WO2000014201A1 - Polypeptides containing modified, non-natural amino acids and methods of production - Google Patents

Abstract

A method for modifying a polypeptide includes providing a polypeptide containing at least one 3,4-dehydroproline and reacting the 3,4-dehydroproline with an epoxidation agent to form a polypeptide containing 3,4-epoxyproline. The method optionally further includes reacting the polypeptide containing 3,4-epoxyproline with a reducing agent to form a polypeptide containing a hydroxyproline. Polypeptides are provided which include an amino acid having an oxirane ring.

Description

POLYPEPTIDES CONTAINING MODIFIED, NON-NATURAL AMINO ACIDS AND METHODS OF PRODUCTION

Background

1. Field

Polypeptides incorporating amino acid analogs, in particular polypeptides incorporating dehydroproline and other proline analogs which have been subsequently modified.

2. Description of the Related Art

The structure and function of biological macromolecules is generally determined by

their constituent chemical units. For polypeptides and proteins, these units are typically drawn

from a pool of twenty naturally occurring free amino acids. The translational component of

the biosynthesis of polypeptides consists of linking these amino acids into linear chains. The

resulting polypeptides in many cases are able to assume stable structures and are fully

functional within the context of their native environment. In other cases, additional "post-

translational" modifications of certain polypeptides occur before the polypeptides become

functional. Post-translational modifications include, for example, the addition of sugar, oligosaccharide, or lipid groups to selected amino acid side chains; insertion of prosthetic groups; and formation of disulfides or other chemical cross-links, and other modifications of selected amino acid side chains.

Post-translational modifications can affect the structure and/or function of the resulting polypeptides. Of particular importance for the structure and/or function of certain

polypeptides is the post-translational conversion of the side chains of naturally occurring amino acids to "non-natural" (otherwise known as "unnatural") amino acids in the sense that

the resulting amino acids are not a part of the pool of twenty free amino acids normally used

during protein translation. Two examples of this process are the post-translational conversion

of proline residues to hydroxyproline residues and the post-translational conversion of lysine

residues to hydroxylysine residues. Post-translational transformations are of particular importance for the structure and function of the extra-cellular matrix protein collagen, and in

the adhesive capacity of mussel adhesive protein isolated from the various mussels such as

Mytilus.

Collagen having low levels of hydroxyproline exhibits poor mechanical properties, as

highlighted by the sequelae associated with scurvy. Hydroxyproline adds stability to the

collagen structure through hydrogen bonding and through restriction of rotation about C-N

bonds in the polypeptide backbone. Type I collagen is the most abundant form of the fibrillar,

interstitial collagens and is the main component of the extracellular matrix. Collagen monomers consist of about 1000 amino acid residues in a repeating array of Gly-X-Y triplets. Approximately 35% of the X and Y positions are occupied by proline and 4-hydroxyproline.

In humans, proline in the Y position is converted to 4-hydroxyproline post-translationally by

the enzyme prolyl-4-hydroxylase. Collagen monomers associate into triple helices which consist of one α2 and two αl chains. The triple helices associate into fibrils which are oriented into tight bundles. The bundles of collagen fibrils are further organized to form the scaffold for extracellular matrix.

The structural attributes of Type I collagen, along with its generally perceived biocompatability make it a desirable surgical implant material. Collagen has been purified

from bovine skin or tendon and used to fashion a variety of medical devices including

hemostats, implantable gels, drug delivery vehicles and bone substitutes. However, when

implanted into humans, bovine collagen can cause acute and delayed immune responses.

Researchers have therefore attempted to produce human recombinant collagen with all of its

structural attributes in commercial quantities through genetic engineering. Unfortunately,

production of collagen by known mass producers of protein such as E. coli has not been successful. A major problem is the extensive post-translational modification of collagen by

enzymes not present in E. coli. Failure of E. coli cells to provide proline hydroxylation of

unhydroxylated collagen proline prevents manufacture of structurally sound collagen in

commercial quantities.

Further, as mentioned above, marine mussels and other sessile invertebrates secrete

adhesive substances in order to affix themselves to underwater objects. Mussels of the genus

Mytilus, for example the species Mytilus edulis and Mytilus califorianus, secrete an adhesive

precursor substance from their foot that upon cure forms a permanent attachment to the substrate. A major component of the adhesive deposited by M. edulis has been identified as a

hydroxylated protein of about 130,000 daltons (Waite, J. H, J. Biol. Chem., Vol. 258, pp. 291 1-2915 (1983)). U.S. Pat. No. 4,496,397 to Waite further discloses that the protein from M. edulis comprises a large number (75-80) of tandem repeats of a decapeptide having the following sequence:

NH₂-Ala-Lys-Pro/Ηyp-Ser/T r-Tyr/I⁾opa-Pro Hyp-Pro/Ηyp-Ser/Thr-Tyr Dopa-Lys-CO₂H This and other bioadhesive precursor proteins have a large number of basic and hydroxylated amino acids, including lysine, serine, threonine, and others. The relatively large numbers of basic and hydroxylated amino acid presumably provide both hydrogen and ionic bonding with

the substrate surface. At least a portion of the hydroxylated amino acids include 3,4-

dihydroxyphenylalanine (also know as dopamine, abbreviated as "Dopa") and hydroxyproline

(abbreviated as "Hyp"). Dopamine and hydroxyproline are unique among amino acids,

because they are incorporated into the protein as tyrosine and proline during protein synthesis

and then later hydroxylated. Post-translational hydroxylation, particularly of the tyrosine

residues, is believed to be important in defining the adhesive properties of the protein. The

hydroxyl groups likely chelate with metals or other ions present on the substrates on which

mussels anchor. Oxidation of the dihydroxy groups of the tyrosine yield ortho-phenols, which

can then cross-link and form or strengthen the adhesive bond. See, Waite, J. H., In Mollusca,

Vol. 1, pp.467-504 (1983); Pizzi, A., et al., Ind. Eng. Chem. Prod. Res. Dev., Vol. 21, pp.

309-369 (1982) and Wake, W.C., "Adhesion and the Formulations of Adhesives", Applied

Science Publish, Ltd. Barking, England (1982). Mussel adhesive proteins have been the subject of intensive study, as they are

anticipated to provide a natural adhesive for use in wet environments, as are found in various

areas of clinical medicine. However, mussel adhesive proteins have thus far failed to live up

to their initial promise for a variety of reasons. One reason has been difficulty in obtaining large quantities of the protein precursor, and another has been in obtaining efficient cure of the

protein precursor. Isolation of the uncured adhesive precursor from mussels for commercial

use is not practical, and reported semi-synthetic methods rely on this native protein.

