Method for the preparation of high molecular weight oligo(alkylene glycol) functionalized polyisocyanopeptides

Abstract

The present invention relates to a process for the preparation of oligo(alkylene glycol)-functionalized polyisocyanopeptides comprising the steps of functionalizing an isocyanopeptide with oligo-(alkylene glycol) side chains and subsequently polymerizing the oligo-alkylene glycol-functionalized isocyanopeptides. Several isocyanopeptides may be functionalized with various linear or non-linear oligo-(alkylene glycol) side chains having variable chain length. The alkylene glycol may be selected from the group consisting of ethylene-, propylene-, butylene- or pentylene glycol. Preferably, the isocyanopeptides are functionalized with at least three ethylene glycol side chains. The peptides may comprise L-amino acids, D-amino acids or D, L-amino acids. The obtained oligoalkylene-functionalized polyisocyanopeptides are a new class of materials with unique thermo-responsive properties.

Claims

1. A poly(oligo(alkylene glycol))isocyanopeptide comprising repeating units of an isocyano di-, tri-, or tetra peptidic motif substituted at its C terminal via an ester linkage to an oligo(alkylene glycol) chain haying from 1 to 10 alkylene glycol repeating units, with the proviso that the poly(oligo(alkylene glycol))isocyanopeptide does not comprise a triazole moiety.

2. The poly(oligo(alkylene glycol))isocyanopeptide of claim 1, wherein the peptidic motif is a dipeptide.

3. The poly(oligo(alkylene glycol))isocyanopeptide of claim 1, wherein the oligo(alkylene glycol) is oligo(ethylene glycol).

4. A poly(oligo(alkylene glycol))isocyanopeptide comprising repeating units of an isocyano di-, tri-, or tetra- peptide motif substituted at its C terminal via an ester linkage to an oligo(alkylene glycol) chain having from one (1) to ten (10) alkylene glycol repeating units, wherein the poly(oligo(alkylene glycol))isocyanopeptide satisfies the formula ##STR00005## wherein n is an integer of from 1 to 4 and m is an integer of up to 100,000.

5. A hydrogel comprising the poly(oligo(alkylene glycol))isocyanopeptide of claim 1.

6. A hydrogel comprising the poly(oligo(alkylene glycol))isocyanopeptide of claim 4.

7. A coating comprising the poly(oligo(alkylene glycol))isocyanopeptide of claim 1.

8. A coating comprising poly(oligo(alkylene glycol))isocyanopeptide of claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The disclosure will now be illustrated by the following examples without being limited thereto.

(2) FIG. 1 shows a schematic representation of the helical oligo-alkylene-functionalized polyisocyano-peptides based on a dialane unit (top middle). The backbone folding is stabilized by a hydrogen bounding network that develops within the helix, between the stacked amide bounds of the side chains (bottom middle). This secondary structure leads to very stiff chains as visualized by AFM (right).

(3) FIG. 2 shows the preparation of polymers poly- 1a-d; a) N-Boc(L)Ala, DMAP, DCC, CH2Cl2, 4 hours, 0 C-RT (75-90%); b) EtOAC/HCl (100%); c) tBuOH (100%); d) N-Boc(D)Ala, DIPEA, DCC, CH2Cl2, 4 hours, 0 C-RT (75-85%); e) NaOOCH/HCOOH reflux, 3 hours (85-95%); f) NMM, diphosgen, CH2Cl2, 78 C. (60-75%); g) Ni(ClO4)2/MeOH, toluene, RT, 4 hours (60-80%).

(4) FIG. 3 depicts conversion of 1c to poly-1c followed by IR spectroscopy. 1c solutions (0.03 mol/L) in toluene (.square-solid.), dichloromethane (.diamond-solid.), and tetrahydrofuran (.box-tangle-solidup.), treated with methanolic aliquots of Ni(ClO4)2.6H2O (final concentration 0.3 mol/mL).

(5) FIG. 4 depicts CD spectra of poly-1a-c solutions in dichloromethane at 25 C.

(6) FIG. 5 depicts CD spectra of poly-1c in dichloromethane (dashed line) and mQ water (solid line) at 25 C.

(7) FIG. 6 shows scattered light intensity of aqueous solutions of poly-1b and poly-1c (1 mg/mL) as a function of temperature. a) poly-1c solutions, DP of 700 (.square-solid.) and 4400 (.square-solid.); b) poly-1c solutions, DP of 2600 (.square-solid.) and 7300 (.square-solid.).

(8) FIG. 7 shows CD spectra of aqueous solutions of poly-1c (1 mg/mL) as a function of the temperature. Left: selected spectral range 25 C. (solid black line); 85 C. (dashed black line). Right: temperature course evolution of CD intensities at 272 (top) and 360 nm (bottom).

(9) FIG. 8 are AFM pictures of 1 mg/mL poly-1b (DP 7300) on a) hydrogel coated HOPG or b) mica and c) when the samples were dried in ambient conditions. Scale bars: 1 m; vertical scale: a) 8 nm, b) and c) 3 nm.

