Synthesis of non-natural sequence-encoded polymers using phosphoramidite chemistry

Article abstract of DOI:10.1021/jacs.5b02639

Sequence-defined non-natural polyphosphates were prepared using iterative phosphoramidite protocols on a polystyrene solid support. Three monomers were used in this work: 2-cyanoethyl (3-dimethoxytrityloxy-propyl) diisopropylphosphoramidite (0), 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dimethyl-propyl) diisopropylphosphoramidite (1), and 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dipropargyl-propyl) diisopropylphosphoramidite (1′). Phosphoramidite coupling steps allowed rapid synthesis of homopolymers and copolymers. In particular, the comonomers (0, 1), (0, 1′), and (1, 1′) were used to synthesize sequence-encoded copolymers. It was found that long encoded sequences could be easily built using phosphoramidite chemistry. ESI-HRMS, MALDI-HRMS, NMR, and size exclusion chromatography analyses indicated the formation of monodisperse polymers with controlled comonomer sequences. The polymers obtained with the comonomers (0, 1′) and (1, 1′) were also modified by copper-catalyzed azide-alkyne cycloaddition with a model azide compound, namely 11-azido-3,6,9-trioxaundecan-1-amine. ¹H and ¹³C NMR analysis evidenced quantitative modification of the alkyne side-chains of the monodisperse copolymers. Thus, the molecular structure of the coding monomer units can be easily varied after polymerization. Altogether, the present results open up interesting avenues for the design of information-containing macromolecules.

Full text of DOI:10.1021/jacs.5b02639

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Article
Synthesis of Non-natural Sequence-encoded
Polymers Using Phosphoramidite Chemistry
Abdelaziz Al Ouahabi, Laurence Charles, and Jean-Francois Lutz
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b02639 • Publication Date (Web): 08 Apr 2015
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Synthesis of Non-natural Sequence-encoded Polymers Using
Phosphoramidite Chemistry
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Abdelaziz Al Ouahabi,1 Laurence Charles,2 Jean-François Lutz1*
1
Precision Macromolecular Chemistry, Institut Charles Sadron, UPR-22 CNRS, BP 84047, 23 rue du Loess, 67034 Stras-
bourg Cedex 2, France. 2 Aix-Marseille Université, CNRS, Institute of Radical Chemistry UMR 7273, Marseille, France.
ABSTRACT: Sequence-defined non-natural polyphosphates were prepared using iterative phosphoramidite protocols on a polysty-
rene solid support. Three monomers were used in this work: 2-cyanoethyl (3-dimethoxytrityloxy-propyl) diisopropylphospho-
ramidite (0), 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dimethyl-propyl) diisopropylphosphoramidite (1) and 2-cyanoethyl (3-
dimethoxytrityloxy-2,2-dipropargyl-propyl) diisopropylphosphoramidite (1’). Phoshoramidite coupling steps allowed rapid synthe-
sis of homopolymers and copolymers. In particular, the comonomers (0, 1), (0, 1’) and (1, 1’) were used to synthesize sequence-
encoded copolymers. It was found that long encoded sequences could be easily built using phosphoramidite chemistry. ESI-HRMS,
MALDI-HRMS, NMR and size exclusion chromatography analyses indicated the formation of monodisperse polymers with con-
trolled comonomer sequences. The polymers obtained with the comonomers (0, 1’) and (1, 1’) were also modified by copper-
catalyzed azide-alkyne cycloaddition with a model azide compound, namely 11-azido-3,6,9-trioxaundecan-1-amine. H and 13C
1
NMR analysis evidenced quantitative modification of the alkyne side-chains of the monodisperse copolymers. Thus, the molecular
structure of the coding monomer units can be easily varied after polymerization. Altogether, the present results open up interesting
avenues for the design of information-containing macromolecules.
is obtained by reaction of a phosphoramidite with a hydroxy
group, followed by the oxidation of the resulting phosphite
into a phosphate. Using AB-type monomers containing a
phosphoramidite function and a dimethoxytrityl (DMT)-
protected hydroxy function, a controlled multi-step growth
polymerization process can be executed on a solid-support.
