L. Pes et al. / Bioorg. Med. Chem. 24 (2016) 1706–1717
1711
13 (170 mg, 0.174 mmol) was dissolved in DCM and 2% v/v of
DBU was added. The reaction was stirred for 30 min and then
water was mixed. The aqueous layer was extracted with DCM
(3 ꢀ 20 mL), the organic layers were combined, washed with acetic
acid 5% and the solvent was removed in vacuo. The product was
purified by prep HPLC from 50% to 100% of phase B over 45 min,
to achieve product 14 as a clear solid (94 mg, 72% yield). Retention
time analytical HPLC from 30% to 100% of phase B over 30 min:
19.295. 1H NMR, d (500 MHz, CDCl3), 8.12 (2H, br s, NH2), 7.94
(1H, d, J = 6.8 Hz, CHNHCO), 7.34–7.14 (21H, m, Ph and NHCH2-
CHPh2), 6.24 (1H, t, J = 5.5 Hz, NHCH2CHPh2), 4.26 (1H, t,
J = 8.1 Hz, CHPh2), 4.22–4.14 (2H, m, CHPh2 and CH Glu), 4.00–
3.95 (1H, m, CH2CHPh2), 3.81 (2H, t, J = 6.5 Hz, CH2CHPh2), 3.76–
3.73 (17H, m, CH2CHPh2 and 16 PEG), 3.12 (2H, br s, NH2CH2
PEG), 2.51–2.38 (2H, m, COCH2 PEG), 1.93–1.83 (2H, m, CH2CO
Glu), 1.78–1.72 (2H, m, CHCH2 Glu). 13C NMR d (126 MHz, CDCl3)
173.23, 172.25 and 171.68 (3 CONH), 142.16, 128.82, 128.76,
128.73, 128.29, 128.25, 128.21, 126.90 and 126.83 (Ph), 70.39,
70.28, 70.11, 69.97, 69.83 and 67.14 (PEG), 53.33 (CH Glu), 50.57
(2CHPh2), 44.06 (CH2CHPh2), 43.95 (CH2CHPh2), 39.92 (NH2CH2
PEG), 35.78 (COCH2 PEG), 32.38 (CH2CO Glu), 28.57 (CHCH2 Glu).
ESI-MS m/z, M+H+ = 753.7, calculated for C44H56N4O7 = 752.4.
Glu), 50.07 (CHPh2), 49.96 (CHPh2), 43.70 (CH2CHPh2), 43.11 (CH2-
CHPh2), 35.79 (COCH2 PEG), 31.53 (CH2CO Glu), 28.22 (CHCH2 Glu).
ESI-MS m/z, M+H+ = 1142.9, calculated for C65H67N5O12S = 1141.5.
2.3.16. Synthesis of MTP
Fmoc-Bip-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(ivDde)-
OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Lys(ivDde)-OH, Fmoc-
Gly-OH, Fmoc-Gly-OH, and Fmoc-b-Ala-OH were coupled sequen-
tially to the solid support, Fmoc-Bip-OH was then coupled to the
free e-amino group of the Lys residues and FITC was grafted to
the peptide on the N-terminal beta alanine as described in previous
work.9 The fully deprotected FITC labeled peptide was cleaved
from the resin with cleaving cocktail A and purified with prep HPLC
from 0% to 100% of phase B over 45 min to achieve the product as a
yellow powder. Retention time analytical HPLC from 0% to 100% of
phase B over 30 min: 18.143 min. MALDI-TOF m/z, M+H+ = 1745.5,
calculated for C93H100N16O17S = 1744.7.
