Hexyl-modified morpholine-2,5-dione-based oligodepsipeptides with relatively low glass transition temperature

Article abstract of DOI:10.1016/j.polymer.2016.10.033

Oligodepsipeptides (oDPs), alternating copolymers of an α-amino acid and an α-hydroxy acid, are typically created by ring-opening polymerization (ROP) of morpholine-2,5-dione derivatives (MDs). In general, oDPs exhibit relatively high glass transition temperatures (T_gs) caused by the strong intermolecular H-bonding between amide and ester bonds. So far, it was not reported that variation at α-amino acid moieties in MDs monomers lead to lower T_g. Here we explored whether the thermal properties of the oDPs can be adjusted by introducing a hexyl side chain in the α-hydroxy acid part of the MDs. By synthesizing a MD with an atactic pendant hexyl group at position 3, the influence of a modification at position 6 compared to a modification at position 3 towards ROP was investigated. In both cases the atactic bulky side groups hindered the H-bonding between chain segments resulting in a significant reduction of the T_gs to a temperature around human body temperature (32 and 36 °C) in contrast to ROP of a MD providing a methyl group at position 3 and a T_g ≈ 65 °C. Such oDPs could be interesting candidate materials for biomedical applications such as degradable implants.

Full text of DOI:10.1016/j.polymer.2016.10.033

Accepted Manuscript
Hexyl-modified morpholine-2,5-diones based oligodepsipeptides with relatively low
glass transition temperature
Xingzhou Peng, Marc Behl, Pengfei Zhang, Magdalena Mazurek-Budzyńska,
Muhammad Yasar Razzaq, Andreas Lendlein
PII:
S0032-3861(16)30945-4
10.1016/j.polymer.2016.10.033
JPOL 19133
DOI:
Reference:
To appear in: Polymer
Received Date: 15 July 2016
Revised Date: 7 October 2016
Accepted Date: 13 October 2016
Please cite this article as: Peng X, Behl M, Zhang P, Mazurek-Budzyńska M, Razzaq MY, Lendlein
A, Hexyl-modified morpholine-2,5-diones based oligodepsipeptides with relatively low glass transition
temperature, Polymer (2016), doi: 10.1016/j.polymer.2016.10.033.
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ACCEPTED MANUSCRIPT
Graphical abstract for
Hexyl-modified morpholine-2,5-diones based oligodepsipeptides with relatively low glass
transition temperature
Xingzhou Peng, Marc Behl, Pengfei Zhang, Magdalena Mazurek-Budzynska, Muhammad Y.
Razzaq, Andeas Lendlein

ACCEPTED MANUSCRIPT
Hexyl-modified morpholine-2,5-diones based oligodepsipeptides with relatively low glass
transition temperature
Xingzhou Peng1,2,3, Marc Behl1,3, Pengfei Zhang1,2,3, Magdalena Mazurek-Budzyńska1,
Muhammad Yasar Razzaq1, Andreas Lendlein1,2,3*
1 Institute of Biomaterial Science and Berlin-Brandenburg Centre for Regenerative Therapies,
Helmholtz-Zentrum Geesthacht, 14513 Teltow, Germany
2 Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany
3Tianjin University-Helmholtz-Zentrum Geesthacht Joint Laboratory for Biomaterials and
Regenerative Medicine, 14513 Teltow, Germany
*Corresponding author. E-mail address: andreas.lendlein@hzg.de
Abstract
Oligodepsipeptides (oDPs), alternating copolymers of an α-amino acid and an α-hydroxy acid,
are typically created by ring-opening polymerization (ROP) of morpholine-2,5-dione derivatives
(MDs). In general, oDPs exhibit relatively high glass transition temperatures (Tgs) caused by the
strong intermolecular H-bonding between amide and ester bonds. So far, it was not investigated
that variation at α-amino acid moieties in MDs monomers did not lead to lower Tg. Here we
explored whether the thermal properties of the oDPs can be adjusted by introducing a hexyl side
chain in the α-hydroxy acid part of the MDs. By synthesizing a MD with an atactic pendant
hexyl group at position 3, the influence of a modification at position 6 compared to a
modification at position 3 towards ROP was investigated. In both cases the atactic bulky side
groups hindered the H-bonding between chain segments resulting in a significant reduction of
the Tgs to a temperature around human body temperature (32 and 36 °C) in contrast to ROP of a
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MD providing a methyl group at position 3 and a Tg ≈ 65 °C. Such oDPs could be interesting
candidate materials for biomedical applications such as degradable implants.
