CAL-B catalyzed regioselective bulk polymerization of l-aspartic acid diethyl ester to α-linked polypeptides

Article abstract of DOI:10.1039/c7cc01300k

This paper reports that the bulk polymerization of l-aspartic acid diethyl ester catalyzed by immobilized CAL-B at 80 °C for 24 h gives primarily (～95%) α-linked poly(l-aspartate) in 70% yield with DP_avg = 50 and regioselectivity (α/β) = 94?:?6. Plots of log{[M]₀/[M]_t} vs. time and DP_avgvs. conversion indicate that this polymerization proceeds in a controlled manner by a chain-growth mechanism up to 90% conversion. Thereafter, competition occurs between chain growth and step mechanisms.

Full text of DOI:10.1039/c7cc01300k

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: F. Totsingan, R.
Centore and R. Gross, Chem. Commun., 2017, DOI: 10.1039/C7CC01300K.
This is an Accepted Manuscript, which has been through the
Volume 52 Number
1 4 January 2016 Pages 1–216
Royal Society of Chemistry peer review process and has been
accepted for publication.
ChemComm
Accepted Manuscripts are published online shortly after
Chemical Communications
www.rsc.org/chemcomm
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
=
3
rsc.li/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 4
View Article Online
DOI: 10.1039/C7CC01300K
Chemical Communications
COMMUNICATION
CAL-B Catalyzed Regioselective Bulk Polymerization of L-Aspartic
Acid Diethyl Ester to α-linked Polypeptides
Filbert Totsingan, Robert Centore, and Richard. A. Gross*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/chemcomm
This paper reports that the bulk polymerization of L-aspartic acid diethyl
ester catalyzed by immobilized CAL-B at 80 oC for 24 h gives primarily
mixture of α- and β-linked repeat units. This work describes for the
first time simple and environmentally friendly route to
a
(~95%) α-linked poly(L-aspartate) in 70% yield with DPavg
= 50 and
enantiopure poly(α-aspartic acid).
regioselectivity (α/β) = 94:6. Plots of log{[M]0/[M]t} vs time and DPavg vs
conversion indicate that this polymerization proceeds in a controlled
manner by a chain-growth mechanism up to 90% conversion. Thereafter, a
competition occurs between chain growth and step mechanisms.
Important advantages of enzyme vs chemically catalyzed
reactions include that enzymes: i) are of natural origin, ii) provide
high enantio- and regio-selectivity, iii) function under mild
conditions (e.g. temperature) and iv) do not require strict exclusion
of water and oxygen.10,11 Immobilized Lipase B from Candida
antarctica (CAL-B) is known to efficiently catalyze polyester
synthesis by polycondensation and ring-opening polymerization
mechanisms.11-15 Immobilized CALB has also been reported to
catalyze the synthesis of polyamides by reaction of aliphatic and
aromatic diesters and diamines.16-18 Of particular interest herein is
the regioselective amidation of aspartic acid at either the α- or β-
carboxyl group. Conde et al. conducted a model study on the
amidation of N-protected (benzyloxycarbonyl, N-Cbz; tert-
butyloxycarbonyl, N-Boc; and acetyl, N-Ac) aspartic acid diethyl
esters.19 Reactions were performed in anhydrous diisopropyl ether
at 60 oC using immobilized CAL-B as catalyst. Generally, the L-
aspartate substrate series yielded α-monoamides whereas the
Poly(aspartic acid) is a water-soluble poly(amino acid) that is
biodegradable in various bioactive environments.1 It has a number
of unique properties that result in its utility in a broad range of
applications such as drug delivery, corrosion inhibitors and
superadsorbants.2-5 Established synthetic routes to poly(α-aspartic
acid) include ring-opening polymerization (ROP) of the
corresponding N-carboxyanhydride (NCA) monomer, protease-
catalysis,6 and solid phase peptide synthesis.7 While protease-
catalyzed polymerizations in bulk6a or organic/aqueous media6b
have several advantages including reduced use of protection-
deprotection steps and toxic chemical reagents, it results in
oligomer mixtures with degree of polymerizations (DP) from about
9-19. Solid phase peptide synthesis (SPPS) is impractical for the
synthesis of high molecular weight homo-polypeptides since it
procedes stepwise using multiple protection-deprotection steps,
solvents and toxic reagents. However, for short chains (2-30
residues), SPPS provides a reliable route to uniform sequence and
chain length peptides. For the synthesis of high molecular weight
poly(α-amino acid) including poly(α-aspartic acid), NCA ROP is the
preferred method.8 Though, NCA ROP requires the use of toxic
reagents such as phosgene, protection-deprotection and high purity
monomers. An alternative route to polyaspartate is by thermal or
acid-catalyzed polymerizations.3,9 However, the harsh conditions
involved result in racemization and chains consisting of a complex
corresponding
D-aspartate substrates showed β-selectivity. The
degree of regioselectivity was a function of the N-protecting group.