The iterative synthesis involves typically a three-step cycle:
phosphoramidite coupling, phosphite oxidation and DMT
deprotection. Despite these steps and the use of protecting
groups, this chemistry is extremely fast and efficient because it
has been optimized by decades of biochemistry.35 Typically,
near-quantitative yields can be obtained for each cycle within
a few minutes. With the help of automated synthesizers, it is
nowadays possible to synthesize sequence-defined polynu-
cleotides containing more than a hundred residues using this
approach.36,37 Thus, phosphoramidite coupling seems very
appealing for the preparation of other polymers than nucleic
acids. A wide variety of non-nucleoside phosphoramidite
monomers has been reported in the literature. However, these
monomers are used, in general, for oligonucleotide modifica-
tion rather than for preparing synthetic polymers.38-43 Ganesan
and coworker have reported the synthesis of peptidomimetic
polyphosphates using phosphoramidite chemistry.44 Häner and
coworkers have described the solid-phase phosphoramidite
synthesis of oligopyrenotides.45,46 These authors have also
studied in details the folding and self-assembly properties of
these non-natural oligophosphates. In a recent publication,
Sleiman and coworkers have described the synthesis of poly-
mer-DNA bio-hybrids using phosphoramidite chemistry.47 In
particular, alternating, periodic and block amphiphilic seg-
ments were prepared in this work. More recently, the same
group has described the relevance of these bio-hybrid struc-
tures for the precise construction of 3D assemblies.48
INTRODUCTION
Sequence-controlled polymers have recently gained increas-
ing attention in chemical sciences.1,2 Indeed, the precise ad-
justment of monomer units in polymer chains opens up inter-
esting perspectives for controlling molecular, supramolecular
and macroscopic properties of polymer materials.3-10 Thus,
many new concepts for monomer sequence-regulation have
been introduced during the last few years.11-13 For example,
strategies for controlling comonomer sequences in classical
polymerization approaches such as chain-growth14-19 and step-
growth20 polymerizations have been described. Yet, polymers
prepared using these strategies exhibit chain-length polydis-
persity.21 In order to design polymers with perfectly-controlled
primary structures, other polymerization tools have to be de-
veloped.22,23 For instance, solid-phase iterative chemistry,
which is a multistep-growth polymerization process, allows
design of monodisperse sequence-defined polymers.1 In such
strategies, monomers are sequentially attached one-by-one to a
growing chain, which is immobilized on a crosslinked or solu-
ble support. These methods are efficient but are limited by the
yields and duration of the coupling steps. Thus, various ap-
proaches have been recently proposed to simplify multistep
growth processes.24-30 In addition, biochemical methodologies,
which have been developed and optimized for bio-oligomer
synthesis, can be adapted for the preparation of non-natural
sequence-defined polymers.1 For example, Fmoc-based proto-
cols that were initially introduced for solid-phase peptide
chemistry have been extensively studied for the preparation of
synthetic oligomers.31-33 On the other hand, the phospho-
ramidite coupling reaction used in oligonucleotide synthesis
has been barely tested in synthetic polymer science.34 This
method has, however, valuable advantages for preparing se-
quence-defined polymers. In this strategy, a phosphate linkage
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In the present work, phosphoramidite chemistry was used was obtained using a dry solvent station GT S100. The 3 %
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for the preparation of non-natural sequence-defined polyphos-
phates (Figure 1). In particular, this methodology was investi-
gated for the synthesis of information-containing macromole-
cules.49 In recent publications,50 our group has shown that
digital information can be encrypted in synthetic polymers
using monomers intentionally defined as 0 and 1 bit. In addi-
tion, analytical methods for reading sequence-encoded infor-
mation have been described.51,52 Thus, this novel type of mac-
romolecules opens up interesting perspectives for applications
in the fields of data storage and molecular identification.49 As
shown in Figure 1, three phosphoramidite monomers were
prepared in this work. With the exception of phosphoramidite
and DMT-protected hydroxy groups, it is important to note
that these monomers do not share any structural feature with
phosphoramidite nucleosides. In order to implement a molecu-
lar code in the polymer chains, two monomers containing a
propyl (0) and a 2,2-dimethyl-propyl (1) spacer were synthe-
sized. Additionally, a monomer containing a 2,2-dipropargyl-
propyl spacer was designed (1’). Such a monomer allows post-
polymerization modification of the polymers using different
chemistries, e.g. azide-alkyne Huisgen cycloaddition, So-
nogashira or Castro-Stephens coupling.53,54
trichloroacetic acid solution in dichloromethane has been
prepared from trichloroacetic acid purchased from Sigma-
Aldrich. 11-Azido-3,6,9-trioxaundecan-1-amine was synthe-
sized as previously reported.50 The synthesis of d-1’ was
adapted from the literature.55 D-0 and 0 were synthesized
according to reported procedures.56 All air-sensitive reactions
have been carried out under argon atmosphere. The phospho-
ramidite compounds have been kept in the freezer at -18°C.
Oligomer syntheses were performed in an equipped ar-
gon/vacuum solid phase extraction glass tube (12 mL with frit
3) and stirred with an IKA HS 260 Basic shaker.
