Design, synthesis and bioevaluation of an EphA2 receptor-based targeted delivery system

Article abstract of DOI:10.1002/cmdc.201400067

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2-targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long-lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate-limiting degradation sites on the peptide-drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide-drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells. Ready, aim, fire! We investigated the chemical determinants responsible for the stability and degradation in plasma of an EphA2-based targeted delivery system that is constituted by receptor-targeting peptides conjugated with paclitaxel. We demonstrate that our agents are both long-lived in plasma and markedly decrease tumor size in a prostate cancer xenograft model.

Full text of DOI:10.1002/cmdc.201400067

Supporting Information
ꢀ Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2014
Design, Synthesis and Bioevaluation of an EphA2
Receptor-Based Targeted Delivery System
Elisa Barile,[a] Si Wang,[a] Swadesh K. Das,[b] Roberta Noberini,[a] Russell Dahl,[c]
John L. Stebbins,[a] Elena B. Pasquale,[a] Paul B. Fisher,[b] and Maurizio Pellecchia*[a]
cmdc_201400067_sm_miscellaneous_information.pdf

Supporting information
Contents
S2.
Synthetic schemes, procedures and analytical data for the synthesis of YSA-L2-
PTX, YNH-L2-PTX and DYP-L2-PTX.
Figure S1: 1D 1H NMR spectrum of Paclitaxel (PTX) in methanol-d4.
Figure S2: 2D 1H-1H COSY NMR spectrum of Paclitaxel (PTX) in methanol-d4.
Figure S3: 1D 13C DEPT-135 NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S7.
S8.
S9.
S10. Figure S4: 2D 1H-13C HMBC NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S11. Figure S5: 1D 1H NMR spectrum of YSA-L1-PTX in methanol-d4.
S12. Figure S6: 1D 1H NMR of YSA-L1-PTX in 18.3% D2O, 4.5% H2O, 0.2% d6-
DMSO, 77% CD3OD
S13. Figure S7: Solubility of YSA-L2-PTX and PTX measured by 1H NMR experiments.
S14. Figure S8: Binding affinity of YSA-L2-PTX with the EphA2 binding domain,
determined by ITC.
S1

Synthetic schemes, procedures and analytical data for the synthesis of YSA-L2-PTX,
dYNH-L2-PTX and DYP-L2-PTX.
General Procedures. Unless otherwise noted, all reagents and anhydrous solvents were
obtained from commercial sources and used without purification, and the peptide motifs were
purchased from Abgent Inc.. All reactions were performed in oven-dried glassware. All
reactions involving air or moisture sensitive reagents were performed under a nitrogen
atmosphere. Silica gel chromatography was performed using prepacked silica gel or C-18
cartridges (RediSep). All final compounds were purified to >95% purity, as determined by a
HPLC Breeze from Waters Co. using an Atlantis T3 5.0 μM 4.6 mm x 150 mm reverse phase
column. 1D and 2D NMR spectra were recorded on a Bruker 600 MHz instruments. Chemical
1
shifts are reported in ppm (δ) relative to H (Me4Si at 0.00 ppm), coupling constant (J) are
1
reported in Hz throughout, and NMR signal assignments were based on DEPT, 2D [1H, H]-
1
13
COSY, 2D [1H, H]-TOCSY, 2D [1H,13C]-HSQC,and 2D [1H, C]-HMBC experiments. Low
resolution and high resolution mass spectral data were acquired on an Esquire LC00066 Mass
Spectrometer, an Agilent ESI-TOF Mass Spetrometer, or a Bruker Daltonic Autoflex Maldi-
Tof/Tof Mass Spectrometer.
Preparation of YSA peptide-paclitaxel conjugate (YSA-L2-PTX)
Compounds 1- 3 of Figure 1B of the manuscript have been prepared starting from PTX
according the previously reported synthetic procedure.1
Preparation of 7-O-alloc-2′-O-(6-azidohexanoyl) - paclitaxel (4)
To a solution of compound 7-O-alloc-paclitaxel (3) (366 mg, 0.391 mmol) in dichloromethane
(DCM, 10 mL) was added N,N-diisopropylcarbodiimide (DIPC, 147 mg, 1.17 mmol) and 4-
4-(dimethylamino) pyridine (DMAP, 143 mg, 1.17 mmol) at room temperature and stirred for 1
h, and then a solution of 6-azido-hexanoic acid (183 mg, 1.17 mmol) in DCM (5.0 ml) was
added and continually stirred for another 12 h. After removal of the solvent, the residue was
purified on a flash silica gel column eluted with ethyl acetate-hexane (10-50%) to give the title
compound (290 mg, 67 %).
S2

