Enzymatically Activated Glyco-Prodrugs of Doxorubicin Synthesized by a Catalysis-Free Diels-Alder Reaction

Article abstract of DOI:10.1021/acs.bioconjchem.8b00314

The severe side effects associated with the use of anthracycline anticancer agents continues to limit their use. Herein we describe the synthesis and preliminary biological evaluation of three enzymatically activatable doxorubicin-oligosaccharide prodrugs. The synthetic protocol allows late stage variation of the carbohydrate and is compatible with the use of disaccharides such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the prodrugs was assayed on MCF-7 breast cancer cells.

Full text of DOI:10.1021/acs.bioconjchem.8b00314

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Article
Enzymatically Activated Glyco-Prodrugs of Doxorubicin
Synthesized by a Catalysis-Free Diels-Alder Reaction
David Bliman, Martine Demeunynck, Pierre Leblond,
Samuel Meignan, Isabelle Baussanne, and Sebastien Fort
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00314 • Publication Date (Web): 07 Jun 2018
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Enzymatically Activated GlycoꢀProdrugs of Doxorubicin Synthesized
by a CatalysisꢀFree DielsꢀAlder Reaction
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David Bliman1,2, Martine Demeunynck2, Pierre Leblond3, Samuel Meignan3, Isabelle
Bausanne2, Sebastien Fort1
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1 Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
2 Univ. Grenoble Alpes, CNRS, DPM, 38000 Grenoble, France.
3 Tumorigenesis and Resistance to Treatment Unit, Centre Oscar Lambret, Lille, FRANCE
INSERM U908, IRCL, Lille, FRANCE
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Abstract
The severe side effects associated with the use of anthracycline anticancer agents continues to
limit their use. Herein we describe the synthesis and preliminary biological evaluation of three
enzymatically activatable doxorubicinꢀoligosaccharide prodrugs. The synthetic protocol
allows late stage variation of the carbohydrate and is compatible with the use of disaccharides
such as lactose as well as more complex oligosaccharides such as xyloglucan oligomers. The
enzymatic release of doxorubicin from the prodrugs by both protease (plasmin) and human
carboxylesterases (hCE1 and 2) was demonstrated in vitro and the cytotoxic effect of the
prodrugs were assayed on MCFꢀ7 breast cancer cells.
Introduction
Despite continuing efforts in oncogenic drug development, surgery remains an important form
of treatment for solid tumours. This approach generally includes a postoperative drug therapy
to prevent reoccurrence of the tumour. Currently used drugs for such adjuvant treatment
include the anthracycline antibiotic Doxorubicin (Dox).1 While the details of the mechanism
of action of anthracyclines are still under investigation,2 the cytotoxic activity is suggested to
be a result of intercalation with DNA3 and inhibition of topoisomerase II.4 Due to the
unselective nature of the anthracyclines, treatments are often associated with severe side
effects such as cardiac toxicity.5 This has led to extensive efforts in reducing the systemic
toxicity of Dox and other anticancer drugs by different types of prodrug approaches.6 In these
strategies, the drug is modified in a way that minimizes its off target activity while allowing
activation at the intended site. Prodrug approaches for Dox include both nonꢀcovalent and
covalent modifications. A notable example of noncovalent prodrugs of Dox is the inclusion of
Dox in liposomes.7 Lipid encapsulation of the drug contributes to decreasing toxic side
effects, since liposomes can penetrate endothelial lesions found in the neovasculature of
tumors but not in normal blood vessels. FDA approved pegylated and nonꢀpegylated
liposomal Dox formulations, namely Doxil® and Myocet® respectively for treating Aids
related Kaposi’s sarcoma, ovarian cancer and breast cancer.8 The covalent approach typically
involves modification either on the primary amine to form carbamates,9, 10 or on the ketone
functionality by forming hydrazones. The pHꢀsensitive hydrazone can be cleaved in the acidic
microenvironment of the tumour11 and the carbamate can be attached to an enzymatically
hydrolyzable oligopeptide. Both approaches have been used to design cellꢀtargeting prodrugs
through the conjugation to antibodies.12, 13
The diverse functionalities of carbohydrates make them potential candidates for use in
prodrugs. Amazingly, there are very few examples of glycosylated prodrugs reported in the
literature to date. Carbohydrates have mainly been used as a triggering group for the drug
release since they can be hydrolyzed by the glycosidases overexpressed by cancer cells. The
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most prominent example is that of glucuronic acid14, 15 that is degraded by βꢀglucuronidase
which have been found at high levels in tumour tissue.16 Assets of carbohydrates are not
restricted to their biocompatibility and biodegradability. They also present a large range of
remarkable biological and physicoꢀchemical properties. Carbohydrateꢀprotein interactions
play an important role for regulating biochemical processes, such as cell differentiation,
proliferation and adhesion, inflammation, and immune response. The feasibility of using
carbohydrate ligands to target protein receptors, termed 'glycotargeting', was first
demonstrated in 1971.17 Since then the potential of using carbohydrates to design drugꢀ
delivering systems has been made clear. However, small molecule drugs, no matter how
heavily glycosylated they are, have the potential to pass into the kidneys, through glomerular
filtration, and be rapidly cleared. For this reason, much effort are devoted to designing
macromolecular glycosylated carriers and there is little work on the development of such
glycotargeting prodrugs.18
Carbohydrates also interact with other carbohydrates through the establishment of an
extensive network of hydrogen bondings and of hydrophobic interactions. In particular,
polysaccharideꢀpolysaccharide interactions play an important role in the control of the
architecture of animal and plant cells. Cellulose and xyloglucan (XG) assemble to form the
cellulose/XG network, which is considered the dominant loadꢀbearing structure in the
growing cell walls of nonꢀgraminaceous land plants. In a biomimetic approach, Brumer et al.
reported the efficient modification of cellulosic materials with different molecules including
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enzymes and the cell adhesion peptide RGD through anchorage of xyloglucanꢀbased
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glycoconjugates at their surface.19,
A particular advantage of such nonꢀcovalent
modification is to circumvent the limited reactivity of insoluble cellulose and to preserve the
fibre’s integrity.
In the present work, we report the synthesis and preliminary biological evaluation of new
enzymatically activated Dox glycoꢀprodrugs aimed at being administered in situ at the tumor
site after resection. The carbohydrates, lactose and XGO, the oligosaccharideꢀrepeating unit
of xyloglucan, were chosen to address two specific roles. Lactose binds to galectins, a family
of βꢀgalactoside binding proteins associated with tumour malignancy.21, 22 Interaction between
lactose and galectins is expected to decrease diffusion of the glycoꢀprodrug and to hamper its
rapid clearance thus allowing a sustained release of the Dox when administered in solution.
On the other hand, the use of XGO could allow the immobilization of the Dox prodrug onto
cellulosic wound dressing. The synthetic route consisted in the conjugation of furanꢀmodified
carbohydrates and maleimideꢀactivacted Dox by a metal free Diels Alder cycloaddition
(Figure 1). We have previously used thiolꢀmaleimide Michaelꢀtype addition23 coupling for
conjugation of reducingꢀend functionalized carbohydrates. While being a metalꢀfree reaction,
this reaction requires handling sulfurylated molecules susceptible to oxidation. Another type
of ligation reaction is the DielsꢀAlder cycloaddition.24 The inverse electron demand Diels–
Alder reaction (iEDDA) between 1,2,4,5ꢀtetrazines and olefins has increasingly gained
importance in chemical biology as a fast and metalꢀfree bioorthogonal ligation reaction.25, 26
Furanꢀmaleimide cycloaddition is another type of popular DielsꢀAlder reaction having the
advantage of using widely available reactants. This conjugation method recently allowed us to
prepare glycosylated chromatographic matrices for the capture of carbohydrateꢀbinding
proteins.27 This reaction allows catalysisꢀfree introduction of the carbohydrate part in water or
in organic solvent at a late stage, avoiding extensive carbohydrate protecting group strategies.
