Synthesis of novel heterobifunctional isocyanato cross-linkers and their applications for the preparation of 10-hydroxycamptothecin and SN-38 conjugates with melanotransferrin p97

Article abstract of DOI:10.1080/10426910701319928

Novel heterobifunctional cross-linkers with an isocyanato group, a protected carboxylic group, and a linear chain spacer are synthesized in high yield by coupling monofunctionalized PEG with 1,6-diisocyanatohexane. The isocyanato groups of those linkers are highly reactive and are efficient reagents to couple with the hydroxy groups of 10-hydroxycamptothecin and SN-38 under mild conditions to give a useful precursor for the synthesis of their bioconjugates with proteins such as melanotransferrin p97. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426910701319928

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Synthesis of Novel Heterobifunctional
Isocyanato Cross‐Linkers and Their
Applications for the Preparation of
10‐Hydroxycamptothecin and SN‐38
Conjugates with Melanotransferrin P97
Zhong Li a , Dingqiao Yang b , Reinhard Gabathuler c & Qingqi Chen d
a Department of Chemistry , University of Alabama , Tuscaloosa, Alabama,
USA
b Department of Chemistry , South China Normal University , Guangzhou,
China
c AngioChem, Inc. , Montreal, Quebec, Canada
d BioMarin Pharmaceutical Inc. , Novato, California, USA
Published online: 30 Jun 2007.
To cite this article: Zhong Li , Dingqiao Yang , Reinhard Gabathuler & Qingqi Chen (2007) Synthesis
of Novel Heterobifunctional Isocyanato Cross‐Linkers and Their Applications for the Preparation of
10‐Hydroxycamptothecin and SN‐38 Conjugates with Melanotransferrin P97, Synthetic Communications:
An International Journal for Rapid Communication of Synthetic Organic Chemistry, 37:11, 1899-1915, DOI:
10.1080/10426910701319928
To link to this article: http://dx.doi.org/10.1080/10426910701319928
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Synthetic Communicationsw, 37: 1899–1915, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/10426910701319928
Synthesis of Novel Heterobifunctional
Isocyanato Cross-Linkers and Their
Applications for the Preparation of
10-Hydroxycamptothecin and SN-38
Conjugates with Melanotransferrin P97
Zhong Li
Department of Chemistry, University of Alabama, Tuscaloosa,
Alabama, USA
Dingqiao Yang
Department of Chemistry, South China Normal University,
Guangzhou, China
Reinhard Gabathuler
AngioChem, Inc., Montreal, Quebec, Canada
Qingqi Chen
BioMarin Pharmaceutical Inc., Novato, California, USA
Abstract: Novel heterobifunctional cross-linkers with an isocyanato group, a protected
carboxylic group, and a linear chain spacer are synthesized in high yield by coupling
monofunctionalized PEG with 1,6-diisocyanatohexane. The isocyanato groups of those
linkers are highly reactive and are efficient reagents to couple with the hydroxy groups
of 10-hydroxycamptothecin and SN-38 under mild conditions to give a useful precursor
for the synthesis of their bioconjugates with proteins such as melanotransferrin p97.
Keywords: bioconjugate, heterobifuncational cross-linker, 10-hydroxycamptothecin,
isocyanato, SN-38
Received in the USA October 19, 2006
Address correspondence to Qingqi Chen, Erimos Pharmaceutical, 930 Main
Campus Drive, Raleigh, NC 27516. E-mail: qqchen@gmail.com
1899

1900
INTRODUCTION
Z. Li et al.
1
Heterobifunctional linkers are one of the most important and widely used
cross-linkers in the areas of bioconjugate synthesis and drug delivery for
cross-linking small molecule drugs, medical image agents, proteins,
enzymes, antibodies, genes, cells, and even living bacteria.[1–3] Heterobifunc-
tional linkers chemically consist of three moieties: a spacer or bridge and two
different functional groups that are able to selectively couple with two
different target molecules. There are a number of commercially available
heterobifuntional linkers, most of which, however, are not efficient for
coupling to 10-hydroxycamptothecin (II) or 7-ethyl-10-hydroxycamptothecin
(SN-38, III). (A number of companies are providing heterobifunctional
linkers, such as Pierce Inc. Sigma-Aldrich, and Solulink Inc.)
10-Hydroxycamptothecin (II) and SN-38 (III) are the semisynthesized
compounds based on camptothecin (I), the natural product first isolated by
Wall et al. in 1966 from the Chinese tree Camptotheca acuminate.[4] Camp-
tothecin (I) and its more potential analogues such as 10-hydroxycamptothecin
(II) and SN-38 (III) have recently undergone extensive clinical evaluation
worldwide and display a broad range of clinical activity against solid
tumors[5,6] through an unique antitumor mechanism of inhibiting DNA topoi-
somerase I, an important nuclear enzyme for various DNA functions including
transcription and replication.[7]
The 10-hydroxy group of 10-hydroxycamptothecin (II) and SN-38 (III) had
been previously modified through the formation of ester bond,[8] carbamate
bond,[9] ether bond,[10] and so forth. In this article, we report the synthesis
of heterobifuncational linkers that contain an isocyanato group, a protected
carboxylic group, and a linear chain spacer. They are highly reactive to the
hydroxyl groups of 10-hydroxycamptothecin and SN-38 to form, in
excellent yields, a carbamate bond between the linker and the target drug
molecules. The resulting compounds are useful precursors for the synthesis
of their conjugates with melanotransferrin p97.
2
RESULTS AND DISCUSSION
We are interested in isocyanato linkers because the isocyanate group is
highly reactive to the hydroxyl and amino groups in the target
compounds[11] to form a stable carbamate and urea linkage, which can be