Recombinant methods for obtaining large quantities of mussel adhesive proteins have also been developed, as disclosed in U.S. Pat. Nos. 5,049,504, 5,149,657, 5,202,236, 5,202,256, and U.S. Pat. No. 5,242,808 to Maugh et al. A significant drawback of recombinant methods

is that none of the produced proteins comprise hydroxyproline, an integral component of

naturally-occurring bioadhesive proteins, and no commercial mussel adhesive-based proteins

are in wide use.

Collagens and mussel adhesive proteins are only two examples of polypeptide systems wherein the presence of non-natural amino acids plays a major role in protein structure and

function. In addition to their role in naturally occurring polypeptides, non-natural amino acids

may be valuable tools in investigating both the structure and function of amino acids and

proteins. Incorporation of non-natural amino acids into polypeptides presents a chemically

unique side chain that may itself be useful as a biophysical probe of the structure and function

of the polypeptide. Alternatively, the side chain may be further selectively modified with

other biophysical probes or ligands that impart desirable properties to the molecule. The non-

natural amino acid side chain may also provide a site for either inter- or intra-molecular

crosslinks, thereby leading to multimeric polypeptides or macromolecular complexes. Such polypeptides may be novel species that exhibit desirable properties, including, for example,

unique biological functions, enhanced stability, and altered solubility or mechanical

properties. Current methods for the incorporation of non-natural amino acids into polypeptides is focused in two areas. In one approach, a side chain of an amino acid in a previously

synthesized polypeptide is enzymatically modified in vitro to give a non-natural side chain.

Although this general approach starts with a polypeptide that can potentially be produced in large quantities, it is limited because only the side chains of the naturally occurring amino

acids are available for modification. Only a limited set of such amino acids have side chains of suitable reactivity. This limits the type of chemical modifications that can be performed

and thus limits the versatility of this method.

The second approach takes advantage of the native cellular protein translation

machinery to insert a no n- natural amino acid into a polypeptide chain either in vivo or in vitro.

Use of this method is only successful where the non-natural amino acid is first chemically linked to a transfer RNA (tRN A), and where the charged tRNA is competent to function in

protein translation. One method in this approach is disclosed by, e.g., Noren et al., in "A

General Method for Site-Specific Incorporation of Unnatural Amino Acids Into Proteins",

Science, Vol. 244, pp. 182-188 (1989) wherein chemically acylated suppressor tRNA is used

to insert an amino acid in response to a stop codon substituted for the codon encoding residue

of interest. Attachment of non-natural amino acids to tRN As in this manner is accomplished

by chemical acylation in vitro. The yield of properly acylated tRNA molecules is thus limited

by the availability of the non-natural amino acid and the tRNA, as well as the efficiency of the

acylation step. Following acylation, the acylated tRNA must be presented to the protein

synthesis machinery. In vivo, this requires entry of the acylated tRNA into the cell. In vitro, the tRNA is added to a preparation of partially purified components of the protein translation machinery. Neither of these processes is particularly efficient, and they are not presently amenable to the production of large quantities of modified polypeptides.

Another method in this area uses aminoacyl-tRNA synthetases. In vivo, aminoacyl- tRNA synthetases link specific free amino acids to tRNA during translation. As a general rule, amino acid-tRNA complexes are quite specific, and normally only a molecule with an

exact stereochemical configuration is acted upon by a particular aminoacyl-tRNA synthetase.

In many living cells, some amino acids are taken up from the surrounding environment and

some are synthesized within the cell from precursors, which in turn have been assimilated

from outside the cell. In certain instances, a cell is auxotrophic, i.e., it requires a specific growth substance beyond the minimum required for normal metabolism and reproduction

which it must obtain from the surrounding environment. Some auxotrophs depend upon the

external environment to supply certain amino acids. This feature allows certain non-natural amino acids to be incorporated into proteins produced by auxotrophs, by taking advantage of

relatively rare exceptions to the above rule regarding stereochemical specificity of aminoacyl-

tRNA synthetases. For example, proline is such an exception, i.e., the amino acid activating

enzymes responsible for the synthesis of the prolyl-tRNA complex are not as specific as

others. As a result, certain proline analogs have been incorporated into bacterial, plant, and

animal cell systems. See Tan et al., "Proline Analogues Inhibit Human Skin Fibroblast

Growth and Collagen Production in Culture", Journal of Investigative Dermatology, Vol. 80, pp. 261-267 (1983). See also, Deming et al., "7n Vitro Incorporation of Proline Analogs into

Artificial Proteins", Poly. Mater. Sci. Engin. Proceed., Vol. 71, p. 673-674 (1994). This

article surveys the potential for incorporation of the proline analogs L-azetidine-2-carboxylic acid, L-γ-thiaproline, 3,4-dehydroproline and trα«5-4-hydroxy-L -proline into artificial

proteins expressed in E. coli. L-azetidine-2-carboxylic acid, L-γ-thiaproline and 3,4- dehydroproline are reported as being incorporated into proteins in E. coli cells in vivo.

Incorporation of hydroxyproline into proteins during protein synthesis has also been disclosed in commonly-assigned U.S. Pat. Application Ser. No. 08/655,086 filed June 3, 1996 to Gruskin et al., which is incorporated by reference herein in its entirety. Hydroxyproline represents a special challenge, because unlike 3,4-dehydroproline and certain other non-

natural amino acids, it is not taken up by most cells even under conditions of proline

starvation. However, Gruskin et al. have discovered that hydroxyproline can by imported into

the cell and incorporated into proteins under conditions of osmotic shock and proline

starvation. Thus, a cell capable of producing the desired protein, when contacted with a

preferably hypertonic growth medium comprising hydroxyproline, assimilates the

hydroxyproline into the cell. The cell then incorporates the hydroxyproline into an amino

acid tRNA complex, thereby producing proteins having hydroxyproline in place of proline.

This assimilation and incorporation is extremely useful when the structure and function of a polypeptide or protein depends on post-translational hydroxylation of proline, but the cellular

machinery for such hydroxylation is not present in the native production system of a

recombinant host, such as E. coli. However, the scope of such assimilation and incorporation

is not yet clear, in that it is unknown how many, or what type, of non-natural amino acids may

be incorporated using this method.

Accordingly, there remains a need for efficient and flexible methods for creating

polypeptides having non-natural amino acids, in order to study polypeptides and the effects of such non-natural amino acids on the structure and function of polypeptides. Efficient

modifications of natural amino acids in polypeptides would be especially useful when such

modifications correspond to naturally occurring post-translational events, e.g., hydroxylation of proline to allow production of collagen or mussel adhesive proteins in commercial

quantities.

