1-Ethoxyethylidene for the SPPS of Aminooxy Peptides
3-5 equiv of diisopropylethylamine (DIPEA) in DMF (10 mL/g
resin) for 30 min except for the first coupling on chlorotrityl resin.
Coupling reactions carried out on the synthesizer were performed
twice. The coupling efficiency in manual synthesis was assessed
by Kaiser and/or TNBS tests. N-R-Fmoc protecting groups were
removed by treatment with a piperidine/DMF solution (1:4) for 10
min (10 mL/g resin). The process was repeated three times and the
completeness of deprotection verified by UV absorption of the
piperidine washings at 299 nm.
Synthetic linear peptides were recovered directly upon acid
cleavage. Before cleavage, the resin was washed thoroughly with
methylene chloride. The linear peptides were then released from
the resin by treatments with a solution of acetic acid/trifluoroethanol/
methylene chloride (1:1:8, 10 mL/mg resin, 2 × 30 min). Hexane
(5-10 volumes) was added to the collected filtrates, and the crude
peptides were isolated after evaporation as white solids. The residue
was dissolved in the minimum of methylene chloride and diethyl
ether was added to precipitate peptides. Then they were triturated
and washed three times with diethyl ether to obtain crude materials
that were used in the next step without further purification.
General Procedure for Cyclization Reactions. All linear
peptides (0.5 mM) were dissolved in DMF and the pH values were
adjusted to 8-9 by addition of DIPEA. PyBOP (1 equiv) was added
and the solution stirred at room temperature for 1 h. as described.15
Solvent was removed under reduced pressure and the residue
dissolved in the minimum of methylene chloride. Diethyl ether was
added to precipitate peptides. Then they were triturated and washed
three times with diethyl ether to obtain crude materials that were
used in the next step without further purification.
N′-Boc-aminooxyacetyl N-Hydroxysuccinimide Ester 5. To a
stirred solution of N-Boc-aminooxyacetic acid (0.500 g, 2.6 mmol)
in ethyl acetate/dioxane (1:1, 10 mL) cooled on an ice bath were
added N-hydroxysuccinimide (0.310 g, 2.7 mmol) and DCC (0.563
g, 2.7 mmol). The resulting mixture was stirred at room temperature
for 5 h and was then filtered through a pad of Celite, and the filtrate
was concentrated under vacuum. The obtained residue was redis-
solved in ethyl acetate (35 mL) and washed with 5% aqueous
NaHCO3 (3 × 5 mL), water (2 × 10 mL), and brine (10 mL). The
organic phase was dried over Na2SO4 and evaporated in vacuo.
The crude solid was re-crystallized from methylene chloride/diethyl
ether/pentane, thereby providing pure compound 5 as a white solid
(0.618 g, 2.14 mmol, 83%): 1H NMR (CDCl3, 300 MHz) δ 7.66
(1H, s), 4.78 (2H, s), 2.87 (4H, s), 1.49 (9H, s); DCI-MS calcd for
C11H16N2O7 288.1, found m/z 305.8 (M + NH4)+.
Fmoc-Lys[Boc-aminooxyacetyl]-OH 1. The ester 5 (0.301 g,
1.05 mmol) in methylene chloride (6 mL) was added dropwise to
a stirred suspension of the TFA salt of Fmoc-lysine (0.409 g, 1.11
mmol) and DIPEA (0.190 mL, 1.09 mmol) in methylene chloride
(6 mL). The resulting suspension was stirred for 7.5 h. The pH of
the resulting reaction mixture was regularly adjusted to pH 8-9
by further additions of DIPEA. Unreacted Fmoc-lysine was then
filtered off and the filtrate concentrated under vacuum. Ethyl acetate
was added to the residue, and the organic solution was washed
with water (2 × 10 mL) and brine (10 mL). The organic phase
was dried over Na2SO4 and evaporated under vacuum. Fmoc-Lys-
[Boc-aminooxyacetyl]-OH was precipitated from ethyl acetate/
hexane. The solvents were removed and the solid rapidly washed
with ice cold ether and dried under vacuum providing compound
1 as a white powder (0.472 g, 0.87 mmol, 84%): analytical HPLC
tR ) 10.5 min; 1H NMR (CDCl3, 300 MHz) δ 8.21 (1H, broad s),
7.75 (2H, d, J)7.5 Hz), 7.66 (1H, broad s), 7.60 (2H, t, J)7.5
Hz), 7.39 (2H, t, J)7.5 Hz), 7.30 (2H, t, J)7.5 Hz), 5.69 (1H, d,
J)7.8 Hz), 4.38-4.31 (5H, m), 4.21 (1H, t, J)7.2 Hz), 3.34 (2H,
m), 1.95-1.54 (6H, m), 1.45 (9H, s); ESI-MS calcd for C28H35N3O8
541.2, found m/z 540.1 (M - H)-.
(0.354 g, 1.72 mmol). The resulting solution was stirred at room
temperature for 3 h and was then filtered through a pad of Celite.
The filtrate was concentrated under vacuum. The obtained residue
was dissolved in ethyl acetate (35 mL) and washed with 5% aqueous
NaHCO3 (3 × 5 mL), water (2 × 5 mL), and brine (5 mL). The
organic phase was dried over Na2SO4 and evaporated in vacuo
affording compound 6 as a white solid (0.649 g, 1.67 mmol,
97%): 1H NMR (CDCl3, 300 MHz) δ 4.87 (2H, s), 2.85 (4H, s),
1.54 (18H, s); ESI-MS calcd for C16H24N2O9 388.1, found m/z 411.1
(M + Na)+, 427.1 (M + K)+.