Heterobifunctional Isocyanato Cross-linkers
1901
Scheme 1. Reagents and conditions: (a) tert-butyl acrylate/Na/THF, rt, 24 h, 40–
88%, (b) Et3N/1,6-diisocyantohexane/dichloromethane, rt, 2 h, 82–95%.
selectively hydrolyzed in some tumor cells.[12,13] The heterobifuncational
linkers 3 were synthesized by two steps in high yield as shown in
Scheme 1. Polyethylene glycols 1 were mono-alkylated via Michael
addition reaction to give compounds 2 in 45–88% by using 1/3 equivalent
of tert-butyl acrylate and a catalytic amount of sodium in THF, a procedure
previously described.[14–16] Compounds 2 were isolated as light yellow oil
1
and fully characterized by H NMR, 13C NMR, IR, MS and combustion
analysis.
Reaction of the monofunctionalized PEG compounds 2 with equimolar
amount of 1,6-diisocyanatohexane gave compounds 3 in 82–90% yield as
1
light yellow oils, which were fully characterized by H and 13C NMR, MS,
IR, and combustion analysis. 13C spectra of 3 showed the characteristic
chemical shifts at ꢀ29 ppm for the methyl groups of the tert-butyl group,
ꢀ80 ppm for OCMe3, ꢀ121 ppm for the isocyanate, ꢀ156 ppm for the
carbamate (NH-CO-O), and ꢀ170 ppm for the COOBut ester. Their IR illus-
trated spectra showed a strong absorption at 2260–2266 cm21, which was
assigned to the isocyanate group. Compounds 3 are sensitive to moisture
and will slowly polymerize upon exposure to air or light. They are stable
for years in the dark, when kept dry and at low temperature (48C).
The novel heterobifunctional linkers were used to conjugate mela-
notransferrin (p97) with anticancer drugs 10-hydroxcamptothecin (II) and
SN-38 (III) (Scheme 2). Melanotransferrin (p97) has been found to localize
in capillary endothelial cells of the human brain and to play an important
role in the transport of iron across the blood brain barrier (BBB).[17] This
provides a base for a vector-mediated approach to delivery of therapeutics
to the brain.
The conjugates 8 and 9 are synthesized in three steps as outlined in
Scheme 2. Reactions of 10-hydroxycamptothecin (II) and SN-38 (III)
with mono-isocyanato linkers 3 under basic conditions give the expected
products 4 and 5 in high yield. Compounds 4 and 5 are semisolid when
freshly prepared and slowly solidified upon storing over months at 48C in
the dark. Upon treatment with trifluoroacetic acid, the expected free acids

1902
Z. Li et al.
Scheme 2. Reagents and conditions: (a) 3/Et3N/DMF, rt, 2 h. 82–92%, (b)
TFA, rt, 20 min, 82–95%, (c) O-benzotriazole-1-yl-N,N,N0,N0tetramethyluronium
tetrafluoroborate(BTTU)/Et3N/DMF, rt, 60 min, then melanotransferrin (p97),
rt, 20 h.
6 and 7 are obtained in 80–95% yield. After activating the carboxy group
in compounds 6 and 7 using BTTU (O-benzotriazole-1-yl-N,N,N 0,N 0-tetra-
methyluronium tetrafluoride), the corresponding intermediates are directly
used to react with melanotransferrin p97 to give the expected conjugates
8 and 9, which were purified by membrane dialysis and characterized by
fast protein liquid chromatography (FPLC), SDS-Page, and Western
blotting against the p97 antibody (L235 anitbody). The results showed
that both compounds 8 and 9 were pure and the conformation of p97
was preserved. The MSR (molar substituted ratio) of the target molecules
attached to p97 were estimated (see Table 1) by UV-vis as previously
described.[3]
Table 1. Preparation of compounds 10 and 11
Compound MSR (molar substituted ratio) Protein p97 recovery (%)
8a
8b
8c
9a
9b
9c
5.4
3.5
4.6
5.3
6.1
7.5
88
90
95
89
91
86

Heterobifunctional Isocyanato Cross-linkers
CONCLUSION
1903
3
We have described an efficient method to synthesize heterobifunctional cross-
linkers, which consist of an isocyanto group, a protected craboxylate group,
and a linear chain spacer. Those linkers are very efficient reagents to react
with the 10-hydroxy group of 10-hydroxycamptotheticin and SN-38 to form
a carbamate bond between the target molecules and the linkers. Carbamate
bond is chemically stable and hydrolysable in some tumor cells, which
suggests that the new heterobifunctional linkers are potentially useful in bio-
conjugate synthesis.
4
EXPERIMENTAL
4.1 General
10-Hydroxycamptothecin (II) and SN-38 (III) were purchased from Qventas,
Inc. Dialysis Cassettes Slide-A-Lyzer (10 K, 10,000 MWCO) and Dialysis
Tubing SnakeSkinTM (10,000 MWCO) were purchased from Pierce Inc. All
other reagents and solvents were purchased from Aldrich, Sigma, and VWR
and used as received. The silica gel used in flash chromatography was
Merck silica gel 60, 230–400 mesh, and Rf values were measured on
Merck silica thin-layer chromatography (TLC) aluminum sheets (silica gel
60 F254). Melting points were determined on a Thomas hot stage or Buchi
1
apparatus and are uncorrected. H and 13C NMR spectra were recorded on
Bruker AC200-, AMX400-, or AMX500-MHz instruments. APT was
recorded on the AMX-400 instrument. Part of 1H and 13C NMR were
assigned based on reported data[8] and APT results. UV-vis spectra were
recorded on a Beckman DU640 photo-diode-array spectrophotometer in the
solvents indicated. IR spectra were recorded on a Shimadzu FTIR 8400.
FPLC was run in a AKTA PurifierTM FPLC (using UNICORNTM version
3.10, Amersham Pharmacia Biotech) using Mono QR HR 10/10 ion
exchange column (from Pharmacia Biotech Inc.) with PBS buffer (0.01 M,
pH ¼ 7.4) and 1M NaCl–0.001 M PBS buffers as the mobile phases or
using a BIOSEPTM size exclusion column (from Phenomenex, Inc) and 0.01
M PBS buffer (pH ¼ 6.80). Elemental analyses were performed by the micro-
analytical laboratory, Department of Chemistry, University of British
Columbia (UBC). The high- and low-resolution mass spectra were obtained
by mass spectrometer service laboratories, Department of Chemistry, UBC.
4.2 General Procedure for the Preparation of Mono-alkylated
PEG Compounds 2
Polyethylene glycol (1.0 mol) was dissolved in THF (500 mL). Sodium
(0.212 g, 9.2 mmol) was cut into small pieces and added under stirring. The