Summary

The above-discussed and other drawbacks and disadvantages are alleviated by a

method for modifying a polypeptide, which includes providing a polypeptide containing at

least one 3,4-dehydroproline and reacting the 3,4-dehydroproline with an epoxidation agent to

form a polypeptide containing 3,4-epoxyproline. The method optionally further includes

reacting the polypeptide containing 3,4-epoxyproline with a reducing agent to form a

polypeptide containing a hydroxyproline. Polypeptides are provided which include an amino

acid having an oxirane ring. This method optionally further includes reacting the polypeptide containing 3,4-

epoxyproline with various reagents to form polypeptides having at least one amino acid with a

non-natural side-chain. Such reagents include nucleophiles and electrophiles. Reaction with

a reducing agent forms a polypeptide containing a hydroxyproline. Polypeptides having 3,4-

dehydroproline and/or 3,4-epoxyproline incorporated therein may also be cross-linked,

optionally in the presence of a cross-linking agent. These methods are used to efficiently provide human collagens and bioadhesive polymeric protein precursors having novel

properties. Brief Description of the Drawings

FIG. 1 depicts synthetic amino acid sequences based on sequences derived from the C- terminal region of the human α, collagen protein and having non-natural amino acids incorporated therein.

FIG. 2 is a mass spectrum showing the presence of an epoxide moiety in a synthetic

peptide.

FIG. 3 is a plasmid map for pUSC-D4.

FIG. 4 depicts the nucleotide sequence and corresponding amino acid sequence of the

D4 gene (the C-terminal 219 amino acids of the human collagen α, chain).

FIG. 5 is a plasmid map for pUSC-74.

FIG. 6 shows the nucleotide sequence and corresponding amino acid sequence of the

human type I ( ,) collagen gene.

FIG. 7 shows the nucleotide sequence and corresponding amino acid sequence of a

decapeptide for a bioadhesive precursor protein.

FIG. 8 is a plasmid map for pLSM-6.

Description of the Preferred Embodiments

A method for the efficient incorporation of non-natural amino acids into polypeptides

is provided herein. The method capitalizes on the potential of in vivo systems to produce large quantities of polypeptides, and combines it with the efficiency of in vitro chemical

modification. As used herein "non-natural" will refer to any amino acid not part of the pool of

the L forms of the twenty free amino acids, namely glycine, proline, alanine, valine, leucine, isoleucine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, histidine, lysine,

arginine, glutamine, asparagine, glutamic acid, aspartic acid, serine, and threonine. As

described in T.S. Papas and A.H. Mehler, "Analysis of the Amino Acid Binding to the Proline

Transfer Ribonucleotide Synthetase of Escherichia coli, J. Biol. Chem. Vol. 245, pp.1588- 1595 (1970), incorporated herein by reference, prolyl tRNA synthetase will misacylate prolyl tRNA with certain proline analogs in vivo. Accordingly, large quantities of polypeptides that

contain 3,4- dehydroproline may be produced as starting materials for use herein.

Incorporation of the appropriate analog into a polypeptide results in a unique non-natural

amino acid side chain which can be further chemically modified in vitro to give rise to a

variety of useful modifications.

In one embodiment, there is provided a method for the production of large quantities

of peptides having amino acids with modified side chains, particularly amino acids having hydroxyl groups appended thereto. Thus, prokaryotic cells, such as E. coli and eukaryotic

cells such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and

Schizosaccharomyces pombe and additional eukaryotes such as insect cells including

lepidopteran cell lines including Spodoptera frugiperda, Trichoplasia ni, Heliothis virescens,

and Bombyx mori infected with a baculovirus; CHO cells, COS cells, and NTH 3T3 cells, can

be made to incorporate 3,4-dehydroproline by, e.g., the method of Deming as disclosed in

Deming et al., "In vivo Incorporation of Proline Analogs into Artificial Proteins, Poly. Mater.

Sci. Εngin. Proceed., Vol. 71, pp. 673-674 (1994), the disclosure of which is hereby

incorporated by reference. The resulting 3,4-dehydroproline-containing polypeptides are purified by standard

techniques. The purified polypeptide is dissolved in a suitable solvent such methanol, and

3,4-dehydroproline is converted to 3,4-epoxyproline by oxidation with a suitable epoxidation

reagent. As will be apparent to one of ordinary skill in the art, suitable chemical reagents within the context of this method are specific only to the non-natural amino acid functionality.

Examples of such epoxidation agents include, but are not limited to, a peroxycarboxylic acid

such a m-chloroperbenzoic acid (MCPBA); treatment with bromine, followed by treatment

with sodium ethoxide in ethanol or treatment with N-bromosucinimide in the presence of

perchlorate in water and dioxane, followed by treatment with potassium acetate in ethanol

(i.e., base-catalyzed cyclization of a halohydrin); treatment with basic peroxide in water or in

a mixture of methanol and acetonitrile; treatment with osmium tetroxide in the presence of

sodium perchlorate, acetic acid, and water; treatment with a nitrile and hydrogen peroxide; or treatment with alkyl peroxides in the presence of a complex of V, Mo, Ti, or Co. Those of

skill in the art will recognize that the foregoing is merely a partial list, and that effective

epoxidation requires use of a reagent which is at least partially soluble in solvents capable of

dissolving the subject peptide or protein, such as water, methanol, ethanol, and the like, and

mixtures thereof with water-miscible solvents such as acrylonitrile, DMSO, and DMF. The

reagent is furthermore preferably non-reactive with other functional groups present in the

polypeptide or protein. Epoxidation of alkenes is reviewed, for example, by Miyaura and

Kochi, J. Am. Chem. Soc. Vol 105, p. 2368 et seq. (1983); Venturello, Alneri, and Ricci, J.

Org. Chem. Vol. 48, p. 3831 et seq. (1983); and de Carvalho and Meunier, Tetr. Lett. Vol. 24,

p. 3621 et seq. (1983), all three of which are incorporated herein by reference. Following epoxidation, excess epoxidation reagent may be removed by standard

techniques such as extraction and/or chromatography. The resulting oxirane ring is then optionally reacted with a variety of reagents as described below to give a range of potentially

useful amino acid side chain modifications. For example, although 4-hydroxyproline is not efficiently incorporated and assimilated into polypeptides in vivo except under conditions of proline starvation and osmotic shock, its incorporation is achieved, in effect, by reduction of

the epoxide with a suitable reductant such as a metal hydride, e.g., lithium borohydride. The

net effect is the efficient production of a polypeptide in which trα 75-4-hydroxyproline has

been incorporated into sites normally occupied by proline. The degree of substitution of

trα«5-4-hydroxyproline for proline can be controlled by regulation of the extent of the reaction

of 3,4-dehydroproline with the epoxidation agent. This is achieved by standard techniques,

including varying the temperature of the reaction, varying the amount of the reactant, or

varying the amount of time the reaction is allowed to proceed. Following conversion of the

epoxide to trα«s-4-hydroxyproline, any unreacted 3,4-dehydroproline can be converted to

proline by reduction with a suitable reductant.

