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twice with diethyl ether precooled to ꢀ788C (dry ice/acetone) to
give cyclo[(-l-Lys-d-Leu-l-Trp-d-Leu)2] as an off-white powder:
124 mg, 96% yield; 1H NMR (400 MHz, [D1]TFA): d=8.13 (d, J=
8 Hz, 2H, 2ꢂTrp-CHaromatic), 7.65 (d, J=8 Hz, 2H, 2ꢂTrp-CHaromatic),
7.55 (s, 2H, 2ꢂTrp-CHaromatic), 7.26–7.42 (m, 4H, 4ꢂTrp-CHaromatic),
5.22 (t, J=8 Hz, 2H, 2ꢂTrp-a-H), 4.60–4.87 (m, 6H, 4ꢂLeu-a-H+
2ꢂLys-a-H), 3.04–3.37 (m, 8H, 2ꢂLys-CH2-N+2ꢂTrp-a-CH2), 0.55–
2.00 ppm (br m, 48H, 2ꢂLys-a-CH2-CH2-CH2 +4ꢂLeu-a-CH2-CH+
8ꢂLeu-CH3); ESI-ToF MS: m/z found=1081.0 (M+H)+; ATR-FTIR:
nmax =3273 (N-Hstr), 1627 cmꢀ1 (C=Ostr).
CH2), 1.42 (s, 64H, 32ꢂpBA-CH2), 0.98 (s, 96H, 32ꢂpBA-CH3), 0.73–
0.89 (m, 24H, 8ꢂLeu-CH3), 0.45 (d, J=10.15 Hz, 3H, PPBTCr-CH3),
0.15–0.22 ppm (m, 3H, PPBTCz-CH3); ATR-FTIR nmax =3274 (NꢀHstr),
3045–2750 (CꢀHstr), 1728 (C=Op(BA)), 1625 cmꢀ1 (C=OCPꢀstr).
CP2-(pBA30)2: CP2 (10 mg, 0.008 mmol), pBA30 (0.071 g,
0.017 mmol), sodium ascorbate (0.016 g, 0.081 mmol) and
CuSO4·5H2O (0.009 g, 0.036 mmol) were suspended in DMF (3 mL),
then the mixture was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure. In an attempt to push the reaction to completion,
r-Terminal alkyne RAFT agent (propynyl 2-propanoate)yl butyl tri-
thiocarbonate (PPBTC) was prepared following a similar protocol to
that described previously.[50] r-Terminal N-succinimidyl ester RAFT
agent (NHS-PABTC) was prepared following a previously described
protocol.[35,41] Polymers with a-alkyne and a-NHS functional group
were synthesised by RAFT polymerisation.
a
second microwave irradiation was performed after adding
sodium ascorbate (0.016 g, 0.081 mmol) and CuSO4·5H2O (0.009 g,
0.036 mmol) in DMF (3 mL). The crude product was dissolved in
CHCl3 and washed with an aqueous solution of EDTA (2ꢂ30 mL,
0.055m, pH 8.5), then water (30 mL), and dried over MgSO4. After
concentration in vacuo, CP2-(pBA30)2 was obtained as a dark-
Poly(n-butyl acrylate) with an alkyne end group was synthesised
by RAFT polymerisation: PPBTC (0.25 g, 0.9 mmol), BA (3.9 g,
31.5 mmol) and AIBN (0.015 g, 0.09 mmol) was mixed and diluted
to 40% (w/w) with dioxane, cooled in an ice bath and purged with
N2(g) for 15 min before stirring at 708C under an atmosphere of
N2(g) until 79% conversion determined by 1H NMR. The polymer
was precipitated and washed with ice-cold water/MeOH (1:9) to
brown film: 54 mg, <100% conversion (ATR-FTIR); ATR-FTIR nmax
=
3272 (NꢀHstr), 3040–2800 (CꢀHstr), 2098 (N3), 1729 (C=Op(BA)),
1624 cmꢀ1 (C=OCPꢀstr).