The presence of a peptideꢀ or esterꢀcontaining spacer arm between the maleimide group and
the anthracycline should allow the enzymatic release of the Dox by endogenous peptidases or
esterases.
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Figure 1. General scheme of the enzymatically activated glycoꢀprodrugs of Dox. D.A.=Diels
–Alder.
Results and Discussion
Synthesis
We sought to obtain two types of glycoꢀprodrugs of Dox with different activating
mechanisms. Our first strategy was to link the drug to the carbohydrate part through ester
formation, permitting the release of the drug by esterases, particularly human carboxylesterase
2 that is commonly expressed in tumor tissues.28 The ester functionality is linked to the
primary amine of Dox by a pꢀaminobenzyl alcohol (PABA) spacer. Hydrolysis of the ester
bond will initiate a cascade reaction of this soꢀcalled selfꢀimmolative spacer, first reported by
Katzenellenbogen,29 ultimately resulting in the release of Dox. Our second strategy sought to
enable Dox release by specific proteases. To this goal, we chose a tripeptide linker ( ꢀAlaꢀ
D
PheꢀLys)30 known to be cleaved by plasmin, which has been identified as a potential target
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protease for prodrug activation of cancer drugs.31 This linker has previously been used in
combination with the PABA selfꢀimmolative linker in the synthesis of Dox32 and Dox
derivative prodrugs.33 The latter were shown to be stable in human plasma and poorly
membrane permeable, thereby limiting activation to locally secreted proteolytic activity and
reducing the likelihood of severe side effects.
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Starting with the linker drug end of the prodrugs, the synthesis of the maleimidyl ester linked
Dox (MalEsterDox 1) was performed starting from ωꢀaminocapronic acid 2 (Scheme 1). The
fiveꢀcarbon spacer length provided by the use of ωꢀaminocapronic acid has previously been
reported for enzymatically cleaved prodrugs of Dox.34
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Scheme 1. Synthesis of 1. a) (Boc)2O (1.1 equiv.) in NaOH(aq.), 1,4ꢀdioxane 1:2, 16 h, 89%.
b) DCC (1 equiv.), DMAP (20 mol%), pꢀmethoxybenzaldehyde (0.95 equiv.) in CH2Cl2, 0
to r.t., 21 h, 86%. c) NaBH4 (0.95 equiv.) in CF3CH2OH, 0 , 1 h 20 min, 90%. d) TFA:
CH2Cl2 1:1, r.t. 20 min, then e) 3ꢀ(Nꢀmaleimido)propionic acid (1.1 equiv.), DCC (1.1 equiv.),
NMM (3 equiv.) in CH2Cl2, 0
to r.t., 17 h, 51% over 2 steps. f) Bis(4ꢀnitrophenyl)
carbonate (1.5 equiv.), DIPEA (3 equiv.) in CH2Cl2, r.t., 4 h, 70%. g) Dox*HCl (0.9 equiv.),
Et3N (2 equiv.) in DMF, r.t., 22h, 59%.
tertꢀButyloxycarbonyl (tBoc) protection of the amine with diꢀ(tertꢀbutyl)dicarbonate in the
presence of sodium hydroxide (NaOH) gave carboxylic acid 3 which was coupled with pꢀ
hydroxybenzaldehyde by Steglich esterification to give ester 4 in 86% yield. The next step
was the reduction of the aldehyde to the corresponding alcohol. Performing the reduction with
NaBH4 in THF at 0°C resulted in multiple products as observed by TLC already after 15 min.
Trifluoroethanol has previously been reported as a coꢀsolvent for NaBH4 reductions.35, 36
Reduction of aldehyde 4 with NaBH4 in trifluoroethanol at 0°C resulted in full conversion
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after one hour and allowed us to isolate hydroxyl 5 in high yield (90%). Next, the maleimide
(Mal) was introduced by tBoc deprotection with trifluoroacetic acid (TFA) followed by
amidation with 3ꢀmaleimidopropionic acid in the presence of dicyclohexylcarbodiimide
(DCC) and Nꢀmethylmorpholine (NMM). MalEsterOH 6 was isolated in 51% over two steps.
To introduce the carbamateꢀlinked Dox, the hydroxyl of 6 was converted to a mixed
anhydride by reaction with bis pꢀnitrophenoxyanhydride in the presence of N,Nꢀ
diisopropylethylamine (DIPEA) in dichloromethane (DCM) (70% yield). Stirring 7 in
dimethylformamide (DMF) at room temperature (r.t.) with Dox in the presence of
triethylamine resulted in MalEsterDox 1 in 59% yield.
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The synthesis of the peptideꢀlinked glycoꢀprodrug started with the synthesis of the ꢀAlaꢀPheꢀ
D
Lys tripeptide. The synthesis of this tripeptide has previously been described both in
solution32 and on solid support.33 Our synthetic strategy for the Dox tripeptide motif started
from NαꢀFmocꢀNεꢀAllocꢀLysine 8 which was activated by isoꢀbutylchloroformate and
functionalized with pꢀaminobenzylalcohol to give 9 in 72 % yield (Scheme 2).
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Scheme 2. Synthesis of 13. a) ibutylchloroformate (1.1 equiv.), pꢀaminobenzylalchohol (1.2
equiv.), NMM (2.3 equiv.) in THF, ꢀ 40 , 72%. b) 1,4ꢀdioxane:MeOH: 2M NaOH (aq.), r.t.,
20 min, then c) DMTMM (1 equiv.), FmocPheOH (1 equiv.), NMM (1.1 equiv.) in CH3CN,
r.t., 19h, then d) 1,4ꢀdioxane:MeOH: 2M NaOH (aq.), r.t., 20 min, then e) DMTMM (1
equiv.), BocꢀDꢀAlaOH (1 equiv.), NMM (1.1 equiv.) in CH3CN, r.t., 14 h, 47% over 4 steps.
f) TFA: CH2Cl2 0.8:1, r.t., 15 min, then g) DMTMMBF4 (1.1 equiv.), 3ꢀ(Nꢀ
maleimido)propionic acid (1.2 equiv.), NMM (1.1 equiv.) in CH3CN, r.t., 22 h, 43% over two
steps. h) bis(4ꢀnitrophenyl) carbonate (5 equiv.), DIPEA (3 equiv.) in DMF, r.t., 46 h then i)
Dox*HCl (0.9 equiv.), Et3N (2 equiv.) in DMF, r.t., 24h, 75%.
The tripeptide linker was synthesized by solution phase peptide synthesis with NꢀFmoc
protected amino acids using 4ꢀ(4,6ꢀDimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀmethylmorpholinium
tetrafluoroborate (DMTMMBF4)37, 38 as coupling reagent followed by deprotection with
NaOH (aq.). The tripeptide 10 was obtained in 47% yield over 4 steps. The maleimide was
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introduced in a similar way as for the previously described linker by TFA deprotection of the
tBoc followed by amidation to give MaltriPep 11 in 43% yield over two steps. The
hygroscopic nature of the tripeptide in combination with the risk of hydrolyzing the
maleimide made it necessary to thoroughly dry (high vacuum pump) and store all following
intermediates below ꢀ15 . As for the esterꢀbased linker described above, the hydroxyl
function of 11 was activated as a mixed anhydride by reaction with five equivalents of bis pꢀ
nitrophenoxy carbonate in the presence of DIPEA. The mixed anhydride 12 was isolated by
precipitation in diethyl ether and was used in the following substitution without further
purification. Stirring 12 with Dox and Et3N in DMF at room temperature gave MaltriPepDox
13 in 75% yield and high purity as confirmed by HPLC (see SI).