Fmoc-Lys[N,N-bis-Boc-aminooxyacetyl]-OH 2. Compound 6
(0.3 g, 0.77 mmol) in methylene chloride (4 mL) was added
dropwise to a stirred suspension of TFA-salt of Fmoc-lysine (0.313
g, 0.85 mmol) and DIPEA (0.150 mL, 0.86 mmol) in methylene
chloride (4 mL). The pH of the resulting reaction mixture was
regularly adjusted to pH 8-9 by further additions of DIPEA. After
3 h, reaction of the excess Fmoc-lysine was filtered off and the
filtrate washed with 5% aqueous NaHCO3 (3 × 5 mL), water (2 ×
5 mL), and brine (2 × 5 mL). The organic phase was dried over
Na2SO4 and evaporated in vacuo. The crude product was purified
by silica gel chromatography with methylene chloride/methanol/
acetic acid (95:4.5:0.5), thereby providing building block 2 as a
white powder (0.247 g, 0.385 mmol, 50%): analytical HPLC tR )
12.1 min; 1H NMR (CDCl3, 300 MHz) δ 7.75 (2H, d, J ) 7.4 Hz),
7.60 (2H, broad d, J ) 7.4 Hz), 7.39 (2H, t, J ) 7.4 Hz), 7.30 (2H,
td, J ) 7.4, 1.2 Hz), 5.55 (1H, d, J ) 7.7 Hz), 4.45 (2H, s), 4.39
(3H, m), 4.22 (1H, t, J ) 6.8 Hz), 3.33 (2H, m), 1.94-1.13 (7H,
m), 1.53 (18H, s); 13C NMR (CDCl3, 75 MHz) δ 175.0, 168.1,
156.1, 150.5, 143.8, 141.3, 127.7, 127.0, 125.1, 119.9, 85.3, 67.0,
53.6, 47.2, 38.6, 33.6, 31.7, 28.8, 28.0, 24.8, 22.2; ESI-MS calcd
for C33H43N3O10 641.3, found m/z 664.2 (M + Na)+, 642.2 (M +
H)+.
2-(1-Ethoxyethylideneaminooxy)acetic Acid 7. To a stirred
solution of iodoacetic acid (9.00 g, 48.4 mmol) in water (19 mL)
at 0 °C was added aqueous NaOH (3.0 mL, 40% w/w). The
resulting solution was allowed to heat to room temperature,
whereafter ethyl N-hydroxyacetimidate (6.0 g, 58.3 mmol) was
added followed by aqueous NaOH (4.5 mL, 40% w/w) and water
(19 mL) (pH of solution >12). After 4.5 h of stirring at 80 °C and
cooling to room temperature, water was added (70 mL) and the
aqueous mixture was washed with methylene chloride (3 × 50 mL).
The water phase was brought to pH 2-3 with a 1 M hydrochloride
solution. The acidified water phase was then extracted with
methylene chloride (4 × 50 mL), and the combined organic phases
from this last extraction was washed with brine (50 mL), dried
over Na2SO4, and concentrated in vacuum, providing compound 7
as a colorless oil (5.69 g, 35.3 mmol, 73%): 1H NMR (CDCl3,
300 MHz) δ 4.48 (2H, s), 4.00 (2H, q, J ) 7.2 Hz), 2.01 (3H, s),
1.27 (3H, t, J ) 7.2 Hz); 13C NMR (CDCl3, 75 MHz) δ 174.5,
165.0, 70.2, 62.8, 14.2, 14.0; DCI-MS calcd for C6H11NO4 161.2,
found m/z 162.0 (M + H)+.
N-Hydroxysuccinimidyl 2-(1-Ethoxyethylideneaminooxy)ac-
etate 8. To a stirred solution of compound 7 (5.69 g, 35.3 mmol)
and N-hydroxysuccinimide (4.06 g, 35.3 mmol) in ethyl acetate/
dioxane (120 mL, 1:1) at 0 °C was added DCC (7.28 g, 35.3 mmol)
in one portion. The resulting mixture was stirred at room temper-
ature for 5 h. The formed DCU was filtered off and the filtrate
concentrated under vacuum. The obtained residue was dissolved
in ethyl acetate (300 mL), and the solution was washed with 5%
aqueous NaHCO3 (2 × 75 mL), water (75 mL), and brine (75 mL).
The organic solution was dried over Na2SO4 and evaporated under
vacuum to oil which was used without further purification (8.69 g,
33.7 mmol, 95%): analytical HPLC tR ) 7.6 min; 1H NMR (CDCl3,
300 MHz) δ 4.78 (2H, s), 4.01 (2H, q, J ) 7.2 Hz), 2.84 (4H, s),
1.98 (3H, s), 1.28 (3H, t, J ) 7.2 Hz).
N′,N′-Bis-Boc-aminooxyacetyl-N-hydroxysuccinimide Ester 6.
To a stirred solution of N,N′-bis-Boc-amino-oxyacetic acid (0.500
g, 1.72 mmol) in ethyl acetate/dioxaen (1:1, 5.5 mL) cooled on ice
were added N-hydroxysuccinimide (0.198 g, 1.72 mmol) and DCC
Fmoc-Lys[N-Eei-Aoa]-OH 3. To a stirred mixture of Fmoc-
lysine (6.22 g, 16.9 mmol) and DIPEA (3.0 mL, 17.3 mmol) in
methylene chloride (100 mL) at room temperature was added
dropwise over 20 min a solution of the above-prepared NHS-ester
J. Org. Chem, Vol. 73, No. 3, 2008 989