(10) FIG. 9, panels a), b), and c) show details of fibrils and of their association into bundles (gel fibers). Scale bars 100 nm, vertical scale 3 nm. Panels a), b), and c) show different images of the same gel at the same magnification. The white arrows in panel a) indicate single polymer chains. Bottom left inset: selected area of a) with contrast enhancement showing the intertwinement of two poly-1b chains to form a fibril.

(11) FIG. 10 depict height versus width plot for 172 random sections on AFM micrographs.

DETAILED DESCRIPTION

(12) Experimental part

(13) Oligo(ethylene glycol)-substituted isocyanopeptides have been synthesized and polymerized with the use of Ni(II) salts. The thermo-responsive properties of these newly prepared non-linear poly(ethylene glycol) analogs have been investigated in details in aqueous conditions. As reported for other oligo(ethylene glycol) decorated polymers, both the length of the side chains and the degree of polymerization (DP) of the poly(isocyanopeptide) core were found to have a great influence on the transition temperature of the materials. In good agreement with previous work, both shortening the length of the oligo(ethylene glycol) substituents and increasing the DP of the poly(isocyanopeptide), resulted in the lowering of the demixing temperature of their aqueous solutions. Remarkably, poly(isocyanopeptide) chains of high DP led to the reversible formation of strong hydrogels above a critical temperature, even at low polymer concentration (0.1 wt %). AFM studies indicate the formation of a highly structured fibrillar network in the gel state, reminiscent of some structures observed for low molecular weight gelators and polysaccharide (hydro)gels. It has been proposed that the stiff and well-defined helical poly(isocyanopeptide) backbone promotes the hierarchical assembly of the polymers into an extended fibrillar network when the oligo(ethylene glycol) corona hydrophilicity is lowered at higher temperature. It is assumed that the gelation ability of this new class of polymer can be extended to other stable, semi-flexible polymers that reach a critical stiffness/chain length (DP) ratio and that bear side chains of tunable hydrophilicity.

(14) Non-linear poly(ethylene glycol) (PEG) analogs have recently attracted a great deal of attention for the development of innovative water-soluble materials..sup.1 Such PEG analogs are classically prepared from the (co)polymerization of macro-monomers bearing oligo(ethylene glycol) substituents. The solution properties of the resulting comb-like polymers arise from the fine balance between the hydrophilic/hydrophobic characteristics of the grafted moieties and of the polymeric core. The introduction of oligo(ethylene glycol) side chains, for which the hydrophilicity is temperature dependent, offers a simple and elegant way to trigger the overall hydrophilic/hydrophobic balance of these materials and, therefore, provides a straightforward approach to the development of a variety of thermo-responsive systems..sup.2 So far, most non-linear PEG analogs have been derived from vinyl,.sup.3,4 (meth)acrylate,.sup.5,6,7,8,9,10 and styrene.sup.5,11 monomers. Only a few examples have been reported on the synthesis of oligo(ethylene glycol)-functionalized poly(isocyanide)s. These have been prepared either by post-modification of a poly(isocyanide) backbone by peptidic coupling.sup.12 and the copper-catalyzed Huisgen 1,3-dipolar cycloaddition,.sup.13 or through the direct polymerization of crown-ether appended precursors..sup.14 The thermo-responsive properties of the resulting materials have, however, not been explored in detail and the direct polymerization of oligo(ethylene glycol)-functionalized isocyanides led to materials with only a limited degree of polymerization..sup.14 In this contribution, an optimized protocol for the preparation of oligo(ethylene glycol)-coated poly(isocyanopeptide)s is described and the basic properties of this class of non-linear PEG analogs is discussed.

(15) Poly(isocyanides) are one of the most studied static helical polymers. They consist of poly(imine) chains in which every carbon atom of the backbone bears a substituent, resulting in an extremely dense comb-like architecture; helical folding permits the minimization of steric repulsion between the pendant side-chains..sup.15,16,17,18,19 The introduction of peptide-containing side chains leads to materials with unprecedented stiffness..sup.20,21 This effect has been attributed to the development of an intramolecular hydrogen-bonding network between the peptide pendants, which adopt a -sheet-like arrangement along the helical poly(imine) core..sup.20 Such a well-defined structure has naturally attracted much attention for the use of these materials as synthetic platforms to order various photo- and electro-active species for optoelectronics applications..sup.22,23,24,25 With the aim of developing non-ionic water-soluble analogs, the improvement of the synthesis of oligo(ethylene glycol)-coated poly(isocyanopeptides) was focused on first.

(16) As alluded to above, the fine balance between the polymer core and the hydrophilic character of the polymer side chains governs the water solubility of non-linear PEG analogs. In the case of acrylate derivatives, it has been shown that side chains composed of two ethylene glycols units were sufficient to lead to fully water-soluble materials,.sup.26 whereas three ethylene glycol units were required in the case of the more hydrophobic styrene derivatives..sup.11 To address the question of glycol substituent length versus water solubility for the densely functionalized poly[oligo(ethylene glycol)isocyanopeptide]s, three isocyano-dialanine derivatives bearing two, three, or four ethylene glycol units have been prepared and polymerized (FIG. 2).