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The next step was the conjugation of the drugꢀlinker part with the carbohydrate moiety. The
furfurylꢀfunctionalized carbohydrates were obtained through a two steps method for
synthesizing Nꢀglycosylamides first introduced by Lokhoff et al.39 and utilized in our group to
synthesize a range of reducingꢀend functionalized oligosaccharides.23, 27 The unprotected
carbohydrate was stirred in neat furfurylamine for 72 hours to afford a transient
glycosylamine that was made nonꢀhydrolyzable by acylation of the nitrogen with acetic
anhydride. The synthesis of furfurylꢀlactose 14 was previously published for the production of
conjugatable glycosaminoglycans.40 The furfurylꢀXGO 15 was synthesized for this study from
the oligosaccharideꢀrepeating unit of tamarind seed xyloglucan. First, the XGO repeating unit,
which consists in a mixture of heptaꢀ, octaꢀ and nonaꢀsaccharides in a ratio of 15/35/50, was
obtained quantitatively by enzymatic hydrolysis of the polysaccharide as reported
previously.41 The furfurylꢀXGO was then obtained in 90% yield following the same protocol
as for lactose. The conjugation of MalEsterDox 1 with furfurylꢀlactose 14 in DMF and water
(1:1) at 37 ºC gave LacEsterDox 16 in high yield (91%, Scheme 3). The XGOEsterDox
prodrug 17 was prepared under similar reaction conditions but was isolated in 35% yield only
after purification on reverse phase C18 cartridges. The yield is lower than typically observed in
this type of cycloaddition and is partly explained by extensive chromatographic purification.
The reaction conditions and the purification procedure should be optimized but sufficient
quantities for drug release and cell toxicity studies were isolated and no attempt to improve
the yield have been done so far.
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Scheme 3. Synthesis of 16 and 17. DMF:H2O 1:1, 37 ºC, 3 and 4 days for 16 and 17,
respectively.
To obtain the tripeptide linked glycoꢀprodrug (LactriPep(Alloc)Dox) (18, Scheme 4), 13 and
furfurylꢀlactose 14 were stirred in a mixture of DMF and H2O (2:1) at 37 for 7 days. 18
(57% yield) was isolated in high purity as confirmed by HPLC (see SI) after purification on
silica eluting with 10% water in acetonitrile.
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Scheme 4. Synthesis of 19. a) DMF:H2O 2:1, 37 , 7 days, 58%. b) Pd(PPh3)4 (1.2 equiv.),
CH2Cl2:AcOH 5:1, r.t., 30 min, 59%.
The last step of removal of the Alloc protecting group from the lysine side chain was more
tricky. Commonly used conditions for Alloc removal include catalytic amounts of Pd0 in the
presence of an allyl scavenger such as morpholine, tributyltin hydride or silane.42 However,
using catalytic amounts of Pd(PPh3)4 (20 mol%) and morpholine as allyl scavenger, we were
unable to isolate the desired product, only observing degradation on TLC. Eliminating the
scavenger and running the reaction with equimolar amount of Pd(PPh3)4 in a mixture of acetic
acid and dichloromethane as described by Barthel et al.33 gave the desired product 19
(LactriPepDox) in 59% yield after precipitation from diethyl ether.
Enzymatic release of Dox from glycoꢀprodrugs
To confirm the intended enzymatic release of Dox, the LactriPepDox 19 (100 µM) was
incubated in vitro in the presence 15 µg/ml of human plasmin at 37 ºC in phosphate buffer
(pH 7.5). The disappearance of prodrug and appearance of Dox was followed by HPLC
(Figure 2). We first checked the stability of the prodrugs in these conditions in the absence of
plasmin (no release after up to 25h at 37°C, data not shown). In the presence of plasmin,
nearly full conversion of the prodrug into Dox was achieved after 2 h.
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Figure 2. In vitro protease mediated release of Dox (•) from LactriPepDox 19 (100µM) using
15µg/ml plasmin in phosphate buffer (pH 7.5) at 37 ºC.
The release of Dox from LacEsterDox 16 was performed in 50 mM Tris buffer (pH 7.4).
Incubating 16 (100µM) at 37 ºC in the presence of human carboxylesterase 1 (hCE1) (100
µg/ml) resulted in only minor (~5%) release of Dox after 22 h (Figure 3).
Figure 3. In vitro esterase mediated release of Dox using 100µg/ml esterase in 50 mM Tris
buffer (pH 7.4) at 37 ºC from LacEsterDox 16 with hCE2(▲), from LacEsterDox 16 with
hCE1(♦), from XGOEsterDox 17 with hCE2(■) and LacEsterDox 16 without enzyme (x).The
values for hCE2 measurements are mean values of duplicate experiments.
In contrast, incubating 16 with human carboxylesterase 2 (hCE2) under identical conditions
resulted in full release after 23h. The rate of hydrolysis for XGOEsterDox 17 was slightly
slower than for 16 (83% after 23h), possibly owing to the steric effect of the larger
oligosaccharide. Nevertheless, after 48h, 98% of 17 had been hydrolyzed. The difference in
rate between hCE1 and hCE2 could be explained by the presence of a phenyl on the alcohol
side since hCE2 is known to hydrolyze esters with bulkier alcohol groups at a higher rate than
hCE1.43 As was observed for the tripeptide prodrug 19, incubating the ester prodrug
(LacEsterDox) in the absence of esterase resulted in less than 2% Dox after 27h (data not
shown). These results confirm that Dox is enzymatically released from prodrugs 16, 17 and
19 and that these three prodrugs are stable in the buffers in absence of enzyme.
Biological evaluation
As a preliminary test for verifying the biological interest of the prodrugs, the cytotoxicity of
compounds 16, 17 and 19 was assayed on MCFꢀ7 breast cancer cells and compared with that
of free Dox (Table 1).
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Compound
IC50 (µM)
0.51 ± 0.07
0.31 ± 0.15
1.11 ± 0.11
0.53
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19
Dox
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Table 1. Inhibition of MCFꢀ7 breast cancer cell growth by the glycoꢀprodrugs and free Dox
as reference. IC50 values were calculated as the concentration of compound needed to reduce
cell growth by 50%. Values are reported as mean
measurements.
± the standard deviation (SD) of triplicate
The plasminꢀactivated prodrug 19 displayed a slightly higher (~2 fold) IC50 compared to Dox
while the esterase activated prodrugs 16 and 17 showed IC50 values similar to that of the
parent drug. Since it has been previously shown that protection of the primary amine of Dox
results in a significant decrease in activity,44 these results are a clear indication of Dox release
from prodrugs 16, 17 and 19 in presence of the MCFꢀ7 cells. One would have probably
expected a more pronounced difference between the IC50 of the ester and amide prodrugs as
plasmin (which is not overꢀexpressed on MCFꢀ7 cells) has not been added in the cell culture
medium), and further experiments are now required to study the mode of action of the glycoꢀ
prodrugs. Nevertheless, the similar IC50 obtained for XGOEsterDox 17 and LacEsterDox 16
are very encouraging since they show that the release of Dox is not dependent on the size and
therefore steric hindrance of the oligosaccharide moiety. In a forthcoming work, we wish to
implement the use xyloglucan glycoconjugates to the development of drugꢀfunctionalized
cellulosic biomaterials.