Introduction
Oligodepsipeptides (oDPs), known as alternating copolymers of an α-amino acid and an α-
hydroxy acid, have shown great promise for biomedical applications such as tissue engineering
and drug delivery systems [1-5] as the degradation of amide bonds in oDPs is inherently buffered
and by this results in lower immune system response in contrast to the degradation of polyester
based materials causing a substantial decrease of pH.
oDPs can be synthesized using ring-opening polymerization (ROP) of morpholine-2,5-dione
(MD) derivatives [6]. A variety of MDs with tunable physical and chemical properties were
synthesized by modification of the amino acid moiety, resulting in oDPs with promising
applications[7-10]. So far MDs were modified by substitutions at position 3 by varying the α-
amino acid moieties; e.g. valine, leucine, isoleucine, and alanine have been used to synthesize
isopropyl, isobutyl, sec-butyl and methyl substituted MDs [11, 12]. From these, oDPs with
controlled molecular weight and designed end functionalities were synthesized by ROP. The
mechanism of ROP catalyzed by tin(II) 2-ethylhexanoate (Sn(Oct)2), especially of lactones and
cyclic esters, was intensively studied and several theories of ROP mechanisms were discussed
[13, 14]. The most commonly accepted mechanism for Sn(Oct)2 catalyzed ROP is the
“coordination-insertion” mechanism, in which at first, a tin alkoxide, which is the active species
for the ROP, is formed from the Sn(Oct)2 and hydroxyl groups of initiator. Afterwards the tin
alkoxide forms a complex with the carbonyl oxygen of the ester group, resulting in a
nucleophilic attack of the acyl carbon. The ester bond cleavage goes along with the formation of
a new tin-oxygen bonds and results in analogue active species. Ester bonds in MDs have a higher
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activity compared to amide bond and act as main functional group during the ROP. In this
context, the “coordination-insertion” mechanism is also applicable for the ROP of MDs
(Scheme 1). Furthermore, the structure of MD influences the efficiency of ROP during the oDPs
synthesis. Recently, the synthesis and polymerization of versatile MDs have been reported,
presenting glass transition temperatures (Tgs) of oDPs ranging from 93 °C to 117 °C [11]. Such
high Tgs can be attributed to the strong H-bond interactions and high crystallinity in the
oligomers.
R1
R2
(3S,6S)-3-isopropyl-6-methyl-2,5-
morpholinedione
(3S)-isopropyl
(6S)-methyl
(6R,S)-6-methyl-2,5-morpholinedione
(3S)-3-methyl-2,5-morpholinedione
(3S)-3-isopropyl-2,5-morpholinedione
H
(6R,S)-methyl
(3S)-methyl
(3S)-isopropyl
H
H
Scheme 1. Coordination insertion mechanism of ROP of MDs catalyzed by Sn(Oct)2 [11, 15].
We hypothesized that introducing pendant bulky groups into the MD would lower the Tg. Our
strategy to achieve such a new class of MDs was to introduce a pendant alkyl group at the
position 6 by a variation of the α-hydroxy acid part of the MDs to explore the effect of such a
MD on the thermal properties of the final oligomer. Nevertheless, modifications at position 6
might influence the steric hindrance and decrease the efficiency of ROP [16]. Therefore, initial
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kinetic studies were performed to compare of the efficiency of the ROP of the two monomers
modified at position 3 and 6, named (3R,S)-hexylmorpholine-2,5-dione (3HMD) and (3S, 6R,S)-
3-methyl-6-hexylmorpholine-2,5-dione (3M6HMD). Furthermore, ROP of MMD was performed
as a reference ROP. The introduction of the atactic bulky side group would hinder the H-bond
interactions resulting in a less regular structure, reduced content of crystalline phase, and
lowered Tgs of oligo3M6HMD (o3M6HMD) and oligo3HMD (o3HMD). After the detailed
characterization of the synthesized MDs, the ROP kinetics were studied study, and thermal
properties of the resulting oDPs were explored.
Experimental
Materials
If not mentioned otherwise, catalyst and solvents were obtained from Sigma Aldrich (Seelze,
Germany) and used as received. 2-bromooctanoic acid (98%) was purchased from Th. Geyer
(Hamburg, Germany), thionyl chloride (97%) and L-alanine were obtained from Roth (Karlsruhe,
Germany). Tin(II) 2-ethylhexanoate (Sn(Oct)2) was distilled three times before usage. Ethylene
glycol (≥ 99%) was dried by stirring over MgSO4, then distilled under inert conditions, and
finally stored above molecular sieves. Solvents were dried by standard methods and distilled
prior to usage.
Methods
Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker DCX-500 spectrometer
at room temperature (r.t.). In most cases, DMSO-d6 was used as deuterated solvent, 2-
aminooctanoic acid was dissolved in D2O/NaOD. The related solvent in deuterated reagent was
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used as an internal standard. Chemical shifts were referenced to DMSO-d6 = 2.5 ppm and D2O =
4.8 ppm, respectively.
The thermal properties of the polymers were determined by differential scanning calorimetry
(DSC) on a Netzsch DSC 204 (Selb, Germany), which was equipped with a low temperature cell.
The cyclic measurements were conducted in the temperature range between -20 °C and 200 °C
with a heating rate of 10 °Cꢀ
min-1 and kept at 200 °C for 2 min. Subsequently, the sample was
cooled with the same rate by liquid nitrogen. After 2 min at the lowest temperature, a second
heating run was performed. The thermal transitions were evaluated from the second heating run.
Gel permeation chromatography (GPC) measurements were performed by universal calibration
(see Supporting Information) on a system consisting of a precolumn, (two 300*8 mm2 linear M
(10 ꢁ) SDV-columns, PSS = Polymer Standards Service GmbH, Mainz, Germany), a degasser
DG-2080-53, a RI-1530 refractive index detector, an isocratic pump PU-1580, an automatic
injector AS-1555 (all JASCO, Groß-Umstadt, Germany) and a viscosimeter T-60A detector
(VISCOTEK, Houston TX, USA). Tetrahydrofuran (THF) was used as eluent at a flow rate of
1.0 mLꢀ
min-1 at r.t. and 0.2 wt% methanol as internal standard. Molecular weight and
polydispersity calculations (see Supporting Information Figure S1 and S2 for GPC traces of
samples listed in table 1 and 2, respectively) were performed using WINGPC 6.2 (PSS) SEC
software (Polymer Standard Service, Mainz, Germany).
Matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS)
was performed on a Biflex III spectrometer (Bruker Daltonik, Leipzig, Germnay). The samples
were prepared as follows: 0.5 mg of the oligomer was dissolved in 1 mL THF, a mixture of
trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) and potassium
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trifluoroacetate in THF was used as matrix. High-resolution mass spectrometry (HRMS) was
recorded on an ESI-Q-TOF maXis mass spectrometer (Bruker Daltonik, Leipzig, Germnay).
IR spectra were obtained using Magna IR 550 series II spectrometer (Nicolet, Germany), which
was equipped with a single beam diamond ATR unit. Melting points were determined on an
electrothermal MEL-TEMP (Bibby Sterilin, Stone, UK) apparatus at a starting temperature of
90 °C and a heating rate of 0.1 °C·min-1.
Synthesis of monomers
Both monomers, 3HMD and 3M6HMD, were synthesized in a three-step synthesis route
according to the procedure described in reference [17]. In brief, in a first step the N-
(halogenacyl)amino acid was synthesized as an intermediate. Afterwards, the ring-closure
reaction to the morpholine-2,5-dione was conducted. The overall yields for 3M6HMD and
3HMD were 15% and 7%, respectively.
Synthesis of (2S)-2-(2-bromooctanamido)propanoic acid
3
Thionyl chloride (3.1314 g, 13.04 mmol) was added dropwise into a 50 mL Schlenk flask
containing 2-bromooctanoic acid (2.5 g, 11.3 mmol), which was kept at 0 °C. The mixture was
heated to 90 °C and refluxed at this temperature for 24 h. The acylation reaction was monitored
by NMR spectroscopy until no signals from residual acid moieties could be observed. The excess
of thionyl chloride was removed by vacuum at ambient temperature.
1.18 g (13.19 mmol) L-alanine was dissolved in 15 mL NaOH solution (1 M) and cooled by an
ice bath (0 °C). The 2-bromooctanoyl chloride was dissolved in diethyl ether (16.8 mL) and
added to the amino acid solution within 7 h simultaneously with 14 mL NaOH solution (1 M)
under vigorous stirring. The pH was held at pH = 12 during the procedure. When the aqueous
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phase was acidified to a pH of 3, a colorless precipitate was formed, which was filtered off,
washed with hexane, and dried overnight at 45 °C until constant weight was achieved.
1H NMR (500 MHz, DMSO-d6):
δ
ppm 12.60 (s, 1H, CO2
H), 8.56 (d,
J = 7.3, 1H,NH), 4.45 -
4.36 (m, 1H, NH-C CH3-CO2H), 4.29 - 4.14 (m, 1H; Br-C
H
HC6H13-CO), 1.97 - 1.52 (m, 2H,
C5H11-CH2-CHBr), 1.52 - 1.00 (m, 11H; (CH2)4-CH3/NH-CHCH3-CO2H), 0.85 (t,
J
= 6.8, 3H,
O-NH),
13
(CH2)5-CH3); C NMR (126 MHz, DMSO-d6):
δ
ppm 173.3 (
C
O2H), 167.9 (CHBr-
H2-CHBr), 30.7 (C2H5-
H2-CH2), 21.6 (CH3-CH2-CH2), 16.9 (NH-CH
C
48.5 (Br-
CH2), 27.6 (C3H7-
CO2H), 13.6 ((CH2)5-
point: 85 2 °C.
C
HC6H13-CO), 47.6 (NH-
C
HCH3-CO2H), 34.4 (C5H11-
C
C
C
H2-
H3-
C
H2-CH2), 26.3 (C4H9-
C
+
C
H3); MS (ESI+): [MH ]determined 294.0714, [M]theoretical 293.0705; melting
±
Synthesis of (3S,6R,S)-3-methyl-6-hexylmorpholine-2,5-dione (3M6HMD) 4
0.50 g (1.7 mmol) of
3 and TEA (0.5 g, 4.82 mmol) were mixed homogeneously in DMF (75 mL)
under vigorous stirring. The reactor was heated to 60 °C and kept at this temperature for 3 h, then
cooled to r.t. and kept at r.t. for another 24 h. The precipitate, triethylamine hydrobromide, was
filtered off and the obtained solution was filtered over silica gel. After removal of the solvent
under vacuum, recrystallization from ethyl acetate for two times gave colorless crystals. The
overall yield was 15%.
