Beruflich Dokumente
Kultur Dokumente
3, 1997
175
1064-7546/97/0700-0175512.50/0 9 1997 PlenumPublishingCorporation
176 Roweton, Huang, and Swift
0 0 O O
f~-~_CH2CrHs ~..(a),
r --,.
o o o o
(a)a-peptide (b)~-peptide
Fig. 2. Schematicrepresentationof the deprotectionof benzylgroups resultingin the formationof fl-aspartyl
linkages [4].
Poly(aspartic Acid) 177
merization method similar to the method described glutamate (3,-BLG). The NCA monomers were formed
above [16]. Polymerization yields a polysuccinimide, as previously described by the phosgenation of the cor-
which can be hydrolyzed in the presence of base to form responding or-amino acids in tetrahydrofuran. Solutions
PAA. The overall process has been shown to yield up (0.1 M) of the 3-BLA and "y-BLG were prepared in both
to 99 % conversion of aspartic acid to polyaspartate. The dioxane and in a 1 : 3 (v/v) mixture of dioxane and meth-
resulting thermal polyaspartate (TPA) was shown to be ylene chloride. The two NCA monomer solutions were
a copolymer having 70% of the amide bonds formed mixed in varying proportions and the copolymerization
from 3-carboxyl groups and 30% from a-carboxyl was initiated using triethylamine. The monomer-to-ini-
groups. tiator ratio used was 50. Following the copolymeriza-
Another method for the thermal polymerization of tion, the copolypeptides formed were precipitated at 4~
aspartic acid has been described in which poly(alkylene by adding four times the amount of methanol by vol-
glycol) is used as a dispersion medium [17, 18]. Aspar- ume. The methanol included 5 vol% of 0.1 N HCI. The
tic acid and poly(alkylene glycol) are heated to an ele- precipitate was washed in pure methanol and dried un-
vated temperature in the range from 120 to 300~ and der reduced pressure at 50~ Anhydrous HBr treatment
maintained at this temperature to form a polysuccinim- was employed for the debenzylation of 3-BLA and
ide. Poly(amino acid)s can then be produced by the hy- 3,-BLG residues. Fianlly, the debenzylated material was
drolysis of the polysuccinimides. dialyzed thoroughly against distilled water, filtered, and
A third method for the synthesis of poly(aspartic freeze-dried.
acid) homopolymers involves the polymerization of ma- Another method for the preparation of a PAA-con-
leamic acid obtained from the reaction of maleic anhy- taining copolymer involves the use of NCAs. This
dride and ammonia. The preparation of high molec- method was used to synthesize amphiphiles with poly(u-
ular weight polysuccinimides and, subsequently, aspartic acid) head groups [26]. These amphiphiles are
poly(aspartic acid) using this method has been described double-chain alkyl compounds with hydrophilic poly(u-
[19-22]. For example, Boehmke describes a process in aspartic acid) head groups. First, L-glutamic acid dido-
which maleic anhydride is melted and heated to 75~ decylamide (L-GAD) is obtained. The structure of the
Subsequently, 1 mol of 25% NHgOH is added to the L-GAD cation is shown in Fig. 4. The L-GAD is ob-
solution and then the solution is dried under vacuum. tained by coupling N-benzyloxycarbonyl-u-glutamic
The resultant maleic acid is reacted with ammonia at a acid with dodecylamine using a method described by
molar ratio of 1 : 1-1.5 at 120-150~ This reaction is Ihara et al. [26]. The L-GAD amine shown in Fig. 4 is
followed by the optional neutralization of the pendant then used as an initiator in the polymerization of poly(3-
acid groups and isolation of the resultant salts. benzyl-g-aspartate). The polymerization is accom-
A patent outlining a similar method for the prepa- plished as previously described, using the NCA 3-ben-
ration of the salt of poly(aspartic acid) is assigned to zyl-u-aspartate monomer. The structure of the resulting
SRchem Incorporated [23-25]. The process outlined in amphiphile following deprotection is shown in Fig. 5.
this patent involves reacting maleic acid and ammonia The common method of polypeptide synthesis de-
at a molar ratio of 1 : 1-2 at 190-350~ for a period of scribed previously, involving the use of NCAs, is an
up to 4 h. The resultant polymer can then be converted effective yet time-consuming approach. It requires prep-
to a salt by adding an alkaline-earth or alkali metal hy- aration of the appropriate NCA prior to polymeriza-
droxide or ammonium hydroxide. tions. This method can be applied only to the polymer-
ization of a-amino acids. An alternative method for
polypeptide synthesis has been described by Nishi et al.