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Instrumentation. H NMR spectra were recorded (in D2O
for polymers or in Acetone and CDCl3 for monomers) on a
Bruker Avance 400 MHz spectrometer. 13C NMR spectra were
recorded at 100.6 MHz and 31P NMR spectra were recorded at
161.92 MHz and were externally referenced to 85% phosphor-
ic acid. Electrospray ionization mass spectrometry (ESI-MS)
experiments were performed on a QStar Elite mass spectrome-
ter (Applied Biosystems SCIEX, Concord, ON, Canada) oper-
ated in the positive mode. The capillary voltage was set at
+5500 V and the cone voltage at +75 V. Samples were diluted
in a methanolic solution of ammonium acetate (3 mM) and
injected into the ESI source at a 5 µL.min-1 flow rate using a
seringe pump. Matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS) experiments were performed on a
Synapt G2 HDMS mass spectrometer (Waters, Manchester,
UK) equipped with a laser emitting at 355 nm. MALDI sam-
ples were prepared by mixing the matrix (2,5-
dihydroxybenzoic acid in methanol) and the analyte (in meth-
anol containing 3 mM ammonium acetate) in a 1000:1 matrix-
to-analyte molar ratio. A 1 µL aliquot of this binary mixture
was deposited on the sample plate and allowed to air dry. Both
instruments allowed accurate mass measurements thanks to
the high resolution capability of the orthogonal acceleration
time-of- flight (oa-TOF) mass analyzer operated in the reflec-
tron mode. Internal calibration of the oa-TOF was performed
with poly(ethylene oxide) oligomers adducted with ammoni-
um in ESI-MS experiments and with red phosphorus aggre-
gates in MALDI-MS experiments. The number-average molar
mass (Mn) and the polydispersity index (Mw/Mn=PDI) were
determined using a DIONEX HPLC system, Ultimate 3000
(degasser, pump, autosampler) equipped with 4 Shodex OH-
pak columns 30 cm (802.5HQ, 804HQ, 806HQ, 807HQ), a
guard column (separation range: 300 to 100 000 000 g·mol-1),
1 diode array UV detector (Dionex), 1 differential refractome-
ter OPTILAB rEX (Wyatt Techn.) and 1 light scattering detec-
tor DAWN HELEOS II (Wyatt Techn.). The measurements
were performed at 30°C in water containing 40% acetonitrile
(ACN) and 0.1M of NaNO3 at a flow rate of 0.5 mL·min-1.
Figure 1. General strategy for the synthesis of sequence-defined
non-natural polyphosphates. (a) Molecular structures of the
comonomers. (b) Iterative polymer synthesis on a solid-support
(red bead). Experimental conditions: (i) DMT deprotection: CCl3-
COOH, CH2Cl2; (ii) coupling step: RT, AcCN, tetrazole; (iii)
oxidation: RT, I2, H2O/Pyridine/THF; (iv) cyanoethyl deprotec-
tion: piperidine, AcCN; (v) cleavage: NH3, H2O, dioxane.
METHODS
General. 2-Cyanoethyl diisopropylchlorophosphoramidite
(95%, Alfa Aesar), 4,4′-dimethoxytriphenylmethyl chloride
Synthesis of monomers. Synthesis of D-1. 2,2-
dimethyl-1,3-propandiol d-1 (0.7 g, 6.7 mmol) was co-
evaporated with 5 mL of anhydrous pyridine in order to elimi-
nate residual water present in the diol. Afterwards, 8 mL of
pyridine and 12 mL of anhydrous THF were introduced and
the diol was reacted with 4,4-dimethoxytrityl chloride
(DMTCl) (2.27 g, 6.7 mmol). The DMTCl was added in three
equal portions at a rate of one portion every hour. After the
three additions, the mixture was stirred at room temperature
for two hours. The reaction was stopped with the addition of 5
(≥97.0%,
Sigma-Aldrich),
N,N-diisopropylethylamine
(DIPEA, 99%, Alfa Aesar), propargyl bromide (solution 80%
w/w in toluene, Alfa Aesar), tetrakis(acetonitrile)copper(I)
tetrafluoroborate
(97%,
Sigma-Aldrich),
4-
(dimethylamino)pyridine (DMAP, 99%, Sigma-Aldrich),
N,N'-dicyclohexylcarbodiimide, (99%, DCC, Alfa Aesar),
aminopolystyrene resin (1.4 mmol.g-1, Novabiochem) were
used as purchased. Anhydrous dichloromethane, pyridine and
acetonitrile were purchased from Aldrich. Anhydrous THF
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mL of methanol and the mixture was evaporated to dryness. mL of anhydrous pyridine at 35°C for 5h. The reaction was
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The residue was dissolved in ethyl acetate (30 mL) and
washed with 5% sodium bicarbonate ice-cold solution. The
aqueous layer was extracted with 30 mL of ethyl acetate. The
combined organic layers were washed with water and brine,
dried with anhydrous Na2SO4 and evaporated. The resulting
product was chromatographed on silica gel (20% ethyl acetate
in cyclohexane with a 1% triethylamine) yielding 2.3 g of D-1
(85%) as a colorless oil. H-NMR [(CD3)2CO, δ, ppm]: 7.50-
7.20 (m, 9H), 6.88-86 (m, 4H), 3.77(s, 6H), 3.43-3.82 (m, 2H),
2.94 (s,2H), 0.91 (m, 6H)
quenched with methanol and the solvent was concentrated.
The resulting oil was diluted with ethyl acetate (20 mL) and
washed with water, 0.1 M sodium phosphate (pH 5.0), and
again water. The organic phase was dried with MgSO4 and the
solvent was evaporated to dryness to give colorless oil.