The smallest protecting group gave the lowest α/β-ratio (3.8). It is
well known that regioselectivity may also vary due to changes in the
reaction media (e.g. solvent vs. bulk reactions) as well as when the
substrate is no longer a simple monomeric substrate.20
Recently, Zhang et al investigated the solvent-free lipase-
catalyzed synthesis of D- and L-polyaspartates.21 At 80 oC for 72
hours using 20%-by-wt immobilized CAL-B, D- and L-polyaspartates
were obtained with DPavg of about 60 in 82% and 72% yield,
respectively. Due to the presence of two different acyl moieties in
aspartic acid diethyl ester, the repeat units may be linked between
the α-amino and either the α- or β-carboxyl group (e.g. α-links, β-
links or a mixture of both) (Figure 1). Based on 1D-1H NMR, Zhang
et al concluded that the polyaspartates obtained were
predominantly β-linked (β/α = 96:4) although the chemical shifts
Department of Chemistry and Chemical Biology, Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), 4005B
Biotechnology Building, 110 Eighth Street, Troy, New York 12180, USA.
Email: grossr@rpi.edu
† Electronic Supplementary Information (ESI) available: Experimental Procedures,
C
1D 1H NMR of poly(Et-L-Asp) in DMSO-d6, 1D 13C NMR, 2D 1H-1H COSY, 2D 1H-13
HSQC, monomer conversion vs reaction time, DPavg vs monomer conversion, DPavg
and conversion vs enzyme loading. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
Chemcomm., 2016, 00, 1-4 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
View Article Online
DOI: 10.1039/C7CC01300K
COMMUNICATION
Journal Name
and CD signs in chloroform were inconsistent with those of poly(β-
aspartates) reported in the same solvent.22
This paper reports a reinvestigatation of the regioselectivity of
this interesting lipase-catalyzed synthesis. This includes full
.
a
structural characterization of the resulting poly(Et-L-aspartate) by
1D and 2D-NMR along with further studies to interrogate the
mechanism of chain growth, reactions were performed at times
from 2 to 24 h and plots of DPavg versus monomer conversion and
log[M]0/[M]t vs reaction time were constructed. Further
characterizations of poly(Et-L-aspartate) synthesized herein was
conducted by trifluoroacetic acid (TFA)-induced conformational
changes monitored by NMR and CD. Also, the thermal properties of
poly(Et-L-aspartate) prepared by lipase catalysis is reported.
Figure 3. 2D 1H-Carbonyl region of HMBC spectrum (600 MHz) in CDCl3 of
poly(β-Et-α-Asp) obtained from 24 h reaction catalyzed by 20 wt.% CAL-B at
80 oC. A schematic representation of different correlations is also shown.
Figure 1. Chemical structures of poly(β-Et-α-aspartate) and poly(α-Et-β-
aspartate) with repeat units linked through the α- (left) and β- (right)
carboxylate moieties, respectively.
