Polynonapeptide bioelastomers having an increased elastic modulus

1. Field of the Invention 
The present invention relates to polynonapeptide bioelastomers having an 
increased elastic modulus, being chemotactic peptides toward which 
fibroblasts and endothelial cells migrate and which are particularly 
suitable for constructing artificial ligaments and vascular walls. 
2. Description of the Background 
Bioelastomeric materials are elastomeric polypeptide biomaterials which 
have as their origins repeating sequences from elastin, the extracellular 
elastic protein of higher animals. Elastin is most prominent in tissues 
such as vascular wall, ligament, lungs and skin. In mammals, elastin 
derives from a single protein, tropelastin, of about 70 kD which on 
crosslinking of lysine side chains becomes the insoluble elastic matrix 
that is fibrous elastin. In the pig and cow, the longest sequence between 
lysine-containing crosslinking sequences is (L-Val.sup.1 -L-Pro.sup.2 
-Gly.sup.3 -L-Val.sup.4 -Gly.sup.5).sub.11 or (VPGVG).sub.11. When high 
molecular weight poly(VPGVG) is synthesized and cross-linked by 
.gamma.-irradiation to form an insoluble matrix, it is found to be elastic 
with physical properties remarkably similar to fibrous elastin, for 
example, with an elastic modulus near 10.sup.6 dynes/cm.sup.2. Yet elastin 
contains some fifteen glycine-rich hydrophobic sequences between 
alanine-rich, lysine derived cross-linking regions and the roles of many 
of the sequences have yet to be determined. Some of these also are seen to 
contain related repeating sequences. In the pig and cow, the second 
longest sequence between cross-links is a repeating hexapeptide 
well-represented by (L-Ala.sup.1 -L-Pro.sup.2 -Gly.sup.3 -L-Val.sup.4 
-Gly.sup.5 -L-Val.sup.6).sub.n or (APGVGV).sub.n where n is approximately 
5. In man, the hexapeptide repeats eight times in a continuous sequence. 
High polymers of (APGVGV) are found on raising the temperature in water to 
form irreversible precipitates and which separate from organic solvents to 
form matrices. These matrices are not elastic. There is also a less 
prominent repeat tetrapeptide (L-Val.sup.1 -L-Pro.sup. 2 -Gly.sup.3 
-Gly.sup.4).sub.n which is elastic. Notably, in all of the above formulae, 
the standard three-letter or one-letter abbreviations for amino acids are 
used. See, for example, Organic Chemistry of Biological Compounds, pages 
56-58 (Prentice-Hall, 1971). 
In humans, the longest sequence between cross-links contains a nonapeptide 
repeat which is well-represented by (L-Val.sup.1 -L-Pro.sup.2 -Gly.sup.3 
-.phi..sup.4 -Gly.sup.5 -L-Val.sup.6 -Gly.sup.7 -L-Ala.sup.8 
-Gly.sup.9).sub.n where .phi..sup.4 may be L-Leu.sup.4 or L-Phe.sup.4 
where n is just greater than four. In cows, the polynonapeptide occurs in 
the third longest sequence where n is four, and in pigs the nonapeptide 
repeats three times and where .phi..sup.4 =L-Phe.sup.4 in each of the 
nonamers. 
Recently, Urry et al prepared synthetic polypentapeptides and 
polytetrapeptides, based on the penta- and tetrapeptide repeating units of 
elastin, and discovered that these peptides could be used to prepare 
bioelastomeric materials having an excellent modulus of elasticity. This 
is disclosed and claimed in U.S. Pat. Nos. 4,132,746 and 4,187,852. 
Moreover, a composite bioelastomeric material based on an elastic 
polypentapeptide or polytetrapeptide and a strength-giving fiber was 
disclosed and claimed in U.S. Pat. No. 4,474,851. Additionally, a 
bioelastomeric material having an increased modulus of elasticity formed 
by replacing the third amino acid in a polypentapeptide with an amino acid 
of opposite chirality was disclosed and claimed in U.S. Pat. No. 4,500,700 
to Urry and to an enzymatically cross-linked polypeptide as disclosed in 
and claimed in U.S. Pat. No. 4,589,882. Furthermore, U.S. Pat. No. 
4,605,413 is directed to a chemotactic peptide, while U.S. Pat. No. 
4,693,718 is directed to a second chemotactic peptide. Also pending is 
Ser. No. 07/180,677, directed to a segmented polypeptide bioelastomer for 
the modulation of elastic modulus. 
Also issued is U.S. Pat. No. 4,783,523 which describes the temperature 
correlated force and structure development of various elastomeric 
polytetrapeptides and polypentapeptides. In that application, Urry et al 
disclosed that the above polypeptides exhibit elastomeric force 
development which can be varied as a function of temperature. In 
particular, it was found that by varying the primary structure of the 
repeating tetrameric or pentameric unit of the polypeptide that it is 
possible to effect the range of temperature over which the elastomer 
develops elastomeric force. 
In related work, Urry et al discovered that polypeptides containing the 
hexapeptide sequence (APGVGV).sub.n and permutations thereof are 
chemotactic toward fibroblasts. However, as cross-linked poly(APGVGV) is 
not elastic, it has not been possible to produce an elastomeric 
polypeptide which is chemotactic toward fibroblasts, and which has an 
elastic modulus as great as 10.sup.7 to 10.sup.8 dynes/cm.sup.2. 
Thus, a need clearly continues to exist for elastomeric polypeptides having 
good elastomeric properties and which also exhibit chemotaxis. Such 
polypeptides would be expected to be particularly useful in the 
construction of artificial ligaments or as a scaffolding for the 
reconstruction of ligament, vascular wall and skin. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide 
polypeptides having increased elastic moduli. 
It is also an object of this invention to provide polypeptides which are 
also chemotactic toward endothelial cells and fibroblasts. 
In particular, it is an object of the present invention to provide 
elastomeric polynonapeptides which have increased elastic moduli and which 
are chemotactic toward endothelial cells and fibroblasts. 
The above and other objects are provided by a polynonapeptide having the 
formula: 
EQU --X (.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
Y-- 
wherein: 
.alpha. is a peptide-forming residue of L-Valine or another peptide-forming 
residue capable of functioning in position i of a .beta.-turn in a 
polypeptide; 
.beta. is a peptide-forming residue of L-Proline or another peptide-forming 
residue capable of functioning in position i+1 of a .beta.-turn in a 
polypeptide; 
.gamma. is a peptide-forming residue of Glycine or another peptide-forming 
residue capable of functioning in position i+2 of a .beta.-turn in a 
polypeptide; 
.delta. is a peptide-forming residue of L-Phenylalanine or another 
peptide-forming residue capable of functioning in position i+3 of a 
.beta.-turn in a polypeptide; 
.epsilon. is a peptide-forming residue of Glycine or D-Alanine, when 
functioning as position i' of a subsequent .beta.-turn in a polypeptide 
when .delta. is as defined above; or .epsilon. is as defined above for 
.alpha., 
when .delta. is Glycine or D-Ala; 
.theta. is a peptide-forming residue of L-Valine or another peptide-forming 
residue as defined above for .alpha., or when functioning as position 
(i+1)' of a subsequent .beta.-turn in a polypeptide, .theta. is a 
peptide-forming residue as defined above for .beta.; 
.lambda. is a peptide-forming residue of Glycine, D-Alanine or another 
peptide-forming residue as defined above for .gamma., when functioning as 
position (i+2)' of a subsequent .beta.-turn in a polypeptide; 
.pi. is a peptide-forming residue of L-Alanine or another peptide-forming 
residue as defined above for .delta., when functioning as position (i+3)' 
in a subsequent .beta.-turn in a polypeptide; or a direct bond; 
.rho. is a peptide-forming residue of L-Glycine or D-Alanine; 
and wherein X is .beta..gamma..delta..epsilon..theta..lambda..pi..rho., 
.gamma..delta..epsilon..theta..lambda..pi..rho., 
.delta..epsilon..theta..lambda..pi..rho., 
.epsilon..theta..lambda..pi..rho., .theta..lambda..pi..rho., 
.lambda..pi..rho., .pi..rho., .rho. or a bond; Y is 
.alpha..beta..gamma..delta..epsilon..theta..lambda..pi., 
.alpha..beta..gamma..delta..epsilon..theta..lambda., 
.alpha..beta..gamma..delta..epsilon..theta., 
.alpha..beta..gamma..delta..epsilon., .alpha..beta..gamma..delta., 
.alpha..beta..gamma., .alpha..beta., .alpha. or a direct bond; and n has a 
value of 1 to about 5,000; and with the proviso that no more than three of 
residues .epsilon., .theta., .lambda., .pi. and .rho. are simultaneously a 
peptide-forming residue of Glycine. 
The present invention will now be further explained by reference to the 
following drawings which are provided only for the purpose of illustration 
.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
A typical biological elastic fiber is comprised of a large elastin core 
covered with a fine surface layer of microfibrillar protein. Elastin is 
formed upon cross-linking of the lysine residues of tropoelastin. The 
repeating elastin pentapeptide has the formula (VPGVG).sub.n, while the 
repeating hexapeptide has the formula (VAPGVG).sub.n, where n varies 
depending upon the species. The repeating polytetrapeptide unit has the 
formula (VPGG).sub.n. These sequences, as noted above, utilize the 
standard one-letter abbreviation for the constituent amino acids. 
It was found that these polypeptides are soluble in water below 25.degree. 
C., but on raising the temperature they associate in the polypentapeptide 
(PPP) and polytetrapeptide (PTP) cases, reversibly to form a viscoelastic 
phase, and in the polyhexapeptide (PHP) case, irreversibly to form a 
precipitate. On cross-linking, the former (PPP) and (PTP) have been found 
to be elastomers. However, cross-linked PHP is not elastic. 
At temperatures above 25.degree. C. in water, PTP and PPP exhibit 
aggregation and form a water-containing viscoelastic phase, which upon 
cross-linking by .gamma.-irradiation forms an elastomer. By contrast, PHP 
forms a granular precipitate, which is not elastomeric. In fact, for 
potential elastomers, such aggregation is readily reversible, whereas for 
non-elastomeric samples, such as PHP, temperature-driven aggregation is 
irreversible and redissolution usually requires the addition of a solvent 
such as trifluoroethanol to the aggregate. 
Cross-linked PPP, PTP and analogs thereof were found to exhibit elastomeric 
force development at different temperatures spanning a range of up to 
about 75.degree. C. depending upon several controllable variables. 
Moreover, for these cross-linked elastomers the development of near 
maximum elastomeric force occurs over a very narrow temperature range. 
Thus, by synthesizing bioelastomeric materials having varying molar 
amounts of the constituent pentamers and tetramers together with such 
units modified by hexameric repeating units, and by choosing a particular 
solvent to support the initial viscoelastic phase which forms, it became 
possible to rigorously control the temperature at which the obtained 
bioelastomer develops elastomeric force. Further modification may now be 
effected using the polynonapeptides disclosed herein. 
For example, by modifying PPP, the temperature of transition may be 
changed. In particular, by increasing the hydrophobicity of the PPP 
repeating unit, the viscoelastic phase transition occurs at lower 
temperatures, while by decreasing the hydrophobicity of the repeating 
unit, this transition occurs at higher temperatures. Importantly, it was 
found possible to modify the hydrophobicity in such a way that elasticity 
is retained. 
For example, modifications of the repeating pentamers have been made which 
destroy the molecular structure required for elasticity, such as the 
Ala.sup.1 and Ala.sup.5 analogs. The Ala.sup.1 and Ala.sup.5 analogs, the 
former decreasing and the latter increasing pentamer hydrophobicity, 
result in the formation of granular precipitates on raising the 
temperature of aqueous solutions rather than forming viscoelastic 
coacervates and .gamma.-irradiation cross-linking of the Ala.sup.5 -PPP 
precipitate results in a hard material that simply breaks upon stretching. 
These analogs apparently fail to produce elastomeric polymers for 
different but consistent reasons First, the Ala.sup.1 analog does not 
appear to allow for important Val.sup.1 . . . .gamma.CH.sub.3 . . . 
Pro.sup.2 .delta.CH.sub.2 intrapentameric intramolecular hydrophobic 
contacts required to form a viscoelastic coacervate. The Ala.sup.5 analog 
appears to interfere with librational motions in the Val.sup.4 -Gly.sup.5 
-Val.sup.1 suspended segment of the proposed PPP molecular structure. 
Similarly, the Ala.sup.9 polynonapeptide analog would be expected to 
interfere with librational motions in the Val.sup.8 -Gly.sup.9 -Val.sup.1 
or Ala.sup.8 -Gly.sup.9 -Val.sup.1 suspended segments. These librational 
motions appear to be essential to the proposed librational entropy 
mechanism of elasticity for these elastomers. 
By contrast, the hydrophobicity of the repeating pentamer has been easily 
increased by introducing a --CH.sub.2 -- moiety, for example, in residue 1 
while maintaining .beta.-branching, that is, to utilize the Ile analog of 
PPP, i.e., (Ile.sup.1 -Pro.sup.2 -Gly.sup.3 -Val.sup.4 -Gly.sup.5).sub.n. 
With a greater than 50,000 molecular weight, Ile.sup.1 -PPP reversibly 
forms a viscoelastic coacervate with the onset of coacervation being near 
8.degree. C. rather than 24.degree. C. as for unsubstituted PPP. It 
appears from circular dichroism data that Ile.sup.1 -PPP and PPP have 
identical conformations both before and after the transitions and that the 
transition to increased intramolecular order on increasing the temperature 
is also shifted by 15.degree. C. or more to lower temperatures. Further, 
the dramatic increase in elastomeric force on raising the temperature of 
the .gamma.-irradiation cross-linked coacervate at fixed extension is 
similarly shifted to a lower temperature for the Ile.sup.1 -PPP analog. 
Thus, with this analog, a coupling of temperature dependent elastomeric 
force development and molecular structure was demonstrated. Hence, it is 
now possible to rationally design polypeptide elastomers that undergo 
transitions at different temperatures and that would function as entropic 
elastomers in different temperature ranges. 
By increasing the hydrophobicity of PPP, such as by substituting Ile.sup.1 
for Val.sup.1 in the pentameric sequence of --(VPGVG).sub.n to form 
--(IPGVG).sub.n --, it became possible to accomplish at least two distinct 
objectives. 
First, the "homopolymeric" polypentapeptide of --(IPGVG).sub.n, can be 
prepared i.e., Ile.sup.1 -PPP, which, as noted dissolves in water at 
4.degree. C., and upon raising the temperature to 8.degree. C., exhibits 
aggregation. After cross-linking the coacervate by .gamma.-irradiation, it 
is observed that essentially full elastic contraction is exhibited at 
about 25.degree. C. for the cross-linked Ile.sup.1 -PPP as opposed to the 
40.degree. C. temperature required for the unsubstituted PPP. Thus, the 
temperature ordered transition for Ile.sup.1 -PPP occurs at a temperature 
approximately 15.degree. C. lower than for PPP. 