Resin modification. The succinate h-0, h-1 or h-1’ (0.73
mmol) was reacted with 0.4 g of amino-polystyrene resin (1.4
mmol.g-1), 0.26 mmol of DMAP and 2.26 mmol of DCC in
5mL of anhydrous dichloromethane following a previously-
reported procedure.57 The unreacted amino groups were
capped with acetic anhydride in pyridine (1:5 v/v). The load-
ing of the support was measured by the removal of the DMT
group of an aliquot and analyzing its absorbance at 504 nm to
find a loading of about 0.8 mmol·g-1.
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Synthesis of D-1’. 2,2-dipropargyl-1,3-propandiol d-1’
(1.5 g, 9.8 mmol) was reacted with four portions of DMTCl (4
x 0.83 g, 9.8 mmol) in 15 mL of pyridine and 30 ml of THF as
described above. The reaction was stirred for 40 min between
each addition. The resulting product was chromatographed on
silica gel (20% ethyl acetate in cyclohexane with a 1% tri-
ethylamine) yielding 4.19 g of D-1’ (94%) as a colorless oil.
1H-NMR [(CD3)2CO, δ, ppm]: 7.49-7.20 (m, 9H), 6.88-86 (m,
4H), 3.77 (s, 6H), 3.63 (s, 2H), 3.20 (s, 2H), 2.40-2.39 (d,4H),
2.30 (t.2H)
Solid-phase synthesis of the oligomers. The couplings
were carried out under vacuum-argon in rigorously dry condi-
tions using a home-made peptide synthesis reaction vessel
equipped with a fritted glass plate and a stopcock. The general
procedure is the following one: 1 molar equivalent of polysty-
rene support R-0 or R-1’ (0.8 mmol·g-1) was introduced in the
reaction vessel, treated with a 3% trichloroacetic acid solution
in dichloromethane and washed with a solution of 2% pyridine
in acetonitrile and again with pure acetonitrile. After a fast
swelling in dichloromethane, the appropriate phosphoramidite
(1.5 eq) dissolved in anhydrous acetonitrile (0.1 M) and te-
trazole (4 eq, 0.45 M solution in acetonitrile) were added
under argon atmosphere. The reactor was shaken at room
temperature for 15 minutes. Afterwards, the solution was
removed and the polystyrene support was washed with ace-
tonitrile and the resulting phosphite-triester was oxidized
during 2 minutes to phosphate-triester with a 0.1 M iodine
solution (I2 in water/pyridine/THF 2/20/80). The excess of
iodine was removed by acetonitrile and dichloromethane
washes. The three steps (i.e. DMT-deprotection, phospho-
ramidite coupling and iodine oxidation) were iteratively re-
peated until the formation of the desired polymer. Finally,
deprotection of the cyanoethyl phosphate protecting group was
performed using a 10% piperidine solution in acetonitrile (5
min), the terminal DMT-protecting group was deprotected
using the 3% trichloroacetic acid solution and the polymers
were cleaved from the resin using 30% aqueous ammonia
solution/dioxane (1:1 v/v) during 12 h at 45ºC. The ammonia
mixture was filtered and the flask was left for 5 min under
argon bubbling in order to eliminate the excess of gaseous
ammonia. After solvent evaporation and drying under vacuum,
Synthesis of 1. D-1 (190 mg, 0.31 mmol) was co-
evaporated with 8 mL of anhydrous dichloromethane and
dried under vacuum over 30 min then 0.1 g of molecular
sieves 3Å were added. 10 mL of anhydrous dichloromethane
and DIPEA (0.33 ml, 1.8 mmol) was added successively under
argon and the system was cooled with an ice-water bath. O-2-
cyanoethyl-N,N-diisopropylchlorophosphor-amidite (0.20 ml,
0.9 mmol) was added dropwise with continuous stirring. The
reaction flask was then allowed to warm to room temperature
and stirred for 1 hr. The mixture was evaporated to dryness
and dissolved in ethyl acetate then chromatographed directly
on silica gel using hexane / ethyl acetate (3:2) + 1% triethyla-
mine as eluent yielding 210 mg of monomer 1 (82%) as a
foam. 31P-NMR [CDCl3, δ, ppm]: 148.4. 1H-NMR [(CD3)2CO,
δ, ppm]: 7.49-7.17 (m, 9H), 6.86-6.89 (m, 4H), 3.78(s, 6H),
3.75-3.77 (m, 2H), 3.57-3.61 (m,3H), 3.43-3.47 (m,1H), 2.91-
2.98 (m, 2H), 2.67 (t, 2H), 1.13-1.19 (dd, 12H), 0.93-0.96-1.19
(d, 6H). 13C-NMR [(CD3)2CO, δ, ppm]: 159.51, 146.63,
137.29, 131.06, 129.12, 128.46, 127.39, 118.91, 113.76,
86.31, 70.65, 69.09, 59.43, 55.48, 43.79, 37.56, 27.53, 24.95,
20.85
Synthesis of 1’. D-1’ (4.47 g, 9.8 mmol) was reacted with
O-2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite (2.5
g, 10 mmol) and DIPEA (11 ml, 60 mmol) as described above.