2.3.17. Synthesis of Bip1
Fmoc-Bip-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser
(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Gly-
OH were coupled sequentially to the solid support as described
in the general procedure. FITC was grafted to the peptide on the
2.3.15. Synthesis of 2-(1-((30,60-dihydroxy-3-oxo-3H-spiro[isoben
zofuran-1,90-xanthen]-5-yl)amino)-1-thioxo-5,8,11,14-tetraoxa-
2-azaheptadecan-17-amido)-N1,N5-bis(2,2-diphenylethyl)pen-
tanediamide (PEG4Bip2, 15)
e-amine of lysine. The fully deprotected FITC labeled peptide was
cleaved from the resin with cleaving cocktail B and purified with
O
H
O
H
N
N
N
H
N
H
FITC (2)
O
O
DIPEA
O
O
FITC
4
4
N
H
O
O
H2N
DMF
overnight
HN
HN
15
14
14 (94 mg, 0.12 mmol) was dissolved in DMF (5 mL) and FITC
was added (0.140 g, 0.36 mmol), followed by DIPEA (1 mL). The
reaction was left stirring in darkness overnight. The solvent was
removed in vacuo, the crude product was redissolved in DCM
and brine was mixed. The aqueous layer was extracted with
DCM (5 ꢀ 20 mL), the organic layers were combined, dried over
NaSO4 and the solvent was removed in vacuo. The crude product
was purified by prep HPLC from 30% to 100% of phase B over
45 min to achieve product 15 as a yellow solid (75 mg, 55% yield).
Retention time analytical HPLC from 30% to 100% of phase B over
30 min: 17.923 min. 1H NMR, d (500 MHz, DMSO), 10.14 (1H, br
s, OH FITC), 10.05 (1H, br. s, OH FITC), 8.30 (1H, br s, FITC), 8.11
(1H, br s, NHCH2CHPh2), 7.90 (1H, d, J = 8.1 Hz, CHNHCO), 7.85
(1H, t, J = 5.6 Hz, NHCH2CHPh2), 7.76–7.71 (2H, m, FITC and NH
PEG), 7.31–7.15 (22H, m, Ph, NH FITC and FITC), 6.70 (2H, d,
J = 2.3 Hz, FITC), 6.63 (2H, d, J = 8.7 Hz, FITC), 6.59–6.57 (2H, dd,
J = 2.3 and 8.7 Hz, FITC), 4.19–4.15 (2H, dd, J = 7.7 and 15.1 Hz,
2CHPh2), 4.13–4.08 (1H, m, CH Glu), 3.78–3.46 (22H, m, 2CH2-
CHPh2 and PEG), 2.40–2.27 (2H, m, COCH2 PEG), 1.84–1.81 (2H,
m, CH2CO Glu), 1.66–1.61 (1H, m, CHCH2 Glu), 1.51–1.49 (1H, m,
CHCH2 Glu). 13C NMR d (126 MHz, DMSO) 180.54 (CS FITC),
171.56, 171.20, 169.88, 168.52, 159.50, 151.89 and 147.10 (FITC),
142.93, 142.71, 141.34, 129.04, 128.39, 128.36, 127.85 and
126.29 (Ph and FITC), 124.06, 116.35, 112.59, 109.72 and 102.24
(FITC), 69.75, 69.65, 69.45, 68.43 and 66.73 (PEG), 51.99 (CH
prep HPLC from 0% to 100% of phase B over 45 min to achieve
the product as a yellow powder. Retention time analytical HPLC
from 0% to 100% of phase B over 30 min: 13.823 min. MALDI-TOF
m/z, M+H+ = 2139.2, calculated for C94H139N37O20S = 2138.1.
2.3.18. Synthesis of Bip2
Fmoc-Bip-OH, Fmoc-Bip-OH, Fmoc-Ser(Bu)-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg
(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(ivDde)-OH,
Fmoc-Gly-OH were coupled sequentially to the solid support as
described in the general procedure. FITC was grafted to the peptide
on the e-amine of lysine. The fully deprotected FITC labeled pep-
tide was cleaved from the resin with cleaving cocktail B and puri-
fied with prep HPLC from 0% to 100% of phase B over 45 min to
achieve the product as a yellow powder. Retention time analytical
HPLC from 0% to 100% of phase B over 30 min: 16.063 min. MALDI-
TOF m/z, M+H+ = 2362.2, calculated for C109H152N38O21S = 2361.2.
2.3.19. Synthesis of RRsBip2
Fmoc-Bip-OH, Fmoc-Bip-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg
(Pbf)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Gly-OH were coupled
sequentially to the solid support as described in the general proce-
dure. FITC was grafted to the peptide on the e-amine of lysine. The