13

ENZYMATIC SYNTHESIS OF POLYAMIDES C NMR spectroscopy.

Comparison with Chemical Synthesis. It may be noted that DETA
Qu-Ming Gu, William W. Maslanka, and H. N. Cheng* adipate polyamide is currently made4 via the chemical route at 180C. The
enzymatic synthesis is carried out at lower temperatures (24-100C). The
Hercules Incorporated Research Center molecular weight ranges for both types of polymerization are comparable (Mn
500 Hercules Road ~ 3800 daltons, Mw ~ 8000-9000 daltons). The polydispersity (Mw/Mn) for the
Wilmington, DE 19808-1599 enzymatic process is lower, around 2.2-2.7, as compared to chemical
polymerization at higher temperature (polydispersity 2.6-3.0).
Introduction Use of Protease. Since amide formation is involved, we also tried to use
The use of lipases for the synthesis of small molecules is well known1. proteases. After several attempts, only lower-molecular-weight materials were
Lipases have also been successfully applied to polyesterification in several achieved. Apparently a protease is not active as a lipase in catalyzing the
cases2 and to polymer modification reactions3. Some of these methods have synthesis of the polyamide. Another advantage of lipase catalysis is that once
relied upon the use of organic solvents as cosolvent or sole media, whereas the amide bond is formed, it cannot be hydrolyzed by any remaining lipase
others are solvent-free processes. activity, so the polyamide product can be dissolved and used in water.
While it is well known that lipase can catalyze reverse reactions and Optimal Reaction Conditions. We found good results by reacting
form polyesters, it is little known that hydrolases can catalyze aminolysis of diester and diamine in approximately equal molar ratio. The reaction may also
diesters to form high-molecular-weight polyamides. In view of industrial be carried out deliberately with stoichiometric imbalance. We have adjusted
interest in water-soluble polyamides4, we explored the use of enzymes as the proportion of the two monomers slightly to produce a polyamide with
catalysts for polyamide synthesis. We found lipases to be good catalysts in terminal amine units. Polyamides having terminal primary amine groups are
this polymerization reaction5. To our knowledge, prior to our work no one has useful in the synthesis of a variety of end-capped polymers.
made a synthetic high-molecular-weight polyamide using a lipase. Several lipases can be used in the polymerization reaction. The preferred
lipases are from the yeast Candida antarctica, Pseudomonas sp., or Mucor
Results and Discussion miehei. The amount of enzyme used is critical. A low level of enzyme results
The goal of this work is to use enzymes to catalyze the synthesis of in a slow reaction and a lower-molecular-weight product. A higher level of
polyamides under mild temperature conditions. The monomers are aliphatic enzyme usually gives a polyamide product having a higher molecular weight.
diesters and diamines with the following general structures: Preferably the enzyme is used at 1-3% by weight.
R1OOC-R-COOR2 H2N-[R3-X-R4-NH2]n The reaction temperature is preferably 50-90C. In general, the higher
where R, R3 and R4 are alkyl groups that may contain olefinic double bonds or the temperature, the higher is the molecular weight. The reactions are usually
heteroatoms, R1 and R2 are methyl or other convenient leaving groups, X may carried out at atmospheric or reduced pressures. The alcohol produced by the
be O, CH2, NH or S, and n is the number of the repeating unit. Frequently X = reaction can be removed during the reaction under reduced pressures or by
NH, and n = 1-5. Many of the examples below involved dimethyl adipate and evaporating in an opened reaction vessel at 70-100C.
diethylene triamine (DETA) as the monomers. The process is carried out either in an organic solvent or in the absence
Use of Lipase. A key discovery was that lipase could catalyze the of solvents. For economic reasons, the solvent-free process is preferred. One
formation of amide bond to give high-molecular-weight polyamides5. In a of the problems of using the solvent-free system is that the resulting
screening study, dimethyl adipate and DETA were mixed together with polyamide tends to solidify at the low temperature range (50-80C), which
different lipases or esterases in the absence of a solvent and incubated at room slows down or terminates the polymerization reaction. It has been found in
temperature for two days. The samples were withdrawn at 24 hrs and 48 hrs some cases that certain solvents such as ethylene glycol and glycerol can
for IR analysis. The extents of the reactions were estimated by the overcome this problem.