The resulting product was purified by chromatography on
silica gel (hexane / ethyl acetate (7:3) + 1% triethylamine)
yielding 5.4 g of monomer 1’ (84%) as a colorless liquid-
foam. 31P-NMR [CDCl3, δ, ppm]: 147.4. 1H-NMR [(CD3)2CO,
δ, ppm]: 7.19-7.51 (m, 9H), 6.87-6.89 (m, 4H), 3.78(s, 6H),
3.75-3.77 (m, 2H), 3.59-3.67 (m,3H), 3.43-3.47 (m,1H),
2.69(t, 2H),, 2.35-2.46 (m, 2H), 1.13-1.19 (dd, 12H). 13C-
NMR [(CD3)2CO, δ, ppm]: 159.60, 146.31, 136.94, 131.13,
129.13, 128.52, 127.48, 118.87, 113.83, 86.81, 81.09, 72.59,
65.62, 64.28, 59.47, 55.50, 43.87, 43.26, 27.53, 24.97, 20.84.
1
the resulting products were characterized by NMR H, 13C,
31P, SEC and ESI-MS.
CuAAC functionalization of the polymers. Functionali-
zation of sequence-encoded oligomer 1’1’1’1’1’1’1’1’ in
solution. The sequence-encoded oligomer 1’1’1’1’1’1’1’1’
(16 ꢀmol) was mixed with 11-azido-3,6,9-trioxaundecan-1-
amine (0.5 mmol, 32 eq.) in 4 mL of water under argon. A 2
mL
freshly
prepared
solution
containing
Cu(MeCN)4,BF4/ligand (THPTA) (1:1) in a mixture of water
and acetonitrile (1:1, 3.2 eq.) was then introduced.58,59 The
mixture was stirred at room temperature overnight. The reac-
tion was diluted with water (10 mL) and dialyzed.
Resin functionalization. Preparation of succinates h-0,
h-1 and h-1’. In a 25 mL round bottom flask, D-0, D-1 and
D-1’ (1mmol) were co-evaporated with 4 mL of pyridine,
dried under vacuum for 30 min and reacted with 1.5 equiva-
lents of succinic anhydride and 1.5 equivalents of DMAP in 4
Functionalization of sequence-encoded oligomer
1’11’111’11’ on the resin. To a polystyrene-resin loaded
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with the deprotected sequence-encoded oligomer 1’11’111’11’ sulting hemisuccinyl esters h-0, h-1 and h-1’were afterwards
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(0.03 mmol) were added (under argon) 11-azido-3,6,9-
trioxaundecan-1-amine (0.5 mmol, 16 eq.), 4 mL of water and
1 mL of the previously described solution containing
coupled to amino-functional polystyrene beads to generate the
corresponding DMT-functionalized resins R-0, R-1 and R-1’.
The formed succinyl linker can be easily cleaved under basic
conditions, e.g. using an aqueous ammonia solution. After
modification, a typical resin loading of about 0.8 mmol·g-1
was obtained. The successful preparation of the three different
types of resins R-0, R-1 and R-1’ imply that coded sequences
can be started by any of the three monomers 0, 1 and 1’.
Cu(MeCN)4, BF4, (THPTA), water and acetonitrile. The mix-
ture was shaken at room temperature overnight. The solution
was removed by filtration and the resin was washed with ace-
tonitrile, water and acetone. Finally, the resulting polymer was
cleaved from the resin using 30% aqueous ammonia/dioxane
(1:1 v/v) during 12 h at 45ºC. The ammonia mixture was fil-
tered and the flask was left for 5 min under argon bubbling in
order to eliminate the excess of gaseous ammonia. After evap-
oration of the solvents and drying under vacuum, the resulting
products were characterized by NMR 1H, 13C, 31P and SEC.
Scheme 2. Resin functionalization (R = H or CH2-C≡C). (i)
Succinic anhydride, DMAP, pyridine; (ii) DCC, CH2Cl2.
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RESULTS AND DISCUSSION
The synthesis of the phosphoramidite monomers is shown in
Scheme 1. They were obtained in two steps from the corre-
sponding 1,3-diol d-0, d-1 and d-1’. First, the 1,3-propanediol
derivatives were monoprotected by dimethoxytrityl (DMT).
The reaction with one molar equivalent of dimethoxy-
tritylchloride (DMTrCl) in the presence of pyridine yielded the
DMT-derivatives D-0, D-1 and D-1’ in relatively high yield.
DMTrCl was added gradually in order to minimize dimer
formation. The resulting DMT-derivatives were reacted with
chloro-N,N-diisopropylamino-O-2-cyanoethoxyphosphine in
anhydrous conditions to yield the corresponding phospho-
ramidites. The use of molecular sieves in this step led to quan-
titative yields. The synthesis of monomer 1’ required first the
preparation of diethyldipropargylmalonate followed by its
reduction using LiAlH4 to afford the 2,2-dipropargylpropan-
1,3-diol derivative, which was afterwards converted into 1’ as
described above.
