Extended scaffold glucuronides: En route to the universal synthesis of O -aryl glucuronide prodrugs

Article abstract of DOI:10.1039/c9ob01384a

We demonstrate that an extended scaffold based on a self-immolative linker (SIL) enables the universal production of O-aryl glucuronide prodrugs: high yield glucuronidation is performed on a precursor substrate (SIL) and the subsequent drug conjugation proceeds via less challenging chemical reactions.

Full text of DOI:10.1039/c9ob01384a

Organic &
Biomolecular Chemistry
View Article Online
View Journal
COMMUNICATION
Extended scaffold glucuronides: en route to
the universal synthesis of O-aryl glucuronide
Cite this: DOI: 10.1039/c9ob01384a
prodrugs†
Received 19th June 2019,
Accepted 3rd July 2019
Raoul Walther,a Morten T. Jarlstad Olesenb and Alexander N. Zelikin
*
a,b
DOI: 10.1039/c9ob01384a
rsc.li/obc
We demonstrate that an extended scaffold based on a self- ation for each specific drug, even within the same class of
immolative linker (SIL) enables the universal production of O-aryl molecules (e.g. phenols).9,10
glucuronide prodrugs: high yield glucuronidation is performed on
In this work, we specifically address this challenge and
a precursor substrate (SIL) and the subsequent drug conjugation develop a modular methodology for the synthesis of glucuro-
proceeds via less challenging chemical reactions.
nide prodrugs, based on the structure of the extended
scaffold glucuronides that includes a self-immolative linker
(SIL).11 We employ an SIL such that the most synthetically
challenging, glucuronidation step becomes universal,
whereas drug conjugation is achieved via less demanding
reactions. We investigate this with O-aryl glucuronides as an
example and demonstrate that the SIL serves to resolve the
synthetic hurdles of glucuronidation and affords straight-
forward, good yield syntheses of diverse extended scaffold
glucuronides. We then compare the natural vs. extended
scaffold glucuronides in terms of kinetics of drug recovery
and their performance in an in vitro EPT. Results of this
study are significant in establishing a modular glucuronida-
tion platform to synthesize these privileged prodrugs for
diverse drug targeting applications.
First, to verify the scope and limitations of “classical”
chemical glucuronidation, we investigated the Koenigs–Knorr
coupling9,10 of phenols with glucuronyl bromide 1 12 (Fig. 1A).
These reactions yielded the O-aryl glucuronides with excellent
β-selectivity, but glycosylation yields were greatly dependent on
the pKa of phenol (Fig. 2, for numerical values of yields, see
Table S1†) and became near zero for p-chlorophenol and ana-
logues with a higher pKa. Our efforts to optimize the Koenigs–
Knorr-based glucuronidation through the variation of the
solvent, the promoters (including Ag2CO3 and AgOTf), and the
additives failed (Table S2†): instead of an improved glycosyla-
tion outcome, we observed the formation of the common glycal
or orthoester as side products. We also considered that glucuro-
nidation of the electron-rich phenols can be performed under
the Lewis acid-catalyzed conditions (using the anomeric acetate
2 13 or trichloroacetimidate 3 14,15). Yet these are not universal
since phenols are ambident nucleophiles and under the Lewis
acid conditions they likely afford C-glycosides.16,17 Based on
these results and the extensive prior work on this subject, we
Targeted drug delivery is rapidly becoming the mainstream of
biomedicine and is expected to revolutionize the treatment of
diverse diseases. Drug targeting can be achieved using anti-
body–drug conjugates (ADCs),1–3 via the organ-specific acti-
vation of prodrugs,4 “enzyme-prodrug therapies” (EPTs),5 or
localized drug synthesis mediated by the diseased tissue
itself.6 In each of these methodologies, the key to success is
the nature of the prodrug. It has to be biocompatible and pre-
ferably devoid of pharmacological activity, yet become a potent
therapeutic upon bioconversion. Of all the prodrugs applied in
the EPTs and the tumor-activated drug syntheses, glucuro-
nides stand out as a privileged scaffold. This is due to the
minimized cell entry of glucuronides, which affords a high
ratio between the toxicity-related IC50 for the prodrug and the
drug.4 Glucuronides are also advantageous in that these typi-
cally afford a significantly enhanced aqueous solubility, a
feature of high importance for hydrophobic drugs. However,
the disadvantage of the glucuronides lies in the synthetic
hurdles for their synthesis.7,8 In nature, glucuronides are
formed enzymatically during the phase II metabolism in the
liver. In the lab, glucuronide syntheses are carried out via
multi-step, laborious routes involving protective groups.