Conclusion
We have synthesized a small series of enzymatically activatable glycoꢀprodrugs of Dox. We
demonstrated the release of the drug by enzymatic cleavage in vitro and in cellulo on MCFꢀ7
cancer cells, as the prodrugs and Dox itself display similar IC50. The late stage metal free
cycloaddition used to ligate the oligosaccharide and the drug allows this methodology to be
used with different oligosaccharides and potentially with different drugs. Current work in our
groups includes extending this ligation to synthesize prodrugs of other drug classes.
Experimental procedures
General considerations
1
All commercial chemicals were used without prior purification. Hꢀ and 13CꢀNMR spectra
were obtained at 400 and 101 MHz respectively, using a Bruker Advance DRX 400
spectrometer. Solvent residual peaks were used as reference and calibrated as follows; CDCl3:
7.26ppm; MeODꢀd4: 3.31ppm; DMSOꢀd6: 2.50 ppm. Reactions were monitored by TLC
(Merck Silica gel 60 F254) and analyzed under UV (254 nm). Reactions involving
carbohydrates were followed by staining TLC plates with H2SO4: MeOH : H2O (3:45:45).
Column chromatography was performed by manual flash chromatography (wetꢀpacked silica,
0.04ꢀ0.063 mm) or by automated column chromatography on a Grace Reveleris instrument
using prepacked silica columns. Analytical high performance liquid chromatography (HPLC)
was performed using a Waters µBondapak C18ꢀcolumn (3.9 x 300mm, 125Å pore size, 10 µm
particle size) connected to a Waters photodiode array detector 996. HPLC method A: 10ꢀ
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100% MeOH in water (phosphoric acid pH 2.5) over 5 min, 2 ml/min. HPLC method B: 0ꢀ
100% MeOH in water (phosphoric acid pH 2.5) over 5 min, then 100% water for 5 min, 2
ml/min. Mass analyses (ESI) were recorded on a Bruker Daltonics Esquire 3000+ instrument
and HRMS analyses were obtained on a Waters Xevo G2ꢀS QTOF instrument.
8
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6ꢀ((tertꢀButoxycarbonyl)amino)hexanoic acid (3)
Following a published procedure,45 NaOH (4.0 g, 0.1 mol) was dissolved in water (130 ml)
and 1,4ꢀdioxane (260 ml) was added. ωꢀAminocapronic acid (13.1 g, 99.9 mmol) was added,
allowed to dissolve and the solution was cooled to 0 °C. Diꢀtertꢀbutyl dicarbonate (24.0 g, 110
mmol) was added in three portions, the reaction mixture was stirred at 0 °C for 30 min and
then at r.t. for 16 h. The 1,4ꢀdioxane was removed by rotary evaporation and the crude was
diluted with water (400 ml) and washed with EtOAc (2 x 200 ml). The aqueous phase was
acidified by addition of 1 M HCl (aq.) and extracted with EtOAc (3 x 300 ml). The organic
phases from the second extraction were collected and dried over MgSO4. Removal of the
solvents gave the expected product 3 as an oil that crystallizes to light pink crystals (20.6 g,
89% yield). Rf : 0.20 (2% MeOH in CH2Cl2). 1H NMR (400 MHz, CDCl3) δ 10.85 (br s, 1H),
5.97 (br s, 0.3H, ꢀCONHꢀ), 4.63 (br s, 0.7H, ꢀCONHꢀ), 3.08 (br s, 2H), 2.32 (t, J = 7.4 Hz,
2H), 1.62 (p, J = 7.5 Hz, 2H), 1.53 – 1.18 (m, 13H) ; ESIꢀMS m/z [M+Na]+ : 254.2.
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4ꢀFormylphenylꢀ6ꢀ((tertꢀbutoxycarbonyl)amino)hexanoate (4)
3 (10.0 g, 43.2 mmol) was dissolved in dry CH2Cl2 (43 ml) in a two necked flask under argon.
DCC (8.9 g, 43.2 mmol) and DMAP (1.0 g, 8.2 mmol) were added, the reaction mixture was
cooled to 0 °C and pꢀhydroxybenzaldehyde (5.0 g, 41.2 mmol) was immediately added. The
reaction was stirred at 0 °C for 30 min and then at r.t. for 21 h. The white precipitate that
formed was filtered off and washed twice with a small volume of CH2Cl2. The solvents were
removed from the filtrate which gave an offꢀwhite solid that was redissolved in CH2Cl2 (200
ml), washed with 0.5 M HCl (50 ml), sat. NaHCO3 (50 ml) and brine (50 ml). The organic
phase was dried over MgSO4 and filtered. Removal of the solvents by rotary evaporation gave
the crude as an off white solid. Purification by column chromatography on silica eluting with
2% MeOH in CH2Cl2 gave 4 as a white solid (12.5 g, 86% yield). Rf : 0.36 (2% MeOH in
1
CH2Cl2); H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 7.95ꢀ7.86 (m, 2H), 7.30ꢀ7.20 (m, 2H),
4.57 (br s, 1H), 3.14 (q, J = 6.7 Hz, 2H), 2.59 (t, J = 7.4 Hz, 2H), 1.77 (p, J = 7.5 Hz, 2H),
1.61 – 1.49 (m, 2H), 1.48 – 1.38 (m, 11H); 13C NMR (101 MHz, CDCl3) δ 191.0, 171.4,
156.1, 155.5, 134.1, 131.3, 122.5, 79.3, 40.5, 34.3, 29.9, 28.5, 26.3, 24.5; MS (ESI) m/z:
[M+Na]+: 358.3 HRMS m/z [M+Na]+ calculated for C18H25NO5Na: 358.1630, found:
358.1627.
4ꢀ(Hydroxymethyl)phenylꢀ6ꢀ((tertꢀbutoxycarbonyl)amino)hexanoate (5)
4 (1.00 g, 2.98 mmol) was dissolved in trifluoroethanol (6 ml) and the flask was cooled on
ice. NaBH4 (107 mg, 2.83 mmol) was added and the reaction was stirred on ice. Full
consumption of 4 was observed after 1 h on TLC (2% MeOH in CH2Cl2). The reaction was
kept on ice and quenched by slow addition of sat. NH4Cl (7 ml) after 1h 20 min. The mixture
was allowed to reach r.t. and stirred for 40 min. Water (5 ml) was added and the aqueous
phase was extracted with EtOAc (3 x 30 ml). The organic phases were pooled, dried over
Na2SO4 and filtered. Removal of the solvents gave a colorless oil, which was purified by flash
column chromatography on silica eluting with 1% MeOH in CH2Cl2 to obtain 5 as a white
solid (900 mg, 90% yield). Rf : 0.32 (2% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ
7.37ꢀ7.29 (m, 2H), 7.06ꢀ6.99 (m, 2H), 4.62 (s, 3H,ꢀCH2ꢀ and –CONHꢀ), 3.10 (q, J = 6.6 Hz,
2H), 2.5 3 (t, J = 7.4 Hz, 2H), 2.49 (br s, 1H), 1.74 (p, J = 7.5 Hz, 2H), 1.56 – 1.46 (m, 2H),
1.45 – 1.35(m, 11H) ; 13C NMR (101 MHz, CDCl3) δ 172.2, 156.1, 150.0, 138.7, 128.0,
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121.6, 79.2, 64.6, 40.4, 34.3, 29.8, 28.5, 26.3, 24.6; MS (ESI) m/z: [M+Na]+: 360.2; HRMS
m/z [M+Na]+ calculated for C18H27NO5Na: 360.1787, found: 360.1786.
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6
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MalEsterOH (6)
5 (400 mg, 1.19 mmol) was dissolved in dry CH2Cl2 (1.5 ml) and TFA (1.5 ml) was added.