The oxirane functionality can optionally be reacted with a number of other reactants,

including reactants characterized by nucleophilic reactivity, such as amines, azide ions,

cyanide ion, enamines, thiols, carboxylic acids, alcohols, and carbon nucleophiles.

Nucleophilic reactants of particular interest include other polypeptides and biophysical probes

or ligands having a nucleophilic functionality. Again, such reactants should be at least

partially soluble in solvents capable of dissolving the subject peptide or protein, such as water,

methanol, ethanol, and the like, and mixtures thereof with water-miscible solvents such as acrylonitrile, DMSO, and DMF. The reagent is furthermore preferably non-reactive with other functional groups present in the polypeptide or protein.

Oxiranes can also be treated under either acidic or basic aqueous conditions to yield, in the case of 3,4-epoxyproline, 3,4-dihydroxyproline wherein the hydroxyl groups are anti to each other. Other known reactions of epoxides include rearrangement to a carbonyl compound, and ring opening to form an allylic alcohol or followed by elimination to form an

allylic alcohol.

The extent of the in vitro chemical modification is limited only by the nature of the chemical group incorporated and the chemistry utilized to generate a unique functionality.

Thus, in general, the application of this method to produce novel functionality within a given

protein sequence is virtually unlimited. In this manner, a discrete set of chemical

manipulations is used to convert a non-natural amino acid functionality incorporated into a

polypeptide into many other useful functionalities.

The procedures described herein are applicable to any polypeptide incorporating

proline or dehydroproline. Indeed, these procedures are especially useful to produce collagen

containing hydroxyproline as is typically found after post-translational modification in native

cells. Proline analogues such as 3,4-dehydroproline are substituted for naturally occurring

proline within repeating Gly-X-Y collagen sequence in accordance with the disclosure herein.

To produce a recombinant collagen polypeptide, plasmid expression vectors encoding for human Type I (α,) collagen are transformed into prokaryotic proline auxotrophs that depend

upon exogenously supplied proline for growth. The E. coli are grown to proline starvation in

a medium containing a limiting amount of proline. The cells are then supplied with fresh growth medium containing an appropriate proline analog (in place of proline), such as 3,4-L-

dehydroproline. Expression of the collagen polypeptide is induced, and after an appropriate

induction period of typically two to 18 hours, the resulting 3,4-dehydroproline-containing polypeptide is purified by standard chromatographic procedures. Thus, recombinant collagen

is produced in E. coli with 3,4-dehydroproline inserted at positions normally occupied by

proline. Following purification, the 3,4-dehydroproline residues in the collagen polypeptide are converted in vitro to chemically distinct side chains as described above, thereby giving rise

to unique polypeptides. This technique provides conversion of 3,4-dehydroproline to trans-4-

hydroxyproline, the naturally occurring functionality in the Y position of a portion of the Gly-

X-Y repeating units of human collagen.

Thus, the purified collagen polypeptide containing the amino acid analog 3,4-

dehydroproline in place of proline is the substrate for in vitro chemical modification. The

olefm functionality of the 3,4-dehydroproline is unique compared to the naturally occurring

pool of 20 amino acids, and allows for chemical modification specific for only the 3,4- dehydroproline-containing positions in the collagen polypeptide. For example, the

recombinant peptide is dissolved in a suitable solvent such as methanol and treated with an

epoxidation reagent such as (MCPBA) which reacts specifically with 3,4-dehydroproline to

produce the 3,4-epoxide. Baldwin et al. have used MCPBA to produce 2S,3R,4S-

Epoxyproline from a protected 3,4-dehydro-L-proline, in "Substrate Specificity of Proline 4-

Hydroxylase: Chemical and Enzymatic Synthesis of 2S,3R,4S-Epoxyproline", Tetrahedron

Letters, Vol. 35, pp. 4649-4652 (1994). However, this reaction has not been reported in the

more complex context of a polypeptide chain. Stereochemical control is achieved by standard chemical techniques, such as variation of solvent, reaction temperature, chemical reagent, or

reaction times. Standard analytical techniques can be used to monitor the progress of the reaction.

Reduction of the 3,4-epoxide to the desired 4-hydroxyproline is carried out using a suitable reductant, such as lithium borohydride in a solvent such as methanol. Hudson et al. have used lithium borohydride to reduce 3,4-epoxy-N-tosylproline methyl ester to the

corresponding hydroxyprolinol as described in "On the Synthesis of 3,4-Dihydroxyproteins. II Synthesis, Stereochemistry and Reactivity of Some 3,4-Epoxy-DL-Proline Derivatives", Aust.

J. Chem., Vol. 28, pp. 2479-2498 (1975). The reduction was highly regioselective in favor of

the 4-hydroxyprolinol versus the 3-hydroxyprolinol. This reaction has not to date been

reported in the context of a polypeptide, but the corresponding stereospecific conversion of

the 3,4-epoxide in a collagen polypeptide leads to the desired trα«^-4-hydroxyproline.

Control of the extent of either the epoxidation or reduction reaction within a collagen

polypeptide allows a desired percentage of the dehydroproline residues to be converted to 4-

hydroxyproline. The procedure described above provides production of human collagen

proteins with various degrees of 4-hydroxyproline incorporation, thus allowing for a more

versatile application of the resulting products. By determining the level, degree, and nature of

chemical conversion, the physical characteristics of the resulting polypeptides can be manipulated to produce biologically active collagen with desired chemical and physical

properties.

Oxirane functionalities in polypeptides can also be used to effect chemical cross-

linking of the polypeptides. Cross-linking between two different polypeptides can be achieved by adjusting the pH of a solution of the modified polypeptides to greater than about

7.0. At this pH and higher, nucleophiles are reactive toward the oxirane moiety. Reactive

nucleophiles include amino acid residues in the polypeptide that have alcohol, sulfhydryl, and

amine functionalities, including the N-terminal amine moiety of polypeptides. Reaction of a

nucleophile on one polypeptide chain with an oxirane group on a second polypeptide chain

would result in the cross-linking of the two chains. Standard conditions for nucleophilic

addition to epoxides may be used, such conditions including but not being limited to acid- or

base-catalyzed addition.

Cross-linking between peptides may also be effected using the olefinic moiety of

dehydroproline itself. For example, alcohols are known to add to olefinic double bonds under

acidic or basic conditions. If the alcohol moiety is a serine or threonine residue on a second

polypeptide, cross-linking is effected. Acid-catalyzed addition of a carboxylic acid

functionality to the double bond is also feasible. If the carboxylic acid moiety is the C-

terminus of a second polypeptide of the side chain of a glutamic acid or aspartic acid residue,

cross-linking is effected. Sulfhydryl moieties will react with olefins under base- or acid-

catalyzed conditions or free-radical conditions. If the sulfhydryl functionality is a cysteine

residue on a second polypeptide, cross-linking is effected. Other chemical reactions can be

envisioned that will effect cross-linking starting from either dehydroproline or 3,4-

epoxyproline.

Cross-linking between dehydropro line-containing or 3,4-epoxyproline-containing

polypeptides can also be achieved using a cross-linking agent comprising at least two

nucleophilic functionalities linked by a tether, again under standard conditions of addition to oxirane or carbon-carbon double bonds. Suitable cross-linking agents include, but are not

limited to agents having at least two nucleophiles which are the same or different. Such

agents may have the formula X-R-Y, wherein X and Y are the same or different and are

selected from the group consisting of an alcohol, amino, carboxyl, or thio group, and R is the

tether. Choice of tether is broad, including for example straight or branched chain alkyls,

cycloalkyls, and olef s; aromatic groups such as substituted and unsubstituted phenyls,

diphenyls, naphtyls, and the like; natural and artificial polymers, including polypeptides,

dextrans, and the like; and molecules which can function as probe or which have biological

activity. Appropriate selection of the nucleophile and the tether enable modification and

adjustment of the degrees of crosslinking and the character of the crosslink, as well as the

relative degrees of cross-linking between the polypeptides themselves and the added

crosslinking reagent.

One area of particular interest with respect to cross-linking is the bioadhesive

precursor proteins and analogs discussed above. The properties of a dehydroproline-

containing or 3,4-epoxyproline-containing bioadhesive can be tailored to specific

requirements by controlling the chemical reactions that are used to initiate cross-linking.

Thus, the extent of cross-linking can be controlled by the presence, nature and concentration

of a crosslinking reagent, by adjustment of the time allowed for cross-linking, or by control of

the temperature or other condition under which cross-linking occurs.

The extent of crosslinking can also be controlled by selection of conditions that allow

competition between reaction of the olefin or oxirane with a nucleophile, and reaction with

solvent. Typical conditions that can be varied to achieve competition include the polypeptide concentration, the reaction time, the reaction temperature, and the reaction pH. In this way,

some of the dehydroproline -derived oxirane moieties can be utilized in the polypeptide cross-

linking reaction and some can be converted to, for example, 3,4-dihydroxyproline by reaction

with water. Thus, cross-linking can be achieved while at the same time the number of

hydrophilic, hydrogen-bonding groups in the bioadhesive is maintained at an amount

appropriate for adhesion. This would result in bioadhesives of varying strength, elasticity, and

rate of bio-absorption. These properties can thus be tailored for specific clinical indications.

The heterogeneity displayed for hydroxylated amino acids in bioadhesive polypeptides

suggests that other amino acids with hydrogen bonding capability may function equally as

well as tr< y-4-hydroxyproline when substituted for proline. Thus other proline analogs such

as .y-4-hydroxyproline, trans-3 -hydroxyproline, cis-3 -hydroxyproline, 3,4-dihydroxyproline,

or 3,4-epoxyproline would also be expected to function in the context of a bioadhesive

polypeptide sequence, resulting in a range of possible protein sequences, each of which would

be expected to possess novel mechanical and adhesive properties. Each of these analogs is

accessible from dehydroproline by variation of the chemistry described above. Also, different

ratios of proline and/or proline analogues may be well tolerated in these proteins, resulting in

novel adhesive or therapeutic properties. Protein polymers containing homogeneous or

heterogeneous substitution of proline analogues in place of proline can be generated that will

recapitulate the chemical and physical properties responsible for maximizing the adhesive

properties of a bioadhesive protein.

Other reactions of the alkene functionality of 3,4-dehydroproline incorporated in

polypeptides or proteins are also possible. The olefm functionality can optionally be reacted with reactants characterized by nucleophilic reactivity, such as amines, azide ions, cyanide

ion. enamines, thiols, carboxylic acids, alcohols, and carbon nucleophiles. Nucleophilic reactants of particular interest include other polypeptides and biophysical probes or ligands having a nucleophilic functionality. Other reagents for reaction with the olefinic moiety of 3,4-dehydroproline include, but are not limited to, electrophilic addition of agents such as

hydrogen halides or halides such a bromine or iodine; oxidation by permanganate-based

reagents, or oxidation by osmium tetroxide and peroxide to yield the corresponding cis-diol.

Again effective reagents are at least partially soluble in solvents capable of dissolving the

subject peptide or protein, such as water, methanol, ethanol, and the like, and mixtures thereof

with water-miscible solvents such as acrylonitrile, DMSO, and DMF. The reagent is

furthermore preferably non-reactive with other functional groups present in the polypeptide or

protein.

The following examples are included as exemplifications and should not be construed

as limitations of the disclosure herein.

EXAMPLE 1 Incorporation of 3,4-Dehydro-L-Proline Into a Synthetic Polypeptide

A series of peptides was generated by standard solid phase synthetic techniques using

an automated protein synthesizer from Applied Biosystems. The synthetic amino acid

sequences as shown in FIG. 1 are derived from the C-terminal region of the human α,

collagen protein. Each obtained peptide is more than 90% pure as characterized by HPLC and mass spectroscopic analysis. The dehydroproline (Dhp) residue is present in either the X or Y position of a Gly-X-Y triplet.

EXAMPLE 2 Conversion of 3,4-Dehydroproline to 3,4-Epoxyproline in a Synthetic Peptide (CC-2)

To a sample of dry CC-2 peptide of Example 1 (FIG. 1) (1 mg, 0.571 μmoles) was added 0.5 mL of dry methanol in a 10 mL pear-shaped flask. To this solution was added 1.1

equivalents (based on dehydroproline) of m-chloroperbenzoic acid in 0.5 mL of methanol,

followed by stirring at room temperature under a nitrogen atmosphere for 15 hours. A 15-fold molar excess of MCPBA was then added and the reaction continued for an additional 15 hours

at room temperature, at which time the temperature was raised to 40 °C and incubation

continued for an additional 3 hours. After cooling and concentration in vacuo, the peptide

was purified by reverse-phase HPLC on a Vydac C18 column from Vydac, Hesperia, CA.

Analysis of the reaction product by mass spectrometry demonstrated the presence of the desired epoxide (FIG. 2).

EXAMPLE 3 Conversion of 3,4-Epoxyproline to Hydroxyproline in a Synthetic Peptide

The dry epoxy-CC-2 peptide obtained in Example 2 and containing 3,4-epoxyproline is redissolved in anhydrous methanol in a round-bottom flask and treated with 5 equivalents

(based on mole amount of epoxide) of lithium borohydride. The reaction is allowed to stir at

room temperature for a period of 1 to 24 hours. The reaction is monitored by reverse phase HPLC and/or Η NMR, and characterized by mass spectrometry. When the desired extent of

reaction is achieved, the peptide is purified by solid phase extraction (Sep-pak C18, Millipore) or by preparative HPLC. The stereochemistry of the hydroxyl group on proline is determined

by acid hydrolysis of the protein and derivatization with 9-fluorenylmethyl chloroformate (FMOC), followed by analytical HPLC analysis, as described by Zukowski et. al. in "Efficient

Enantioselective Separation and Determination of Trace Impurities in Secondary Amino

Acids", J. Chromatogr. Vol. 623, pp. 33-41 (1992), incorporated herein by reference.

EXAMPLE 4

Incorporation of 3,4-Dehydro-L-Proline into a Fragment of Human Type 1 (α,) Collagen

Plasmid pUSC-D4 (FIG. 3) containing DNA encoding the C-terminal 219 amino acids

of the human collagen , chain (FIG. 4) is used to transform proline auxotrophic E. coli strain

JM109(DE3)pLysS(pro^"). Two 1-liter cultures of the transformed E. coli strain are grown in M9 minimal medium (M9 salts, 2% glucose, 0.01 mg/mL thiamine, 200 μg/mL ampicillin, 34

μg mL chloramphenicol, supplemented with all 20 amino acids at 20 μg/mL except for

proline which is supplemented at 1 μg/mL) until a constant OD₆₀₀ of about 0. 8 is reached

(indicating proline starvation). The cells are harvested by centrifugation, and the media

replaced by an equal amount of the original medium except lacking proline and containing

3,4-dehydro-L-proline at 10-100 μg/mL. After equilibration for 10 minutes at 37 °C, the cells

are induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a concentration of

1.0 mM. The cells are incubated for an additional 2-18 hours at 37 °C with vigorous shaking (250 rpm). The cells are harvested by centrifugation, resuspended in one-fiftieth of the original culture volume of 10 mM Tris-HCL (pH 7.5), 1 mM EDTA, 100 mM NaCl (TEN buffer), and lysed by freeze/thaw and sonication. The 219-amino acid fragment of human

type I (α,) collagen protein containing 3,4-dehydroproline in place of proline (D4) is then purified by standard chromatographic techniques.

EXAMPLE 5

Conversion of 3,4-Dehydroproline in a Fragment of Human Type I(α,) Collagen to 3,4- Epoxyproline A sample of the dry D4 protein obtained as in Example 4 and containing 3,4- dehydroproline is dissolved in dry methanol to a concentration of 0. 1 mg/mL in a round- bottom flask equipped with a 24/40 ground glass joint. To this solution is added 1.1

equivalents of MCPBA (based on mole amount of dehydroproline). The solution is allowed

to stir at 40 °C for a period of 1-24 hours and the reaction is monitored by reverse phase

HPLC and/or 'H NMR. When the reaction is complete, the peptide is purified by solid phase extraction (Sep-pak C18, Millipore) or by preparative HPLC, and characterized as necessary

by mass spectrometry, amino acid analysis, 'H NMR analysis, and/or ^l3C NMR analysis.

EXAMPLE 6

Conversion of 3,4-Epoxyproline in a Fragment of Human Type I(αl)Collagen to 4- Hydroxyproline

The dry D4 protein containing 3,4-epoxyproline from Example 5 is dissolved in

methanol in a round bottom flask and treated with 5 equivalents (based on mole amount of epoxide) of lithium borohydride. The reaction is allowed to stir at room temperature for a

period of 1-24 hours and monitored by reverse phase HPLC and/or 'H NMR, and the product

characterized by mass spectrometry. When the desired extent of reaction is achieved, the protein is purified by solid phase extraction (Sep-pak C 18, Millipore) or by preparative HPLC. The stereochemistry of the hydroxyl group on proline is determined by acid hydrolysis of the protein and derivatization with 9-fluorenylmethyl chloroformate (FMOC), followed by analytical HPLC analysis, as described by Zukowski et al., supra.

EXAMPLE 7 Incorporation of 3,4-Dehydro-L-Proline into Human Type I (α,) Collagen

Plasmid pUSC-74 (FIG. 5) containing DNA encoding the human collagen Type 1 (α,)

chain (FIG. 6) is used to transform proline auxotrophic E. coli strain JM109(DE3)pLysS(pro^"). Two one-liter cultures of the transformed E. coli strain are grown in M9 minimal medium

(M9 salts, 2% glucose, 0.01 mg/mL thiamine, 200 μg/mL ampicillin, 34 μg/mL chloramphenicol, supplemented with all 20 amino acids at 20 μg/mL except for proline which

is supplemented at 1 μg/mL) until a constant OD₆₀₀ of about 0. 8 is reached (indicating proline starvation). The cells are harvested by centrifugation, and the media replaced by an equal amount of the original medium except lacking proline and containing 3,4-dehydro-L-proline

at 10-100 μg/mL. After 10 minutes equilibration at 37 °C, the cells are induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at 1.0 mM. The cells are incubated for an

additional 2-18 hours at 37 °C with vigorous shaking (250 rpm). The cells are harvested by centrifugation, resuspended in one-fiftieth the original culture volume of 10 mM Tris-HCL (pH 7.5), 1 mM EDTA, 100 mM NaCl (TEN buffer), and lysed by freeze/thaw and sonication.

The human Type I (α,) collagen polypeptide containing 3,4-dehydroproline in place of proline is purified by standard chromatographic techniques.

EXAMPLE 8

Conversion of 3,4-Dehydroproline in Human Type I (α,) at Collagen to 3,4-Epoxyproline

A sample of human Type I (α,) collagen protein containing 3,4-dehydroproline as

obtained in Example 7 is dissolved in methanol to a final concentration of 0. 1 mg/mL in a

round-bottom flask equipped with a 24/40 ground glass joint. To this solution is added 1.1 equivalents of MCPBA (based on mole amount of dehydroproline). The solution is allowed

to stir at 40 °C for a period of at least 1 hour and monitored by reverse phase (HPLC) analysis and/or 'H NMR analysis. When the reaction is complete, the protein is purified by solid phase

extraction (Sep-pak C18, Millipore) or by preparative HPLC, and characterized as required by

mass spectrometry, amino acid analysis, ^lH NMR analysis and/or ¹³C NMR analysis.

EXAMPLE 9 Conversion of 3,4-Epoxyproline in Human Type I (α,) Collagen to Hydroxyproline A sample of the dry type I (o ,) collagen protein containing 3,4-epoxyproline as

obtained in Example 8 is dissolved in methanol in a round bottom flask and treated with 5

equivalents (based on mole amount of epoxide) of lithium borohydride. The reaction is allowed to stir at room temperature for a period of 1-24 hours and monitored by reverse phase HPLC and/or 'H NMR, and the product characterized by mass spectrometry. When the desired extent of reaction is achieved, the protein is purified by solid phase extraction (Sep- pak C18, Millipore) or by preparative HPLC. The stereochemistry of the hydroxyl group on proline is determined by acid hydrolysis of the protein and derivatization with 9-

fluorenylmethyl chloroformate (FMOC), followed by analytical HPLC analysis, as described by Zukowski et al., supra.

EXAMPLE 10 Synthesis of a Gene Encoding a Bioadhesive Precursor Protein A DNA sequence was synthesized using codon preferences favorable for expression in

E. coli as shown in FIG. 7 (corresponding to the mussel adhesive protein analog decapeptide

amino acid sequence shown in FIG. 7, hereinafter the "MAP analog") using a Beckman, Inc, (Fullerton, CA) automated synthesizer, and purified by preparative gel electrophoresis and

reverse-phase high-pressure liquid chromatography (HPLC). The two DNA strands were

synthesized to contain a three base overhang following annealing. Each DNA oligomer (800

pmol) was 5'-phosphorylated in a 25 μL reaction mixture containing T4 polynucleotide

kinase and 1 mM ATP (37 °C, 2.5 hrs). The top and bottom strands were annealed by

mixing, heating to 80 °C for 15 minutes, and allowing to slow-cool to 30°C, followed by self-

ligation via the addition of 1 mM ATP, T4 DNA ligase buffer, and T4 DNA ligase (60 mL total) and incubation at 16 °C for 16 hours. The ligated multimers were then capped by the addition of synthetic linker oligonucleotide duplexes (200 pmol) containing restriction sites for cloning. The ligation reaction was allowed to continue for an additional 2 hours and the

ligase inactivated by heating at 65 °C for 20 min and slow cooling to 30 °C. The mixture of capped oligonucleotides encoding the MAP analog was cleaved with

Nco l and Hind III (37 °C, 3 hours), gel purified on a 2% agarose gel (IX TPE buffer, 1.5

hours, 60 V), and ligated into the Nco I and Hind III restriction sites of expression vector pET- 20b . Clones containing various lengths of polymeric sequence were selected from a population of transformant clones containing the MAP analog DNA insert. A clone

containing 25 repeats of the bioadhesive precursor protein analog DNA sequence was isolated (pLSM-6, FIG. 8), and verified by standard sequencing methods. This clone contains the bioadhesive precursor protein analog as a C-terminal fusion to the periplasmic localization sequence encoded by pET-20b.

EXAMPLE 1 1

Incorporation of 3,4-Dehydro-L-Proline Into a Bioadhesive Precursor Protein in E. coli

Plasmid pLSM-6 (FIG. 8) containing DNA encoding 25 repeats of the bioadhesive precursor protein decapeptide sequence of FIG. 7 and obtained in Example 10 is used to

transform proline auxotrophic E. coli strain JM109F^"(DE3pLysS)(pro^~). A one-liter culture of

the transformed E. coli strain is grown in LB medium until an OD₅₀₀ of 0.75 - 1.00 is reached. The cells are harvested by centrifugation, washed with M9 minimal medium (M9 salts, 2%

glucose, 0.01 mg/mL thiamine, 200 μg/L ampicillin, 34 μg/mL chloramphenicol, supplemented with the 20 natural amino acids minus proline, at 50 μg/mL), and the media is replaced by an equal amount of the identical M9 minimal medium, also lacking proline. After incubation for 30 minutes at 37 °C, the cells are induced by addition of isopropyl-β-D-

thiogalactopyranoside (IPTG) to a concentration of 1.5 mM and 3,4-dehydro-L-proline is added to provide a final concentration of 0.1 - 10 mM. The cells are incubated for an

additional 4 hours at 37 °C with vigorous shaking (250 rpm). The cells are harvested by centrifugation and an aliquot of the total cell lysate was electrophoresed on a 10% SDS-PAGE gel to verify expression.

EXAMPLE 12

Purification of 3,4-Dehydro-L-Proline-Containing Bioadhesive Precursor Protein

The cell pellet from Example 1 1 is resuspended in one-fiftieth of the volume of the original culture in lysis buffer (50 mM sodium phosphate, pH 6.5, 100 mM NaCl, 10 mM EDTA, 5 mM DTT) and lysed by standard methods. Protein in the insoluble cell fraction is solubilized by addition of 8.0 M urea, and dialyzed against 10 mM sodium phosphate, pH 6.5.

The protein is further purified by cation exchange chromatography, dialyzed into 0.05 M

ammonium acetate buffer at pH 6.0, and lyophilized.

EXAMPLE 13

Conversion of 3,4-Dehydroproline in Bioadhesive Precursor Protein to 3,4-Epoxyproline A sample of the adhesive precursor protein containing 3,4-dehydroproline as obtained in Example 1 1 and purified in Example 12 is dissolved in methanol to a final concentration of

0. 1 mg/mL in a round-bottom flask equipped with a 24/40 ground glass joint. To this

solution is added 1.1 equivalents of MCPBA (based on mole amount of dehydroproline). The solution is allowed to stir at about 37 °C for a period of at least 1 hour and monitored by reverse phase (HPLC) analysis and/or 'H NMR analysis. When the reaction is complete, the peptide is purified by solid phase extraction (Sep-pak Cl 8, Millipore) or by preparative

HPLC, and characterized as required by mass spectrometry, amino acid analysis, 'H NMR

analysis and/or ¹³C NMR analysis.

EXAMPLE 14

Conversion of 3,4-Epoxyproline in Bioadhesive Precursor Protein to Hydroxyproline A sample of the bioadhesive precursor protein containing 3,4-epoxyproline as obtained

in Example 13 is dissolved in methanol in a round bottom flask and treated with 5 equivalents (based on mole amount of epoxide) of lithium borohydride. The reaction is allowed to stir at

room temperature for a period of 1-24 hours and monitored by reverse phase HPLC and/or Η

NMR, and the product characterized by mass spectrometry. When the desired extent of

reaction is achieved, the protein is purified by solid phase extraction (Sep-pak C18, Millipore)

or by preparative HPLC. The stereochemistry of the hydroxyl group on proline is determined by acid hydrolysis of the protein and derivatization with 9-fluorenylmethyl chloroformate

(FMOC), followed by analytical HPLC analysis, as described by Zukowski et al., supra.

EXAMPLE 15

Cure of 3,4-Epoxyproline-Containing Bioadhesive Precursor Protein The dry bioadhesive precursor protein containing 3,4-epoxyproline obtained in Example 13 is dissolved in a weak buffer at neutral pH (e.g., 10 mM ammonium acetate, pH

7) and cure is initiated by addition of a solution effective to increase the pH (e.g., 500 mM Tris buffer at pH 9.0). Extent of cure is controlled by altering the temperature and/or length of time of the cure. Cure is terminated by re-adjustment of the reaction pH to neutrality.

It will be understood that various modifications may be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but

merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.

What is claimed is:

Claims

1. A method for incorporating a non-natural amino acid into a polypeptide, comprising

providing a polypeptide having at least one 3,4-dehydroproline residue; and

reacting the 3,4-dehydroproline with an epoxidation agent to form a polypeptide having at least one 3,4-epoxyproline residue.

2. The method of claim 1 , wherein the epoxidation agent is selected from the group consisting of trifluoroperacetic acid, MCPBA, bromine and sodium ethoxide in ethanol,

N-bromosucinimide/perchlorate and potassium acetate, basic peroxide, osmium tetroxide /perchlorate/acetic acid, a nitrile/hydrogen peroxide and an alkyl peroxides/complex of V, Mo,

Ti, or Co.

3. The method of claim 1, further comprising reacting the polypeptide having at

least one 3,4-epoxyproline residue with a reducing agent to form a polypeptide having

hydroxyproline.

4. The method of claim 3, wherein the reducing agent is a metal hydride.

5. The method of claim 3, wherein the reducing agent is selected from the group

consisting of lithium borohydride and sodium borohydride.

6. The method of claim 1, further comprising reacting the polypeptide having at least one 3,4-epoxyproline with a nucleophile to form a polypeptide having an amino acid with a modified side chain.

7. The method of claim 6, wherein the nucleophile is selected from the group consisting of amines, azide ions, cyanide ion, enamines, thiols, carboxylic acids, alcohols, and carbon nucleophiles.

8. The method of claim 7, wherein the nucleophile further comprises a biological probe or a therapeutic ligand.

10. The method of claim 7, wherein the nucleophile is selected from the group

consisting of a second polypeptide chain, a cross-linking agent or a combination thereof.

1 1. The method of claim 10, wherein the crosslinking agent has the formula X-R-

Y, wherein X and Y are the same or different and are selected from the group consisting of an

alcohol, amino, carboxyl, or thio group and R is selected from the group consisting of straight

and branched-chain alkyls, cycloalkyls and olefins, substituted and unsubstituted phenyls,

diphenyls, naphthyls, peptides, dextrans, molecules which can function as a probe and molecules which have biological activity.

12. The method of claim 1, wherein the polypeptide includes a plurality of Gly-X-

Y units wherein X and Y are the same or different and are selected from the group consisting of proline, hydroxyproline and 3,4-dehydroproline.

13. The method of claim 12, wherein the crosslinking agent has the formula X-R- Y, wherein X and Y are the same or different and are selected from the group consisting of an alcohol, amino, carboxyl, or thio group and R is selected from the group consisting of straight and branched-chain alkyls, cycloalkyls and olefins, substituted and unsubstituted phenyls,

diphenyls, naphthyls, peptides, dextrans, molecules which can function as a probe and molecules which have biological activity.

14. The method of claim 1, wherein the polypeptide is a bioadhesive precursor

protein.

15. A method for crosslinking a polypeptide, comprising providing a solution of a polypeptide having at least one residue selected from the group comprising 3,4-dehydroproline, 3,4-epoxyproline residue, wherein the polypeptide

optionally further comprises at least one nucleophilic residue; and

adjusting the pH, temperature, or composition of the solution to effect crosslinking between the 3,4-dehydroproline residue or 3,4-epoxyproline residue of the polypeptide and the at least one optional nucleophilic residue, between the 3,4-dehydroproline or 3,4-epoxyproline

residue and a crosslinking agent, or a combination thereof.

16. A polypeptide comprising an amino acid which has an oxirane ring.

17. The polypeptide of claim 16 wherein the amino acid is 3,4-epoxyproline.

18. The polypeptide of claim 16, wherein the polypeptide includes a plurality of Gly-X-Y units wherein X and Y are the same or different and are selected from the group consisting of proline, hydroxyproline, 3,4-dehydroproline, and 3,4-epoxyproline.

19. The polypeptide of claim 16, wherein the polypeptide is a bioadhesive

precursor protein.

20. The polypeptide of claim 19, wherein the bioadhesive precursor protein is

crosslinked.

21. A polypeptide having at least one crosslink between a 3,4-dehydroproline residue and a nucleophile or between a 3,4-epoxyproline residue and a nucleophile.

22. The polypeptide of claim 21, wherein the nucleophile is a residue within a second polypeptide, or wherein the nucleophile is a crosslinking agent having the formula X- R-Y, wherein X and Y are the same or different and are selected from the group consisting of

an alcohol, amino, carboxyl, or thio group and R is selected from the group consisting of straight and branched-chain alkyls, cycloalkyls and olefins, substituted phenyls, unsubstituted phenyls, diphenyls, naphthyls, molecules which can function as a probe and molecules which have biological activity.

23. A method for incorporating a non-natural amino acid into a polypeptide,

comprising providing a polypeptide having at least one 3,4-dehydroproline residue; and

reacting the 3,4-dehydroproline with a reagent to form a polypeptide having an amino

acid with a modified side chain.

24. The method of claim 23, wherein the reagent is selected from the group consisting of a nucleophile, and electrophile, a hydrogen halide, bromine, iodine,

permanganate-based reagents, or osmium tetroxide/peroxide.

25. The method of claim 24, wherein the nucleophile is selected from the group

consisting of amines, azide ions, cyanide ion, enamines, thiols, carboxylic acids, alcohols, and

carbon nucleophiles.

26. The method of claim 5, wherein the nucleophile further comprises a biological probe or a therapeutic ligand.