In order to test the viability of our approach, model homo-
polymers of the monomer 0 were first prepared on the modi-
fied resin R-0 (samples H1-H3 in Table 1). These polymers
were prepared following the experimental conditions shown in
Figure 1. The resin was first detritylated using a solution of
trichloroacetic acid in dichloromethane, washed and swelled.
Afterwards, coupling with 0 was performed using the com-
mercial activator 1H-tetrazole. The formed phosphite bond
was then oxidized to phosphate by adding a 0.1M iodine solu-
tion. Detrytilation/coupling/oxidation cycles were repeated
until a desired chain-length was reached. After the multi-step
growth process, the 2-cyanoethyl phosphate protecting groups
were removed by treatment with a piperidine/acetonitrile
solution. Finally, the terminal DMT-protecting group was
removed and the polymers were cleaved from the resin using
an ammonia solution. It should be noted that this final proce-
dure employing three steps is not standard. In conventional
oligonucleotide chemistry, two steps are usually performed:
the DMT group is first removed and afterwards both phos-
phate-deprotection and cleavage are performed in a single step
in the presence of ammonia. However, the final polymer has
usually to be purified by HPLC. The three-step strategy shown
in Figure 1, in which cyanoethyl groups are removed before
polymer cleavage, allow an easier purification of the polymer
while it is still attached to the resin.
Scheme 1. Synthesis of the phosphoramidite monomers 0 (R =
H) 1 (R = CH3) and 1’ (R = CH2-C≡C). (i) DMTrCl, DIPEA,
CH2Cl2; (ii) (iPr)2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2.
The phosphoramidite monomers 0, 1 and 1’ were then in-
vestigated for the synthesis of sequence-defined copolymers
on a solid-support. Phosphoramidite oligonucleotide chemistry
is often performed on porous glass beads.60,61 However, such
solid supports do not swell in the reaction solvent and large
pore sizes are required to synthesize long sequence-defined
polynucleotides. As mentioned above, the non-natural mono-
mers 0, 1 and 1’ have little in common with phosphoramidite
nucleosides. Thus, more conventional cross-linked polystyrene
supports were used to synthesize the polymers in the present
work. These hydrophobic supports are useful for moisture-
sensitive phosphoramidite chemistry. The functionalization of
the polystyrene solid-supports is shown in Scheme 2. The
DMT-monoprotected hydroxy-functional precursors D-0, D-1
and D-1’ were first reacted with succinic anhydride. The re-
Figure 2. Characterization of homopolymers of 0. (a) Size exclu-
sion chromatography recorded in ACN/H20 for homopolymers of
different chain length (Table 1, Entries H1-H3). (b) ESI mass
spectrum recorded for the homopolymer H1.
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Table 1. Characterization of polyphosphodiester homopolymers (H) and copolymers (C) synthesized by solid-phase chemistry.
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b
m/zexp
Sequence
Yield [%]a
Mn [g·mol-1]b
Mw/Mn
m/zth
H1
H2
00000
≥ 98
650
1.04
629.0925 [M + H]+
629.0916c [M + H]+
629.0923d [M + H]+
522.0610c [M + 2H]2+
1043.1155d [M + H]+
2147.1779d [M + H]+
685.1564c [M + H]+
685.1544d [M + H]+
522.0622 [M + 2H]2+
1043.1171 [M + H]+
2147.1827 [M + H]+
685.1551 [M + H]+
00000000
≥ 98
1040
1.03
H3
C1
0000000000000000
01010
≥ 95
≥ 98
1850
730
1.01
1.05
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2315.3705 [M + H]+
2259.3079 [M + H]+
3363.3734 [M + H]+
826.1874 [M + 2H]2+
1651.3675 [M + H]+
674.1248 [M + 2H]2+
1347.2428 [M + H]+
730.1874 [M + 2H]2+
e
C2
C3
C4
0100101001010010
≥ 95
≥ 98
≥ 87
≥ 98
2000
1850
2950
1.07
1.07
1.08
-
0100100010000100
2259.3050d [M + H]+
e
001000100001000001000000
-
f
f
H1’ 1’1’1’1’1’1’1’1’
C1’ 1’01’001’01’
C2’ 1’11’111’11’
-
-
826.1861c [M + 2H]2+
1651.3652d [M + H]+
674.1248c [M + 2H]2+
1347.2392d [M + H]+
730.1863c [M + 2H]2+
≥ 98
≥ 98
-
-
1799
1.02
a Percentage of the total isolated polymer; b Measured by size-exclusion chromatography in H2O/ACN; Measured by ESI- HR-MS.