1H NMR (500 MHz, DMSO-d6):
CO), 4.46 - 4.28 (m, 1H; NH-C
11H, (CH2)4-CH3/NH
DMSO-d6): ppm 170.6 (CHC6H13-
48.6 (NH- HCH3-CO), 31.4 (C5H11-
δ ppm 8.49 (s, 1H; NH), 4.96 (dd, J = 7.7, 1H; O-CH(C6H11)-
H
CH3-CO), 1.94 - 1.63 (m, 2H, C5H11-CH2-CH), 1.47 - 1.18 (m,
13
-
CHCH3-CO), 0.87 (t,
O-NH), 168.6 (CHCH3-
H2-CH), 29.6 (C2H5- H2-CH2), 28.7 (C3H7-
J
= 6.8, 3H; (CH2)5-CH3); C NMR (126 MHz,
δ
C
CO-O), 77.5 (O-CHC6H13-CO),
C
C
C
C
H2
-CH2),
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24.5 (C4H9-
C
H2-CH2), 22.3 (CH3-
C
H2-CH2), 16.2 (NH-CH
C
H3-CO), 14.2 ((CH2)5-CH3); IR
+
(ATR): 3199, 2922, 1749, 1693, 1530 cm-1; MS (ESI+): [MH ]determined 214.1447, [M]theoretical
213.1443; melting point: 117
± 2 °C.
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Synthesis of 2-aminooctanoic acid
5
In a 100 mL round-bottomed flask, 2-bromooctanoic acid (5 g, 22.6 mmol), sodium azide (3.6 g,
55 mmol), and 50 mL of DMSO were mixed homogeneously by stirring at 80 °C for 72 h. Then
the solvent was removed by application of vacuum whereby the NaBr precipitated. After
dissolving the residue in 30 mL dichloromethane (DCM), the precipitate was filtered off.
Afterwards, the remaining solution was extracted several times with water. The organic phase
was dried overnight over sodium sulfate. Once the sodium sulfate was filtered off, a yellow
solution was obtained, which was used without further purification.
In a 1 L round flask, 10% palladium on carbon (100 mg) was added into the yellow solution and
the mixture was vigorously stirred under a H2 atmosphere. Once a colorless solid precipitated,
additional palladium on carbon (30 mg) and DCM were added. Hydrogenation was continued for
another 48 h. Afterwards, the residual solvent was decanted. Extraction with boiling water and
ethanol for three times gave colorless crystals, which were further dried in an oven at 45 °C until
constant weight was achieved.
1H NMR (500 MHz, D2O/NaOD):
δ ppm 3.20 (dd, J = 7.0, 1H,CO2H-CHC6H13-NH2), 1.65 -
1.46 (m, 2H, C5H11-CH2-CH), 1.38 - 1.22 (m, 8H, (CH2)4-CH3), 0.92 - 0.81 (m, 1H, (CH2)4-
13
C
H3),; C NMR (126 MHz, D2O/NaOD):
δ
ppm 181.2 (
C
O2H), 53.3 (CO2H-
C
HC6H13-NH2),
32.0 (C5H11-
19.2 (CH3-
159.1336; melting point: 268
C
H2-CH), 28.2 (C2H5-
H2-CH2), 10.6 ((CH2)5-
2 °C.
C
H2-CH2), 25.7 (C3H7-
C
H2-CH2), 22.2 (C4H9-
C
H2-CH2),
+
C
C
H3); MS (ESI+): [MH ]determined 160.1336, [M]theoretical
±
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Synthesis of 2-(2-chloroacetamido)octanoic acid
6
3 g (19 mmol) of
5
was dissolved in 20 mL NaOH solution (1M) and cooled by an ice bath
(0 °C). 2-chloroacetyl chloride (20 mmol) was dissolved in diethyl ether (20 mL) and added to
the amino acid solution within 7 h whereby 20 mL NaOH solution (1 M) was added
simultaneously under vigorous stirring. The pH was held at pH = 12 during the procedure.
Afterwards, acidification of the aqueous phase with HCl resulted in a colorless solid precipitate.
The precipitate was filtered off, extracted with hexane, and dried overnight at 45 °C until
constant weight was achieved.
1H NMR (500 MHz, DMSO-d6):
= 8.3, 1H, NH-C
δ ppm 12.68 (s, 1H, CO2H), 8.44 (d, J = 7.8, 1H, NH), 4.18 (td,
J
H
C6H13-CO), 4.10 (m, 2H, Cl-CH2-CO), 1.65 (m, 2H, C5H11-CH2-CH), 1.25
= 6.8, 3H, (CH2)4-CH3); 13C NMR (126 MHz, DMSO-d6):
(m, 8H, (CH2)4-CH3), 0.85 (t,
173.4 (CO2 ), 166.1 (CH2Cl-
(C5H11- H2-CH), 31.1 (C2H5-
(CH3- H2-CH2), 14.1 ((CH2)5-
melting point: 87 1 °C.
J
δ ppm
H
C
O-NH), 52.3 (NH-
C
HC6H13-CO), 42.5 (Cl- H2-CO), 31.2
C
C
C
C
H2-CH2), 28.3 (C3H7-
C
H2-CH2), 25.3 (C4H9-CH2-CH2), 22.1
+
C
H3); MS (ESI+): [MH ]determined 236.1055, [M]theoretical 235.1053;
±
Synthesis of (3R,S)-hexylmorpholine-2,5-dione (3HMD) 7
1.00 g (4.3 mmol) of
6 and TEA (1.0 g, 5.64 mmol) were mixed homogeneously in DMF (150
mL) under vigorous stirring. The reactor was heated to 60 °C, kept at this temperature for 3 h,
then cooled to r.t. and kept at this temperature for another 24 h. After removal of triethyl
hydrochloride and passing through silica gel, the solvent was decanted. Recrystallization from
ethyl acetate for two times gave white crystals. The overall yield was 7%.
