Expedient synthesis of trifunctional oligoethyleneglycol-amine linkers and their use in the preparation of PEG-based branched platforms

Article abstract of DOI:10.1039/C8OB02097C

We designed a convergent synthesis pathway that provides access to trifunctional oligoethyleneglycol-amine (OEG-amine) linkers. By applying the reductive coupling of a primary azide to bifunctional OEG-azide precursors, the corresponding symmetrical dialkylamine bearing two homo-functional end chain groups and a central nitrogen was obtained. These building blocks bear minimal structural perturbation compared to the native OEG backbone which makes them attractive for biomedical applications. The NMR investigations of the mechanism process reveal the formation of nitrile and imine intermediates which can react with the reduced free amine form. Additionally, these trifunctional OEG-amine linkers were employed in a coupling reaction to afford branched multifunctional PEG dendrons which are molecularly defined. These discrete PEG-based dendrons (n = 16, 18 and 36) could be useful for numerous applications where multivalency is required.

Full text of DOI:10.1039/C8OB02097C

Organic &
Biomolecular Chemistry
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Expedient synthesis of trifunctional
oligoethyleneglycol-amine linkers and their use in
the preparation of PEG-based branched platforms†
Cite this: Org. Biomol. Chem., 2018,
16, 8579
Sylvain Ursuegui, *a Jérémy P. Schneider,a Claire Imbs,a Florian Lauvoisard,a
a
Marta Dudek,a,b Michel Mossera and Alain Wagner
We designed a convergent synthesis pathway that provides access to trifunctional oligoethyleneglycol-amine
(OEG-amine) linkers. By applying the reductive coupling of a primary azide to bifunctional OEG-azide precur-
sors, the corresponding symmetrical dialkylamine bearing two homo-functional end chain groups and a
central nitrogen was obtained. These building blocks bear minimal structural perturbation compared to the
native OEG backbone which makes them attractive for biomedical applications. The NMR investigations of the
mechanism process reveal the formation of nitrile and imine intermediates which can react with the reduced
free amine form. Additionally, these trifunctional OEG-amine linkers were employed in a coupling reaction to
afford branched multifunctional PEG dendrons which are molecularly defined. These discrete PEG-based
dendrons (n = 16, 18 and 36) could be useful for numerous applications where multivalency is required.
Received 27th August 2018,
Accepted 22nd October 2018
DOI: 10.1039/c8ob02097c
rsc.li/obc
multivalency required by most biological applications, these
linear hetero-bifunctional OEGs are assembled via stepwise
Introduction
Polyfunctional molecularly defined short-chain PEGs called chemical coupling onto a multi-reactive core structure, such as
oligoethylene glycols (OEG) are becoming an increasingly glycerol, 2-(2-aminoethoxy)
triazine heterocycle,9
a
a
a
important class of linkers. Thanks to their particular biophysi- ethanol,10 a bicine,11 a trimethylolpropane allyl ether12 or a
cal characteristics (i.e., being uncharged, water soluble, bio- lysine structure13–15 (Fig. 1a). In addition, due to often being
stable, and biocompatible), these building blocks are omnipre- tedious, this synthetic strategy loses the intrinsic benefit of
sent in every application where biological systems have to using an OEG backbone by introducing a central structural
interact with exobiotic, synthetic moieties. For example, they motif that can lead to biological instability, pH sensitivity, or
are considered as the privileged linker backbone for immobi- non-specific interaction with the biological media.
