ꢀ3.67 (m, 1H), 3.24 (ddd, J = 23.7, 15.5, 7.2 Hz, 2H), 3.11 (t, J = 6.8
Hz, 2H), 3.02ꢀ2.95 (m, 2H), 2.76ꢀ2.69 (m, 1H), 2.68 (t, J = 6.8 Hz, 2H),
2.56ꢀ2.43 (m, 2H), 2.41ꢀ2.22 (m, 2H), 2.13ꢀ1.54 (m, 18H), 1.52ꢀ1.31
(m, 8H), 1.21 (m, 1H), 0.98ꢀ0.85 (m, 23H); 13C NMR (151 MHz, H2O
þ D2O, 20 °C) δ 179.9, 179.2, 176.8, 176.6, 176.3, 176, 174.8, 174.7, 174.1,
173.9, 173.8, 172.1, 165.8, 165.6, 137, 136.9, 136, 135.9, 121.9, 121.4, 120.5,
120, 118.1, 116.1, 103.8, 63, 62.9, 58.6, 58.6, 58.6, 58.5, 56.2, 56.1, 56.1, 55.3,
55.2, 55.2, 53.5, 45.3, 45.1, 42.1, 39.5, 39.2, 39, 37.9, 36.9, 36.8, 36.5, 36.1,
35.3, 33.1, 33.1, 32.3 32.2, 32.2, 31.9, 29.9, 29.4, 29.1, 28.8, 27.9, 27.4, 27.2,
26.8, 26.7, 26.3, 26, 25.3, 24.8, 19.8, 19.3, 13.5, 12.9.
Purification was performed by RP-HPLC on a C18 Nucleosil 120 Å,
5 μm column (215 nm, 6 mL/min, rt, buffer A water containing 0.05%
TFA, buffer B CH3CN/water 4/1 by volume containing 0.05% TFA,
0ꢀ25% B in 30 min) to give 495 mg of peptide 5 (69%).
MALDI-TOF matrix: R-cyano-4-hydroxycinnaminic acid. Calcd for
C49H84N14O12S [M þ H]þ 1093.6, found 1093.4; [M þ Na]þ 1115.6,
found 1115.4.
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3c. Synthesis was done on a 8.5 μmol scale.
Purification was done by RP-HPLC on a C18 nucleosil 120 Å, 5 μm
column (215 nm, 6 mL/min, rt, buffer A water containing 0.05% TFA,
buffer B CH3CN/water 4/1 by volume containing 0.05% TFA, linear
gradientof 0ꢀ30%Bin40min) togive10.9mgof peptide 3c(87%yield).
Chiral GC-MS analysis: 0.6% of D-Tyr.
Synthesis of Peptide 6c. 4-Mercaptophenylacetic acid (MPAA,
33.63 mg, 0.2 mmol) and tris(2-carboxyethyl)phosphine hydrochloride
(TCEP. Cl, 459 mg, 1.6 mmol) were solubilized in 0.2 M sodium
3
phosphate buffer pH 7.3 (10 mL). NaOH (6 M) was added to adjust the
pH to 7.2. Then, peptide 3c (5.3 mg, 3.6 μmol) and peptide 5 (7.7 mg,
5.4 μmol, 1.5 equiv) were dissolved in the pH 7.3 phosphate buffer
(1.8 mL). The two solutions were mixed and the reaction mixture was
agitated at 20 °C. Final reaction conditions: 1 mM for peptide 3c,
1.5 mM for peptide 5, 10 mM MPAA, 80 mM TCEP, 0.1 M phosphate,
pH 7.2. Purification was performed by RP-HPLC on a C18 Atlantis
column 120 Å, 5 μm (215 nm, 25 mL/min, rt, buffer A water containing
0.05% TFA, buffer B CH3CN/water 4/1 by volume containing 0.05%
TFA, 0ꢀ35% B in 40 min) and afforded 5.2 mg of peptide 6c (54%).