Secondly, mixed "copolymers", for example, of the polypentapeptides 
--X.sup.1 --(IPGVG).sub.n --Y.sup.1 -- and --X.sup.2 --(VPGVG--).sub.n 
--Y.sup.2 -- can be prepared which exhibit variable and controllable 
transition temperatures which are in between the separate transition 
temperatures of PPP and Ile.sup.1 -PPP. Further, a great degree of control 
became possible inasmuch as the transition temperature obtained is 
directly proportional to the molar ratios of the respective pentapeptides 
incorporated therein. 
As noted above, it was recently discovered that hexapeptide segments of the 
formula VGVAPG and permutations thereof are chemotactic toward fibroblasts 
and endothelial cells. However, because cross-linked PHP is not elastic, 
it has not been possible to prepare polypeptides which are, at once, both 
elastomeric and chemotactic toward fibroblasts and endothelial cells and 
which have a modulus of elasticity of about 10.sup.7 to 10.sup.8 
dynes/cm.sup.2. 
The present invention, therefore, provides, for the first time, 
polypeptides which have both increased elastomeric properties and which 
are chemotactic toward fibroblasts and endothelial cells. 
In general, the present invention provides a polynonapeptide of the 
formula: 
EQU --X (.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
Y-- 
wherein: 
.alpha. is a peptide-forming residue of L-Valine or another peptide-forming 
residue capable of functioning in position i of a .beta.-turn in a 
polypeptide; 
.beta. is a peptide-forming residue of L-Proline or another peptide-forming 
residue capable of functioning in position i+1 of a .beta.-turn in a 
polypeptide; 
.gamma. is a peptide-forming residue of L-Glycine or another 
peptide-forming residue capable of functioning in position i+2 of a 
.beta.-turn in a polypeptide; 
.delta. is a peptide-forming residue of L-Phenylalanine or another 
peptide-forming residue capable of functioning in position i+3 of a 
.beta.-turn in a polypeptide; 
.epsilon. is a peptide-forming residue of Glycine or D-Alanine, when 
functioning as position i' of a subsequent .beta.-turn in a polypeptide 
when .delta. is as defined above, or .epsilon. is as defined above for 
.alpha. when .delta. is glycine or D-Alanine; 
.theta. is a peptide-forming residue of L-Valine or another peptide-forming 
residue as defined above for .alpha. or a direct bond; 
.lambda. is a peptide-forming residue of Glycine, D-Alanine or another 
peptide-forming residue as defined above for .alpha.; or when functioning 
as position (i+1)' of a subsequent .beta.-turn in a polypeptide, .theta. 
is a peptide-forming residue as defined above for .beta.; 
.pi. is a peptide-forming residue of L-Alanine or another peptide-forming 
residue as defined above for .delta. when functioning as position (i+2)' 
in a subsequent .beta.-turn in a polypeptide or a direct bond; 
.rho. is a peptide-forming residue of L-Glycine, D-Alanine; 
wherein X is .beta..gamma..epsilon..delta..theta..lambda..pi..rho., 
.gamma..delta..epsilon..theta..lambda..pi..rho., 
.delta..epsilon..theta..lambda..pi..rho., 
.epsilon..theta..lambda..pi..rho., .theta..lambda..pi..rho., 
.lambda..pi..rho., .pi..rho., .rho. or a direct bond; Y is 
.alpha..beta..gamma..delta..epsilon..theta..lambda..pi., 
.alpha..beta..gamma..delta..epsilon..theta..lambda., 
.alpha..beta..gamma..delta..epsilon..theta., 
.alpha..beta..gamma..delta..epsilon., .alpha..beta..gamma..delta., 
.alpha..beta..gamma., .alpha..beta., .alpha. or a direct bond; and n has a 
value of 1 to about 5,000; and with the proviso that no more than three of 
residues .epsilon., .theta., .lambda., .pi. and .rho. are simultaneously a 
peptide-forming residue of Glycine. 
The polynonapeptides of the present invention can be produced as 
"homopolymer" nonapeptides or one or more of the present nonapeptide 
sequences may be chemically bonded to other elastomeric sequences such as 
the PPP or PTP sequences described above or those sequences with the 
appropriate substitutions as may be required to modify the temperature of 
transition. Additionally, polyhexapeptide sequence, PHP and modifications 
as will be described, can also be included. 
For purposes of comprehending the present invention, the preparation and 
use of various PPP or PTP sequences, with and without substitution, will 
first be briefly discussed. Thereafter, the preparation and use of the 
polynonapeptides will be described. 
PPP cross-linked analogs having increased hydrophobicity were found to 
develop full elastomeric force over a very narrow temperature range. For 
example, for crosslinked Ile.sup.1 -PPP, it was found that the elastomeric 
force thereof shows an abrupt increase from essentially zero at 8.degree. 
C. to three-quarters of full force at 10.degree. C., and essentially full 
force by 20.degree.-25.degree. C. Such an increase in elastomeric force 
over only a 2.degree. C. temperature differential is, indeed, 
unprecedented and can be controlled by the percent extension in relation 
to swelling of the elastomer on lowering the temperature. 
Although Ile.sup.1 -PPP is an excellent example of an increased 
hydrophobicity PPP analog, any PPP analog, which increases the 
hydrophobicity of the repeating pentameric unit, while retaining the 
elasticity of the polypeptide, and without interfering with either the 
formation of the viscoelastic coacervate or the librational motion may be 
used. 
For example, in addition to repeating unit sequences of (IPGVG).sub.n, 
using Ile.sup.1, is also possible to effect a variety of other 
substitutions. In general, a pentapeptide repeating unit of the formula: 
EQU --(R.sub.1 PR.sub.2 R.sub.3 G).sub.n -- 
can be used, wherein R.sub.1 may be Phe, Leu, Ile, Val, Tyr and Trp; 
R.sub.2 may be Ala and Gly; R.sub.3 may be Phe, Leu, Ile, Met, Ala and 
Val; and n is an integer from 1 to 5,000, and P is L-proline and G is 
glycine. 
Notably, the above substitutions modify the hydrophobicity of the repeating 
unit so as to attenuate the transition temperature for near maximum 
elastomeric force development, of course, without destroying the 
elasticity of the bioelastomer. 
In the above formula, it is noted that the amino acid Leu is, of course, 
Leucine. R.sub.1, R.sub.2 and R.sub.3 correspond to positions 1, 3 and 4 
in the numbered sequence as described herein. 
Interestingly, with about 50% Phe.sup.1 -PPP in water, it is possible to 
shift the temperature of transition initiation from 25.degree. C. for PPP 
to about 0.degree. C. Furthermore, this shift can be driven to even lower 
temperatures by utilizing mixed solvent systems of water/ethylene glycol 
or water/dimethyl sulfoxide (DMSO). For example, by using the about 50% 
Phe.sup.1 -PPP/water-ethylene glycol system, a transition temperature of 
as low as about -25.degree. C. can be obtained. Of course, a range of 
transition temperatures can be obtained between 0.degree. C. and about 
-25.degree. C. for the Phe.sup.1 -PPP/water-ethylene glycol system 
depending upon the amount of ethylene glycol added. It has been found that 
very low transition temperatures are obtained using approximately 50/50 
mixtures of water/ethylene glycol. 
Conversely, the maximum shift to higher transition temperatures is limited 
by further structural change to the polypeptide. With the present 
elastomeric polypeptides, this upper limit appears to be about 50.degree. 
C. with transition to a less elastic state beginning at about 60.degree. 
C. 
However, as noted previously, not only are PPP analogs contemplated, such 
as Ile.sup.1 -PPP, Phe.sup.1 -PPP or Ala.sup.3 -PPP, but all PPP analogs, 
and bioelastomers containing the same, which have transition temperatures, 
and, hence, temperatures of near maximum elastomeric force development, 
which are different from PPP; while retaining elasticity are contemplated. 
Given, the present disclosure, one skilled in the art could clearly 
ascertain additional PPP analogs, and bioelastomers incorporating the same 
which meet the above criteria. 
As noted above, the increased hydrophobicity analog, such as Ile.sup.1 -PPP 
may be synthesized as a "homopolymer", or a "copolymer" of --X.sup.2 
--(VPGVG--).sub.n --Y.sup.2 -- and --X.sup.1 --(IPGVG--).sub.n --Y.sup.1 
may be synthesized with the molar ratio of the constituent pentamers being 
dependent upon the desired temperature for elastomeric force development. 
However, in general, in such "copolymers", the --X.sup.1 --(IPGVG--).sub.n 
--Y.sup.1 -- pentameric component is present in about 1-99% of the total 
pentameric molar content, while the --X.sup.2 --(VPGVG--).sub.n --Y.sup.2 
-- pentameric component is present in about 99-1% of the total pentameric 
content. More preferably, the --X.sup.1 --(IPGVG).sub.n --Y.sup.1 -- 
component is present in about 5-95% of the total pentameric molar content, 
while the --X.sup.2 --(VPGVG--).sub.n --Y.sup.2 -- component is present in 
about 95-5% of the total pentameric molar content. However, any 
combination of relative molar amounts can be used as dictacted by the 
desired transition temperature. 
Thus, bioelastomers can be prepared which contain repeating units 
containing elastomeric tetrapeptide, or pentapeptide or units thereof 
modified by hexapeptide repeating units and mixtures thereof, wherein said 
repeating units contain amino acid residues selected from the group 
consisting of hydrophobic amino acid and glycine residues, wherein the 
repeating units exist in a conformation having a .beta.-turn which 
contains a polypentapeptide unit of the formula: 
EQU --X.sup.1 --(IPGVG--).sub.n --Y.sup.1 -- 
wherein I is a peptide-forming residue of L-isoleucine; 
P is a peptide-forming residue of L-proline; 
G is a peptide-forming residue of glycine; 
V is a peptide-forming residue of L-valine; and 
wherein X is PGVG, GVG, VG, G or a covalent bond; Y is IPGV, IPG, IP or I 
or a covalent bond; and n in both formulas is an integer from 1 to 5,000; 
or n is 0, with the proviso that X.sup.1 and Y.sup.1 together constitute a 
repeating pentapeptide unit, in an amount sufficient to adjust the 
development of elastomeric force of the bioelastomer to a predetermined 
temperature. 
However, bioelastomers can also be prepared which contain elastomeric units 
comprising tetrapeptide, or pentapeptide or units thereof modified by 
hexapeptide repeating units and mixtures thereof, wherein said repeating 
units comprise amino acid residues selected from the group consisting of 
hydrophobic amino acid and glycine residues, wherein the repeating units 
exist in a conformation having a .beta.-turn which comprises (A) a 
polypentapeptide unit of the formula: 
EQU --X.sup.1 --(IPGVG--).sub.n --Y.sup.1 -- 
and (B) a polypentapeptide unit of the formula: 
EQU --X.sup.2 --(VPGVG--).sub.n --Y.sup.2 -- 
wherein for the above formulas, 
I is a peptide-forming residue of Lisoleucine; 
P is a peptide-forming residue of L-proline; 
G is a peptide-forming residue of glycine; 
V is a peptide-forming residue of L-valine; and 
wherein X.sup.1 and X.sup.2 are each PGVG, GVG, VG, G or a covalent bond: 
Y.sup.1 is IPGV, IPG, IP or I or a covalent bond; Y.sup.2 is VPGV, VPG, 
VP, V or a covalent bond; and n in both formulas an integer from 1 to 
5,000; or n in both formulas is 0, with the proviso that X.sup.1 and 
Y.sup.1 together, and X.sup.2 and Y.sup.2 together constitute a repeating 
pentapeptide unit, in relative amounts sufficient to adjust the 
development of elastomeric force of the bioelastomer to a predetermined 
temperature. 
It should be noted that bioelastomeric polypeptide chains containing either 
one or both of the above pentapeptide repeating units can be synthesized 
using any of the pentapeptide "monomers" that are permutations of the 
basic sequence. However, if the polymer is not synthesized using the 
pentapeptide "monomers", but rather is synthesized by sequential adding of 
amino acids to a growing peptide, such as in the case of an automatic 
peptide synthesizer, the designation of the repeating unit is somewhat 
arbitrary. For example, the peptide H--V(PGVGVPGVGVPGVGVPGVGV)P--OH can be 
considered to consist of any of the following repeating units and end 
groups: H--(VPGVG).sub.4 --VP--OH, H--V--(PGVGV).sub.4 --P--OH, HVP(GVGVP) 
.sub.4 --OH, H--VPG--(VGVPG).sub.3 --VGVP--OH, or H--VPGV--(GVPGV).sub.3 
--GVP--OH, for example. 
Furthermore, it is entirely possible that mixed repeating units such as 
those of the formula --VPGVGIPGVG--.sub.n can be incorporated into the 
bioelastomers. 
Synthesis of the elasticity promoting and modifying segments, which are 
incorporated into the final elastomeric polypeptide, is straightforward 
and easily accomplished by a peptide chemist. The resulting intermediate 
peptides generally have the structure, B.sup.1 -(repeating unit).sub.n 
-B.sup.2 where B.sup.1 and B.sup.2 represent any chemically compatible end 
group on the amino and carboxyl ends of the molecule, respectively, and n 
is an integer of from 1 to about 5,000. of course, when B.sup.1 is --H and 
B.sup.2 is --OH, and n is 1, the compound is either the pentapeptide 
H--VPGVG--OH or HIPGVG--OH. When n is greater than 1, the compound 
intermediate is a polypentapeptide. The same will hold true when utilizing 
tetrameric repeating units in the present bioelastomers. 
It should be noted that the term "hydrophobic amino acid" refers to amino 
acids which have appreciably hydrophobic R groups as measured on a 
hydrophobicity scale generated by measuring the relative solubilities of 
the amino acids in organic solvent. In this respect, see Arch. Biochem. 
Biophy, Bull and Breese Vol. 161, 665-670 (1974). By this method, all 
amino acids which are more hydrophobic than glycine may be used. More 
specifically, preferable hydrophobic amino acids are Ala, Val, Leu, Ile, 
Pro, Met, Phe, Tyr and Trp. 
Further, in order to allow the present elastomers to be switched "on" and 
"off" at fixed length, it is not necessary to restrict the amino acids 
utilized to only those having hydrophobic R groups. While one or more 
amino acids having polar R groups are preferable for using the switching 
mechanism, it is only necessary to maintain an appropriate mean or average 
hydrophobicity. Of particular interest, however, are amino acids having 
ionizable R groups, such as Glu, Asp, His, Lys or Tyr or even 
hydroxyl-containing or sulfhydryl-containing R groups which can be 
phosphorylated or otherwise chemically modified, such as Ser, Thr, Tyr, 
Hyp and Cys. 
Additionally, it is permissible that one or more amino acid residues or 
segments of amino acid residues not present in the normal pentapeptide or 
tetrapeptide sequence may be interspersed within a polypentapeptide or 
polytetrapeptide portion of an elastomeric polypeptide chain. 