disappearance of the ester absorption (1745 cm-1) and the appearance of amide The molecular weight of the polyamides synthesized in the present work
absorption (1650 cm-1). The results are summarized in Table I. ranges from 2000 to 9000 daltons and depends on the amount of enzyme used,
O H O the reaction temperature, and monomers used for the reaction. As indicated
OCH3 Lipases
H3CO + H2N N HN NH
NH2 o
60-100 C N earlier, a typical polymer has Mn ~ 3800, and Mw ~ 8000-9000 daltons. The
O O H n 13
Dimethyl adipate DETA
C NMR spectrum of a typical product appears relatively clean, suggesting
Polyamidoamine (MW = 9K)
the absence of significant extraneous reactions.
Other Polyamides. The lipase employed in the polymerization reaction
Table I. Enzyme-Catalyzed Amidation between Dimethyl Adipate and appears to be non-specific. The same reaction can be readily extended to other
DETA diesters and diamines. For example, the amide of DETA and dimethyl
# Lipase sources Form Yield4 Form Yield4 succinate can be made using the same procedure.
(24hr) (24hr) (48hr) (48hr) O
Control Liquid <2% Liquid 2-6% H3CO
OCH3
2 Candida antarctica1 Solid 30-40% Solid 60-70% O
H O
2 Dimethyl succinate N
3 Pseudomonas sp. Liquid 5-10% Solid 20-30% o
HN N
Candida lipases, 80-95 C H O n
4 Candida rogasa3 liquid - Semi-solid 10-15%
MW = 4200
5 Porcine panceas3 liquid - Liquid - O O
2
6 Mucor javanicus solid 20-30% Solid 30-40% H3CO OCH3
H Dimethyl malonate H O O
7 Penicillium liquid - Liquid - H2N
N
NH2 N
HN NH
camemberti2 DETA
Novozym 435, 60-80 C
o
n

8 Aspergillus niger2 liquid - Semi-solid ~10% O O MW = 8000

1 H3CO OCH3
From Novozymes. 2From Sigma. 3From Amano. 4Amide/[Amide+Ester],
estimated by IR.
Dimethyl phenylmalonate HN NH
N
In another experiment, dimethyl adipate, DETA and Novozym 435 Novozym 435, 80-100 C
o H O O n
MW = 3600
lipase (from Novozymes A/S) were mixed in a 250-ml flask and heated at
90C for 16 hrs under nitrogen. 150 ml of methanol was added to dissolve the
polyamide product. The immobilized enzyme was insoluble in the methanol
solution and was removed by filtration. Methanol was removed by
evaporating using a rotary evaporator under a reduced pressure to give the
product as a yellowish solid. The yield was ca. 87%: Mw 8,400; Mw/Mn 2.73.
The chemical structure of the polyamide was characterized by infrared, 1H and

Proceedings Published 2006 by the American Chemical Society

 This enzymatic reaction can be used to make some polyamides that may
be difficult (or impossible) to do by chemical methods. For example, malonic
acid and diethylene triamine do not readily form a high-molecular-weight
polymer using high-temperature chemical synthesis because cyclization can
occur at chain terminus. This polymer can be readily made by lipase catalysis.
Similarly, a new polyamide can be made from diethyl phenylmalonate and
DETA. The procedures are given in the Experimental Section.
Fumaric (or maleic acid) and diethylene triamine also do not polymerize
well in a high-temperature chemical reaction because the amine (in diethylene
triamine) can add to the double bond of the fumaric and maleic acid (Michael
reaction). Yet, these reactions can be carried out using the enzyme process
described herein.
O O
OCH3 +
H
N HN NH
H3CO H2N NH2 N
O O H n
Dimethyl Fumarate DETA
Variations can also be made in the diamine structure. For example, the
enzyme-catalyzed reaction can be conducted with dimethyl adipate and
triethylene tetraamine or tetraethylene pentaamine. Moreover, heteroatoms
can be placed in the diester or diamine without much effect on the reaction.