10

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1H NMR (500 MHz, DMSO-d6):
δ
ppm 8.56 (s, 1H, NH), 4.73 (q, 2H, O-CH2-CO), 4.16 (td,
J
=
6.0, 1H, NH-C
H
C6H13-CO), 1.73 (m, 2H, C5H11-
C
H2-CH), 1.32 (m, 8H; (CH2)4-CH3), 0.86 (t,
J
13
= 6.8, 3H; -(CH2)4-CH3); C NMR (126 MHz, DMSO-d6):
δ
ppm 168.1 (CH2-
HC6H13-CO), 31.1 (C5H11-
H2-CH2), 23.9 (C4H9- H2-CH2), 21.7 (CH3-
C
O-NH), 165.5
H2-CH), 30.7
(CHC6H13-
(C2H5- H2-CH2), 27.9 (C3H7-
((CH2)5-
199.1205, [M]theoretical 199.1208; melting point: 100
CO-O), 67.2 (O-CH2-CO), 52.0 (NH-C
C
C
C
C
C
H2-CH2), 13.6
+
C
H3); IR (ATR): 3196, 2914, 1740, 1691, 1546 cm-1; MS (ESI+): [MH ]determined
±
2 °C.
Synthesis of (3S)-methylmorpholine-2,5-diones (MMD)
The synthesis of (3S)-methylmorpholine-2,5-diones was performed according to the synthesis
route described in reference [18].
1H NMR (500 MHz, DMSO-d6):
δ
(ppm) 8.53 (s, 1H, NH), 4.92 - 4.54 (q, 2H, O-CH2-CO), 4.30
(q, 1H, NH-C CH3-CO), 1.33 (d, 3H, CHCH3). melting point: 138
H
±
1 °C.
Synthesis of oDPs
oDPs were synthesized by ROP of the morpholine-2,5-dione (MD) as shown in Scheme 2.
A general procedure was based on polymerization of MDs catalyzed by Sn(Oct)2 using ethylene
glycol (EG) as initiator. Polymerization reactions were carried out with approximate 1 g
monomer (nMMD = 7.8 mmol; n3M6HMD = 4.7 mmol; n3HMD = 5.0 mmol, respectively). Monomer,
predefined amount of initiator, and catalyst were filled into a flame-dried Schlenk reactor (10 mL)
under a dry nitrogen atmosphere. Afterwards, the reaction mixture was heated to the
polymerization temperature and stirred under inert conditions for time periods according to Table
1and 2. The targeted molecular weights for oDPs were 6 kg·
mol-1.
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The ROP kinetics were studied at 140 °C. Samples were taken at appropriate time intervals and
the ROP was quenched by freezing in liquid nitrogen. Afterwards, the crude samples were
directly analyzed by GPC measurements without any further purification and removal of
unreacted monomer. The monomer conversions were determined from GPC eluograms via
comparing the integrals from monomer peaks with the sum of integrals of the peaks from
monomers and oligomers.
R2
O
O
OH
m+n
HO
N
H
O
R1
Morpholineꢂ2,5ꢂdione
Ethylene glycol
140 °C, bulk
Sn(Oct)2
O
R1
O
R2
H
O
m
H
O
N
N
H
n
O
O
H
R2
O
R1
O
Oligodepsipeptide (oDP)
Scheme 2. ROP of morpholine-2,5-diones.
Results and discussion
Monomer synthesis
In general, monomers were synthesized in a three-step synthesis route according to scheme 3. 2-
bromooctanoic acid was transferred to 2-bromooctanoyl chloride as well as to 2-aminooctanoic
acid to create related acetyl chloride in case of 3M6HMD and the
α-amino acid in case of 3HMD.
The related -amino acid was reacted with the appropriate acyl halide in a modified Schotten-
α
Baumann reaction at the interphase between diethyl ether and water. The presence of NaOH in
the aqueous phase neutralizes the generated carboxylic acid and shifts in this way the equilibrium
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reaction to the corresponding intermediate products. The monomers (3M6HMD and 3HMD),
which were created in a subsequently performed ring-closure reaction, were purified by
recrystallization from ethyl acetate. All synthesized compounds were confirmed by 1H NMR and
13C NMR spectroscopy as well as mass spectrometry.
Scheme 3. Synthesis of morpholine-2,5-diones.
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Table 1. Comparision of ROP of MMD and 3M6HMD catalysed by Sn(Oct)2.
b
Time
[h]
Conversionc
[%]
Mn GPC
,
a
Run Monomer [M]0:[EG]0:[Sn(Oct)2]0
500:18:1
ROP condition
Mw/Mn
[kg
·
mol-1]
1
2
3M6HMD
3M6HMD
3M6HMD
3M6HMD
MMD
Toluene 80 °Cd
Toluene 110 °Cd
Bulk 140 °C
3
3
n.de
n.d
3.2
4.8
n.d
n.d
4.3
5.3
4.3
5.6
5.4
n.d
n.d
1.1
1.2
n.d
n.d
1.1
1.4
1.6
1.2
1.3
7
500:18:1
500:18:1
500:18:1
500:11:1
500:11:1
500:11:1
500:11:1
500:18:1
500:11:1
500:17:1
21
36
49
15
29
47
83
49
83
79
3
3
4
Bulk 140 °C
15
3
5
Toluene 80 °Cd
Toluene 110 °Cd
Bulk 140 °C
6
MMD
3
7
MMD
3
8
MMD
Bulk 140 °C
15
24
24
24
9f
10f
11f
3M6HMD
MMD
Bulk 140 °C
Bulk 140 °C
3HMD
Bulk 140 °C
a Mn of oligomers aimed at was 6 kg mol-1.