SYNTHESIS OF POLY(ASPARTIC ACID)- and involves the use of dipbenylphosphoryl azide
CONTAINING COPOLYMERS (DPPA) and an adequate tertiary amine [27, 28]. This
method can be used to prepare poly(ot-amino acid)s and
Various types of amino acid-containing copoly- sequential polypeptides by direct polycondensation of
mers have been synthesized including copolymers con- peptides. In general, this method of polypeptide synthe-
taining aspartic acid. Copolymers of L-aspartic acid and
L-glutamic acid have been used to investigate the rate of
CH3(CH2)11---NHCO-'~H-NH3 +
simulated in vivo degradation of these materials as a
function of copolymer composition [3, 5]. In this study, ~H~ X_
copolypeptides were prepared using the NCA mono- CH2(CH2)ll--NHCO-CH2
mers of 3-benzyl-L-aspartate (3-BLA) and "y-benzyl-L- Fig. 4. Structureof the L-GAD[26].
178 Roweton, Huang, and Swift
n = 4, 7, and 13
Fig. 5. A representative amphiphile with a poly(L-aspartic acid) BIODEGRADATION
headgroup [26].
The biodegradation of poly(aspartic acid) and as-
sociated materials has been investigated. Recently,
sis involves 1.3 mol of DPPA and 2.3 mol of trieth- characterization of the biodegradation of poly(aspartic
ylamine per mol of amino acid. A solvent, such as di- acid) has been reported by Swift et al. [1] and Alford et
methyl sulfoxide, N,N-dimethylformamide, 1,4- al. [33]. Swift et al. describe the preparation of sodium
dioxane, or pyridine, is also required. This method often salts of poly(aspartic acid) from initial thermal poly-
eliminates the likelihood of racemization during synthe- merization of aspartic acid alone or with an acid catalyst
sis. or from the thermal polymerization of maleamic acid.
Copoly(amino acid)s have also been synthesized by The resulting polysuccinimides were subsequently hy-
direct polycondensation using the method described by drolyzed to form the sodium salts of poly(aspartic acid).
Nishi et al. [29-31]. DPPA was used as a condensation Semicontinuous activated sludge (SCAS) testing and
agent for the synthesis of these polymers. The random subsequent CO2 evolution testing were used to evaluate
copolymers produced were characterized by bimodal the rate and degree of biodegradation of the aspartic acid
molecular weight distributions. The effects of monomer polymers. It was determined that the acid-catalyzed
concentration, reaction time, solvents used, and tem- poly(aspartic acid) materials were significantly more
perature on molecular weight were investigated. Bulk biodegradable than the poly(aspartic acid) materials pre-
polymerization proved most effective in producing pared via thermal polymerization without an acid cata-
poly(amino acid)s with relatively high molecular lyst or those samples prepared from maleic anhydride
weights. DPPA has also been used to synthesize random and ammonia. Swift et al. report that a linear polyamide
copolymers consisting of a-amino acid and nylon units backbone is essential for polyaspartates to be totally
via direct copolycondensation [29-31]. biodegradable.