(17) Compounds 1a-c were derived from di-, tri-, and tetraethylene glycol monomethyl ether, respectively. A classical two-step dicyclohexyl carbodiimide coupling strategy was used for the successive introduction of the (L)- and (D)-N-Boc protected alanine moieties. After the introduction of the desired dialanine motifs, the Boc-protecting end-group was cleaved off. The compounds were formylated and subsequently dehydrated with diphosgene, using N-Methylmorpholine as a base, to yield the desired isocyanides 1a-c in acceptable yields (overall yields 30-60%).

(18) Polymerization

(19) Acid-induced polymerization is the historical method for the preparation of poly(isocyanide)s..sup.27 In the case of dialananyl-isocyanides, chains with an exceptionally high degree of polymerization (DP>10000) can be obtained, but this is strongly dependent on the stereochemistry of the dipeptide fragments;.sup.20,28 either LD or DL diastereomers are required. Despite presenting a proper stereochemistry (i.e., the DL form), compounds 1a-c could not be polymerized in the presence of acid, but were hydrolyzed over time under all the conditions tried ([1a-c] 30-300 mM in dichloromethane, chloroform, and toluene, [H.sup.+] 1.5-12.5 mol %, 25 C.). Subtle steric factors were proposed to explain the low reactivity of inadequate diastereomeric forms of the dipeptide derivatives..sup.28 Similarly, the introduction of flexible and sterically demanding oligo(ethylene glycol) side chains on the isocyanides 1a-c may greatly lower their reactivity toward acid-induced polymerization. Therefore, the more robust nickel catalyzed polymerization introduced by Drenth and Nolte.sup.29 was focused on. During the preliminary experiments, poly-1a was easily obtained with the classical conditions described for related isocyano(dipeptide)s derivatives (dichloromethane, [Ni(ClO.sub.4).sub.2.Math.6H.sub.2O], 1 mol %). Poly-1b-c were, however, not obtained in satisfactory yields using the same protocol. Extensive tests were carried out to improve the polymerization conditions of isocyanides 1b-c.

(20) The nickel-catalyzed polymerization of isocyanides can be greatly influenced by the solvent used, both in terms of yields.sup.17 and final polymer structure..sup.30,31 To investigate these aspects with the newly prepared oligo(ethylene glycol) isocyanopeptides, isocyanide 1c was treated with nickel(II) salts at room temperature in toluene, dichloromethane, tetrahydrofurane, and methanol. In these experiments, toluene was found to systematically lead to the highest yields, but no clear solvent effect regarding the polymer conformation could be evidenced with circular dichroism (CD) spectroscopy. Besides the marked difference in polymerization efficiency, the solvents were found to have a drastic influence on the polymerization rate. By following the disappearance of the isocyanide stretching band using IR spectroscopy, the conversion of 1c into poly-1c could be monitored over time (FIG. 3). The relative polymerization rates (Kp) according to the different solvents were found to follow the order Kp[toluene]>Kp[dichloromethane]>Kp[tetrahydrofuran]. Unfortunately, the polymerization kinetics diverge from simple first order in all cases and no rate constant could be extracted. Furthermore, the reaction could not be followed by IR spectroscopy in methanol due to the broad absorption spectrum of this solvent.

(21) A similar solvent dependence on the rate of polymerization was observed for isocyanide 1b). Moreover, 1b was found to polymerize faster than 1c in all cases. Although not optimal to solubilize poly-1a, toluene was preferred on the basis of the above-described IR experiments for the polymerization of all three oligo(ethylene glycol) isocyanide monomers.

(22) Monomers 1a-c were dissolved in toluene (0.030-0.300 mol/L) and subsequently treated with methanolic aliquots of Ni(ClO.sub.4).sub.2.Math.6H.sub.2O under aerobic conditions at room temperature. After the required reaction time (2-12 hours, according to the monomer/catalyst ratio; the reactions were followed by TLC), the polymers were precipitated against diethyl ether and further purified by precipitation in THF/diethyl ether mixtures to afford the desired materials in satisfactory yields (75%-90%). The degree of polymerization (DP) of the resulting materials could be roughly controlled according to the initial monomer/catalyst ratio (Table 1). For identical monomer/catalyst ratios, the DPs of the prepared polymers varied significantly between the different isocyanides (DP.sub.poly-1c<DP.sub.poly-1a<DP.sub.poly-1b, Table 1). In the case of 1a, the reaction mixture turned rapidly into a gel when a monomer/catalyst ratio of 100/1 was used; therefore, higher ratios were not tested. Since the influence of the gelation of the reaction mixture on the polymerization of 1a has not been explored in detail, it is difficult to draw a conclusion on the observed monomer-DP relationship.