The reaction was stirred at r.t. for 20 min. Full consumption of 5 was observed by TLC (10%
MeOH in CH2Cl2). Solvents were removed and coevaporated four times with CH2Cl2. The
resulting clear oil was redissolved in dry CH2Cl2 (2 ml) and basified (pH approx. 8) by
addition of Nꢀmethylmorpholine (400 µl). 3ꢀ(Maleimidyl)propanoic acid (220 mg, 1.30
mmol) was added, the reaction was cooled on ice and DCC (268 mg, 1.30 mmol) was added.
The ice bath was removed and the reaction was stirred at r.t. for 17 h. The white precipitate
that had formed was filtered off and the filtrate was concentrated. The yellow oil that resulted
was redissolved in CH2Cl2 (20 ml), washed with 0.5 M HCl (5 ml) and dried over MgSO4 and
filtered. Removal of the solvents gave a white solid, which was purified by flash column
chromatography on silica eluting with 25% cyclohexane in EtOAc to obtain 6 as a white solid
(236 mg, 51% yield). Rf : 0.52 (10% MeOH in CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 7.41ꢀ
7.34 (m, 2H), 7.09ꢀ7.02 (m, 2H), 6.68 (s,2H), 5.74 (br s, 1H), 4.67 (s, 2H), 3.81 (t, J = 7.2 Hz,
2H), 3.28ꢀ3.19 (m, 2H), 2.55 (t, J = 7.3 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 1.86 (br s, 1H), 1.75
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(p, J = 7.4 Hz, 2H), 1.58 – 1.48 (m, 2H), 1.46 – 1.36 (m, 2H); C NMR (101 MHz, CDCl3)
δ172.3, 170.7, 169.6, 150.2, 138.7, 134.3, 128.2, 121.8, 64.8, 39.4, 34.9, 34.4, 34.2, 29.2,
26.4, 24.5; MS (ESI) m/z: [M+Na]+: 411.3; HRMS m/z [M+H]+ calculated for C20H25N2O6:
389.1713, found: 389.1714.
MalEsterPNP (7)
6 (178 mg, 0.46 mmol) and bis(pꢀnitrophenyl)carbonate (210 mg, 0.69 mmol) were added to a
dry flask filled with argon which was capped, evacuated and refilled with argon (repeated 3
times). Dry CH2Cl2 (4.6 ml) was added followed by diisoproylethylamine (245 µl, 1.38
mmol). The yellow reaction mixture was stirred at r.t. for 4 h in the dark. Full consumption of
6 was observed on TLC (2% MeOH in CH2Cl2). The reaction mixture was diluted with
CH2Cl2 (25 ml) and washed with aq. citric acid (10 % w/v). The organic phase was dried over
Na2SO4 and filtered. Removal of the solvents gave a yellow oil. DEE (25 ml) was added and
brief sonication resulted in a white precipitate. The precipitate was filtered off, washed with
DEE repeatedly, collected and redissolved in a minimum of CH2Cl2. The solution was added
dropwise to stirred DEE (25 ml). The white precipitate that formed was collected by filtration
and dried to give 7 as a white solid (178 mg, 70% yield). Rf : 0.28 (2% MeOH in CH2Cl2); 1H
NMR (400 MHz, CDCl3) δ 8.31ꢀ8.23 (m, 2H), 7.50ꢀ7.43 (m, 2H), 7.41ꢀ7.35 (m, 2H), 7.16ꢀ
7.09 (m, 2H), 6.70 (s, 2H), 5.64 (br s, 1H), 5.28 (s, 2H), 3.84 (t, J = 7.2 Hz, 1H), 3.31ꢀ3.20
(m, 2H), 2.58 (t, J = 7.4 Hz, 1H), 2.51 (t, J = 7.1 Hz, 1H), 1.82ꢀ1.72 (m, 2H), 1.58 – 1.50 (m,
2H), 1.49 – 1.38 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 172.0, 170.7, 169.6, 155.6, 152.6,
151.3, 145.6, 134.4, 131.9, 130.2, 125.5, 122.2, 121.9, 70.4, 39.4, 34.9, 34.4, 34.3, 29.3, 26.4,
24.5; MS (ESI) m/z: [M+H]+: 554.2 and [M+Na]+: 576.2; HRMS m/z [M+H]+ calculated for
C27H28N3O10: 554.1775, found: 554.1776.
MalEsterDox (1)
7 (132 mg, 0.24 mmol) was dissolved in dry DMF (2.2 ml) and the solution was added to
Doxorubicin hydrochloride (128 mg, 0.22 mmol). Et3N (61 µl, 0.44 mmol) was added and the
dark red reaction mixture was stirred under argon. After 22 h, the reaction mixture was added
dropwise to stirred DEE (75 ml) resulting in a red precipitate which was collected by filtration
and further purified by flash column chromatography eluting with 2ꢀ5% MeOH in CH2Cl2. 1
1
(124 mg, 59% yield) was isolated as a red foam. Rf : 0.29 (5% MeOH in CH2Cl2); H NMR
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(400 MHz, CDCl3) δ 13.96 (s, 1H), 13.23 (s, 1H), 8.02 (dd, J = 7.7, 1.1 Hz, 1H), 7.82 – 7.73
(m, 1H), 7.39 (dd, J = 8.6, 1.1 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H),
6.68 (s, 2H), 5.73 (s, 1H), 5.49 (d, J = 3.8 Hz, 1H), 5.28 (br s, 1H), 5.22 (d, J = 8.6 Hz, 1H),
5.08ꢀ4.91 (m, 2H), 4.75 (dd, J = 5.0, 1.4 Hz, 2H), 4.57 (s, 1H), 4.18 – 4.08 (m, 1H), 4.07 (s,
3H), 3.85 (br s, 1H), 3.81 (t, J = 7.2 Hz, 2H), 3.66 (d, J = 7.2 Hz, 1H), 3.32 – 3.16 (m, 3H),
3.08ꢀ2.94 (m, 2H), 2.53 (t, J = 7.3 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H), 2.39ꢀ2.27 (m, 1H), 2.25ꢀ
2.10 ( m, 2H), 1.91 – 1.68 (m, 4H), 1.52 (p, J = 7.1 Hz, 2H), 1.46ꢀ1.33 (m, 2H), 1.28 (d, J =
6.5 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 214.0, 187.2, 186.8, 172.2, 170.7, 169.7, 161.2,
156.3, 155.8, 155.6, 150.6, 135.9, 135.6, 134.3, 134.1, 133.7, 129.5, 121.8, 121.0, 120.0,
118.6, 111.7, 111.6, 110.1, 100.8, 76.8, 69.7, 69.6, 67.5, 66.2, 65.7, 56.8, 47.2, 39.4, 35.8,
34.9, 34.4, 34.2, 34.1, 30.3, 29.2, 26.4, 24.5, 17.0; MS (ESI) m/z: [M+Na]+ : 980.3; HRMS
m/z [M+H]+ calculated for C48H52N3O18: 958.3246, found: 958.3235.
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FmocLys(Alloc)PABOH (9)
Following a published procedure,32 FmocLys(Alloc)OH (1.44g, 3.18 mmol) was dissolved in
dry THF (20 ml) under argon in a twoꢀnecked flask equipped with an addition funnel. The
solution was cooled to ꢀ40 °C and Nꢀmethylmorpholine (0.39 ml, 3.54 mmol) and isobutyl
chloroformate (0.45 ml, 3.47 mmol) were added. The turbid mixture was stirred at ꢀ40 °C for
3 h after which a solution of pꢀaminobenzylalcohol (470 mg, 3.82 mmol) and Nꢀ
methylmorpholine (0.42 ml, 3.82 mmol) in dry THF (6 ml) was added dropwise over 10 min.