Figure SI-1) due to amide bond formation. The appearance of two
carbonyl signals (amide and ester) in the 13C NMR spectrum of
poly(L-Et-Asp) (Figure SI-2) at 166.5 ppm and 170.8 ppm,
respectively, confirms the formation of an amide bond. In addition,
the integral ratio of the CH3 1H NMR signal (A) of ethyl moieties to
the β-CH2 signal (D) decreased from 3 to ~ 1.5 (Figure 2). This
corresponds to consumption of about 50% of ethyl ester moieties.
Control reactions (without enzyme) were also carried out for 24
hours at 80 oC and no polymer/oligomer was detected by NMR
(Figure SI-5).
B
a)
A
A
D
C
E
A
B
B
E
D
C
L-Asp(OEt)2 has ethyl ester groups linked to the α- and β-
B
carbonyl carbons.
monomer could result in either poly(β-Et-α-Asp) with α-carboxylate
moieties, poly(α-Et-β-Asp) with β-carboxylate moieties, or
Hence, enzymatic polymerization of this
b)
A
C(α)
A
D
C
A
a
B
B
E
C(β)
mixture of both amide bond linkage chemistries (Figure 1). Figure
2b shows that there is a prominent peak at 4.38 ppm along with a
less intense resonance at 4.82 ppm. Analysis by 1H-1H COSY and 1H-
13C HSQC, as well previously reported data on poly(β-aspartate)s,22
confirm that both of these signals correspond to protons (C) (see
Figures SI-3 and SI-4). The assignment of NMR signals was further
E
C(α)
D
C(β)
1
Figure 2. 1D H NMR (600 MHz) in CDCl3 of: (a) L-aspartic acid diethyl ester
(L-Asp[OEt]2) and (b) poly(Et-L-aspartate) obtained after 24 h reaction
catalyzed by 20% immobilized CAL-B at 80 C. [Can you improve the clarity
1
analyzed by conducting a 2D H-13C HMBC (Heteronuclear multiple-
o
bond correlation spectroscopy) experiment. The 1H-carbonyl
correlation region from the 2D 1H-13C HMBC is displayed in Figure 3.
Of the two carbonyls present, the up-field resonance at 166.5 ppm
has a strong correlation with the –NH proton (E) and, therefore, is
assigned to the –CO– next to the –NH amide (2J(CO-NH), strong).
of the x-axis ppm numbers]
The polymerization of L-aspartic acid diethyl ester (L-Asp(OEt)2),
using immobilized CAL-B (20%-by-wt), was carried out at 80oC for
24 hours. One-dimensional proton (1H) NMR as well as 2D 1H-13C
HMBC were used to assign the relative content of α- and β-links in
Also, this carbonyl strongly correlates with the α-H (C) (2J(COamide
-
the product. All signals were assigned based on 2D H-1H COSY and
1
1H-13C HSQC (Figures SI-3 and SI-4). The 1D 1H NMR spectrum of
poly(Et-L-aspartate) obtained from the 24 hour reaction (Figure 2b)
showed several signal shifts relative to the corresponding signals of
the monomer (Figure 2a). Prominent examples include: i) the α-H
(C) at 3.79 ppm of L-Asp(Et)2 shifted downfield to 4.38 ppm (Cα) and
4.82 ppm (Cβ), respectively (see below for discussion on
assignments); ii) the free amine proton signal (E) of L-Asp(OEt)2 at
1.83 ppm shifted downfield by ~ 7 ppm (8.18 ppm in DMSO-d6, see
HC), strong) at 4.38 ppm and displays a weaker correlation with β-
CH2 protons (D) (3J(COamide-HD), weak) at 3.00 ppm. This reveals
unambiguously that the amide bond is formed through the α-acyl
group. The high preference by CAL-B for formation of α-linked
repeat units agrees with work by Conde et al on CAL-B catalyzed
19
regioselective amidation of Asp(OEt)2 but contrasts with the
report by Zhang et al.21 Further confirmation is derived from that
2 | Chemcomm., 2016, 00, 1-4
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 3 of 4
View Article Online
DOI: 10.1039/C7CC01300K
Journal Name
COMMUNICATION
the more downfield carbonyl at 170.8 ppm exhibits a strong chemical environment in immobilized CAL-B bulk L-Asp(Et)2