1904
Z. Li et al.
mixture was warmed at 408C until all sodium dissolved. Then tert-butylacrylate
(51 mL, 0.34 mmol) was added at room temperature. The mixture was stirred
overnight at room temperature. Solvent was removed under vacuum (30–
40 mm Hg). The residue oil was mixed with brine (100 mL), and the mixture
was extracted with ethyl acetate (6 ꢀ 150 mL). The combined extracts were
washed with brine and dried over anhydrous sodium sulfate. After removing
the solvent under reduced pressure (30–40 mm Hg), the crude product was
obtained after drying under vacuum overnight (0.1–0.5 mm Hg), which was
pure enough and directly used for the synthesis of compound 3. Analytical
samples were purified through a flash silica-gel chromatographic column
using dichloromethane–methanol (95/5, v/v) as eluent.
4.2.1 Tert-butyl 3-(ethylene glycol)propanoate (2a)
Yield 45%. IR, n ¼ 3450, 2877, 1728, 1369, 1325, 1240, 1178, 1116, 1051,
1
954, 927, 883, 846, 812, 605, 542, 516 cm21. H NMR (200 MHz, CDCl3),
d ¼ 1.40 (s, 9H, 3 CH3), 2.30 (t, J ¼ 7.0 Hz, 2H, CH2), 3.20 (brs, 1H, OH),
3.80 (m, 4H, 2 OCH2), 4.20 (m, 2H, OCH2) ppm. 13C NMR (50 MHz,
CDCl3), d ¼ 27.5 (CH3), 34.2 (CH2), 61.1, 66.5, 70.1, 80.6 (OCMe3), 170.3
(COO) ppm. LC-MS, m/z ¼ 213 [M þNa]þ. Anal. calcd. for C9H18O4
(190.24): C, 56.82; H, 9.54. Found: C, 56.65; H, 9.22.
4.2.2 Tert-butyl 3-[di(ethylene glycol)]propanoate (2b)
Yield 72%. IR, n ¼ 3452, 2872, 1726, 1452, 1392, 1367, 1332, 1251, 1157,
1112, 1062, 933, 846, 889, 754, 548, 517 cm21 1H NMR (200 MHz,
.
CDCl3), d ¼ 1.35 (s, 9H, 3 CH3), 2.35 (t, J ¼ 7.0 Hz, 2H, CH2), 3.80
(m, 11H, 5 OCH2, OH) ppm. 13C NMR (50 MHz, CDCl3), d ¼ 27.8 (CH3),
34.5 (CH2), 61.4, 66.6, 70.2 (multiple peaks), 72.5, 80.3 (OCMe3), 170.5
(COO) ppm. LC-MS, m/z ¼ 257 [M þNa]þ. Anal. calcd. for C11H22O5
(234.29): C, 56.39; H, 9.46. Found: C, 56.55; H, 9.32.
4.2.3 Tert-butyl 3-[tri(ethylene glycol)]propanoate (2c)
Yield: 80.5%. IR, n ¼ 3450, 2870, 1726, 1456, 1392, 1365, 1330, 1251, 1111,
1
1064, 943, 847, 888, 756, 532 cm21. H NMR (200 MHz, CDCl3), d ¼ 1.45
(s, 9H, 3 CH3), 2.52 (t, J ¼ 7.0 Hz, 2H, CH2), 3.75 (m, 15H, 7 OCH2, OH)
ppm. 13C NMR (50 MHz, CDCl3), d ¼ 27.5 (CH2), 28.0 (CH3), 36.5 (CH2),
61.3, 65.8, 70.5 (multiple peaks), 72.3, 80.2 (OCMe3), 170.4 (COO) ppm,
LSIMS (matrix: thioglycerol), m/z ¼ 279 [M þH]þ. Anal. calcd. for
C13H26O6 (278.34): C, 52.92; H, 9.48. Found: C, 52.55; H, 9.22.

Heterobifunctional Isocyanato Cross-linkers
1905
4.2.4 Tert-butyl 3-[tetra(ethylene glycol)]propanoate (2d)
Yield 88%. IR, n ¼ 3450, 2870, 1726, 1451, 1365, 1329, 1249, 1105, 1068,
945, 887, 847, 756, 528 cm21
.
1H NMR (200 MHz, CDCl3), d ¼ 1.50
(s, 9H, 3 CH3), 2.50 (t, J ¼ 7.0 Hz, 2H, CH2), 3.80 (m, 19H, 9 OCH2, OH)
ppm. 13C NMR (50 MHz, CDCl3), d ¼ 27.5 (CH3), 27.8 (CH2), 36.0 (CH2),
62.3, 66.6, 70.3 (multiple peaks), 72.3, 76.4, 80.2 (OCMe3), 170.6 (COO)
ppm. LSIMS (matrix: thioglycerol), m/z ¼ 333 [M þH]þ. Anal. calcd. for
.
C15H30O7 H2O (340.41): C, 52.92; H, 9.48. Found: C, 52.88; H, 9.12.
4.3 General Procedure for the Preparation of Mono-isocyanato
Heterobifunctional Linkers 3
1,6-Diisocyanatohexane (5 mL, 0.03 mol) was dissolved in anhydrous
dichloromethane (50 mL). Then a solution of compound 2 (0.03 mol) in
dichoromethane (50 mL) and triethylamine (1.5 mL) was added dropwise
under stirring over a period of 30 min. The mixture was stirred at room temp-
erature for 2 h and was protected from moisture. The solvent was removed
under vacuum. The oily residue was dried overnight under vacuum to give
the crude products 3, which were purified through flash silica-gel chromato-
graphic column using dichloromethane as eluent.
4.3.1 Compound 3a
Yield 91%. IR, n ¼ 3458, 2933, 2862, 2260, 1722, 1526, 1458, 1365, 1240,
1157, 1118, 1053, 954, 846, 777, 756, 582 cm21 1H NMR (200 MHz,
.
CDCl3), d ¼ 1.40 (s, 9H, 3 CH3), 1.55 (m, 6H, 3 CH2), 2.43 (t, J ¼ 7.0 Hz,
2H, CH2), 2.55 (t, J ¼ 7.0 Hz, 2H, CH2), 3.20 (m, 2H, CH2), 3.30 (m, 2H,
CH2), 3.80 (m, 4H, 2 OCH2), 4.20 (t, J ¼ 6.8 Hz, 2H, OCH2), 5.10 (s, 1H,
NH) ppm. 13C NMR (50 MHz, CDCl3), d ¼ 27.1 (CH2), 27.3 (CH2), 28.8
(CH3), 31.6 (CH2), 32.0 (CH2), 34.9 (CH2), 43.6 (CH2), 45.4 (CH2), 70.5
(m, 3 OCH2), 80.1 (OCMe3), 122.3 (N55C55O), 156.5 (NH-CO-O), 170.3
(COO) ppm. LSIMS (matrix: thioglycerol), m/z ¼ 358 [M þH]þ. Anal.
calcd. for C17H30N2O6 (358.43): C, 56.97; H, 8.44; N, 7.81. Found: C,
56.88; H, 8.45; N, 7.50.
4.3.2 Compound 3b
Yield 94%. IR, n ¼ 3485, 2933, 2866, 2262, 1724, 1523, 1458, 1365, 1392,
1
1247, 1157, 1114, 1062, 950, 846, 777, 756, 729, 582 cm21. H NMR (200
MHz, CDCl3), d ¼ 1.42 (s, 9H, 3 CH3), 1.55 (m, 8H, 4 CH2), 2.50
(t, J ¼ 7.0 Hz, 2H, CH2), 3.20 (m, 2H, CH2), 3.30 (m, 2H, CH2), 3.80
(m, 8H, 4 OCH2), 4.20 (t, J ¼ 6.8 Hz, 2H, OCH2), 5.20 (s, 1H, NH) ppm.
13C NMR (50 MHz, CDCl3), d ¼ 26.1 (CH2), 27.3 (CH2), 28.1 (CH3), 29.7

