Bioconjugate Chemistry
ARTICLE
1H, J = 10.7, 3.3 Hz), 5.97ꢀ5.86 (m, 1H), 5.85 (d, 1H, J = 8.2 Hz),
5.65 (d, 1H, J = 3.1 Hz), 5.46 (dd, 1H, J = 10.8, 6.0 Hz), 5.35 (d, 1H,
J = 17.1 Hz), 5.27 (d, 1H, J = 10.4 Hz), 4.73 (ddd, 1H, J = 8.2, 5.2,
5.2 Hz), 4.64 (d, 2H, J = 5.6 Hz), 4.60 (dd, 1H, J = 6.4, 6.4 Hz), 4.39
(d, 2H, J = 7.1 Hz), 4.24 (dd, 1H, J = 7.0, 7.0 Hz), 4.10 (dd, 1H,
J = 11.3, 6.6 Hz), 4.00 (dd, 1H, J = 11.3, 6.5 Hz), 3.32 (d, 2H,
J= 5.1 Hz), 2.19 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.88 (s, 3H). 13C
NMR (100 MHz, CDCl3): δppm 170.6, 170.6, 170.3, 170.0, 169.4,
155.9, 143.8, 143.7, 142.0, 141.3, 141.2, 131.4, 127.8, 127.1, 127.1,
125.1, 124.5, 120.0, 119.2, 81.7, 70.5, 67.7, 67.5, 67.2, 67.2, 66.2, 61.2,
53.4, 47.1, 28.0, 20.61, 20.61, 20.6, 20.3. ESI-MS m/z calcd
for C37H40N4O13 [MþH]þ: 749.2665; [MþNa]þ: 771.2490.
Found: 749.22, 771.20.
(2S)-1-Allyl-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-
3-(4-(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosylethyl)-2,3,
4-triazol-1-yl)-propanoate (17). Compounds 13 (10 mg,
0.027 mmol) and 16 (11 mg, 0.027 mmol) were treated with
the same conditions as the preparation of 15. The crude
product 17 was purified by preparative TLC (7:3 ethyl acetate:
hexanes). 1H NMR (400 MHz, CDCl3): δppm 7.76 (d, 2H, J =
7.5 Hz), 7.59 (t, 2H, J = 7.4 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.37
(s, 1H), 7.31 (t, 2H, J = 7.5, 7.5 Hz), 5.94 (d, 1H, J = 8.9 Hz),
5.89 (m, 1H), 5.41 (t, 1H, J = 2.8, 2.8 Hz), 5.32 (d, 1H, J = 17.1
Hz), 5.25 (dd, 1H, J = 10.4, 1.1 Hz), 5.20 (d, 1H, J = 4.5 Hz),
5.17 (dd, 1H, J = 8.7, 3.1 Hz), 4.73 (dd, 1H, J = 13.3, 5.4 Hz),
4.66 (d, 2H, J = 5.7 Hz), 4.46 (ddd, 1H, J = 12.9, 7.9, 4.8 Hz),
4.41ꢀ4.27 (m, 4H), 4.23 (dd, 1H, J = 7.2, 7.2 Hz), 4.19ꢀ4.13 (m,
1H), 4.11ꢀ4.02 (m, 2H), 3.31 (d, 2H, J = 4.9 Hz), 2.31ꢀ2.17 (m,
1H), 2.10 (s, 3H), 2.07 (s, 6H), 2.03 (s, 3H). 13C NMR (100
MHz, CDCl3): δppm 170.8, 170.6, 169.9, 169.7, 169.7, 156.0,
143.8, 143.8, 142.7, 141.2, 141.2, 131.6, 127.7, 127.1, 125.2,
125.2, 122.8, 120.0, 118.7, 77.5, 77.2, 76.9, 69.2, 68.6, 68.0,
67.7, 67.1, 66.1, 61.1, 53.5, 47.1, 46.4, 28.1, 27.1, 20.77, 20.74,
20.71, 20.63. ESI-MS m/z calcd for C39H44N4O13 [MþH]þ:
777.2978. Found: 777.21.
protocols.34 5 mg of resin was directly cleaved and the crude
peptide was purified through preparatory TLC (30% water in
methanol) to yield 0.8 mg of 18 as a white powder (91% based on
resin loading). H NMR (400 MHz, D2O): δppm 3.94ꢀ3.78
1
(m, 5H), 3.59 (d, 2H, J = 1.35 Hz), 2.64 (d, 2H, J = 3.23 Hz, 2H),
2.36 (t, 1H, J = 2.21). 13C NMR (100 MHz, D2O): δppm 171.4,
171.32, 171.31, 168.9, 76.2, 74.4, 51.4, 42.6, 42.4, 42.3, 21.0. ESI-MS
m/z calcd for C11H16N4O5 [MþH]þ: 285.1199. Found: 285.072.
Resin-Supported Peptide 20. Peptide 20 was prepared using
115 mg of Wang resin using standard Fmoc-based SPS
protocols.34 18 mg of resin was cleaved directly and purified by
reversed-phase HPLC to yield 3.0 mg of 20 as a white powder in
78% yield (based on resin loading). 1H NMR (400 MHz, D2O):
δppm 7.40ꢀ7.15 (m, 5H), 4.60 (dd, 1H, J = 8.2, 6.6 Hz), 4.30
(q, 1H, J = 7.3 Hz), 4.19 (t, 1H, J = 6.1 Hz), 3.91 (q, 2H, J =
16.9 Hz), 3.11 (dd, 1H, J = 13.9, 6.5 Hz), 2.96 (dd, 1H, J = 13.9,
8.3 Hz), 2.84 (td, 2H, J = 3.3, 2.9 Hz), 2.52 (t, 1H, J = 2.7 Hz),
1.33 (d, 3H, J = 7.3 Hz). 13C NMR (125 MHz, D2O): δppm
176.7, 173.2, 171.0, 169.3, 136.8, 129.9, 129.4, 127.8, 83.2, 75.1,
55.4, 52.0, 49.3, 42.869, 42.861, 37.9, 21.7. ESI-MS m/z calcd for
C19H24N4O5 [MþH]þ: 389.182; [MþNa]þ: 411.164. Found:
389.19, 411.17.
Resin-Supported Peptide 23. Peptide 23 was prepared using
115 mg of Wang resin according to standard Fmoc-based SPS
protocols.34 10 mg of resin was cleaved directly and purified by
reversed-phase HPLC to yield 1.8 mg of 23 as a white powder in
70% yield (based on resin loading). 1H NMR (400 MHz, D2O):
δppm 4.58 (m, 3H), 4.29 (t, 1H, J = 5.9 Hz), 4.19ꢀ3.79 (m, 18H),
2.93 (dd, 2H, J = 5.9, 2.8 Hz), 2.76 (bd, 6H, J = 6.1 Hz), 2.60 (t,
1H, J = 2.5 Hz), 2.46 (t, 3H, J = 2.4 Hz). 13C NMR (125 MHz,
D2O): δppm 172.7, 171.8, 171.5, 171.3, 168.8, 79.2, 76.2, 74.3,
72.2, 52.4, 52.3, 51.3, 42.5, 42.4, 42.2, 20.8. ESI-MS m/z calcd for
C38H49N13O14 [MþH]þ: 912.3595; [MþNa]þ: 934.3420.
Found: 912.34, 934.32.