The reaction mixture was stirred at ꢀ40 °C for 2 h and then at room temperatrure for 1 h. The
solvents were removed and the yellow residue was taken up in CH2Cl2 (250 ml). The organic
phase was washed with 0.5 M NaHCO3 solution (100 ml), 0.5 M NaHSO4 solution (100 ml)
and brine (100 ml). The organic solvents were removed under reduced pressure and the
yellow residue was purified by flash column chromatography on silica (2ꢀ10% methanol in
1
CH2Cl2) to provide 9 as a white solid (1.28 g, 72% yield). H NMR (400 MHz, dmsoꢀd6) δ
9.96 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (dd, J = 7.5, 4.9 Hz, 2H), 7.62 (d, J = 7.9 Hz, 1H),
7.55 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.37ꢀ7.29 (m, 2H), 7.24 (d, J = 8.3 Hz, 2H),
7.19 (t, J = 5.8 Hz, 1H), 5.89 (ddt, J = 17.2, 10.5, 5.3 Hz, 1H), 5.30ꢀ5.20 (m, 1H), 5.18 – 5.12
(m, 1H),5.10 (t, J = 5.7 Hz, 1H) 4.52ꢀ4.39 (m, 4H), 4.34 – 4.06 (m, 4H), 3.07 – 2.91 (m, 2H),
1.75ꢀ1.56 (m, 2H), 1.51 – 1.21 (m, 4H). 13C NMR (101 MHz, DMSOꢀd6) δ 171.0, 156.1,
155.9, 143.9, 143.8, 140.7, 137.5, 137.4, 133.8, 127.6, 127.0, 126.9, 125.3, 120.1, 118.9,
116.8, 65.6, 64.1, 62.6, 55.4, 46.7, 31.6, 29.1, 22.9. HRMS m/z [M+H]+ calculated for
C32H36N3O6: 558.2604, found: 558.2606.
Tripep (10)
Compound 9 (560 mg, 1.00 mmol) was dissolved in a premade mixture of 1,4ꢀ
dioxane:MeOH:2M NaOH(aq.) (14:5:1, 20 ml).46 After 30 min (TLC, CH2Cl2:MeOH 90:10),
the solution was neutralized by addition of acetic acid. The solvents were removed by
evaporation and coevaporated with 1,4ꢀdioxane (2 x 20 ml). The oily solid residue was
washed with DEE (3 x 10 ml) and then with CH2Cl2 (10 x 10 ml). The CH2Cl2 fractions were
collected and the solvent was removed to yield the free amine as a pale yellow oil (326 mg)
which was used in the next step without further purification.
A solution of DMTMMBF4 (365 mg, 1.11 mmol), FmocPheOH (390 mg, 1.01 mmol) and Nꢀ
methyl morpholine (121 µl, 1.10 mmol) in CH3CN (5 ml) was stirred for 10 min and then
added to a solution of the crude form the previous step (326 mg) in CH3CN (5 ml). The
reaction mixture was stirred at r.t for 19 h (TLC, CH2Cl2:MeOH 90:10). The solvents were
removed by evaporation and CH2Cl2 (100 ml) was added and the organic phase was washed
with water (25 ml), 0.5 M NaHCO3 (25 ml), 0.5 M NaHSO4 (3 x 25 ml) and water (50 ml).
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The solvents were removed from the organic phase to give an offꢀwhite solid (665 mg) which
was used in the next step without further purification.
A premade mixture of 1,4ꢀdioxane:MeOH:2M NaOH(aq.) (14:5:1, 20 ml) was added to the
crude from the previous step and the reaction mixture was stirred at r.t. for 50 min (TLC,
CH2Cl2:MeOH 90:10). The reaction was neutralized by addition of acetic acid. The solvents
were removed by evaporation and coevaporated with 1,4ꢀdioxane (20 ml). The resulting white
residue was washed with DEE (3 x 5 ml) and then with CH2Cl2 (10 x 20 ml). The white
precipitate formed in the DEE phase was collected by filtration, dissolved in CH2Cl2 and
pooled with the other CH2Cl2 fractions. The CH2Cl2 fractions were collected and the solvent
was removed to yield the free amine as an off white solid (463 mg) which was used in the
next step without further purification.
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A solution of DMTMMBF4 (360 mg, 1.10 mmol), BocꢀDꢀAlaOH (190 mg, 1.00 mmol) and
Nꢀmethyl morpholine (121 µl, 1.10 mmol) in CH3CN (5 ml) was stirred for 10 min and then
added to a solution of the crude from the previous step (463 mg) in CH3CN (6 ml). The
reaction mixture was stirred at r.t for 14 h (TLC, CH2Cl2:MeOH 90:10). The solvents were
removed by evaporation and CH2Cl2 (70 ml) was added and the organic phase was washed
with water (40 ml), 0.5 M NaHSO4 (2 x 20 ml), 0.5 M NaHCO3 (3 x 20 ml) and water (20 ml).
The solvents were removed from the organic phase to give an offꢀwhite solid, which was
purified by flash column chromatography on silica eluting with 2ꢀ5% methanol in CH2Cl2.
The product was dryloaded on to the column after being dissolved in 10% methanol in
CH2Cl2 and dried on to silica. Compound 10 was isolated as a white solid (309 mg, 47% over
4 steps). 1H NMR (400 MHz, MeODꢀd4) δ 7.61 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H),
7.28 – 7.09 (m, 5H), 5.91 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.28 (dd, J = 17.1, 1.7 Hz, 1H),
5.16 (d, J = 10.5 Hz, 1H), 4.60 (dd, J = 9.8, 4.5 Hz, 1H), 4.56 (s, 2H), 4.50 (d, J = 5.0 Hz,
2H), 4.43 (dd, J = 9.6, 4.9 Hz, 1H), 4.00 (q, J = 7.1 Hz, 1H), 3.30ꢀ3.23 (m, 1H), 3.11 (t, J =
6.7 Hz, 2H), 2.95 (dd, J = 14.2, 9.8 Hz, 1H), 2.02 – 1.78 (m, 2H),1.59ꢀ1.40 (m, 4H) 1.33 (s,
13
9H), 1.13 (d, J = 7.1 Hz, 3H). C NMR (101 MHz, MeODꢀd4) δ 176.9, 173.6, 172.2, 158.8,
157.9, 138.8, 138.5, 138.4, 134.5, 130.2, 129.5, 128.5, 127.8, 121.4, 117.4, 80.7, 66.3, 64.8,
56.4, 55.7, 51.8, 41.5, 37.9, 32.3, 30.5, 28.7, 24.5, 17.7. HRMS m/z [M+H]+ calculated for
C34H48N5O8: 654.3503, found: 654.3496.