1906
Z. Li et al.
(CH2), 30.9 (CH2), 34.9 (CH2), 40.7 (CH2), 42.7 (CH2), 66.8 (OCH2), 68.8
(OCH2), 70.5 (m, 3 OCH2), 80.1 (OCMe3), 121.9 (N55C55O), 156.4 (NH-
CO-O), 170.8 (COO) ppm. LSIMS (matrix: thioglycerol), m/z ¼ 403 [M
þH]þ. Anal. calcd. for C19H34N2O7 (402.48): C, 56.70; H, 8.51; N, 6.96.
Found: C, 56.48; H, 8.35; N, 6.80.
4.3.3 Compound 3c
Yield 95%. IR, n ¼ 3445, 2933, 2864, 2262, 1722, 1525, 1458, 1392, 1365,
1
1247, 1109, 1070, 947, 846, 777, 756, 729, 580 cm21. H NMR (200 MHz,
CDCl3), d ¼ 1.40 (s, 9H, 3 CH3), 1.50 (m, 8H, 4 CH2), 2.50 (t, J ¼ 7.0 Hz,
2H, CH2), 3.20 (m, 2H, CH2), 3.30 (m, 2H, CH2), 3.80 (m, 12H, 6 OCH2),
4.20 (t, J ¼ 6.8 Hz, 2H, OCH2), 5.20 (s, 1H, NH) ppm. 13C NMR (50 MHz,
CDCl3), d ¼ 26.5 (CH2), 27.6 (CH2), 28.0 (CH3), 29.7 (CH2), 31.1 (CH2),
34.8 (CH2), 39.8 (CH2), 42.7 (CH2), 63.6 (OCH2), 66.7 (OCH2), 69.1
(OCH2), 69.9 (m, 4 OCH2), 80.2 (OCMe3), 121.8 (N55C55O), 156.4 (NH-
CO-O), 170.7 (COO) ppm. LSIMS (matrix: thioglycerol), m/z ¼ 447 [M
þH]þ. Anal. calcd. for C21H38N2O8 (446.54): C, 56.48; H, 8.58; N, 6.27.
Found: C, 56.28; H, 8.25; N, 6.00.
4.3.4 Compound 3d
Yield 82%. IR, n ¼ 3337, 2931, 2866, 2266, 1720, 1627, 1535, 1458, 1365,
1247, 1107, 949, 846, 775, 756 cm21 1H NMR (200 MHz, CDCl3),
.
d ¼ 1.60 (s, 9H, 3 CH3), 1.65 (m, 8H, 4 CH2), 2.45 (t, J ¼ 7.0 Hz, 2H,
CH2), 3.20 (m, 2H, CH2), 3.32 (m, 2H, CH2), 3.60 (m, 14H, 8 OCH2), 4.20
(t, J ¼ 6.8 Hz, 2H, OCH2), 5.10 (s, 1H, NH) ppm. 13C NMR (50 MHz,
CDCl3), d ¼ 25.7 (CH2), 27.7 (CH2), 29.8 (CH3), 31.1 (CH2), 36.2 (CH2),
40.8 (CH2), 42.8 (CH2), 45.4 (CH2), 63.7 (OCH2), 66.8 (OCH2), 70.5 (br, 7
OCH2), 80.7 (OCMe3), 122.3 (N55C55O), 156.4 (NH-CO-O), 170.8 (COO)
ppm. LSIMS (matrix: thioglycerol), m/z ¼ 491 [M þH]þ. Anal. calcd. for
.
C23H42N2O9 0.5H2O (499.59): C, 55.29; H, 8.68; N, 5.61. Found: C,
54.98; H, 8.45; N, 5.40.
4.4 General Procedure for the Preparation of Compounds 6 and 7:
Coupling Reactions of 3 with 10-Hydroxycamptothecin, SN-38
In a 100-mL, round-bottomed flask equipped with a magnetic stirrer bar, 10-
hydroxycamptothecin (II) [or SN-38 (III), 1.37 mmol], anhydrous DMF
(30 mL) and triethylamine (1.0 mL) were added. The flask was placed in an
ultrasonic bath until all the solid was completely dissolved. A solution of
monocyanato linker 3 (2.75 mmol, 2.0 equivalent) in dichloromethane
(10 mL) was added under vigorous stirring. The flask was wrapped with
aluminum foil to protect if from light. The reaction was monitored by TLC

Heterobifunctional Isocyanato Cross-linkers
1907
(dichloromethane/methanol, 95/5, V/V). After 2 h, TLC confirmed that the
reaction was finished. The solvent was removed under vacuum until dry.
The residue was taken up by methanol (5 mL) and then mixed with
anhydrous ether (40 mL). The resulting suspension mixture was placed in
the ultrasonic bath for 30 s and then kept at 48C for 3 h. The solid was
collected by suction filtration to give the expected products 6 (or 7) as light
yellow powder.
4.4.1 Compound 4a (10-CPT, n ¼ 2)
Yield: 92%, mp 159–1648C. IR, n ¼ 3321, 2925, 2854, 1733, 1642, 1611,
1523, 1485, 1452, 1354, 1241, 1201, 1134, 1063, 1029, 920, 830, 810,
1
760 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.83 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.25 (m, 2H, CH2), 1.29 (m, 4H, 2 CH2), 1.40 (s, 9H, 3 CH3), 1.48
(m, 2H, CH2), 1.79 (t, J ¼ 7.2 Hz, 2H, CH2), 2.38 (t, J ¼ 6.0 Hz, 2H, CH2),
2.78 (m, 2H, CH2), 3.10 (m, 2H, CH2), 3.40 (obscured with water peak, m,
2H, CH2) 3.60 (m, 6 H, 3 OCH2), 4.12 (m, 2H, OCH2), 5.30 (m, 2H, CH2),
5.50 (s, 2H, CH2), 6.60 (s, 1H), 7.10 (m, 1H), 7.33 (m, 1H), 7.61 (m, 1H),
7.88 (m, 1H), 8.45 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.5
(CH3), 24.8, 28.1 (CH3), 28.9, 29.5, 30.3, 30.6, 34.7, 49.8, 62.1, 65.1, 66.0,
69.0 (multiple peaks, 5 OCH2), 72.0, 79.1, 96.4 (CH), 118.0 (CH), 119.4,
126.4 (CH), 128.5, 119.4 (CH), 130.6, 131.4 (CH), 145.6, 145.6, 149.2,
150.5, 152.8, 154.7 (C55O), 155.8 (C55O), 156.8 (C55O), 170.7 (COO),
172.9 (COO) ppm. UV-vis (DMF) l(1) ¼ 289 (12 500), 333 (14 900), 379
(33 600), 395 (29 700) nm. UV-vis (methanol) l(1) ¼ 294 (11 600), 335
(14 800), 369 (29 900), 382 (29 300) nm. UV-vis (DMSO) l(1) ¼ 291 (27
800), 335 (12 500), 371 (27 500), 380 (26 300) nm. LSIMS (matrix: thiogly-
cerol), m/z ¼ 767 [Mþ þH]. Anal. calcd. for C39H50N4O12: C, 61.08; H,
6.57; N, 7.31. Found: C, 60.95; H, 6.64; N, 7.21.
4.4.2 Compound 4b (10-CPT, n ¼ 3)
Yield: 89%, mp 175–1788C. IR, n ¼ 3319, 2922, 2830, 1728, 1658, 1604,
1544, 1484, 1455, 1344, 1225, 1190, 1101, 1042, 996, 920, 837, 801,
1
763 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.86 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.21 (m, 2H, CH2), 1.33 (m, 4H, 2 CH2), 1.45 (s, 9H, 3 CH3), 1.51
(m, 2H, CH2), 1.82 (t, J ¼ 7.2 Hz, 2H, CH2), 2.40 (t, J ¼ 6.0 Hz, 2H, CH2),
2.91 (m, 2H, CH2), 3.12 (m, 2H, CH2), 3.35 (obscured with water peak, m,
2H, CH2) 3.56 (m, 10H, 5 OCH2), 4.10 (m, 2H, OCH2), 5.28 (m, 2H, CH2),
5.46 (s, 2H, CH2), 6.51 (s, 1H), 7.21 (m, 1H), 7.39 (m, 1H), 7.62 (m, 1H),
7.91 (m, 1H), 8.59 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.8
(CH3), 25.8, 27.3 (CH3), 29.0, 29.2, 30.0, 30.1, 35.5, 50.0, 63.1, 65.3, 66.1,
69.4 (multiple peaks, 7 OCH2), 72.1, 79.3, 96.1 (CH), 117.9 (CH), 119.2,
126.1 (CH), 128.2, 119.3 (CH), 130.1, 131.1 (CH), 146.1, 145.3, 150.2,
150.1, 151.8, 153.7 (C55O), 156.2 (C55O), 157.1 (C55O), 169.9 (COO),