Measured by MALDI-HR-MS; e not detected. f This polymer precipitates in the SEC solvent mixture.
c
d
The formed homopolymers were characterized by elec-
trospray ionization high resolution mass spectrometry (ESI-
HR-MS), matrix-assisted laser desorption/ionisation high
resolution mass spectrometry (MALDI-HR-MS), size exclu-
sion chromatography (SEC) in water/acetonitrile, as well as 1H
and 13C NMR. 31P NMR was also used to follow the oxidation
of the trivalent phosphorous to the pentavalent state and to
monitor phosphate deprotection. Figure 2 shows SEC and ESI-
MS data measured for different homopolymers. All these
analyses account for the formation of monodisperse non-
natural polyphosphodiesters. SEC chromatograms suggest an
excellent control of chain-length on resin R-0 (Figure 2a). The
number average molecular weights Mn and the molecular
weight distribution Mw/Mn of the different homopolymers are
listed in Table 1. In all cases, the measured experimental Mn
values were found to be in very good agreement with the
theoretical values. Moreover, monomodal peaks and narrow
molecular weight distributions were observed for all samples.
It should be however noted that monodisperse samples never
lead to polydispersity index values of 1 in SEC. Due to axial
dispersion and other peak broadening effects, apparent values
above 1 are usually observed.62 This aspect is particularly true
in the case of charged polyelectrolytes that are never trivial to
analyze by SEC. This assumption was confirmed by MS
measurements, which showed the formation of monodisperse
polymers with defined molecular weights. Accurate mass
measurements were performed using ammonium acetate to
promote the formation of protonated molecules. Although both
ESI-MS and MALDI-MS allowed homopolymer detection,
ESI-MS led to cleaner spectra (Figure 2b). Indeed, MALDI-
MS measurements of these polyacids were complicated by
proton exchanges with sodium impurities coming from the
matrix (Figure S1). The quality of MS analysis also depended
on the chain-length of the analyzed polymers. For example,
H3 was detected by MALDI-MS but not by ESI-MS. Never-
theless, the expected species were observed in all cases. The
formation of pure homopolymers was further confirmed by
NMR analysis (Figure S2). It should be specified that all pol-
ymers were synthesized without capping step and using only
1.5 Eq. of phosphoramidites in coupling steps. These results
clearly show the efficiency of phosphoramidite chemistry for
preparing non-natural polymers.
Afterwards, the synthesis of coded polymers using comon-
omers (0, 1) was investigated on R-0. Table 1 shows the char-
acterization of these copolymers (samples C1-C4). At first, a
model alternating pentamer C1 was prepared in order to opti-
mize all iterative steps with comonomers 0 and 1. SEC (Table
1 and Figure S3), ESI-HR-MS (Table 1), MALDI-HR-MS
(Table 1), and NMR (Figure S4) confirmed the formation of a
monodisperse sequence-defined oligomer. Based on these
encouraging results, longer sequences were prepared on R-0.
Copolymers C2 and C3 have a similar chain-length (i.e. 16
monomer units) but contain different sequences. SEC analysis
(Table 1) evidenced the formation of polymers of controlled
molecular weight. As expected, sample C2 that contains 6
units of 1 eluted at lower elution volume than C3, which con-
tains only 4 (Figure S2). This controlled comonomer composi-
tion was further confirmed by the integration of methyl groups
1
signals in H NMR (Figure S5). The synthesis of a coded 24-
mer C4 was also achieved in this work (Table 1 and Figure
S6). This particular chain-length is certainly not the upper
limit of our approach. Since all polymers shown in this work
were prepared manually, longer sequences were not investi-
gated. Nevertheless, based on existing literature knowledge
about sequence-defined oligonucleotides,36,37 it is almost cer-
tain that longer non-natural sequences are achievable.
The synthesis of copolymers using comonomers (0, 1’) was
then studied. As mentioned in the introduction, monomer 1’ is
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interesting because it allows synthesis of homopolymers and sequence-encoded polyphosphates. This approach allowed
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copolymers that can be post-modified after polymerization.
Thus, it enables to bypass the use of complex functional mon-
omers. A homopolymer of 1’ (sample H1’ in Table 1) and a
coded copolymer of 1’ and 0 (sample C1’ in Table 1) were
prepared on resin R-1’. The presence of terminal alkynes in
the final polymers was confirmed by 1H and 13C NMR analysis
(Figure S7). In addition, ESI-MS analysis confirmed for-
mation of the expected macromolecules. The modification of
polymers H1’ and C1’ by copper-catalyzed azide-alkyne
cycloaddition (CuAAC) was then investigated. 11-azido-3,6,9-
trioxaundecan-1-amine was used as a model organic azide in
these reactions. This model compound is interesting because it
allows synthesis of modified polyelectrolytes bearing hydro-
philic oligo(ethylene glycol) side chains terminated by prima-
ry amine groups. CuAAC was performed in water in the pres-
ence of Cu(MeCN)4,BF4 and THPTA.58,59 For both H1’ and
C1’, a quantitative conversion of the terminal alkyne functions
was evidenced by 1H and 13C NMR (Figure S7). In 13C NMR,
the alkyne peaks at 72 ppm fully vanished after CuAAC, and
were replaced by two new peaks at 125 ppm and 143 ppm that
correspond to the secondary and tertiary carbon of the newly
formed triazole ring, respectively. Quantitative polymer modi-
synthesis of near-monodisperse homopolymers and sequence-
1
encoded copolymers. SEC, ESI-MS and H, 13C and 31P NMR
confirmed the formation of macromolecules with precisely
controlled chain-lengths (i.e. from pentamer to 24-mer) and
primary structures. It was also shown that these polymers can
be modified after their synthesis. Using a phosphoramidite
monomer bearing terminal alkyne functions, it was possible to
prepare coded macromolecules that can be modified by Cu-
AAC. As a proof of concept, the PEGylation of sequence-
defined polyphosphates was reported.