Thus, these bioelastomers, regardless of the particular functional 
repeating unit incorporated therein, may have these repeating units 
incorporated either in the form of block or random copolymers as long as 
the desired shift in temperature of elastomeric force development of the 
bioelastomer is obtained. As noted above, by considering the transition 
temperatures and temperatures of elastomeric force development for two PPP 
or PTP analogs, or even for a PPP analog and a PTP analog, it is possible 
to attain a desired intermediate transition temperature and temperature of 
elastomeric force development by directly correlating the molar ratios of 
each analog component therewith. For example, a 50/50 molar ratio of two 
analog components would give rise to a bioelastomer "copolymer" having a 
transition temperature and temperature of elastomeric force development 
approximately in between those of the analog components. 
Additionally, it is also noted that the elastomeric units used in 
conjunction with all aspects of the present invention, i.e., whether the 
repeating unit is PPP, PTP or analogs thereof, may also comprise those 
described in U.S. Pat. Nos. 4,132,746, 4,187,852; 4,474,851; 4,500,700, 
4,589,882, 4,605,413, 4,693,718 and 4,783,523 and U.S. patent application 
Ser. Nos. 07/180,677, 07/062,557, 07/163,388 and 07/184,147 all of which 
patents and patent applications are incorporated herein in their entirety. 
The preparation of PPP and analogs thereof will now be illustrated by 
Examples, which are provided only for the purpose of illustration and are 
not intended to be limiting. 
EXAMPLES 
Peptide Synthesis 
The synthesis of Ile.sup.1 -PPP was carried out by the classical solution 
methods as shown in Scheme I. 
In the following Examples, the following abbreviations will be used: Boc, 
tert-butyloxycarbonyl; Bzl, benzyl; DMF, dimethylformamide; DMSO, 
dimethylsulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide; 
HOBt, 1-hydroxybenzotriazole; IBCF, isobutyl-chloroformate; NMM, 
N-methylmorpholine; ONp, p-nitrophenylester; TFA, trifluoroacetic acid; 
PPP, (VPGVG).sub.n ; Ile.sup.1 -PPP, (IPGVG).sub.n ; V, valine; I, 
isoleucine; P, proline; G, glycine. 
##STR1## 
The sequence of the starting pentamer for polymerization is preferably 
Gly-Val-Gly-Ile-Pro rather than Ile-Pro-Gly-Val-Gly, because the 
permutation with Pro as the C-terminal amino acid produces high molecular 
weight polymers in better yields. The approach to the synthesis entailed 
coupling the tripeptide Boc-GVG-OH (II) with H-IP-OBzl, each in turn being 
synthesized by the mixed anhydride methodology of J. R. Vaughan et al, J. 
Am. Chem. Soc., 89, 5012 (1967). The possible formation of the urethane as 
a by-product during the reaction of Boc-Ile-OH with H-Pro-OBzl by the 
mixed anhydride method was avoided by carrying out the reaction in the 
presence of HOBt. The dipeptide was also prepared using EDCI for 
confirmation of the product. The pentapeptide benzylester (III) was 
hydrogenated to the free acid (IV) which was further converted to the 
p-nitrophenylester (V) on reacting with bis(p-nitrophenyl)carbonate. On 
removing the Boc-group, a one molar solution of the active ester in DMSO 
was polymerized in the presence of 1.6 equiv. of NMM. The polypeptide was 
dialyzed against water using a 50,000 dalton cut-off dialysis tubing and 
lyophilized. The purity of the intermediate and final products was checked 
by carbon-13 nuclear magnetic resonance, elemental analyses and thin layer 
chromatography (TLC). 
Elemental analyses were carried out by Mic Anal, Tuscon, AZ. All amino 
acids are of L-configuration except for glycine. Boc-amino acids were 
purchased from Bachem, Inc., Torrance, CA. HOBt was obtained from Aldrich 
Chemical Co., Milwaukee, WI. TLC was performed on silica gel plates 
purchased from Whatman, Inc., Clifton, NJ in the following solvent 
systems: R.sub.f.sup.1, CHCl.sub.3 (C):CH.sub.3 OH(M):CH.sub.3 COOH(A), 
95:5:3; R.sub.f.sup.2, CMA (85:15:3); R.sub.f.sup.3, CMA (75:25:3); 
R.sub.f.sup.4, CM (5:1). Melting points were determined with a Thomas 
Hoover melting point apparatus and are uncorrected. 
Boc-Ile-Pro-OBzl (mixed anhydride method) (I): Boc-Ile-OH (12.01 g, 0.05 
mole) in DMF (50 ml) was cooled to 0.degree. C. and NMM (5.49 ml) was 
added. After cooling the solution to -15.degree. C. isobutylchloroformate 
(6.48 ml) was added slowly while maintaining the temperature at 
-15.degree. C. and stirred for 10 minutes at which time HOBt (7.65 g) was 
added and stirring was continued for additional 10 minutes. A pre-cooled 
solution of HCl-H-Pro-OBzl (12.09 g, 0.05 mole) in DMF (50 ml) and NMN 
(5.49 ml) was added to the above solution and the completeness of the 
reaction was followed by TLC. The reaction mixture was poured into a cold 
saturated NaHCO3 solution and stirred for one hour. The peptide was 
extracted into CHCl.sub.3 and washed with acid and base (0.5 N NaOH to 
remove HOBt), and on evaporating the solvent the product was obtained as 
an 92% yield. R.sub.f.sup.1, 0.65. Anal. Calcd. for C.sub.23 H.sub.34 
N.sub.2 O.sub.5 : C 66.00, H 9.19, N 6.69%. Found: C 65.58, H 8.28, N 
7.13%. 
Boc-Ile-Pro-OBzl (using EDCI): Boc-Ile-OH (7.20 g, 0.03 mole) and HOBt 
(5.05 g, 0.033 mole) in DMF (30 ml) was cooled to -15.degree. C. and EDCI 
(6.32 g, 0.033 mole) was added. After stirring for 20 minutes, a 
pre-cooled solution of HCL-HPro-OBzl (7.25 g, 0.103 mole) in DMF (30 ml) 
and NMM (3.3 ml) was added and stirred overnight at room temperature. 
After evaporating DMF, the residue was taken into CHCl3 and extracted with 
20% citric acid and 0.5 N NaOH. The solvent was removed and the product 
was obtained as an oil in almost quantitative yield which was identical to 
the product obtained by the mixed anhydride method. 
Boc-Gly-Val-Gly-Ile-Pro-OBzl (III): Boc-GVG-OH (II) (20) (5.6 g, 0.017 
mole) was coupled with H-Ile-Pro-OBzl (6.7 g, 0.019 mole) (obtained by 
deblocking I with HCl/Dioxane) in the presence of EDCI (3.65 g, 0.019 
mole) and HOBt (2.9 g, 0.019 mole) and the product was worked up as 
described above to obtain 8.8 g of III (yield: 82.4%), m.p. 
107.degree.-108.degree. C. (decomp.) R.sub.f.sup.2, 0.75 Anal. calcd. 
C.sub.32 H.sub.49 N.sub.5 O.sub.10 : C 60.83, H 7.81, N 11.08%. Found: C 
61.12, H 8.06, N 11.06%. 
Boc-Gly-Val-Gly-Ile-Pro-OH (IV): III (7.8 g, 0.0123 mole) was taken in 
acetic acid (80 ml) and hydrogenated in the presence of 10% Pd-C (1 g) at 
40 psi. After filtering the catalyst with the aid of celite, the solvent 
was removed under reduced pressure, triturated with ether, filtered, 
washed with ether then pet. ether and dried to obtain 6.5 g of the product 
(yield: 97.3%), m.p. shrinks at 127.degree. C. and decomp. at 3 
145.degree. C. R.sub.f.sup.3, 0.24; R.sub.f.sup.4, 0.11 Anal. Calcd. for 
C.sub.25 H.sub.43 N.sub.5 O.sub.10.1/2H.sub.2 O: C 54.52, H 8.05, N 
12.71%. Found: C 54.32, H 8.02, N 12.59%. 
Boc-Gly-Val-Gly-Ile-Pro-ONp (V): IV (5.41 g, 0.01 mole) in pyridine (40 ml) 
was reacted with bis(p-nitrophenyl)carbonate (4.56 g, 0.015 mole) 
following the completeness of the reaction by TLC. Pyridine was removed; 
the residue was taken into CHCl.sub.3 and extracted with acid and base. 
The p-nitrophenyl ester obtained was chromatographed over a silica gel 
(200-400 mesh) column. After initial washing with CHCl.sub.3 4.8 g of V 
was obtained when eluted with 35% acetone in CHCl.sub.3 (yield: 71.4%), 
m.p. 97.degree.-100.degree. C. R.sub.f.sup.2, 0.72; R.sub.f.sup.4, 0.75; 
Anal. Calcd. for C.sub.31 H.sub.46 N.sub.6 O.sub.12.2H.sub.2 O: C 53.28, H 
7.21, N 12.02%. Found: C 53.76, H 6.83, N 12.01%. 
H-(Gly-Val-Gly-Ile-Pro).sub.n -OH(VI): The Boc-group was removed from V 
(3.8 g, 0.0057 mole) by reacting with TFA (35 ml) for 45 min. TFA was 
removed under reduced pressure, triturated with ether, filtered, washed 
with ether, pet. ether and dried. The TFA salt (3.3 g, 0.0049 mole) in 
DMSO (4.9 ml) was stirred for 14 days in the presence of NMM (0.86 ml, 
0.0078 mole). After diluting with water in the cold, the polypeptide was 
dialyzed using a 50 kD cut-off dialysis tubing changing the water daily 
for 15 days. The retentate was lyophilized to obtain 1.81 g of the 
Ile.sup.1 -polypentapeptide (yield: 88%). The carbon-13 NMR spectrum is 
presented in FIG. 1 along with that of the regular polypentapeptide for 
comparison. 
In addition to the above synthetic methods for synthesizing the PPP 
polypeptide elastomers of the present invention, the elastomers of the 
present invention may also be prepared by microbial biosynthesis. In 
particular, microbial biosynthesis may be effected by using well-known 
techniques of genetic engineering using suitable host organisms such as E. 
coli and plasmid vectors capable of expression therein. Of course, by 
using the gene splicing technique, a gene sequence corresponding to the 
desired elastomeric polypeptide sequence is inserted into a suitable 
plasmid vector using known techniques, which hybrid plasmid is then 
inserted into a suitable host organism, such as E. coli. The resultant 
transformed microorganism is then cultured in accordance with known 
fermentative techniques to afford the product bioelastomer. 
Notably, recombinant microbial synthesis can also be used to synthesize 
PTP, PTP/PPP and PTP/PPP/PHP combinations and any or all of these 
combinations with a polynonapeptide (PNP). Of course, it can also be used 
to synthesize all of the various polar-substituted polypeptide elastomers 
involved in chemomechanical transduction. 
As noted above, E. coli expression systems for amino acid and peptide 
synthesis are well known. U.S. Pat. Nos. 4,278,765, 4,321,325 and 
4,264,731 are hereby incorporated herein in the entirety. 
Temperature Profiles for Coacervation 
The temperature dependence for aggregation of the polypentapeptide is 
followed as the development of turbidity at 300 nm using a Cary 14 
spectrophotometer. The sample cell is placed within a chamber vibrating at 
300 Hz in order to facilitate equilibrium and to keep the aggregates from 
settling. The scan rate is 30.degree. C./hour and the temperature was 
controlled with a Neslab ETP-3 programmer and monitored with an Omega 199A 
thermocouple monitor placed at the cell. The turbidity as a function of 
temperature provides a temperature profile for coacervation which is found 
to be concentration dependent. As the concentration is raised, the profile 
shifts to lower temperatures until further increases in concentration 
cause no further lowering of the temperature for aggregation. This defines 
the high concentration limit. The temperature for the onset of 
coacervation at the high concentration limit coincides with the 
temperature for the onset of the transition within the coacervate itself, 
even when there is no appreciable change in water content of the 
coacervate. The temperature for the midpoint of the temperature profile 
for the high concentration limit has been shown to correlate with the 
molecular weight of the polypentapeptide. When the midpoint is 25.degree. 
C. for the PPP, the molecular weight is close to 100,000 daltons as 
calibrated by dialysis. For the Ile.sup.1 -PPP with a midpoint of 
9.degree. C., the molecular weight is greater than 50,000 daltons, as the 
synthetic polypeptide was retained by a 50,000 daltons dialysis membrane. 
The dialysis was carried out at 4.degree. C. where the Ilel-PPP is in 
solution. 
Circular Dichroism Measurements 
The circular dichroism studies were carried out on a Cary 60 
spectropolarimeter equipped with a Model 6001 CD accessory modified for 
330 Hz modulation of the left and right circularly polarized light. A 
concentration of 0.025 mg Ile.sup.1 -PPP/ml of doubly distilled water was 
characterized in a 10 mm path length cell. The low concentration was used 
to keep the size of the aggregate sufficiently small as not to cause light 
scattering distortions of the CD spectra. Even at this low concentration 
with this more hydrophobic polypentapeptide, above 35.degree. C. the size 
of the aggregates was sufficient to cause particulate distortions as was 
apparent with the red shifting and dampening of the long wavelength 
negative band. The temperature was controlled and monitored from the cell 
as for the temperature profiles for coacervation. 
Formation of the Elastomeric Matrix 
In preparation for .gamma.-irradiation cross-linking (the means of forming 
the elastomeric matrix), 130 milligrams of peptide Ile.sup.1 -PPP were 
dissolved in 220 milligrams of water in a cryotube. The sample was then 
shear oriented at 0.degree. C. in a previously described pestle-cryotube 
arrangement. Gamma-irradiation was carried out at the Auburn University 
Nuclear Science Center at a dose rate of approximately 8,000 Roentgen/min 
and for sufficient time to achieve a 20 .times.10.sub.6 radiation absorbed 
dose (20 Mrad). 
Thermoelasticity Studies 
Thermoelasticity studies were carried out on a stress-stain instrument 
built in this Laboratory. The sample is mounted in two Delrin clamps. The 
top clamp is attached to a Statham UTC strain-gauge and the assembly is 
fixed. The bottom clamp is attached to a moving platform driven by a 
variable speed motor. Both clamps are enclosed in a thermostated water 
jacket. An inner chamber contains the solvent in which the elastomer is 
immersed which in this case is doubly distilled water. The sample was 
fixed in the top clamp and equilibrated in water at 60.degree. C. for 
about an hour. The strain-gauge signal conditioner was balanced for zero 
force and the bottom clamp was attached to the sample. The sample was left 
to set overnight at room temperature. The bottom clamp was then adjusted 
for zero force and the distance between the clamps was measured. The 
elastomer was elongated to 40% extension at 5.degree. C. and elastomeric 
force was then determined as a function of temperature. Equilibrium time 
to achieve constant force at a given temperature was typically twenty-four 
hours. Force measurements were made in 2.degree. C. increments through the 
sharp rise in force and 5.degree. C. increments at higher temperatures. 
RESULTS 
Temperature Profiles for Coacervation 
The Ile.sup.1 -PPP can be dissolved in water on standing below 8.degree. C. 