1908
Z. Li et al.
172.2 (COO) ppm. UV-vis (DMF) l(1) ¼ 296 (11 500), 329 (13 900), 371 (32
700), 385 (28 600) nm. UV-vis (methanol) l(1) ¼ 293 (10 300), 331 (14 500),
363 (29 500), 372 (28 500) nm. UV-vis (DMSO) l(1) ¼ 289 (27 700), 335 (11
500), 366 (27 400), 379 (25 300) nm. LC-MS (ESI), m/z ¼ 811 [Mþ þH].
Anal. calcd. for C41H54N4O13: C, 60.73; H, 6.71; N, 6.91. Found: C, 60.59;
H, 6.64; N, 6.81.
4.4.3 Compound 4c (10-CPT, n ¼ 4, C43H58N4O14, FW ¼ 854)
Yield: 85%, mp 180–1838C. IR, n ¼ 3313, 2929, 2860, 1718, 1654, 1600,
1541, 1489, 1446, 1348, 1227, 1195, 1103, 1043, 1001, 916, 835, 800,
1
760 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.85 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.20 (m, 2H, CH2), 1.30 (m, 4H, 2 CH2), 1.40 (s, 9H, 3 CH3), 1.50
(m, 2H, CH2), 1.80 (t, J ¼ 7.2 Hz, 2H, CH2), 2.41 (t, J ¼ 6.2 Hz, 2H, CH2),
2.90 (m, 2H, CH2), 3.10 (m, 2H, CH2), 3.35 (obscured with water peak, m,
2H, CH2), 3.55 (m, 14H, 7 OCH2), 4.05 (m, 2H, OCH2), 5.25 (m, 2H,
CH2), 5.45 (s, 2H, CH2), 6.50 (s, 1H), 7.20 (m, 1H), 7.38 (m, 1H), 7.65
(m, 1H), 7.95 (m, 1H), 8.62 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz),
d ¼ 7.7 (CH3), 25.9, 27.5 (CH3), 29.1, 29.3, 30.0, 30.3, 35.8, 50.2, 62.9,
65.3, 66.2, 69.7 (multiple peaks, 9 OCH2), 72.4, 79.7, 96.6 (CH), 118.6
(CH), 118.9, 126.2 (CH), 128.4, 130.1 (CH), 130.2, 130.9 (CH), 145.4,
145.5, 149.7, 150.0, 152.1, 154.0 (C55O), 156.1 (C55O), 156.8 (C55O),
170.4 (COO), 172.4 (COO) ppm UV-vis (DMF) l(1) ¼ 295 (11 400), 332
(13 400), 368 (32 600), 382 (28 400) nm. UV-vis (methanol) l(1) ¼ 292 (9
350), 330 (14 400), 362 (29 300), 375 (28 200) nm. UV-vis (DMSO)
l(1) ¼ 286 (27 600), 332 (11 200), 368 (27 900), 385 (25 000) nm. LSIMS
(matrix: thioglycerol), m/z ¼ 855 [Mþ þH], 799, 365. HRMS (LSIMS,
matrix: thioglycerol): found 855.40251, required 855.40278 for
[C43H59N4O14]þ. Anal. calcd. for C43H58N4O14 1/2H2O: C, 59.78; H,
.
6.88; N, 6.49. Found: C, 60.04; H, 7.04; N, 6.21.
4.4.4 Compound 5a (SN-38, n ¼ 2)
Yield: 88%, mp 89–958C. IR, n ¼ 3311, 2935, 2864, 1743, 1652, 1621, 1521,
1483, 1442, 1334, 1231, 1213, 1144, 1069, 1035, 930, 836, 823, 790 cm21. 1H
NMR (DMSO-d6, 400 MHz), d ¼ 0.95 (t, J ¼ 7.2 Hz, 3H, CH3), 1.45 (m, 22
H, 4 CH3, 5 CH2), 1.81 (m, 2H, CH2), 2.40 (m, 4H, 2 CH2), 3.05 (m, 2H, CH2),
3.50 (obscured with water peak, m, 8H, CH2, 3OCH2), 4.22 (m, 2H, OCH2),
5.35 (m, 2H, CH2), 5.45 (s, 2H, CH2), 7.20 (m, 1H), 7.65 (m, 1H), 7.90
(m, 1H), 8.15 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.7
(CH3), 13.7 (CH3), 22.2, 24.5, 28.4 (CH3), 28.9, 29.1, 30.2, 30.8, 34.5, 49.9,
62.6, 64.1, 67.3, 70.3 (multiple peaks, 5 OCH2), 71.8, 79.5, 96.4 (CH),
114.5 (CH), 118.8, 125.8 (CH), 127.0 128.3, 128.4, 131.0 (CH), 145.0,
146.0, 149.8, 151.5, 154.1, 156.8 (C55O), 158.1 (C55O), 156.8 (C55O),
170.7 (COO), 172.9 (COO) ppm. UV-vis (DMF) l(1) ¼ 291 (12 400), 335