Resin-Supported Peptide 25. Peptide 25 was prepared using
150 mg of Wang resin using standard Fmoc-based SPS protocols
with amino acid 26 (Scheme 3b).34 20 mg of resin was directly
cleaved and the crude peptide was purified by reversed-phase
HPLC (isocratic flow with 10% acetonitrile in water for 5 min
followed by a linear gradient from 10% to 60% acetonitrile in
water) to yield 4.1 mg of 25 as a white powder in 48% yield
(based on resin loading). 1H NMR (500 MHz, D2O, presatura-
tion on HOD): δppm 4.36 (m, 3H), 3.90ꢀ3.75 (m, 17H), 3.60
(s, 2H), 2.28ꢀ2.09 (m, 12H), 1.91 (m, 4H), 1.80 (m, 4H). 13C
NMR (125 MHz, D2O): δppm 176.4, 174.2, 174.2, 174.1, 171.9,
171.8, 171.7, 171.6, 171.5, 170.9, 83.3, 82.6, 70.8, 70.3, 52.9, 52.8,
52.8, 52.6, 43.1, 42.5, 42.4, 42.3, 29.3, 29.2, 14.4, 13.9. ESI-MS
m/z calcd for C42H58N13O14 [MþH]þ: 968.42. Found: 968.76.
(S)-2-(9-Fluorenylmethoxycarbonylamino)-hex-5-ynoic
Acid (26). Amino acid 26 was synthesized as indicated in Scheme
2 in the Supporting Information. 1H NMR (CDCl3/CD3OD 2/
1, 400 MHz): δppm 7.71 (d, 2H, J = 7.6 Hz), 7.58 (dd, 2H, J = 7.1,
3.9 Hz), 7.35 (t, 2H, J = 7.6 Hz), 7.27 (t, 2H, J = 7.6 Hz), 4.35
(m, 3H), 4.18 (t, 1H, J = 6.8 Hz), 2.24 (m, 2H), 2.09 (m, 1H),
2.01 (t, 1H, J = 2.6 Hz), 1.86 (m, 1H). 13C NMR (CDCl3/
CD3OD 2/1, 100 MHz): δppm 173.9, 156.7, 143.8, 143.7, 141.2,
141.2, 127.7, 127.0, 125.0, 119.8, 82.6, 77.5, 69.3, 66.9, 53.0, 47.1,
30.9, 14.9. ESI-MS m/z calcd for C21H19NO4 [M þ Na]þ:
372.12. Found: 372.24.
General Method for Peptide Synthesis. The syntheses of
the peptides were carried out using an Advanced Chem Tech
Apex 396 automated peptide synthesizer (40 wells), equipped
with a dual-arm system and argon atmosphere controlled from an
IBM PC using Advanced Chem Tech v 1.6 software. Preloaded
Fmoc-Gly-Wang or Fmoc-Ala-Wang resins were swollen in
DMF for 1 h followed by filtration, and then were subjected to
20% piperidine in DMF twice successively for 30 min. Peptide
couplings were performed using standard conditions for Fmoc
solid-phase synthesis using HCTU as coupling agent.34 Follow-
ing the coupling of each residue, deprotection of the Fmoc
moiety was accomplished by treatment with 20% piperidine in
DMF. Between each deprotection and coupling, and after every
coupling, the beads were shaken 4 times with 4 mL of DMF
followed by filtration. Upon completion of the synthesis, the
beads were washed extensively with DMF (6 ꢁ 4 mL), MeOH
(6 ꢁ 4 mL), DCM (6 ꢁ 4 mL), hexanes (6 ꢁ 4 mL), and
ethanol (3 ꢁ 6 mL) and then removed from the synthesizer
and stored in a desiccator under vacuum in the presence of
P2O5 until required. A small amount of the peptide was
cleaved from the resin for characterization using 92.5:5:2.5
(v/v/v) TFA:triisopropylsilane:water. The peptides were
purified using reversed-phase HPLC (ramp from 0% to 18%
acetonitrile in water over 5 min followed by isocratic flow for
25 min) unless stated otherwise.
General Protocol for the Solid-Phase Organic Synthesis
Microwave “Click” Reaction. A CEM Mars X Microwave
System with 300 W maximum power and quartz reaction vessels
Resin-Supported Peptide 18. Peptide 18 was prepared
using 100 mg of Wang resin using standard Fmoc-based SPS
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dx.doi.org/10.1021/bc100394k |Bioconjugate Chem. 2011, 22, 605–616