Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: Potential applications in bioconjugation and targeted drug delivery

Article abstract of DOI:10.1039/c2ob26968f

Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH₂, -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG ₁₆ and PEG₂₄) by employing a Cu(i)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α_vβ₃) receptor targeting peptide, cyclo-(Arg-Gly-Asp-d-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

Full text of DOI:10.1039/c2ob26968f

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Efficient synthesis of diverse heterobifunctionalized
clickable oligo(ethylene glycol) linkers: potential
applications in bioconjugation and targeted drug
delivery†
Cite this: DOI: 10.1039/c2ob26968f
Lalit N. Goswami, Zachary H. Houston, Saurav J. Sarma, Satish S. Jalisatgi and
M. Frederick Hawthorne*
Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible hetero-
bifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of
these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one
of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on
the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (–OMs) group. The –OMs func-
tional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers contain-
ing different highly reactive end groups, e.g., iodo, –NH2, –SH and maleimido, that were orthogonal to
the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generat-
ing larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG16 and PEG24) by employing a Cu(I)-cata-
lyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in
bioconjugation chemistry was demonstrated by attachment of the integrin (αvβ3) receptor targeting
peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The
synthetic methodology presented herein is suitable for the large scale production of several novel hetero-
bifunctionalized OEGs from readily available and inexpensive starting materials.
Received 9th October 2012,
Accepted 20th December 2012
DOI: 10.1039/c2ob26968f
www.rsc.org/obc
Introduction
therapeutic payload, and the other for attaching a targeting
ligand (e.g., peptides, proteins, or antibodies).6 Most commer-
PEGylation of drug molecules plays an important role in the
delivery of certain therapeutic agents. PEG-drug conjugates
exhibit more favorable in vivo behavior than the unconjugated
forms. They are less susceptible to degradation by metabolic
enzymes, and they exhibit prolonged circulation times in the
blood stream. PEGylation can also reduce or even eliminate
antigenicity and immunogenicity.1–3
Short-chain PEGs or oligo(ethylene glycol)s (OEGs) have
found widespread use as spacers or linkers for targeted drug
delivery systems because they are inexpensive, water soluble,
biostable, and available in a wide range of molecular weight
distributions.4,5 A targeted drug delivery system requires two
distinct reactive termini on the linker, one for attaching the
cially available OEGs are monofunctional, containing only a
single reactive hydroxyl, amine, thiol, aldehyde or carboxylic
acid terminal group or activated variants of these. However,
few heterobifunctional OEGs are commercially available con-
taining two different reactive groups at the distal ends. There-
fore, there is a need to develop efficient and diverse methods
to synthesize heterobifunctionalized OEGs with highly reactive
end groups (Fig. 1). These termini must additionally be reac-
tive under mild, aqueous reaction conditions to permit conju-
gation of delicate peptide and antibody targeting moieties.
Although the synthesis of heterobifunctional PEGs with
azide or alkyne groups at their termini has been accomplished
using a ring-opening polymerization of ethylene oxide, this
method employed polydisperse PEGs.7,8 Although polydisperse
PEGs are suitable for macromolecular drug delivery systems
based on polymers, micelles, liposomes, or nanoparticles, they
have limited use in the synthesis of small, discrete, multi-
modal theranostic agents. In our ongoing research on the utility
of closo-boranes as multifunctional targeted drug delivery
scaffolds,9,10 we needed a diverse collection of monodisperse
International Institute of Nano and Molecular Medicine, School of Medicine,
University of Missouri, 1514 Research Park Drive, Columbia, Missouri 65211-3450,
USA. E-mail: hawthornem@missouri.edu; Fax: (+)573 884 6900;
Tel: (+)573 882 7016
†Electronic supplementary information (ESI) available: Experimental
and characterization details, copies of 1H, 13C NMR and HRMS. See DOI:
10.1039/c2ob26968f
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Fig. 1 Schematic illustration of the synthesis of clickable heterbifunctionalized OEGs and their potential applications in targetable/traceable delivery of drugs or
imaging agents.
Scheme 1 Synthesis of alkyne terminated heterobifunctionalized OEG linkers. Reagents and conditions: (a) Propargyl bromide, NaH, THF, 0 °C–RT–60 °C, 15 h; (b)
4-nitrophenyl chloroformate, pyridine, MeCN, RT, 15 h; (c) MsCl, Et3N, DCM, 0 °C–RT, 3.5 h; (d) NaI, acetone, 65 °C, 15 h; (e) KSAc, DMF, RT, 15 h; (f) LAH, THF,
−10 °C–RT, 3 h; (g) Potassium phthalimide, DMF, 110 °C, 15 h; (h) Hydrazine, EtOH, 60 °C, 3 h; (i) N-methoxycarbonyl maleimide, Sat. aq NaHCO3, 0 °C–RT, 2 h.
heterobifunctional OEG linkers having the ability to undergo chemically equivalent terminal diols. Many previous studies
click reactions. Therefore, the objective of this study was to find addressed the desymmetrization of homofunctional OEGs
simple and efficient synthetic routes to generate a diverse using the Williamson ether synthesis,15,16 in which the benzyl
library of heterobifunctional OEG linkers with one terminal and p-methoxybenzyl ether groups were most commonly used
alkyne or azide functionality, which would render them suitable for the monoprotection of symmetrical OEGs. However,
for biocompatible azide-alkyne [3 + 2] dipolar cycloaddition reports on the synthesis of alkyne-terminated heterobifunctio-
reactions with bioactive ligands, leaving the remaining reactive nalized OEGs are rare.17 In one such report, Gill et al.18 syn-
terminus available for subsequent conjugation to payloads such thesized propargyl-PEG6–OH in 54% yield from hexa(ethylene
as drugs and imaging probes (Scheme 1).11–14
glycol) (OH–PEG6–OH) using equimolar amounts of sodium
hydride (NaH) and propargyl bromide. In our case, tetra(ethyl-
ene glycol) (OH–PEG4–OH, 1a), hexa(ethylene glycol) (OH–
PEG6–OH, 1b) and octa(ethylene glycol) (OH–PEG8–OH, 1c)
were used to synthesize 2a–c in moderate yields (37–69%)
using a similar procedure. In contrast to traditional monoalkyl-
ations, propargyl bromide was added to 1b and 1c at room
Results and discussion
A crucial requirement while working with homobifunctional
OEGs is the ability to differentiate the reactivity of two
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temperature to prevent the solidification of the starting
Because the mesylate group is an effective leaving group in
materials at 0 °C in THF. Minor amounts (∼5%) of dialkylated nucleophilic substitution reactions, the alkyne–OEG–OMs
OEGs were also formed, but they were easily separated by silica compounds 4a–c served as starting materials for the synthesis
gel column chromatography. Unreacted starting OEGs (1a–c) of several heterobifunctional OEG linkers commonly used in
were recovered from the aqueous layer during the workup pro- bioconjugation reactions. For example, the reaction of NaI
cedures. The 1H NMR spectra of OEGs 2a–c exhibited a very with 4a–c in refluxing acetone produced 5a–c, which have
characteristic triplet for one proton at ∼δ 2.4 ppm assigned to alkyne and iodo-groups at their distal ends. The iodo-termi-
the propargyl group.
nated OEG linkers 5a–c were produced in quantitative yields
To construct useful heterobifunctional OEG linkers, and were readily purified by a simple silica gel filtration
the remaining hydroxyl group on 2a–c was converted into column. 1H NMR, 13C NMR, and HRMS were used to verify
several highly reactive, ready-to-use functional groups their structures. Absence of the characteristic sharp –OMs
1
(Scheme 1 and Table 1). First, the hydroxyl group was con- singlet at ∼δ 3.0 ppm from the H NMR spectra of 5a–c con-
verted into an amine-reactive 4-nitrophenyl carbonate firmed conversion of the mesylate groups. This is the first pub-
(PNPC) group using 4-nitrophenyl chloroformate under basic lished report of the synthesis of 5b–c. Synthesis of 5a has been
conditions to produce 3a–c. Yields were greater than 71%, previously reported; however, with a lower yield (52%) than
and the 1H NMR spectra of 3a–c exhibited distinct doublets our method (74%).20
for the four protons assigned to the 4-nitrophenyl moiety.