On raising the temperature of the solution above 8.degree. C., the 
solution becomes cloudy; on standing at the elevated temperature settling 
occurs and a viscoelastic phase forms in the bottom of the vial; on 
placing the vial in an ice bath the cloudiness immediately clears and the 
viscoelastic phase readily dissolves. Thus the Ile.sup.1 -PPP coacervates 
when dissolved in water. The temperature profiles for As the concentration 
is raised, the temperature profile shifts to lower temperature. At 40 
mg/ml, the high concentration limit (i.e., the lower concentration for 
which further increases in concentration cause no further lowering of the 
temperature for the onset of aggregation), the midpoint for the 
temperature profile for coacervation of Ile.sup.1 -PPP is 9.degree. C. 
It was observed that the simple addition of a CH.sub.2 moiety to the 409 
dalton repeating unit causes the onset of aggregation to shift to lower 
temperatures by 16.degree. C. 
Circular Dichroism 
Determined are the circular dichroism curves for Ile.sup.1 -PPP in water 
(0.025 mg/ml) at 2.degree. C. and at 35.degree. C. The low concentration 
was chosen in order that the size of the aggregate formed on association 
at 35.degree. C. would have limited particulate distortions in the CD 
spectrum. At low temperature there is a large negative band near 195 nm. 
Such a negative band is characteristic of disordered proteins and 
polypeptides, though a standard value for this negative peak for complete 
disorder is -4.times.10.sup.4 rather than the observed value of 
-1.2.times.10.sup.4. Also the negative band near 220 nm, rather than zero 
ellipticity or a positive band which are taken as indicative of complete 
disorder, suggests elements of order at low temperature. Furthermore, 
Raman studies have shown the presence of the .beta.-turn at temperatures 
below the transition. Thus, as far as the CD spectra are concerned what is 
observed is an unmasking of the .beta.-turn spectrum. The decrease in 
intensity of the negative CD band near 195 nm on raising the temperature 
of Ile.sup.1 -PPP in water indicates an increase in intramolecular order 
on raising the temperature, that is, there is an inverse temperature 
transition in an aqueous system. This indicates that hydrophobic 
interactions are developing as the ordered state develops. The 
intramolecular increase in order begins just above 0.degree. C. and is 
complete by about 30.degree. C. for a concentration of 0.025 mg/ml. It was 
observed that Ile.sup.1 -PPP and PPP have essentially identical 
conformations below the onset temperature for the transition and that they 
have essentially identical conformations after the transition is mostly 
completed. Thus while maintaining essentially identical conformations, 
which is assisted by the retention of .beta.-branching, the addition of a 
CH.sub.2 moiety lowers the transition toward increased order by about 
15.degree. C. 
Characterization of Elasticity 
The elastic (Young's) modulus determined for 20 MRAD cross-linked Ilel-pPP 
coacervate was 4.times.105 dynes/cm.sup.2 which is within the range of 
values obtained for 20 Mrad cross-linked PPP. The range of values is due 
to variable vacuolization occurring during .gamma.-irradiation which makes 
difficult accurate measurement of cross-sectional area. It should be 
appreciated, however, that .gamma.-irradiation causes no detectable 
polymer breakdown when measured by carbon-13 and nitrogen-15 NMR. 
The results obtained demonstrate with three different physical methods that 
the addition of a CH.sub.2 moiety (the replacement of Val by Ile) shifts 
the transition to lower temperatures by 15.degree. C. without changing the 
conformation of the polypentapeptide before and after the transition. 
While the previously reported data on the naturally occurring PPP of 
elastin demonstrate a correlation of increased structural order with 
increased elastomeric force, the Ile.sup.1 -PPP data with the transition 
shifted by 15.degree. C. appear to confirm an obligatory coupling of 
increased order with increased elastomeric force at fixed extension. 
The above described hydrophobic effect upon transition temperatures was 
also observed for the elastin polytetrapeptide, (Val.sup.1 -Pro.sup.2 
-Gly.sup.3 -Gly.sup.4).sub.n. That is, it was also shown that high 
molecular weight PTP undergoes a reversible temperature elicited 
aggregation with an onset of aggregation at 48.degree. C., rather than 
24.degree. C. as for high molecular weight PPP. 
However, it also been found that the inverse temperature transition for PTP 
is only complete at about 70.degree. C. This high temperature of 
transition is due to the lower hydrophobicity of PTP, and of poly VPGAG, 
as compared to PPP. 
For example, utilizing the Bull-Breese hydrophobicity scales with the 
hydrophobicity of the Gly residue taken as zero, the free energy of 
transfer for the pentamer, VPGVG, would be -4100 cal/mole whereas that of 
the tetramer, VPGG, would be -2540 cal/mole. Thus, if hydrophobicity of 
the repeating unit is the determining factor, then the inverse temperature 
transition for the PTP would be at a higher temperature than that of the 
PPP. Furthermore if the inverse temperature transition (the increase in 
intramolecular order) is required for the development of elastomeric 
force, then the temperature dependence of elastomeric force of the PTP 
matrix would be expected to show a similar shift to higher temperature 
relative to that of the PPP matrix. 
This inverse temperature transition is actually centered at near 50.degree. 
C. for PTP, shifted some 25.degree. C. higher than that of PPP. For 
Ile.sup.1 -PTP, it is shifted some 40.degree. C. lower in temperature than 
that of PTP. Also, it has been found that the development of elastomeric 
force upon raising the temperature is similarly shifted about 25.degree. 
C. higher for the PTP matrix (20 Mrad cross-linked) as compared to the PPP 
matrix (20 Mrad cross-linked). 
Accordingly, in view of the above, it is possible, by selecting the 
appropriate combination of PTP and PPP matrices or analogs thereof to 
shift the transition temperature of a bioelastomer containing elastin PTP, 
PPP and analogs thereof and PHP over a range of about 75.degree. C. 
Furthermore, wherever this transition would occur in the range of about 
-25.degree. C. for about 50% Phe.sup.1 -PPP in water/ethylene glycol or 
about 50.degree. C. for PTP, in water, for example, there is a large 
change in elastomeric force which accompanies a relatively small change in 
temperature. 
Thus, bioelastomers are available having incorporated therein repeating 
units having decreased hydrophobicity, such as --(VPGG).sub.n --or 
--(VPGAG)--.sub.n. 
For example, bioelastomers were provided containing elastomeric units 
comprising tetrapeptide, or pentapeptide or units thereof modified by 
hexapeptide repeating units and mixtures thereof, wherein the repeating 
units comprise amino acid residues selected from the group consisting of 
hydrophobic amino acid and glycine residues, wherein said repeating units 
exist in a conformation having a .beta.-turn which comprises a 
tetrapeptide of the formula: 
EQU --X.sup.3 --(VPGG).sub.n --Y.sup.3 -- 
wherein 
X.sup.3 is PGG, GG, G or a covalent bond; 
Y.sup.3 is VPG, VP, V or a covalent bond; and 
V is a peptide-producing residue of L-valine; 
P is a peptide-producing residue of L-proline; 
and 
G is a peptide-producing residue of glycine; and n is an integer from 1 to 
5,000, or n is 0, with the proviso that X.sup.3 and Y.sup.3 together 
constitute a repeating tetrameric unit in an amount sufficient to adjust 
the development of elastomeric force of the bioelastomer to a 
predetermined temperature. 
Moreover, bioelastomers were provided containing elastomeric units 
comprising tetrapeptide, or pentapeptide or units thereof modified-by 
hexapeptide repeating units and mixtures thereof, wherein the repeating 
unit comprises amino acid residues selected from the group consisting of 
hydrophobic amino acid and glycine residues, wherein said repeating units 
exist in a conformation having a .beta.-turn which comprises 
(A) a polypentapeptide of the formula: 
EQU --X.sub.1 --(IPGVG).sub.n --Y.sup.1 -- 
wherein X.sup.1, Y.sup.1, P, G, I, V and n are as defined above; and 
(B) a polypentapeptide of the formula: 
EQU --X.sup.2 --(VPGVG)--.sub.n Y.sup.2 --or --X.sup.2 --(VPGAG)--.sub.n 
Y.sup.2 -- 
wherein X.sup.2, Y.sup.2, P, G, V, A and n are as defined above; or 
(C) a polytetrapeptide of the formula: 
EQU --X.sup.3 --(VPGG).sub.n --Y.sup.3 --or --X.sup.3 --(FPGG)--.sub.n Y.sup.3 
-- 
wherein X.sup.3, Y.sup.3, P, G, V, F and n are as defined above, and F is 
phenylalanine, in relative amounts sufficient to adjust the development of 
elastomeric force of said bioelastomer to a predetermined temperature. 
Thus, any PTP-analog can be used in the preparation of bioelastomers which 
suffices to attenuate the hydrophobicity of the functional repeating unit, 
such as --IPGG-- and --FPGG-- while retaining the elasticity of the 
bioelastomer. 
Thus, bioelastomers are provided containing elastomeric units comprising 
tetrapeptide, or pentapeptide or units thereof modified by hexapeptide 
repeating units and mixtures thereof, wherein the repeating units comprise 
hydrophobic amino acid and glycine residues, wherein the repeating units 
exist in a conformation having a .beta.-turn which comprises a 
tetrapeptide of the formula: 
EQU --X.sup.4 --(IPGG).sub.n --Y.sup.4 -- 
wherein 
X.sup.4 is PGG, GG, G or a covalent bond; 
Y.sup.4 is IPG, IP, I or a covalent bond; and 
I is a peptide-producing residue of L-isoleucine; 
P is a peptide-producing residue of L-proline; and 
G is a peptide-producing residue of glycine; 
and n is an integer from 1 to 5,000, or n is 0, with the proviso that 
X.sup.4 and Y.sup.4 together constitute a repeating tetrameric unit, in an 
amount sufficient to adjust the temperature of which the elastomeric force 
of the bioelastomer develops. 
Also provided are bioelastomers having the above-recited structural 
features, but which have any combination of the repeating units 
--IPGVG--.sub.n, --VPGVG--.sub.n, --VPGAG--.sub.n, --VPGG--.sub.n, 
--IPGG--.sub.n, or other analogs thereof, such as Ala.sup.3 -PPP or 
Phe.sup.1 -PPP. 
In fact, the present invention may include, in addition to 
polynonapeptides, in general, all bioelastomers containing elastomeric 
units comprising tetrapeptide, or pentapeptide or units thereof modified 
by hexapeptide repeating units and mixtures thereof, wherein the repeating 
units comprise hydrophobic amino acid residues and glycine residues, 
wherein the repeating units exist in a conformation having a .beta.-turn 
which comprises a tetrapeptide or pentapeptide unit or repeating unit 
thereof, in an amount sufficient to adjust the development of elastomeric 
force at fixed length of said bioelastomer to a predetermined temperature, 
with the proviso that the elasticity of the bioelastomer is retained. 
The following Examples and discussion are provided to exemplify the 
preparation of PTP. Of course, the Examples are for purposes of 
illustration only and are not intended to limit the present invention. 
EXAMPLES 
Peptide Synthesis 
General Approach: The synthesis of polytetrapeptide, (VPGG).sub.n, can be 
achieved using any of the following permutations as the starting tetramer 
unit: Val-Pro-Gly-Gly, Gly-Val-Pro-Gly, Gly-Gly-Val-Pro, or 
Pro-Gly-Gly-Val. The first sequence (VPGG) was used in this laboratory 
both with the pentachlorophenyl ester (OPcp) activation and with the 
p-nitrophenyl ester (ONp) activation methods, and the latter method 
yielded polymer of significantly higher molecular weight. The sequence 
(GVPG) was utilized with -OPcp activation but no mention was made about 
the size of the polymer. In synthesizing the polypentapeptide, 
(VPGVG).sub.n, using different permutations of the pentamer unit with 
different activating groups for polymerization, it was observed that the 
pentamer having Pro as the C-terminal amino acid and -Onp for activation 
gave high molecular weight polymers. Similar results have been experienced 
in the case of the preparation of polyhexapeptide, (VAPGVG).sub.n. Hence, 
a similar approach was determined to be reasonable in the case of PTP 
also, i.e., sequence (GGVP) with -ONp activation. For comparison, 
H-VPGG-ONp, H-GVPG-ONp and H-GGVP-ONp were all tried for polymerization. 
As expected, the latter tetramer sequence gave a very high molecular 
weight polymer when determined by the TPI studies and here is described 
the synthesis of this latter material as shown in the Scheme II. The 
sequence (PGGV) wa not attempted because it has an optically active and 
bulky amino acid, Val, at its C-terminal. 
##STR2## 
Boc-GG-OBzl (I) was prepared using EDCI for coupling and was hydrogenated 
to give acid (II). Boc-VP-OBzl (III) was synthesized by the mixed 
anhydride method in the presence of HOBt, deblocked, and coupled with II 
using EDCI-HOBt to obtain Boc-GGVP-OBzl (IV). After hydrogenating to the 
acid, V, it was converted to -ONp (VI) by reacting with 
bis(p-nitrophenyl)carbonate. After removing the Boc-group, the active 
ester was polymerized, dialyzed against water using a 50,000 molecular 
weight cut-off dialysis tubing and lyophilized. The intermediate and the 
final products were checked by carbon-13 nuclear magnetic resonance, 
thin-layer chromatography (TLC) and elemental analyses. 
Details of Syntheses: Valine and Proline are of L'configuration. Boc-amino 
acids were purchased from Bachem, Inc., Torrance, Calif. HOBt was obtained 
from Aldrich Chemical Co., Milwaukee, Wis., and Bio-sil silica gel 
(200-400 mesh) was purchased from Bio-Rad Laboratories, Richmond, Calif. 
TLC plates were obtained from Whatman, Inc., Clifton, N.J. and the 
following solvent systems were used for determining the homogeneity of the 
products: R.sub.f.sup.1, CHCl.sub.3 (C):MeOH (M):CH.sub.3 COOH (A), 
95:5:3, R.sub.f.sup.2, CMA (85:15:3); R.sub.f.sup.3, CMA (75:25:3); 
R.sub.f.sup.4, CM (5:1). Elemental analyses were carried out by Mic Anal, 
Tuscon, Ariz. Melting points were determined with a Thomas Hoover melting 
point apparatus and are uncorrected. 
Boc-Gly-Gly-OBzl (I): Boc-Gly-OH (17.52 g, 0.1 mole) in a mixture of 
CHCl.sub.3 (50 ml) and acetonitrile (50 ml) was cooled to -15.degree. C. 
and EDCI (19.17 g, 0.1 mole) was added and stirred for 20 minutes. To 
this, a pre-cooled solution of H-Gly-OBzl.tosylate (37.1 g, 0.11 mole), 
NMM (12.09 ml, 0.11 mole) in CHCl.sub.3 (100 ml) was added and stirred 
overnight at room temperature. After removing the solvent, the residue was 
taken in CHCl.sub.3 and extracted with acid and base. Chloroform was 
removed under reduced pressure, triturated with pet. obtain 30.2 g of I 
(yield: 93.7%), m.p. 82.degree.-83.degree. C. R.sub.f.sup.2, 0.52; 
R.sub.f.sup.4, 0.82. Anal. Cald. for C.sub.16 H.sub.22 N.sub.2 O.sub.5 : 
C, 59.61; H, 6.88, N, 8.69%. Found: C, 59.43; H, 6.88; N, 8.35%. 
