Bioconjugate Chemistry
Article
yl)-methyl]-carbamic Acid tert-Butyl Ester (6a). {2-[2-(2-tert-
Butoxycarbonylamino-acetylamino)-acetylamino]-acetylami-
no}-acetic acid (4; 70 mg, 0.20 mmol) was first sonicated to
completely dissolve in 20 mL DMF, followed by submerging
the reaction mix in an ice−water bath. EDC (40 mg, 0.40
mmol) and HOBt (65 mg, 0.4 mmol) were then added to the
reaction mixture to activate 4 for 15 min, followed by addition
of a solution of mono-t-Boc-ethylenediamine (5a; 80 mg, 0.50
mmol) and DIPEA (0.035 mL, 0.40 mmol) in 5 mL of DMF to
initiate the coupling reaction at rt for 6 h. Compound 5a was
previously synthesized according to similar reported procedures
for 5b synthesis.20 The final reaction solution was filtrated,
evaporated in vacuo, resuspended in 25% MeOH in DCM, and
loaded to a silica column for further purification. The products
were separated by eluting with mobile phases of 25% MeOH in
colored oil-like TFA salt of 7a. The obtained TFA salt of 7a was
further dissolved in 5 mL of a DCM/MeOH mixture (DCM/
MeOH = 1:1) and mixed with 10 equiv of Amberlyst A-21
(0.14 mg, 1.1 mmol) while rocking for 30 min.20 After filtering
to remove Amberlyst A-21, the filtrate of the reaction products
was concentrated under reduced pressure to give white solid 7a
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(30 mg, 95%). H NMR (400 MHz) (DMSO-d6) δ: 8.65 (t,
1H, NHCH2CH2), 8.32 (t, 1H, Gly4-NH), 8.17 (t, 1H, GIy3-
NH), 8.06 (t, 1H, Gly2-NH), 7.80−8.02 (br, 4H, Gly1-NH2,
CH2CH2NH2), 3.85 (d, 2H, Gly4α-H), 3.77 (d, 2H, Gly3α-H),
3.71 (d, 2H, Gly2α-H), 3.62 (s, 2H, Gly1α-H), 3.30 (q, 2H,
CH2NH), 2.85 (q, 2H, NHCH2). 13C NMR (DMSO-d6) δ:
169.6, 169.1, 168.8, 166.4, 42.0, 42.0., 42.0, 39.0, 38.5, 36.3.
HRMS (ESI) calculated for C10H21N6O4, [M+H]+ 289.1624
(calcd.), 289.1626 (found).
1
2-Amino-N-{[({[(6-amino-hexylcarbamoyl)-methyl]-carba-
moyl}-methyl)-carbamoyl]-methyl}-acetamide (7b). [6-(2-
{2-[2-(2-tert-Butoxycarbonylamino-acetylamino)-acetylamino]-
acetylamino}-acetylamino)-hexyl]-carbamic acid tert-butyl ester
(6b; 60 mg, 0.11 mmol) was dissolved in a TFA/DCM solution
(1:1; 4 mL), stirred at 0 °C for 1 h, and then reacted at rt for 1
h. The product was evaporated under reduced pressure, washed
by ether and chloroform, sequentially, and again concentrated
under reduced pressure to remove ether and chloroform to
obtain the brown oil-like TFA salt of 7b. The acquired TFA salt
of 7b (0.52 g, 0.88 mmol) was further dissolved in 5 mL of
DCM/MeOH mixture (DCM/MeOH = 1:1) and mixed with
10 equiv of Amberlyst A-21 (0.14 mg, 1.1 mmol) while rocking
for 30 min.20 After filtering to remove Amberlyst A-21, the
filtrate of the reaction products was concentrated under
DCM to afford 6a (50 mg; 50%) as light yellowish solid. H
NMR (400 MHz) (DMSO-d6) δ: 8.17 (t, 1H,
CONHCH2CH2), 8.10−8.04 (m, 2H, Gly1-NH, CH2CH2NH
CO2 C(CH3)3), 7.80 (t, 1H, Gly4-NH), 6.99 (t, 1H, GIy3-NH),
6.80 (t, 1H, Gly2-NH), 3.74 (t, 4H, Gly4α-H, Gly1α-H), 3.66 (d,
2H, Gly3α-H), 3.58 (d, 2H, Gly2α-H), 3.08 (q, 2H,NHCH2),
2.98 (q, 2H, CH2NH), 1.37 [d, 18H, CO2C(CH3)3 ]. 13C
NMR (DMSO-d6) δ: 169.9, 169.4, 169.0, 168.8, 155.8, 155.6,
78.2, 77.7, 48.6, 43.3., 42.1, 42.1, 42.0, 40.1, 28.2, 28.2. HRMS
(ESI) calculated for C20H36N6O8 Na, [M+Na]+ 511.2492
(calcd.), 511.2490 (found).