correlation with β-CH2 protons (D) (2J(COester-HD, strong) at 3.00
polymerizations changes with conversion, the regioselectivity
ppm and weakly correlates with both α-H (C) (3J(COester-HC), weak)
during chain growth may also vary. Regioselectivity (ratio of α- to β-
linked repeat units) was determined as described above from the
at 4.38 ppm and the CH2 ethyl ester (B) (3J(COester-HB), weak)
integral ratio of signals at 4.38 ppm (C(α), α-H in α-linkage) to that
protons at 4.19 ppm. The signal at 4.82 ppm is assigned to the α-H
of β-linked repeat units. This assignment agrees with published data
at 4.82 ppm (C(β), α-H in β-linkage). The ratio of α/β-linked repeat
units varies irregularly and to small extents with monomer
(4.8-5.0 ppm in CDCl3) for poly(α-alkyl-β-aspartate)s prepared by conversion with an average value of about 95:5. Hence, high
regioselectivity was retained throughout the polymerization. The
effect of enzyme loading was also studied (Figure SI-8). Decrease in
enzyme loading from 20 to 10 and 5 wt% resulted in decreases in
conversion (98%, 81% and 68%, respectively) and DPavg values (50,
25 and 18, respectively).
ring-opening polymerization of optically pure (S)-4-alkoxycarbonyl-
2-azetidinones.22
Table 1. Immobilized CAL-B catalyzed bulk polymerization of L-
aspartate diethyl estera
Poly(β-Et-α-L-Asp) is known to form a right-handed α-helix
conformation.24 Hence, circular dichroism (CD) and NMR
measurements were performed to further confirm that repeat units
are indeed α-linked. The CD spectrum of poly(β-Et-α-L-Asp) in
methanol (Figure 4B, inset) displays a typical trough at 223 nm,
consistent with the right-handed α-helix conformation reported in
the literature for poly(β-Et-α-L-Asp) synthesized by ethanolysis of
poly(β-benzyl-α-L-Asp).24 In chloroform (Figure 4B), the negative
peak is slightly shifted to 231 nm, suggesting the presence of an
additional side-chain ordered structure linked to the helix. This
phenomenon was also observed by Tsujita et al when studying
other poly(β-alkyl-α-Asp)s.25 A loss of CD intensity occurred upon
addition of TFA in the 1-5% range, suggesting a helix-coil transition.
Furthermore, 1H NMR spectra in CDCl3 showed a down-field shift of
α-H by about 0.25 ppm upon addition of 1-10% (v/v) TFA (Figure
4A). This is consistent with the down-field shift found in the helix-
coil transition of poly(α-amino acid)s.26 Furthermore, the down-
field displacement observed for α-H is opposite to the upfield shift
displayed by α-H in poly(α-alkyl-β-Asp)s.22
d
Entry
Time
(h)
Conversion
(%)b
Yield
(%)c
DPavg
Regioselectivity
(α/β)d
1
2
3
4
5
6
7
24
18
15
12
8
98
95
92
90
47
39
17
70
66
62
60
30
26
9
50
44
41
37
18
10
6
94:6
93:7
94:6
95:5
97:3
98:2
93:7
4
2
a reactions were conducted at 80 C in bulk under vacuum by immobilized
o
b
1
CAL-B (20 wt-% relative to monomer). Estimated from H NMR of crude
products. c Isolated yields. d Calculated from 1H NMR of pure products.
The effect of reaction time on the progress of immobilized CAL-
B-catalyzed condensation polymerizations of L-Asp(Et)2 at 80 oC was
investigated. To assess the change in molecular weight with time,
1
the DPavg = n was calculated from 1D H NMR spectra by measuring
the integral ratio of methyl proton signals (A) of esters at 1.28 ppm
to methylene β-CH2 protons signals (D) at 2.75-3.16 ppm
[
3(n+1)/2n = A/D]. Alternatively, the DPavg was calculated from the
integral ratio of the internal methyne α-H protons (Cα) at 4.38 ppm
to the N-terminal α-H (Cα-end) at 3.74 ppm [n-1 = Cα/Cα-end].