MaltriPep (11)
10 (150 mg, 0.23 mmol) was suspended in dry CH2Cl2 (1ml) under argon, TFA (0.8 ml) was
added and the solution was stirred for 15 min. Full consumption of 10 was confirmed by TLC
(10% methanol in CH2Cl2). The solvents were removed and coevaporated with CH2Cl2 (3 x 1
ml). The resulting residue was taken up in CH3CN (3.5 ml) and neutralized by addition of
NMM (150 µl). DMTMMBF4 (82 mg, 0.25 mmol), 3ꢀ(maleimidyl)propanoic acid (46 mg,
0.27 mmol) and NMM (27 µl, 0.25 mmol) were stirred in CH3CN (1.5 ml) at r.t. until all
material dissolved (~5 min). The solution of deprotected 10 was then added and the reaction
mixture was stirred at r.t. for 22 h. The formed precipitate was filtered off and washed with
ice cold CH3CN and DEE. The filtrate was collected, diluted in CH2Cl2 (100 ml) and washed
with 0.5 M NaHSO4 (30 ml) and water (30 ml). The organic phases were dried over MgSO4
and the solvents were then removed to give a white solid which was pooled with the
precipitated material. The crude was purified by flash column chromatography on silica
eluting with 5% methanol in CH2Cl2. The crude product was dryloaded on to the column after
being dissolved in methanol and dried on to silica. The expected product 11 was isolated as a
1
white solid (72 mg, 43% over 2 steps). H NMR (400 MHz, MeODꢀd4) δ 7.60 (app d,
2H),7.29 (app d, 2H), 7.28ꢀ7.14 ( m, 5H), 6.76 (s, 2H) 5.91 (ddt, J = 16.3, 10.6, 5.4 Hz, 1H),
5.28 (dd, J = 17.2, 1.8 Hz, 1H), 5.20 – 5.12 (m, 1H), 4.62 – 4.52 (m, 3H), 4.50 (dt, J = 5.6, 1.6
Hz, 2H), 4.40 (dd, J = 9.6, 5.0 Hz, 1H), 4.14 (q, J = 7.1 Hz, 1H), 3.73ꢀ3.57 (m, 2H), 3.30ꢀ3.25
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(m, 1H, partly overlapping with solvent peak), 3.13 (t, J = 6.7 Hz, 2H), 2.95 (dd, J = 14.2,
10.0 Hz, 1H), 2.47ꢀ2.27 (m, 2H), 2.00 – 1.84 (m, 2H), 1.62 – 1.36 (m, 4H), 1.14 (d, J = 7.1
Hz, 3H). C NMR (101 MHz, MeODꢀd4) δ 176.2, 173.7, 173.2, 172.4, 172.1, 158.8, 138.8,
13
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138.5, 135.4, 134.6, 130.2, 129.5, 128.5, 127.8, 121.4, 117.4, 112.9, 66.3, 64.8, 56.7, 55.8,
51.1, 41.5, 37.8, 35.2, 35.1, 32.3, 30.5, 24.5, 17.1. MS (ESI) m/z: [M+H]+: 705.4 and
[M+Na]+: 727.4; HRMS m/z [M+H]+ calculated for C36H45N6O9: 705.3248, found: 705.3253.
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MaltriPepOPNP (12)
11 (65 mg, 0.09 mmol) and bis(pꢀnitrophenyl)carbonate (82 mg, 0.27 mmol) were dissolved
in dry DMF (1.5 ml) under argon. The flask was capped, evacuated and refilled with argon
several short turns before DIPEA (47 µl, 0.27 mmol) was added by syringe. The reaction
mixture was stirred at r.t. for 23 h at which time a second portion of bis(pꢀ
nitrophenyl)carbonate (55 mg, 0.18mmol) was added. The reaction was stirred for an
additional 23 h after which all 11 was consumed as verified by TLC (5% methanol in
CH2Cl2). The reaction mixture was diluted in CH2Cl2 (40 ml), washed with citric acid solution
(10 wt%, 2 x 15 ml) and dried over MgSO4. The solvents were removed resulting in a yellow
oil, which was dissolved in a small amount of CH2Cl2 and added dropwise to DEE (40 ml)
while stirring. The product precipitated as a white solid (60 mg) containing ~15% pꢀ
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nitrophenol (estimated from HꢀNMR integrals). The identity of the expected product was
1
confirmed by HꢀNMR, ESIꢀMS and HRMS and the material was used without further
purification in the following step. 1H NMR (400 MHz, MeODꢀd4) δ 8.31 (app d, 2H), 7.69 (d,
J = 8.6 Hz, 2H), 7.46 (app d, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.31ꢀ7.23 (m, 4H), 7.23ꢀ7.16 (m,
1H), 6.74 (s, 2H), 5.97ꢀ5.84 (m, 1H), 5.33ꢀ5.23 (m, 3H), 5.16 (d, J = 10.5 Hz, 1H), 4.56 (dd, J
= 10.0, 4.4 Hz, 1H), 4.53 – 4.46 (m, 2H), 4.41 (dd, J = 9.6, 5.0 Hz, 1H), 4.14 (q, J = 7.1 Hz,
1H), 3.73 – 3.44 (m, 3H), 3.14 (t, J = 6.7 Hz, 2H), 2.95 (dd, J = 14.2, 10.1 Hz, 1H), 2.46ꢀ2.25
(m, 2H), 2.08 – 1.81 (m, 1H), 1.63 – 1.37 (m, 4H), 1.15 (d, J = 7.1 Hz, 3H). MS (ESI) m/z:
[M+Na]+: 892.3; HRMS m/z [M+H]+ calculated for C43H48N7O13: 870.3310, found: 870.3318.
MaltriPepDox (13)
12 (53 mg, 0.061 mmol) was dissolved in dry DMF (1.7 ml) and the hydrochloride salt of
doxorubicin (35 mg, 0.061 mmol) was added followed by Et3N (18 µl, 0.12 mmol). The
reaction mixture was stirred at r.t. and followed by TLC (5% methanol in CH2Cl2). After 24 h,
the reaction mixture was added dropwise to DEE (35 ml) under stirring which resulted in a
red precipitate. The precipitate was collected by filtration and isolated by washing the filter
paper with a solution of 5% methanol in CH2Cl2. The solvents were removed to give a red
solid, which was purified by flash column chromatography on silica eluting with 5% methanol
in CH2Cl2. The product was dryloaded on to the column after being dissolved in
methanol:CH2Cl2 mixture and dried on to silica. The expected product 13 was isolated as a red
solid (59 mg, 75%). HPLC method A. MS (ESI) m/z: [M+Na]+: 1296.5; HRMS m/z [M+Na]+
calculated for C64H71N7O21Na: 1296.4601, found: 1296.4608. Characteristic peaks of
maleimidyl (6.96 ppm, s, 2H), Vinyl proton from Alloc protecting group (5.86 ppm, m, 1H),
methoxy methyl of doxorubicin (3.97 ppm, s, 3H), Alaninyl methyl (1.12 ppm, d, 3H) and
1
doxorubicin glycon methyl (0.91ppm, d, 3H) with matching integrals found on HꢀNMR
(DMSOꢀd6) (see SI).
NꢀAcetamidoꢀNꢀfurfurylꢀXGO (15)
A solution of XGO41 (1.015 g, 8.3 mmol based on the octasaccharide) in furfurylamine (4
mL) was stirred for 72h at 25°C. The solution was poured into ethyl acetate (25 mL), cooled
to 0°C and centrifuged for 20 min at 6000 rpm. The solid was washed with ethyl acetate and
centrifuged again. The crude solid of glycosylamine was then dissolved in methanol (15 mL)
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and acetic anhydride (7 mL) was slowly added at 0°C to the solution. The reaction mixture
was then allowed to warm up to room temperature and stirred a further 16h. The solution was
then concentrated and coꢀevaporated with toluene. The residue was dissolved in 14% aqueous
ammonia (50 mL), stirred 1h at room temperature to hydrolyze potential Oꢀacetyl groups.