·
b Mn was determined by GPC using THF as an eluent.
c Monomer conversion was determined by GPC using THF as an eluent.
d Monomer concentration was 0.94 mol
e n.d : no data.
·
L-1.
f Polymerizations were performed for kinetic study, see Figure 2.
Determination of reaction parameters by ROP of 3M6HMD
The capability of the newly created compounds acting as monomers in ROP was explored in
preliminary homopolymerizations catalyzed by Sn(Oct)2. In a feasibility study, the optimum
reaction conditions for the ROP were determined by ROP of MMD. These optimized parameters
were used for ROP of 3M6HMD. In this way, the influence of modification at position 6 was
investigated. The efficiency of the reactions was evaluated by determining the monomer
conversions after appropriate time intervals (Table 1). In initial experiments, ROP was conducted
in solution (toluene) and at 80 °C to avoid side reactions. The monomer conversions with 7%
and 15% were relatively low after 3 h at 80 °C for both 3M6HMD and MMD, respectively. A
slight increase of monomer conversion was achieved when the temperature was raised from
14

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80 °C to 110 °C. The monomer conversion increased tremendously when the ROP was
performed as bulk polymerization of the melt at 140 °C. In case of 3M6HMD conversions of 36%
and 49% were achieved after 3 h and 15 h. When MMD was polymerized, the conversion
increased to 47% after 3 h and to 83% after 15 h at the same conditions. The difference in the
monomer conversions between 3M6HMD and MMD might be attributed to the steric hindrance
caused by hexyl group substitute.
Kinetic study of ROP
In the ROP kinetic study experiments of 3M6HMD, 3HMD, and MMD (Table 1, runs 9-11), the
polymerizations were catalyzed by Sn(Oct)2. Samples were collected from the reaction mixture,
dissolved in THF, and analyzed by GPC.
100
(a)
7000
(b)
6000
80
5000
60
4000
3000
40
2000
20
1000
0
0
0
10
20
30
0
10
20
30
Time / h
Time / h
Figure 2. Homopolymerization of MDs catalyzed by Sn(Oct)2: (a) dependence of monomer
conversion on time; (b) dependence of Mn on time. MMD ; 3M6HMD ; 3HMD
.
Figure 2a displays the dependence of the monomer conversion as a function of time. MMD
achieved the highest monomer conversion at any given time during the reaction, which indicates
that MMD have the highest activity towards the ROP. The ring-chain equilibria were reached for
15

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both MMD and 3M6HMD after 12 h, while a slight increase was still observed for 3HMD after
16 h. When the reactions reached the equilibria, 3M6HMD displayed the lowest monomer
conversion of around 50% among the three monomers in the given reaction conditions. In
contrast, MMD and 3HMD presented a reasonable conversion of around 80% after 24 h. The
obtained results show clearly the differences in the reactivity of each monomer in the ROP,
which is supposed to be caused by the steric hindrance of the hexyl group substitute.
Figure 2b presents the obtained Mn as a function of the reaction time. A similar tendency of
increase as observed for the conversion can be noted. The highest Mn was observed for ROP of
MMD. After 12 h and 16 h of the reaction time, almost constant Mns were reached for MMD and
3HMD, respectively. In case of 3M6HMD, the Mn decreased after reaching a maximum value
after 12 h. Due to the presence of the hexyl group at position 6 in the 3M6HMD, the efficiency
of ROP should be relatively low, which was also observed in a kinetic study. The decrease of Mn
,
as presented in Figure 2b, after reaching the maximum value indicates that the intermolecular
side reactions became the favored reaction versus the competing chain growth reaction.
Analogous observations were reported by former research work on ROP of morpholine-2,5-
diones [15]. The decrease of Mn was attributed to the chain fragmentation reaction, which means
that the polymer chains were partially transferred to the Sn(Oct)2 via intermolecular
transesterification resulting in polymer chains terminated with tin residue. Meanwhile, back-
biting leading to macrocycle can also contribute to the reduction of Mn via intramolecular
transesterification, which can be confirmed by MALDI-TOF.
Figure 3 shows the kinetic plots of the polymerization of the monomers in semilogarithmic scale.
The time law for polymerization with ring-chain equilibrium can be expressed according to
equation (1)[19, 20]:
16

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ꢂꢃ − ꢂꢄ
ꢂꢅ − ꢂꢄ
ꢈ
ꢌꢈ ꢌ∗
= ꢆꢇ ꢉꢊꢋ ꢂ ꢋ = ꢆꢍꢇꢇꢋ (1)
ꢀꢁ
ꢂꢃ is the monomer concentration of the starting mixture, ꢂꢄ represents the monomer
concentration at the moment when reaction reaches the ring-chain equilibrium, ꢂꢅ is the actual
monomer concentration after various reaction time period, and ꢂ∗ is the concentration of the
ꢈ
ꢌꢈ ꢌ∗
(ꢆꢍꢇꢇ = ꢆꢇ ꢉꢊꢋ ꢂ ),
active species. The slope of the plot is the apparent rate constant Kapp
which is a direct parameter for the reaction rate at the given reaction conditions.