(23) TABLE-US-00001 TABLE 1 Principal characteristics of poly-1a-c critical temper- [].sup.c [monomer]/ Ln.sup.a ature (10.sup.1 deg polymer [Ni] (nm) DP.sup.a Mw/Mn.sup.a ( C.) cm.sup.2 g.sup.1) poly-1a 100 207 1760 1.5 nd.sup.d +76 poly-1b 100 309 2630 1.6 35.sup.e +105 poly-1b 10000 859 7310 1.5 22.sup.e nd.sup.f poly-1c 100 81 690 1.3 52.sup.b, 50.sup.e +175 poly-1c 10000 518 4410 1.7 42.sup.e nd.sup.f .sup.aDetermined on the basis of AFM micrograph analysis, the mean DP values were calculated from Ln values assuming an identical helical pitch (0.47 nm) for all three polymers; .sup.bdetermined from absorption measurements at 450 nm (UV, solution at 1 mg/mL); .sup.cfrom solutions in chloroform; .sup.dnot water soluble; .sup.edetermined from scattering intensity (DLS, solution at 1 mg/mL); .sup.fnot determined.
Backbone Conformation

(24) Poly(isocyanodialaninyl methyl ester) possesses a very characteristic signature (i.e., an intense Cotton effect centered at =310 nm) in circular dichroism (CD) spectroscopy. The classical signal is negative for poly((D)-isocyanoalanyl-(L)-alanyl methyl ester) (poly-(DL)-IAA). This signal has been attributed to the n-* transition of the imine groups, which are trapped in a pseudo 4.sub.1 helical symmetry, and interact with the strong dipole that can develop along the polymer backbone (as a result of the ideal -sheet-like packing of the peptidic pendants). .sup.20,32 The presence of oligo(ethylene glycol) moieties in poly-1a-c resulted in materials possessing altered CD spectra. In all cases, a bisignate curve presenting a main positive component centered at =272 nm and a smaller negative component centered at =360 nm was observed. The three polymers presented signals of comparable shape regardless of the oligo(ethylene glycol) chain length, which suggests similar backbone conformations and side chain orientations in poly-1a-c (FIG. 4).

(25) Comparable CD signals have previously been reported for the polymerization of heptyne-functionalized (D)-isocyanoalanyl-(L)-alanyl esters..sup.33 Although very pronounced, this signal alteration has not been related to major backbone conformational changes. Rather, it has been assigned to a perturbation of the permanent dipole, which interacts with the n-* imine dipole transitions due to a slight reorientation of the peptidic pendants to accommodate the steric constraints introduced by the heptyne side chains. IR spectroscopy supports a similar interpretation in the case of poly-1a-c. As shown in Table 2, NH and CO.sub.amide I stretching bands are strongly red-shifted after the polymerization of isocyanides 1a-c. This effect is an unambiguous signature of the development of hydrogen bonds between the poly-1a-c side chains. The IR signatures are very similar for all three polymers, suggesting an identical hydrogen bounding pattern, that is, an identical core structure for poly-1a-c, in good agreement with CD spectroscopy measurements. The NH stretching bands are slightly less shifted than expected for the optimal -sheet-like packing, as observed in poly-(DL)-IAA (vNH3268 vs. 3252 cm.sup.1, respectively) and are very similar to the values observed for the heptyne-funcationlized polymers synthesized previously..sup.33 It is, therefore, proposed that poly-1a-c present the classical 4.sub.1 helical conformation of poly(isocyanopeptide)s with a slightly perturbed orientation of the side chains due to the steric constraints introduced by the oligo(ethylene glycol) substituents.

(26) TABLE-US-00002 TABLE 2 Selected IR data for 1a-c monomers and poly-1a-c solutions (dichloromethane, 30 mM, 25 C.). NH stretch NH stretch CO amide I CO amide I Com- (cm.sup.1) (cm.sup.1) (cm.sup.1) (cm.sup.1) pounds monomer polymer monomer polymer (DL)-IAA .sup.3416.sup.a .sup.3252.sup.a .sup.1688.sup.a .sup.1657.sup.a 1a 3421 3268 1689 1656 1b 3420 3267 1690 1656 1c 3421 3269 1690 1657 .sup.aExtracted from ref..sup.20
Water Solubility and Thermo-responsive Behavior

(27) As expected, the water solubility of poly-1a-c is directly related to the length of the oligo(ethylene glycol) fragments. Poly-1b-c were found to be highly water soluble, whereas poly-1a could not be dissolved or swollen, even in cold water (1-2 C.). Poly-1b-c exhibit almost identical CD spectra in aqueous and organic media, which confirms the stability of the helical poly(isocyanopeptide) core in water (FIG. 5).

(28) In good agreement with examples reported in the literature for non-linear PEG analogs, aqueous solutions of poly-1b-c presented thermally induce phase separations, in which the transition temperatures were related to the length of the ethylene glycol side chains and to the degree of polymerization of the isocyanopeptides. Due to the marked differences in the DP values of the polymers, direct comparison between the poly-1b and poly-1c systems is limited to general considerations. Only major trends will be discussed in the following paragraphs.