Determinations of DPavg for the 24 h reaction by both of the above
integral ratios are in excellent agreement (50 and 48.5,
respectively).
Both conversion and yield gradually increase from 17% and 9%,
respectively, at 2 h to 98% and 70%, respectively, at 24 h (Table 1).
Furthermore, from 2 to 24 h the DPavg increases from 6 to 50. The
semilogarithmic plot of conversion vs. time (Figure SI-6), when fit
by linear regression analysis, has an R2 of 0.9597. Hence, the
polymerization does not proceed by a conventional step-growth
mechanism but, instead, by chain growth. Furthermore, the DPavg
linearly increases with conversion up to 90% (Figure SI-7).
Thereafter, a large linear increase in DPavg from 37 to 50 occurs with
a small increase in conversion (from 90% to 98%). This corresponds
to a change in the dominant mechanism from chain to step-growth
at high monomer conversions (>90%).23
Figure 4. Trifluoroacetic acid (TFA)-induced conformational changes in
poly(β-Et-α-Asp): (A) α-H signal shift in the presence of 1-10% TFA (v/v) in
CDCl3; (B) circular dichroism (CD) spectra in 0-5% (v/v) TFA in chloroform.
The CD spectrum in methanol is also shown (inset).
Thermal analysis was performed on three poly(β-Et-α-Asp)s
with Dpavg of 18, 37 and 50 prepared at reaction times 8, 12 and 24
h, respectively. Thermogrametric analysis (TGA) (Figure SI-9)
showed similar decomposition temperatures (Td = 391, 393, 393 oC,
CAL-B has been reported to catalyze a wide range of organic
transformations with high stereo- and regioselectivities.19 As the
This journal is © The Royal Society of Chemistry 20xx
Chemcomm., 2016, 00, 1-4 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
View Article Online
DOI: 10.1039/C7CC01300K
COMMUNICATION
Journal Name
respectively) for all three polyaspartates. Interestingly, the 17 P. Steunenberg, M. Uiterweerd, M. Sijm, E. L. Sott, H. Zuilhof, J. P.
decomposition process occurs in a single step in contrast to the
two-step process (formation of imides followed by chain cleavage)
usually observed for β-polyaspartates [poly(α-Et-β-Asp): Td1 = 261
M. Sanders and M. C. R. Franssen, Curr. Org. Chem., 2013, 17,
682-690.
18 B. Sharma, A. Azim, H. Azim, R. A. Gross, E. Zini, M. L. Focarete
and M. Scandola, Macromolecules, 2007, 40, 7919-7927.
19 S. Conde and P. Lopez-Serrano, Eur. J. Org. Chem., 2002, 5, 922-
929.
o
oC and Td2 = 368 C].27 The DSC traces of all three polymers (Figure
SI-10) exhibited a clear endotherm in the range of 175-187 oC.
Furthermore, the melting temperature (Tm) decreases as the DPavg
increases. This is likely due to increasing chain flexibility with
20 A. M. Klibanov, Nature, 2001, 409, 241-246.
21 Y. Zhang, B. Xia, Y. Li, Y. Wang, X. Lin and Q. Wu,
Biomacromolecules, 2016, 17, 362-370.
increased DPavg
.
22 A. M. de Ilarduya, C. Alemán, M. García-Alvarez, F. López-
Carrasquero and S. Muñoz-Guerra, Macromolecules, 1999, 32,
3257-3263.
ACKNOWLEDGEMENT
23 Y. Mei, A. Kumar and R. Gross, Macromolecules, 2003, 36, 5530-
5536.
The authors are grateful for funding received from the NSF Division
of Materials Research Biomaterials (BMAT) Program (award no.
1508422).