After concentration of the solution to half of its initial volume under vacuum, the aqueous
phase was extracted 3 times with ethyl acetate to remove free Nꢀacetyl furfurylamine and
concentrated. NꢀacetamidoꢀNꢀfurfurylꢀXGO was isolated in 90% yield (998 mg) after
1
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purification by flash chromatography on silica gel (CH3CN/H2O 7:3 v/v). H NMR (400
MHz, D2GOlc)Nδ 7.51 and 7.45 (2 x s, rotamers, 1H), 6.46ꢀ6.36 (m, 2H), 5.53 (d, J = 9.0 Hz,
GlcN
0.3H, H1
rotamer), 5.18 (m, 1H), 5.13 (d, J = 9.0 Hz, 1H, H1
rotamer), 4.96 (m, 2H),
4.62ꢀ4.55 (m, 5H), 4.06ꢀ3.33 (m, 51H), 2.30 and 2.26 ( 2 x s, rotamers, 3H, NCOCH3). The
presence of the amide rotamers was confirmed by their disappearance at elevated temperature
(323K) as observed by VTꢀNMR (see SI). (ESI) m/z: [M+Na]+ : 1206.4 (DP7), 1368.5 (DP8),
1530.5 (DP9). HRꢀMS m/z [M+Na]+ calculated for DP7; C46H73NO34Na; 1206.3912 Found
1206.3876, m/z [M+Na]+ calculated for DP8; C52H83NO39Na; 1368.4440 Found 1368.4401,
m/z [M+Na]+ calculated for DP9; C58H93NO44Na; 1530.4968 Found 1530.4930.
LacEsterDox (16)
1 (21 mg, 0.022 mmol) was dissolved in DMF (60 µl) and NꢀacetamidoꢀNꢀfurfurylꢀLactose40
(28 mg, 0.06 mmol) was added followed by water (40µl). The reaction mixture was stirred at
37 °C for 3 days. The solvents were removed by rotary evaporation and the crude material
was dissolved in CH2Cl2/MeOH, loaded on to silica and purified by flash column
chromatography eluting with 10ꢀ20% H2O in CH3CN. 16 was isolated as a red solid (30 mg,
91%) after lyophilization. HPLC method A (see SI); MS (ESI) m/z: [M+Na]+ : 1443.6; HRꢀ
MS m/z [M+Na]+ calculated for C67H80N4O30Na 1443.4755; Found 1443.4757.
XGOEsterDox (17)
1 (10 mg, 0.01 mmol) was dissolved in DMF (60 µl) and NꢀacetamidoꢀNꢀfurfurylꢀXGO 15
(66 mg, 0.05 mmol) was added followed by water (60µl). The reaction mixture was stirred at
37 °C for 4 days. The solvents were removed by rotary evaporation and the crude was
purified by two consecutive C18ꢀcolumns (1g) eluting with a gradient of 0ꢀ100% MeOH in
water. The expected product 17 was isolated as a red solid (8 mg, 35%) after lyophilization.
HPLC method A (see SI); (ESI) m/z: [MꢀH]ꢀ : 2140.9 (DP7) , 2302.9 (DP8), 2464.9 (DP9).
HRꢀMS m/z [M+Na]+ calculated for DP7; C94H124N4O52Na; 2163.7029 Found 2163.7079, m/z
[M+Na]+ calculated for DP8; C100H134N4O57Na; 2325.7607 Found 2325.7495, m/z [M+Na]+
calculated for DP9; C106H144N4O62Na; 2487.8135 Found 2487.8136.
LactriPep(Alloc)Dox (18)
13 (15 mg, 0.012 mmol) was dissolved in DMF (80 µl) and NꢀacetamidoꢀNꢀfurfurylꢀLactose
(16 mg, 0.035 mmol) was added followed by water (40µl). The reaction mixture was stirred at
37 °C for 7 days. The solvents were removed by rotary evaporation and the crude material
was dissolved in CH2Cl2/MeOH, loaded on to silica and purified by flash column
chromatography eluting with 10% H2O in CH3CN. The expected product 18 was isolated as a
red solid (12 mg, 58%) after lyophilization. HPLC method A (see SI). MS (ESI) m/z:
[M+Na]+: 1759.7; HRMS m/z [M+Na]+ calculated for C83H100N8O33Na: 1759.6290, found:
1759.6302.
LactriPepDox (19)
Following a modified procedure for Alloc deprotection,33 18 (7 mg, 4.0 µmol) was dissolved
in degassed CH2Cl2:AcOH (5:1, 600 µl). Pd(PPh3)4 (6 mg, 5.2 µmol) was added. The reaction
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was stirred at rt in the dark and followed by TLC (CH2Cl2:MeOH:NH4OH 80:18:2). After 30
min, the CH2Cl2 was removed and DEE (1 ml) was added which resulted in a red precipitation
which was washed with DEE (1 ml), EtOAc (2 x 1 ml) and then DEE (1 ml) again.
Ultrasonication and quick centrifugation was used in each wash cycle. The resulting red pellet
was dried in a dessicator under low pressure. The resulting red solid (4 mg, 59%) had a mass
on ESIꢀMS and HRMS corresponding with the expected product 19 and consisted of one
main compound with minor impurities on HPLCꢀUV. HPLC method A. MS (ESI) m/z:
[M+H]+: 1653.6 HRMS m/z [M+H]+ calculated for C79H97N8O31: 1653.6260, found:
1653.6244.
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Enzymatic release of Doxorubicin
Plasmin and human carboxylesterases were purchased from SigmaꢀAldrich.
Plasmin
Following a protocol from the enzyme provider,47 a 100 µM solution of 19 in phosphate
buffer (pH 7.5) containing 100mM Lysine was incubated at 37 with plasmin from human
plasma (15µg/ml). The reaction was followed by reversed phase HPLC (method B) by
injecting 15µl aliquots from the reaction. The ratio of released doxorubicin over prodrug was
calculated from integration of the peaks at 480 nm and plotted against time.
Carboxylesterase
A 100 µM solution of ester linked prodrug (16 or 17) in Tris buffer (50 mM, pH 7.4)48 was
incubated at 37 with either recombinant human carboxylesterase 1 isoform b (100µg/ml) or
recombinant human carboxylesterase 2 (100µg/ml) expressed in baculovirus infected BTI
insect cells . The reaction was followed by reversed phase HPLC (method B) by injecting 15
µl aliquots from the reaction. The ratio of released doxorubicin over prodrug was calculated
from integration of the peaks at 480 nm and plotted against time.
Growth inhibition assay
The breast cancer cells MCFꢀ7 were seeded at a density of 2×103 cells per well in 96ꢀwell
plates. After 24 h, cells were treated with an increasing concentration of compounds, from 0
to 100 µM. Cells were incubated for 72 h, and then, 20 ꢁL of MTS (Promega) was added per
well for one hour at 37 °C. The absorbance was read at 492 nm using a spectrophotometer
(SPECTRAmax, Molecular Devices). IC50 values were obtained from the cytotoxicity curves
using SOFTmax PRO software.
Supporting Information description
The SI contains NMR spectra and HPLC chromatograms.
Acknowledgments
This work was supported by the French National Research Agency (ANR) under the research
grants Arcane (ANRꢀ11ꢀLABXꢀ0003ꢀ01), Cellical (ANRꢀ15ꢀCE08ꢀ0033) and Glyco@Alps
(ANR 15 IDEX 02). We acknowledge ICMG (FR 2607) platforms for granting access to
the mass spectrometry and NMR facilities.
Abbreviations
Doxorubicin, Dox; Xyloglucan, XG; trifluoroacetic acid, TFA; dicyclohexylcarbodiimide,
DCC;
Nꢀmethylmorpholine,
NMM;
N,Nꢀdiisopropylethylamine,
DIPEA;
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(dimethoxy)triazinylmethylmorpholonium
carboxylesterase 2, hCE2; pꢀaminobenzyl alcohol, PABA; maleimidyl ester linked
tetrafluoroborate,
DMTMMBF4;
human
Doxorubicin, MalEsterDox; maleimidyl tripeptidyl linked Doxorubicin, MaltriPepDox
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