(29) As expected, demixing generally occurred at lower temperatures for poly-1b than for poly-1c and increasing the DP values of the chains resulted in a lowering of the transition temperatures (Table 1). In the case of 1 mg/mL aqueous solutions of poly-1c, chains with a DP of 700 start to aggregate above 50 C., leading to the precipitation of poly-1c. This transition could easily be followed by UV-Vis spectroscopy due to the increase in turbidity of the medium. The precipitation was found to be fully reversible; that is, a clear solution was recovered upon the cooling of the medium. Interestingly, for chains of higher DP (4400), the medium remained optically transparent over the whole range of temperatures explored. A clear transition could, however, be evidenced with the help of DLS measurements, which showed an abrupt change in the scattered light intensity above 42 C. (FIG. 6, Panel a). This observation was associated to the formation of an optically transparent hydrogel. Similar increases in scattered light intensities were observed for 1 mg/mL aqueous solutions of poly-1b, above 35 C. and 22 C. for chains of DP 2600 and 7300, respectively (FIG. 6, Panel b). These transitions were also associated with the formation of hydrogels.

(30) These sol-gel transitions were again fully reversible; that is, upon decreasing the temperature, fluid solutions were recovered from all hydrogels. Cycles could be repeated several times without noticeable changes in the gelation abilities of the polymer or without significant shifts in phase transition temperatures.

(31) Characterization of the hydrogels

(32) Due to the optical transparency of the hydrogels, CD spectra could be measured at an extended range of temperatures. In the case of poly-1c (DP4400), the intensity of the CD signal slightly decreased above 40 C. and reached a stable value around 50 C. (FIG. 7, I.sub.CD360 6.4%, I.sub.CD272 5.6%). Although irreversible, these very limited modifications in the CD spectrum of poly-1c support the idea of a global preservation of the helical polymeric structure within the hydrogel (which is formed above 42 C. for the considered chains). After increasing the temperature to 70 C., a marked change in the CD signal was observed and could be associated to the gel syneresis. This effect was only reversible for samples that were kept for a short period above 80 C. (<10 minutes). Longer annealing periods at 85 C. or higher, resulted in a drastic lowering of the CD intensity after cooling of the medium to room temperature. Similar behaviors were found for derivatives of 1b; the helical structure of the polyisocyanides was preserved in the gel phases up to 70 C.

(33) For poly-1b chains with the highest degree of polymerization (DP7300), the formation of a stable hydrogel at room temperature (22 C.-25 C.) allowed further exploration of its structure by using Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). As shown in FIG. 8, AFM samples prepared from 1 mg/mL poly-1b hydrogels exhibited an extended fibrillar network at room temperature. Such structures were observed both on Highly Ordered Pyrrolitic Graphite (HOPG) and on mica, indicating their surface-independent nature, and were reminiscent of the features observed in TEM pictures. On HOPG, a thick deposit unambiguously showed a collapsed fibrillar network, which proves the self-standing nature of the fibers (FIG. 8, Panel a). On mica, thinner layers of material could be deposited, which permitted the observation of isolated fibers (FIG. 8, Panels b and c). The lateral dimension of these structures is polydispersed, ranging from a few nm to several tens of nm.

(34) As shown in FIG. 9, these fibers resulted from the lateral association and the intertwinement of thinner fibrils, which seemed to be relatively homogeneous in their widths (regardless of the tip broadening effect; the apparent widths of the isolated fibrils are mostly between 20 nm and 25 nm). A closer look at these structures showed that the fibrils themselves resulted from the association of thinner chains, presumably the elemental poly-1b chains (FIG. 9, Panel a).

(35) When plotting heights versus widths (h,w) of the fibrillar structures observed on mica for 172 random sections, two groups of coordinates can be distinguished on the graph (FIG. 10). The sections were taken from four different pictures, each section was measured perpendicularly to the main axis of the structure considered. The height is defined as the highest point measured in the cross-section (even if it was not the center of the structure cross-section). Random accumulation of matter on certain points was not taken into account for this analysis (white dots in FIGS. 8 (panel c) and 9).

(36) The first is narrowly distributed around the couple (h,w=0.46 nm, 15.51 nm) and presumably corresponds to single poly-1b chain cross-sections (standard deviation: h=0.09 nm, w=2.92 nm). The lateral tip broadening effect is not corrected; therefore, the widths are exaggerated. In samples prepared by the spin coating of diluted chloroform solutions, isolated chains of poly-1c and poly-1b present similar apparent heights of 0.4 nm to 0.5 nm. The second group of coordinates is more dispersed with apparent heights comprised between 0.8 nm and 1.4 nm and with a rather broad range of width distributions, which corresponds to both the fibrils and the fiber cross-sections. Most of the isolated fibrils exhibit heights comprised between 0.8 nm and 1.1 nm and apparent widths inferior to 30 nm. Interestingly, the apparent height of these isolated fibrils is close to twice the medium apparent height of the supposed single polymer chains (0.46 nm). This suggests that the fibrils result from the close intertwining of at least two poly-1b chains and that these fibrils further aggregate into bundles to form the fibrous gel network. The highest cross-sections (>1.2 nm), which are mainly observed for bundles, may be due to the overlapping of several fibrils on the substrate or to their local intertwinement in the fibers.