Boc-Gly-Gly-OH (II): I (10 g, 0.31 mole) in acetic acid (100 ml) was 
hydrogenated at 40 psi in the presence of 10% Pd-C catalyst (1 g). The 
catalyst was filtered with the aid of celite and solvent removed under 
reduced pressure. The residue was triturated with EtOAC, filtered, washed 
with EtOAC, pet. ether and dried to yield 6.3 g of II (yield: 87.5%), m.p. 
118.degree.-120.degree. C. (decomp.). R.sub.f.sup.2, 0.28; R.sub.f.sup.3, 
0.44. Anal. Calcd. for C.sub.9 H.sub.16 N.sub.2 O.sub.5 H.sub.2 O: C, 
43.19; H, 7.25; N, 11.19%. Found; C, 43.53; H, 7.40; N 10.90% 
Boc-Gly-Gly-Val-Pro-OBzl (IV): III (6.0 g, 0.0148 mole) was deblocked with 
HCl/Dioxane and solvent removed under reduced pressure. The residue was 
triturated with ether, filtered, washed with ether, then pet. ether and 
dried. A very hygroscopic material was obtained (4.2 g, 0.0123 mole) which 
was coupled in DMF with II (2.86 g, 0.0123 mole) in the presence of 10% 
excess of EDCI (2.60 g) and HOBt (2.07 g). The reaction was worked up as 
described for I to obtain IV as a white foam in a quantitative yield, no 
sharp m.p. 54.degree.-62.degree. C. R.sub.f.sup.2, 0.42, R.sub.f.sup.3, 
0.74. Anal Calcd. for C.sub.26 H.sub.38 N.sub.4).sub.7 ; C, 60.21; H, 
7.38; N, 10.805. Found: C, 60.0; H, 7.46; N, 10.81%. 
Boc-Gly-Gly-Val-Pro-OH (V): IV (6.2 g, 0.012 mole) in acetic acid was 
hydrogenated and worked up as for II to obtain V quantitatively, no sharp 
m.p. 743 Calcd. for 83.degree. C. R.sub.f.sup.3, 0.25; R.sub.f.sup.4, 
0.15. Anal. Calcd. for C.sub.19 H.sub.32 N.sub.4 O.sub.7 : C, 51.10; H, 
7.67; N, 12.54%. Found: C, 51.28: H, 7.50, N, 12.38%. 
Boc-Gly-Gly-Val-Pro-ONp (VI): V (5.3 g, 0.0123 mole) in pyridine (30 ml) 
was reacted with bis(pnitrophenyl)carbonate (5.64 g, 0.0185 mole). After 
removing the solvent, the residue was taken in CHCl.sub.3 and extracted 
with acid and base. The peptide was chromatographed over a silica-gel 
column and eluted with 35% acetone in CHCl.sub.3 after initially eluting 
with CHCl.sub.3 ' to obtain 4.7 g of VI (yield: 69.2%), no sharp 
74.degree.-79.degree. C. R.sub.f.sup.2, 0.76; R.sub.f.sup.4, 0.75. Anal. 
Calcd. for C.sub.25 H.sub.35 N.sub.5 O.sub.9.1/2H.sub.2 O: C, 53/75 H, 
6.49; N, 12.53%. Found: C, 53.69; H, 6.44; N, 12.34%. 
H-(Gly-Gly-Val-Pro).sub.n -OH (VII): VI (4.5 g, 0.0082 mole) in CHCl.sub.3 
(20 ml) was treated with TFA (35 ml) for 30 minutes and solvent removed 
under reduced pressure. The residue was triturated with ether, filtered, 
washed with ether, then with pet. ether and dried. The TFA salt (3.9 g, 
0.0069 mole) in DMSO (7.6 ml) and NMM (1.22 ml, 1.6 equiv) was stirred for 
14 days. After diluting with cold water, the polymer was dialyzed in a 50 
kD cut-off dialysis tubing, changing water daily for 15 days, and the 
retentate was lyophilyzed to yield 1.65 g of the polytetrapeptide (yield: 
77%). The carbon-13 NMR spectrum has been determined. The assignments have 
all been made and there were no extraneous peaks thereby verifying the 
synthesis. 
Attention is here directed to the remarks made above regarding the use of 
microbial biosynthesis for the preparation of the present bioelastomers. 
Temperature Profiles for Coacervation 
Polypeptide coacervation in water is reversible aggregation to form a new 
phase with a distinct composition. Association occurs on raising the 
temperature, disassociation on lowering the temperature. The process of 
coacervation was followed by monitoring the turbidity as a function of 
temperature using a Cary 14 spectrophotometer set at 300 nm, a Neslab 
ETP-3 temperature programmer with a 30.degree. C./hour scan rate and an 
Omega 199A thermocouple monitor. The sample cell was placed in a vibrating 
chamber (300 Hz) to keep the aggregates from settling and to facilitate 
equilibrium. The temperature profiles for coacervation are concentration 
dependent. Dilution from a high concentration, after the high 
concentration limit is reached (approximately 40 mg/ml for high molecular 
weight elastomeric polypeptides), results in a shift of the turbidity 
profile to higher temperature. 
Circular Dichroism Measurements 
A Cary 60 spectropolarimeter equipped with a Model 6001 circular dichroism 
accessory with 330 Hz modulation of the left and right circular polarized 
beams was used to determine the circular dichroism patterns of 5 mg PTP in 
one ml of deionized-distilled (quartz immersion heater) water. Because of 
the smaller size or the relative transparency of the PTP aggregates (as 
with the cross-linked PTP matrix with a relatively small change in 
refractive index between solution and matrix) when compared to that of the 
PPP system, it was possible to use the 5 mg/ml concentration for the CD 
studies without being compromised by light scattering (particulate) 
distortions of the CD spectra. This is apparent from monitoring the 
negative band near 220 nm which becomes damped and red-shifted as the 
particulate distortions become significant. 
Preparation of the Cross-linked PTP Matrix 
The PTP was prepared for .gamma.-irradiation cross-linking by dissolving 
130 milligrams of th peptide in 220 milligrams of water in a cryotube. The 
material was shear oriented overnight at 40.degree. C. in a previously 
described pestle-cryotube assembly. The sample was exposed to 
approximately 8,000 Roentgen/min .gamma.-irradiation at the Auburn 
University Nuclear Science Center. Exposure was of sufficient time to 
achieve a 20.times.10.sup.6 radiation absorbed dose (20 Mrad). 
Thermoelasticity Measurements 
Thermoelasticity studies were carried out on a stress-strain apparatus. 
Clamping of the sample in the holder was done in two stages to prevent 
damage to the material at the clamp edge. The sample was first gripped 
lightly with the top clamp, raised to 60.degree. C. while submerged in 
water within the temperature jacket and allowed to equilibrate for about 2 
hours. The measured force consisting of the weight of the sample and grips 
in water were set to zero. The bottom grip was then attached to the sample 
and both grips tightened to hold the sample firmly. The bottom clamp was 
driven as in a stress-strain measurement and stopped at 40% elongation. 
Force data were recorded in 5.degree. C. steps starting at 70.degree. C. 
and continuing to 40.degree. C. where the force approached zero. 
RESULT 
Temperature Profiles for Coacervation 
The polytetrapeptide is soluble in water in all proportions below 
40.degree. C. On raising the temperature above 40.degree. C. the solution 
becomes turbid; on standing settling occurs to form a dense viscoelastic 
phase called a coacervate. The process is readily reversible; on lowering 
the temperature cloudiness clears and coacervate readily redissolves. By 
following the turbidity as a function of temperature, temperature profiles 
for coavervation are obtained which are concentration dependent. As more 
concentrated solutions are used, the onset of turbidity occurs at lower 
temperatures until further increases of concentration cause no further 
lowering of the temperature for onset of turbidity. The lower 
concentration above which raising the concentration no further lowers the 
temperature for onset of turbidity is called the high concentration limit. 
For this high molecular weight PTP the high concentration limit is 40 
mg/ml as 100 mg/ml gives the same profile. Dilution from 40 mg/ml causes a 
shift to higher temperature for the onset. are compared to similar data 
for the PPP. The midpoint for the high concentration limit of PTP is 
49.degree. C. whereas the value for the high concentration limit of PPP is 
25.degree. C. The decreased hydrophobicity of the tetramer results in a 
24.degree. C. increase in the temperature required to bring about the 
hydrophobic interactions attending aggregation. 
Circular Dichroism 
At the lower temperature there is a negative band near 220 nm and a second 
negative band in the 195-200 nm range. This latter band is considered to 
be indicative of polypeptides with limited order as fully disordered 
polypeptides are considered to have a negative band near 195 nm with an 
ellipticity of -4.times.10.sup.4. The lower magnitude of the short 
wavelength negative band for PTP and the negative band near 220 nm 
indicate some order in the PTP at 35.degree. C. On raising the temperature 
the short wavelength negative band decreases in magnitude indicative of a 
transition toward greater intramolecular order. Interestingly, its 
midpoint corresponds approximately to the midpoint in the temperature 
profile for coacervation for a comparable concentration. It is important 
to note for the PTP that the change in intramolecular order precedes the 
intermolecular interactions, i.e., begins at a substantially lower 
temperature than the aggregational process. The temperature midpoint for 
the PTP intramolecular transition is shifted some 25.degree. C. to higher 
temperatures from that of the PPP. Thus, the intramolecular ordering of 
the PTP is shifted to higher temperature due to the decreased 
hydrophobicity of the tetramer as compared to the pentamer. 
Thermoelasticity Data 
The temperature dependence of elastomeric force (thermoelasticity data) is 
plotted in FIG. 8B for 20 Mrad cross-linked PTP at an extension of 40%. 
There is very little elastomeric force exhibited by this matrix below 
40.degree. C. As the temperature is raised above 40.degree. C., however, 
the elastomeric force develops to a maximal value near 70.degree. C. Also 
included for comparison in FIG. 8B are the thermoelasticity data for a 20 
Mrad cross-linked PPP matrix which exhibit a similar transition but 
shifted some 20.degree. to 25.degree. C. to lower temperatures. The 
development of elastomeric force, just as the temperature dependence of 
coacervation and of ellipticity for the PTP, is shifted by about 
25.degree. C. from that of the PPP. These properties are a function of the 
hydrophobicity of the repeating unit. Of particular interest is the 
comparison of the ellipticity data for the PTP with the thermoelasticity 
for the PTP. The transition as followed by ellipticity, which is a measure 
of intramolecular order, begins in the range 35.degree. to 40.degree. C., 
and similarly the elastomeric force begins, to develop just below 
40.degree. C. By both physical measurements the transition is essentially 
complete by 70.degree. C. There is a close parallel between-increase in 
intramolecular order and increase in elastomeric force. As the 
aggregational intermolecular processes, followed by turbidity, do not 
become significantly until nearly 50.degree. C., it appears that the PTP 
matrix allows a delineation between intramolecular and intermolecular 
processes as related to origins of elastomeric force. 
The bioelastomeric materials of the present invention exhibit an inverse 
temperature transition resulting in the development of a regular 
non-random structure, unlike typical rubbers, which utilize, as a 
characteristic component, hydrophobic intramolecular interactions. The 
regular structure appears to be a .beta.-spiral, a loose water-containing 
helical structure with .beta.-turns as spacers between turns of the helix 
which provides hydrophobic contacts between helical turns and has 
suspended peptide segments. These peptide segments are relatively free to 
undergo large amplitude, low frequency rocking motions called liberations. 
This produces the librational entropy mechanism of elasticity. 
An essential feature of the elastomeric pentapeptide, tetrapeptide, 
hexapeptide and nonapeptide-containing elastomeric units of the present 
invention is the existence of a sequence of regularly recurring 
.beta.-turns in the protein's secondary structure, i.e., the conformation 
of its peptide chain. A .beta.-turn is characterized by a ten atom 
hydrogen bonded ring of the following formula showing residues i, i+1, i+2 
and i+3: 
##STR3## 
In this formula, R.sub.1 -R.sub.5 represent the side groups of the 
respective amino residues. Notably, R.sup.5 of (i+4) can also be i' of a 
subsequent .beta.-turn. Likewise, subsequent residues (i+1)', (i+2)' and 
(i+3)' are analogous and as defined for residues i, i+1, i+2 and i+3 of 
the first .beta.-turn. 
The spiral structures produced by a series of .beta.-turns are more open 
than the more common .alpha.-helix. As a result, the atoms between the 
atoms that participate in hydrogen bonding have a relatively greater 
freedom of movement, more so than in an .alpha.-helix. This is 
particularly true of librational motions involving peptide moieties. A 
libration is a torsional oscillation involving simultaneous rotational 
motions of the two single bonds on each side of a librating moiety. The 
moiety involved in a libration may be a single peptide bond or several 
peptide residues. For adequate freedom of motion to exist, it is 
important, however, that the carbonyl oxygen and that amino hydrogen of 
the peptide bond not be involved in a motional restricting hydrogen 
bonding to other parts of the molecule or to other peptide molecules. 
Otherwise a greater energy barrier to the libration exists and motion will 
be restricted. Since non-hydrogen-bonded segments having freedom of motion 
exist in the .beta.-spiral between the points of hydrogen bonding for the 
.beta.-turns, these segments may be said to be librationally suspended 
with librational capabilities. Librationally active suspended segments 
associated with glycine resides are, therefore, a necessary structural 
feature that exists in the elastomeric polymer between the repeating 
.beta.-turns. 
Another factor leading to the high librational freedom of such molecules is 
the absence of polar interactions between the amino acid residues, either 
interchain or interchain, other than the previously mentioned hydrogen 
bond. The amino acid residues present are generally all hydrophobic or 
glycine and accordingly do not exert significant forces on one another 
through space. If charged or polar groups are present, electrostatic 
interactions would limit librational freedom and restrict the number of 
available states in the related (non-extended) form of the molecules. 
Polar and charged amino acid residues are not strictly prohibited, 
however, if their presence does not destroy the elasticity of the 
polypeptide molecule as a whole. For example, an occasional serine residue 
is present in the polypentapeptide sequence of naturally occurring porcine 
tropoelastin without destroying elasticity. Accordingly, hydrophobic amino 
acid residues and glycine are preferred in forming elastomeric 
polypeptides of the present type although other amino acids may be present 
to a small extent. 
The size of the repeating unit of the elastomeric component is important in 
determining the stability and dynamic properties of the .beta.-spiral. 
Repeating units having fewer than four or more than five amino acid 
residues do not easily form .beta.-spirals having the required librational 
motions. Three amino acid residues are too few for both a .beta.-turn and 
a dynamic suspended segment. 