1H NMR (600 MHz, CDCl3): δ 8.13 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.63 (t, J =
7.8 Hz, 1H), 7.52 (m, 3H), 7.45 (m, 4H), 7.36 (m, 3H), 6.90 (d, J = 9.0 Hz, 1H), 6.40 (s, 1H),
6.25 (t, J = 9.0 Hz, 1H), 5.98 (m, 2H), 5.70 (d, J = 7.2 Hz, 1H), 5.56 (d, J = 3.0 Hz, 1H), 5.53
(dd, J = 10.2, 7.8 Hz, 1H), 5.36 (d, J = 15.6 Hz, 1H), 5.25 (d, J = 15.6 Hz, 1H), 4.98 (d, J = 9.6
Hz, 1H), 4.72 (m, 1H), 4.67 (m, 1H), 4.35 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H), 3.98 (d,
J = 7.2 Hz, 1H), 3.23 (t, J = 7.2 Hz, 2H), 2.65 (m, 1H), 2.46 (s, 3H), 2.39 (m, 2H), 2.20 (m,
1H), 2.16 (s, 3H), 2.05 (s, 3H), 2.04 (t, J = 7.2 Hz, 1H), 1.83 (s, 3H), 1.65 (m, 4H), 1.54 (m,
13
2H), 1.37 (m, 2H), 1.22 (s, 3H), 1.18 (s, 3H); C NMR (150 MHz, CDCl3): δ 210.72, 172.26,
169.60, 169.80, 168.17, 167.03, 166.93, 144.27, 136.97, 133.72, 133.66, 132.60, 132.03,
131.90, 130.19, 129.04, 128.74, 128.44, 127.05, 126.51, 118.75, 83.91, 80.79, 78.76, 76.34,
75.34, 75.25, 74.46, 73.71, 71.73, 69.09, 56.06, 52.83, 51.07, 46.91, 43.23, 35.33, 33.47, 33.33,
28.42, 26.43, 25.95, 24.17, 22.61, 21.29, 20.73, 14.54, 10.71; HRMS (ESI-TOF, m/z): [M+Na]+
calcd for C57H65N4O17Na, 1099.4158; found, 1099.4148.
Preparation of 2′-O-(6-azidohexanoyl) - paclitaxel (5)
To a stirred solution of compound 4 (150 mg, 0.139 mmol) in THF (5.0 mL) was added
Pd(0)(Ph3P)4 (5.0 mg, 0.007 mmol) and 1,3-dimethylbarbituric acid (47 mg, 0.167 mmol) at
room temperature. The reaction completion was monitored by TLC which showed the starting
material was consumed. The solvent was removed under vacuum to provide a residue, which
was purified on a flash silica gel column eluted with ethyl acetate-hexane (15-60%) to provide
121 mg (88%) of the title compound.
1H NMR (600 MHz, DMSO-d6): δ 9.20 (d, J = 8.4Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.86 (d, J =
7.8 Hz, 2H), 7.74 (t, J = 7.8 Hz, 1H), 7.66 (t, J = 7.8 Hz, 2H), 7.57 (t, J = 7.8 Hz, 1H), 7.51 (t, J
= 7.8 Hz, 2H), 7.44 (m, 4H), 7.19 (m, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.29 (s, 1H), 5.83(t, J = 9.0
Hz, 1H), 5.56 (t, J = 9.0 Hz, 1H), 5.42 (d, J = 7.2 Hz, 1H), 5.36 (d, J = 9.0 Hz, 1H), 4.92 (m,
2H), 4.62 (s, 1H), 4.10 (m, 1H), 4.01 (m, 3H), 3.59 (d, J = 7.2 Hz, 1H), 3.20 (t, J = 7.2 Hz, 2H),
2.42 (m, 2H), 2.32 (m, 1H), 2.24 (s, 3H), 2.10 (s, 3H), 1.98 (m, 1H), 1.83 (m, 1H), 1.79 (s, 3H),
1.64 (t, J = 7.2 Hz, 1H), 1.54 (m, 3H), 1.52 (s, 3H), 1.42 (m, 2H), 1.28 (m, 2H), 1.02 (s, 3H),
0.99 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 202.39, 172.36, 169..69, 169.30, 168.78,
166.31, 165.22, 139.35, 137.31, 134.18, 133.55, 133.40, 131.59, 130.27, 129.95, 128.74,
128.39, 128.25, 127.66, 127.42, 83.61, 80.25, 76.71, 75.29, 74.71, 74.51, 74.48, 70.83, 70.41,
59.79, 57.39, 54.01, 50.36, 46.08, 42.92, 36.54, 34.38, 33.00, 27.85, 26.34, 25.44, 23.96, 22.60,
21.40, 20.70, 13.89, 9.81; HRMS (ESI-TOF, m/z): [M+H]+ calcd for C53H60N4O15, 993.4128;
found, 993.4119.
S3