(37) Most rigid and rod-like polymers are able to form gels in adequate conditions (liquid crystalline gel phase at high concentration)..sup.34 The low concentration (0.1 wt %) at which poly-1b-c formed hydrogels is, however, remarkable for macromolecules. A few examples of block polymers presenting critical gelation concentration of about 0.1 wt % have been described,.sup.35,36 but generally synthetic homopolymers rarely form physical gels below 1 wt %. In the case of the newly prepared poly-1b-c, the above-described hierarchical assembly of the poly[oligo(ethylene glycol)isocyanide]s explains the formation of strong hydrogels at rather low polymer concentrations. This assembly process is probably related to the secondary structure of the polymer chains and their associated stiffness. It is interesting to note that poly-1c chains with DP700 precipitate above the transition temperature, whereas chains with higher DP lead to the formation of hydrogels at the same polymer concentration. Therefore, it was assumed that a critical parameter for the formation of the hydrogel at low poly-1b-c concentrations would be the ratio between the polymer DP (i.e., the chain lengths) and the chain stiffness or the related persistence length. It is proposed that the gel network can only be formed if a significant amount of polymer chains reach sizes well above the persistence length of the polymers. This would permit the polymer chains to intertwine and to form elongated micro-fibrils, which further aggregate into bundles that are the base of the gel network. If it is considered as a first approximation that the persistence length of the oligo(ethylene glycol) coated poly(isocyanopeptide) poly-1b-c are similar to the persistence length of the parent poly-(DL)-IAA (76 nm), it is interesting to note that precipitation occurred for chains having a mean length close to that value (81 nm), whereas all the other materials exhibited mean lengths at least three times higher and also formed hydrogels. A more detailed study of the gelation mechanism of the oligo(ethylene glycol)-functionalized polyisocyanopeptides is currently under progress.

(38) Conclusion

(39) A new family of non-linear poly(ethylene glycol) analogs has been prepared based on a polyisocyanopeptide backbone. Using these stiff helical polymers, water-soluble materials could be obtained starting from triethylene glycol side chains. Due to the thermo-sensitive behavior of ethylene glycol side chains, these materials present a clear thermo-induced phase separation. An unexpected effect of the polymer chain length has been shown for these materials; the longest chains were found to be able to gelate water at low polymer concentration (0.1 wt %), the shortest chains simply precipitated in solution, unable to develop extended 3D networks at low polymer concentration. It is proposed that this is a general behavior for long, stiff (or semi-flexible) polymers for which the hydrophilicity can be tuned without modifying the general structure of the chains (i.e., in rigid structures, the chains do not collapse but rather aggregate laterally with other chains to form extended fibers) and might be used to design other low concentration, synthetic macro-gelators. The post-modification of functionalized analogs of these polymers with biomolecules is now being investigated.

EXAMPLES

(40) General

(41) Dichloromethane and chloroform were distilled over CaCl.sub.2. Tetrahydrofuran, diethyl ether and toluene were distilled from sodium, in the presence of benzophenone. Water was purified with a MILLIPORE MILLI-Q system, (mQ water 18.2 M). All other chemicals were used as received from the suppliers. Column chromatography was performed using silica gel (40 m to 60 m) purchased from Merck or silica gel (0.060 mm to 0.200 mm) provided by Baker. TLC analyses were carried out on silica 60 F.sub.254 coated glass obtained from Merck and the compounds were visualized using Ninhydrine or basic aqueous KMnO.sub.4 solutions. .sup.1H NMR and .sup.13C NMR spectra were recorded on a BRUKER AC-300 MHz instrument operating at 200 or 300 MHz and 75 MHz, respectively. FT-infrared spectra of the pure compounds were recorded on a ThermoMattson IR300 spectrometer equipped with a Harrick ATR unit. Solution IR spectroscopy was carried out in sealed KBr cuvette (1 mm) on a BRUKER Tensor 27 spectrometer operated with Opus software. Solutions of poly-1a-c and the respective isocyanides 1a-c were prepared in chloroform, tetrahydrofurane, or toluene at a concentration of 30 mM. Melting points were measured on a BCHI B-545 and are reported uncorrected. Mass spectrometry measurements were performed on a JEOL ACCUTOF instrument (ESI). Optical rotations were measured on a PERKINELMER 241 Polarimeter at room temperature and are reported in 10.sup.1 deg cm.sup.2 g.sup.1. CD spectra were recorded on a JASCO 810 instrument equipped with a Peltier temperature control unit. The cell was thermostated at 20 C. or heated/cooled within the desired temperature range at a temperature gradient of 1 C/minute. DLS measurements were carried out on a ZETASIZER Nano (Malvern Instruments) on non-filtered aqueous solutions (1 mg/mL) in mQ water. All the solutions were degassed (ultrasound bath 315 s). The measurement cell was heated/cooled within the desired temperature range at a temperature gradient of 1 C/minute. The polymers were dissolved and AFM experiments were performed using a dimension 3100 or Multimode microscope operated with nanoscope III or nanoscope IV control units (Digital Instruments). Solutions of poly-1a-c (10.sup.6 M in CHCl.sub.3) were spin-coated (1600 RPM) onto freshly cleaved Muscovite Mica to determine the contour length (Ln) of isolated polymers chains. Poly-1b hydrogels were deposited by direct contact with freshly cleaved HOPG or muscivite mica. All images were recorded with the AFM operating in TAPPING MODE in air at room temperature, with a resolution of 10241024 pixels, using moderate scan rates (1-1.5 lines/second). Commercial tapping-mode golden-coated silicon tips (NT-MDT) were used with a typical resonance frequency around 300 kHz. Polymer chain lengths were evaluated using NeuronJ plugin (v1.4.1 by E. Meijering) run on ImmageJ (v1.43I) software (W.S. Rasband, ImageJ, U. S. National Institutes of Health, Bethesda, Md., USA, on the World Wide Web at rsb.info.nih.gov/ij/, 1997-2009).