[6-(2-{2-[2-(2-tert-Butoxycarbonylamino-acetylamino)-
acetylamino]-acetylamino}-acetylamino)-hexyl]-carbamic
Acid tert-Butyl Ester (6b). {2-[2-(2-tert-Butoxycarbonylamino-
acetylamino)-acetylamino]-acetylamino}-acetic acid (4; 100
mg, 0.29 mmol) was first sonicated to completely dissolve in
20 mL DMF, followed by immersing the reaction mix in an
ice−water bath. EDC (110 mg, 0.58 mmol) and HOBt (78 mg)
were initially added to the reaction mix to activate 4 for 15 min,
followed by addition of a solution of mono-t-Boc-hexanedi-
amine (5b; 63 mg, 0.29 mmol)20 and DIPEA (0.1 mL, 0.29
mmol) in 5 mL of DMF to commence the coupling reaction at
rt for 6 h. The final reaction mixture was filtrated, evaporated in
vacuo, redissolved in 10% MeOH in DCM, and loaded to a
silica column for further purification. The products were
separated by eluting with mobile phases of 10% MeOH in
DCM to afford 6b (85 mg; 54%) as a yellowish solid. 1H NMR
(400 MHz) (DMSO-d6) δ: 8.16 (t, 1H, CONHCH2CH2),
8.04−8.08 [m, 2H, Gly4-NH, NH CO2C(CH3)3], 7.70 (t, 1H,
Gly3-NH), 7.00 (t, 1H, Gly1-NH), 6.75 (t, 1H, GIy2-NH),, 3.75
(d, 2H, Gly1α-H), 3.72 (d, 2H, Gly4α-H), 3.65 (d, 2H, Gly3α-H),
3.58 (d, 2H, Gly2α-H), 3.03 (q, 2H, NHCH2), 2.88 (q, 2H,
CH2NH), 1.37 [d, 22H, CO2C(CH3)3, NHCH2CH2 ], 1.21−
1.23 (m, 4H, CH2). 13C NMR (DMSO-d6) δ: 169.9, 169.4,
169.0, 168.4, 155.9, 155.6, 78.2, 77.3, 43.3, 42.2., 42.1, 42.0,
38.9, 38.5, 29.5, 29.1, 28.3, 28.2, 26.1, 26.0. HRMS (ESI)
calculated for C24H44N6O8 Na, [M+Na]+ 567.3118 (calcd.),
567.3115 (found).
1
reduced pressure to afford white solid 7b (50 mg, 95%). H
NMR (400 MHz) (DMSO-d6) δ: 8.68 (t, 1H, NHCH2CH2),
8.33 (t, 1H, Gly4-NH), 8.13 (t, 1H, Gly3-NH), 7.84−8.08 (br,
4H, GIy1-NH2, CH2CH2NH2), 7.79 (t, 1H, Gly2-NH), 3.84 (d,
2H, Gly4α-H), 3.75 (d, 2H, Gly3α-H), 3.66 (d, 2H, Gly2α-H),
3.62 (s, 2H, Gly1α-H), 3.04 (q, 2H, CH2NH), 2.76 (t, 2H,
CH2NH2), 1.18−1.60 (m, 8H, CH2). 13C NMR (DMSO-d6) δ:
169.0, 168.9, 168.5, 166.5, 42.1, 42.5, 42.0, 40.2, 38.8, 38.4,
28.9, 27.0,25.8,25.5. HRMS (ESI) calculated for C14H29N6O4,
[M+H]+ 345.2250 (calcd.), 345.2251 (found).
Synthesis of a N-Maleoyl Amino Acid Succinimidyl
Ester, AMAS (9). (2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-acetic
acid 2,5-dioxo-pyrrolidin-1-yl ester (9) was synthesized from
(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-acetic acid (8) which was
prepared by a previously reported method.22 The acquired 8
(69 mg, 0.45 mmol) and NHS (103 mg, 0.89 mmol) were first
dissolved in THF (10 mL), followed by dropwise addition of
DCC (184 mg, 0.89 mmol) in THF (10 mL) while stirring.
After overnight reaction at rt, the mixture was quenched by
addition of three drops of glacial acetic acid and stirring for 1 h
and filtered to remove suspension. The acquired filtrate was
concentrated under reduced pressure, washed by EtOH twice,
resuspended in 2-propanol (30 mL) while stirring for 1 h, and
filtered to separate the suspension from its filtrate. The final
solid was washed by 2-propanol and dried to afford a white-
colored 9 (96 mg; 86%). 1H NMR (400 MHz) (DMSO-d6) δ:
7.20 (s, 2H, CHCH), 4.73 (s, 2H, CH2), 2.81 (s 4H, CH2CH2)
13C NMR (DMSO-d6) δ: 169.7, 169.7, 164.4, 135.1, 36.3, 25.4.
HRMS (ESI) calculated for C10H8N2O6Na, [M+Na]+ 275.0280
(calcd.), 275.0279 (found).
2-Amino-N-{[({[(2-amino-ethylcarbamoyl)-methyl]-carba-
moyl}-methyl)-carbamoyl]-methyl}-acetamide (7a).
[({[({[(2-tert-Butoxycarbonylamino-ethylcarbamoyl)-methyl]-
carbamoyl}-methyl)-carbamoyl]-methyl}-carbamoyl)-methyl]-
carbamic acid tert-butyl ester (6a; 50 mg, 0.12 mmol) was
dissolved in a TFA/DCM solution (1:1; 4 mL), stirred at 0 °C
for 1 h, and then reacted at rt for 1 h. The product was
evaporated under reduced pressure, washed by ether and
chloroform, sequentially, and again concentrated under reduced
pressure to remove ether and chloroform to give the yellow-
Synthesis of the Thiol-Containing Biotin Derivative
(13). 5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-penta-
noic acid (2-{2-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-
C
dx.doi.org/10.1021/bc300444y | Bioconjugate Chem. XXXX, XXX, XXX−XXX