Mesylates 4a–c also were reacted with potassium thioacetate
PNPC-functionalized PEG linkers are useful for biomedical (KSAc) in dimethylformamide (DMF) at room temperature to
applications because they can readily react with the amine afford nearly quantitative yields of previously unpublished
functionality of amino acids present in bioactive peptides or thioacetates 6a–c. These compounds were reduced to the novel
antibodies to form carbamate bonds under very mild reaction set of OEG thiols 7a–c following a reduction procedure
conditions.
reported by Stefanko et al.21 Thioacetates 6a–c and thiols 7a–c
To the best of our knowledge, no alkyne-terminated OEG were characterized using 1H NMR, 13C NMR, and HRMS.
derivatives of 1a–c with aryl or alkyl carbonates at their distal 1H-NMR spectral analysis confirmed the conversion of –OMs
ends have been reported previously in the literature.
to a thioacetate group by the appearance of a singlet at ∼δ
The remaining hydroxyl group on 2a–c was converted to a 2.3 ppm attributed to the three –SCOCH3 protons along with
mesylate (–OMs) group using 1.5 eq. of methanesulfonyl chlor- the absence of the characteristic –OMs peak at ∼δ 3.0 ppm.
ide and 2.0 eq. of Et3N in DCM, giving mesylates 4a–c in quan- 1H-NMR analysis of 7a–c showed loss of the singlet for the
1
titative yields. The structures of 4a–c were analyzed using H, three –SCOCH3 protons at ∼δ 2.3 ppm and the appearance of a
13C NMR and HRMS. The 1H NMR spectra of the products triplet at ∼δ 1.5 ppm assigned to the –SH functional group.
showed a characteristic triplet for one proton at ∼δ 2.4 ppm, Thiol-terminated heterobifunctional OEG linkers have fre-
which was assigned to the propargyl group, and a sharp quently been used to form disulfide linkages with peptides,
singlet at ∼δ 3.0 ppm, which was assigned to the three –OMs antibodies, and other bioactive molecules.22,23 Moreover, the
protons.
alkyne–OEG–SH linkers 7a–c are excellent substrates for dual
This is the first report of the synthesis of 4b–c. Although click reactions in which a Cu(I)-catalyzed alkyne-azide cyclo-
the synthesis of 4a has been previously published,19 the addition4 is followed by a thiol-maleimide click reaction.24
method reported here requires fewer steps and gives a 1.5-fold
The utility of mesylate intermediates 4a–c was further
higher yield. It should be noted that 4a–c required no further demonstrated by the synthesis of alkyne–OEG–NH2 com-
purification for their use in the subsequent reactions pounds 9a–c via the Gabriel reaction. The mesylate groups on
described below.
4a–c were first transformed into phthalimides 8a–c in nearly
quantitative yields, and were then deprotected using hydrazine
in ethanol to produce 9a–c in excellent yields (≥95% over two
steps). The structures of 8a–c and 9a–c were confirmed using
1H, 13C NMR and HRMS.
Table 1 Yields of alkyne-terminated heterobifunctionalized OEG linkers 2–10
Compound
n
Yield (%)
Compound
n
Yield (%)
To the best of our knowledge, no OEG linkers with dual
alkyne- and amino-functionalities at their distal ends have
been produced from 1a–c via Gabriel synthesis. Other investi-
gators have synthesized alkyne-OEG-amines using the Staudin-
ger reduction of an azide, but their yields for 9a–b were 3- and
4-fold lower than what we were able to obtain.25–27 Purification
of 9a–b on a multi-gram scale is difficult when using Staudin-
ger reduction, and thus Gabriel synthesis has proven to be a
much more efficient means of obtaining these molecules in
high purity. Yields for 9c via Staudinger reduction are poten-
tially higher; however, no synthetic route has been reported
starting from 1c for accurate comparison.
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
2
4
6
2
4
6
2
4
6
2
4
6
2
4
58
37
69
75
71
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
10a
10b
10c
6
2
4
6
2
4
6
2
4
6
2
4
6
65
92
83
98
99
92
97
99
99
99
49
63
42
87
100
100
99
74
78
98
99
97
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Scheme 2 Sequential synthesis of azide terminated heterobifunctionalized OEG linkers. Reagents and conditions: (a) Method A: (i) MsCl, Et3N, DCM, 0 °C–RT, 3 h;
(ii) NaN3, DMF, 65 °C, 15 h; Method B: (i) MsCl, Ag2O, DCM, 0 °C–RT, 24 h; (ii) NaN3, DMF, 65 °C, 15 h; (b) (i) MsCl, Et3N, DCM, 0 °C–RT, 4 h; (ii) NaN3, DMF, 65 °C,
15 h; (c) 4-nitrophenyl chloroformate, pyridine, MeCN, RT, 15 h; (d) MsCl, Et3N, DCM, 0 °C–RT, 3.5 h; (e) NaI, acetone, 65 °C, 15 h; (f) potassium phthalimide, DMF,
110 °C, 15 h; (g) Hydrazine, EtOH, 60 °C, 3 h; (h) Ph3P, Et2O-5% aq HCl, RT, 24 h.
Finally, another set of dual click-compatible OEG linkers,
alkyne–OEG–maleimides 10a–c, were prepared in good yield by
reacting 9a–c with N-methoxycarbonyl maleimide using a
recently reported procedure.28 The 1H NMR spectra of OEG
linkers 10a–c exhibited a very characteristic sharp singlet at ∼δ
6.6 ppm, which was assigned to the two equivalent protons of
the maleimido moiety.
Azido-terminated heterobifunctionalized OEG linkers were
also prepared (Scheme 2 and Table 2) by using a synthetic
strategy that paralleled the one used to produce the alkyne-ter-
minated heterobifunctionalized OEG linkers (Scheme 1). First,
the desymmetrization of OEGs 1a–c was achieved by formation
of the monoazide products 11a–c in moderate yields (Method
A, 36–46%). During this transformation, a small amount of
diazido OEGs 12a–c was also formed, but they were easily sep-
arated by silica gel chromatography. The mono-azido products
11a–c were characterized by 1H NMR, 13C NMR, and mass
spectrometry. The 1H NMR spectra showed a characteristic
triplet at ∼δ 3.1 ppm for the –CH2– bound to the azide func-
tional group.‡
Table 2 Yields of various azido-heterobifunctionalized OEG linkers
Compound
n
Yield (%)
Compound
n
Yield (%)
11a
11b
11c
12a
12b
12c
13a
13b
13c
14a
14b
2
4
6
2
4
6
2
4
6
2
4
36(69)a
37(54)a
46(51)a
100
100
100
94
87
95
99
14c
15a
15b
15c
16a
16b
16c
17a
17b
17c
6
2
4
6
2
4
6
2
4
6
99
96
61
94
96
99
98
92(88)b
93(86)b
91(83)b
99
a Yields in parentheses are obtained using Method B. b Yields in
parentheses are obtained using Route 2.
literature, nor have any aryl or alkyl carbonates of OEGs with 4,
6, or 8 ethylene glycol units.