Preparation of YSA peptide-L2-paclitaxel conjugate (YSA-L2-PTX)
To a stirred solution of compound 5 (80 mg, 0.0805 mmol) and YSA motif, prepared as
1
previously reported (151 mg, 0.0962 mmol) in DMF-water (4: 1, 3.0 mL) was added the
solutions of CuSO4 (1.0 M, 50 uL) and sodium ascorbate (1.0 M, 50 uL) and continually stirred
for another 2 days. The product was purified on a reverse phase C-18 column by HPLC with a
gradient of 10-90 % acetonitrile-water to give the title compound trifuoroacetic acid salt (137
mg, 65%).
1H NMR (600 MHz, DMSO-d6): δ 9.21 (d, J = 7.8 Hz, 1H), 8.65 (d, J = 7.2 Hz, 1H), 8.14 (d, J
= 7.2 Hz, 2H), 8.07 (m, 2H), 7.97 (m, 9H), 7.85 (d, J = 7.8 Hz, 2H), 7.78 (m, 2H), 7.73 (t, J =
7.2 Hz, 1H), 7.66 (t, J = 7.2 Hz, 2H), 7.58 (d, J = 7.2 Hz, 1H), 7.48(t, J = 7.2 Hz, 2H), 7.44 (m,
4H), 7.18 (brs, 1H), 7.07 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 7.8 Hz, 2H), 6.68 (d, J = 7.8 Hz, 2H),
6.65 (d, J = 7.8 Hz, 2H), 6.28 (s, 1H), 5.81 (t, J = 9.0Hz, 1H), 5.54 (t, J = 9.0 Hz, 1H), 5.41 (d,
J = 8.4 Hz, 1H), 5.35 (d, J = 8.4 Hz, 1H), 4.91 (d, J = 8.4 Hz, 1H), 4.55 (m, 2H), 4.41 (m, 2H),
4.35 4.21-4.35 (m, 8H), 4.18 (m, 3H), 4.10 (m, 1H), 4.01 (m, 2H), 3.67 (m, 1H), 3.53-3.61 (m,
9H), 3.47 (m, 1H), 3.00 (m, 4H), 2.88 (m, 1H), 2.78 (m, 2H), 2.64 (m, 1H), 2.57 (m, 2H), 2.51
(m, 1H), 2.43 (m, 4H), 2.32 (m, 1H), 2.22 (s, 3H), 2.10 (s, 3H), 2.09 (m, 2H), 2.02 (s, 3H), 2.01
(s, 3H), 1.97 (m, 5H), 1.90 (m, 8H), 1.79 (s, 3H), 1.77 (m, 3H), 1.52 (m, 6H), 1.49 (s 3H), 1.36
(m, 2H), 1.28 (m, 2H), 1.18 (m, 2H), 1.15 (d, J = 7.2 Hz, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.89
13
(d, J = 7.2 Hz, 3H), 0.85 (d, J = 7.2 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 202.36 s,
173.45 s, 172.31 s, 171.87 s, 171.76 s, 171.67 s, 171.59 s, 171.10 s, 170.87 s, 170.29 s, 170.16
s, 169.75 s, 169.66 s, 169.63 s, 169.55 s, 169.25 s, 169.03 s, 168.78 s, 168.15 s, 166.35 s,
165.21 s, 158.17 (q, J = 37 Hz), 156.57 s, 155.82 s, 146.44 s, 139.34 s, 137.30 s, 134.19 s,
133.52 d, 133.40 s, 131.53 d, 130.51 d, 130.24 d, 129.95 s, 129.58 d, 128.71 d, 128.36 d, 128.23
d, 127.66 s, 127.64 d, 127.40 d, 124.77 s, 121.63 d, 115.64 (q, J = 293 Hz), 115.31 d, 114.95d,
83.61 d, 80.26 s, 76.71 s, 75.28 t, 74.72 d, 74.49 (d, 2×CH), 70.84 d, 70.40 d, 61.85 t, 61.82 t,
59.70 d, 59.22 d, 57.39 s, 55.78 d, 55.04 d, 54.80 d, 54.63 d, 53.98 d, 53.56 d, 52.57 d, 52.00 d,
51.83 (d, 2×CH), 49.90 d, 48.87 t, 48.60 d, 48.18 d, 47.11 d, 46.80 d, 46.08 d, 42.95 s, 38.25 t,
36.53 t, 35.83 t, 34.88 t, 34.40 t, 32.92 t, 32.01 t, 31.87 t, 30.82 t, 30.11 d, 29.48 t, 29.39 t, 29.31
t, 29.03 t, 28.91 t, 28.79 t, 26.33 q, 25.26 t, 25.19 t, 24.70 t, 24.52 t, 24.40 t, 23.78 t, 22.70 t,
22.56 q, 20.68 q, 19.10 q, 18.31 q, 18.06 q, 14.66 q, 14.59 q, 13.87 q, 9.79 q; HRMS (ESI-TOF,
m/z): [M+H]+ calcd for C124H165N18O37S2, 2562.10; found, 2562.26.
S4