Furthermore, chemical glucuronidation lacks
methodology and glucuronidation requires tedious optimiz-
a universal
aDepartment of Chemistry, Aarhus University, Aarhus, Denmark
biNano Interdisciplinary Nanoscience Centre, Aarhus University, Aarhus, Denmark.
E-mail: zelikin@chem.au.dk
†Electronic supplementary information (ESI) available: Detailed procedures for
syntheses and characterization (NMR, HR-MS and HPLC; quantification of the
optical purity of the prodrugs was beyond the scope of this study). See DOI:
10.1039/c9ob01384a
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
PHBA as a unique tool for the design of O-aryl glucuronides.
The vision put forward is that, in this case, the most syntheti-
cally challenging, glucuronidation step becomes universal,
whereas drug conjugation is achieved via less demanding
reactions.
The synthesis of O-aryl glucuronide was accomplished
using 4-hydroxylbenzaldehyde and the o-nitro analogue hereof
(phenols d and f, Fig. 1). The two molecules exhibited high
yields of glycosylation (Fig. 2, Table S1†) and were conveniently
reduced to the corresponding benzyl alcohols (5, 6) with near-
quantitative yields. The nitro group may be preserved in the
structure of the prodrug or, to avoid the risk of immune reac-
tions,22 can be reduced at this stage to the corresponding
amine and optionally acetylated 7 (Fig. 1, Fig. S1†). The latter
method is specifically important with the view of enhancing
synthetic possibilities, for e.g., bioconjugation of glucuronides
to antibodies,23,24 albumin,6 etc.
Conjugation of phenols to the extended glucuronide
scaffold was optimized using resorufin as a model turn-on fluo-
rescent probe. Conventional synthesis of the key alkyl–aryl
ether bond through the Williamson ether approach using
benzyl halides/mesylates S4 or under Lewis-acidic conditions
through benzyl 2,2,2-trichloroacetimidate intermediate S5 was
unsatisfactory (Fig. S2†). The modified Mitsunobu reaction25
on the other hand gave encouraging results (Table S3, entries a
and b†), shortened the synthetic sequence, and circumvented
the transformation of the benzyl alcohol glucuronide inter-
mediate into a suitable precursor for an SN2 reaction. However,
it afforded a high yield of the unwanted side product, namely
the Δ4,5-dehydro compound.7,8 Nevertheless, under optimized
conditions (Table S3†), the yield of the Δ4,5-dehydro compound
was minimized to ca. 2.5%, whereas the formation of the
desired product conjugate was maximized to 47%.
Next, we investigated the substrate scope of the optimized
reaction conditions (Fig. 1D). Derivatives of 4-nitrophenol 8e,
2-naphthol 8i, 6-hydroxyquinoline 8j and vanillin 8h were suc-
cessfully isolated with good yields (50–70%), already extending
well beyond the substrate scope of the Koenigs–Knorr glucuro-
nidation conditions in terms of the pKa of phenol.
Furthermore, the extended scaffold glucuronide of a tyrosine
derivative (8o, pKa over 10) was also obtained in good isolated
Fig. 1 (A) Schematic representation of the existing methodologies for
O-aryl glucuronidation and (B) the herein proposed “extended scaffold”
approach towards establishing a universal glucuronidation method; (C)
chemical formulas of the phenolic compounds tested in this work as
substrates for traditional and extended scaffold glucuronidation; (D)
scatter plot representation of the scope and limitations of the traditional
O-aryl glycosylation methods, with the reaction yield being greatly
dependent on the pKa of phenol, and the herein proposed extended
scaffold glucuronidation method in which case glucuronidation yields
are virtually constant across the range of pKa of the phenols.
conclude that a “universal” glucuronidation pathway may not yield paving the way to the prodrugs of biological drugs and
be identified through the variation of glycosylation conditions. tubulysin A. Finally, a glycosylation product for a marketed
Instead, we propose that such a synthetic opportunity can be anticancer drug SN-38 (with a pKa of phenol of 9.7) was deli-
engineered using a cunning tool of chemistry, namely SILs.