Nucleophilic substitution of the mesylate groups on the
azide-substituted OEGs occurred just as readily as it did for
the mesylate groups of the alkyne-substituted OEGs. Com-
pounds 14a–c were readily converted to iodo-substituted pro-
ducts 15a–c; likewise, the mesylate group could also readily be
replaced by phthalimide to give 16a–c in high yield (96–99%).
1H NMR analysis confirmed the formation of the phthalimide
derivatives 16a–c by the presence of characteristic aromatic
peaks at ∼δ 7.80 and 7.70 ppm. Reacting 16a–c with hydrazine
produced the amine derivatives 17a–c in appreciable yields
(91–93%). Although preparation of 17a via the Gabriel syn-
thesis has been reported by others,31 the synthesis of 17b–c
from 1b–c has not.
The di-azide OEGs 12a–c were obtained as minor products
(15–20%) during the synthesis of 11a–c. However, increasing
the amount of MsCl to 3.0 eq. in the reaction with 1a–c gave
12a–c in 100% yield. We used the di-azide products to investi-
gate whether 17a–c could be produced by selective reduction
of one of the azido groups to an amine via Staundinger
In order to determine the best reaction conditions for the
formation of 11a–c, an alternative Method B was employed in
which silver oxide (Ag2O) was used during the monomesylation
step.16,29,30 Use of Ag2O resulted in significantly higher yields
of 11a–c (51–69%) compared to Method A.
Functionalization of the remaining hydroxyl group on 11a–
c was achieved through a series of reactions similar to those
used for the alkyne-terminated OEGs 2a–c. The carbonates
1
13a–c were prepared in high yield (87–95%). H NMR verified
the formation of the carbonates by the presence of aromatic
doublets at ∼δ 8.30 and 7.40 ppm. All three compounds 13a–c
are novel and have not been previously reported in the
‡Synthetic procedures and characterization details for the heterofunctionalized
OEG linkers obtained from tetraethylene glycol and hexaethyelene glycols are
included in the ESI.†
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Scheme 3 Synthesis of discrete PEGs via alkyne-azide click reactions.
reduction in a biphasic reaction medium (Et2O-5% aq HCl).32
Conjugation of the peptide cRGDfK to 3c via a carbamate
Interestingly, this attempt produced 17a–c from 1a–c in a more linkage was achieved by reaction of the amino group on the
facile manner than the five-step method using the Gabriel syn- lysine moiety of cRGDfK with the 4-nitrophenyl carbonate
thesis described above. The overall yield of 17a–c from 1a–c moiety of 3c in the presence of Et3N in DMF. A high yield
was also considerably higher, with yields ranging from (86%) of the OEG-cRGDfK conjugate 20 was obtained after
83–88% when using the 12a–c route compared to 30–37% size-exclusion chromatography on a lipophilic Sephadex LH-20
when using the 11a–c route. Formation of 17a–c introduces column. The alkyne-terminated OEG-fluorophore conjugate 21
heterofunctionality that allows the OEG linkers to be readily was synthesized by reacting sulfo-rhodamine B acid chloride
used in bioconjugation reactions. Moreover, the multiple path- with 9c in the presence of N,N-diisopropylethylamine (DIPEA)
ways for producing 17a–c demonstrate the versatility of these in THF. Sulfo-rhodamine B conjugate 21 was obtained in good
synthetic schemes for addition of both alkyne and azide func- yield (71%) following alumina column chromatography. The
tionalities on discretely sized OEGs.
structures of both conjugates were verified by 1H NMR and
The OEGs produced by these various methods make them mass spectrometry.
useful for azide-alkyne click reactions. An example of this
utility is demonstrated in high-yield production of discrete
long-chain PEGs. Although expensive, PEGs having 6 or 8
ethylene glycol units are commercially available in 85–95%
Conclusions
purity. However, long-chain PEGs (e.g., with 16 or 24 ethylene
glycol units), are extremely difficult to acquire and are usually
of low purity. We overcame this barrier by developing a very
simple and efficient methodology to synthesize discrete PEG16
(18) and PEG24 derivatives (19), in high yield by employing a
Cu(I)-catalyzed azide-alkyne click reaction (Scheme 3). The
PEG16 derivative 18 was synthesized in 98% yield by reacting
1.0 eq. of alkyne-terminated OEG 2c with 1.0 eq. azide-functio-
nalized OEG 11c. The PEG24 derivative 19 was synthesized by
reacting 2.0 eq. of alkyne-terminated OEG 2c with 1.0 eq of
diazide 12c in 96% yield. PEG products 18 and 19 were charac-
terized using 1H, 13C NMR and HRMS. The 1H NMR spectra of
both compounds showed a characteristic singlet at ∼δ 7.6 ppm
that was assigned to the –CH– of the triazole ring formed via
the azide-alkyne [3 + 2] dipolar cycloaddition reaction.
This manuscript describes the efficient and high-yield syn-
thesis of a variety of discretely sized heterobifunctional oligo-
(ethylene glycol) linkers useful for click chemistry applications.
To demonstrate the broad utility of these heterobifunctional
linkers in bioconjugate chemistry, we developed a reliable,
high-yield protocol for the synthesis of linker conjugates with
a tumor-targeting peptide (cRGDfK) and the fluorophore sulfo-
rhodamine B. The rapid covalent linkage of two dissimilar
components under biocompatible conditions is useful for the
creation of bifunctional agents for diagnostic and therapeutic
applications.36,37 The methods presented here will be useful
for achieving diverse and efficient bioconjugation strategies in
biomedical and drug discovery research. Specific biological
applications of bioconjugates produced by the methods
described here will be reported in future publications.
The utility of the clickable OEG linkers in bioconjugation
reactions was demonstrated by their successful attachment to
the peptide cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGDfK, 20) and the
fluorophore sulfo-rhodamine B (21) (Scheme 4). The peptide
cRGDfK is able to selectively target integrin receptors (αvβ3)
that are commonly present in the neovasculature of a variety of
tumor types and has been used successfully in tumor-targeting
strategies.33,34 Fluorescent dyes such as sulfo-rhodamine B are
of increasing interest in biomedical research because of their
ability to be traced in vivo.35
Experimental section
Materials and methods
Common reagents and chromatographic solvents were pur-
chased from commercial suppliers (Sigma-Aldrich, Fisher
Scientific and Acros Organics) and used without any further
purification. Lipophilic Sephadex LH-20 was obtained from GE
Healthcare. NMR spectra were recorded on Bruker Avance 400
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Scheme 4 Conjugation of alkyne-terminated OEGs with the tumor-targeting peptide cRGDfK and the fluorescent probe, sulfo-rhodamine B.
and 500 MHz spectrometers. All NMR chemical shifts (δ) are General procedure for the preparation of 3
reported in parts per million (ppm). The high-resolution mass
spectrometry analysis was performed using Applied Biosystems
Mariner ESI-TOF.