CP2-(pBA30)1.5
.
CP2: (5 mg, 0.004 mmol), pBA30 (0.024 g,
0.006 mmol), sodium ascorbate (0.008 g, 0.040 mmol) and
CuSO4.5H2O (0.005 g, 0.020 mmol) were suspended in DMF
(2.5 mL), then the mixture was placed in a microwave reactor and
irradiated (dynamic mode) at 1008C for 15 min with a flow of N2(g)
and delivering 200 W in the initial ramp. The DMF was removed
under reduced pressure, then the crude product was dissolved in
CHCl3 and washed with an aqueous solution of ethylenediaminete-
traacetic acid (EDTA) (2ꢂ30 mL, 0.055m, pH 8.5), then water
(30 mL), and dried over MgSO4. After concentration in vacuo, CP2-
(pBA30)1.5 was obtained as a brown film: 20 mg, ꢂ100% conver-
sion (ATR-FTIR); ATR-FTIR nmax =3270 (NꢀHstr), 3040–2800 (CꢀHstr),
2097 (N3), 1732 (C=Op(BA)), 1625 cmꢀ1 (C=OCPꢀstr).
CP3-(pBA30)2: CP3 (10 mg, 0.008 mmol), pBA30 (0.085 g,
0.021 mmol), sodium ascorbate (0.020 g, 0.098 mmol) and
CuSO4.5H2O (0.010 g, 0.039 mmol) were suspended in DMF (2 mL)
then the mixtrue was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure and the crude product was dissolved in DCM and
washed with an aqueous solution of EDTA (2ꢂ30 mL, 0.055m,
pH 8.5), then water (30 mL), and dried over MgSO4. After concen-
tration in vacuo, CP3-(pBA30)2 was obtained as a dark-brown film:
52 mg, ꢂ100% conversion (ATR-FTIR); ATR-FTIR nmax =3279 (N-Hstr),
3050–2800 (C-Hstr), 1733 (C=Op(BA)), 1631 cmꢀ1 (C=OCPꢀstr).
yield pBA30 as a yellow viscous liquid: Mn (1H NMR)=4200 gmolꢀ1
;
1H NMR (400 MHz, CDCl3): d=4.78–4.88 (m, 1H, pBA-S-CH), 4.58–
4.72 (m, 2H, PPBTCR-COO-CH2), 3.80–4.30 (m, 30ꢂ2H, pBA-COO-
CH2), 3.34 (t, J=6 Hz, 2H, S-CH2), 0.70–2.60 ppm (m, 311H, PPBTCZ-
CH2-CH2-CH3 +30ꢂpBA-CH2-CH2-CH3 +30ꢂpBAbackbone-CH-CH2 +
PPBTCR-CH3 +PPBTCR-CꢁCH);
SEC-DRI(DMF+LiBr):
Mn =
3500 gmolꢀ1, ꢀ=1.16. MALDI-ToF MS: (M+Na)+ observed; repeat-
ing unit m/z=128, end group m/z=276.
Poly(n-butyl acrylate) with an N-succinimidyl ester end group was
synthesised by RAFT polymerisation. NHS-PABTC (0.075 g,
0.22 mmol), BA (1.04 g, 7.8 mmol), ACVA (0.018 g, 0.0065 mmol) in
dioxane (0.94 g) was purged with N2(g) for 15 min before stirring at
708C under an atmosphere of N2(g) until 94% conversion deter-
mined by 1H NMR. The polymer was twice precipitated and
washed with water/MeOH (1:9) precooled to ꢀ788C (dry ice/ace-
tone) to yield pBA33 as a yellow viscous liquid: Mn (1H NMR)=
;
4600 gmolꢀ1 1H NMR (300 MHz, CDCl3): d=4.76–4.89 (br t, 1H,
pBA-S-CH), 3.86–4.20 (m, 33ꢂ2H, pBA-COO-CH2), 3.34 (t, J=6 Hz,
2H, S-CH2), 2.67–2.89 (m, 5H, NHS-CH2-CH2-+R-CH-COO), 0.66–
2.67 ppm (m, 339H, Z-CH2-CH2-CH3 +33ꢂpBA-CH2-CH2-CH3 +33ꢂ
pBAbackbone-CH-CH2 +R-CH3);
4700 gmolꢀ1, ꢀ=1.09.