vered in a yield of 68% for the nitro-containing linker 8m and
Historically, SILs have been developed to optimize the trig- 46% for the acetamide-containing linker 8n, the yield being
gered drug release by virtue of increasing accessibility of the significantly better than 5% yield of the direct glucuronidation
scissile bond for the enzyme for bioconversion.18 In recent in either case (Table S1†). Taken together, the results in Fig. 1
years, we and others also recognized the power of SILs to illustrate that the proposed synthetic pathway involving (nitro)
enable “unnatural” chemistry within the existing molecules, PHBA SIL as the glucuronidation partner with a subsequent
such as the use of thiol–disulfide chemistry in thiol-free mole- synthesis of aryl ether delivers a considerable synthetic flexi-
cules.19 Common SILs include p-hydroxylbenzyl alcohol PHBA bility and affords glucuronide derivatives for a broad range of
and its close analogues, p-aminobenzyl alcohol20 and p-mer- phenols (Fig. 1). Suitable substrates include phenols across the
captobenzyl alcohol,21 which are well-established tools of med- range of pKa for phenol, as high as 10.5 for tyrosine, and
icinal chemistry, most notably as spacers for the enzyme-acti- accommodate a straightforward synthesis of derivatives for
vated prodrug decomposition. In this work, we propose to use SN-38.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Organic & Biomolecular Chemistry
Communication
Fig. 2 (A) Schematic illustration of the bioconversion and ensuing immolation of the extended scaffold glucuronides; (B) evolution of the fluor-
●
■
▲
escence intensity (λex = 570 nm; λem = 585 nm) in solutions of prodrugs reso–GlcA ( ), 9 ( ), and 10 ( ) at a final concentration of 13 µM in PBS in
the presence of 1 mg L−1 β-Glu at 37 °C; (C) fluorescence intensity in solutions of reso-GlcA, 9, and 10 (13 µM in PBS) upon incubation over 2 h in
the presence or absence of β-Glu (1 mg L−1); (D) representative normalized analytical HPLC traces illustrating the bioconversion of the extended
scaffold glucuronides of SN-38 (100 µM prodrug, 1 mg L−1 enzyme, PBS, 2 h); (E) quantitative data on the release of SN-38 from its extended
scaffold glucuronides (100 µM prodrug, 1 mg L−1 enzyme, PBS, 2 h).
Deprotection conditions were validated for the resorufin (Fig. 2C). Fluorescence evolution profiles were used to estimate
turn-on fluorescent probes 9 and 10 and the derivatives of the parameters of enzymatic catalysis, namely Km and kcat
,
SN-38 (11 and 12). For resorufin, deprotection was carried out Table 1. In applying the Michaelis–Menten equations, we
via Zémplen deacetylation and subsequent saponification of assumed that enzymatic bioconversion (not the self-immola-
the methyl ester was done with 2 M NaOH (Fig. S3†). For tion) is the rate limiting step in the overall drug release. Km
SN-38, saponification was optimized well beyond the reported values for the extended scaffold prodrugs were similar to those
yields (typically 12–27%)3,26 and using Ba(OH)2·8H2O27 in of the native glucuronides with direct linkage, which illustrates
anhydrous MeOH, prodrugs 11 and 12 were obtained in 86% that the extended glucuronide scaffold does not result in an
and 48% yields, respectively, with the lactone moiety of SN-38 inferior prodrug design compared to the natural glucuronide
still in place (Fig. S3†).
scaffold. Furthermore, incorporation of a nitro-substituent
Next, we aimed at validating whether the extended scaffold ortho into the glycosidic linkage (compound 10) drastically
glucuronides are accepted by the enzyme for bioconversion, lowered the Km suggesting that the electron-withdrawing
and if so, at quantitating the kinetics of enzymatic catalysis.
For the prodrugs of resorufin (9, 10) and SN-38 (11, 12), enzy-
matic scission of the glycosidic linkage affords a phenolic
Table 1 Michaelis–Menten kinetic parameters and quantitative in vitro
compound that undergoes a 1,6-elimination with the for-
mation of resorufin or SN-38, respectively (Fig. 2A). Resorufin
prodrugs were designed as fluorogenic probes, for which the
scission of the glucuronide bond and the ensuing self-immola-
tion of PHBA afford resorufin, accompanied by a drastic
increase in fluorescence. The latter phenomenon provides a
convenient means to monitor and quantify the enzymatic con-
version of the substrate. Incubation of resorufin-β-D-glucuro-
nide (reso-GlcA) and the two extended scaffold resorufin pro-
drugs 9 and 10 in the presence of β-Glu afforded very similar
fluorescence evolution profiles (Fig. 2B) and levels of total
attained fluorescence after 2 h of enzymatic bioconversion
cytotoxicity data for the prodrugs of resorufin and SN-38. IC50 values
were determined in human cervical cancer HeLa cells over 72 h cell
culture (Fig. 2F). QIC50 = IC50(prodrug)/IC50(prodrug in the presence of
β-Glu)
IC50
(−Enz) (+Enz)
[nM]
IC50
Km
[µM]
kcat
kcat/Km
Entry
[s−1
]
[s−1 µM−1
]
[nM]
QIC50
reso-GlcA
9
36
33
8
5
14.2 2.3 0.39 0.02
20.7 2.4 0.62 0.02
—
—
—
—
10
3.6 0.2 4.7 0.1 1.31 0.04
—
1528
—
SN-38-GlcA
11
12
—
42
50
—
—
15.2
58.7
81.9
100.5
10.9
23.4
4
5
263 14 6.26 0.30 642.1
273 14 5.45 0.25 1921
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem.