A stirred mixture of 4-nitrophenyl chloroformate (1.5 eq.) and
pyridine (2.0 eq.) in acetonitrile was allowed to cool to 0 °C for
15 min. A solution of 2 (1.0 eq.) in acetonitrile was added
slowly to the mixture. The mixture was allowed to warm to
room temperature and reacted for 15 h. Then, the reaction
mixture was concentrated to dryness, re-dissolved in DCM, and
washed with brine. The organic layer was concentrated and
dried in vacuo to give the crude product as a yellow oil. The
pure product was then obtained by size-exclusion chromato-
graphy on lipophilic Sephadex LH-20 using acetonitrile as the
mobile phase.
Synthesis of 3c. By using the general procedure above with
2c (0.50 g, 1.22 mmol), 4-nitrophenyl chloroformate (0.37 g,
1.84 mmol), and pyridine (0.19 g, 2.45 mmol), the pure
product was obtained as a yellow oil (0.61 g, 87%). 1H NMR
(400 MHz, CDCl3) δ = 8.20 (d, J = 9.20 Hz, 2H, Ar–H–NO2), 7.32
(d, J = 9.20 Hz, 2H, Ar–H–OCO–), 4.36 (t, J = 4.4 Hz, 2H,
–O–CH2CH2–OCO–), 4.11 (d, J = 2.0 Hz, 2H, HCCCH2O–),
3.74 (m, 2H, –O–CH2CH2–OCO–), 3.61–3.57 (m, 28H,
–OCH2CH2O–), 2.42 ppm (m, 1H, HCCCH2O–). 13C NMR
(100.6 MHz, CDCl3) δ = 156.28 (1C), 153.18 (1C), 146.10 (1C),
126.02 (2C), 122.58 (2C), 80.47 (1C), 75.45 (1C), 71.41–71.29
(12C), 71.10 (1C), 69.81 (1C), 69.32 (1C), 69.06 (1C), 59.07 ppm
(1C). HRMS (ESI): m/z calcd for C26H39O13 + Na+ [M + Na]+
596.2319; found 596.0696.
General procedure for the preparation of 2
A mixture of NaH (0.7 eq.) in THF (30 mL) was prepared in a
two-neck round-bottom flask with extensive stirring, and a
solution of OEG 1 (1.0 eq.) in THF (40 mL) was added over
30 min. The reaction was stirred for 1 h at room temperature,
and a solution of propargyl bromide (1.0 eq.) in THF (30 mL)
was then added by syringe pump over 1 h. The solution was
stirred for 1 h at room temperature and then an additional
15 h at 60 °C. The reaction mixture was quenched with 3%
HCl (100 mL), and the organic layer was removed by rotary
evaporation. The crude product was extracted from the acidic
aqueous layer with DCM (100 mL) and washed with brine
(100 mL). The organic layer was dried over Na2SO4 and concen-
trated in vacuo to obtain the crude product as a colorless oil.
The pure product was obtained by silica gel column chromato-
graphy (gradient eluent: 0–1–2–3–4–5% MeOH in DCM). The
OEG starting material was easily recovered by extraction from
the concentrated aqueous layer with diethyl ether.
Synthesis of 2c. By using the general procedure described
above with OEG 1c (2.00 g, 5.40 mmol), NaH (0.91 g,
3.78 mmol), and propargyl bromide (0.64 g, 5.40 mmol), the
1
pure product was obtained as a colorless oil (1.07 g, 69%). H
NMR (400 MHz, CDCl3) δ = 4.21 (d, J = 2.4 Hz, 2H,
HCCCH2O–), 3.74–3.60 (m, 30H, –OCH2CH2O–), 3.10 (m, 1H,
General procedure for the preparation of 4
HCCCH2O–), 2.45 ppm (t, J = 2.0 Hz, 2H, –CH2CH2OH). 13C A mixture of Et3N (2.0 eq.) and 2 (1.0 eq.) in DCM (35 mL) was
NMR (100.6 MHz, CDCl3) δ = 80.47 (1C), 75.33 (1C), 73.44 (1C), stirred at 0 °C, and a solution of MsCl (1.5 eq.) in DCM
71.40–71.31 (11C), 71.19 (1C), 71.08 (1C), 69.91 (1C), 62.50 (20 mL) was added slowly over 30 min. After the addition was
(1C), 59.19 ppm (1C). HRMS (ESI): m/z calcd for C19H36O9
Na+ [M + Na]+ 431.2257; found 431.2579.
+
complete, the reaction was allowed to proceed at 0 °C for
90 min and then at room temperature for an additional 2 h.
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The reaction mixture was concentrated to dryness, re-dissolved General procedure for the preparation of 7
in DCM (100 mL), and washed with 3% HCl (100 mL) and
In a dry round bottom flask, LAH (4.0 eq.) and THF were com-
bined under cooling in an NH4Cl ice bath (−10 °C). A solution
of 6 (1.0 eq.) in THF was added slowly to the reaction vessel.
The reaction mixture was stirred for 3 h allowing it to come to
room temperature. The mixture was then quenched with a
saturated aqueous solution of NH4Cl. The crude product was
extracted with dichloromethane, dried over sodium sulfate,
and concentrated in vacuo. The pure product was obtained by
silica gel column chromatography (gradient-eluent: 0–1–2–3–
4% MeOH in DCM) as a pale yellow oil.
Synthesis of 7c. By using the general procedure above with
6c (0.09 g, 0.19 mmol) and LAH (0.29 g, 0.77 mmol), the pure
product was obtained as a pale yellow oil (0.08 g, 98%). 1H
NMR (400 MHz, CDCl3) δ = 4.20 (d, J = 2.4 Hz, 2H,
HCCCH2O–), 3.70–3.60 (m, 30H, –OCH2CH2O–), 2.70 (m, 2H,
–OCH2CH2SH), 2.45 (t, J = 2.4 Hz, 1H, HCCCH2O–), 1.60 ppm
(t, J = 8.2 Hz, 1H, –OCH2CH2SH). 13C NMR (100.6 MHz, CDCl3)
δ = 80.47 (1C), 75.35 (1C), 73.68 (1C), 71.43–71.33 (11C), 71.20
(1C), 71.03 (1C), 69.91 (1C), 59.18 (1C), 25.05 ppm (1C). HRMS
(ESI): m/z calcd for C19H36O8S + Na+ [M + Na]+ 447.2023; found
447.1960.
brine. The organic layer was separated, dried over Na2SO4 and
then concentrated in vacuo to give the pure product as a pale
yellow oil.
Synthesis of 4c. By using the general procedure above with
2c (1.45 g, 3.54 mmol), MsCl (0.61 g, 5.31 mmol), and Et3N
(0.72 g, 7.08 mmol), the pure product was obtained as a pale
yellow oil (1.71 g, 99%). 1H NMR (400 MHz, CDCl3) δ = 4.31
(m, 2H, HCCCH2O–), 4.13 (d, J = 2.40 Hz, 2H, –OCH2CH2–
SO2CH3), 3.70 (m, 2H, –OCH2CH2–SO2CH3), 3.64–3.57 (m,
28H, –O–CH2CH2–O–), 3.02 (s, 3H, –S–(CH3), 2.43 ppm (m, 1H,
HCCCH2O–). 13C NMR (100.6 MHz, CDCl3) δ = 80.47 (1C),
75.44 (1C), 71.35–71.23 (12C), 71.11 (1C), 70.15 (1C), 69.83
(1C), 69.73 (1C), 59.09 (1C), 38.44 ppm (1C). HRMS (ESI): m/z
calcd for C20H38O11S + Na+ [M + Na]+ 509.2033; found
509.3040.