For the PTP, the dynamic segment is simply the residue 4- residue 1 
bridging peptide moiety. Further, for the PPP, the dynamic segment 
includes both peptde moieties flanking the fifth residue. We now describe 
elastomeric repeats wherein the dynamic segments involve entire 
pentameric, tetrameric or trimeric bridging segments. Clearly the key 
elements are structural hydrophobic spacers, e.g., the .beta.-turn, 
between which are segments capable of dynamic motion wherein the dynamic 
motion becomes of lower amplitude upon chain extension. 
In the specific case of the polynonapeptide it can be a combination of two 
.beta.-turns, the common .beta.-turn with Pro.sup.2 -Gly.sup.3 as residues 
i+1 and i+2, and an additional .beta.-turn with Val.sup.6 -Gly.sup.7 as 
residues (i+1)' and (i+2)'. In this case, the dynamic suspended segment is 
best defined as the Ala.sup.8 -Gly.sup.9 -Val.sup.1 segment or the 
Val.sup.8 -Gly.sup.9 -Val.sup.1 segment by direct analogy to the Val.sup.4 
-Gly.sup.5 -Val.sup.1 suspended segment of poly(VPGVG). 
The choice of individual amino acids from which to synthesize the bridging 
sections of the elastomeric repeating units and resulting polypeptide is 
unrestricted so long as the resulting structure comprises librationally 
suspended segments in a .beta.-spiral. The amino acids are not restricted 
to .alpha.-amino acids, although these are preferred since it has recently 
become possible to predict the occurrence of .beta.-turns from the 
.alpha.-amino acid sequence of a polypeptide A review article discussing 
the prediction of protein conformation, including the prediction of 
.beta.-turns, was published by Chou and Fasman, Ann. Rev. Biochem., 47, 
251 (1978), which is herein incorporated by reference. The size of the 
side chains present in the hydrophobic amino acids does not greatly affect 
the .beta.-spiral since the side chains generally extend outward from the 
surface of the spiral with some important but non-restrictive interturn 
hydrophobic interactions. However,,in order to minimize interchain 
interactions, it is preferred that the side chain contain no more than 10 
carbon atoms. Preferred hydrophobic side chains are the same as those 
previously described for position .delta., which is position i+3. In 
addition, it appears from the studies leading to the present invention 
that preferred side chains of the amino acids are hydrogen or hydrocarbon 
chains having 1-4 carbon atoms. Examples of especially preferred residues 
are glycine and the naturally occuring L-amino acids alanine, valine, 
leucine, and isoleucine as well as closely related molecules such as 
2-methyl-2-aminopropanoic acid, L-2-aminobutanoic acid, and 
L-2-methyl-2-aminobutanoic acid, although it is preferred that the 
.alpha.-carbon have an .alpha.-hydrogen. Proline is also a preferred amino 
acid. 
It is also possible to use .alpha.-hydroxy acids, such as 
.alpha.-hydroxy-isovaleric acid or even glycolic acid, at any positions 
except position 4, which are positions i+3 and (i+3)' which are .delta. 
and .pi., respectively. 
For clarity, the sequence: 
EQU (.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.) 
is numbered such that .alpha. is position i, .beta. is position i+1, 
.gamma. is position i+2 and .delta. is position i+3. 
.epsilon.,.theta.,.lambda.,.pi.and .rho. might then be considered 
positions i+4, i+5, i+6, i+7 and i+8, respectively. For the subsequent 
.beta.-turn, i+4 is i', i+5 is (i+1)', i+6 is (i+2)', and i+7 is (i+3'). 
This numbering sequence is also used above in the illustration of the 
.beta.-turn in order to show the residues which are important in the 
.beta.-turn formation. However, in accordance with the present invention, 
each of the above residues, i.e, .alpha. through .rho. may be a variety of 
peptide-forming residues as will now be discussed. 
.alpha. is generally any L-hydrophobic peptide-forming residue. However, 
.alpha. is generally L-Valine or another peptide-forming residue capable 
of functioning in position i of a .beta.-turn in a polypeptide. By the 
term "capable of functioning in position i" is meant peptide-forming 
residues of L-Valine, L-isoleucine, L-leucine, L-phenylalanine, 
L-tryptophan and L-tryosine, for example. L-Valine, however, is a 
preferred such peptide-forming residue. 
.beta. is generally also any L-hydrophobic peptide-forming residue. 
However, .beta. is generally L-Proline or another peptide-forming residue 
capable of functioning in position i+1 of a .beta.-turn in a polypeptide. 
By the term "capable of functioning in position i+1" is meant 
peptide-forming residues of L-Proline and residues which sterically 
constrain the peptide-bond torsion angle. These may be the same residues 
as defined for .alpha.. Such residues are, for example, L-Valine, 
L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan and L-tyrosine, 
L-Proline, however, is a preferred such peptide-forming residue. 
.gamma. is any hydrophobic peptide-forming residue which has a relatively 
small steric requirement .gamma. is generally Glycine, however other 
peptide-forming residues capable of functioning in position i+2 of a 
.beta.-turn in a polypeptide may be used. By the term "capable of 
functioning in position i+2" is meant peptide-forming residues of Glycine, 
D-alanine, L-alanine, D-valine, D-isoleucine, D-leucine, D-phenylalanine, 
D-tryptophan and D-tyrosine. Other residues of similar steric requirements 
may also be used. The only exception appears to be L-hydrophobic residues 
other than glycine and L-alanine which appear to interfere with 
.beta.-turn formation. However, it is preferred that .gamma. is glycine. 
.delta. is generally any L-hydrophobic peptide-forming residue. However, 
.delta. is generally as defined above for .alpha.. Thus, by the term 
"capable of functioning in position i+3" is meant peptide-forming residues 
of L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan and 
L-tyrosine, for example. It is preferred that .delta. is L-phenylalanine 
or L-leucine. 
.epsilon. is generally any hydrophobic peptide-forming residue of small 
steric requirements. In this case, generally .epsilon. is a 
peptide-forming residue of Glycine or D-alanine. .epsilon. is preferably 
Glycine. 
However, .epsilon. may also be a hydrophobic peptide-forming residue of 
large steric requirement as defined above for .alpha., in which case 
.delta. is necessarily a hydrophobic peptide-residue of small steric 
requirement as defined above for .epsilon.. In this case, .delta. is then 
as defined for .epsilon., i.e., a peptide-forming residue of Glycine or 
D-alanine. 
For the subsequent .beta.-turn, .epsilon. is position i', and .theta., 
.lambda., and .pi. are positions (i+1)', (i+2)' and (i+3)'. 
When .epsilon. functions as position i' of the subsequent .beta.-turn in 
the polypeptide, it may be Glycine or D-alanine when .delta. is as defined 
above. Alternatively, .epsilon. may be defined as .alpha., when .delta. is 
Glycine or D-ala. 
.theta. is generally any L-hydrophobic peptide-forming residue. However, 
generally .theta. is a peptide-forming residue such as L-valine, 
L-alanine, or as defined above for .alpha.. Alternatively, when .theta. 
functions as position (i+1)' of a subsequent .beta.-turn in a polypeptide, 
.theta. may also be a peptide-forming residue as defined above for .beta.. 
.lambda. is generally Glycine or any peptide-forming residue as defined 
above for .gamma. or such a residue of D-alanine. Notably, when .lambda. 
functions as position (i+2)' of a subsequent .beta.-turn in a polypeptide, 
.lambda. is as defined above for .gamma.. 
.pi. is a generally any L-hydrophobic peptide-forming residue. However, it 
is preferably such a residue of L-valine or L-alanine or such a residue as 
defined for .alpha.. However, .pi. may also be a direct bond. If .pi. is a 
direct bond, octameric sequences are described. One specific case of 
--(VPGG)-- or --(VPGGVPGG)-- has already been described in issued U.S. 
patents. 
Finally, .rho. is either glycine or D-alanine. 
Notably moieties X and Y are as defined above. However, a further proviso 
to the defined sequences is that for residues .epsilon., .theta., 
.lambda., .pi. and .rho. no more than three of the same should be glycine 
simultaneously. 
However, in another principal aspect of the present invention are provided 
polynonapeptides having an increased elastic modules and which exhibit 
chemotoxis toward fibroblastics and endothelial cells. When chemotaxis is 
also required, it is important that either of the pentameric sequences 
--(GFGVG)-- or --(GLGVG)-- be incorporated as five of the nine residues 
are preferably linked sequentially in the peptide sequence. 
Inasmuch as residue .pi. be a direct bond, the present invention also 
specifically contemplates octameric sequences. However, the same rules 
noted above for nonameric sequenes will also apply to the octameric 
sequences when designing bioelastomeric sequences and such sequences which 
are also chemotactic. 
As mentioned above, the present invention provides polynonapeptide and 
other elastomeric polypeptides containing nonapeptide sequences. 
In accordance with the present invention, the present nonapeptide 
sequences, i.e., 
--X--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.)--.sub. 
n Y-- may be incorporated as only one unit, where n is 1, in any one of the 
elastomeric polypeptides disclosed above or as many repeating units where 
n is up to about 5,000 units. The repeating polynonapeptide units may be 
part of a "homopolymer" polynonapeptide where n may have a value of as 
high as 5,000, or the nonapeptide units may be interspersed throughout 
other elastomeric polypeptide units to form a copolymeric elastomer. 
For example, the sequence: 
EQU --X--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.)--.sub.n 
Y-- 
as defined above, may be incorporated as part of a repeating unit: 
EQU --(A--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.)--.sub. 
n B)--.sub.m 
where A and B may be any of the elastomeric polypentapeptide or 
polytetrapeptide sequences described above, including those sequences 
incorporating any amino acid substitutions for the purpose of modifying 
the temperature of transition for the elastomer. In the above formula, 
both m and n have a value of 1 to about 5,000. 
For example, A and B may be the same or different and may each represent 
unit or repeating sequences of PPP or PTP or --(IPGVG)-- or --(IPGG)--, 
for example. A and B may represent random or block assemblages of any of 
the aforementioned elastomeric sequences. 
Also, A and B may each represent repeating sequences of --(APGVGV)--, which 
are not elastic and which function to align polypeptide chains by an 
interlocking mechanism and which also function to modulate the elastic 
modulus. 
It is also specifically contemplated by the present invention that the 
present nonapeptide and polynonapeptide sequences be used in conjunction 
with other elastomeric sequences, i.e., A and B, wherein the other 
elastomeric sequences have been modified for the purpose of attenuating 
the inverse temperature transition. Any of the above-described 
substitutions may be used and are herein considered to be within the 
definition of the terms "A" and "B". Thus, the moieties "A" and "B" are 
defined as elastomeric moieties which develop elastomeric force when at 
fixed length by an inverse temperature transition. 
It is important to note that any elastomeric sequence disclosed herein may 
be used as moieties A and B. Any combination of the above-described PTP, 
PPP or PHP sequences or those sequences substituted to modulate the 
temperature of the inverse temperature transition may be used in order to 
set the overall elastic modulus at a desired value. The phrase "moiety 
which is capable of modulating elastomeric force by an inverse temperature 
transition" concisely defines moieties A and B as the present inventors 
are unaware of any other polymeric materials which so function. 
Although PHP, as noted above, is not elastomeric, either moieties A or B 
may include such a sequence provided that the overall elastic modulus of 
the matrix is maintained at a desired value. 
For example, moieties A and B may each be --APGVGV).sub.m, --VPGVG).sub.m, 
--IPGVG).sub.m, --VPGAG).sub.m, --VPGG.sub.m, --IPGG).sub.m and 
--FPGG).sub.m, wherein V, P, G, I, A and F are peptide-forming residues of 
the respective amino acids as defined by the standard one-letter 
abbreviations, and m has a value of from 1 to about 5,000. 
Notably, all of the earlier above-described U.S. patents and pending patent 
applications are specifically incorporated herein by reference. 
The polynonapeptide of the present invention, whether homopolymer or 
copolymeric in nature, may be easily synthesized according to the methods 
previously described for PTP and PPP. 
Having now fully described the present invention, the same will now be more 
fully understood in view of the following examples which are provided 
solely for the purpose of illustration and are not intended to limit the 
present invention. 
EXAMPLE 
Peptide Synthesis: Elemental analyses were carried out by Desert Analytics, 
Tucson, Ariz. All amino acids were of the L-configuration. Thin layer 
chromatography (tlc) was performed on silica gel plates obtained from 
Whatman, Inc., New Jersey. tert-Butyloxycarbonyl (BOC)-amino acids and 
amino acid benzyl esters (-OBzl) were purchased from Bachem, Inc., 
Torrance, Calif. 
The synthesis of the nonapeptide and its polymer was carried out by the 
classical solution methods and is presented in Scheme III. Briefly, the 
synthetic approach was to synthesize the tetrapeptide unit BOC-AGVP-OH and 
the pentapeptide unit H-GFGVG-OBzl and couple them together using 
1-ethyl-3-dimethylaminopropyl carbodiimide (EDCI) and 
1-hydroxybenzotriazole (HOBt) to obtain BOC-AGVPGFGVG-OBzl, which 
represents a different permutation of VPGFGVGAG. The former sequence was 
selected since it represents sterically hindered amino acids at the 
N-terminus as well as at the C-terminus for possibly enhancing 
polymerization yields. The tetra- and penta-peptides were synthesized 
using the mixed anhydride method, or the EDCI coupling method as described 
previously. The nonapeptide benzyl ester was hydrogenated to the free acid 
and converted to the p-nitrophenyl ester (--ONp) using bis(p-nitrophenyl) 
carbonate. The N.sup..alpha. Boc-group was removed, and the active ester 
was polymerized for 14 days in dimethylsulfoxide (DMSO) at a one molar 
concentration in the presence of 1.6 equiv. of N-methylmorpholine (NMM) as 
the base. After diluting with water, the polymer was transferred into a 
3500 dalton cut-off dialysis tubing and dialyzed for one week changing 
water everyday. The polymer was lyophilized, treated with base, to remove 
any unreacted --ONp esters and redialyzed using a 50 kD cut-off dialysis 
tubing and lyophilized. The intermediate products and the final polymer 
were characterized by tlc, elemental analyses, carbon-13 and proton NMR 
spectroscopic methods. 
##STR4## 
Abbreviations: Boc, tert-butyloxycarbonyl; OBzl, benzyl ester; DMF, 
dimethylformamide; DMSO, dimethylsulfoxide; EDCI, 1 
ethyl-3-dimethylaminopropyl carbodiimide; HOBt, 1-hydroxybenzotriazole; 
IBCF, isobutylchloroformate; NMM, N-methylmorpholine; ONp, p-nitrophenyl 
ester; TFA, trifluoroacetic acid; A, alanine; G, glycine, F, 
phenylalanine; P, proline; V, valine. 