Poly(amino acid)s, including fl-benzyl-L-aspartate Alford et al. [33] studied the biodegradation of
(BLA), have been grafted onto a polystyrene copolymer thermally synthesized polyaspartate prepared via a dry
[32]. To accomplish this, a copolymer of styrene and thermal polymerization. Such a polymerization requires
1,4-divinylbenzene (DVB) is initially prepared. This is no reagents other than aspartic acid. This type of poly-
accomplished via a radical copolymerization of DVB merization is convenient for large-scale production and
and styrene at 45 ~ using 2,2'-azobis(2,4-dimethylval- is characterized by the fact that its only significant prod-
eronitrile) as an initiator. Benzene is used as the solvent. ucts are the polymer and the water produced by conden-
After the reaction is complete, the polymer is precipi- sation reactions. As described previously, the conden-
tated in petroleum ether and subsequently redissolved in sation polymerization produces a polysuccinimide that
benzene. Lyophilization is then used to isolate the poly- is subsequently hydrolyzed with base to form a polyam-
mer product. ide. A racemic mixture of aspartic acid as well as co-
Once the styrene/DVB copolymer has been synthe- polymer with amide bonds can be formed from both the
sized, the poly(/3-benzyl-L-aspartate) can be radically a- and the/3-carboxyl groups. Alford et al. investigated
grafted onto it. This reaction is initiated by mixing a the biodegradation of poly(aspartic acid) prepared via
THF solution of BLA/NCA with a dichloromethane so- dry thermal polymerization using biochemical oxygen
lution of the styrene/DVB copolymer at 35~ The re- demand (BOD) analysis, SCAS and CO2 evolution test-
action is complete after several days. The polymer prod- ing, TLC and GPC analysis, and adsorption assays in-
uct is precipitated in petroleum ether and then filtered volving various particulate adsorbents. Adsorbents in-
and dried thoroughly. cluded calcium carbonate, acid-washed kaolin, activated
In addition to the examples described above, a va- sludge, and subsoil.
riety of additional novel aspartic acid copolymers has From this investigation, Alford et al. concluded
been synthesized for a range of uses. Examples include that polyaspartate synthesized using thermal methods,
poly(aspartic acid)/citric acid and poly(aspartic acid)/ referred to as thermal polyaspartate (TPA), has a desir-
succinic acid copolymers useful in water treatment ap- able degradative character and would essentially be ad-
Poly(aspartic Acid) 179
sorbed and at least partially mineralized when subjected than similar products that are currently available and can
to the environment of a typical wastewater treatment be used as agricultural, sanitary, and building materials.
plant. The mechanism of TPA hydrolysis has not been The superabsorbant is made when poly(anhydroaspartic
definitively determined but could potentially involve acid) is hydrolyzed at a pH of 10 and 60~ to form
both exo- and endopeptidase activity. Enzymatic activ- poly(aspartic acid), which is subsequently combined
ity associated with TPA is unique due to the fact that with L-aspartic acid and lysine-HC1. Heating at 220~
TPA does not have the structure of a typical protein. for 24 h under a nitrogen environment produces a su-
Unlike other polypeptides, poly(aspartic acid) com- perabsorbant material.
prises only one amino acid. Racemization during poly- Polyaspartates and other poly(amino acid)-contain-
merization may also affect the degradative profile of the ing materials have current and potential applications as
polymer as well as side reactions occurring during poly- biomedical materials including the use of these mate-
merization. It is possible that such side reactions could dais as selective membranes [32]. One example of this
produce structures resistant to degradation. is the use of poly(L-aspartic acid) as a pH-sensitive
The in vitro biodegradation of copoly(L-aspartic transmembrane domain in a synthetic polymer mem-
acid/L-glutamic acid) has been studied [3, 5]. Copoly- brane [40]. A natural biomembrane comprises a contin-
mers of L-aspartic acid and L-glutamic acid were pre- uous nonpolar hydrocarbon matrix that is impenetrable
pared to investigate the influence of molar composition to most polar substances. Incorporated in the biological
on the rate of degradation in vivo. To simulate this deg- phospholipid bilayer are protein molecules that are able
radation in vitro, the copolymers were treated with pa- to bind substrates reversibly and transport them across
pain in a pseudo-extracellular fluid solution at a tem- the cell membrane, irrespective of the concentration
perature of 37 ~ and at pH levels of 4.75 and 7.40. This gradient. Such protein molecules are capable of revers-
study showed that the copolymers under investigation ible conformation changes that facilitate the creation of
degraded by random-chain scission with papain. Also, channels in the biomembrane. Synthetic biomembrane
the degradation profile was consistent with Michaelis- models must have analogous pathways for facilitated
Menten kinetics. The side chain structure of the co- transport of otherwise impermeable molecules.
polymers was found significantly to influence the rate of Poly(aspartic acid) can be used in the preparation of
degradation. pathway protein analogs.