General procedure for the preparation of 5
A mixture of 4 (1.0 eq.) and NaI (4.0 eq.) in acetone was stirred
for 15 h at 65 °C. The reaction mixture was concentrated to
dryness and filtered over celite with DCM. The filtrate was con-
centrated and purified by silica gel chromatography (gradient-
eluent: 0–1–2–3–4% MeOH in DCM) to obtain the product as a
pale yellow oil.
General procedure for the preparation of 8
Compound 4 (1.0 eq.) was dissolved in DMF with potassium
phthalimide (1.5 eq.) and reacted at 110 °C for 15 h. The
mixture was concentrated under high-vacuum to remove DMF,
and the resulting solid was dissolved in diethyl ether. This
solution was filtered with a sintered funnel and concentrated
to give the pure product as a yellow oil.
Synthesis of 8c. By using the general procedure above with
4c (2.68 g, 5.51 mmol) and potassium phthalimide (1.53 g,
8.27 mmol), the pure product was obtained as a yellow oil
(2.87 g, 97%). 1H NMR (400 MHz, CDCl3) δ = 7.87 (m, 2H,
Ar–H), 7.74 (m, 2H, Ar–H), 4.22 (d, J = 2.0 Hz, 2H, HCCCH2O–),
3.92 (t, J = 5.6 Hz, 2H, –OCH2CH2–NPhth), 3.77–3.60 (m, 30H,
–OCH2CH2O–), 2.46 ppm (t, J = 2.4 Hz, 1H, HCCCH2O–). 13C
NMR (100.6 MHz, CDCl3) δ = 169.05 (2C), 134.90 (2C), 132.97
(2C), 124.20 (2C), 80.49 (1C), 75.31 (1C), 71.41–71.36 (11C),
71.22 (1C), 70.91 (1C), 69.93 (1C), 68.71 (1C), 59.21 (1C),
38.08 ppm (1C). HRMS (ESI): m/z calcd for C27H39NO10 + Na+
[M + Na]+ 560.2466; found 560.2863; for C27H39NO10 + K+
[M + K]+ 576.2206; found 576.2664.
Synthesis of 5c. By using the general procedure above with
4c (0.60 g, 1.22 mmol) and NaI (0.73 g, 4.90 mmol), the pure
product was obtained as a pale yellow oil (0.62 g, 98%).
1H NMR (400 MHz, CDCl3) δ = 3.98 (d, J = 2.4 Hz, 2H,
HCCCH2O–), 3.54 (t, 2H, J = 6.8 Hz, –OCH2CH2I), 3.48–3.43 (m,
28H, –OCH2CH2–O), 3.06 (t, J = 6.8 Hz, 2H, –OCH2CH2I),
2.38 ppm (t, J = 2.4 Hz, 1H, HCCCH2O–). 13C NMR (100.6 MHz,
CDCl3) δ = 80.43 (1C), 75.72 (1C), 72.52 (1C), 71.26–71.20
(11C), 70.99 (1C), 70.82 (1C), 69.68 (1C), 58.96 (1C), 4.08 ppm
(1C). HRMS (ESI): m/z calcd for C19H35IO8 + H+ [M + H]+
519.1455; found 519.1453.
General procedure for the preparation of 6
A mixture of 4 (1.0 eq.) and KSAc (1.5 eq.) in DMF was stirred
for 15 h at room temperature. The reaction mixture was
washed with a saturated aqueous solution of NH4Cl, extracted
with DCM, dried over Na2SO4, and concentrated to dryness
in vacuo. The pure product was then obtained by silica gel
column chromatography (gradient-eluent: 0–1–2–3–4% MeOH
in DCM) as a colorless oil.
General procedure for the preparation of 9
Synthesis of 6c. By using the general procedure above with
4c (0.24 g, 0.49 mmol) and KSAc (0.08 g, 0.74 mmol), the pure Hydrazine (17.0 eq.) was added to a solution of 8 (1.0 eq.) in
1
product was obtained as a colorless oil (0.15 g, 65%). H NMR ethanol, and the mixture was reacted for 3 h at 60 °C. The reac-
(400 MHz, CDCl3) δ = 4.17 (d, J = 2.4 Hz, 2H, HCCCH2O–), tion mixture was concentrated to dryness, and the product was
3.67–3.55 (m, 30H, –OCH2CH2O–), 3.06 (t, J = 6.4 Hz, 2H, isolated from the resulting phthalyl hydrazide by filtering over
–OCH2CH2–SCOCH3), 2.44 (t, J = 2.4 Hz, 1H, HCCCH2O–), 2.31 celite and washing with diethyl ether. The solution was then
(s, 3H, –OCH2CH2–SCOCH3). 13C NMR (100.6 MHz, CDCl3) δ = concentrated to give the pure product as a pale yellow oil.
196.21 (1C), 80.46 (1C), 75.36 (1C), 71.41–71.29 (13C), 71.17
Synthesis of 9c. By using the general procedure above with
(1C), 71.09 (1C), 70.52 (1C), 69.88 (1C), 59.15 ppm (1C). HRMS 8c (2.77 g, 5.16 mmol) and hydrazine (2.77 g, 86.5 mmol), the
(ESI): m/z calcd for C21H38O9S + Na+ [M + Na]+ 489.2129; found pure product was obtained as a pale yellow oil (2.08 g, 99%).
489.2032.
1H NMR (400 MHz, CDCl3) δ = 4.23 (m, 2H, HCCCH2O–),
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3.73–3.66 (m, 30H, –OCH2CH2O–), 3.53 (m, 2H, –OCH2CH2- then reacted with NaN3 (5.0 eq.) in DMF for 12 h at 65 °C. The
NH2), 2.88 (m, 2H, –OCH2CH2NH2), 2.45 ppm (m, 1H, mixture was again filtered over celite, concentrated, dried
–HCCCH2O–). 13C NMR (100.6 MHz, CDCl3) δ = 75.31 (1C), in vacuo, and then purified via silica gel chromatography (gra-
74.30 (1C), 71.40 (12C), 71.22 (1C), 71.12 (1C), 69.93 (1C), 59.21 dient-eluent: 0–1–2–3–4% MeOH in DCM) as a pale yellow oil.
(1C), 42.65 ppm (1C). HRMS (ESI): m/z calcd for C19H37NO8 +
H+ [M + H]+ 408.2592; found 408.2108.