LC-MS analysis of 6c: C99H160N26O24S [M þ H]þ calcd 2131.6,
found 2132.01; [M þ 2H]2þ calcd 1066.3, found 1066.5.
MALDI-TOF matrix: 2,5-dihydroxybenzoic acid. Calcd for C53H82-
N12O14S [M þ H]þ1143.6, found 1143.4; [M þ Na]þ 1165.6, found
1165.4.
1H and 13C NMR analysis of 3c (selected NMR data): 1H NMR (300
MHz, H2O þ D2O) δ 8.59ꢀ8.5 (m, 3H), 8.47 (t, J = 7.1 Hz, 2H), 8.36 (m,
2H), 8.24 (d, J = 6.9 Hz, 1H), 7.26 (s, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.83 (d,
J= 8.6 Hz, 2H), 4.49ꢀ4.38 (m, 2H), 4.31 (q, J= 6.6 Hz, 1H), 3.98 (t, J= 7.2
Hz, 1H), 3.93ꢀ3.82 (m, 4H), 3.82ꢀ3.73 (m, 1H), 3.72 (s, 1H), 3.63 (d, J =
10.2 Hz, 1H), 3.28ꢀ3.05 (m, 7H), 2.94 (m, 4H), 2.63 (t, J = 6.7 Hz, 2H),
2.45 (t, J = 7.2 Hz, 2H), 2.32ꢀ1.10 (m, 28H), 1.,02ꢀ0.85 (m, 24H); 13C
NMR (75 MHz, H2O þ D2O) δ 205.5, 180.3, 179.7, 176.8, 176.6, 176.3,
176, 174.6, 174.1, 173.8, 172, 169.7, 157.4, 136.4, 133.4, 131.2, 130.6, 121,
120.1, 118.3, 117.1, 103.8, 63.8, 62.9, 62.7, 60.4, 56.1, 55.2, 55.1, 53.6, 50.7,
45, 42.5, 42.1, 39.2, 39, 36.8, 33.1, 32.7, 32.1, 29.1, 28.6, 27.4, 27, 26.8, 26.7,
24.8, 24.6, 23.9, 21, 20.7, 16.8, 13.2.
Synthesis of Peptide 6d. Peptide 6d was synthesized by using the
same procedure as described above on a 2.2 μmol scale of peptide 3d
(3.1 mg). Purification by RP-HPLC on a C18 Atlantis column 120 Å, 5
μm (215 nm, 25 mL/min, rt, buffer A water containing 0.05% TFA,
buffer B CH3CN/water 4/1 by volume containing 0.05% TFA, 15ꢀ35%
B in 40 min) afforded 1.9 mg of peptide 6d (33%).
LC-MS analysis of 6d: C95H160N26O23S [M þ H]þ m/z calcd
2067.5, found 2068.02; [M þ 2H]2þ m/z calcd 1034.3, found 1034.6.
Synthesis of 4e
Moreover, COSY, HSQC, and ROESY NMR experiments were in
accord with the proposed structure (see the Supporting Information).
Synthesis of Peptide 3d. Synthesis was done on a 7.4 μmol scale.
Purification was done by RP-HPLC on a C18 nucleosil 120 Å, 5 μm
column (230 nm, 6 mL/min, rt, buffer A water containing 0.05%
TFA, buffer B CH3CN/water 4/1 by volume containing 0.05% TFA,
linear gradient of 0ꢀ35% B in 40 min) to give 6.5 mg of peptide 3d
(62% yield).
Preparation of bis(2-sulfanylethyl)amino trityl polystyr-
ene resin: Bis({2-[(triphenylmethyl)sulfanyl]ethyl})amine47 (933 mg,
1.5 mmol) was deprotected by treatment with TFA/TIS (97.5/2.5,
v/v, 150 mL) during 30 min. The solvent was evaporated under
reduced pressure and residual TFA was coevaporated with cyclohexane
3200
dx.doi.org/10.1021/jo200029e |J. Org. Chem. 2011, 76, 3194–3202