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These results indicate that phosphoramidite chemistry is a
valid option for the preparation of synthetic polymers. Very
interestingly, experimental protocols (e.g. HPLC purification,
automated synthesis) that have been optimized among past
years for the synthesis of oligonucleotides can also be utilized
for the synthesis of non-natural monodisperse polymers. Thus,
it is very realistic to imagine that long sequence-defined pol-
ymers can be synthesized using phosphoramidite chemistry,
although slightly different experimental conditions than those
studied herein (e.g. higher monomer excess, capping steps)
might be required for reaching longer chain-lengths.
In addition, the coded primary structure of these new poly-
phosphates can be most probably analyzed using DNA se-
quencing technologies. For instance, such water-soluble non-
natural macromolecules are interesting analytes for protein-
based nanopore sequencing.52 Since their side-chains can be
easily modified, their molecular structure can be possibly
adapted for optimal pore readability. Hence, the results of this
article open up interesting perspectives for the emerging field
of information-containing macromolecules.
1
fication was further confirmed by H NMR analysis. Indeed,
the peaks due to the alkyne protons at 2.44-2.46 ppm disap-
peared after CuAAC. In addition, new peaks due to the tria-
zole ring proton, to the methylene protons of the oli-
go(ethylene glycol) spacer in alpha of the triazole and to the
repeat units of oligo(ethylene glycol) appeared in the spectra
of the modified polymers at 7.99 ppm, 4.54-4.58 ppm and
3.60-3.63 ppm, respectively. Thus, NMR clearly confirmed
the PEG functionalization of H1’ and C1’. A change in the
physical appearance of these polymers was also observed, i.e.
varying from a solid white powder before functionalization to
a viscous oil after oligo(ethylene glycol) modification. The
CuAAC post-modification has been tested on the support R-1’
(i.e. after deprotection but before cleavage) and on a cleaved
deprotected polymer. In both cases, near-quantitative yields
were obtained. However, resin-supported modification al-
lowed a better removal of CuAAC catalyst and reactants.
a.
c
The monomer alphabets (0, 1) and (0, 1’) used in this paper
were, of course, arbitrarily chosen. The iterative synthesis and
the post-modification concept studied in the present work can
be potentially extended to other types of comonomers. For
instance, during the evaluation of this article, one reviewer
asked if the synthesis of copolymers based on comonomers (1,
1’) was feasible. Other comonomer combinations and se-
quences are indeed attainable. As a proof of concept, Table 1
shows the characterization of copolymer C2’ that contain the
sequence 1’11’111’11’. This copolymer was synthesized and
cleaved from resin R-1’. SEC and ESI-HR-MS confirmed the
formation of a monodisperse sequence-defined oligomer (Ta-
ble 1). Moreover, this copolymer could be easily post-
modified by CuAAC as described in the previous paragraph.
NMR analysis (Figure 3) indicated quantitative side-chain
modification.
fd
a b
ee'
g
hi
8
7
6
5
4
3
2
1
0
b.
fl
c
m
ee'
k
a
j
d
CONCLUSION
8
7
6
5
4
3
2
1
0
In summary, phosphoramidite chemistry was found to be a
rapid and convenient method for the synthesis of non-natural
ppm
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Figure 3. H NMR spectra recorded in D2O for copolymer C2’
before (a) and after (b) modification by copper-catalyzed azide-
alkyne cycloaddition with 11-azido-3,6,9-trioxaundecan-1-amine.
(17) Hibi, Y.; Ouchi, M.; Sawamoto, M. Angew. Chem., Int.
Ed. 2011, 50, 7434.
(18) Chan-Seng, D.; Zamfir, M.; Lutz, J.-F. Angew. Chem.,
Int. Ed. 2012, 51, 12254.
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ASSOCIATED CONTENT
Supporting Information. Supplementary Figures S1-S7. This
information is available free of charge via the Internet at
http://pubs.acs.org/.
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AUTHOR INFORMATION
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Corresponding Author
*Jean-François Lutz, E-mail: jflutz@unistra.fr
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The post-doctoral position of AAO was funded by the IDEX
program of the University of Strasbourg. The CNRS is also
acknowledged for financial support. The authors also thank Méla-
nie Legros and Catherine Foussat for the SEC measurements,
Laurence Oswald for the synthesis of 11-Azido-3,6,9-
trioxaundecan-1-amine and Jean-Marc Strub for some preliminary
ESI-MS characterization.
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Table of Contents artwork
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