Synthesis of 11c. By using the general procedure above for
Method A with OEG 1c (4.98 g, 13. 5 mmol), MsCl (1.39 g,
12.1 mmol), Et3N (2.04 g, 20.2 mmol), and NaN3 (4.37 g,
67.3 mmol), the pure product was obtained as a pale yellow oil
General procedure for the preparation of 10
Compound 9 (1.0 eq.) was dissolved in a saturated aqueous (2.20 g, 46%). By using the general procedure above for
solution of NaHCO3 at 0 °C, and N-methoxycarbonyl male- Method B with OEG 1c (1.00 g, 2.70 mmol), Ag2O (938 mg,
imide (1.0 eq.) was added slowly. The mixture was reacted for 4.05 mmol), and MsCl (340 mg, 2.97 mmol), the pure product
1 h at 0 °C and then for an additional 1 h at room temperature. was obtained as a pale yellow oil (545 mg, 51%): 1H-NMR
The product was extracted from the aqueous mixture with (400 MHz, CDCl3) δ = 3.56–3.50 (m, 28H, –OCH2CH2O–), 3.24
DCM, and the organic layer was dried over Na2SO4 and concen- (t, J = 5.2 Hz, 2H, –CH2CH2OH), 3.07 ppm (t, J = 5.9 Hz, 2H,
trated in vacuo. The pure product was then obtained by silica N3CH2CH2O–). 13C (100.6 MHz, CDCl3) δ
gel column chromatography (gradient eluent: 0–1–2–3% 71.30–71.21 (11C), 70.99 (1C), 70.67 (1C), 62.21 (1C),
= 73.29 (1C),
MeOH in DCM) as a pale yellow oil.
51.32 ppm (1C). HRMS (ESI): m/z calcd for C16H33N3O8 + Na+
Synthesis of 10c. By using the general procedure above with [M + Na]+ 418.2160; found 418.3255.
9c (1.00 g, 2.45 mmol) and N-methoxycarbonyl maleimide
General procedure for the preparation of 12
(0.38 g, 2.45 mmol), the pure product was obtained as a pale
1
yellow oil (0.50 g, 42%). H NMR (400 MHz, CDCl3) δ = 6.67 (s, A solution of MsCl (3.0 eq.) in DCM (15 mL) was added slowly
2H, Mal-H), 4.14 (d, J = 2.4 Hz, 2H, –HCCCH2O–), 3.68–3.53 over 30 min to a mixture of Et3N (3.0 eq.) and OEG 1 (1.0 eq.)
(m, 32H, –OCH2CH2O–), 2.42 ppm (t, J = 2.4 Hz, 1H, in DCM (15 mL) stirring at 0 °C. After addition was complete,
HCCCH2O–). 13C NMR (100.6 MHz, CDCl3) δ = 170.62 (2C), the reaction was allowed to proceed at 0 °C for 2 h and then at
134.15 (2C), 79.65 (1C), 74.60 (1C), 70.56–70.49 (11C), 70.35 room temperature for a further 2 h. The reaction mixture was
(1C), 69.99 (1C), 69.06 (1C), 67.75 (1C), 58.34 (1C), 37.07 ppm concentrated to dryness and washed first with 3% HCl
(1C). HRMS (ESI): m/z calcd for C23H37NO10 + Na+ [M + Na]+ (100 mL) and then brine (100 mL), extracting with DCM
510.2310; found 510.2936.
(100 mL). This procedure was repeated thrice and the organic
layer was dried over Na2SO4 and then in vacuo. The resulting
dimesylated product was reacted with NaN3 (5.0 eq.) in DMF at
General procedure for the preparation of 11 – Method A
A solution of MsCl (0.9 eq.) in DCM (20 mL) was added by 65 °C for 15 h. DMF was removed in vacuo and then excess
syringe pump over 1 h to a mixture of Et3N (1.5 eq.) and OEG 1 NaN3 removed via filtration over celite, washing with Et2O to
(1.0 eq.) in DCM (35 mL) stirring at 0 °C. After addition, the give the pure product as a pale yellow oil.
reaction was allowed to proceed at 0 °C for 1 h, and then at
Synthesis of 12c. By using the general procedure above with
room temperature for a further 2 h. The reaction mixture was OEG 1c (500 mg, 1.35 mmol), MsCl (466 mg, 4.05 mmol), Et3N
concentrated to dryness and washed first with 3% HCl (409 mg, 4.05 mmol), and NaN3 (438 mg, 6.75 mmol), the pure
(100 mL) and then with brine (100 mL), extracting with DCM product was obtained as a pale yellow oil (568 mg, 100%):
(100 mL). This procedure was repeated thrice and the organic 1H-NMR (400 MHz, CDCl3) δ = 3.63 (m, 28H, –OCH2CH2O–),
layer was dried over Na2SO4 and then in vacuo to give a 3.36 ppm (t,
mixture of di- and mono-mesylated polyethylene glycol. This (100.6 MHz, CDCl3)
J
=
5.2 Hz, 4H, N3CH2CH2O–). 13C-NMR
71.46–71.36 (12C), 70.79 (2C),
δ
=
mixture was reacted with NaN3 (5.0 eq.) in DMF for 15 h at 51.46 ppm (2C). HRMS: m/z calcd for C16H32N6O7 + Na+
65 °C. The crude product was concentrated to dryness and [M + Na]+ 443.2225; found 443.2072.
filtered over celite, washing with Et2O. The mixture was then
General procedure for the preparation of 13
concentrated and purified via silica gel chromatography (gradi-
ent-eluent: 0–1–2–3–4% MeOH in DCM) as a pale yellow oil.
A stirring mixture of 4-nitrophenyl chloroformate (1.5 eq.) and
pyridine (2.0 eq.) in MeCN was cooled to 0 °C for 15 min, and
a solution of 11 (1.0 eq.) in MeCN was added slowly. The
General procedure for the preparation of 11 – Method B
Note: For the reaction with hexa(ethylene glycol), KI was used as mixture was allowed to warm to room temperature and then
an additive. KI was not required for tetra(ethylene glycol) or octa- react for 15 h. The reaction mixture was then concentrated to
(ethylene glycol). MsCl (1.1 eq.) was added over a period of dryness, washed with brine, and extracted with DCM. The
30 min with a syringe pump (0.333 mL min−1) to a solution of organic layer was concentrated and dried in vacuo to give the
OEG 1 (1.0 eq.) and Ag2O (1.5 eq.) in DCM at 0 °C. The mixture crude product as a pale yellow oil. Pure product was obtained
was then allowed to slowly warm to room temperature and by size-exclusion chromatography on LH-20 using MeCN as
react for 24 h. The reaction mixture was filtered over celite, the mobile phase.
washing with ethyl acetate. The organic phase was concen-
Synthesis of 13c. By using the general procedure above with
trated and thoroughly dried in vacuo. This crude mixture was 11c (100 mg, 0.25 mmol), 4-nitrophenyl chloroformate
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(76.5 mg, 0.38 mmol), and pyridine (40.0 mg, 0.51 mmol), the mixture was concentrated under high vacuum to remove the
pure product was obtained as a pale yellow oil (135 mg, 95%): DMF. The resulting solid was filtered through a sintered
1H-NMR: (500 MHz, CDCl3) δ = 8.33 (d, J = 5.0 Hz, 2H, Ar–H– funnel, washing with Et2O, and then concentrated to give the
NO2), 7.45 (d, J = 5.0 Hz, 2H, Ar–H–OCO–), 4.49 (m, 2H, pure product as a yellow oil.