Heterobifunctional Isocyanato Cross-linkers
1909
(14 700), 380 (34 600), 398 (29 200) nm. UV-vis (methanol) l(1) ¼ 293 (12
600), 331(14 200), 370 (29 500), 380 (29 500) nm. UV-vis (DMSO)
l(1) ¼ 297 (27 300), 338 (13 400), 377 (28 600), 384 (27 800) nm. LSIMS
(matrix: thioglycerol), m/z ¼ 795 [Mþ þH]. Anal. calcd. for C41H54N4O12:
C, 61.95; H, 6.85; N, 7.05. Found: C, 61.75; H, 6.64; N, 7.21.
4.4.5 Compound 5b (SN-38, n ¼ 3)
Yield: 85%, mp 75–808C. IR, n ¼ 3291, 2945, 2875, 1735, 1642, 1611, 1511,
1451, 1438, 1345, 1233, 1223, 1154, 1069, 1025, 890, 847, 833, 765 cm21. 1H
NMR (DMSO-d6, 400 MHz), d ¼ 0.95 (t, J ¼ 7.2 Hz, 3H, CH3), 1.42 (m, 22
H, 4 CH3, 5 CH2), 1.79 (m, 2H, CH2), 2.44 (m, 4H, 2 CH2), 3.10 (m, 2H, CH2),
3.50 (obscured with water peak, m, 12H, CH2, 5OCH2), 4.20 (m, 2H, OCH2),
5.30 (m, 2H, CH2), 5.50 (s, 2H, CH2), 7.25 (m, 1H), 7.40 (m, 1H), 7.85
(m, 1H), 8.20 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.8
(CH3), 13.6 (CH3), 23.1, 24.5, 27.9 (CH3), 28.5, 29.3, 30.1, 30.9, 33.9, 50.3,
61.9, 63.5, 66.8, 70.5 (multiple peaks, 7 OCH2), 72.1, 80.5, 95.9 (CH),
114.3 (CH), 118.9, 125.2 (CH), 127.3 129.1, 129.5, 131.1 (CH), 145.1,
145.8, 149.3, 150.5, 153.1, 155.9 (C55O), 157.9 (C55O), 158.8 (C55O),
171.7 (COO), 172.5 (COO) ppm. UV-vis (DMF) l(1) ¼ 289 (12 100), 333
(15 200), 385 (35 100), 397 (28 900) nm. UV-vis (methanol) l(1) ¼ 290
(13 400), 331 (14 500), 371 (29 600), 381 (28 700) nm. UV-vis (DMSO)
l(1) ¼ 295 (28 100), 335 (14 500), 378 (28 700), 386 (26 600) nm. LSIMS
(matrix: thioglycerol), m/z ¼ 839 [Mþ þH]. Anal. calcd. for C43H58N4O13:
C, 61.56; H, 6.97; N, 6.68. Found: C, 61.75; H, 6.74; N, 6.41.
4.4.6 Compound 5c (SN-38, n ¼ 4)
Yield: 82%, mp 73–788C. IR, n ¼ 3285, 2930, 2845, 1729, 1643, 1601, 1521,
1435, 1440, 1340, 1243, 1243, 1165, 1059, 1036, 910, 856, 832, 745 cm21. 1H
NMR (DMSO-d6, 400 MHz), d ¼ 0.90 (t, J ¼ 7.2 Hz, 3H, CH3), 1.46 (m, 22
H, 4 CH3, 5 CH2), 1.81 (m, 2H, CH2), 2.45 (m, 4H, 2 CH2), 3.12 (m, 2H, CH2),
3.55 (obscured with water peak, m, 16H, CH2, 7 OCH2), 4.25 (m, 2H, OCH2),
5.35 (m, 2H, CH2), 5.45 (s, 2H, CH2), 7.20 (m, 1H), 7.38 (m, 1H), 7.79
(m, 1H), 8.18 (m, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.7
(CH3), 13.8 (CH3), 22.9, 24.1, 28.1 (CH3), 28.3, 29.0, 29.9, 30.2, 32.3, 49.6,
62.1, 62.9, 67.1, 71.0 (multiple peaks, 9 OCH2), 72.6, 80.8, 96.1 (CH),
114.4 (CH), 118.2, 125.5 (CH), 126.9, 128.9, 129.1, 131.9 (CH), 146.0,
146.8, 148.3, 151.5, 152.9, 156.1 (C55O), 156.9 (C55O), 159.8 (C55O),
171.3 (COO), 172.1 (COO) ppm. UV-vis (DMF) l(1) ¼ 291 (12 400), 334
(15 500), 380 (36 100), 396 (28 700) nm. UV-vis (methanol) l(1) ¼ 294
(12 900), 333 (14 900), 379 (31 300), 391 (29 200) nm. UV-vis (DMSO)
l(1) ¼ 291 (28 800), 334 (14 200), 380 (29 700), 391 (27 900) nm. LSIMS
(matrix: thioglycerol), m/z ¼ 883 [Mþ þH]. Anal. calcd. for C45H62N4O14:
C, 61.21; H, 7.08; N, 6.35. Found: C, 61.15; H, 6.84; N, 6.41.

1910
Z. Li et al.
4.5 General Procedure for Synthesis of Compounds 6 and 7
Compound 4 (or 5, 0.585 mmol) was placed in a 50-mL, round-bottomed flask
equipped with a magnetic stirrer. Trifluoroacetic acid (10 mL) was added. The
mixture was stirred at room temperature for 20 min. Then anhydrous ether
(60 mL) was added slowly over a period of 5 min. The suspension was then
placed over an ultrasonic bath for 2 min. The yellow solid was collected by
suction filtration. The crude product is then redissolved in a minimum
amount of DMF and precipitated by anhydrous ether. The expected
compound 6 (or 7) was obtained after filtration and drying under vacuum
overnight.
4.5.1 Compound 6a (n ¼ 2)
Yield 88%, mp 132–1388C. IR, n ¼ 3335, 2932, 2823, 1735, 1642, 1611,
1530, 1450, 1430, 1340, 1240, 1178, 1140, 1120, 1045, 1028, 912,
1
812 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.85 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.35–1.45 (m, 8H, 4 CH2), 1.80 (q, J¼7.2 Hz, 2H, CH2), 2.95
(t, J ¼ 6.9 Hz, 2H, CH2), 3.10 (t, J ¼ 6.9 Hz, 2H, CH2), 3.50 (m, 12 H, 4
OCH2), 4.05 (m, 2H, OCH2), 5.35 (m, 2H, CH2), 5.45 (s, 2H, CH2), 6.60
(s, 1H), 7.25 (m, 1H), 7.55 (m, 1H), 8.15 (m, 1H), 8.20 (m, 1H), 8.60
(m, 1H), 12.20 (brs, COOH, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz),
d ¼ 7.8 (CH3), 24.7, 28.9, 29.6, 30.3, 34.7, 50.8, 63.5, 64.9, 65.9, 68.8, 70.5
(multiple peaks, 5 OCH2), 72.8, 96.9 (CH), 118.5 (CH), 119.5, 125.6 (CH),
127.1, 1291 (CH), 130.8, 132.3 (CH), 133.6, 142.1, 146.9, 149.9, 153.8,
154.7 (C55O), 156.4 (C55O), 156.9 (C55O), 170.8 (COO), 172.7 (COO)
ppm. UV-vis (DMF) l(1) ¼ 283 (12 100), 333 (12 300), 363 (29 900), 386
(25 300) nm. UV-vis (methanol) l(1) ¼ 281 (15 700), 334 (13 100), 370
(28 600), 390 (26 800) nm. UV-vis (DMSO) l(1) ¼ 290 (11, 800), 332
(12 300), 371 (25 900), 391 (25 800) nm. LSIMS (matrix: thioglycerol),
]m/z ¼ 711 [Mþ þH]. Anal. calcd. for C35H42N4O12: C, 59.15; H, 5.96; N,
7.88. Found: C, 59.00; H, 6.05; N, 7.75.
4.5.2 Compound 6b (n ¼ 3)
Yield 95%, mp 148–1528C. IR, n ¼ 3345, 2930, 2850, 1731, 1652, 1601,
1525, 1449, 1433, 1338, 1236, 1186, 1136, 1121, 1040, 1008, 920,
1
830 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.90 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.30–1.50 (m, 8H, 4 CH2), 1.85 (q, J ¼ 7.2 Hz, 2H, CH2), 2.90
(t, J ¼ 6.9 Hz, 2H, CH2), 3.15 (t, J ¼ 6.9 Hz, 2H, CH2), 3.50 (m, 12 H, 6
OCH2), 4.10 (m, 2H, OCH2), 5.30 (m, 2H, CH2), 5.40 (s, 2H, CH2), 6.55
(s, 1H), 7.20 (m, 1H), 7.60 (m, 1H), 8.10 (m, 1H), 8.25 (m, 1H), 8.50
(m, 1H), 12.15 (brs, COOH, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz),
d ¼ 7.9 (CH3), 25.4, 29.3, 29.5, 30.8, 34.3, 50.3, 64.5, 65.1, 66.1, 68.2, 69.1
(multiple peaks, 7 OCH2), 72.3, 96.5 (CH), 118.8 (CH), 119.4, 125.9 (CH),

Heterobifunctional Isocyanato Cross-linkers
1911
127.6, 129.5 (CH), 130.1, 132.8 (CH), 133.6, 142.5, 147.9, 150.6, 152.8, 154.1
(C55O), 155.4 (C55O), 156.2 (C55O), 171.8 (COO), 172.6 (COO) ppm. UV-
vis (DMF) l(1) ¼ 285 (11 500), 334 (12 500), 365 (29 500), 386 (25 900) nm.
UV-vis (methanol) l(1) ¼ 284 (15 900), 335 (12 800), 369 (29 900), 389 (25
700) nm. UV-vis (DMSO) l(1) ¼ 289 (17 800), 334 (11 300), 370 (24 900),
389 (25 100) nm. LSIMS (matrix: thioglycerol), m/z ¼ 755 [Mþ þH]. Anal.
calcd. for C37H46N4O13: C, 58.88; H, 6.14; N, 7.42. Found: C, 58 60; H, 6.05;
N, 7.25.
4.5.3 Compound 6c (n ¼ 4)
Yield 92%, mp 155–1598C. IR, n ¼ 3311, 2920, 2858, 1730, 1655, 1600,
1539, 1498, 1443, 1348, 1226, 1195, 1151, 1103, 1043, 1001, 914,
1
833 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.85 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.25–1.50 (m, 8H, 4 CH2), 1.84 (q, J ¼7.2 Hz, 2H, CH2), 2.95
(t, J ¼ 6.9 Hz, 2H, CH2), 3.13 (t, J ¼ 6.9 Hz, 2H, CH2), 3.55 (m, 16H, 8
OCH2), 4.05 (m, 2H, OCH2), 5.25 (m, 2H, CH2), 5.44 (s, 2H, CH2), 6.55
(s, 1H), 7.26 (m, 1H), 7.60 (m, 1H), 8.08 (m, 1H), 8.16 (m, 1H), 8.67
(m, 1H), 12.10 (brs, 1H) ppm. 13C NMR (DMSO-d6, 100 MHz), d ¼ 7.8
(CH3), 25.9, 29.1, 29.3, 30.2, 34.7, 50.2, 64.9, 65.2, 66.2, 68.9, 69.7
(multiple peaks, 9 OCH2), 72.4, 96.6 (CH), 118.6 (CH), 119.0, 126.2 (CH),
128.4, 130.2 (CH), 130.8, 131.1 (CH), 131.6, 145.4, 149.7, 150.0, 152.1,
154.0 (C55O), 156.1 (C55O), 156.8 (C55O), 172.4 (COO), 172.6 (COO)
ppm. UV-vis (DMF) l(1) ¼ 283 (10 600), 332 (11 800), 367 (28 900), 385
(25 000) nm. UV-vis (methanol) l(1) ¼ 283 (15 800), 332 (11 800), 367
(28 900), 385 (25 000) nm. UV-vis (DMSO) l(1) ¼ 286 (17 100), 332 (9
300), 368 (23 700), 386 (21 100) nm. LSIMS (matrix: thioglycerol), m/
z ¼ 799 [Mþ þH], 429, 365. HRMS (LSIMS, matrix: thioglycerol): found
799.34053, required 799.34018 for [C39H51N4O14]þ. Anal. calcd. for
.
C39H50N4O14 1/3H2O: C, 58.20; H, 6.35; N, 6.96. Found: C, 58.10; H,
6.27; N, 7.05.
4.5.4 Compound 7a (n ¼ 2)
Yield 87%, mp 122–1278C. IR, n ¼ 3343, 2935, 2866, 1734, 1635, 1620,
1545, 1489, 1433, 1332, 1234, 1175, 1143, 1123, 1025, 1032, 915,
1
746 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.93 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.35–1.50 (m, 11 H, CH3, 4 CH2), 1.90 (q, J ¼ 7.2 Hz, 2H, CH2),
2.55 (m, 4H, 2 CH2), 3.10 (m, 4H, 2 CH2), 3.85 (m, 8H, 4 OCH2), 4.20
(m, 2H, OCH2), 5.35 (m, 2H, CH2), 5.45 (s, 2H, CH2), 7.25 (m, 1H), 7.50
(m, 1H), 7.85 (m, 1H), 8.20 (m, 1H), 11.25 (brs, 1H) ppm. 13C NMR
(DMSO-d6, 100 MHz), d ¼ 7.9 (CH3), 13.2 (CH3), 22.8, 26.5, 26.8, 29.2,
29.9, 30.4, 34.8, 62.0, 63.1, 66.4, 67.3, 68.7 (multiple peaks, 5 OCH2), 72.6,
96.5 (CH), 118.2, 119.3, 127.1 (CH), 128.6, 130.4 (CH), 130.7, 131.6 (CH),
132.7, 146.2, 149.4, 150.7, 152.1, 153.9 (C55O), 157.1 (C55O), 157.9

1912
Z. Li et al.
(C55O), 170.2 (COO), 172.1 (COO) ppm. UV-vis (DMF) l(1) ¼ 282 (13
700), 330 (13 500), 370 (26 600), 385 (27 300) nm. UV-vis (methanol)
l(1) ¼ 289 (12 900), 340 (13 100), 365 (25 200), 382 (23 400) nm. UV-vis
(DMSO) l(1) ¼ 284 (13 400), 340 (13 900), 374 (24 400), 390 (22 300)
nm. LSIMS (matrix: thioglycerol), m/z ¼ 739 [Mþ þH]. Anal. calcd. for
C37H46N4O12: C, 60.15; H, 6.28; N, 7.58. Found: C, 59.90; H, 6.15; N, 7.45.
4.5.5 Compound 7b (n ¼ 3)
Yield 85%, mp 143–1478C. IR, n ¼ 3332, 2945, 2875, 1724, 1634, 1626,
1535, 1490, 1422, 1312, 1245, 1185, 1153, 1120, 1020, 1036, 910,
1
736 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.90 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.35–1.50 (m, 11 H, CH3, 4 CH2), 1.85 (q, J ¼ 7.2 Hz, 2H, CH2),
2.50 (m, 4H, 2 CH2), 3.15 (m, 4H, 2 CH2), 3.70 (m, 12H, 6 OCH2), 4.10
(m, 2H, OCH2), 5.30 (m, 2H, CH2), 5.40 (s, 2H, CH2), 7.35 (m, 1H), 7.55
(m, 1H), 7.80 (m, 1H), 8.25 (m, 1H), 11.20 (brs, 1H) ppm. 13C NMR
(DMSO-d6, 100 MHz), d ¼ 7.8 (CH3), 13.5 (CH3), 23.0, 26.4, 26.7, 29.6,
29.8, 30.2, 35.4, 62.5, 63.4, 66.1, 67.8, 68.9 (multiple peaks, 7 OCH2), 72.9,
96.8 (CH), 118.5, 119.4, 127.3 (CH), 128.7, 130.1 (CH), 130.5, 131.8 (CH),
132.6, 146.0, 149.1, 150.1, 151.8, 154.8 (C55O), 156.9 (C55O), 157.8
(C55O), 170.8 (COO), 172.9 (COO) ppm. UV-vis (DMF) l(1) ¼ 283 (12
600), 336 (12 300), 375 (27 300), 390 (28 100) nm. UV-vis (methanol)
l(1) ¼ 283 (14 500), 344 (14 500), 369 (27 200), 392 (24 400) nm. UV-vis
(DMSO) l(1) ¼ 280 (17 400), 341 (15 300), 370 (24 000), 395 (22 900)
nm. LSIMS (matrix: thioglycerol), m/z ¼ 783 [Mþ þH]. Anal. calcd. for
C39H50N4O13: C, 59.84; H, 6.44; N, 7.16. Found: C, 59.70; H, 6.55; N, 7.35.
4.5.6 Compound 7c (n ¼ 4)
Yield 82%, mp 132–1378C. IR, n ¼ 3322, 2925, 2866, 1727, 1652, 1610,
1542, 1492, 1434, 1326, 1237, 1175, 1148, 1109, 1048, 1011, 912,
1
836 cm21. H NMR (DMSO-d6, 400 MHz), d ¼ 0.95 (t, J ¼ 7.2 Hz, 3H,
CH3), 1.25–1.50 (m, 11 H, CH3, 4 CH2), 1.90 (q, J ¼ 7.2 Hz, 2H, CH2),
2.55 (m, 4H, 2 CH2), 3.10 (m, 4H, 2 CH2), 3.75 (m, 16H, 8 OCH2), 4.05
(m, 2H, OCH2), 5.25 (m, 2H, CH2), 5.44 (s, 2H, CH2), 7.25 (m, 1H), 7.65
(m, 1H), 7.90 (m, 1H), 8.20 (m, 1H), 11.10 (brs, 1H) ppm. 13C NMR
(DMSO-d6, 100 MHz), d ¼ 7.7 (CH3), 13.8 (CH3), 22.33, 26.1, 26.4, 29.5,
29.4, 30.0, 35.8, 62.9, 63.7, 66.2, 68.8, 69.7 (multiple peaks, 9 OCH2), 72.4,
96.5 (CH), 118.8, 119.0, 127.0 (CH), 128.4, 130.2 (CH), 130.8, 131.1 (CH),
131.6, 145.0, 149.9, 150.0, 151.5, 154.1 (C55O), 156.1 (C55O), 156.8
(C55O), 170.4 (COO), 172.6 (COO) ppm. UV-vis (DMF) l(1) ¼ 285 (11
800), 340 (12 900), 374 (29 300), 394 (28 200) nm. UV-vis (methanol)
l(1) ¼ 290 (14 700), 354 (13 800), 379 (26 200), 399 (25 800) nm. UV-vis
(DMSO) l(1) ¼ 279 (16 400), 342 (14 300), 376 (24 600), 390 (23 100)

Heterobifunctional Isocyanato Cross-linkers
1913
nm. LSIMS (matrix: thioglycerol), m/z ¼ 827 [Mþ þ H]. Anal. calcd. for
C41H54N4O14: C, 59.55; H, 6.58; N, 6.78. Found: C, 59 30; H, 6.75; N, 6.55.
4.6 General Procedure for Synthesis of Conjugates 8 and 9
A mixture of compounds 6 (or 7, 0.007 mmol), O-benzotriazole-1-yl-N,N,N0,N0-
tetramethyluronium tetrafluoroborate (BTTU, 6.9 mg, 0. 021 mmol, 3.0 equiv.),
triethylamine (0.01 mL, 0.07 mmol, 10 equiv.), and DMF (0.5 mL) was stirred at
room temperature for 60 min. The solution was saved for the following reaction.
Melanotransferrin p97 (in PBS, pH 7.4, c ¼ 1.23 mg/mL, 11 mL,
1.433 ꢀ 1024 mM) was placed in a 50-mL, round-bottomed flask. To this
solution, BTTU-activated camptothecin compound prepared from the
previous mixture (0.007 mmol, 50 equiv., mixed with 1.5 mL of DMF) was
added dropwise over a period of 5 min under vigorous stirring. The mixture
was stirred at room temperature for 20 h, then purified by dialysis using
Slide-A-Lyzer dialysis cassette (WMCO ¼ 10 K) against PBS (10 mM,
pH ¼ 7.4) to give the expected conjugates 8 and 9.
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