An example is shown below for triethylene glycol diamine.
O
OCH3 + O O
H3CO H2N O NH2
O 3.12-3.30 ppm, corresponding to central methylene in the adipic moiety,
Dimethyl adipate Amine-ended triethylene glycol
O diethylene triamine, and methylene adjacent to amide nitrogen, respectively.
Lipases
o
HN
O
O
O
NH
60-100 C
O n
Linear polyamide (MW = 4K)
The enzymatic polyamides made via DETA and diesters have linear
backbone structures because the secondary amines are not reactive under the
mild reaction conditions used. These secondary amines can be further reacted
with epichlorohydrin to produce resins that impart wet strength when applied
to paper 5. Thus, these polyamides are potentially wet strength resins in paper
applications.
Experimental
Materials. All chemical compounds were obtained from commercial
sources (e.g., Sigma-Aldrich). The enzymes were obtained from Novozymes
A/S, Amano Enzyme USA Co. Ltd., and Sigma-Aldrich.
Synthesis of DETA-Adipate Polyamide. A typical synthetic
procedure is shown herein. Dimethyl adipate (6.97 g, 0.04 mol), diethylene
triamine (4.12 g, 0.04 mol) and Novozym 435 lipase (0.5 g) were mixed in a
100 ml flask and heated in an oil bath to 90C. The flask containing the
viscous mixture was kept in the oil bath at 90C for 16 hrs in an open vessel
with a stream of nitrogen. The product turned into a yellowish solid at the end
of the reaction. 60 ml of methanol was added to dissolve the polyamide
product. The immobilized enzyme was insoluble in the methanol solution and
was removed by filtration. Methanol was removed by evaporation using a
rotary evaporator under reduced pressures to give the product as a yellowish
solid. The yield was 8.4 g: Mw, 6,700; Mw/Mn, 2.20.
Synthesis of DETA-Malonate Polyamide. Dimethyl malonate (39.40
g, 0.30 mol), diethylene triamine (30.90g, 0.30 mol) and Novozym 435 (2 g)
were mixed in a 250 ml flask and heated in an oil bath to 80C. The mixture
was stirred at 80C. After being stirred at this temperature for 16 hrs in an
open vessel, the reaction mixture solidified. 150 ml of methanol was added to
dissolve the polyamide product. The immobilized enzyme was insoluble in the
methanol solution and was removed by filtration. Methanol was removed by
evaporation using a rotary evaporator under reduced pressures to give the
product as a brown solid. The yield was 50 g: Mw, 8000; Mw/Mn, 2.10.
Alternatively, we can use Palatase lipase available from Novozymes
A/S, which is a lipase from Rhizomucor miehei. 20 g of analytical grade
Celite and 100 ml of 25% Palatase solution in 0.1M phosphate buffer at pH
7.0 were mixed and stirred at 10C for 3 hours. The mixture was lyophilized
to provide 37.6 g of immobilized Palatase powder. The synthesis of the
polyamide can then be done using the same procedure as before.
Proceedings Published 2006 by the American Chemical Society
Polymerization of DETA-Phenylmalonate. Diethyl phenylmalonate
(23.6 g, 0.10 mol), diethylene triamine (10.3 g, 0.10 mol) and Novozym 435
lipase (1 g) were mixed in a 500-ml flask and heated in an oil bath to 100C.
The viscous mixture was stirred at 90-100C for 24 hrs in an open vessel with
a stream of nitrogen. The mixture solidified at the end of the reaction. The
product was not soluble in most organic solvents and in water at neutral pH. It
was soluble in water at pH 3. 150 ml of water was added and the pH was
adjusted to 3 by adding concentrated HCl. The immobilized enzyme (being
insoluble in water) was removed by filtration. The aqueous solution was
lyophilized to give the product as a white solid. The yield was 26.9 g.: Mw,
3600; Mw/Mn, 2.70.