5
4
3
2
1
0
0
10
20
30
Time / h
Figure 3. Homopolymerization of MDs in bulk catalyzed Sn(Oct)2: 1st order plot. MMD
3M6HMD ; 3HMD
The monomer reaction activities for the ROP are comparable to the apparent rate constants Kapp
;
.
s
obtained from the slopes of the semilogarithmic-time curves. In case of MMD the rate constant
obtained a value of 0.28 ± 0.01 h-1, which is 2 times higher than for the ROP of 3M6HMD with a
rate constant of 0.14 ± 0.02 h-1. This can be attributed to the steric hindrance introduced by
hexyl-group substitute at position 6. The rate constant of ROP of 3HMD was 0.20 ± 0.03 h-1.
This lower Kapp compared to ROP of MMD can also be explained by the steric hindrance caused
by the substitution of methyl group with the hexyl group at position 3. Compared with 3M6HMD,
17

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the higher Kapp of 3HMD can be explained by a slight electron pushing effect (+I-effect) of the
hexyl group compared to the hydrogen in addition to the steric hindrance.
In general, the amide group of depsipeptide is considered to be more stable than an ester group
and less active towards ROP. However, attempts to synthesize polydepsipeptides from the
corresponding N-alkyl substituted morpholine-2,5-diones via ROP have not been successful until
now [21]. A possible explanation might be that the amide group could form a chelating
intermediate at the ester group. In this context, the reduction of the monomer activity for ROP in
case of 3HMD, could be caused by the steric hindrance introduced by hexyl-substitute at position
3.
Investigation of MD homopolymerization
The homopolymerization of MDs was studied by ROP. The reactions were quenched after
appropriate time periods before intermolecular side reactions became the favored reactions. The
obtained oligomers were characterized by NMR, GPC, MALDI-TOF, and DSC (see Supporting
Information Figure S3 for full MALDI-TOF spectra of o3M6HMD and o3HMD, Figure S4 for
DSC curves, and Figure S5 for 13C NMR spectra of o3M6HMD and o3HMD).
Table 2. ROP of MMD, 3M6HMD, and 3HMD at 140 °C in melt catalysed by Sn(Oct)2
a
Monomer
[M]0:[EG]0:[Sn(Oct)2]0
(equiv.)
Reaction
Timec
[h]
Yield
[%]
DP
Mn
Aimedd
Measurede
[kg·mol-1]
NMR
GPC Mw/Mn
MMD
3M6HMDb
3HMDb
500:11:1
500:18:1
500:17:1
16
62
23
58
46
28
30
44
25
28
1.4
1.2
1.2
6.5 ± 0.3 5.7 ± 0.6
12
18
6.1 ± 0.3 5.4 ± 0.5
5.8 ± 0.3 5.6 ± 0.6
a Mn of oligomers aimed at was 6 kg·mol-1.
b Purified by chromatography.
c Reaction time periods were defined with contribution of the results from kinetic study.
d Aimed degree of polymerization (DP) was based on the mole ratio of starting compounds.
e Determined by GPC on the purified product.
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(a)
k
j
d-g; k
h
*
i
p
a
b
i
**
I2
I1
c
I1,I2
i
b j
d
f
h
a
p
c
e
g
8
6
4
2
0
δ (ppm)
(b)
j
k
a
p
b
k
K2
K1
d
f
h
c
e
g
1
Figure 4. 500 MHz H NMR spectra in DMSO-d6 of (a) o3M6HMD and (b) o3HMD. One
asterisk indicates the DMSO peak, and two asterisks indicate the H2O peak.
1
When the molecular weights of the oligomers were determined by H NMR, the ratio of the
signal intensities of the repeating units and initiator were calculated according to equation (2):
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ꢏꢐꢑ
ꢒ
ꢓ
3
ꢂꢎ =
∗ ꢂꢕꢖꢗꢇꢘꢖꢙꢚꢎꢄ ꢛꢚꢖꢎꢄ + ꢂꢜꢎꢚꢅꢚꢍꢅꢖꢗ (2)
ꢏꢐꢑ
ꢔ
ꢓ
4
ꢏꢝꢞ and ꢏꢝꢞ are integrals of the signals originating from the initiator at
δ
= 3.80 - 3.88 ppm and
= 0.76 - 0.89 ppm for o3M6HMD and at = 0.68 - 0.91 ppm
for o3HMD, respectively (shown in Figure 4a and Figure 4b).
ꢒ
ꢔ
from the oDPs repeating unit at
δ
δ
The molecular mass of the oDPs were also determined by GPC and the data are summarized in
Table 2. The Mw Mn of oMMD was 1.4, which was slightly higher than 1.2 of o3M6HMD and
/
o3HMD. This slightly higher value can be explained by a lower solubility of the oligomer in the
melted monomer resulting in precipitation and by this in a loss of control of the polymerization
reaction.