lizing biomolecules on various surfaces,1–3 designing a drug
Thus, on one hand, our goal was to devise a straightforward
delivery system,4 or preparing neutral surfactants and water/ access to trifunctional OEG bearing minimal structural pertur-
bio-compatible polymers. Regarding pharmaceutical appli-
cations, engineered OEGs with controlled molecular structures
are often used as spacers for the conjugation of small drugs to
antibodies5,6 and nanoparticles.7 Because of this pivotal role,
intensive research efforts have been dedicated to developing a
variety of OEGs, which differ in their shapes and functional-
ities. Hence, a large panel of reactive functional groups has
been introduced at the opposite end of these linear OEGs such
as activated carboxylic esters, free or protected amines,
azides, alkynes, or thiols for example.8 In order to achieve the
aBio-Functional Chemistry (UMR 7199), LabEx Medalis, University of Strasbourg,
74 Route du Rhin, 67401 Illkirch-Graffenstaden, France. E-mail: ursuegui@unistra.fr
bAdvanced Materials Engineering and Modelling Group, Faculty of Chemistry,
Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27,
50-370 Wroclaw, Poland
Fig. 1 (a) Illustration of current strategies to access trifunctional bis-
OEG constructs. (b) The proposed alternative approach toward unal-
tered trifunctional OEG-amine linkers and their use for the preparation
of discrete multifunctional PEG-based dendrons.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02097c
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bation compared to the native OEG backbone (Fig. 1, first in good yields (≥70%). Thus, tetra-ethylene glycol 1a and hexa-
objective: structural simplification). We hypothesized that ethylene glycol 1b gave mono-tosyl-OEGs 2a and 2b, respect-
introducing a central nitrogen atom in the OEG skeleton ively, which were converted quantitatively to their corres-
would create a molecularly defined analogue bearing an ponding azido-OEGs 3a and 3b (Scheme 1). The tert-butyl
additional derivatization center. These OEG linkers could then esters 4a and 4b were subsequently obtained by alkylation of
replace the traditional bifunctional building blocks for bio- OEG azides 3a and 3b with tert-butyl acrylate. After quantitat-
medical applications. On the other hand, due to their trifunc- ive acidic deprotection, the resulting carboxylic acids 5a and
tional properties, these linkers can also be incorporated into 5b were reacted with commercially available N-Boc ethylene
the traditional multifunctional cores to increase the multiva- diamine, using EDC and HOBt as coupling reagents, cleanly
lency of the resulting chemical platforms offering molecularly delivering bifunctional OEGs 6a and 6b in 75 and 78% yields,
defined PEG-based dendrons (Fig. 1b, second objective: respectively.
expanded multivalency).
The optimization of the reductive azide dimerization step
Examination of the literature showed that such molecules was then performed using the hetero-bifunctional OEG4 4a as
have only been scarcely described. For instance, bis-chloro- the model substrate. In 2004, I. H. An et al. reported a study on
OEG3-amine has been synthesized by alkylation of N-Boc the reductive dimerization of 3-phenyl propyl azide and
diethanolamine with 1,1′-dichloro ethyl ethers16 and converted showed that this process was preferred over the simple
to the corresponding bis-azide derivative after NaN3 treat- reduction, especially at elevated temperature and high concen-
ment.17 This synthetic approach appears limited, due to the trations.21 With these considerations in mind, we screened
low availability of bis-chloro OEG derivatives and its narrow several parameters, including the catalyst loading, the reactant
scope.
concentration, the temperature, and the solvent, and com-
In order to design a versatile synthesis of such a highly pared by 1H-NMR spectroscopy the ratios between the expected
functionalized hydrosoluble compound in a limited number of dimer 8a and the amine side-product 7a resulting from the
steps, we turned our attention to the reductive coupling of simple reduction (see ESI Table 1†). Briefly, the reductive
primary azides, a reaction first described by M. Lange et al.18 dimerization reaction was found to be dependent on the cata-
Interestingly, this reaction seemed to be compatible with the lyst loading, reaction temperature and substrate concen-
ethylene glycol motif as demonstrated by the synthesis of a trations. For instance 8a was obtained in 42% yield when
simple amino bis-OEG6 albeit in a poor 30% yield.19 We thus 5 mol% of Pd/C was used, as opposed to only 18% with a
decided to investigate this key reaction further and evaluate its 1 mol% Pd loading. Also the yield of the desired product
application to the synthesis of N,N-bis-oligoethyleneglycol- increased when the substrate concentration increased from
amines by testing it on a readily available azido OEG. In order 100 mM to 500 mM. As previously reported for 3-phenyl propyl
to further demonstrate the potency of these new trifunctional azide, we also found that the dimerization process was more
OEG-amine linkers we engaged them in the preparation of new efficient at elevated temperatures with yields of 39% at 0 °C
branched multifunctional discrete PEG-dendrons.
and 79% at 60 °C. Finally, ethyl acetate, 1,4-dioxane or THF
gave a better yield (90–93%) than EtOH (79%).