SEC-DRI(CHCl3+TEA):
Mn =
CP3-(pBA30)1.5
:
CP3 (10 mg, 0.008 mmol), pBA30 (0.062 g,
CP1-(pBA30)2 was synthesised following a previously described
protocol.[29] CP1 (40 mg, 0.035 mmol) was suspended in 2,2,2-tri-
fluoroethanol (TFE) (2 mL) by sonication for 10 min. PBA30 (0.31 g,
0.074 mmol), sodium ascorbate (0.14 g, 0.71 mmol) and
CuSO4·5H2O (0.037 g, 0.14 mmol) in DMF (3 mL) were added to the
cyclic peptide solution, then the mixture was placed in a microwave
reactor and irradiated (dynamic mode) at 1008C for 15 min with
a flow of N2(g) and delivering 200 W in the initial ramp. TFE and
DMF were removed under reduced pressure. The crude product
was dissolved in DCM and washed with an aqueous solution of
EDTA (2ꢂ30 mL, 0.055m, pH 8.5), then water (30 mL), and dried
over MgSO4. After concentration in vacuo, CP1-(pBA30)2 was ob-
tained as a brown film: 145 mg, 100% conversion (ATR-FTIR);
1H NMR (300 MHz, [D1]TFA): d=8.48 (s, 2H, 2ꢂtriazole-CH), 7.60 (s,
2H, 2ꢂTrp-CHaromatic), 7.34 (s, 2H, 2ꢂTrp-CHaromatic), 7.02–7.26 (m,
6H, 6ꢂTrp-CHaromatic), 5.46 (s, 4H, 2ꢂO-CH2-triazole), 5.18 (s, 2H, 2ꢂ
Trp-a-H), 4.52–4.87 (m, 6H, 4ꢂLeu-a-H+2ꢂLys-a-H), 4.24 (s, 64H,
32ꢂpBA-O-CH2), 3.95 (s, 2H, PPBTCz-CH2), 1.71 (s, 64H, 32ꢂpBA-
0.015 mmol), sodium ascorbate (0.021 g, 0.11 mmol) and
CuSO4.5H2O (0.011 g, 0.042 mmol) were suspended in DMF (2 mL),
then the mixture was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure. The crude product was dissolved in DCM and
washed with an aqueous solution of EDTA (2ꢂ30 mL, 0.055m,
pH 8.5), then water (30 mL), and dried over MgSO4. After concen-
tration in vacuo, CP3-(pBA30)1.5 was obtained as a dark brown film:
49 mg, ꢂ100% conversion (ATR-FTIR); ATR-FTIR nmax =3280 (Nꢀ
Hstr), 3060–2800 (CꢀHstr), 1733 (C=Op(BA)), 1631 cmꢀ1 (C=OCPꢀstr).
CP-(pBA33)2 by NHS conjugation: Cyclo[(-l-Lys-d-Leu-l-Trp-d-
Leu)2] (0.020 g, 0.015 mmol), pBA33 (0.202 g, 0.044 mmol) and
NMM (0.093 g, 0.092 mmol) were suspended in a mixture DMF/
DMSO (1:3 vol, 4 mL) and stirred for 4 days at room temperature.
Solvents were removed with a stream of N2(g) and the product was
redissolved in DCM, then precipitated into dry ice/acetone-cold
Chem. Eur. J. 2016, 22, 1 – 11
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ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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