Supplementary information
for
Terpolymerisation kinetics of amino acid N-carboxy anhydrides
by
Mischa Zelzer,a‡* Andreas Heisea,b*
a
Technical University Eindhoven, Department of Chemical Engineering and Chemistry, Den Dolech 2,
5612 AZ, Eindhoven, The Netherlands, m.zelzer@nottingham.ac.uk.
b
Dublin City University, School of Chemical Sciences, Glasnevin, Dublin 9, Ireland,
andreas.heise@dcu.ie.
‡ Present Address: University of Nottingham, School of Pharmacy, Boots Science Building, University
Park, Nottingham, NG7 2RD, UK.
Synthesis of Tyr(Bzl)-NCA
10.1 g H-Tyr(Bzl)-OH were dissolved in 100 ml ethyl acetate and 14 ml α-pinene (2.4 equ.) were
added. The suspension was heated to 90 °C and stirred under reflux and N2 for 15 min. A solution of
6.2 g triphosgene (0.6 equ.) in 40 ml ethyl acetate was added dropwise. The mixture was stirred
under N2 at 90°C under reflux for 5 h until the reaction turned into a clear, yellow solution.
The reaction was allowed to cool down, filtered and the solvent was reduced to approximately 30%
of its original volume. The product was precipitated through addition of n-heptane. The
recrystallization was repeated twice. The product was dried in a vacuum oven at 50 °C for over night
to yield 7.8 g of off-white crystals (70% yield).
1
H NMR (400 MHz, CDCl3): δ = 2.95 (dd, J = 14.2 Hz / 8.2 Hz, 1 H, CH2-CH), 3.21 (dd, J = 14.2 Hz / 4.1
Hz, 1 H, CH2-CH), 4.48 (dd, J = 8 Hz / 3.9 Hz, 1 H, NH-CH-C=O), 5.04 (s, 2 H, CH2-O), 5.97 (bs, 1 H, NH),
6.94 (d, J = 8.6 Hz, 2 H, 2 x CHaromatic), 7.10 (d, J = 8.6 Hz, 2 H, 2 x CHaromatic), 7.29-7.45 (m, 5 H, 5 x
CHaromatic).
13
C NMR (400 MHz, CDCl3): δ = 37.1 (1 C, CH2), 59.1 (1 C, CH), 70.2 (1 C, CH2), 115.7 (2 C, 2 x
CHaromatic), 126.1 (1 C, CH-CH2-Caromatic), 127.6 (2 C, 2 x CHaromatic) 128.2 (1 C, CHaromatic), 128.8 (2 C, 2 x
CHaromatic), 130.5 (2 C, 2 x CHaromatic), 136.8 (O-CH2-Caromatic), 151.9 (1 C, NH-C(=O)-O), 158.4 (1 C, OCaromatic), 168.8 (1 C, CH-C(=O)-O).
m/z (theoretical): 297.10
m/z (MALDI-ToF): 298.42 [M+H]+, 320.17 [M+Na]+
1
Figure S1. Chromatogram of Tyr(Bzl) at 10 mg ml-1 (top) and the quenched Tyr(Bzl)-NCA at 100 mg
ml-1 (middle) obtained from HPLC measurements. Mass spectra of the quenched Tyr(Bzl)-NCA
(bottom) obtained from LC-MS analysis.
Figure S2. Calibration curves for Tyr(Bzl)-NCA. The average values are shown and have been used to
obtain the linear calibration line (n = 3, error bars represent standard deviations).
2
Figure S3. HPLC chromatograms for time-points at 60 min intervals during the co-polymerisation of
Glu(Bzl)-NCA / Tyr(Bzl)-NCA = 3:1 (top) and Lys(Z)-NCA / Tyr(Bzl)-NCA = 3:1 (bottom).
3
Figure S4. Comparison of the course of the polymerisation of binary mixtures of Glu(Bzl) / Tyr(Bzl
and Lys(Z) / Tyr(Bzl) at two different monomer ratios using either IR spectroscopy or HPLC.
Figure S5. Determination of the reaction rate constants (k) for the copolymerisation of Glu(Bzl)-NCA
/ Lys(Z)-NCA (kinetic data reported in Ref 15 was used), Glu(Bzl)-NCA / Tyr(Bzl)-NCA and Lys(Z)-NCA /
[𝑁𝐶𝐴]
Tyr(Bzl)-NCA using the integrated rate law for a first order reaction: 𝑙𝑛 ([𝑁𝐶𝐴] ) = −𝑘𝑡 ([NCA] –
0
concentration of the respective amino acid NCA, [NCA]0 – theoretical NCA starting concentration; t –
reaction time). k was determined from the slope of the linear regression lines. Datapoints originate
from single kinetic experiments.
4
Figure S6. 1H-NMR spectrum of the E/Y copolypeptide in TFA obtained after reaction of Glu(Bzl)-NCA
and Tyr(Bzl)-NCA for 20 min at a ratio of 1:1.
Figure S7. 1H-NMR spectrum of the K/Y copolypeptide in TFA obtained after reaction of Lys(Z)-NCA
and Tyr(Bzl)-NCA for 20 min at a ratio of 1:1.
5
Table S1. Data used for the calculation of the copolymerisation parameters for the Glu(Bzl) / Tyr(Bzl)
system.
feed
feed
polymer composition
calculated values
compo- ratio (f)
peak area
peak area
ratio
f2/F
f(1-F)/F
ξ
a
b
c
sition
Glu(Bzl)
Tyr(Bzl)
(F)
1:1
1.00
121292407 119947459
2.02
0.49
0.51
0.31
11:9
1.22
106416870
75630933
2.81
0.53
0.79
0.32
3:2
1.50
260800898 172810428
3.02
0.75
1.00
0.40
13:7
1.86
178440191
92551873
3.86
0.89
1.38
0.44
7:3
2.33
196718090
88089033
4.47
1.22
1.81
0.52
3:1
3.00
183332738
55401857
6.62
1.82
2.39
0.62
4:1
4.00
151365192
47646267
6.35
2.52
3.37
0.69
a
obtained from 1H-NMR spectra by fitting peaks to the glutamic acid backbone CH region
b
obtained from 1H-NMR spectra by fitting peaks to tyrosine’s CH2 region
c
calculated after division of the Tyr(Bzl) peak areas by two
η
0.31
0.48
0.54
0.68
0.78
0.81
0.93
Table S2. Data used for the calculation of the copolymerisation parameters for the Lys(Z) / Tyr(Bzl)
system.
feed
feed
polymer composition
calculated values
compo- ratio (f)
peak area
peak area
ratio
f2/F
f(1-F)/F
ξ
η
a
b
c
sition
Lys(Z)
Tyr(Bzl)
(F)
1:1
1.00
52384067
81779244
0.65
1.56
-0.56
0.41
-0.15
11:9
1.22
22718009
22711780
1.00
1.49
0.00
0.39
0.00
3:2
1.50
21775580 19278802.5
1.13
1.99
0.17
0.47
0.04
13:7
1.86
32757442
20788679
1.58
2.19
0.68
0.49
0.15
7:3
2.33
29837926 16996000.5
1.76
3.10
1.00
0.58
0.19
3:1
3.00
64564100
25063594
2.58
3.49
1.83
0.60
0.32
4:1
4.00
61248795
17397989
3.52
4.54
2.86
0.67
0.42
1:1
1.00
20660327
54053565
0.76
1.31
-0.31
0.36
-0.09
11:9
1.22
29603282
49574736
1.19
1.25
0.20
0.35
0.06
3:2
1.50
21773347
32678375
1.33
1.69
0.37
0.42
0.09
13:7
1.86
22636043
29926022
1.51
2.28
0.63
0.50
0.14
7:3
2.33
86971562
98529637
1.77
3.08
1.01
0.57
0.19
3:1
3.00
51639933
45912325
2.25
4.00
1.67
0.64
0.26
4:1
4.00
27821233
14901292
3.73
4.28
2.93
0.65
0.45
a
1
obtained from H-NMR spectra by fitting peaks to the lysine backbone CH region
b
obtained from 1H-NMR spectra by fitting peaks to tyrosine’s CH2 region
c
calculated after division of the Tyr(Bzl) peak areas by two
6
Figure S8. Determination of the copolymerisation parameters for the Glu(Bzl) / Tyr(Bzl)
copolymerisation using the approach by Fineman-Ross (a) and Kelen-Tüdös (b).
7
Figure S9. Determination of the copolymerisation parameters for the Lys(Z) / Tyr(Bzl)
copolymerisation using the approach by Fineman-Ross (a) and Kelen-Tüdös (b).
8
Figure S10. GPC data for the terpolymers obtained from Glu(Bzl)-NCA, Lys(Z)-NCA and Tyr(Bzl)-NCA
at various ratios.
9
Figure S11. Concentrations of monomers obtained from HPLC measurements during the course of
the terpolymerisation using seven different ratios of Glu(Bzl)-NCA, Lys(Z)-NCA Tyr(Bzl)-NCA and total
conversion of monomers over time.
10
Figure S12. Determination of the reaction rate constants (k) for the terpolymerisation of Glu(Bzl)NCA / Lys(Z)-NCA / Tyr(Bzl)-NCA at various monomer starting concentration using the integrated rate
[𝑁𝐶𝐴]
law for a 1st order reaction: 𝑙𝑛 ([𝑁𝐶𝐴] ) = −𝑘𝑡 ([NCA] – concentration of the respective amino acid
0
NCA, [NCA]0 – theoretical NCA starting concentration; t – reaction time). k was determined from the
slope of the linear regression lines. Datapoints originate from single kinetic experiments.
11
Figure S13. 1H-NMR spectrum of the terpolymer in TFA obtained after reaction for 20 min at a
monomer ratio of 1:1:1 in the feed.
12