MALDI-TOF MS was performed on o3M6HMD and o3HMD to characterize the terminal
groups of the obtained oligomers (Figure 5). Four series of signals corresponding to three
different species could be identified for the telechelic o3M6HMD as shown in Figure 5a. The
main series 2 and 2´corresponding to the oligomers are the series with terminal hydroxyl groups
having 7 or 8 repeating units, respectively. Series 1 represents the polymer chain ionized by
accidentally introduced sodium ions. Series 3 can be attributed to the formation of macrocycles
by intramolecular transesterification. The molar mass of series 4 is 44 g·
mol-1 less than the molar
mass of series 2´ and results from the polymer chains providing carboxylic terminal groups,
which were initiated by traces of water or other active hydroxyl-containing species in the starting
mixture.
20

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20000
15000
10000
5000
0
8000
(b)
(a)
1
1´
2
2´
6000
4000
2000
1
3
4
1´
2
0
1600
1650
1700
1750
1800
1500
1550
1600
m / z
1650
1700
+
+
m / z
Figure 5. Details of MALDI-TOF mass spectra, reflector negative ion mode: (a) o3M6HMD and
(b) o3HMD.
The structures of the corresponding species identified from the o3M6HMD mass spectrum are
presented in Figure 6. A lower number of signal series can be observed compared to the spectra
of o3M6HMD in the MALDI-TOF spectrum of o3HMD (Figure 5b). Series 1 and 1´ can be
correlated to oligomers with repeating units 7 or 8. The structures of the corresponding species,
which can be identified from the o3HMD MALDI-TOF mass spectra, are displayed in Figure 7.
Series 2 is caused by water initiation as discussed before. The low content of additional series in
the spectrum of o3HMD can be explained by higher activity of 3HMD towards the chain growth
reaction, resulting in a better reaction control.
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Figure 6. Structures of the species as assigned from the o3M6HMD mass spectrum.
Figure 7. Structures of the species as assigned from the o3HMD mass spectrum.
22

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In order to determine efficiency to reduce the glass transition temperature (Tg) by introducing
atactic bulky side group, the thermal properties of oligomers with comparable molecular weights
were studied by DSC (Table 3). The o3HMD exhibited the lowest Tg of 32 °C, which is below
human body temperature. The o3M6HMD showed a close value of 36 °C around body
temperature. In contrast, the Tg of oMMD was 65 °C, which is approximately 30 °C higher than
the Tgs for both other oDPs in which hexyl side groups were introduced. This behavior could be
caused by bulky atactic pendant groups, resulting in a plasticizing effect by disturbing the H-
bonding between polymer chains. Meanwhile, the higher Tg. of oMMD can also be attributed to
more polymer chains due to the lower monomer mass, which contributed a higher number of
terminal hydroxy groups. As a result, the H-bonding was relatively enhanced leading to a
stronger limitation of polymer chain movements. Furthermore, the absence of a second transition
temperature (Tm) in both o3M6HMD and o3HMD strongly suggested amorphous polymers
resulting in no significant crystalline domains. Both factors, the reduction of H-bonding and the
weakening of crystalline behavior, could contribute to the enhancement of polymer chain
movement.
Table 3 Thermal properties of synthesized oDPs
a
oDPs
Tg
[°C]
65
Tm
[°C]
142
oMMD
o3M6HMD
o3HMD
36
32
n.d
n.d
a No Tm is detected in o3M6HMD and o3HMD
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Conclusion
Two morpholine-2,5-diones, named 3M6HMD and 3HMD with atactic hexyl groups, were
successfully synthesized via acylation reaction and subsequent ring-closure reaction. In initial
studies, the ROP capability of 3M6HMD and 3HMD using ethylene glycol as initiator and
Sn(Oct)2 as coordination insertion catalyst was explored. In both oDPs no Tm could be detected
suggesting the presence of only amorphous phases. The glass transition temperatures of
o3M6HMD and o3HMD were approximately 30 °C lower than that of oMMD. All oligomers
provided the presence of hydroxyl groups whereas in ROP of 3HMD less side reactions seemed
to occur as evidenced by a lower number of series in MALDI-TOF measurements compared to
that of o3M6HMD. Kapp of 3M6HMD was slightly lower compared to 3HMD, which was a hint
that in addition to the steric hindrance also the electron influence of the substitutions needs to be
considered. Nevertheless, the oligomers from MDs providing terminal hydroxyl groups can be
considered as valuable building blocks for degradable, elastic scaffold materials e.g. for cell
culture systems.
Acknowledgments
:
This work was financially supported by the Helmholtz-Association through programme-oriented
funding, the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research
Centre 1112 “Nanocarriers”, subproject A03, the Tianjin University-Helmholtz-Zentrum
Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, which is financed by
the German Federal Ministry of Education and Research (BMBF, Grant No. 0315496) and the
Chinese Ministry of Science and Technology (MOST, 2008DFA51170), and X.P. gratefully
acknowledges funding by the China Scholarship Council (CSC) (grant No. 201206250098).
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Highlights for
Hexyl-modified morpholine-2,5-diones based oligodepsipeptides with relatively low glass
transition temperature
Xingzhou Peng, Marc Behl, Pengfei Zhang, Magdalena Mazurek-Budzynska, Muhammad Y. Razzaq,
Andeas Lendlein
•
•
•
Morpholine-2,5-diones with hexyl-side chains (HMMDs) have been prepared
Kinetics of the ring-opening polymerization of HMMDs were understood
Significant reduction of Tg of oligodepsipeptides by hexyl-side chains introduction