View Article Online
Communication
Organic & Biomolecular Chemistry
groups positively modulate enzyme affinities for PHBA-con- such as amines (as is already realized in, e.g., brentuximab
taining prodrugs, as has been previously observed for other vedotin) and aliphatic hydroxyls.
phenyl glycosides.28,29
For the prodrugs of SN-38, the main characterization tool
was HPLC (Fig. 2D and E). In the presence of β-Glu, the pro-
Conflicts of interest
drugs readily released the constituting toxin and this process
was near-complete within 2 h, whereas incubation of prodrugs
We declare no conflicts of interest.
in PBS without the enzyme showed negligible drug release
over 24 h (Fig. 2D; Fig. S4† for 12). We also observed that Acknowledgements
prodrug bioconversion for SN-38 was accompanied by an
ANZ acknowledges funding from the European Research Council
(ERC-2013-CoG 617336 BTVI). T. Poulsen Lab (Aarhus University)
is acknowledged for providing access to a preparative HPLC and
Dr T. Breitenbach for providing us with the HeLa cells.
increase in fluorescence (Fig. S5†), a phenomenon useful for
quantitative kinetics measurements. The Km values for the two
SIL-containing SN-38 prodrugs (42 4 and 50 5 µM for 11
and 12, respectively, Table 1) were similar to the prodrugs
of resorufin and the glucuronide prodrugs of camptothecin
and analogues.3,26 In turn, the catalytic efficiency was 10-fold
higher than that for resorufin, yet similar for prodrugs 11
References
and 12.
Lastly, we aimed at characterizing the native and the
1 P. J. Carter and G. A. Lazar, Nat. Rev. Drug Discovery, 2017,
17, 197.
extended scaffold glucuronides of SN-38 in cell culture. The
anti-proliferative properties of the prodrugs were evaluated in
the human cervical cancer HeLa cells (Fig. S6† and Table 1). In
short, the growth medium was supplemented with prodrugs in
a dilution series ranging from 5.1 × 10−12 M to 10 × 10−6 M, in
the presence or absence of β-Glu. Without enzymatic acti-
vation, the natural metabolite SN-38-β-D-glucuronide (SN-38-
GlcA) and the two glucuronide prodrugs 11 and 12 exhibited
IC50 values between 0.6 and 2 µM. In other words, extended
scaffold glucuronides were successful in masking the toxicity
of the incorporated anticancer drug (for SN-38, IC50 = 5.5–16
nM). Notably, the least toxic prodrug was the acetylated
extended scaffold product 12. Enzymatic bioconversion shifted
the IC50 values to 59 and 82 nM for prodrugs 11 and 12,
respectively (Table 1). Toxicity data reveal that the QIC50 values
(i.e. the ratio between the non-activated and activated prodrug
in terms of IC50) for the extended scaffold glucuronide was
somewhat lower than that for the native glucuronide (QIC50 of
2 T. Rodrigues and G. J. L. Bernardes, Angew. Chem., Int. Ed.,
2018, 57, 2032–2034.
3 Z. M. Prijovich, P.-A. Burnouf, H.-C. Chou, P.-T. Huang,
K.-C. Chen, T.-L. Cheng, Y.-L. Leu and S. R. Roffler, Mol.
Pharm., 2016, 13, 1242–1250.
4 R. Walther, J. Rautio and A. N. Zelikin, Adv. Drug Delivery
Rev., 2017, 118, 65–77.
5 B. Stadler and A. N. Zelikin, Adv. Drug Delivery Rev., 2017,
118, 1.
6 B. Renoux, F. Raes, T. Legigan, E. Peraudeau, B. Eddhif,
P. Poinot, I. Tranoy-Opalinski, J. Alsarraf, O. Koniev,
S. Kolodych, S. Lerondel, A. Le Pape, J. Clarhaut and
S. Papot, Chem. Sci., 2017, 8, 3427–3433.
7 A. V. Stachulski and G. N. Jenkins, Nat. Prod. Rep., 1998, 15,
173–186.