BOC-Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly-OBzl: BOC-AGVP-(OH(5.26 g, 11.8 
mmol) and HOBt (1.99 g, 13 mmol) were dissolved in dimethylformamide (35 
ml) and cooled with ice-water. EDCI (2.49 g, 13 mmol) was added and 
stirred for 20 min. To this solution was then added an ice cold solution 
of HCl-H-GFGVG-OBzl (7.6 g, 11.8 mmol) and NMM (1.3 ml, 11.8 (mmol) in DMF 
(35 ml). The reaction mixture was stirred for 2 days at room temperature 
and the solvent was removed under reduced pressure. The residue was taken 
into CHCl.sub.3 and extracted with water, 10% citric acid, water, 0.5N 
NaOH, water, dried over anhyd. Na.sub.2 SO.sub.4 and solvent removed under 
reduced pressure. The residue was triturated with ether, filtered, washed 
with ether, petroleum ether and dried. The peptide was characterized and 
compared with the sample previously prepared by a different method. 
BOC-Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly-ONp: After hydrogenating the above 
peptide benzyl ester, to produce the free acid, and amount of 7.5 g, 8.69 
mmol in pyridine (100 ml) was treated with bis(p-nitrophenyl) carbonate 
(3.69 g, 13 mmol) for several days. Pyridine was removed under reduced 
pressure, triturated with ether, filtered washed with ether, petroleum 
ether and dried to yield 7.3 g (yield: 85.38%) of the desired product. 
Further purification was carried out by silica gel column chromatography 
using 13% EtOH in CHC.sub.13. All of the fractions which showed single 
spots on tlc were pooled and concentrated. The R.sub.f in CHCl.sub.3 (85): 
CH.sub.3 OH(15): HOAC(3) was 0.58. Anal. calcd. for C.sub.46 H.sub.64 
N.sub.10 O.sub.014.2H.sub.2 O, C, 54.15; H, 6.71; N, 13.73%. Found: C, 
54.46; H, 6.48; N, 13.75%. 
Poly[Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly]-OH: The 
N.sup..alpha. -BOC-group from the above peptide-ONp was removed by 
treatment with TFA for 45 min. TFA was evaporated under reduced pressure, 
triturated with ether, filtered, washed with ether and dried. The TFA salt 
(1.0 g, 1 mmol) was taken in DMSO (1 ml); NMM (0.18 ml, 1.6 equiv.) was 
added, and the sample was stirred for 14 days. After 4 days of stirring, 
the solution turned very viscous and 1.5 ml more of DMSO were added and 
stirring continued. The reaction mixture was diluted with water, 
transferred into dialysis tubing and dialyzed with frequent exchanges of 
bathing water for 10 days. The insoluble material in the tubing was 
treated with 1N NaOH overnight in the cold room, neutralized and 
redialyzed using a 50 kD cut-off dialysis tubing. The polymer was 
solubilized by lyophilizing from trifluoroethanol-water mixtures. 
Nuclear Magnetic Resonance Verification of Synthesis: Both carbon-13 and 
proton nuclear magnetic resonance (NMR) spectra of poly(VPGFGVGAG) are 
given in FIGS. 1 and 2, respectively. The assignment of resonances are all 
indicated and these with the absence of extraneous peaks verify the 
synthesis and purity of the product. 
Formation of Insoluble Matrices: The polynonapeptide, poly(VPGFGVGAG), can 
be solubilized in water below 5.degree. C. On raising the temperature, the 
solution becomes cloudy as shown in the temperature profiles for turbidity 
development (aggregation) in FIG. 3 for 40 mg/ml and 1 mg/ml solutions. As 
the aggregates grow in size, they settle out to form a viscoelastic phase. 
When the viscoelastic phase is collected in a tube and a pestle with a 
channel cut in it is inserted into the tube, the viscoelastic phase flows 
into and fills the channel On .gamma.-irradiation with a 20.times.10.sup.6 
radiation absorbed dose (20 Mrad) from a Cobalt-60 source, a cross-linked 
elastomeric matrix is produced which is suitable for mechanical studies. 
The resulting matrix is referred to as X.sup.20 -PNP or X.sup.20 
-poly(VPGFGVGAG). 
Stress/Strain Studies: The stress/strain studies were performed on an 
apparatus built in this laboratory as previously described with the 
exception that a Sensotec Model 31 force transducer was used allowing 
force levels up to 250 grams. 
The carbon-13 and proton NMR data of FIGS. 1 and 2 evidence the obtainment 
of the polynonapeptide sequence poly(VPGFGVGAG). The sequence was also 
confirmed using both correlation and nuclear Overhauser enhancement 
spectroscopy. 
When greater than 50,000 molecular weight polynonapeptide is prepared by 
means of equilibrium dialysis with a 50 kD cut-off dialysis membrane, the 
material is found to be solubilized below 5.degree. C. in water. As seen 
from the temperature profiles of turbidity formation (TP.tau.) in FIG. 3, 
the midpoint of the TP.tau. for a concentration of a 40 mg/ml solution is 
about 8.degree. C. whereas for a 1 mg/ml solution, the midpoint is raised 
only to about 11.degree. C. The slope of the profile has decreased on 
dilution consistent with a cooperative nature to the aggregational process 
which has been extensively demonstrated to be an inverse temperature 
transition for poly(VPGVG). 
The stress/strain data for two .gamma.-irradiation cross-linked samples of 
X.sup.20 -PNP are given in FIG. 4. After the initial cycle (not shown), 
there is a readily reproducible curve obtained with a regular amount of 
hysteresis. The elastic modulus at 4% extension was approximately 6 to 
7.times.10.sup.7 dynes/cm.sup.2 for the sample cycled at 5% extensions. 
Cyclic extensions up to &gt;60% have been demonstrated. When a 25 Mrad 
cross-linking dose was used, elastic moduli of 10.sup.8 dynes/cm.sup.2 
have been obtained. This elastic modulus is two orders of magnitude 
greater than that obtained with similarly treated poly (VPGVG). Thus, the 
present polynonapeptide sequences afford bioelastomers having excellent 
elastic properties. 
The present polynonapeptides also exhibit chemotaxis toward fibroblasts and 
endothelial cells. Chemotaxis is the vectorial translocation of cells in 
response to an increasing chemical gradient. Thus, with present 
polynonapeptide, it is now possible to prepare elastomeric polypeptide 
biomaterials which, with the appropriate mix of PPP and PTP, can be made 
to match vascular wall compliance. Such chemotactic sequences within the 
prepared biomaterials support the development of an endothelial lining in 
the case of vascular walls, or the invasion of fibroblasts in the case of 
a ligament or a scaffolding for a ligament. 
The chemotaxis of the present polynonapeptide towards fibroblasts and 
endothelial cells can be determined and verified by the well known 
checkerboard assay of Zigmond and Hirsch. See S. H. Zigmond and J. G. 
Hirsch, "Leukocyte Locomotion and Chemotaxis: New Methods for Evaluation 
and Demonstration of a Cell Derived Chemotactic Factor", J. Exp. Med. 137, 
387-410 (1973). Checkerboard analysis of AGVPGFGVG and GFGVGAGVP sequences 
with fibroblasts using the procedure as described above afforded the 
following results which are represented graphically in Tables I and II. 
TABLE I 
__________________________________________________________________________ 
Checkerboard Analysis of AGVPGFGVG with Fibroblasts 
__________________________________________________________________________ 
Peptide concentration above filters (M) 
0 10.sup.-10 
10.sup.-9 
10.sup.-8 
Peptide conc. 
0 (10) 0 .+-. 1.3 
-2 .+-. 1.1 
-2 .+-. 0.5 
-3 .+-. 0.9 
below filters 
.sup. 10.sup.-10 
6 .+-. 1.3 
-3 .+-. 1.0 
-4 .+-. 0.8 
2 .+-. 1.8 
(M) 10.sup.-9 
25 .+-. 1.6 
3 .+-. 1.7 
-4 .+-. 0.6 
-2 .+-. 1.1 
10.sup.-8 
9 .+-. 2.3 
5 .+-. 1.6 
5 .+-. 1.2 
-2 .+-. 1.1 
__________________________________________________________________________ 
Results are expressed as mean .+-. S.E.M. where n = 15. Positive control, 
PDGF at 60 .mu.g/ml = 21. 
TABLE II 
__________________________________________________________________________ 
Checkerboard Analysis of GFGVGAGVP with Fibroblasts 
__________________________________________________________________________ 
Peptide concentration above filters (M) 
0 10.sup.-10 
10.sup.-9 
10.sup.-8 
Peptide conc. 
0 (9) 0 .+-. 1.0 
-1 .+-. 1.0 
-3 .+-. 1.2 
-2 .+-. 1.4 
below filters 
.sup. 10.sup.-10 
13 .+-. 1.6 
-1 .+-. 0.8 
-3 .+-. 0.7 
-1 .+-. 0.8 
(M) 10.sup.-9 
45 .+-. 3.4 
7 .+-. 1.8 
-2 .+-. 1.0 
-1 .+-. 1.1 
10.sup.-8 
21 .+-. 1.5 
12 .+-. 1.6 
7 .+-. 1.5 
0 .+-. 1.50 
__________________________________________________________________________ 
Results are expressed as mean .+-. S.E.M. where n = 15. Positive control, 
PDGF at 30 .mu.g/ml = 59. 
The various polynonapeptide permutations of the present invention, in order 
to exhibit chemotactic behavior generally preferably have a nonapeptide 
sequence concentration in the range of 10.sup.-5 to 10.sup.-12 M. However, 
for both endothelial cell and fibroblast chemotaxis, it is more preferred 
that the present nonapeptide sequences have a concentration releaseable 
form the elastomeric polypeptide in the range of 10.sup.-7 to 10hu -10 M. 
The nonapeptide or octameric sequences as described above, either as unit 
or repeating sequences may be either chemically bonded, i.e., covalently 
bonded within an elastomeric matrix or the nonapeptide, or octameric 
sequences described above may be "doped", i.e., physically intermingled, 
within the elastomeric matrix. By either approach, the peptide sequence of 
interest is gradually released from the elastomeric matrix. 
In addition to all of the sequence modifications described above which are 
used to modify the temperature of the inverse temperature transition, it 
is also possible to modify the nonapeptide sequences disclosed herein. In 
particular, it is possible to modify the .alpha., .delta. or .pi. 
positions, i.e., positions i+3 and (i+3)', with a residue such as Glu. 
However, other modifications may be made at the .alpha., .delta., or .pi. 
positions in accordance with the above description with respect to PPP and 
PTP. 
All of the polypeptide sequences described herein must, of necessity, be 
cross-linked in order to be utilized as elastomeric materials. Specific 
procedures which may be used to effect cross-linking and various 
side-chain moieties and chemical agents which may be used therefore have 
been described in some of the U.S. patents already incorporated herein by 
reference. 
In accordance with another important aspect of the present invention, the 
nonapeptide and polynonapeptide sequences disclosed herein may be 
advantageously used in the construction of vascular prosthetic materials 
and ligaments. This will now be discussed in detail. 
The present nonapeptide and polynonapeptide sequences can be used in 
conjunction with other elastomeric polypeptides as described in copending 
application Ser. No. 07/184,873, which is incorporated herein by reference 
in the entirety in the construction of vascular prosthetic materials. 
These elastomeric polypeptides can be constructed to match the compliance 
of small vessels in mammals, particularly humans. 
In the most basic form, an elastomeric vessel may be prepared which 
contains a single-layer wall containing the nonapeptide or polynonapeptide 
sequences of the present invention. However, the elastomeric vessel may be 
constructed to have a wall with at least three layers, or even more. 
In perhaps the most fundamental form, the elastomeric prostheses of the 
present invention consists of an elastomeric tube or vessel containing 
predominantly repeating units of elastin pentapeptide (VPGVG), with the 
remainder being the repeating hexapeptide (VAPGVG) and/or the nonapeptide 
or the polynonapeptide of the present invention. Thus, the formula of the 
bulk matrix may be generally written as 
A. [(VPGVG).sub.m 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
].sub.l 
B. [(VPGVG).sub.m 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
--(VAPGVG).sub.o ].sub.l 
C. [(VPGVG).sub.m --(VAPGVG).sub.o 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
].sub.l 
wherein m may have a value of 0 to about 20, n may have a value of from 2 
to 20, preferably 3 to 15 for both m and n, and o has a value of from 
about 0 to 20, preferably 3 to 9. l has a value such that upon 
.gamma.-irradiation the bulk matrix will have an appropriate elastic 
modulus. Generally, a molecular weight in the range of 10,000-50,000 
daltons will be adequate as a minimal molecular weight. However, molecular 
weights of up to about 1,000,000 daltons may be used. 
The elastomeric prostheses may be constructed so as to have one or more 
layers, each layer having a specialized function. For example, in 
accordance with the present invention as elastomeric vessel may be 
designed to contain one or more layers having cell attachment sequences 
for endothethial cells or smooth muscle cells. Alternatively or 
additionally, a layer may be designed for attachment to surrounding 
connective tissue and for selective interactions with fibroblasts. 
Elastomeric prostheses may also be constructed which contain several layers 
each with a particular and different function. For example, a 
three-layered elastomeric vessel may be constructed, the vessel wall of 
which contains (1) an intimal-inner layer specialized for cell attachment 
of endothelial cells, (2) a medial-middle layer specialized for cell 
attachment of smooth muscle cells, and (3) an adventitial-outer layer 
specialized for attachment to surrounding connective tissue and for cell 
attachment of fibroblasts. The particular elastomeric components for each 
of these layers and their properties will now be discussed. 
PROPERTIES OF THE ELASTOMERIC PROSTHESIS COMPONENTS 
A basic elastomeric polypeptide is the polypentapeptide, 
(Val-Pro-Gly-Val-Gly).sub.n abbreviated (VPGVG).sub.n or simply PPP. A 
viscoelastic state of the PPP which is approximately 40% peptide and 60% 
water by weight may be .gamma.-irradiation cross-linked to form an 
elastomeric matrix. The elastic modulus (Young's modulus, YM) obtained on 
preparation of the cross-linked PPP matrix is dependent on the 
.gamma.-irradiation cross-linking dose. Stress-strain data obtained on a 
band of 20 Mrad cross-linked polypentapeptide (X.sup.20 -PPP) for a 
sequence of cycles at 10% increments up to 90% illustrates the low degree 
of hysteresis for these initial extensions. For such a sample, rupture 
occurs on approaching 100% extension, though extensions of greater than 
200% are routinely obtained when the sample is prepared without 
vacuolization. The material is significantly strengthened and is given 
more body for ease of handling by compounding with a polyhexapeptide, 
(VAPGVG).sub.o, PHP. A series of stress/strain curves for the X.sup.20 
-(PHP:PPP, 1:6) taken at larger percent increments illustrates the extent 
of hysteresis and a rupture of this sample on approaching 200% extension. 
When the sequential multicomponent models are prepared, i.e., 
[(VPGVG).sub.m --(VAPGVG).sub.o ].sub.1, and cross-linked properties yet 
more favorable will be obtained than simply mixing PHP and PPP in 
parallel. 