Preparation of dYNH peptide-L2-paclitaxel conjugate (dYNH-L2-PTX)
The title compound was prepared using a method similar to that described for YSA-L2-PTX
dYNH-L2-PTX
1H NMR (600 MHz, DMSO-d6): δ 9.21 (d, J = 7.8 Hz, 1H), 8.57 (d, J = 7.2 Hz, 1H), 8.26 (d, J
= 7.2 Hz, 1H), 8.16 (d, J = 7.2 Hz, 1H), 8.03 (m, 4H), 7.98 (m, 4H), 7.87 (m, 5H), 7.78 (m, 2H),
7.73 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 7.2 Hz, 1H), 7.48 (t, J = 7.2 Hz,
2H), 7.44 (m, 4H), 7.18 (m, 2H), 7.10 (d, J = 7.8 Hz, 2H), 7.06 (d, J = 7.8 Hz, 2H), 6.70 (d, J =
7.8 Hz, 2H), 6.65 (d, J = 7.8 Hz, 2H), 6.28 (s, 1H), 5.81 (t, J = 9.0Hz, 1H), 5.54 (t, J = 9.0 Hz,
1H), 5.41 (d, J = 8.4 Hz, 1H), 5.35 (d, J = 8.4 Hz, 1H), 4.91 (d, J = 8.4 Hz, 1H), 4.55 (m, 2H),
4.41 (m, 1H), 4.21-4.35 (m, 7H), 4.18 (m, 3H), 4.10 (m, 2H), 4.01 (m, 3H), 3.67 (m, 1H), 3.62
(m, 1H), 3.53-3.61 (m, 6H), 3.47 (m, 3H), 3.47 (m, 2H), 3.00 (m, 2H), 2.95 (m, 1H), 2.90 (m,
1H), 2.88 (m, 1H), 2.73 (m, 1H), 2.64 (m, 1H), 2.55 (m, 3H), 2.39 (m, 2H), 2.32 (m, 1H), 2.24
(s, 3H), 2.10 (s, 3H), 2.09 (m, 2H), 1.97 (m, 3H), 1.88 (m, 3H), 1.81 (m, 7H), 1.77 (s, 3H), 1.69
(m, 4H), 1.63 (m, 3H), 1.52 (m, 5H), 1.49 (s 3H), 1.36 (m, 2H), 1.23 (m, 8H), 1.15 (d, J = 7.2
Hz, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.89 (d, J = 7.2 Hz, 3H), 0.84 (d, J = 7.2 Hz, 3H), 0.83 (d, J
13
= 7.2 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 202.38 s, 173.73 s, 172.33 s, 172.09 s,
171.90 s, 171.71 s, 171.69 s, 171.61 (s, 2×C=O), 171.59 s, 170.32 s, 170.15 s, 169.84 s, 169.69
(s, 2×C=O), 169.58 s, 169.27 s, 169.23 s, 168.80 s, 168.03 s, 166.37 s, 165.23 s, 158.24 (q, J =
37 Hz), 156.58 s, 155.84 s, 146.45 s, 139.36 s, 137.31 s, 134.44 s, 133.55 s, 133.41 s, 132.21 d,
130.79 d, 130.27 d, 129.96 s, 129.59 d, 128.74 d, 128.38 d, 128.23 d, 127.70 s, 127.66 d, 127.42
d, 126.22 s, 121.66 d, 115.56 (q, J = 293 Hz), 115.28 d, 114.96 d, 83.62 d, 80.27 s, 76.73 s,
75.30 t, 74.73 d, 74.49 d, 70.85 d, 70.41 d, 61.85 t, 61.66 t, 59.71 d, 59.20 t, 57.58 t, 57.40 s,
55.78 d, 55.08 d, 55.01 d, 54.81 d, 54.32 d, 54.01 d, 53.47 d, 52.76 d, 52.63 d, 52.58 d, 50.32 d,
49.52 d, 48.89 t, 48.62 d, 48.21 d, 47.11 t, 46.82 t, 46.09 d, 42.96 s, 38.37 t, 36.54 t, 36.46 t,
34.89 t, 34.41 t, 32.93 t, 31.54 t, 30.80 t, 30.09 d, 29.33 t, 28.96 t, 28.50 t, 27.38 t, 26.34 q,
25.27 t, 25.20 t, 24.71 t, 24.51 t, 24.41 t, 23.79 t, 22.89 t, 22.58 q, 21.96 t, 21.40 q, 20.69 q,
19.13 q, 18.23 q, 17.79 q, 13.89 q, 13.87 q, 9.80 q: MS (MALDI-TOF, m/z): [M+Na]+ calcd for
C124H164N18NaO38, 2536.13; found, 2536.09.
S5