(42) The polymer chain heights were measured using the Nanoscope software (v6.14r1) from Digital Instruments. TEM micrographs were recorded on a JEOL JEM-1010 instrument.

Example 1

Materials and Reaction Conditions

Synthesis of 2-methyloxyethyloxyethyl-N-boc-(L)-alaninate (2a)

(43) Unless mentioned, CD spectra were taken from 1 mg/ml samples in freshly distilled dichloromethane or mQ water (18.2 M) on a JASCO J-810 spectrometer.

(44) The cell was thermostated at 25 C. with the JASCO peltier module or heated/cooled on the desired range of temperatures with a gradiant rate of 1 C/minute.

(45) NMR spectra were taken on BRUKER AVANCE 200 or 300 MHz. When recorded in deuterated chloroform, the chemical shifts were calibrated on TMS signal.

(46) IR spectra were taken on a TENSOR 27 spectrometer run with Opus (BRUKER Optics, Marne la Vallee, France), from 1 mg/ml solutions in chloroform.

(47) TAPPING MODE AFM measurements were conducted on a Dimension 3100 microscope (Digital Instruments, Santa Barbara, Calif.) controlled with nanoscope IV controller (Digital Instruments, Santa Barbara, Calif.).

(48) The measurements were done on mica samples prepared by spin coating solution of poly 1a-d in chloroform (0.5 mg/ml; 25 l) on freshly cleaved mica support.

(49) NSG-10 golden coated tips (NT-MDT, Moscov, Russia) were used to take the micrograph.

(50) 2-(2-Methoxyethoxy)ethanol (1.28 g, 10.5 mmol), 4-N,/N-(dimethyl)aminopyridine (128 mg, 1 mmol) and N-Boc-(L)-alanine (2 g, 10.5 mmol) were dissolved in freshly distilled CH.sub.2Cl.sub.2 (25 mL). The reaction mixture was cooled to 0 C. (ice bath) and dicyclohexylcarbodiimide (DCC, 2.39 g, 1.1 mmol) was added portionwise. The reaction mixture was stirred for 1 hour at 0 C. and then warmed up to room temperature over 2 hours. The dicyclohexyl urea was filtered off, washed with ethyl acetate (220 mL) and the solvents evaporated. Column chromatography (SiO.sub.2, 0.060 mm to 0.200 mm/1% MeOH in CH.sub.2Cl.sub.2) yielded compound 2a as a colorless to pale yellow oil (2.40 g, 8.3 mmol, 79%).

(51) .sup.1H NMR (CDCl.sub.3, 300 MHz): 5.14 (br s, 1H, NH); 4.32-4.28 (br m, 3H, CH(CH.sub.3), COOCH.sub.2); 3.71 (t, J=4.5 Hz, COOCH.sub.2CH.sub.2); 3.66-3.63 (m, 2H, CH.sub.2CH.sub.2OCH.sub.3); 3.56-3.53 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.45 (s, 9H, (CH.sub.3).sub.3C); 1.39 (d, J=7.2 Hz, 3H, CH(CH.sub.3)) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.8 (CH(CH.sub.3)COO); 154.5 (NHCOO); 79.2 (OC(CH.sub.3).sub.3); 71.4; 70.0 (COOCH.sub.2CH.sub.2); 68.4 (OCH.sub.2CH.sub.2O); 63.7 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 48.7 (CH(CH.sub.3)); 27.8 (OC(CH.sub.3).sub.3); 18.1 (CH(CH.sub.3)) FT-IR (cm.sup.1, ATR): 3352 (br s, NH); 2977, 2932, 2881, 2825 (CH); 1742 (CO ester); 1712 (CO carbamate); 1518 (NH carbamate); 1248 (CO carbamate), 1164 (CO carbamate, ester); 1109, 1067 (CO ethers) MS (ESI) : m/z ([M +Na].sup.+: C.sub.13H.sub.25NO.sub.6Na), calcd 291.17; found 291.1 [].sub.D.sup.20: 7.7 (c 0.81; CHCl.sub.3).