8 A. V. Stachulski and X. Meng, Nat. Prod. Rep., 2013, 30,
806–848.
9 K. J. Jensen, J. Chem. Soc., Perkin Trans. 1, 2002, 2219–2233.
23 for the acetylated prodrug 12 vs. 100 for SN-38-GlcA), poss- 10 M. Jacobsson, J. Malmberg and U. Ellervik, Carbohydr. Res.,
ibly due to the increased hydrophobicity of the linker. 2006, 341, 1266–1281.
Taken together, the results of this study demonstrate that 11 A. Alouane, R. Labruère, T. Le Saux, F. Schmidt and
extended scaffold design comprising a self-immolative linker L. Jullien, Angew. Chem., Int. Ed., 2015, 54, 7492–7509.
delivers the highly warranted “universal” strategy for the syn- 12 W. Koenigs and E. Knorr, Chem. Ber., 1901, 34, 957–981.
thesis of glucuronide prodrugs. The SIL is used such that the 13 B. Helferich and E. Schmitz-Hillebrecht, Chem. Ber., 1933,
synthetically challenging glucuronidation step is performed
with high yield on the same precursor molecule, whereas drug 14 R. R. Schmidt and J. Michel, Angew. Chem., Int. Ed. Engl.,
conjugation is performed via less demanding, readily available 1980, 19, 731–732.
syntheses. Extended scaffold glucuronides undergo enzymatic 15 R. R. Schmidt and J. Michel, Tetrahedron Lett., 1984, 25,
bioconversion with kinetic parameters similar or even favor- 821–824.
66, 378–383.
able when compared to the bioconversion of native glucuro- 16 S. Weck and T. Opatz, Synthesis, 2010, 2393–2398.
nides. In cell culture, extended scaffold glucuronides reversibly 17 C. Jaramillo and S. Knapp, Synthesis, 1994, 1–20.
mask the toxicity of the incorporated toxin, as is used in the 18 F. Kratz, I. A. Müller, C. Ryppa and A. Warnecke,
enzyme prodrug therapies for localized drug synthesis.5 This
ChemMedChem, 2008, 3, 20–53.
strategy for designing glucuronide prodrugs is directly appli- 19 C. F. Riber, A. A. A. Smith and A. N. Zelikin, Adv. Healthcare
cable to a wide range of phenols with varied pKa and includes Mater., 2015, 4, 1887–1890.
tyrosine, towards the glucuronidation of peptides, and is 20 P. L. Carl, P. K. Chakravarty and J. A. Katzenellenbogen,
readily extendable to other functionalities common in drugs
J. Med. Chem., 1981, 24, 479–480.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Organic & Biomolecular Chemistry
Communication
21 T. Sun, A. Morger, B. Castagner and J.-C. Leroux, Chem. 25 T. Tsunoda, Y. Yamamiya and S. Itô, Tetrahedron Lett.,
Commun., 2015, 51, 5721–5724. 1993, 34, 1639–1642.
22 P. J. McEnaney, C. G. Parker, A. X. Zhang and D. A. Spiegel, 26 Y.-L. Leu, C.-S. Chen, Y.-J. Wu and J.-W. Chern, J. Med.
ACS Chem. Biol., 2012, 7, 1139–1151. Chem., 2008, 51, 1740–1746.
23 R. P. Lyon, T. D. Bovee, S. O. Doronina, P. J. Burke, 27 K. Inoue and K. Sakai, Tetrahedron Lett., 1977, 18, 4063–
J. H. Hunter, H. D. Neff-LaFord, M. Jonas, M. E. Anderson,
J. R. Setter and P. D. Senter, Nat. Biotechnol., 2015, 33, 733.
24 R. V. Kolakowski, K. T. Haelsig, K. K. Emmerton,
C. I. Leiske, J. B. Miyamoto, J. H. Cochran, R. P. Lyon,
4066.
28 T. Duo, K. Robinson, I. R. Greig, H.-M. Chen, B. O. Patrick
and S. G. Withers, J. Am. Chem. Soc., 2017, 139, 15994–
15999.
P. D. Senter and S. C. Jeffrey, Angew. Chem., Int. Ed., 2016, 29 J. B. Kempton and S. G. Withers, Biochemistry, 1992, 31,
55, 7948–7951.
9961–9969.
This journal is © The Royal Society of Chemistry 2019
Org. Biomol. Chem.