Polymerization of DETA-Fumarate. The dimethyl fumarate (14.4 g,
0.10 mol), diethylene triamine (10.3 g, 0.10 mol) and 1.0 g of Novozym 435
lipase were mixed and stirred at 50C for 16 hrs under nitrogen. 50 ml of
methanol was added and the immobilized enzyme was removed by filtration.
The methanol was removed by evaporation using a rotary evaporator under
reduced pressures. The polymer was obtained as a sticky semi-solid. The
yield was 23.0 g: Mw, 3060; Mw/Mn, 1.85.
Characterization. The polyamides were analyzed by infrared and 1H
and 13C NMR. IR clearly showed the formation of secondary amide by a
strong absorption at 3300 cm-1 (N-H stretch), 1650 cm-1 (O=CNHR stretch
band I) and 1560 cm-1 (O=CNHR stretch band II). 1H NMR (in D2O) showed
only four multiplets at 1.35-1.55 ppm, 2.05-2.20 ppm, 2.55-2.70 ppm and
methylene adjacent to carbonyl, methylene adjacent to the central amine of
13
C NMR showed five peaks at 25.4, 35.8, 39.1, 47.5 and 177.0 ppm,
corresponding to central carbons in the adipic moiety, carbons adjacent to
carbonyl, carbons adjacent to the central amine of diethylene triamine, carbons
adjacent to amide nitrogen, and amide carbons, respectively. The data
provided confirmation of the polyamide structure.
Conclusions
Polyamides are well-known industrial products having applications in a
variety of areas. The current work produces polyamide resins by enzyme-
catalyzed polymerization of diester and diamine monomers. For example, a
water-soluble polyamide can be produced via lipase-catalyzed polymerization
of dimethyl adipate and ethylene triamine at 24-1000C. An equivalent
chemical reaction would require elevated temperatures (180C). The
enzymatic reaction is easy to do and applicable to a range of diacids and
diamines. In addition, this enzymatic reaction permits the synthesis of several
new high-molecular-weight polyamides that cannot be made through the
chemical, high-temperature method. Examples are the polyamides from
diethylene triamine and malonate, and diethylene triamine and fumarate.
Acknowledgements. The authors would like to thank Robert G. Nickol
and Ronald R. Staib for helpful discussions and Sadhana Mital and Gordon
Tozer for technical assistance.
References:
(1) For example, a) Djeghaba, Z.; Deleuze, H.; DeJeso, B.; Messadi, D. and
Maillard, B. Tetrahedron Lett., 1991, 32, 761-762. b) Kanerva, L. T.;
Csomos, P.; Sundholm, O.; Bernath, G. and Fulop, F., Tetrahedron.
Asymm., 1996, 7, 1705-1716. c) Vorde, C.; Hogberg, H.-E. and
Hedenstrom, E. Tetrahedron. Asymm., 1996, 7, 1507-1513.
(2) a) Chaudhary, A.K.; Lopes, J.; Beckman; E. J. and Russell, A. J.
Biotechnol. Prog., 1997, 13, 318-325. b) Brazwell, E. M.; Filos D. Y.;
and Morrow, C. J., J. Polym. Sci., Part A: Polym. Chem. 1995, 33, 89. c)
Linko, Y. Y;. Wang, Z. L. and Seppala, J., Enzyme Microb. Technol.,
1995, 17, 506-511. d) Binns, F.; Roberts, S. M.; Taylor, A.; Williams, C.
F., J. Chem. Soc., Perkin Trans. 1993, 1. e) Geresh, S. and Gilboa,,Y.
Biotechnol. Bioeng., 1991, 37, 883-888. f) Taylor, A.; Binnis, F., WO
94/12652.
(3) For example, Gu, Q.-M.; Cheng, H. N. ACS Symp. Ser. 2005, 900, 427.
(4) For example, a) Espy, H. H. TAPPI J. 1995, 78, 90. b) Riehle, R. J. ACS
Symp. Ser. 2005, 900, 302.
(5) Cheng, H. N.; Gu, Q.-M.; Maslanka, W. W. US Patent 6,677,427.
January 13, 2004.