24 (a) E. M. Bradbury, B. G. Carpenter and H. Goldman,
Biopolymers, 1968, 6, 837-850. (b) M. De Zotti, F. Formaggio, M.
Crisma, C. Peggion, A. Moretto and C. Toniolo, J. Pept. Sci.,
2014, 20, 307-322. (c) S. León, C. Alemán and S. Muñoz-Guerra,
Macromolecules, 1997, 30, 6662-6667.
REFERENCES
1 R. A. Gross and B. Kalra, Science, 2002, 297, 803-807.
2 H. Uchida, K. Miyata, M. Oba, T. Ishii, T. Suma, K. Itaka, N.
Nishiyama and K. Kataoka, J. Am. Chem. Soc., 2011, 133, 15524-
15532.
25 Y. Tsujita, Biophys. Chem., 1988, 31, 11-20.
26 (a) E. M. Bradbury, P. D. Cary, C. Crane-Robinson P. G. Hartman,
Pure Appl. Chem., 1973, 36, 53-92. (b) F. A. Bovey, J. Polym. Sci.,
Macromol. Rev., 1974, 9, 1-81.
3 S. Roweton, S. J. Huang and G. Swift, J. Environ. Polym. Degrad.,
1997, 5, 175-181.
4 Y. Sanada, I. Akiba, S. Hashiba and K. Sakurai, J. Phys. Chem. B,
2012, 116, 8241-8250.
27 F. López-Carrasquero, M. García-Alvarez, and S. Muñoz-Guerra,
Polymer, 1994, 35, 4502-4510.
5 M. Maeda, M. Kimura, Y. Hareyama and S. Inoue, J. Am. Chem.
Soc., 1984, 106, 250-1.
6 (a) S. Matsumura, Y. Tsushima, N. Otozawa, S. Murakami, K.
Toshima and G. Swift, Macromol. Rapid Commun., 1999, 20, 7-11.
(b) Y. Soeda, K. Toshima and S. Matsumura, Biomacromolecules,
2003, 4, 196-203.
7 (a) B. K. Yoo, M. A. J. Miah, E.-S. Lee and K. Han, Arch. Pharm.
Res. 2005, 28, 756-760. (b) K. Ogawa, A. Ishizaki, K. Takai, Y.
Kitamura, T. Kiwada, K. Shiba and A. Odani, Plos One, 2013, 8, 1-
9 (e84335).
8 H. R. Kricheldorf, Angew. Chem. Int. Ed., 2006, 45, 5752-5784.
9
(a) E. Kokufuta, S. Suzuki and K. Harada, Bull. Chem. Soc. Jpn.,
1978, 51, 1555-1556. (b) T. Nakato, A. Kusuno and T. Kakuchi, J.
Polym. Sci. Part A: Polym. Chem., 2000, 38, 117-122.
10 S. Kobayashi and A. Makino, Chem. Rev., 2009, 109, 5288-5353.
11 R. A. Gross, M. Ganesh and W. Lu, Trends in Biotechnology, 2010,
28, 435-443.
12 S. Shoda, H. Uyama, J. Kadokawa, S. Kimura and S. Kobayashi,
Chem. Rev., 2016, 116, 2307-2413.
13 C. Robert and C. M. Thomas, Chem. Soc. Rev., 2013, 42, 9392-
9402.
14 A.-C. Albertsson and R. K. Srivastava, Adv. Drug Delivery. Rev.,
2008, 60, 1077-1093.
15 (a) S. Spinella, M. Ganesh, G. Lo Re, S. Zhang, J.-M. Raquez, P.
Dubois, and R. A. Gross, Green Chem. 2015, 17, 4146-4150. (b) R.
A. Gross, A. Kumar and B. Kalra, Chem. Rev., 2001, 101, 2097-
2124.
16 E. Stavila, G. O. R. Alberda van Ekenstein and K. Loos,
Biomacromolecules, 2013, 14, 1600-1606.
4 | Chemcomm., 2016, 00, 1-4
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins