WO2022261181A1 - Preparation of amino acid functionalized homocysteine residues - Google Patents

Abstract

The present disclosure relates to polypeptides comprising methionine residues functionalized with amino acid moieties. The present disclosure also relates to compositions comprising the polypeptides disclosed herein. The present disclosure also relates to methods of using the compositions disclosed herein.

Description

PREPARATION OF AMINO ACID FUNCTION AT HOMOCYSTEINE RESIDUES

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 63/209,225, filed June 10, 2021, the contents of which are incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Number 1412367, awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND

Polyelectrolyte complex coacervation, where oppositely charged polymer chains aggregate and separate as a polymer-rich liquid phase upon mixing in aqueous media, has been the subject of much attention. Recent interest in these systems stems from their similarity to “membraneless organelle” (MLO) coacervate domains found within cells that are rich in intrinsically disordered proteins (IDPs), and are linked to activation of many transient biological processes. Protein coacervates have also been identified as key components in moisture-resistant adhesives of marine organisms. There is also considerable interest in use of coacervate phase separation to form aqueous block copolymer assemblies, such as micelles, vesicles, and hydrogels, where coacervate fluidity can assist rapid complex formation and equilibration. Currently, most of the synthetic systems, especially those based on polypeptides, employ simple poly electrolytes, such as poly (ly sine HC1) and poly(glutamate Na), which offer limited opportunity to tune coacervate properties at the molecular level. An exception to this is the variation of optical purity in polypeptides, where the inclusion of at least one racemic polypeptide component was found to favor coacervate formation, as opposed to solid precipitates that form with pure homochiral components. However, since optical purity is encoded into the polypeptide backbone, it cannot be reversibly switched by external stimuli. Thus, there is an ongoing need for tunable polypeptide systems having coacervate properties that can be reversibly switched by external stimuli. SUMMARY

In one aspect, the present disclosure provides polypeptides comprising one or more R- C^HAA residues having a structure represented by formula la, formula lb, formula Ila, or formula lib:

or a salt thereof, or a stereoisomer or enantiomer thereof, wherein:

R¹, R², R³, R⁵, R^7a and R^7b are each independently selected from H, alkyl, or aralkyl; each R⁴ is independently the side chain of a naturally occurring amino acid, alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, amidoalkyl, ester, acyl, cycloalkyl, (cylcoalkyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl;

R⁵ is H,

X¹⁺ is a cation;

R⁶ is alkyl;

A¹ and A² are each independently anions;

Z is amino, urealyl, or HN⁺(R^7a)(R^7b).A² ;

Z¹ is OR⁵ or 0 X¹⁺; m is 0-5; n is 1-5; and p is 1-5.

In another aspect, the present disclosure provides compositions comprising a polypeptide disclosed herein and an excipient.

In yet another aspect, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof comprising administering the compositions disclosed herein to the subject.

In yet another aspect, the present disclosure provides methods of administering a therapeutic agent to a subject in need thereof comprising administering the compositions disclosed herein to the subject. In yet another aspect, the present disclosure provides methods of treating fine lines or superficial wrinkles in the skin of a subject, comprising administering a composition into a dermal region of the subject which displays the fine lines or superficial wrinkles, thereby treating the fine lines or superficial wrinkles, wherein the composition comprises a polypeptide disclosed herein.

In yet another aspect, the present disclosure provides methods of treating a skin condition, comprising administering to an individual suffering from the skin condition a composition, wherein the administration of the composition improves the skin condition, thereby treating the skin condition, wherein the composition comprises a polypeptide disclosed herein.

In yet another aspect, the present disclosure provides methods of preventing skin wrinkles in a subject, comprising administering to the subject a composition, thereby preventing skin wrinkles, wherein the composition comprises a polypeptide disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an optical micrograph of coacervate droplets from 5c and pyrophosphate, pH 7.0, 30 °C.

FIG. 2 shows the reversibility of coacervation measured by solution turbidity. 5c (3.0 mg/mL) in pH 7.0 PBS. Temperature cycled from 15 to 45 °C and back at 1 °C/min.

FIG. 3 shows zeta potential measurements of polypeptide samples as a function of solution pH. Measurements of polypeptides 5a-5d and reference polypeptides were performed at 5.0 mg/mL in 20 mM NaCl at 20 °C. 5a = black; 5b = red; 5c = blue; 5d = green; poly(L- lysine HCl)₆o = orange; poly(S-methyl-L-methionine sulfonium chloride)₆o = grey; poly(L- methionine sulfoxide)₆o = magenta. Data was not recorded above pH 9 for sample 5d due to phase separation. Error bars represent standard deviations of triplicate measurements.

FIGs. 4A-4D are circular dichroism spectra of polypeptide samples 5a-5d at different solution pH. A) 5a; B) 5b; C) 5c; D) 5d. Samples 5a-5c were prepared at 0.5 mg/mL in pH 5.5, 7.0 or 9.0 phosphate buffer (100 mM) at 20 °C. Sample 5d was prepared at 0.1 mg/mL in pH 5.5 phosphate buffer (20 mM). Sample 5d is not soluble in phosphate buffer at pH 7.0 or 9.0. FIGs. 5A-5D show temperature dependent coacervate formation in solutions of polypeptides 5b, 5c and 5d as functions of pH and counterions. Panels show optical transmittance at 500 nm for 3.0 mg/mL solutions of polypeptides in 150 mM PBS buffer measured at different pH over a range of temperature. A) 5b; B) 5c; C) 5d. D) Optical transmittance at 500 nm for 3.0 mg/mL solutions of 5c in 150 mM NaCl measured in the presence of different counterions (12 mM) at pH 7.0 over a range of temperature.

FIGs. 6A-6D are optical micrographs of polypeptide mixtures with sodium tripolyphosphate. Solutions of polypeptides 5a-5d at 3.0 mg/mL in 150 mM NaCl at 20 °C and pH 7.0 were mixed with sodium tripolyphosphate (12 mM final concentration) and the resulting turbid suspensions were allowed to settle onto glass slides. A) 5a; B) 5b; C) 5c; D) 5d. Scale bars = 20 pm.

FIG. 7A shows the reversibility of temperature dependent coacervate formation for polypeptide 5c in PBS buffer as determined by optical transmittance at 500 nm. Sample was prepared at 3.0 mg/mL in 150 mM PBS buffer at pH 8.0 and subjected to repeated thermal cycling between 17 and 44 °C. Sample was heated and cooled at the rate of 3 °C/min.

FIG. 7B shows coacervate formation of 5c mixed with sodium tripolyphosphate as a function of sodium chloride concentration. Coacervate Fraction = (mass of polypeptide in coacervate phase)/ (total mass of polypeptide). Samples were prepared by mixing 5c (3.0 mg/mL) in different concentrations of NaCl at 20 °C and pH 7.0 with sodium tripolyphosphate (12 mM final concentration).

FIG. 8 is a circular dichroism spectrum of coacervate phase of 5c after mixing with sodium tripolyphosphate. The spectrum was taken using a sample cell with a path length of 0.1 mm and accumulation of 20 scans. Sample was prepared by mixing 5c (3.0 mg/mL) in 150 mM NaCl at 20 °C and pH 7.0 with sodium tripolyphosphate (12 mM final concentration).

FIG. 9A is a circular dichroism spectra of 5c and oxidized polypeptide 6c both at 0.5 mg/mL in DI water and 20 °C.

FIG. 9B shows temperature dependent coacervate formation of polypeptides 5c and 6c in 150 mM PBS buffer as determined by optical transmittance at 500 nm. Each sample was prepared at 3.0 mg/mL polypeptide and pH 8.0.

FIG. 9C are images of coacervate phase of 5c mixed with sodium tripolyphosphate before (left) and after oxidation to 6c using sodium periodate (0.5 M, 1.05 eq over 2 additions) at 0 °C (right). Initial sample was prepared by mixing 5c (3.0 mg/mL) in 150 mM NaCl at 20 °C and pH 7.0 with sodium tripolyphosphate (12 mM final concentration). FIG. 10A shows a family of cationic poly(S-alkyl-L-homocysteine) derivatives bearing different amino acids at their side-chain termini.

FIG. 10B shows an optical micrograph of a mixture of polypeptide 5c with sodium tripolyphosphate.

FIGs. 11A & 11B show circular dichroism spectra of polypeptide samples 8a and 8b at different solution pH. Samples 8a and 8b were prepared at 0.5 mg/mL in pH 5.0, 7.0 or 9.0 phosphate buffer (12 mM) at 20 °C.

FIG. llC shows a comparison of circular dichroism spectra of polypeptides 8a and 8b at 0.5 mg/mL in phosphate buffer (12 mM) with 5b at 0.5 mg/mL in pH 7.0 phosphate buffer (100 mM) all at 20 °C. Polypeptide 5b was found to be 77% a-helical under these conditions.

FIGs. 12A & 12B show optical micrographs of mixtures of polypeptides 8a and 8b with sodium tripolyphosphate. The sample in FIG. 11 A was prepared by mixing a solution of polypeptide 8a at 3.0 mg/mL in 150 mM NaCl with sodium tripolyphosphate (12 mM final concentration). The sample in FIG. 1 IB was prepared by mixing a solution of polypeptide 8b at 5.0 mg/mL in 150 mM NaCl at 20 °C and pH 7.0 with sodium tripolyphosphate (12 mM final concentration). The resulting turbid suspensions were allowed to settle onto glass slides before imaging. Scale bars = 20 pm.

FIGs. 13A & 13B show circular dichroism spectra of polypeptide samples 13a and 13b at different solution pH. Samples 13a and 13b were prepared at 0.83 mg/mL in pH 5.0, 7.0 or 9.0 phosphate buffer (12 mM) at 20 °C.

FIG. 13C shows a comparison of circular dichroism spectra of polypeptides 13a and 13b at 0.83 mg/mL in phosphate buffer (12 mM) with 5c at 0.5 mg/mL in pH 7.0 phosphate buffer (100 mM) all at 20 °C. Polypeptide 5c was found to be 96% a-helical under these conditions.

FIGs. 14A & 14B show optical micrographs of polypeptide mixtures with sodium tripolyphosphate. Solutions of polypeptides 13a (FIG. 14A) and 13b (FIG. 14B) at 5.0 mg/mL in 150 mM NaCl at 20 °C and pH 7.0 were mixed with sodium tripolyphosphate (13 mM final concentration), and the resulting turbid suspensions were allowed to settle onto glass slides before imaging.

FIGs. 15A & 15B show temperature-dependent coacervate formation in solutions of polypeptides 13a and 13b as functions of pH. Panels show optical transmittance at 500 nm for 2.5 mg/mL solutions of polypeptides in 150 mM PBS buffer measured at different pH over a range of temperatures. 13a (FIG. 15 A) and 13b (FIG. 15B). FIG. 15C shows a comparison of optical transmittance at 500 nm of polypeptides 13a and 13b at 2.5 mg/mL with polypeptide 5c at 3.0 mg/mL all in 150 mM PBS buffer at pH 7.4. Polypeptide 5c was found to have a Tcp of 49 °C under these conditions.

FIGs. 16A-16D show circular dichroism spectra of aqueous solutions of polypeptides 18a-d at different solution pH. 18a (FIG. 16A), 18b (FIG. 16B), 18c (FIG. 16C), and 18d (FIG. 16D). Samples polypeptides 18a-d were prepared at 0.5 mg/mL in pH 5.0, 7.0 or 9.0 phosphate buffer (100 mM) at 20 °C.

FIGs. 17A-17H show optical micrographs of aqueous solutions of polypeptides 18a-d mixed with multivalent cations of different valency. Solutions of polypeptides at 5 mg/mL in 150 mM NaCl at 20 °C and pH 7.0 were mixed with CaCb (final concentrations of 13 mM) or [CO(NH3)6]C13 (final concentrations of 13 mM) resulting in the formation of turbid suspensions that were allowed to settle on glass slides before imaging. 18a with CaCb (FIG. 17A), 18a with [Co(NH₃)6]Cb (FIG. 17B), 18b with CaCb (FIG. 17C), 18b with [Co(NH₃)6]Cb (FIG. 17D), 18c with CaCb (FIG. 17E), 18c with [Co(NH₃)₆]Cb (FIG. 17F), 18d with CaCb (FIG. 17G), 18d with [Co(NH₃)6]Cb (FIG. 17H). Scale bars = 20 pm.

FIGs. 18A-18C show optical micrographs of solutions of 18c mixed with lysozyme. Solutions of polypeptide 18c at 5 mg/mL final concentration in 150 mM NaCl Milli-Q water at 20 °C and pH 7.0 were mixed with lysozyme solutions to give final lysozyme concentrations of 0.25 (FIG. 18 A), 5 (FIG. 18B), and 10 (FIG. 18C) mg/mL. Scale bars = 20 pm.

FIGs. 18D shows an optical micrograph of a solution of 18d mixed with lysozyme. Solution of polypeptide 18d at 5 mg/mL final concentration in 150 mM NaCl Milli-Q water at 20 °C and pH 7.0 mixed with lysozyme at a final concentration of 5 mg/mL. The resulting turbid suspensions that formed upon mixing were allowed to settle on glass slides before viewing. Scale bars = 20 pm.

FIGs. 19A & 19B show optical micrograph of polypeptide 18c mixed with oppositely charged polypeptides. Solutions of polypeptide 18c in 150 mM NaCl at 20 °C and pH 7.0 at final concentration of 5 mg/mL were mixed with solutions of oppositely charged polypeptides (final concentration of 5 mg/mL). The resulting turbid suspensions were allowed to settle on glass slides before viewing. Scale bars = 20 pm. Mixture of 18c with 5c (FIG. 19A). Mixture of 18c with 6c (FIG. 19B).

FIG. 20A shows an inner addition approach to load the PIC hydrogels into syringes for subsequent injection. FIG. 20B shows a simultaneous addition approach to load the PIC hydrogels into syringes for subsequent injection.

FIG. 20C shows a successive addition approach to load the PIC hydrogels into syringes for subsequent injection.

FIG. 20D shows a manual loading approach to load the PIC hydrogels into syringes for subsequent injection.

FIG. 21 shows an example photograph from an animal of Animal Study #1 in group 2 at day 30 with no observable erythema or irritation at the site of injection (marked with the dark circle).

FIG. 22 shows an example photo of a palpable lump on the dorsum of an animal of Animal Study #2 in Group A (Hyaluronic Acid Control) on day 7.

FIG. 23 shows day 0 transillumination from left ear of rabbit 1 (Hyaluronic acid control) demonstrates impeded blood flow and embolus in the central auricular artery (Animal Study #3).

FIG. 24 shows day 0 transillumination from right ear of rabbit 1 (M°A;JJ(E/K)65 at 7 wt% in 0.9%NaCl) demonstrates intact blood flow in the central auricular artery without apparent emboli. These results were representative of the day 0 transillumination experiments. All of the hyaluronic acid ears showed emboli with impeded blood flow, while all of the claimed hydrogel filler injected ears remained patent (Animal Study #3).

FIG. 25 shows a photo from day 7 from animal 1 depicting ischemic changes in the left ear (hyaluronic acid) compared to right ear (M°A;JJ(E/K)65 at 7wt% in 0.9%NaCl). The ischemic changes are clearly seen as the dusky coloration in the auricular tissue (Animal Study #3).

FIG. 26 shows a photo from day 7 of rabbit 3 demonstrates ischemic changes in the right ear (hyaluronic acid), but no ischemic changes in the left ear (M°A;so(E/K)75 at 7wt% in 0.9%NaCl). As with the transillumination studies, these changes were consistent and reproducible in the animals. All of the hyaluronic acid ears demonstrated ischemic changes at day 7, while none of the claimed hydrogel filler injected ears demonstrated ischemic changes (Animal Study #3).

FIG. 27 depicts a schematic representation of the assembly process for preparation of polyion complex (M°A)i55E/K_x diblock copolypeptide hydrogels, which are employed in the animals studies recited in Example 3. RETAIT,ER DESCRIPTION

Synthetic polypeptide based coacervates consist mainly of highly charged, simple sequences (e.g. homopolymers of lysine or glutamic acid), which offer few opportunities to tune or actively modify coacervate properties at the molecular level. These systems possess limited response to pH, temperature, and ionic strength within physiological ranges. One type of model system relies primarily on polyelectrolyte complexation between two oppositely charged polypeptides, i.e. complex coacervation. In these models, it has been reported that at least one macromolecular component must possess a disordered conformation to prevent precipitation of solids. Another well-studied model system is based on coacervation of cationic homopolypeptides (e.g. poly(L-lysine)) with anionic nucleotides and polynucleotides. Noteworthy achievements in these models include the demonstration of active control of coacervation by enzymatic interconversion of ATP and ADP nucleotides in mixtures with poly(L-lysine), as well as formation of temperature dependent liquid crystalline coacervate phases in mixtures of poly(L-lysine) with short dsDNA containing complimentary sticky ends that are able to self-assemble in the coacervate phase. Contrary to the active control observed in protein coacervates that is due to enzymatic post-translational modifications, the responsive behavior in these examples does not arise from the polypeptide components, which function primarily as disordered polycations.

To bridge the gap between polypeptide and peptide/protein coacervate models, the inventors sought to develop synthetic polypeptides that could replicate most of the features found in coacervate forming proteins, such as the ability to respond to changes in pH, ionic strength and temperature within physiologically relevant ranges. The ability to actively control coacervation via reversible polypeptide modification, mimicking post-translational modifications in protein coacervates, was also deemed important. In addition, the inventors desired a modular design with many readily adjustable functional elements to allow deconvolution of molecular requirements for coacervation and for fine tuning of coacervate properties for downstream applications. In order to decouple the presence of multiple functional groups from concomitant peptide sequence complexity, the inventors avoided copolypeptide designs and instead pursued a new concept centered on homopolypeptide chains that contain multiple, precisely located functional elements in each side-chain group.

Scheme 1. Exemplary properties of amino acid functionalized poly(S-alkyl-L-homocysteine)s

(C^HAA)

Specifically, the design incorporates amino acid components into the side-chains of a poly(S-alkyl-L-homocysteine) scaffold (Scheme 1). This homopolypeptide backbone was chosen because it favors an a-helical conformation in water that can be readily and reversibly converted to a disordered conformation upon mild oxidation to the sulfoxide derivative. The incorporation of amino acids as side-chain functionality was enables many adjustable molecular features commonly found in proteins, such as H-bonding from amide groups, charges from carboxylate or ammonium groups, chirality, as well as hydrophobicity or other functionality from the amino acid “R” groups (Scheme 1). Through designed combinations of charge, hydrophobicity, hydrophilicity and side-chain flexibility, the inventors sought to develop a homopolypeptide based, coacervate system with tunable properties that can respond to multiple biologically relevant stimuli and chemical modifications.

In one aspect, the present disclosure provides polypeptides comprising one or more R- C^HAA residues having a structure represented by formula la, formula lb, formula Ila, or formula lib:

la

or a salt thereof, or a stereoisomer or enantiomer thereof, wherein:

R¹, R², R³, R⁵, R^7a and R^7b are each independently selected from H, alkyl, or aralkyl; each R⁴ is independently the side chain of a naturally occurring amino acid, alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, amidoalkyl, ester, acyl, cycloalkyl, (cylcoalkyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl;

R⁵ is H, X is S, S(O), S(O)2, or S⁺-R⁶.A^1-; X¹⁺ is a cation; R⁶ is alkyl; A^1- and A^2- are each independently anions; Z is amino, urealyl, or HN⁺(R^7a)(R^7b).A^2-; Z¹ is OR⁵ or O-X¹⁺; m is 0-5; n is 1-5; and p is 1-5. In certain embodiments, Z¹ is O-X¹⁺. In other embodiments, Z¹ is OR⁵. In certain embodiments, X¹⁺ is Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, or NH4⁺. In certain preferred embodiments, X¹⁺ is NH4⁺. In one aspect, the present disclosure provides polypeptides comprising one or more R- C^HAA residues having a structure represented by formula Ia*, formula Ib*, formula IIa*, or formula IIb*:

or a salt thereof, or a stereoisomer or enantiomer thereof, wherein:

R¹, R², R³, R⁵ R^7a and R^7b are each independently selected from H, alkyl, or aralkyl; each R⁴ is independently the side chain of a naturally occurring amino acid, alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, amidoalkyl, ester, acyl, cycloalkyl, (cylcoalkyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl;

X is S, S(O), S(0)₂, or S⁺^.A^1';

R⁶ is alkyl;

A^1' and A^2' are each independently anions;

Z is amino, urealyl, or HN⁺(R^7a)(R^7b).A^2'; m is 0-5; n is 1-5; and p is 1-5.

In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula I or formula II. In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula la or a salt thereof.

In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula lb or a salt thereof.

In other embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula Ila or a salt thereof.

In other embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula lib or a salt thereof.

In yet other embodiments, the polypeptide comprises R-C^HAA residues represented by formula la and formula Ila or salts thereof.

In yet other embodiments, the polypeptide comprises R-C^HAA residues represented by formula lb and formula lib or salts thereof.

In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula I* or formula II*.

In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula la* or a salt thereof.

In certain embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula lb* or a salt thereof.

In other embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula Ila* or a salt thereof.

In other embodiments, each R-C^HAA residue in the polypeptide has a structure represented by formula lib* or a salt thereof.

In yet other embodiments, the polypeptide comprises R-C^HAA residues represented by formula la* and formula Ila* or salts thereof.

In yet other embodiments, the polypeptide comprises R-C^HAA residues represented by formula lb* and formula lib* or salts thereof.

In certain embodiments, R¹ is H.

In certain embodiments, X is S⁺-R⁶.A^1'.

In certain embodiments, R⁶ is methyl or ethyl. In certain preferred embodiments, R⁶ is methyl.

In certain embodiments, A^1' is a pharmaceutically acceptable anion. In certain preferred embodiments, A^1' is halide, sulfate, phosphate, citrate, pyrophosphate, or tripolyphosphate.

In certain preferred embodiments, R² is H.

In certain preferred embodiments, R³ is H.

In certain embodiments, R⁴ is the side chain of a naturally occurring amino acid ( e.g the side chain of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine).

In certain embodiments, R⁴ is the side chain of glycine, valine, leucine, or phenylalanine. In certain preferred embodiments, R⁴ is the side chain of glycine. In other preferred embodiments, R⁴ is the side chain of valine. In yet other preferred embodiments, R⁴ is the side chain of leucine. In yet other preferred embodiments, R⁴ is the side chain of phenylalanine.

In certain embodiments, wherein Z is amino. In certain preferred embodiments, Z is N⁺(R^7a)(R^7b) . A^2' .

In certain embodiments, R^7a is H.

In certain embodiments, R^7b is H.

In certain embodiments, A^2' is a pharmaceutically acceptable anion. In certain preferred embodiments, A^2' is halide, sulfate, phosphate, citrate, pyrophosphate, or tripolyphosphate.

In certain embodiments, R⁵ is H.

In certain embodiments, m is 1.

In certain embodiments, n is 2.

In certain embodiments, p is 1. In certain embodiments, the R-C^HAA residues are a salt of formula la, formula lb, formula Ila, or formula lib. In certain embodiments, the R-C^HAA residues are a salt of formula la. In certain embodiments, the R-C^HAA residues are a salt of formula Ila.

In certain embodiments, the R-C^HAA residues are a salt of formula la*, formula lb*, formula Ila*, or formula lib*. In certain embodiments, the R-C^HAA residues are a salt of formula la*. In certain embodiments, the R-C^HAA residues are a salt of formula Ila*.

In certain embodiments, the polypeptide comprises 5-500 R-C^HAA residues. In certain embodiments, the polypeptide consists essentially of 5-500 R-C^HAA residues. In certain embodiments, the polypeptide consists of 5-500 R-C^HAA residues. In certain preferred embodiments, the polypeptide comprises 10-200 R-C^HAA residues. In other preferred embodiments, the polypeptide consists essentially of 10-200 R-C^HAA residues. In yet other preferred embodiments, the polypeptide consists of 10-200 R-C^HAA residues.

In certain embodiments, the R-C^HAA residues are the same. In other embodiments, the R-C^HAA residues are different.

In certain embodiments, the N-terminus of the polypeptide is capped with alkyl or alkoxy. In certain embodiments, the N-terminus of the polypeptide is capped with alkoxy. In certain preferred embodiments, the N-terminus of the polypeptide is capped with poly (ethylene glycol).

In certain preferred embodiments, the N-terminus of the polypeptide is capped with poly(ethylene glycol) having a molecular weight of 50-20,000 Da. In certain preferred embodiments, the N-terminus of the polypeptide is capped with poly(ethylene glycol) having a molecular weight of 100-10,000 Da.

In another aspect, the present disclosure provides compositions comprising the polypeptides disclosed herein and an excipient.

In certain embodiments, the composition further comprises a therapeutic agent. In certain embodiments, the therapeutic agent is an anti-cancer agent, an anti-inflammatory agent, an anti-biotic agent, an anti-viral agent, an anti-fungal agent, or an analgesic. In certain embodiments, the therapeutic agent is a vaccine. In certain embodiments, the therapeutic agent is an antibody, a protein, a nucleic acid, or a small molecule.

In yet another aspect, the present disclosure provides methods of treating a disease or disorder in a subject in need thereof comprising administering the compositions disclosed herein to the subject. In certain embodiments, the disease or disorder is cancer. In yet another aspect, the present disclosure provides methods of administering a therapeutic agent to a subject in need thereof comprising administering the compositions disclosed herein to the subject.

In yet another aspect, the present disclosure provides methods of treating fine lines or superficial wrinkles in the skin of a subject, comprising administering a composition into a dermal region of the subject which displays the fine lines or superficial wrinkles, thereby treating the fine lines or superficial wrinkles,

In certain embodiments, the dermal region is a tear trough region, a glabellar line, a periorbital region, or a forehead region.

In yet another aspect, the present disclosure provides methods of treating a skin condition, comprising administering to an individual suffering from the skin condition a composition, wherein the administration of the composition improves the skin condition, thereby treating the skin condition, wherein the composition comprises a polypeptide disclosed herein.

In certain embodiments, the skin condition is skin dehydration. In certain embodiments, the composition rehydrates the skin of the subject. In other embodiments, the skin condition is skin elasticity. In certain embodiments, the composition increases the elasticity of the skin of the subject. In yet other embodiments, the skin condition is skin roughness. In certain embodiments, the composition decreases skin roughness in the subject. In yet other embodiments, the skin condition is a lack of skin tautness. In certain embodiments, the composition increases skin tautness in the subject. In yet other embodiments, the skin condition is a skin stretch line or mark. In certain embodiments, the composition reduces or eliminates the skin stretch line or mark in the subject. In yet other embodiments, the skin condition is skin paleness. In certain embodiments, the composition increases skin tone or radiance in the subject. In yet other embodiments, the skin condition is skin wrinkles. In certain embodiments, the composition reduces or eliminates skin wrinkles in the subject.

In yet another aspect, the present disclosure provides methods of preventing skin wrinkles in a subject, comprising administering to the subject a composition, thereby preventing skin wrinkles, wherein the composition comprises a polypeptide disclosed herein. In certain embodiments, the composition makes the skin of the subject resistant to skin wrinkles. In certain embodiments, the administration occurs at a depth of less than about 1 mm below the surface of the skin.

In certain embodiments, the method does not result in arterial occlusion.

In certain embodiments, the method does not result in unpredictable augmentation.

In certain embodiments, the method does not result in irritation, for example, chronic irritation.

In certain embodiments, the composition is soluble in blood.

In certain embodiments, administration of the composition results in limited swelling.

In certain embodiments, the administration of the composition results in low immunogenicity.

Pharmaceutical Compositions

A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a polypeptide such as a polypeptide of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a polypeptide of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

The phrase "pharmaceutically acceptable" is employed herein to refer to those polypeptide, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The polypeptide may also be formulated for inhalation. In certain embodiments, a polypeptide may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

Methods of preparing these formulations or compositions include the step of bringing into association an active polypeptide, such as a polypeptide of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a polypeptide of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

The present disclosure includes the use of pharmaceutically acceptable salts of polypeptides of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2- (diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, lH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1 -(2-hydroxy ethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1, 5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1- pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid acid salts.

The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et ah, “Molecular Cell Biology, 4th ed ”, W. H. Freeman & Co., New York (2000); Griffiths et ah, “Introduction to Genetic Analysis, 7th ed ”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed ”, Sinauer Associates, Inc., Sunderland, MA (2000).

Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.

A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.

Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

It is understood that substituents and substitution patterns on the polypeptides of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable polypeptides which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to the replacement of one to six hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2- O-alkyl, -0P(0)(0-alkyl)2 or -CH2-0P(0)(0-alkyl)2. Preferably, “optionally substituted” refers to the replacement of one to four hydrogen radicals in a given structure with the substituents mentioned above. More preferably, one to three hydrogen radicals are replaced by the substituents as mentioned above. It is understood that the substituent can be further substituted.

As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to C1-C1₀ straight-chain alkyl groups or C1-C1₀ branched-chain alkyl groups. Preferably, the “alkyl” group refers to C1-C₆ straight-chain alkyl groups or C1-C₆ branched- chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1 -pentyl, 2-pentyl, UCLA Ref. No.: [UCLA 2021-312-2] WO 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1- octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted. The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-. The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-. The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-. The term “alkoxy” refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl. The term “alkyl” refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C_1- 30 for straight chains, C3-30 for branched chains), and more preferably 20 or fewer. Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2- trifluoroethyl, etc. The term “Cx-y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. C₀alkyl indicates a hydrogen where the group is in a terminal position, a bond if internal. A C_1-6alkyl group, for example, contains from one to six carbon atoms in the chain. The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group. The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-. The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen or hydrocarbyl group, or

R⁹ and R¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by

wherein R⁹, R¹⁰, and R¹⁰’ each independently represent a hydrogen or a hydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group.

The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7- membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl group.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group. The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro- lH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group -OCO2-.

The term “carboxy”, as used herein, refers to a group represented by the formula -CO2H.

The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R¹⁰⁰) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, denzodioxane, tetrahydroquinoline, and the like.

The term “ester”, as used herein, refers to a group -C(0)0R⁹ wherein R⁹ represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O- heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heteroaryl” and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocyclyl” and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the poly cycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group -OSChH, or a pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the group represented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl. The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

The term “sulfonate” is art-recognized and refers to the group SCbH, or a pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group -S(0)2-.

The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

The term “thioester”, as used herein, refers to a group -C(0)SR⁹ or -SC(0)R⁹ wherein R⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the general formula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as cell proliferation) as well as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base polypeptides represented by Formula I or Formula II. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of polypeptides of Formula I or II are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of polypeptides of Formula I or II for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid polypeptides represented by Formula I or II. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

Many of the polypeptides useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the polypeptides, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain polypeptides which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

Some of the polypeptides may also exist in tautomeric forms. Such forms, although not explicitly indicated in the formulae described herein, are intended to be included within the scope of the present disclosure.

The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

EXAMPLES

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1: Preparation and Characterization of C^HAA Derivatives

Methionine alkylation and demethylation methodology has been employed to add amino acids to the side-chains of polypeptides. The introduction of many structural and functional features, such as amide linkages, H-bonding, chiral centers, charges, and a variety of possible “R” groups (Scheme 1) produces polypeptides with new properties. The initial design was based on the epoxides 3a-c (Scheme 2), which were then incorporated into M₆₀ polypeptides via published processes. N-trifluoroacetyl-amino acids with varying hydrophobicity (i.e., L-phenylalanine (Phe), L-valine (Val), and glycine (Gly)) were coupled with allyloxyethylamine (1) to give carboxy-terminal linked amino amide derivatives (2a-c), which were then oxidized to give the amine-protected epoxides (overall yields ca.60-70%). Protection of amines was necessary to avoid their reaction with the epoxides. These initial epoxides were each reacted with M₆₀ as described above (ca.3 equivalents epoxide/methionine residue) to yield the desired C^HAA derivatives, 5a-c (Scheme 2).¹H and ¹³C NMR analysis showed that all methionine residues were fully converted to the corresponding functionalized products in all three samples.

Scheme 2. Synthesis of C^HAA polypeptides.5a-d were purified by dialysis against 3 mM HCl. a = marked stereocenter. Samples 5a-5d were purified by dialysis against aqueous HCl (3 mM) followed by deionized water to give the polypeptides as white powders after lyophilization. The resulting protonated polypeptides 5a-5d with Cl- counterions were all found to be soluble in deionized water at 20 °C. Since a-amine groups on peptides are known to have substantially lower p/C values compared to aliphatic amines, the degree of protonation for 5a-5d in 20 mM aqueous NaCl as a function of pH was examined. Measurement of Zeta potential for solutions of 5a-5d as a function of pH revealed transitions from positive to moderately negative values between pH 6 and 9, indicative of a change from protonated to non-protonated a-amine groups over this pH range (FIG. 3). This transition correlates well with the pAa range of a-amino amide groups ( ca . 7 to 8), and was corroborated by analysis of other control samples that included non-ionic poly(L-methionine sulfoxide)6o, pH invariant cationic poly(S-methyl-L-methionine sulfonium chloride)6o, and poly(L-lysine HCl)6o that undergoes a protonated to non-protonated transition at a much higher pH (pAa ca. 9.5 to 10) (FIG. 1). These results show that 5a-5d side-chains can undergo protonation and deprotonation within a physiologically pH range, which is advantageous for biologically relevant adjustment of physical properties ( vide infra ) in contrast to other amine containing polymers used in coacervation models that have much higher p/Cs ( e.g poly(L-lysine).

To determine how the degree of protonation of 5a-5d influences their chain conformations in water, each polypeptide was analyzed using circular dichroism (CD) spectroscopy over a range of pH in aqueous phosphate buffer (FIG. 4). At pH 5.5, where all chains were highly charged, CD spectra revealed that 5a-5d predominantly adopt a-helical conformations {ca. 70% a-helical content). As solution pH was increased, the a-helical contents of 5a-5c were all found to increase to ca. 85-90% as side-chain amine groups were deprotonated and chains were neutralized. The highly hydrophobic 5d was not soluble in phosphate buffer above pH 5.5 ( vide infra). The less hydrophobic samples, especially 5a and 5b, were soluble and a-helical up to pH 9.0, where the chains are expected to contain few charges. Overall, these CD data show that 5a-5d predominantly adopt a-helical conformations, which are stable over a biologically relevant range of pH in water.

The aqueous phase behavior of 5a-5d as functions of varying pH, temperature and counterions was evaluated. For the initial study, dilute aqueous solutions of 5a-5d (3.0 mg/mL) were prepared in 150 mM PBS buffer at pH values ranging from 6.5 to 8.0, which were chosen to include varying degrees of polypeptide protonation. These solutions were heated at a controlled rate up to 90 °C, and optical transmittance at 500 nm was measured as a function of temperature. The least hydrophobic sample 5a remained soluble up to 90 °C, while the more hydrophobic 5b-5d were found to phase separate in a pH and temperature dependent manner (FIGs. 5A-C). Reversible cloud points were observed for 5b-5d upon increase in temperature, indicative of lower critical solution temperature (LCST) induced phase separation, with cloud point temperatures (T_Cp) that decreased with increasing hydrophobicity of side-chain amino acids in the order Val < Leu < Phe. Cloud point temperatures for 5b-5d also decreased as pH was increased, which is consistent with other charged thermoresponsive polymers where T_Cp decreases substantially as charge is removed from the polymer chains. The results of this study show that 5b-5d in 150 mM PBS buffer can undergo phase separation from aqueous solution at physiologically relevant temperatures, ionic strength and solution pH, where T_Cp can be predictably tuned by variation of polypeptide side-chain amino acid hydrophobicity.

Having observed that the substitution of phosphate for chloride decreases the solubility of 5d in water (FIG. 4), investigated the effects of counterion valency on the phase separation of 5a-5d were further investigated. Aqueous samples of 5a-5d (3.0 mg/mL) were prepared in 150 mM NaCl at pH 7 and then mixed with sodium salts of anionic counterions with different valency (12 mM final concentration, Table 1). Note that the most hydrophobic sample, 5d, did not dissolve in water with any counterion at pH 7 and 20 °C. In general, as anion valency was increased, all samples became less soluble in water. This result is explained by stronger ion complexation between the cationic polypeptides and anions with higher valency. Even the more hydrophilic samples, 5a and 5b, were found to undergo phase separation in the presence of pyrophosphate or tripolyphosphate anions at 20 °C. Intermediate hydrophobicity sample 5c was the most interesting since its temperature dependent water solubility was found to vary with anion valency. While high valent tripolyphosphate caused 5c to phase separate at 20 °C, other anions were found to give cloud point temperatures that in general varied inversely with anion valency (FIG. 3D, Table 1). Hence, in addition to pH and side-chain amino acid hydrophobicity, counterions provide another means to fine tune phase transition temperature of these polypeptides.

Table 1. Coacervate transition temperatures for solutions of polypeptides 5a-5d containing different counterions. Cloud point temperatures (T_Cp) were measured for individual 3.0 mg/mL solutions of 5a-5d in the presence of different counterions (12 mM) in 150 mM NaCl at pH 7.0. sol = polypeptide was soluble up to 90 °C. — = polypeptide formed a coacervate at 20 °C. ppt = polypeptide formed a solid precipitate, not a coacervate, at 20 °C.

To investigate the nature of phase separation in 5a-5d samples described above, the suspensions formed in the presence of tripolyphosphate anions were allowed to settle onto glass slides and were examined using differential interference contrast (DIC) optical microscopy (FIG. 6). Images of the complex formed with least hydrophobic 5a showed irregular edges and clusters consistent with formation of a solid precipitate. However, images of tripolyphosphate complexes with 5b-5d all showed smooth outlines and coalescing droplets consistent with formation of liquid coacervate phases. For additional conformation of coacervate formation, centrifugation of the 5c-tripolyphosphate suspension resulted in the formation of two separate liquid layers. Examination of the phase separated complexes of 5d with different counterions, including chloride (Table 1), also showed coacervate formation in all cases. In addition to coacervation with simple counterions, 5c was also able to form a complex coacervate with polyadenylic acid at 20 °C, which is advantageous for potential downstream applications.

Although the images described above show 5b-5d form coacervates at 20 °C, the phase separation of 5c in PBS buffer at pH 8 was also imaged see if coacervation occurs at elevated temperature as well (FIG. 5). Differential interference contrast (DIC) microscopy of this sample at 45 °C confirmed the formation of coacervate droplets. Coacervate formation in this sample was also found to be highly reversible with no signs of irreversible polypeptide aggregation. Multiple heating and cooling cycles were applied to the mixture of 5c in PBS buffer at pH 8 where coacervation and dissolution with each thermal cycle were monitored by optical transmittance (FIG. 7A). Finally, for an initial study of salt stability, complexes of 5c with tripolyphosphate were prepared in aqueous solutions containing 0 to 500 mMNaCl. As ionic strength was increased, the fraction of 5c that partitioned into the coacervate phase decreased up to 250 mM NaCl, and no phase separation occurred at higher salt concentrations (FIG. 7B). These results are consistent with trends observed for many coacervate systems, and show that this 5c coacervate is stable at physiological ionic strength.

The 5b-5d coacervates are stable a-helices. Most coacervate forming proteins are highly disordered, and all other synthetic polypeptide-based coacervates rely on conformational disorder in at least one of the macromolecular components. The current dogma of polypeptide coacervation demands a conformationally disordered component to prevent solid precipitate formation. However, 5b-5d with chloride counterions, possess stable a-helical conformations in dilute aqueous solution over a range of pH (FIG. 5). To confirm that the polypeptides retain their a-helical conformations in coacervates, the polymer-rich coacervate phase was isolated from a mixture of 5c and tripolyphosphate in 150 mM NaCl at pH 7.0. The CD spectrum collected from a thin film of this coacervate showed the presence of the characteristic double minima consistent with a predominantly a-helical conformation (FIG. 8 A).

An additional feature of poly(S-alkyl-L-homocysteine) derivatives is that their stable a-helical conformations in water can be disrupted to disordered conformations upon oxidation of side-chain thioether groups to sulfoxides. Such oxidation can occur under mild conditions, with the intracellular oxidation of methionine residues in proteins being a well- known example. Also, ubiquitous reductase enzymes can readily reduce intracellular methionine sulfoxide residues in proteins; this can also be accomplished with synthetic poly(L-methionine sulfoxide) substrates in vitro. As such, reversible oxidation of side-chain thioether groups in 5a-5d was investigated as a means to actively control their coacervation, akin to how post-translational modifications are used to control protein coacervation within cells. A sample of 5c was oxidized to give the corresponding sulfoxide derivative 6c using tert- butyl hydroperoxide in water (Scheme 3). Polypeptide 6c was soluble in deionized water, and CD spectroscopy analysis showed that it possessed a highly disordered chain conformation as

Scheme 3. Synthesis of sulfoxide derivative 6c from 5c. TBHP = /er/-butyl hydroperoxide. CSA = camphorsulfonic acid.

The increased hydrophilicity of 6c relative to 5c had a striking effect on its temperature-dependent phase separation. When 6c was dissolved in 150 mM PBS buffer at pH 8, the polypeptide remained soluble as the sample was heated and only started to phase separate above 80 °C. This contrasts drastically with 5c, which phase separated at 28 °C under the same conditions (FIG. 9B). Hence, at moderate temperatures ( e.g 37 °C) coacervation can be actively controlled by interconversion of 5c and 6c. Such active control was demonstrated by addition of one equivalent of NaI04 oxidant at 0 °C to a coacervate suspension of 5c mixed with tripolyphosphate (FIG. 9C). Within 10 minutes, the coacervate droplets were observed to completely dissolve as 5c was oxidized to 6c. Note that coacervate dissolution occurred at ca. 73% oxidation of 5c residues, indicating that complete oxidation is not required to dissolve the coacervate phase. Reduction of 6c back to 5c was also accomplished using sodium thioglycolate, which confirmed the reversibility of this process. The 5c/6c system reported here is the first actively controlled polypeptide coacervate that utilizes non-destructive, biologically relevant chemical modification akin to post-translational modifications in proteins.

Disclosed herein are new, side-chain amino-acid functionalized homopolypeptides that reversibly form coacervate phases in aqueous media. The multifunctional nature of the side-chains provides means to actively control coacervation via mild, biomimetic redox chemistry, as well as respond to physiologically relevant environmental changes in pH, temperature and counterions. The modular design, uniform nature, and ordered chain conformation of these polypeptides provides a well-defined platform that can be used to tune coacervate properties and deconvolute molecular elements that affect coacervation, as exemplified here by variation of amino acid hydrophobicity. These homopolypeptides were found to possess properties that mimic many of those observed in natural coacervate forming proteins, including IDPs. Accordingly, these new polymers may be amenable for use as mimics of IDPs, as tunable models to help understand their properties, or in applications involving therapeutic delivery, protein separation, or as stimuli controlled microreactors.

Example 2: Preparation and Characterization of Further C^HAA Derivatives

Materials and Methods

Tetrahydrofuran (THF) and di chi orom ethane (DCM) were each degassed with dinitrogen and passed through an alumina column before use. Unless otherwise specified, all post-polymerization modification chemistry was performed in glass vials under ambient atmosphere. Co(PMe3)4, L-methionine N-carboxyanhydride (Met NCA), L-homomethionine (Hmt) NCA, and 6-(methylthio)-L-norleucine (Mtn) NCA monomers were prepared according to the literature procedures. Poly(L-methionine)₆o (Mbo) , poly(L- homomethionine)₆o (Hmt₆o), and poly(6-(methylthio)-L-norleucine)₆o (Mtn₆o) were prepared according to literature procedures. Reactions of Mbo, Hthίbo, and Mtn₆o with epoxides, 5b, 11a, lib and 13a-d were performed in scintillation vials under ambient atmosphere and temperature, unless otherwise specified. Small molecule chemistry was performed in heat- dried glassware under a nitrogen atmosphere, unless otherwise specified. All other reagents and solvents were used as received. In-house deionized water was used for all aqueous chemistry and dialysis unless otherwise specified. Thin-layer chromatography was performed with EMD gel 60 F254 plates (0.25 mm thickness) and spots were visualized using a UV lamp or KMnCri stain. Silicycle Siliaflash G60 silica (60-200 pm) was used for all column chromatography. Silica used for chromatographic purification of NCA monomers was dried under vacuum at 250 °C for 48 h and then stored in a dinitrogen-filled glovebox. Compositions of mobile phases used for chromatography are given in volume percentages. Dialysis was performed with regenerated cellulose tubing obtained from Spectrum laboratories with 2000 Da molecular weight cutoff. NMR spectra of solution samples were recorded on Bruker AV400, Bruker AV300 and Bruker AV500 instruments with chemical shifts reported relative to the deuterated solvent used. Samples for circular dichroism (CD) spectroscopy were prepared using deionized water. CD spectra were collected between 0.1 and 0.83 mg/mL solutions of polypeptide on a JASCO J-715 or Chirascan VIOO spectrophotometer using a 0.1 cm path length quartz cuvette. The % a-helical content of polypeptides was estimated using the formula: % a-helix = 100 ^c (— [q]222 + 3000)/39000), where [0]222 is the measured molar ellipticity at 222 nm in (deg x cm² /dmol).⁴⁷ Cloud point temperature measurements were recorded at a wavelength of 500 nm on an HP 8453 spectrophotometer equipped with an Agilent 8909 A temperature control. Differential interface contrast (DIC) images were taken using a Zeiss Axiovert 200 DlC/fluorescence inverted optical microscope.

Coacervate preparation

Stock solutions of 8a, 8b, 13a and 13b (all with chloride counterions) and 18a-d (all with ammonium counterions) were prepared at 10 mg/mL in DI water, and separate stock solutions of 18c and 18d were prepared in Milli-Q water at 10 mg/mL containing 300 mM NaCl for mixing with lysozyme. Stock solutions of 5c and 6c were prepared at 10 mg/mL DI water containing 300 mM NaCl for mixing with 18c, and stock solutions of chloride salts of different counterions (26 mM) were prepared in DI water containing NaCl (300 mM). A stock solution of 2x PBS buffer (300 mM) was also prepared, as well as stock solutions of lysozyme prepared at 5, 10 and 20 mg/mL Milli-Q water. A stock solution of 5c, 6c, counterions, PBS or lysozyme was then mixed with a stock solution of polypeptide in a 1 : 1 (v/v) ratio.

Circular dichroism spectroscopy

Samples of 8a, 8b, 13a, 13b, and 18a-d, were prepared at concentrations between 0.5 - 0.83 mg/mL in phosphate buffer (12-100 mM). Average degree of polymerization for all polypeptides was 60. The buffers were adjusted to pH of 5.0, 7.0 or 9.0 using HC1 (0.1 M) or NaOH (0.1 M) before preparing the polymer solutions, and then the final pH of each solution was confirmed using a pH meter. The data were reported in units of molar ellipticity [Q] (deg-cnri-dmoT¹), which was calculated using [Q] = (Q x 100 x Mw)/(c x I) where Q is the measured ellipticity (millidegrees), Mw is the average residue molecular mass (g/mol), c is the polypeptide concentration (mg/mL), and / is the cuvette path length (cm). Percent a- helical content was calculated using % helicity = ((-[Q222] + 3000)/39000) x 100 where [Q222] is the molar ellipticity at 222 nm.⁴⁷

Cloud point temperature measurements

Samples of 13a and 13b were prepared at 2.5 mg/mL in DI water lx PBS buffer (150 mM). Separate stock solutions of polypeptide (5.0 mg/mL) in DI water and lOx PBS (1500 mM) at pH 5.0, 7.0 and 9.0 were used in sample preparation. Samples were prepared by mixing a polypeptide sample solution and pH 7.0 stock solutions of a sodium counterion and NaCl in a 1:0.2:0.15 (v/v/v) ratio, then diluting with DI water to achieve a final polypeptide concentration of 2.5 mg/mL. The samples in PBS buffer were prepared by mixing the polypeptide sample solution and lOx pH 7.0 PBS buffer in a 1 :0.2 (v/v) ratio, and the mixture was then diluted with DI water to obtain a final polypeptide concentration of 2.5 mg/mL. The final pH of these samples was adjusted as desired by addition of small volumes of the same sample prepared using either pH 5.0 or 9.0 stock solutions. Transmittance of each sample at 500 nm was measured on a HP 8453 spectrophotometer equipped with an Agilent 8909A temperature control. In initial runs, temperature was increased at a rate of 10 °C/min from 20 °C to 90 °C, followed by additional experiments within a selected temperature range at a heating rate of 1 °C/min. Cloud point temperatures (T_cp) were determined as the temperature at 50% transmission.

General procedure for synthesis of N-TFA-amino acid (2/3/4-allyloxy)alkyl amides

A round bottom flask was charged with a stirbar, the desired TFA-amino acid (1 eq, TFA-AA), THF (to give 0.38 M solution), and hydroxybenzotriazole (1 eq, HOBt). The mixture was cooled to 0 °C in an ice bath; then a 3.6 M solution of N,N'- dicyclohexylcarbodiimide (DCC) (1.2 eq) in THF was added dropwise to the stirred mixture. The mixture was let stir on ice for 30 minutes, after which a 3.3 M solution of the desired (allyloxy)alkyl ammonium trifluoroacetate (1.1 eq) in THF was added to the mixture dropwise, followed by dropwise addition of TEA (2 eq). The mixture was let stir in the ice bath, which was allowed to warm to ambient temperature overnight. The next day, the resulting suspension in THF was filtered into a round bottom flask and the THF was removed in vacuo. The residue was dissolved in EtOAc to give a 0.30 M solution with respect to the starting quantity of TFA-AA. This solution was left in a freezer overnight, and any further precipitate was removed by filtration. The resulting solution was purified using flash chromatography at a solvent ratio of 2: 1 hexanes (hex) to EtOAc to afford the product as a white solid.

General procedure for synthesis of N-TFA-amino acid (2/3/4-glvcidyloxy)alkyl amides

The desired TFA-amino acid (allyloxy)alkyl amide (1 eq) was dissolved in DCM (3.3 mL/mmol alkene). Commercial 70 % mCPBA (1.5 eq) was then added and the mixture was stirred. After full conversion of alkene was confirmed by TLC (24 - 36 h), the suspension was cooled in an ice bath. The mixture was treated with 10% Na2SCb (aq) (1.5 eq) followed by 10% Na2CCb (aq) (1.3 eq), and stirred for 5 min. The reaction mixture was diluted with EtOAc and washed 2x with sat. NaHCCb (aq) followed by brine. The organic layer was dried over Na2S04, concentrated in vacuo and purified by flash chromatography with a solvent ratio of 1 : 1 hex to EtOAc to afford the product as a white solid.

General procedure for synthesis of N-(allyloxyacetvD-amino acid methyl esters

A round bottom flask was charged with a stirbar, 2-(allyloxy)acetic acid (1 eq, allyl- acid), and dichloromethane (DCM) to give a final concentration of 0.83 M. To this mixture was added hydroxybenotriazle (1.1 eq, HOBt) followed by l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (1.1 eq, EDC) added as slowly as possible, and the mixture was permitted to stir for 30 minutes at ambient temperature. In a separate flask, the desired amino acid methyl ester (1.5 eq, MeO-AA) was mixed with DCM to give a MeO-AA concentration of 0.62 M, and triethylamine (2 eq with respect to allyl-acid, TEA). The resulting slurry was added dropwise to the reaction flask containing the allyl-acid, HOBt and EDC. The mixture was let stir overnight before being quenched with sat. MECl, and was then extracted 3x with DCM, lx NaHC03, and lx brine. The organic layer was dried over Na2S04, concentrated in vacuo , and purified by flash chromatography with a hexanes (hex) and ethyl acetate (EtOAc) solvent system to afford the product as an oil.

General procedure for synthesis of N-(glvcidyloxyacetvD-amino acid methyl esters

The desired N-(allyloxyacetyl)-amino acid methyl ester (1 eq) was dissolved in DCM (3.3 mL/mmol alkene). Commercial 70 % mCPBA (1.5 eq) was then added, and the mixture was stirred. After full conversion of alkene was confirmed by TLC (24 - 36 h), the suspension was cooled in an ice bath. The mixture was treated with 10% Na2S03(aq) (1.5 eq) followed by 10% Na2C0₃ (aq) (1.3 eq) and stirred for 5 min. The reaction mixture was then diluted with EtOAc and washed 2x with sat. NaHC0₃ (aq) followed by brine. The organic layer was dried over Na2S04, concentrated in vacuo , and purified by flash chromatography with a hex and EtOAc solvent system to afford the product as an oil.

N-TFA-L-leucine (S-allyloxy)propyl amide, 10a

Prepared from TFA-Leu and 9a using the General procedure for synthesis ofN-TFA- amino acid (2/3/4-allyloxy)alkyl amides. Yield is given in Table 4. 'H NMR (300 MHz, Chloroform -7) d 7.12 (br d, J= 8.0 Hz, 1H), 6.57 (br s, 1H), 5.91 (ddt, J= 17.2, 10.4, 5.7 Hz, 1H), 5.32 - 5.19 (m, 2H), 4.44 - 4.35 (m, 1H), 4.00 - 3.96 (m, 2H), 3.57 (t, J= 5.6 Hz, 2H), 3.46 - 3.38 (m, 2H), 1.81 (p, 7= 6.1 Hz, 2H), 1.71 - 1.56 (m, 3H), 0.95 (d, J= 5.0 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-7) d 170.32, 157.06 (q, J= 37.5 Hz), 134.51, 117.46, 115.88 (q, J= 287.5 Hz), 72.19, 69.39, 52.38, 41.84, 38.73, 28.81, 24.85, 22.79, 22.34.

N-TFA-L-leucine (4-allyloxy)butyl amide, 10b

Prepared from TFA-Leu and 9b using the General procedure for synthesis ofN-TFA- amino acid (2/3/4-allyloxy)alkyl amides. Yield is given in Table 4. 'H NMR (300 MHz, Chloroform -7) d 7.00 (br d, J= 7.3 Hz, 1H), 6.22 (br s, 1H), 5.92 (ddt, 7= 17.2, 10.4, 5.7 Hz, 1H), 5.32 - 5.18 (m, 2H), 4.45 - 4.36 (m, 1H), 3.99 (dt, J= 5.6, 1.3 Hz, 2H), 3.47 (t, J= 5.7 Hz, 2H), 3.34 - 3.27 (m, 2H), 1.64 (m, 7H), 0.96 (dd, J= 6.3, 1.9 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-7) d 170.68, 157.26 (q, J= 37.6 Hz), 134.79, 117.16, 115.88 (q, J= 287.4 Hz), 71.98, 69.87, 52.49, 41.54, 39.61, 27.17, 26.28, 24.84, 22.76, 22.33.

N-TFA-L-leucine (3-glycidyloxy)propyl amide, 11a

Prepared from 10a using the General procedure for synthesis of N-TFA-amino acid (2/3/4-glycidyloxy)alkyl amides. Yield is given in Table 4. 'H NMR (300 MHz, Chloroform- d) d 7.20 - 7.11 (br s, 1H), 6.67 (br d, J= 18.0 Hz, 1H), 4.53 - 4.44 (m, 1H), 3.84 (dd,7= 11.3, 2.2 Hz, 1H), 3.72 - 3.55 (m, 2H), 3.53 - 3.31 (m, 3H), 3.22 - 3.16 (m, 1H), 2.85 (ddd, J= 6.0, 4.9, 4.2 Hz, 1H), 2.69 (ddd, J= 11.6, 4.9, 2.7 Hz, 1H), 1.79 (m, 2H), 1.73 - 1.57 (m, 3H), 0.96 (d, J = 6.2 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-J) d 170.46, 157.04 (q, J= 37.2 Hz), 115.91 (q, J= 287.7 Hz), 71.04, 70.93, 52.49, 51.03, 44.52, 41.82, 38.78, 28.50, 24.91, 22.92, 22.21. DART-MS m/z = 341.16994 [m+H]⁺ (calculated 340.16099 for C14H23F3N2O4)

N-TFA-L-leucine (4-glycidyloxy)butyl amide, lib

Prepared from 10b using the General procedure for synthesis of N-TFA-amino acid (2/3/4-glycidyloxy)alkyl amides. Yield is given in Table 4. 'H NMR (300 MHz, Chloroform- d) d 7.06 (br s, 1H), 6.31 (br s, 1H), 4.51 - 4.42 (m, 1H), 3.81 (ddd, J= 11.5, 2.4, 1.3 Hz, 1H), 3.53 (m, 2H), 3.39 - 3.27 (m, 3H), 3.18 (m, 1H), 2.84 (dd, J= 4.9, 4.2 Hz, 1H), 2.67 (ddd, J = 10.4, 5.0, 2.7 Hz, 1H), 1.70 - 1.56 (m, 7H), 0.96 (d, J = 6.0 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-J) d 170.59, 157.10 (q, J= 37.5 Hz), 115.89 (q, 7= 287.6 Hz), 71.36, 71.10, 52.41, 51.06, 44.40, 41.76, 39.62, 26.99, 26.33, 24.87, 22.89, 22.30. DART-MS m/z = 355.18557 [m+H]⁺ (calculated 354.17664 for C15H25F3N2O4)

2-(allyloxy)acetic acid, 14

A round bottom flask was charged with a magnetic stirbar and ethyl 2-(allyloxy)acetate (1 lg, 77 mmol). The material was dissolved in 500 mL of MeOH then cooled to 0 °C in an ice bath before adding 250 mL of 1 M NaOH. The reaction was let stir overnight and permitted to come to room temperature. The next day the MeOH was removed by rotary evaporation until only the H2O remained in the flask. This solution was diluted with DI H2O to ~2x the volume and then the solution was washed 5x with 150 mL Et20. The aqueous solution was then adjusted to a pH of 1.0 using 3 M HC1 and then the product was extracted 5x with 150 mL Et20. The organic layers were combined and dried with Na2S04, and then the solvent was removed by rotary evaporation to afford 6.1 g (68% yield) of crude product as a clear oil, which was used directly for subsequent reactions.

N-(allyloxyacetyl)-L-alanine methyl ester , 15a

Prepared from MeO-Ala using the General procedure for synthesis of N- (allyloxyacetyl) -amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroforme d 7.05 (br d, J= 7.3 Hz, 1H), 5.88 (ddt, J= 17.2, 10.4, 5.7 Hz, 1H), 5.33 - 5.20 (m, 2H), 4.61 (p, J= 7.2 Hz, 1H), 4.04 (dt, J= 5.7, 2.6 Hz, 2H), 3.92 (d, J= 3.1 Hz, 2H), 3.72 (s, 2H), 1.40 (d, J= 7.2 Hz, 3H). ¹³C NMR (126 MHz, Chloroforme d 173.17, 169.33, 133.47,

118.34, 72.42, 69.23, 52.53, 47.42, 18.50.

N-(allyloxyacetyl)-L-valine methyl ester, 15b

Prepared from MeO-Val using the General procedure for synthesis of N- (allyloxyacetyl) -amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroforme d 6.98 (br d, J= 8.6 Hz, 1H), 5.86 (ddt, J= 17.2, 10.4, 5.6 Hz, 1H), 5.31 - 5.17 (m, 2H), 4.52 (dd, J= 9.2, 5.0 Hz, 1H), 4.02 (ddt, J= 5.6, 2.8, 1.4 Hz, 2H), 3.92 (d, J= 6.0 Hz, 2H), 3.68 (s, 3H), 2.14 (dtd, J= 13.7, 6.9, 5.0 Hz, 1H), 0.88 (dd, J= 10.7, 6.9 Hz, 6H). ¹³C NMR (126 MHz, Chloroforme d 172.09, 169.65, 133.44, 118.10, 72.33, 69.21, 56.41, 52.13, 31.22, 18.99, 17.73.

N-(allyloxyacetyl)-L-leucine methyl ester, 15c

Prepared from MeO-Leu using the General procedure for synthesis of N- (allyloxyacetyl) -amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroforme d 6.86 (br d, 7= 8.4 Hz, 1H), 5.86 (ddt, J= 17.2, 10.4, 5.7 Hz, 1H), 5.31 - 5.17 (m, 2H), 4.63 (td, J= 8.8, 5.3 Hz, 1H), 4.04 - 3.99 (ddt, J= 6.0, 3.3, 1.4 Hz, 2H), 3.91 (d, J = 3.3 Hz, 2H), 3.68 (s, 3H), 1.67 - 1.49 (m, 3H), 0.89 (d, J= 5.9 Hz, 6H). ¹³C NMR (126 MHz, Chloroform -J) d 173.13, 169.53, 133.45, 118.24, 72.38, 69.19, 52.29, 50.02, 41.55, 24.87,

22.83, 21.89.

N-(allyloxyacetyl)-L-phenylalanine methyl ester, 15d

Prepared from MeO-Phe using the General procedure for synthesis of N- (allyloxyacetyl) -amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroform -J) d 7.33 - 7.08 (m, 5H), 6.98 (br d, J= 7.7 Hz, 1H), 5.81 (ddt, J= 17.2, 10.4, 5.6 Hz, 1H), 5.27 - 5.17 (m, 2H), 4.90 (dt, J= 8.3, 6.1 Hz, 1H), 3.96 (ddt, J= 5.7, 2.3, 1.4 Hz, 2H), 3.92 (s, 3H), 3.71 (s, 3H), 3.20 - 3.06 (m, 2H). ¹³C NMR (126 MHz, Chloroform-J) d 171.76, 169.55, 135.81, 133.42, 129.28, 128.70, 127.26, 118.11, 72.33, 69.22, 52.54, 38.06.

N-(glycidyloxyacetyl)-L-alanine methyl ester, 16a

Prepared from 15a using the General procedure for synthesis of N-(glycidyloxyacetyl)- amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroform-r/) d 7.16 (br s, 1H), 4.62 (p, J= 7.3 Hz, 1H), 4.11 - 3.94 (m, 2H), 3.88 (m, 1H), 3.75 (s, 3H), 3.48 (m, 1H), 3.24 - 3.14 (m, 1H), 2.84 (m, 1H), 2.68 (m, 1H), 1.44 (d, J= 7.2 Hz, 3H). ¹³C NMR (126 MHz, Chloroform -J) d 173.24, 169.14, 71.99, 70.51, 52.60, 50.53, 47.54, 44.22, 18.38. DART-MS m/z = 218.10283 [m+H]⁺ (calculated 217.09502 for C9H15NO5)

N-(glycidyloxyacetyl)-L-valine methyl ester, 16b

Prepared from 15b using the General Procedure for synthesis of N-(glycidyloxyacetyl)- amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroform-r/) d 7.14 (br s, 1H), 4.61 (dd, J= 9.1, 5.1, Hz, 1H), 4.19 - 3.99 (m, 2H), 3.93 (m, 1H), 3.78 (s, 3H), 3.55 (m, 1H), 3.25 (m, 1H), 2.89 (m, 1H), 2.74 (m, 1H), 2.25 (m, 1H), 0.99 (td, J= 6.8, 1.5 Hz, 6H). ¹³C NMR (126 MHz, Chloroform-J) d 172.25, 169.55, 72.09, 70.59, 56.70, 52.29, 50.46, 44.33, 31.24, 19.16, 17.92. DART-MS m/z = 246.13466 [m+H]⁺ (calculated 245.12632 for C11H19NO5)

N-(glycidyloxyacetyl)-L-leucine methyl ester, 16c

Prepared from 15c using the General Procedure for synthesis of N-(glycidyloxyacetyl)- amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroform-r/) d 6.98 (br s, 1H), 4.67 - 4.49 (m, 1H), 4.05 - 3.88 (m, 2H), 3.83 (m, 1H), 3.66 (s, 3H), 3.43 (m, 1H), 3.14 (m, 1H), 2.77 (m, 1H), 2.63 (m, 1H), 1.65 - 1.50 (m, 3H), 0.88 (dd, J= 4.4, 6H). ¹³C NMR (126 MHz, Chloroform-J) d 173.10, 169.31, 71.77, 70.32, 52.23, 50.39, 50.08, 44.02, 41.22, 24.82, 22.79, 21.75. DART-MS m/z = 260.15043 [m+H]⁺ (calculated 259.14197 for C12H21NO5)

N-(glycidyloxyacetyl)-L-phenylalanine methyl ester, 16d

Prepared from 15d using the General Procedure for synthesis of N-(glycidyloxyacetyl)- amino acid methyl esters. Yield is given in Table 3.6. 'H NMR (300 MHz, Chloroform-r/) d 7.35 - 7.11 (m, 5H), 7.07 (br s, 1H), 4.89 (m, 1H), 4.07 - 3.90 (m, 2H), 3.85 - 3.65 (m, 1H), 3.72 (s, 3H), 3.41 (m, 1H), 3.24 - 3.05 (m, 3H), 2.82 - 2.56 (m, 2H). DART-MS m/z = 294.13487 [m+H]⁺ (calculated 293.12632 for C15H19NO5)

General procedure for alkylation of Mόo

Acidic dialysis conditions: The sample was dialyzed against 3 mM HCl(aq) (24 h, 3 changes) followed by DI water (24 hrs, 3 changes). The retentate was lyophilized to provide the amino acid functionalized polypeptides 7a, 7b, 12a and 12b.

Neutral dialysis conditions: The sample was dialyzed against 6 mM NaCl (24 h, 3 changes) followed by DI water (24 hrs, 3 changes). The retentate was lyophilized to provide the amino acid functionalized polypeptides 17a-d.

General procedure for demethylation and deprotection of alkylated M60 sulfonium salts

Acidic dialysis conditions: The sample was dialyzed against 50% MeOH(aq) containing 3 mM HC1 (24h, 3 changes), followed by 3 mM HC1 (24h, 3 changes) and then DI H2O (12h, 3 changes). The retentate was lyophilized to provide the amino acid functionalized polypeptides 8a, 18b, 13a and 13b.

Basic dialysis conditions: The sample was dialyzed against 50% MeOH(aq) containing 3 mM NH4OH (24h, 3 changes), followed by 3 mM HC1 (24h, 3 changes) and then DI H2O (12h, 3 changes). The retentate was lyophilized to provide the amino acid functionalized polypeptides 18a-d.

For polypeptides 8a, 8b, 13a and 13b, additional processing was performed to enhance their dissolution in H2O. A minimal amount of TFA was added to lyophilized solids of 8a, 8b, 13a, and 13b to obtain complete polypeptide dissolution. The solutions were then diluted by addition of 10 volumes of DI H2O and were then transferred to 2000 Da MWCO dialysis bags, after which they were dialyzed against 1 M NaCl containing 3 mM HC1 (24h, 1 change), followed by 3 mM HC1 (24h, 2 changes), and then DI H2O (24h, 3 changes). ¹⁹F NMR was used to confirm complete removal of TFA counterions.

Poly(S-(3-(2-(N-trifluoroacetyl-L-valine amido)ethoxy)-2-hydroxypropyl)-L-homomethionine sulfonium chloride), 7a

Prepared from Hmt₆o using the General procedure for alkylation of Mr, o and purified using Acidic dialysis conditions. Yield is given in Table 3.1. 'H NMR (400 MHz, TFA-r/) d 4.84 (br s, 1H), 4.63 (br s, 1H), 4.42 (d, J= 8.2 Hz, 1H), 4.00 - 3.37 (br m, 10H), 3.15 (br m, 3H), 2.47 - 2.16 (br m, 5H), 1.22 - 1.02 (m, 6H).

Poly(S-(3-(2-(N-trifluoroacetyl-L-valine amido)ethoxy)-2-hydroxypropyl)-6-(methylthio)-L- norleucine sulfonium chloride), 7b

Prepared from Mtn₆o using the General procedure for alkylation of Mbo and purified using Acidic dialysis conditions. Yield is given in Table 3.1. 'H NMR (400 MHz, TFA-r/) d 4.75 (br s, 1H), 4.61 (br s, 1H), 4.42 (d, J= 12.2 Hz, 1H), 4.01 - 3.32 (br m, 10H), 3.11 (m, 3H), 2.29 (m, 1H), 2.15 - 1.63 (br m, 6H), 1.13 (dd, J= 6.6, 3.4 Hz, 6H).

Poly(S-(3-(2-L-valine amido)ethoxy)-2-hydroxypropyl)-5-(thia)-L-norvaline hydrochloride), 8a

Prepared from 7a using the General procedure for demethylation and deprotection of alkylated Mbo sulfonium salts and purified using Acidic dialysis conditions. Yield is given in Table 3.1. ¾ NMR (400 MHz, TFA-J) d 4.74 (br s, 1H), 4.25 (br s, 2H), 4.03 - 3.58 (br m, 6H), 2.85 (br s, 2H), 2.74 (br s, 2H), 2.42 (br s, 1H), 2.24 - 1.65 (br m, 4H), 1.22 (d, J= 6.6 Hz, 6H).

Poly(S-(3-(2-L-valine amido)ethoxy)-2-hydroxypropyl)- 6-(thia)-L-nor leucine hydrochloride), 8b

Prepared from 7b using the General procedure for demethylation and deprotection of alkylated Mbo sulfonium salts and purified using Acidic dialysis conditions. Yield is given in Table 3.1. ¾ NMR (400 MHz, TFA-J) d 4.71 (br s, 1H), 4.28 (bs s, 2H), 4.09 - 3.57 (br m, 6H), 2.87 (br s, 2H), 2.71 (br s, 2H), 2.43 (br s, 1H), 1.96 - 1.61 (br m, 6H), 1.23 (d, J= 6.6 Hz, 6H).

Poly(S-( 3-( 3-(N-trifluoroacetyl-L-leucine amido)propoxy)-2-hydroxypropyl)-L-methionine sulfonium chloride), 12a

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Acidic dialysis conditions. Yield is given in Table 4. 'H NMR (400 MHz, TFA-r/) d 5.06 (br s, 1H), 4.81 (s, 1H), 4.69 (br s, 1H), 3.94 - 3.53 (br m, 10H), 3.28 - 3.16 (m, 3H), 2.72 (br d, J = 93.4 Hz, 2H), 2.04 (s, 2H), 1.96 - 1.72 (m, 3H), 1.07 (dd, J= 12.1, 6.0 Hz, 6H).

Poly(S-(3-(4-(N-trifluoroacetyl-L-leucine amido)butoxy)-2-hydroxypropyl)-L-methionine sulfonium chloride), 12b

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Acidic dialysis conditions. Yield is given in Table 4. 'H NMR (400 MHz, TFA-r/) d 5.07 (br s, 1H), 4.84 (s, 1H), 4.71 (br s, 1H), 3.84 (br m, 8H), 3.52 (s, 2H), 3.25 (s, 3H), 2.72 (br d, J = 90.7 Hz, 2H), 1.81 (m, 7H), 1.08 (dd, J = 10.5, 4.7 Hz, 6H).

Poly(S-(3-(3-L-leucine amido)propoxy)-2-hydroxypropyl)-L-homocysteine hydrochloride), 13a Prepared from 12a using the General procedure for demethylation and deprotection of alkylated M₆₀ sulfonium salts and purified using Acidic dialysis conditions. Yield is given in Table 4. ¹H NMR (400 MHz, TFA-d) δ 4.95 (br s, 1H), 4.43 (t, J = 7.1 Hz, 1H), 4.35 (br s, 1H), 3.97 – 3.76 (br m, 4H), 3.63 (br s, 2H), 3.02 – 2.71 (br m, 4H), 2.36 – 2.17 (br m, 2H), 2.13 – 2.03 (br m, 2H), 2.03 – 1.80 (m, 3H), 1.19 – 1.03 (m, 6H).

Poly(S-(3-(4-L-leucine amido)butoxy)-2-hydroxypropyl)-L-homocysteine hydrochloride), 13b Prepared from 12b using the General procedure for demethylation and deprotection of alkylated M₆₀ sulfonium salts and purified using Acidic dialysis conditions. Yield is given in Table 4. ¹H NMR (400 MHz, TFA-d) δ 4.94 (br s, 1H), 4.42 (t, J = 7.2 Hz, 1H), 4.35 (br s, 1H), 3.97 - 3.79 (br m, 4H), 3.60 - 3.45 (br m, 2H), 2.96 - 2.77 (br m, 4H), 2.25 (s, 2H), 2.01 - 1.73 (m, 7H), 1.10 (t, 7= 6.0 Hz, 6H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-alanine methyl ester)-L-homocysteine hydrochloride), 17a

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Neutral dialysis conditions. Yield is given in Table 3.6. ¹HNMR (500 MHz, TFA-r/) d 4.92 (br s, 1H), 4.74 (p, J= 12 Hz, 1H), 4.56 (br s, 1H), 4.34 - 4.23 (m, 2H), 3.85 (s, 3H), 3.83 - 3.59 (br m, 6H), 3.17 - 3.06 (br m, 3H), 2.57 (br d, J= 106.9 Hz, 2H), 1.50 (d, J= 7.2 Hz, 3H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-valine methyl ester)-L-methionine sulfonium chloride, 17b.

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Neutral dialysis conditions. Yield is given in Table 3.6. ¹HNMR (500 MHz, TFA-r/) d 4.95 (br s, 1H), 4.69 - 4.63 (m, 1H), 4.60 (br s, 1H), 4.38 - 4.26 (m, 2H), 3.88 (s, 3H), 3.86 - 3.64 (br m, 6H), 3.19 - 3.08 (br m, 3H), 2.60 (br d, J= 110.4 Hz, 2H), 2.29 - 2.22 (br m, 1H), 0.99 (t, J= 7.0 Hz, 6H).

Poly(S-( N-((2-hydroxypropoxy)acetyl)-L-leucine methyl ester )-L-methionine sulfonium chloride, 17c.

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Neutral dialysis conditions. Yield is given in Table 3.6. ¹HNMR (500 MHz, TFA-r/) d 4.93 (br s, 1H), 4.83 - 4.75 (m, 1H), 4.57 (br s, 1H), 4.35 - 4.22 (m, 2H), 3.85 (s, 3H), 3.83 - 3.59 (br m, 6H), 3.16 - 3.07 (br m, 3H), 2.58 (br d, J= 111.6 Hz, 2H), 1.78 - 1.59 (m, 3H), 0.93 (dd, J = 9.4, 6.6 Hz, 6H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-alanine methyl ester)-L-methionine sulfonium chloride, 17 d

Prepared from Mbo using the General procedure for alkylation ofMeo and purified using Neutral dialysis conditions. Yield is given in Table 3.6. 'H NMR (500 MHz, TFA-r/) d 7.32 - 7.07 (m, 5H), 5.06 (s, 1H), 4.95 (br s, 1H), 4.64 - 4.43 (br m, 1H), 4.20 (s, 2H), 3.86 (s, 3H), 3.80 - 3.55 (br m, 6H), 3.23 - 2.99 (m, 5H), 2.60 (br d, J= 112.4 Hz, 2H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-alanine anion)-L-homocysteine ammonium salt, 18a Prepared from 17a using the General procedure for demethylation and deprotection of alkylated Mbo sulfonium salts and purified using Basic dialysis conditions. Yield is given in Table 3.6. ¾ NMR (300 MHz, TFA-J) d 4.77 (br s, 2H), 4.26 (br s, 2H), 3.97 - 3.61 (br m, 3H), 2.93 - 2.62 (br m, 4H), 2.20 - 2.04 (br m, 2H), 1.56 (s, 3H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-valine anion)-L-homocysteine ammonium salt, 18b.

Prepared from 17b using the General procedure for demethylation and deprotection of alkylated Mr,o sulfonium salts and purified using Basic dialysis conditions. Yield is given in Table 3.6. ¾ NMR (300 MHz, TFA-J) d 4.82 (br s, 1H), 4.68 (br s, 1H), 4.36 - 4.20 (br m, 3H), 3.91 - 3.70 (br m, 2H), 2.94 - 2.61 (br m, 4H), 2.40 - 2.05 (br m, 3H), 1.03 (s, 6H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-leucine anion)-L-homocysteine ammonium salt, 18c

Prepared from 17c using the General procedure for demethylation and deprotection of alkylated Mr,o sulfonium salts and purified using Basic dialysis conditions. Yield is given in Table 3.6. ¾ NMR (300 MHz, TFA-J) d 4.78 (br s, 1H), 4.35 - 4.14 (br m, 2H), 3.94 - 3.65 (br m, 3H), 2.95 - 2.59 (br m, 4H), 2.10 (br s, 2H), 1.83 - 1.66 (br m, 3H), 0.94 (s, 6H).

Poly(S-(N-((2-hydroxypropoxy)acetyl)-L-phenylalanine anion)-L-homocysteine ammonium salt, 18d

Prepared from 17d using the General procedure for demethylation and deprotection of alkylated Mr,o sulfonium salts and purified using Basic dialysis conditions. Yield is given in Table 3.6.¾NMR (300 MHz, TFA-J) d 7.36 - 7.08 (m, 5H), 5.03 (br s, 1H), 4.81 (br s, 1H), 4.25 - 3.53 (br m, 5H), 3.43 - 3.12 (br m, 2H), 2.70 (br s, 4H), 2.10 (br s, 2H).

Results and Discussion

A family of poly(S-alkyl-L-homocysteine) derivatives bearing cationic charges at their periphery underwent liquid-liquid phase separation (LLPS) when exposed to different stimuli. These polymers were made by reacting poly(L-methionine) (Mbo) chains with epoxide alkylating agents that contained different amino acids, allowing polymer properties to be tuned by varying the amino acid side-chains (FIG. 10A). The resulting samples were found to have stimuli-responsive behavior dependent on amino acid side-chain and counterion identity. While this initial study resulted in useful biomimetic coacervation with well-defined homopolypeptides (FIG. 10 A), it was not clear if coacervation was dependent on the side-chain lengths or cationic charges used in these samples. An important advantage of this system is its modular nature, which enables modification of a common Mbo precursor with a range of different side-chain functionalities while still retaining a common backbone structure. Furthermore, coacervates-forming behavior of analogous anionic polypeptides was also explored, since these would allow polyelectrolyte complexation with cationic polyelectrolytes and the bioencapsulation of a wider range of cargos.

Poly(S-alkyl-L-homocysteine)s with increased distance of side-chain ammonium groups from the peptide backbone

To prepare samples where the distance of the side-chain ammonium groups from the polypeptide backbone was varied we explored two different strategies: (1) increasing the number of carbons between side-chain thioether groups and the polypeptide backbone using Mr, (I homologue (Scheme 4); and (2) increasing the number of carbons in the alkyl linker between epoxide groups and side-chain amino acids (Scheme 5). By using two different methods to increase the distance of the side-chain ammonium groups from the polypeptide backbone, we hoped to better understand the roles played by side-chain molecular components.

Previously, it was found that the side-chain lengths of oxidized Mbo homologs were found to influence the secondary structure adopted by these polypeptides in aqueous solutions, where increased distance of thioether groups from the backbone was found to increase stability of a-helical conformations. Using these results as inspiration for the studies here, Mbo precursor polymer in the synthetic scheme was replaced with the homologous polypeptides poly(L- homomethionine) (Hmt₆o) and poly(6-(methylthio)-L-norleucine) (Mtn₆o) (Scheme 4). In an effort to ensure solubility of the final polypeptides when using more hydrophobic precursors, the less hydrophobic epoxide 3b was used to functionalize Hmt₆o and Mtn₆o , thus creating polypeptides 8a and 8b which were homologous to cationic polypeptide 5b (Scheme 4). The homologs Hmt₆o and Mtn₆o were successfully functionalized with alkylating agent 3b and subsequently fully converted to polypeptides 8a and 8b in isolated yields of ca. 44-50% for overall polypeptide modification (Scheme 4 and Table 2). The polypeptides 8a and 8b were purified by dialysis against aqueous HC1 (3 mM) followed by DI water to ensure complete protonation of terminal amine groups. Solid samples of polypeptides 8a and 8b were dissolved in a minimal amount of trifluoroacetic acid (TFA) to facilitate complete solubilization, and then were subjected to another round of dialysis against 1 M NaCl, HC1 (3 mM) and then DI water. The resulting white solids obtained after freeze drying were found to be soluble in DI water at 20 °C.

Scheme 4. Synthesis of 8a and 8b as homologs of 5b.

Table 2, Yields of intermediates and polypeptides 8a and 8b shown in Error! Reference source not found..

The chain conformations of polypeptides 8a and 8b in aqueous media were evaluated over a range of pH to determine if the increased distance of the charged side-chain groups from the backbone in 8a and 8b compared to 5b affected their a-helical conformations. Both polypeptides 8a and 8b were observed by CD spectroscopy to be predominantly a-helical {ca. 75-93%) over a pH range of 5.0 to 9.0 (FIGs. 11 A-l 1C Error! Reference source not found.and Table 3). Compared to their homolog, 5b, the polypeptides 8a and 8b possessed a greater a-helical content, which is likely due to their increased hydrophobicity and increased distance of ammonium groups from the backbone (FIGs. 11 A-l 1C).

Table 3, Percent a-helical content of polypeptides 8a and 8b at 0.5 mg/mL in 12 mM phosphate buffer as a function of pH, Samples are those shown in FIGs. 11 A-l 1C. a-Helical content was derived from the intensity of minima at 222 nm in CD spectra.

After confirming that polypeptides possessed a-helical conformations similar to polypeptide 5b, the complexes of 8a and 8b were formed with the model multivalent counterion sodium tripolyphosphate (TPP) to see if complex coacervates would form. Solutions of polypeptides 8a (3 mg/mL) and 8b (5 mg/mL) in 150 mM aqueous NaCl were each mixed with TPP (final concentration of 12 mM) dissolved in 150 mM aqueous NaCl, which resulted in formation of turbid suspensions that were examined using differential interference contrast (DIC) microscopy. In contrast to the behavior of cationic polypeptide samples 5b-d under the same conditions, the complexes of 8a and 8b were observed by DIC microscopy to phase separate as irregular clusters of solid precipitates. The formation of precipitates with 8a and 8b contrasts the formation of coacervates observed with 5b under the same conditions, which indicated that the distance of side-chain thioether groups from the polypeptide backbone may dictate the nature of complex coacervate phase separation. More specifically, only the spacing found in the homocysteine repeat unit was found to result in successful coacervate formation for these particular model polypeptides.

Although increasing the distance of side-chain thioether groups from the polypeptide backbone resulted in loss of coacervate formation, we also wanted to investigate if modifications to chain length in other parts of the tether would be better tolerated. To test this hypothesis, the overall distance between side-chain ammonium groups and the polypeptide backbone at the lengths found in 8a and 8b was maintained and the thioether to backbone spacing that is found in coacervate forming polypeptides 5a-d was preserved. Synthesis of these target molecules was accomplished via slight modifications of the procedure for synthesis of epoxide 3c in order to prepare two new epoxide alkylating agents. Homologs of 3c were thus prepared that contained either one or two additional methylene units in the linkage. The increased number of methylene units in the new epoxides, 11a and lib, were obtained via use of propanolamine and butanolamine starting materials in place of ethanolamine (Scheme 5 and Table 4). The alkylating agents 11a and lib were each used to successfully functionalize the backbone of Mbo. After subsequent demethylation and deprotection, the resulting polypeptides, 13a and 13b, were isolated in isolated yields for overall polypeptide modification of ca. 74 - 83% (Scheme 5 and Table 4). Samples of 13a and 13b were purified by dialysis against aqueous HC1 (3 mM) and DI water to ensure complete protonation of side-chain amine groups. As with 8a and 8b, samples were further processed by dissolving solid polymer in a minimal amount of TFA to facilitate complete solubilization followed by another round of dialysis against NaCl (1 M), HC1 (3 mM) and DI water. The final products were obtained as white solids after freeze drying, and both were found to be soluble in DI water at 20 °C.

Scheme 5. Synthesis of intermediates 11a and lib and polypeptide samples 13a and 13b. Table 4 Yields of the intermediates and polypeptides shown in Scheme 5

The CD spectra of polypeptides 13a and 13b were also recorded to see how increasing the linker lengths in these samples affected chain conformations relative to 5c. Both 13a and 13b were found to be primarily a-helical (percent a-helical content = 49-99%) over a range of pH (FIGs. 13A-13C and Table 5) with CD spectral features that were quite similar to the parent 5c. The apparent loss of a-helical content for 13a and 13b at pH 9.0 can be attributed to precipitation of samples upon neutralization, which prevented collection of quality CD data. While the a-helical conformations of 13a and 13b were consistent with 5c (FIGs. 13A-13C), the conformations varied less with pH compared to 5c within the same range. These results suggest that placement of charged ammonium groups further from the backbone via side-chain extension results in less disruption of the a-helical conformation, which has also been observed in other systems.

Table 5, Percent a-helical content of polypeptides 13a and 13b at 0.5 mg/mL in 12 mM phosphate buffer as a function of pH, Samples are those from FIGs. 13A-13C. a-Helical content was derived from intensity of minima at 222 nm in CD spectra.

After confirming that polypeptides possessed a-helical conformations similar to polypeptide 5b, complexes of 13a and 13b were formed with TPP to see if complex coacervates would form. Solutions of polypeptides 13a and 13b at 5 mg/mL in aqueous 150 mM NaCl were each mixed with TPP (13 mM) in aqueous 150 mM NaCl, both samples were found to form coacervate droplets as verified by DIC microscopy (FIGs. 14A & 14B). This result demonstrated that 13a and 13b retain the ability to undergo coacervate formation even with extended alkyl linker lengths compared to 5c. Hence, the spacer in the epoxide linker was identified as a suitable candidate for fine-tuning of coacervate formation.

Once it was found that 13a and 13b were capable of forming coacervates, coacervate formation as a function of pH was also evaluated. Cationic polypeptides 5b-d in water possess lower critical solution temperatures (LCST) that depended on the nature of side-chain amino acid groups as well as the pH of the solution. Similar temperature dependent solubility is frequently studied for protein sequences to identify those that are responsible for driving phase separation in MLOs. Here, solutions of polypeptides 13a and 13b (2.5 mg/mL) separately prepared in 150 mM phosphate-buffered saline (PBS) over a pH range of 6.7-7.4 were heated, and were found to possess pH dependent phase separation as observed by decreases in light transmission at 500 nm (FIGs. 15A & 15B and Table 6). When comparing samples 13a and 13b to our parent sample 5c at a pH of 7.0 in 150 mM PBS, it was possible to observe a correlation between linker length and cloud point temperatures (Tcp) where samples with longer hydrocarbon linkers (increased hydrophobicity) possessed lower T_cp (FIG. 15C). This correlation between linker length and T_cp shows that the temperature and pH-dependent phase behavior of these polypeptides can be finely tuned through molecular modifications. Table 6. Coacervate transition temperatures for solutions of polypeptides 13a and 13b at different pH. Cloud point temperatures (Tcp) were measured for individual 2.5 mg/mL solutions of 13a and 13b in 150 mM PBS buffer at different solution pH for samples shown in Error! Reference source not found..

In addition to retaining the ability to form coacervates, polypeptides 13a and 13b exhibit pH dependent aqueous solubility similar to that seen for polypeptides 5a-d. These results create an opportunity to introduce further modifications of polypeptide side-chains away from the backbone to fine tune coacervate properties. Poly(S-alkyl-L-homocysteine)s with anionic side-chain groups Being able to form coacervates using either anionic or cationic polypeptides would provide many advantages in using these materials as biomimetic models of MLOs and in downstream applications. The incorporation of side-chain carboxylate groups in polypeptides is expected to provide aqueous solubility and pH dependent ionization similar to the previously used ammonium groups. Anionic, carboxylate-functionalized poly(S-alkyl-L- homocysteine) derivatives should also facilitate complexation with cationic polyelectrolytes and proteins. Using the synthetic platform based on M₆₀, it is possible readily change side- chain functionality via synthesis of new, small molecules epoxide alkylating agents. Straightforward reversal of how the terminal amino acids are connected to the epoxides (i.e., via N-terminus instead of C-terminus) will result in carboxylate groups at the termini of side- chains in the final polypeptides (Scheme 5). To prepare anionic poly(S-alkyl-L-homocysteine) derivatives, new epoxide alkylating reagents were prepared containing amino acids with their orientation reversed from previous studies. First, L-alanine (Ala), Val, Leu, and L-phenylalanine (Phe) were protected as methyl esters. These amino acid esters were then each coupled to allyl glycolate (14) and converted to the epoxides 16a-d (Scheme 6Error! Reference source not found, and Table 7). The resulting epoxides were used to fully alkylate all side-chain thioether groups in Mbo, and the resulting products were then fully demethylated and deprotected to provide the series of anionic polypeptides 18a-d as their ammonium salts in good yields (overall isolated yields for polypeptide modification were ca. 72-86%) (Scheme 6Error! Reference source not found, and Table 7). ¹HNMR analysis of these polypeptides showed complete conversion of all Met residues, although a small amount of oxidation instead of alkylation was found to occur at some thioether groups (~5 mol%). Polypeptides 18a-d were purified by dialysis against aqueous MLOH (3 mM) followed by DI water to give the purified polypeptides as white solids. The ammonium salts of polypeptides 18a-d were all soluble in DI water at pH greater than 3.0. Insolubility below pH 3.0, which correlates roughly with the pK_a of glutamate residues, was due to protonation of side-chain carboxylate functional groups.

Scheme 6. Synthesis of intermediates and polypeptide samples 18a-d.

Circular dichroism spectra (CD) 18a-d at concentrations of 0.5 mg/mL in aqueous phosphate buffer (100 mM) over a range of different pH was used to identify the chain conformations of these polypeptides. It was expected that the terminal side-chain carboxylate groups would remain completely charged between pH 5.0 and 9.0.

Table 8, Percent a-helical content of polypeptides 18a-d at 0.5 mg/mL in 100 mM phosphate buffer as a function of pH as shown in FIGs. 16A-16D q-Helical content was derived from intensity of minima at 222 nm in CD spectra.

Polypeptides 18a-d were found to adopt stable a-helical conformations with a-helical contents ranging from 65-88% FIGs. 16A-16D and Table. 8Table ). The % a-helical conformation was observed to increase as side-chain amino acids became more hydrophobic, similar to the behavior of the cationic polypeptides 5a-d. Unlike 5a-d, the conformations of 18a-d samples showed little change, which was likely due to the carboxylate groups being fully ionized across this range of pH. These results indicate that reversal of the amino acid linkages in polypeptides 18a-d resulted in minimal perturbation of the polypeptide chain conformations.

To test for the ability of anionic polypeptides 18a-d to form coacervates they were mixed with multivalent cations in aqueous solution. Calcium chloride (CaCk) and hexammine cobalt ([Co(NH3)₆]Cb) was selected as the model multivalent cations where the metal ions possessed +2 and +3 charges, respectively. Solutions of each of the four polypeptides, 18a-d, at 5 mg/mL was complexed with solutions of each metal ion (13 mM final concentration) in 150 aqueous mM NaCl followed by examination using DIC microscopy. When mixed with CaCh at pH 7.0, solutions of polypeptides 18a-c all showed minimal phase separation (FIGs. 17A, 17C, and 17E), while the mixture of CaCh with 18d resulted in the formation of small droplets (FIG. 16G). When combined with hexammine cobalt, solutions of the most hydrophobic polypeptides 18c and 18d were observed to form phase separated coacervates (FIGs. 17F & 17H). In comparison, the two more hydrophilic derivatives 18a and 18b remained mostly soluble with only a small amount of coacervate formation (FIGs. 17B & 17D). Gentle heating of the mixtures of hexammine cobalt with 18a and 18b to 40 °C did not induce any additional coacervate formation. These data showed that coacervate formation of 18a-d was dependent on both side-chain hydrophobicity and counterion multivalency in these systems, which was similar to results observed for coacervate formation with cationic 5a-d.

The ability to encapsulate proteins with coacervates either in binary or ternary complexes has many applications that include drug delivery and protein purification, due to the potential to drive encapsulation of a specific protein cargo and protect it from denaturation. Certain polypeptides have been shown to form coacervates upon complexation with lysozyme due to its overall positive charge over a range of pH (the pi of lysozyme = 11). The overall positive charge of lysozyme at neutral pH makes it a good partner for complexation with anionic polypeptides. For this reason, we studied the complexation of the model protein lysozyme with 18c and 18d to see if complex coacervates would form. Solutions of lysozyme at different concentrations were prepared at pH 7.0 in Milli-Q water and mixed in 1 : 1 (v/v) ratios with solutions of either polypeptide 18c or 18d in Milli-Q water to give final polypeptide concentrations of 5 mg/mL and final lysozyme concentrations of 2.5, 5 and 10 mg/mL in 150 mM NaCl. The range of concentrations used for lysozyme were chosen based on what had been used in previously reported examples of protein- poly electrolyte complex coacervation. Concentrations of lysozyme were varied to assess if different charge ratios between lysozyme and polypeptide would have any influence on the nature of the phase separation formed upon complexation. Based on a presumed net positive charge of +8 for each lysozyme molecule at a pH of 7.0, the mixtures of lysozyme at 2.5, 5 and 10 mg/mL with 18c or 18d at polypeptide concentrations of 5 mg/mL in Milli-Q water in the presence of 150 mM NaCl, resulted in overall anion to cation charge ratios of ~9: 1,

-4.5:1, and -2.3:1, respectively. Turbidity was observed in all mixtures, and optical microscopy revealed structures with jagged outlines that indicated the formation of precipitates for all samples (FIGs. 18A-18D). In a similar study of protein-polyelectrolyte coacervation performed by Obermeyer and coworkers, it was found that the model protein used for complexation played a significant role in determining the nature of the phase upon complex formation. This suggests that the lack of coacervate formation in our polypeptide- lysozyme complexation study with 18c and 18d might be related to using lysozyme, and we cannot rule out other routes to complex coacervation with proteins until other model proteins are evaluated.

In polypeptide-polypeptide and polypeptide-polynucleotide complex coacervates, the secondary structure of the polyelectrolytes can control the nature of the phase separation that occurs upon complexation. The mild oxidation of cationic polypeptide 5c to the cationic sulfoxide 6c (Scheme 7Error! Reference source not found.) resulted in disruption of the a- helical conformation of the polypeptide chain in aqueous solution and significantly altered coacervate forming properties. Mixture of aqueous solutions of a-helical 18c and a-helical 5c in DI water (5 mg/mL final polypeptide concentration for each polypeptide) in the presence of 150 mM NaCl was found to result in the formation of precipitates (FIG. 19A) as observed by DIC microscopy. This result is consistent with other reports of polyelectrolyte complexation of homochiral cationic and anionic polypeptides where their ability to adopt ordered chain conformations results in more closely associating complexes that form solids instead of liquid coacervates. When aqueous solutions of a-helical 18c and disordered 6c (5 mg/mL final concentration of each polypeptide) were mixed in the presence of 150 mM NaCl, liquid coacervate droplets were observed to form by DIC microscopy instead of solid precipitates (FIG. 19B). This result contrasts results seen with other polypeptide based complex coacervates that require one of the components to consist of a racemic mixture of residues to prohibit the formation of an ordered chain conformation. Here, the disorder in 6c appears to be substantially resistant to ordering, likely due to the hydrophilicity of the side- chains. Overall, this result showed that the phase behavior of polyelectrolyte complexes of polypeptides can be controlled in by mild chemical modification of one of the polypeptides involved in complexation.

Scheme 7. Synthesis of sulfoxide derivative 6c from 5c. TBHP = tert-butyl hydroperoxide, CSA = camphorsulfonic acid.

The new series of anionic polypeptides 18a-d were found to adopt stable a-helical chain conformations in aqueous solution and also possessed the ability to undergo LLPS in the presence of oppositely charged multivalent counterions. However, when anionic 18c was complexed with an oppositely charged model protein, lysozyme, a precipitate was formed instead of a coacervate. Given the ability to control the nature of the phase formed upon complexation by altering polypeptide secondary structure, it’s possible that further tuning of anionic polypeptide chain conformations could be used to overcome these challenges.

Summary

The understanding of the molecular features required in side-chain amino acid- functionalized polypeptides to form coacervates upon phase separation from aqueous solutions has been expanded. Here, the modular nature of the disclosed synthetic methodology was employed by modifying a variety of molecular features of these polypeptides by altering the structures of readily prepared small molecule reagents. Disclosed herein are two types of modifications to previously reported family of cationic polypeptides 5a-d and tested their effects on coacervate formation: (1) increasing the distance of the side- chain amino acids from the polypeptide backbone, and (2) reversing the charge side-chain termini from cationic to anionic. Altogether, these results show that side-chain amino acid containing poly(S-alkyl-L-homocysteine)s can be prepared with a range of structural changes to their side-chains, such as increased distance of charged groups from polypeptide backbone and anionic charges at their termini, while retaining their aqueous LLPS behavior. Example 3: Exemplary Animal Studies

Animal Study #1: Preliminary Biocompatibilitv Rat Subcutaneous Model Protocol:

To assess the biocompatibility of the claimed hydrogel fillers in a preclinical in vivo model, subcutaneous injection in rats was performed as previously described ( Hillel , Alexander T, et al. “Validation of a small animal model for soft tissue filler characterization. ’’Dermatologic surgery 38.3 (2012): 471-478., n.d.). Fifteen Male Sprague-Dawley rats (250-300gm) were injected into the dorsal subcutaneous pocket with the following representative compound: 0.5 mL (M° A) /55-E/IG5 at 9wt% in 0.9%NaCl.

The rats were then scheduled for necropsy with histology according to the following schedule:

Table 9.

Results (Clinical Examination):

Animals were assessed at days 7, 30, and 62 for clinical irritation or erythema according to the following scale: 1 - No erythema (normal); 2 - Mild erythema; 3 - Moderate erythema; and 4 - Severe erythema. FIG. 21 provides an example photograph from an animal in group 2 at day 30 with no observable erythema or irritation at the site of injection (marked with the dark circle).

Table 10. Median Clinical (Visual) Assessment Values of Erythema by Study Day

Results (Histology):

Dermal filler study, group 2-3 injected with test material 30 and 62 days ago ANIMAL SPECIES: Rattus norwegicus / white rat / animals 6-15 day 30-62 GROSS DESCRIPTION: 10 skin samples measuring approximately 4x4cm Microscopic diagnosis:

Animal 6: Subcutis: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (minimal) and rare hair shafts (drag-in from injection);

Animal 7: Subcutis: Fascial infiltrates, mastocytic (minimal), lymphoplasmacytic (minimal), histiocytic (minimal) and fibrocytic (moderate), focally extensive, with fibrosis (mild), panni cuius myocyte loss (mild) and rare injection drag-in material

Animal 8: Subcutis: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal), histiocytic (minimal) and fibrocytic (moderate), focally extensive, with fibrosis (minimal) panniculus myocyte loss (mild);

Animal 9: Subcutis: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal), histiocytic (minimal) and fibrocytic (moderate), focally extensive, with fibrosis (mild) and panniculus myocyte loss (mild);

Animal 10: Subcutis: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal), histiocytic (minimal) and fibrocytic (moderate), focally extensive, with fibrosis (mild) and panniculus myocyte loss (mild);

Animal 11: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (mild) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (mild), panniculus myocyte loss (moderate) and rare hair shafts (drag-in from injection);

Animal 12: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (mild) and panniculus myocyte loss (minimal);

Animal 13: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (mild) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (mild) and panniculus myocyte loss (mild);

Animal 14: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (minimal) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (mild) and panniculus myocyte loss (moderate); and

Animal 15: Fascial infiltrates, mastocytic (mild), lymphoplasmacytic (mild) and histiocytic (minimal) and fibrocytic (mild), focally extensive with fibrosis (mild), panniculus myocyte loss (moderate) and rare hair shafts (drag-in from injection).

Summary: Animals 6-10: These cases had mild to minimal mastocytosis, minimal lymphoplasmacytic inflammation, minimal histiocytic inflammation and minimal to mild fibrosis in the superficial subcutis with multifocal loss of panniculus muscle. No filler material was identified. There are rare cross sections of hair shafts and refractile foreign material (standard injection drag-in with surrounding granulomatous inflammation). Loss of the panniculus muscle is a common finding with subcutaneous injections and is likely unrelated to the test material.

Animals 11-15: These cases had mild to minimal mastocytosis, minimal to mild lymphoplasmacytic inflammation, minimal histiocytic inflammation and minimal to mild fibrosis in the superficial subcutis with multifocal loss of panniculus muscle. No filler material was identified. There are rare cross sections of hair shafts and refractile foreign material (standard injection drag-in with surrounding granulomatous inflammation). Loss of the panniculus muscle is a common finding with subcutaneous injections and is likely unrelated to the test material.

Animal Study #2: Effectiveness Rat Subcutaneous Model Protocol:

To assess the effectiveness and further assess biocompatibility of the claimed hydrogel fillers in a preclinical in vivo model, subcutaneous injection in rats was performed as previously described ( Hillel , Alexander T, et al. “Validation of a small animal model for soft tissue filler characterization. ” Dermatologic surgery 38.3 (2012): 471-478., n.d.) . Sprague-Dawley rats (150-200gm) were separated into six groups (with 7 animals per group) and each animal was injected directly in the subcutaneous space with one of the following compounds based on group:

Group A: 0.5 mL Juvederm Voluma (Hyaluronic Acid)

Group B: 0.5 mL M⁰A1₅₅(E/K)65 at 7wt% in 0.9%NaCl Group C: 0.5 mL M°Ai55(E/K)75 at 7wt% in 0.9%NaCl Group D: 0.5 mL M⁰Ai55(E/K>5 at 7wt% in 0.9%NaCl Group E: 0.5 mL M°Ai8o(E/K)75 at 7wt% in 0.9%NaCl Group F: 0.5 mL M°Ai8o(E/K)75 at 5wt% in 0.9%NaCl

Two rats in each group were sacrificed at day 7 to assess for histologic response to the test material. The remaining five rats in each group were planned to be followed until complete clinical resorption of the test material. Clinical erythema, irritation, and adverse events were similarly observed and recorded. FIG. 22 is an example photo of a palpable lump on the dorsum of an animal in Group A (Hyaluronic Acid Control) on day 7.

Results (Day 7):

Table 11. Gel Persistence

While no photographs were taken, it was noted that the gel deposits in groups B-F persisted for roughly 5 days before complete disappearance of the palpable lump.

Host Response:

On day 7 there was no detectable erythema, irritation, inflammation, drainage, or infection in any of the study animals in any of the study groups.

Results (Later Time Points):

Given the lack of remaining palpable lumps in the study groups with PIC gels, the remaining animals in each group were sacrificed at the two-week time point.

Animal Study #3 : Rabbit Arterial Occlusion Study

Protocol:

A significant advantage of the claimed hydrogel filler is the vascular safety if inadvertently injected intra-arterially. To test the concept that the claimed hydrogel filler does not obstruct arteries, rabbit ears were directly injected intra-arterially with 0.15cc of material according as previously described in the literature (Nie, Fangfei, et al. "Risk comparison of filler embolism between polymethyl methacrylate (PMMA) and hyaluronic acid (HA). "Aesthetic plastic surgery 43.3 (2019): 853-860.), according to the following groups: Table 12.

At day 0, the ears were transilluminated to assess for apparent filler emboli in the vessels. The ears were then clinically assessed at day 7 for ischemic changes.

Results (Transillumination):

Day 0 transillumination from left ear of rabbit 1 (Hyaluronic acid control) demonstrates impeded blood flow and embolus in the central auricular artery (FIG. 23).

Day 0 transillumination from right ear of rabbit 1 (M°Ai55(E/K)65 at 7wt% in 0.9% NaCl) demonstrates intact blood flow in the central auricular artery without apparent emboli. These results were representative of the day 0 transillumination experiments. All of the hyaluronic acid ears showed emboli with impeded blood flow, while all of the claimed hydrogel filler injected ears remained patent (FIG. 24).

Results (Clinical Ischemia):

Animals were then assessed at day 7 for clinically visible ischemic changes.

Photo from day 7 from animal 1 depicts ischemic changes in the left ear (hyaluronic acid) compared to right ear (M°Ai55(E/K)65 at 7wt% in 0.9%NaCl). The ischemic changes are clearly seen as the dusky coloration in the auricular tissue (FIG. 25).

Similarly, photo from day 7 of rabbit 3 demonstrates ischemic changes in the right ear (hyaluronic acid), but no ischemic changes in the left ear (M°Ai8o(E/K)75 at 7wt% in 0.9%NaCl) (FIG. 26).

As with the transillumination studies, these changes were consistent and reproducible in the animals. All of the hyaluronic acid ears demonstrated ischemic changes at day 7, while none of the claimed hydrogel filler injected ears demonstrated ischemic changes.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMS We claim:

1. A polypeptide comprising one or more R-C^HAA residues, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula la, formula lb, formula Ila, or formula lib:

or a salt thereof, or a stereoisomer or enantiomer thereof, wherein: R¹, R², R³, R⁵, R^7a and R^7b are each independently selected from H, alkyl, or aralkyl; each R⁴ is independently the side chain of a naturally occurring amino acid, alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, amidoalkyl, ester, acyl, cycloalkyl, (cylcoalkyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl; R⁵ is H, X is S, S(O), S(O)₂, or S⁺-R⁶.A^1-; X¹⁺ is a cation; R⁶ is alkyl; A^1- and A^2- are each independently anions; Z is amino, urealyl, or HN⁺(R^7a)(R^7b).A^2-; Z¹ is OR⁵ or O-X¹⁺; m is 0-5; n is 1-5; and p is 1-5.

2. The polypeptide of claim 1, wherein Z¹ is O-X¹⁺.

3. The polypeptide of claim 1 or 2, wherein X¹⁺ is Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, or NH₄ ⁺.

4. The polypeptide of claim 1 or 2, wherein X¹⁺ is NH4⁺.

5. The polypeptide of claim 1, wherein Z¹ is OR⁵.

6. A polypeptide comprising one or more R-C^HAA residues, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula la, formula lb, formula Ila, or formula lib:

or a salt thereof, or a stereoisomer or enantiomer thereof, wherein: R¹, R², R³, R⁵ R^7a and R^7b are each independently selected from H, alkyl, or aralkyl; each R⁴ is independently the side chain of a naturally occurring amino acid, alkyl, alkenyl, alkynyl, hydroxyalkyl, aminoalkyl, amidoalkyl, ester, acyl, cycloalkyl, (cylcoalkyl)alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclyl, or heterocyclylalkyl; X is S, S(O), S(O)2, or S⁺-R⁶.A^1-; R⁶ is alkyl; A^1- and A^2- are each independently anions; Z is amino, urealyl, or HN⁺(R^7a)(R^7b).A^2-; m is 0-5; n is 1-5; and p is 1-5.

7. The polypeptide of any one of claims 1-5, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula Ia or formula Ib or a salt thereof.

8. The polypeptide of any one of claims 1-5, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula IIa or formula IIb or a salt thereof.

9. The polypeptide of claim 6, wherein the polypeptide comprises R-C^HAA residues represented by formula la* and formula Ila* or salts thereof.

10. The polypeptide of claim 6, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula la* or formula lb* or a salt thereof.

11. The polypeptide of claim 6, wherein each R-C^HAA residue in the polypeptide has a structure represented by formula Ila* or formula lib* or a salt thereof.

12. The polypeptide of claim 6, wherein the polypeptide comprises R-C^HAA residues represented by formula la* and formula Ila* or salts thereof.

13. The polypeptide of any one of claims 1-12, wherein R¹ is H.

14. The polypeptide of any one of claims 1-13, wherein X is S⁺-R⁶.A¹ .

15. The polypeptide of claim 14, wherein R⁶ is methyl or ethyl.

16. The polypeptide of claim 14, wherein R⁶ is methyl.

17. The polypeptide of any one of claims 14-16, wherein A¹ is a pharmaceutically acceptable anion.

18. The polypeptide of any one of claims 14-17, wherein A¹ is halide, sulfate, phosphate, citrate, pyrophosphate, or tripolyphosphate.

19. The polypeptide of any one of claims 1-18, wherein R² is H.

20. The polypeptide of any one of claims 1-19, wherein R³ is H.

21. The polypeptide of any one of claims 1-20, wherein R⁴ is the side chain of a naturally occurring amino acid ( e.g the side chain of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine).

22. The polypeptide of any one of claims 1-21, wherein R⁴ is the side chain of glycine, valine, leucine, or phenylalanine.

23. The polypeptide of any one of claims 1-7, 9, 10, or 12-22, wherein Z is amino.

24. The polypeptide of any one of claims 1-7, 9, 10, or 12-22, wherein Z is N⁺(R^7a)(R^7b) . A^2' .

25. The polypeptide of claim 24, wherein each R^7a is H.

26. The polypeptide of claim 24 or 25, wherein each R^7b is H.

27. The polypeptide of any one of claims 24-26, wherein A^2' is a pharmaceutically acceptable anion.

28. The polypeptide of any one of claims 24-27, wherein A^2' is halide, sulfate, phosphate, citrate, pyrophosphate, or tripolyphosphate.

29. The polypeptide of any one of claims 1, 5, 6, 8, 9, or 11-28, wherein R⁵ is H.

30. The polypeptide of any one of claims 1-29, wherein m is 1.

31. The polypeptide of any one of claims 1-30, wherein n is 2.

32. The polypeptide of any one of claims 1-31, wherein p is 1.

33. The polypeptide of any one of claims 1-32, wherein the R-C^HAA residues are a salt of formula la, formula lb, formula Ila, or formula lib.

34. The polypeptide of any one of claims 1-32, wherein the R-C^HAA residues are a salt of formula la.

35. The polypeptide of any one of claims 1-32, wherein the R-C^HAA residues are a salt of formula Ila.

36. The polypeptide of any one of claims 1-32, wherein the R-C^HAA residues are a salt of formula la*, formula lb*, formula Ila*, or formula lib*.

37. The polypeptide of any one of claims 1-25, wherein the R-C^HAA residues are a salt of formula la*.

38. The polypeptide of any one of claims 1-25, wherein the R-C^HAA residues are a salt of formula Ila*.

39. The polypeptide of any one of claims 1-38, wherein the polypeptide comprises 5-500 R-C^HAA _residues_.

40. The polypeptide of any one of claims 1-38, wherein the polypeptide consists essentially of 5-500 R_C^HAA residues.

41. The polypeptide of any one of claims 1-38, wherein the polypeptide consists of 5-500 R-C^HAA _residues_.

42. The polypeptide of any one of claims 1-38, wherein the polypeptide comprises 10-200 R-C^HAA _residues_.

43. The polypeptide of any one of claims 1-38, wherein the polypeptide consists essentially of 10-200 R_C^HAA residues.

44. The polypeptide of any one of claims 1-38, wherein the polypeptide consists of 10- 200 R-C^HAA residues.

45. The polypeptide of any one of claims 1-44, wherein the R-C^HAA residues are the same.

46. The polypeptide of any one of claims 1-44, wherein the R-C^HAA residues are different.

47. The polypeptide of any one of claims 1-46, wherein the N-terminus of the polypeptide is capped with alkyl or alkoxy.

48. The polypeptide of any one of claims 1-46, wherein the N-terminus of the polypeptide is capped with alkoxy.

49. The polypeptide of any one of claims 1-46, wherein the N-terminus of the polypeptide is capped with poly(ethylene glycol).

50. The polypeptide of any one of claims 1-46, wherein the N-terminus of the polypeptide is capped with poly(ethylene glycol) having a molecular weight of 50-20,000 Da.

51. The polypeptide of any one of claims 1-46, wherein the N-terminus of the polypeptide is capped with poly(ethylene glycol) having a molecular weight of 100-10,000 Da.

52. A composition comprising the polypeptide of any one of claims 1-51 and an excipient.

53. The composition of claim 52, wherein the composition further comprises a therapeutic agent.

54. The composition of claim 53, wherein the therapeutic agent is an anti-cancer agent, an anti-inflammatory agent, an anti-biotic agent, an anti-viral agent, an anti-fungal agent, or an analgesic.

55. The composition of claim 53, wherein the therapeutic agent is a vaccine.

56. The composition of any one of claims 53-55, wherein the therapeutic agent is an antibody, a protein, a nucleic acid, or a small molecule.

57. A method of treating a disease or disorder in a subject in need thereof, comprising administering the composition of any one of claims 53-56 to the subject.

58. The method of claim 57, wherein the disease or disorder is cancer.

59. A method of administering a therapeutic agent to a subject, comprising administering the composition of any one of claims 53-56 to the subject.

60. A method of treating fine lines or superficial wrinkles in the skin of a subject, comprising administering a composition into a dermal region of the subject which displays the fine lines or superficial wrinkles, thereby treating the fine lines or superficial wrinkles, wherein the composition comprises the polypeptide of any one of claims 1-51.

61. The method of claim 60, wherein the dermal region is a tear trough region, a glabellar line, a periorbital region, or a forehead region.

62. A method of treating a skin condition, comprising administering to an individual suffering from the skin condition a composition, wherein the administration of the composition improves the skin condition, thereby treating the skin condition, wherein the composition comprises the polypeptide of any one of claims 1-51.

63. The method of claim 62, wherein the skin condition is skin dehydration.

64. The method of claim 63, wherein the composition rehydrates the skin of the subject.

65. The method of claim 62, wherein the skin condition is skin elasticity.

66. The method of claim 65, wherein the composition increases the elasticity of the skin of the subject.

67. The method of claim 62, wherein the skin condition is skin roughness.

68. The method of claim 67, wherein the composition decreases skin roughness in the subject.

69. The method of claim 62, wherein the skin condition is a lack of skin tautness.

70. The method of claim 69, wherein the composition increases skin tautness in the subject.

71. The method of claim 62, wherein the skin condition is a skin stretch line or mark.

72. The method of claim 71, wherein the composition reduces or eliminates the skin stretch line or mark in the subject.

73. The method of claim 62, wherein the skin condition is skin paleness.

74. The method of claim 73, wherein the composition increases skin tone or radiance in the subject.

75. The method of claim 62, wherein the skin condition is skin wrinkles.

76. The method of claim 75, wherein the composition reduces or eliminates skin wrinkles in the subject.

77. A method of preventing skin wrinkles in a subject, comprising administering to the subject a composition, thereby preventing skin wrinkles, wherein the composition comprises the polypeptide of any one of claims 1-51.

78. The method of claim 77, wherein the composition makes the skin of the subject resistant to skin wrinkles.

79. The method of any one of claims 57-78, wherein the administration is by subcutaneous injection.

80. The method of any one of claims 57-79, wherein the administration occurs at a depth of less than about 1 mm below the surface of the skin.

81. The method of any one of claims 57-80, wherein the method does not result in arterial occlusion.

82. The method of any one of claims 57-81, wherein the method does not result in unpredictable augmentation.

83. The method of any one of claims 57-82, wherein the method does not result in irritation, for example, chronic irritation.

84. The method of any one of claims 57-83, wherein the composition is soluble in blood.

85. The method of any one of claims 57-84, wherein administration of the composition results in limited swelling.

86. The method of any one of claims 57-5, wherein the administration of the composition results in low immunogenicity.