To more clearly illustrate the advantageous properties of the present 
elastomeric prostheses, comparison with natural vessels is noted. For the 
dog femoral artery, there is an irreversible hysteresis for extensions 
above 30% presumably because collagen fibers came into tension and 
rupture. In an elastomeric vascular prosthesis, the purpose of which is to 
provide a scaffolding for regeneration of the natural artery, a low 
extension elasticity is required. Notably, the basic synthetic elastomeric 
matrices have the requisite property which for X.sup.20 -PPP falls between 
the values for the internal mammary and femoral arteries. This property 
can be fine tuned to match well the stress/strain characteristics in the 0 
to 30% extension range. It, of course, is to be appreciated that the 
radial dilatations and changes in length in situ normally are less than 
20% with the special exception of the pulmonary artery of man where the 
radial dilatation is 60 to 70% and the static elastic moduli 
(Ep=.DELTA.PR/.DELTA.R) vary from about 0.4.times.10.sup.6 dynes/cm.sup.2 
for the carotid artery of man to about 3.times.10.sup.6 dynes/cm.sup.2 for 
the femoral artery. Also, the active contractile force developed by 
vascular muscle is in the range of 2 to 3.times.10.sup.6 dynes/cm.sup.2 
for the carotid and pulmonary arteries. These elastic properties of 
vascular wall are attainable with the described elastomeric polypeptide 
vascular materials. 
Preliminary stress/strain studies have also been used to assess suture 
pull-out characteristics. Comparison has been made with the internal 
mammary artery of dog where single suture pull-out occurred at a stress of 
11 grams/mm.sup.2 ; using the same Prolene.TM. suture, value for X.sup.20 
-(PHP:PPP, 1:6) occurred at 4.3 grams/mm.sup.2. This was with a sample 
that was formed before methods for improving uniformity of the PHP and PPP 
mix and while vacuolization was yet a significant factor. It is expected 
that suture pull-out values similar to those of the internal mammary 
artery can be obtained for the synthetic material. 
The polypentapeptide when formed as an elastomeric matrix results in a very 
interesting biomaterial. For vascular wall, however, a matrix of greater 
strength and easier handling characteristics could be desired. This has 
been achieved by combining polypentapeptide (PPP) with polyhexapeptide 
(PHP) to obtain a matrix. The more gelatinous-like X.sup.20 -PPP (20 Mrad 
cross-linked polypentapeptide matrix) on addition of PHP becomes more 
teflon-like. One approach is making a section of synthetic elastomeric 
tube is to dissolve PPP and PHP in water at low temperature in a suitable 
tube. On raising the temperature, a coacervate phase is formed and the 
excess water is removed. A pestle can then be inserted into the 
viscoelastic phase which flows into and fills the channel in the pestle. 
On .gamma.-irradiation cross-linking, the pestle is removed from the tube 
and the cross-linked matrix is removed from the pestle to obtain an 
elastomeric tube. 
The stress/strain curves of the composite PHP/PPP matrix are temperature 
dependent. The elastic modulus is seen to increase with temperature and 
for X.sup.20 -(PHP:PPP, 1:6) the elastic modulus is in the range of 2 to 
4.times.10.sup.6 dynes/cm.sup.2 near physiological temperatures. Above 
55.degree. C., the matrix begins to destructure as reflected by the loss 
of elastic modulus. Accordingly, a small caliber vessel (e.g., the 4.8 mm 
ID tube shown) can be formed for consideration as a synthetic elastomeric 
polypeptide vascular material and it contains within it polypeptide that 
has been found to be chemotactic toward vascular wall cells. 
VASCULAR WALL LAYERING 
As noted above, the properties of the sequential polypeptides of elastin 
may be varied considerably and may be used in linear or cross-linked 
combination in a single layer of material or various layers, each having a 
different composition, which may be combined advantageously. 
The present invention will now be further illustrated by the following 
example which is provided for purposes of illustration only and is not 
intended to limit the present invention. 
EXAMPLE 
Formation of an Intimal Layer 
The intimal layer may contain PPP, the polynonapeptide (PNP) or composition 
A as indicated above which is [(VPGVG).sub.m 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
].sub.l, where m, n and l are as defined. Also, cell attachment sequences 
for endothelial cells may also be included within the primary structure. 
For example, cell attachment sequence concentrations of up to about 1 
sequence per 100 matrix residues are suitably used. Preferably, however, 
one or more of the polynonapeptides are used in this layer. 
The polypentapeptide in the precross-linked state at 37.degree. C. is a 
sticky material which adheres to all but hydrophobic surfaces. The 
37.degree. C. state is 38% peptide and 62% water by weight, but on drying, 
it hardens and becomes glass-like. Accordingly, it is a simple process to 
apply a thin layer of polypentapeptide on a rotating pestle; the PPP or 
any variant thereof as described previously, would contain, within its 
primary structure, cell attachment sequences for endothelial cells and as 
well could contain dissolved within the viscoelastic mass an appropriate 
concentration of chemotactic peptides for endothelial cells. Additionally, 
as noted previously, the composition of the polypentapeptide can be varied 
to compensate for the effect of adding cell attachment sequences and 
chemotactic peptides and the temperature of the inverse temperature. While 
the pestle rotates, the sample dries forming a layer of dried glue of 
desired thickness on the pestle. 
Addition of a Medial Layer: The pestle with the dried intimal layer can be 
inserted into the tube of desired diameter which, in its bottom, contains 
the viscoelastic mass that is to be the medial layer. This would be a 
PHP/PPP/PNP or PPP/PNP coacervate at its 37.degree. C. water composition. 
Once the pestle is inserted, the coacervate fills the space between the 
intimal layer and the tube wall. The intimal layer slowly hydrates but 
does not lose its identity due to the extremely slow diffusion of the high 
molecular weight polypeptides at compositions of 40% peptide by weight. 
When an additional layer is desired then a lower cross-linking dose, say 
10 Mrad would result in an insoluble matrix that could be further layered. 
Generally, for the medial layer, elastomeric matrices are used of the 
formula: 
##EQU1## 
Generally, in the above formulae, m may have a value of from about 0 to 
about 20, preferably 3 to 15, n may have a value of from 2 to about 20, 
preferably 3 to 15, and o has a value of 2 to about 20, preferably 3 to 
9.0 is defined previously. However, l can have a high value corresponding 
to a value of (m+n) of as high as 5,000. 
In addition, to being the primary structural component of the vascular wall 
the medial layer is to be specialized for smooth muscle cells by inclusion 
of the appropriate cell attachment and chemotactic peptide sequences. 
As noted above, a dose of .gamma.-irradiation is used which is, generally, 
the lowest possible dose required to match the compliance of the vascular 
wall. However, doses in excess of 30-40 Mrad are not generally used. 
Addition of an Adventitial Layer: Using a larger tube, the cylinder with 
intimal and medial layers surrounding the pestle at 37.degree. C. is 
inserted into a larger tube in which a lysine containing PPP coacervate 
has been placed. This material then flows around the medial layer to form 
a third layer. On exposing this tube to another 10 Mrad, the inner layers 
will have received the desire 20 Mrad and an outer, looser layer will have 
been added. As the lysine-containing polypentapeptide or polytetrapeptide 
is a substrate for the extracellular enzyme, lysyl oxidase, the potential 
exists for the adventitial layer to become covalently cross-linked to the 
extracellular matrix surrounding the implant site. This is because 
oxidation of lysine by lysyl oxidase to form the aldehyde is the natural 
mechanism for cross-linking in the extracellular matrix. 
As noted, any additional method of chemical cross-linking using 
cross-linking units capable of being cross-linked by lysyl oxidase may be 
used as described in U.S. Pat. No. 4,589,882. For the intimal and 
adventitial layers, the sequences as pentameters (VPGVG), tetramers (VPGG) 
or hexamers (VAPGVG) may be added in the desired ratio and then 
polymerization is carried out such that the incorporation along the 
sequence is random to obtain a polymer of greater than 50 kD. For the 
medial layer, the sequences may be incorporated as desired in a single 
long sequence of the order of 100 residues, and then this unit is 
polymerized to a greater molecular weight. As an example of a medial 
layer, can be mentioned a layer which is obtained by appropriately mixing 
polynonapeptide and polypentapeptide, each of greater than 50 kD, which 
are cross-linked by .gamma.-irradiation. 
The adventitial layer may also contain cell attachment sequences for 
fibroblasts such as the sequences GRGDSP or RGD, using the standard one 
letter amino acid abbreviation. Concentrations of up to 1 cell attachment 
sequence per 100 matrix residues may be used. 
Further, at the junction between the intimal and medial layers or layered 
within the medial matrix or at the outer surface of the medial layer, can 
be included a porous layer to facilitate migration of smooth muscle cells. 
This can be achieved by addition of a fine coral or sized particulate 
carbonate powder to a semi-dry, tacky surface of the coated pestle at any 
desired radius as long as the .gamma.-irradiation dose is 10 Mrad or less. 
On drying, the treated pestle with its layers would be introduced into a 
tube of desired diameter with the PHP/PPP/PNP or PPP/PNP viscoelastic 
coacervate of the proper volumn in the bottom. On completing the 20 to 25 
Mrad dose, the synthetic tube could be removed and treated with acid of 
sufficient strength to dissolve the carbonates but not of a strength to 
hydrolyze the peptide. 
Additionally, it is noted that instead of using --(VPGVG)-- in the basic 
sequential polypeptide, it is also possible to use generally a 
pentapeptide of the formula --(R.sub.1 PR.sub.2 R.sub.3 G)-- as already 
defined previously. Similarly, instead of using --(VAPGVG)-- in the basic 
sequential polypeptide, the variations described previously may also be 
utilized. 
STIMULATION OF CELLS ON THE SURFACE OF ELASTOMERIC MATRICES 
The elastomeric materials described herein may be advantageously used as 
vascular prostheses for the following additional reason. In particular, 
when cells, such as smooth muscle cells and/or endothelial cells, are 
attached to the surface of elastomeric matrices and subjected to 
stretch/relaxation cycles, it is found that the cells are stimulated to 
produce the macromolecules which make up the vascular wall. In other 
words, the cells are stimulated to produce their own vascular wall. 
The above aspect of the present invention is, in part, based upon the 
earlier work of Glagov who demonstrated that elastic substrates might be 
used to stimulate cell synthesis of matrix components. See Exp. Cell 
Research, 109, 285-298 (1977); Science, 191, 75-477 (1976); and Circ. 
Res., 14, 400-413 (1964) by Glagov et al. However, none of this work was 
conducted with the present elastomeric materials. 
Further, the biodegradability and biocompatibility of the present vascular 
prostheses also appear to be advantageous in that it has been demonstrated 
that compliance and biodegradation of vascular grafts stimulates the 
regeneration of elastic laminae in neoarterial tissue. See Cell Tissue 
Res. 242, 569-578 (1985), by van der Lei et al. Thus, the present vascular 
prostheses have the advantageous effect of stimulating the synthesis of 
cell matrix components, such as collagen and new elastic fibers, to 
thereby regenerate vascular walls. 
LIGAMENT CONSTRUCTION 
In addition to constructing vascular prosthesis, the elastomeric 
polypeptides can be advantageously used in the construction of artificial 
ligaments. 
Although any of the above-described elastomeric materials can be utilized, 
in conjunction with the present nonapeptide and polynonapeptide sequences, 
in the construction of artificial ligaments, the artificial ligaments of 
the present invention are most advantageously constructed from any one of 
the following three compositions: 
A. [(VPGVG).sub.m 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
].sub.l or 
B. [(VPGVG).sub.m 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n 
--(VAPGVG).sub.o).sub.l or 
C. [(VPGVG).sub.m --(VAPGVG).sub.o 
--(.alpha..beta..gamma..delta..epsilon..theta..lambda..pi..rho.).sub.n).su 
b.l 
wherein m may have a value of 0 to about 20, preferably 3 to 15, n may have 
a value of from 2 to about 20, preferably 3 to 15, and o has a value of 
from about 2 to about 20, preferably 3 to 9, and l has a value such that 
upon .gamma.-irradiation the bulk matrix will have an increased elastic 
modulus. 
Generally, however, a molecular weight in the range of 10,000-50,000 
daltons will be adequate as a minimal molecular weight. However, a 
molecular weight of up to about 1,000,000 daltons may be used. 
The artificial ligaments of the present invention may also contain cell 
attachment sequences for fibroblasts such as the sequences GRGDSP or RGD. 
These same cell attachment sequences may also be used for the vascular 
prosthesis described above. Further, as an example of a chemotactic 
peptide which may be used with either the described vascular prosthesis or 
artificial ligament is the sequence --VGVAPG-- or permutations thereof 
such as --GVGVAP-- or --PGVGVA--. Concentrations of up to 1 cell 
attachment sequence per 100 matrix residues may be used. 
Although the artificial ligaments can be constructed from any of the 
above-mentioned elastomeric materials, it is preferred that a mixture of 
one or more of the present polynonapeptides be incorporated in the 
elastomeric matrix. For example, it is advantageous to construct a 
ligament from a mixture of a polynonapeptide and a polyhexapeptide (PHP) 
to afford both high elastic modules and mechanical strength. The 
proportions of each may be 1 to 99 molar % to 99 to 1 molar % with the 
precise combination being determined by the elasticity and mechanical 
strength desired. 
The artifical ligament of the present invention may be utilized in 
accordance with techniques described in U.S. Pat. Nos. 4,773,910, 
4,731,084, 4,642,119, 4,246,660 and 4,149,277; all of which are 
incorporated herein in the entirety. 
It should be emphasized, however, that the present elastomeric polypeptide 
materials are used as a temporary functional scaffolding for ligament 
reconstruction wherein cellular in growth and elaboration of natural 
ligaments is an integral aspect of the present invention. As such, one 
skilled in the art would easily be able to utilize the present elastomeric 
polypeptide materials in accordance with any or all of the incorporated 
U.S. patents in fashioning ligaments. 
The vascular prosthesis and artificial ligaments of the present invention 
are constructed of elastomeric materials which are all capable of 
reversibly contracting and relaxing by an inverse temperature transition. 
These elastomeric materials are preferably substantially cross-linked. The 
vascular prosthesis is preferably in the form of a vessel suitable for use 
as a portion of an artery, vein or lymphatic vessel. 
The elastomeric matrices of which the vascular prosthesis and artificial 
ligaments are constructed are generally made of repeating units of 
elastomeric tetrapeptide and pentapeptide repeating units and units 
thereof modified by hexapeptide units and mixtures thereof, wherein the 
repeating units contain amino acid residues, i.e., peptide-forming 
residues, of hydrophobic amino acid residues and glycine residues, and 
wherein the repeating units exist in a conformation having a .beta.-turn. 
Such matrices may, for example, be used as moieties A and B defined above. 
Of course, the above generalized compositions are modified as specifically 
described above in order to contain the disclosed polynonapeptide 
sequences, or the specific compositions disclosed for the various vascular 
prosthetic layers. 
Having fully described the present invention, it will be apparent to one 
skilled in the art that many changes and modifications may be made to the 
same without departing from either the spirit or scope thereof.