–O–CH2CH2–OCO–), 3.87 (m, 2H, –O–CH2CH2–OCO–), 3.72
Synthesis of 16c. By using the general procedure above with
(m, 26H, –OCH2CH2O–), 3.40 ppm (t, J = 5.0 Hz, 2H, 14c (400 mg, 0.85 mmol) and potassium phthalimide (234 mg,
N3CH2CH2O–). 13C-NMR (100.6 MHz, CDCl3) δ = 155.51 (1C), 1.27 mmol), the pure product was obtained as a yellow oil
152.45 (1C), 145.35 (1C), 125.28 (2C), 121.80 (2C), 70.68–70.55 (434 mg, 98%): 1H-NMR (500 MHz, CDCl3) δ = 7. 85 (m, 2H,
(12C), 70.00 (1C), 68.59 (1C), 68.31 (1C), 50.65 ppm (1C). Ar–H), 7.74 (m, 2H, Ar–H), 3.91 (t, J = 10.0 Hz, 2H,
HRMS (ESI): m/z calcd for C23H36N4O12 + Na+ [M + Na]+ –OCH2CH2NPhth), 3.75 (t, J = 5.0 Hz, 2H, –OCH2CH2NPhth),
583.2222; found 583.1295.
3.68 (m, 26H, –OCH2CH2O–), 3.40 ppm (t, J = 5.0 Hz, 2H,
N3CH2CH2O–). 13C-NMR (125.8 MHz, CDCl3) δ = 168.15 (2C),
133.90 (2C), 132.06 (2C), 123.16 (2C), 70.61–70.45 (11C), 70.02
General procedure for the preparation of 14
A solution of MsCl (1.5 eq.) in DCM (20 mL) was added slowly (1C), 69.96 (1C), 67.82 (1C), 50.61 (1C), 37.19 ppm (1C). HRMS:
over 30 min to a mixture of Et3N (2.0 eq.) and 11 (1.0 eq.) in m/z calcd for C24H36N4O9 + Na+ [M + Na]+ 547.2374; found
DCM (35 mL) stirring at 0 °C. After this, the reaction was 547.0832; for C24H36N4O9 + K+ [M + K]+ 563.2114; found
allowed to proceed at 0 °C for 90 min, and then at room temp- 563.0738.
erature for a further 2 h. The reaction mixture was concen-
trated to dryness and washed first with 3% HCl (100 mL) and
General procedure for the preparation of 17 from 16 – Route 1
then brine (100 mL), extracting with DCM (100 mL). This pro- Hydrazine (17.0 eq.) was added to a solution of 16 (1.0 eq.) in
cedure was repeated thrice and the organic layer was dried ethanol, and the mixture was reacted for 3 h at 60 °C. The reac-
over Na2SO4 and then in vacuo. The organic layer was dried tion mixture was concentrated to dryness, and the pure
over Na2SO4 and then in vacuo to give the pure product pale product was separated from the formed phthalyl hydrazide by
yellow oil.
filtration over celite, washing with Et2O. The solution was then
Synthesis of 14c. By using the general procedure above with concentrated to give the pure product as a yellow oil.
11c (2.39 g, 6.05 mmol), MsCl (1.04 g, 9.07 mmol), and Et3N
General procedure for the preparation of 17 from 12 – Route 2
(1.22 g, 12.1 mmol), the pure product was obtained as a pale
yellow oil (2.83 g, 99%): 1H-NMR (400 MHz, CDCl3) δ = 4.34 To 12 (1.0 eq.), 5% HCl was added with vigorous stirring at
(m, 2H, –OCH2CH2OMs), 3.73 (m, 2H, N3CH2CH2O–), 3.63 (m, room temperature. A solution of triphenyl phosphine (0.9 eq.)
26H, –OCH2CH2O–), 3.35 (m, 2H, N3CH2CH2O–), 3.06 ppm (m, in Et2O was added to this mixture over 3 hours. The mixture
3H, –OSO2CH3). 13C-NMR (100.6 MHz, CDCl3) δ = 71.40–71.26 was then allowed to react at room temperature for a further
(11C), 70.76 (1C), 70.16 (1C), 69.76 (1C), 51.44 (1C), 38.46 (1C), 24 hours. The reaction mixture was washed with ethyl acetate
32.38 ppm (1C). HRMS: m/z calcd for C17H35N3O10S + Na+ (50 mL × 3) to remove the unreacted starting materials and tri-
[M + Na]+ 496.1935; found 496.1485.
phenylphosphine oxide that was formed during the reaction.
The aqueous layer was collected, cooled to 0 °C, and concen-
trated potassium hydroxide was added to it until the pH of the
General procedure for the preparation of 15
A mixture of 14 (1.0 eq.) and sodium iodide (4.0 eq.) in solution was basic (∼12). The product was extracted as a pale
acetone was stirred for 15 h at 65 °C. The reaction mixture was yellow oil by washing the aqueous layer with DCM thrice,
then concentrated to dryness and filtered over celite with drying over Na2SO4 and then in vacuo.
DCM. The filtrate was concentrated and purified by silica gel
chromatography (gradient-eluent: 0–1–2–3–4% MeOH in DCM) Route 1 with 16c (410 mg, 0.78 mmol) and hydrazine (410 mg,
to obtain the product as a pale yellow oil. 12.8 mmol), the pure product was obtained as a yellow oil
Synthesis of 17c. By using the general procedure above for
Synthesis of 15c. By using the general procedure above with (281 mg, 91%). By using the general procedure above for Route
14c (1.85 g, 3.91 mmol) and sodium iodide (2.34 g, 2 with 12c (2.00 g, 4.76 mmol), triphenyl phosphine (1.12 g,
15.6 mmol), the pure product was obtained as a pale yellow oil 4.28 mmol), 5% HCl (19 mL), and Et2O (14 mL), the pure
(1.86 g, 94%): 1H-NMR (400 MHz, CDCl3) δ = 3.67 (t, J = 6.8 Hz, product was obtained as a pale yellow oil (1.55 g, 83%):
2H, –OCH2CH2I), 3.58 (m, 26H, –OCH2CH2O–), 3.31 (t, J = 1H-NMR (500 MHz, CDCl3) δ = 3.62 (m, 26H, –OCH2CH2O–),
5.2 Hz, 2H, N3CH2CH2O–), 3.18 ppm (t, J = 6.8 Hz, 2H, 3.49 (t, J = 5.0 Hz, 2H, N3CH2CH2O–), 3.36 (t, J = 5.0 Hz, 2H,
–OCH2CH2I). 13C-NMR (100.6 MHz, CDCl3) δ = 72.68 (1C), –OCH2CH2NH2), 2.85 ppm (t, J = 5.0 Hz, 2H, N3CH2CH2O–).
71.41–71.32 (11C), 70.95 (1C), 70.76 (1C), 51.41 (1C), 3.87 ppm 13C-NMR (125.8 MHz, CDCl3) δ = 73.03 (1C), 70.57–70.43 (11C),
(1C). HRMS (ESI): m/z calcd for C16H32IN3O7 + Na+ [M + Na]+ 70.17 (1C), 69.92 (1C), 50.56 (1C), 41.56 ppm (1C). HRMS (ESI):
528.1177; found 528.0790.
m/z calcd for C16H34N4O7 + H+ [M + H]+ 395.2500; found
395.1928.
General procedure for the preparation of 16
Synthesis of PEG16 (18) (click reaction). In an oven-dry
Compound 14 (1.0 eq.) was dissolved in DMF with potassium 50 mL round-bottom flask, 2c (0.21 g, 0.51 mmol), 11c (0.20 g,
phthalimide (1.5 eq.) and reacted at 110 °C for 15 h. The 0.51 mmol) and copper(I) iodide (0.10 g, 0.51 mmol) were
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dissolved in a 50 : 50 mixture of THF and MeCN (15 mL). (0.28 g, 0.77 mmol) in 10 mL THF was added slowly. The
DIPEA (0.07 g, 0.51 mmol) was added and the reaction mixture reaction mixture was gradually warmed to room temperature
was vigorously stirred at room temperature for 12 h under an and stirred for 15 h under an argon atmosphere. The reaction
argon atmosphere. The reaction mixture was concentrated to mixture was concentrated via rotary evaporation, and the
dryness, re-dissolved in ethyl acetate, and filtered through a pure product was obtained as a pink oil by column chromato-
celite pad. The filtrate was concentrated, and the pure product graphy on alumina(IV) using MeOH/DCM gradient as the
1
was obtained by silica gel column chromatography as a color- mobile phase. Yield: 0.35 g (71%). H NMR (400 MHz, CDCl3)
less oil (0.40 g, 98%). 1H NMR (400 MHz, CDCl3) δ = 7.68 δ = 8.66 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H),
(s, 1H, CH-triazole), 4.57 (s, 2H, N–CH2–O–), 4.44 (t, J = 5.2 Hz, 7.12 (d, J = 9.2 Hz, 2H), 6.76 (d, J = 9.2 Hz, 2H), 6.65 (s, 2H),
2H, –NCH2–CH2O–), 3.77 (t, J = 5.2 Hz, 2H, –NCH2–CH2O–), 4.13 (d, J = 2.4 Hz, 2H), 3.62–3.48 (m, 39H), 3.16 (t, J = 5.2 Hz,
3.63–3.48 (m, 60H, –OCH2CH2O–), 3.19 (m, 2H, 1H), 2.43 (s, 1H), 1.24 ppm (t, J = 6.8 Hz, 12H). 13C NMR
–HOCH2CH2O–). 13C NMR (100.6 MHz, CDCl3) δ = 145.56 (1C), (100.6 MHz, CDCl3) δ = 158.65, 158.53, 156.31, 147.78, 143.08,
124.56 (1C), 73.33 (1C), 71.29–71.02 (27C), 70.33 (1C), 70.16 134.39, 133.70, 130.87, 128.20, 127.67, 114.92, 114.34,
(1C), 65.26 (1C), 62.28 (1C), 50.91 ppm (1C). HRMS (ESI): m/z 96.44, 80.22, 75.42, 71.18, 71.13, 71.03, 70.96, 70.53,
calcd for C35H69N3O17 + Na+ [M + Na]+ 826.4519; found 69.76, 59.05, 46.57, 43.64, 13.18 ppm. HRMS (ESI): m/z calcd
826.5635.
for C46H65N3O14S2 + Na+ [M + Na]+ 970.3800; found 970.4205;
Synthesis of PEG24 (19) (click reaction). In an oven-dry for C46H65N3O14S2 + 2Na+ [M + 2Na+]2+ 496.6849; found
50 mL round-bottom flask, 2c (0.49 g, 1.19 mmol), 12c (0.25 g, 496.7605.
0.59 mmol) and copper(I) iodide (0.23 g, 1.19 mmol) were dis-
solved in a 50 : 50 mixture of THF and MeCN (20 mL). DIPEA
(0.15 g, 1.19 mmol) was added, and the reaction mixture was
Abbreviations
vigorously stirred at room temperature for 12 h under an argon
atmosphere. The reaction mixture was concentrated to
dryness, re-dissolved in ethyl acetate, and filtered through a
celite pad. The filtrate was concentrated, and the pure product
was obtained by silica gel column chromatography as a color-
MeCN Acetonitrile
NH4Cl Ammonium chloride
DCM
Et2O
Dichloromethane
Diethyl ether
1
less oil (0.71 g, 96%). H NMR (400 MHz, CDCl3) δ = 7.67 (s,
DIPEA N,N-diisopropylethylamine
2H, 2-CH-triazole), 4.59 (s, 4H, 2-N–CH2–O–), 4.44 (t, J = 4.8 Hz,
4H, 2-NCH2–CH2O–), 3.78 (t, J = 5.2 Hz, 4H, 2-NCH2–CH2O–),
3.63–3.48 (m, 88H, –OCH2CH2O–), 2.99 (m, 2H,
–HOCH2CH2O–). 13C NMR (100.6 MHz, CDCl3) δ = 145.59 (1C),
124.51 (1C), 73.29 (1C), 71.31–71.06 (46C), 70.34 (1C), 70.17
(1C), 65.29 (1C), 62.32 (1C), 50.91 ppm (1C). HRMS (ESI): m/z
calcd for C54H104N6O25 + H+ [M + H]+ + Na+ 1260.7027; found
1260.7263.
DMF
HCl
LAH
MsCl
N,N′-dimethylformamide
Hydrochloric acid
Lithium aluminum hydride
Methanesulfonyl chloride
MeOH Methanol
KSAc
NaN3
NaI
Potassium thioacetate
Sodium azide
Sodium iodide
Synthesis of 20. In an oven-dry 50 mL round-bottom flask,
c(RGDfK) (10.0 mg, 16.5 μmol; Peptides International, Inc.,
Cat. PCI-3661-PI), and 3c (11.4 mg, 19.8 μmol) were dissolved in
10 mL DMF. Et3N (50.0 μl, excess) was added and the mixture
stirred for 15 h at room temperature under an argon atmos-
phere. The reaction mixture was concentrated under reduced
pressure, and the pure product was obtained as a pale yellow
oil by size-exclusion chromatography on lipophilic Sephadex
LH-20 using MeOH as the mobile phase. Yield: 15.0 mg (86%).
1H NMR (400 MHz, CDCl3) δ = 7.29–7.23 (m, 5H), 4.66 (t, J =
4.0 Hz, 1H), 4.57 (t, J = 6.4 Hz, 1H), 4.49 (t, J = 6.0 Hz, 1H), 4.30
(d, J = 15.2 Hz, 1H), 4.19 (m, 5H), 4.10 (dd, J = 4.8 and 9.6 Hz,
1H), 3.75 (m, 38H), 3.43 (d, J = 14.8 Hz, 1H), 3.29–2.96 (m, 6H),
2.88 (m, 1H), 2.67 (m, 1H), 2.56 (m, 1H), 1.89 (m, 1H),
1.78–1.73 (m, 2H), 1.68–1.50 (m, 3H), 1.41 (m, 2H), 1.15 (t, J =
6.8 Hz, 1H), 1.05 ppm (m, 2H). HRMS (ESI): m/z calcd for
C47H74N8O18 + Na+ [M + Na]+ 1061.5008; found 1061.3796.
Synthesis of 21. In an oven-dry 100 mL round-bottom flask,
sulfo-rhodamine B acid chloride (0.30 g, 0.51 mmol) was dis-
solved in 10 mL THF followed by the addition of DIPEA
(0.20 g, 1.56 mmol). The mixture was cooled to 0 °C, and 9c
Na2SO4 Sodium sulfate
THF
Et3N
Tetrahydrofuran
Triethylamine
Acknowledgements
Authors are thankful to Brett Meers of the International Insti-
tute of Nano and Molecular Medicine for the mass spectro-
metry results and to Pamela Cooper for manuscript editing.
Financial support from National Cancer Institute-NIH (grant
no. R21 CA114090) is greatly appreciated.
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