Applying these optimized conditions to precursors 4a–b
and 6a–b (500 mg, 5% Pd/C, 500 mM in dioxane at 60 °C), we
were able to synthesize the desired N,N-bis-OEG-amines 8a,
Results & discussion
We first synthesized the required linear bifunctional OEG 8b, 9a and 9b in one step and in good isolated yields (72–84%)
building blocks bearing an azide group and either an ester or (Scheme 2). It is noteworthy that these reactions were per-
an amino group on the opposite end of the chain. To this end, formed at a 2 g scale and they proved to be highly reproducible
mono-activation of one of the hydroxyl-ends of the parent OEG affording high yields (>70%) each time.
was performed according to the procedure of Bouzide A. and
The reductive azide dimerization was also directly applied
Sauvé G.20 The use of a stoichiometric amount of tosyl chlor- to azido-OEG4-alcohol 3a, affording the corresponding bis-
ide in the presence of silver(I) oxide and a catalytic amount of OEG-amine 10. The resulting secondary amine was then Boc-
potassium iodide led selectively to the monotosyl derivatives protected before the alcohol groups were converted to their
Scheme 1 Synthesis of bifunctional OEG azides.
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leading to a primary amine complexed with Pd. Ultimately this
amine would react with a neighboring imine Pd complex to
form an aminal intermediate which is further reduced to
afford the final dimerized compound. In our reactions, we had
sometimes observed traces of a nitrile derivative. Since other
reports describe that an azide can be converted into a nitrile,
and that the nitrile can be reduced into an amine, we wonder
whether this pathway could be predominant in azide dimeriza-
tion of an OEG-based substrate. To evaluate this alternative
mechanism, the reduction of azide-OEG 4a was monitored by
1H NMR spectroscopy. The reaction was performed at room
temperature, for reducing the kinetics of the reaction and
allowing isolation and characterization of the intermediates.
Aliquots were taken from the reaction mixture at regular inter-
vals (5 min, 30 min, 1 h, 3 h and 22 h), filtered through a
Celite pad, concentrated under reduced pressure and analyzed
Scheme 2 Reductive dimerization of OEG azides to bis-OEG amines.
corresponding tosyl derivatives (Scheme 3). The bis-azido-
OEG4-N-Boc amine 13 was finally obtained after treatment
with sodium azide and was subsequently deprotected under
acidic conditions to afford bis-azido-OEG-amine 14. It is worth
mentioning that this clickable linker was prepared on a 3 g
scale, demonstrating the robustness of our synthetic strategy.
We then turned our attention towards the investigation of
the mechanism of this reaction. In their early work, Lange
et al. proposed as a first catalytic act the formation of an imine
intermediate that is complexed with palladium via the expul-
sion of N2.18 This intermediate undergoes on-site reduction
1
by H NMR spectroscopy (Fig. 2). The complete conversion of
4a was observed after 1 h leading first, as suspected, to the for-
mation of the corresponding nitrile 15 and primary amine 7a
Scheme 3 Synthesis of a bis-azide OEG4 amine. * Isolated yield after dimerization and N-Boc protection.
Fig. 2 1H NMR monitoring of reductive azide dimerization.