Vinyl polymers that have domains of poly(amino
acid) are similar to those vinyl polymers that have
grafted poly(amino acid) branches [32]. A butyl meth-
APPLICATIONS acrylate-L-aspartic acid graft copolymer was prepared
and hydrolyzed in the form of a membrane to produce
One important area of application for polyaspar- a synthetic biomembrane material [40]. Experiments
tates is the use of these polymer,,~as a totally biodegrad- monitoring Na + ion transport through the synthetic
able water-soluble component in detergents and other biomembrane indicated that the polyaspartate domain
cleaning products [1, 34-36]. Poly(ct, /~-DL-aspartate)s does facilitate ion transport via a pH-dependent revers-
have been shown to adsorb on sewage sludge and have ible conformational change.
proven to be degradable by organisms present in munic- Other research has been done on poly(aspartic
ipal treatment plants. Rhone-Poulenc Chimie holds a acid)-containing membranes [26]. As discussed previ-
patent concerning imidated polymers prepared by poly- ously, amphiphiles with poly(L-aspartic acid) head-
condensation of aspartic acid and/or glutamic acid that groups have been prepared. These amphiphiles were
can be used as builders in laundry and dishwashing de- dispersed via ultrasound in aqueous media and the mor-
tergents [36]. These imidated polymers are stable in de- phologies of the resulting membranes in dilute solution
tergent compounds and are biodegradable. Poly(aspartic were studied using electron microscopy. Characteriza-
acid) and its copolymers are also important in other tion of the amphiphilic bilayers revealed the ability of
water-related applications [13, 14, 24, 25, 37]. These these structures to self-assemble in dilute aqueous so-
materials are known to inhibit the scale deposition and, lutions to form helices. These helices are capable of dra-
therefore, valuable water treatment agents in a range of matic morphological changes such as a change from a
capacities. globular structure to a twisted filament, double helix, or
Superabsorbant materials ahve been produced from twisted ribbon. The superstructures formed were in-
poly(amino acid)s, including poly(aspartic acid) [38, duced by the interaction of two or more microfilaments
39]. These materials have a higher absorption ability formed from bilayer membranes. Use of amphiphiles
180 Roweton, Huang, and Swift
28. N. Nishi et al. (1991) Makromol. Chem. 192, 1799-1809. 40. M. Maeda et al. (1984) J. Am. Chem. Soc. 106, 250-251.
29. J. Z. Yang et al. (1993) Polym. Preprints 34(1), 536-537. 41. M. Yokoyama etal. (1990)Makromol. Chem. 191, 301-311.
30. J. Z. Yang et al. (1993) Polym. Preprints 34(1), 538-539. 42. B. K. Kishore et al. (1992) J. Pharmacol. Exp. Ther. 262, 424-
31. J. Z. Yang et al. (1993) Polym. Preprints 34(2), 420-421. 432.
32. M. Kimura et al. (1982)Makromol. Chem. 183, 1393-1400. 43. S. J. Kohlhepp et al. (1992)J. Pharmacol. Exp. Ther. 263, 1464-
33. D. D. Alford et al. (1994) J. Environ. Polym. Degrad. 2, 225- 1470.
236. 44. M. K. Reinhard et al. (1994)Antimicrob. Agents Chemother. 38,
34. B. Potthoff-Karl et al., DE 43 10 995 A1. 79-82.
35. A. Du Vosel et al., EP 0 612 842 A2. 45. G. Kwon et al. (1993) in Takagi, Toshinori et al. (Eds.), Pro-
36. A. Ponce and F. Tournilhac, EP 0 511 037 A1. ceedings of the International Conference on Intelligent Mate-
37. M. Kroner et al., DE 43 00 020 A1. rials, 1st Naka-gun, Japan, March 23-25, 1992.
38. J. Donachy and S. C. Sikes, Water-Insoluble Crosslinked 46. M. Yokoyama and S. Inoue (1989) Makromol. Chem. 19tl, 2041-
Poly(amino Acid) Superabsorbants, WO 9320856 A!. 2054.
39. A. Nagatomo et al., Superabsorbent Polymer and Process for 47. F. Zunino et al. (1982) Int. J. Cancer 30, 465-470.
Producing Same, U.S. Patent 5,461,085.