Preparation of DYP peptide-L2-paclitaxel conjugate (DYP-L2-PTX)
The title compound was prepared using a method similar to that described for YSA-L2-PTX
DYP-L2-PTX
1H NMR (600 MHz, DMSO-d6): δ 9.21 (d, J = 7.8 Hz, 1H), 8.70 (d, J = 7.2 Hz, 1H), 8.20 (m,
1H), 8.07 (m, 5H), 7.97 (m, 6H), 7.85 (d, J = 7.8 Hz, 2H), 7.78 (m, 2H), 7.73 (t, J = 7.2 Hz,
1H), 7.66 (t, J = 7.2 Hz, 2H), 7.54 (d, J = 7.2 Hz, 1H), 7.48(t, J = 7.2 Hz, 2H), 7.44 (m, 4H),
7.18 (brs, 1H), 7.08 (d, J = 7.8 Hz, 2H), 6.98 (d, J = 7.8 Hz, 2H), 6.67 (d, J = 7.8 Hz, 2H), 6.60
(d, J = 7.8 Hz, 2H), 6.28 (s, 1H), 5.81 (t, J = 9.0Hz, 1H), 5.54 (t, J = 9.0 Hz, 1H), 5.41 (d, J =
8.4 Hz, 1H), 5.35 (d, J = 8.4 Hz, 1H), 4.91 (d, J = 8.4 Hz, 1H), 4.57 (m, 2H), 4.41 (m, 5H), 4.35
4.21-4.35 (m, 5H), 4.17 (t, J = 7.2 Hz, 2H), 4.09 (m, 2H), 4.01 (m, 2H), 3.62 (m, 1H), 3.55-3.61
(m, 9H), 3.51 (m, 1H), 3.00 (m, 4H), 2.98 (m, 1H), 2.85 (m, 1H), 2.64 (m, 3H), 2.57 (m, 3H),
2.43 (m, 4H), 2.32 (m, 1H), 2.25 (s, 3H), 2.10 (s, 3H), 2.09 (m, 2H), 2.02 (s, 3H), 2.01 (s, 3H),
1.87 (m, 5H), 1.90 (m, 8H), 1.79 (s, 3H), 1.77 (m, 3H), 1.52 (m, 4H), 1.49 (s 3H), 1.36 (m, 2H),
1.28 (m, 2H), 1.18 (m, 2H), 1.18 (d, J = 7.2 Hz, 3H), 1.02 (s, 3H), 0.99 (s, 3H), 0.86 (d, J = 7.2
13
Hz, 3H), 0.82 (d, J = 7.2 Hz, 3H); C NMR (150 MHz, DMSO-d6): δ 202.36 s, 173.37 s,
172.31 s, 172.01 s, 171.63 s, 171.61 s, 171.59 s, 171.01 s, 170.89 s, 170.77 s, 170.56 s, 170.34
s, 169.89 s, 169.67 s, 169.53 s, 169.25 s, 169.04 s, 168.78 s, 167.77 s, 166.36 s, 165.22 s,
158.21 (q, J = 37 Hz), 156.02 s, 155.80 s, 146.44 s, 139.35 s, 137.30 s, 134.19 s, 133.53 d,
133.41 s, 131.54 d, 130.16 d, 130.06 d, 129.95 s, 129.59 d, 128.72 d, 128.36 d, 128.36 d, 128.21
d, 127.64 d, 127.40 d, 121.65 d, 115.53 (q, J = 293 Hz),115.11 d, 114.76 d, 83.61 d, 80.26 s,
76.72 s, 75.29 t, 74.72 d, 74.49 (d, 2×CH), 70.84 d, 70.40 d, 61.53 t, 61.50 t, 59.47 d, 57.39 s,
57.00 d, 55.11 d, 55.01 d, 53.99 d, 53.79 d, 53.27 d, 52.95 d, 51.94 d, 90.87 d, 50.76 d, 48.88 t,
48.65 d, 48.60 d, 46.94 d, 46.78 d, 46.08 d, 42.95 s, 38.22 t, 36.89 t, 36.53 t, 35.78 t, 35.49 t,
34.88 t, 34.40 t, 32.92 t, 32.06 t, 31.87 t, 30.81 d, 30.48 t, 29.40 t, 29.31 t, 29.21 t, 28.93 t, 28.82
t, 26.33 q, 25.25 t, 25.19 t, 24.69 t, 24.51 t, 24.21 t, 23.78 t, 22.90 t, 22.56 q, 21.38 q, 20.68 q,
19.10 q, 17.65 q, 14.62 q, 13.88 q, 9.78 q; HRMS (ESI-TOF, m/z): [M+H]+ calcd for
C124H165N18O37S2, 2562.10; found, 2562.21.
References
1. Wang, S.; Placzek, W. J.; Stebbins, J. L.; Mitra, S.; Noberini, R.; Koolpe, M.; Zhang, Z.; Dahl,
R.; Pasquale, E. B.; Pellecchia, M. Novel targeted system to deliver chemotherapeutic drugs to
EphA2-expressing cancer cells. J Med Chem 2012, 55, 2427-36.
S6

1H (ppm)
Figure S1: 1D 1H NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S7

1H (ppm)
Figure S2: 2D 1H-1H COSY NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S8

13C (ppm)
Figure S3: 1D 13C DEPT-135 NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S9

1H (ppm)
Figure S4: 2D 1H-13C HMBC NMR spectrum of Paclitaxel (PTX) in methanol-d4.
S10

*
*
1H (ppm)
Figure S5: 1D 1H NMR spectrum of YSA-L1-PTX in methanol-d4. Proton signals
corresponding to methanol and buffer are labeled with asterisks.
S11

*
*
1H (ppm)
Figure S6: 1D 1H NMR of YSA-L1-PTX in 18.3% D2O, 4.5% H2O, 0.2% d6-DMSO, 77%
CD3OD. Proton signals corresponding to methanol and buffer are labeled with asterisks.
S12

Figure S7: Solubility of YSA-L2-PTX a
nd PTX
measured by H NMR experiments.
1
Bottom and top spectra belong to PTX and to YSA-L2-PTX, respectively, in PBS, pH=7.4 (+
10 % D2O, 1 % d6-DMSO). Differently from PTX, at the test concentration (50 M), YSA-L2-
PTX is soluble in aqueous buffer. Representative signals belonging to the PTX moiety
resonating between 5 and 7 ppm are included in brackets, while proton signals corresponding to
d6-DMSO, buffer and internal standard, are labeled with asterisks.
S13

Figure S8: Binding affinity of YSA-L2-PT
X with th
e EphA2 bi
nding dom
ain, determined
by ITC. The derived Kd is 2.1 M. Isothermal titration calorimetry (ITC) was performed on an
ITC200 calorimeter from Microcal/GE Healthcare (Northampton, MA, USA). The direct
titration was performed by injecting 2 L of 0.5 M compound into 50 M EphA2 (5%
DMSO). Experimental data were analyzed using Microcal Origin software provided by the ITC
manufacturer (Microcal/GE Healthcare, Northampton, MA, USA).
S14