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derivatives, which were isolated and characterized. With pro- thesis of nitrile and imine compounds from the corresponding
longed reaction times, we observed the formation of the bis- azide derivatives using Pd catalysts.22 Moreover the direct syn-
alkylamine 8a, while the concentration of both nitrile and thesis of secondary or tertiary amines via catalytic reduction of
primary amine decreased, suggesting that the reductive azide nitriles has been widely described in the literature affording
dimerization involves indeed a nitrile substrate as an inter- secondary23–29 and tertiary amines.30
mediate. A transient compound whose chemical shifts match
To assess this hypothesis, the isolated nitrile derivative 15 was
those of the imine derivative could also be observed. This subjected to the reductive dimerization conditions. We were
study thus suggests that the reductive dimerization involves delighted to notice the formation of the corresponding bis-alkyl
the formation of a nitrile and an imine intermediate, which amine 8a that was isolated in 50% yield, thus demonstrating that
can then react with the reduced free amine derivative to form the nitrile derivative is a key intermediate in this reaction.
the corresponding symmetrical dialkylamine compound.
Finally, we demonstrated that these trifunctional OEG-
Interestingly, the ratio between the nitrile and imine forms amine linkers can be used for the preparation of poly-func-
remains constant during the reaction suggesting an equili- tional platforms increasing the multivalency of chemical plat-
brium between the two intermediates. These observations are forms particularly when these building blocks are introduced
in agreement with Díez-González’s work describing the syn- on multi-functional cores such as triazine (Scheme 4). Starting
Scheme 4 Preparation of compact tri- and hexa-functional OEG platforms.
Scheme 5 Preparation of hetero-functional OEG platforms.
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from bis-N-Boc-OEG6-amine 9b, an additional OEG chain was
introduced on the central secondary amine by coupling it with
Conflicts of interest
various linear OEG6 carboxylic acids. The corresponding tri- There are no conflicts to declare.
OEG derivatives 16 and 18 were isolated in 78% and 86%
yields, respectively. With the aim of obtaining clickable OEG
platforms, the N-Boc derivatives 16 and 18 were deprotected to
yield the corresponding free amines, which were immediately
Acknowledgements
The authors gratefully acknowledge Dr Guilhem Chaubet (Bio-
Functional Chemistry UMR 7199, University of Strasbourg) for
proofreading the manuscript, Estefania Oliva & Delphine
Garnier (PACSI platform, University of Strasbourg) for HRMS
analyses and ANR ClickReal for financial support.
reacted with an activated BCN31 carbonate to give the tri-OEG6
scaffolds 17 and 19 bearing two or three stained alkyne moi-
eties in 57% and 67% yields, respectively. Amine 9b was then
reacted with cyanuric chloride to afford the homo hexa-func-
tional OEG platform 20 in 76% yield (Scheme 4). Subsequent
N-Boc deprotection delivered the hexa-OEG6 platform 21,
bearing six strained alkyne moieties, in a good 71% yield.
In order to obtain hetero-tetra-functionalized derivatives,
double addition of bis-OEG amine on cyanuric chloride was
performed by controlling temperature conditions (Scheme 5).
Cyanuric chloride was thus disubstituted at room temperature
with either bis-azide OEG 14 or bis-NHBoc-OEG 9a to yield
tetra-azide-OEG 22 or tetra-NHBoc-OEG 24, respectively, in 73
and 66% yields. These tetra-OEG platforms were then sub-
jected to a third nucleophilic substitution at a higher tempera-
ture (80 °C). The introduction of ethylene diamine into 22
afforded the tetra-azide-OEG amine 23 in 79% yield. On the
other hand, OEG-platform 24 was reacted with β-alanine
methyl ester, giving access to the tetra-NHBoc-OEG-methyl
ester 25 in 85% yield. After N-Boc deprotection, the resulting
free amines were coupled to activated BCN derivatives to
deliver tetra-BCN-OEG-methyl ester 26 in 70% yield.
Interestingly, the use of our new bis-functional OEG amine
instead of the classical heterobifunctional OEG enabled the
doubling of the density of the reactive groups per generation.
These hetero-functionalized OEG platforms are suitable for
both click reaction and peptidic coupling and could find
various applications in bioconjugation and surface functionali-
zation processes.
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