Example 2

Synthesis of 2-methyloxyethyloxyethyloxyethyloxyethyl-N-boc-(L)-alaninate (2b)

(52) This compound was synthesized according to the same procedure as described in Example 1.

(53) Compound 2b was prepared using the same procedure as described for the synthesis of 2a with the following reactants and solvents: 2-(2-(2-methoxyethoxy)ethoxy)ethanol (1.32 g, 8 mmol), 4-N,N-(dimethyl)aminopyridine (100 mg, 0.81 mmol), N-Boc-(L)-Alanine (1.51 g, 8 mmol) and DCC (1.67 g, 8.1 mmol) in dichloromethane (25 mL). Column chromatography (SiO.sub.2, 0.060 mm to 0.200 mm/1% MeOH in CH.sub.2Cl.sub.2) yielded compound 2b as a colorless to pale yellow oil (2.35 g, 7.1 mmol, 86%).

(54) .sup.1H NMR (CDCl.sub.3, 300 MHz): 5.17 (br s, 1H, NH); 4.35-4.27 (br m, 3H, CH(CH.sub.3), COOCH.sub.2); 3.71 (t, J=4.5 Hz, 2H, COOCH.sub.2CH.sub.2); 3.67-3.63 (br s, 6H, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.57-3.53 (m, 2H, CH.sub.2CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.45 (s, 9H, (CH.sub.3).sub.3C); 1.39 (d, J=7.2 Hz, CH(CH.sub.3)) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.8 (CH(CH.sub.3)COO); 154.6 (NHCOO); 79.2 (OC(CH.sub.3).sub.3); 71.4; 70.1; 70.0; 68.4; 63.7 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 48.7 (CH(CH.sub.3)); 27.8 (OC(CH.sub.3).sub.3); 18.1 (CH(CH.sub.3)) FT-IR (cm.sup.1, ATR): 3341 (NH); 2976, 2876 (CH); 1744 (CO ester); 1714 (CO carbamate); 1556 (NH carbamate); 1249 (CO carbamate); 1164 (CO carbamate, ester); 1108, 1068 (CO ethers) MS (ESI): m/z ([M +Na].sup.+: C.sub.15H.sub.29NO.sub.7Na), calcd 358.18; found 358.1 [].sub.D.sup.20: 0.97 (c 0.30; CHCl.sub.3).

Synthesis of 2-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)ethyl-N-Boc-(L)-alaninate (2c)

(55) Compound 2c was prepared using the same procedure as described for the synthesis of 2a with the following reactants and solvents: 2-(2-(2-methoxyethoxy)ethoxy)ethanol (4.04 g, 19.4 mmol), 4-N,N-(dimethyl)aminopyridine (0.27 g, 2,22 mmol), N-Boc-(L)-Alanine (3.66 g, 19.4 mmol) and DCC (4.0 g 19.4 mmol) in dichloromethane (25 mL). Column chromatography (SiO.sub.2, 0.060 mm to 0.200 mm/1% MeOH in CH.sub.2Cl.sub.2) yielded compound 2c as a pale yellow oil (6.88 g, 18.1 mmol, 93%).

(56) .sup.1H NMR (CDCl.sub.3, 300 MHz): 5.18 (br s, 1H, NH); 4.33-4.27 (br m, 3H, CH(CH.sub.3), COOCH.sub.2); 3.71 (t, J=4.8 Hz, 2H, COOCH.sub.2CH.sub.2); 3.66-3.63 (m, 10H, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.56-3.53 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.44 (s, 9H, C(CH.sub.3).sub.3); 1.39 (d, J=7.0 Hz, 3H, CH(CH.sub.3)) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.8 (CH(CH.sub.3)COO); 154.6 (NHCOO); 79.2 (OC(CH.sub.3).sub.3); 71.4; 70.1; 70.0; 68.4; 63.7 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 48.7 (CH(CH.sub.3)); 27.8 (OC(CH.sub.3).sub.3); 18.1 (CH(CH.sub.3)) FT-IR (cm.sup.1, ATR): 3337 (NH); 2975, 2935, 2879 (CH); 1743 (CO ester); 1712 (CO carbamate); 1520 (NH carbamate); 1249 (CO carbamate); 1165 (CO carbamate, ester); 1107, 1069 (CO ethers) MS (ESI): m/z ([M +Na].sup.+: C.sub.17H.sub.33NO.sub.8Na), calcd 402.21; found 402.1 [].sub.D.sup.20: 5.4 deg (c 1.04; CHCl.sub.3).

Example 3

Synthesis of 2-methyloxyethyloxyethyl-(L)-alaninamido-(D)-alaninate (3a)

(57) Compound 2a (2.0 g, 6.8 mmol) was treated with HCl (20 mL, 2 M) in ethyl acetate at room temperature. The deprotection was followed by TLC. When no protected compound remained (1 hour), the solvent was evaporated under reduced pressure. The crude material was dissolved in tertbutyl alcohol (10 mL), which was subsequently evaporated (two times). The residual tertbutyl alcohol was removed by azeotropic distillation with CH.sub.2Cl.sub.2 and the crude product was used without further purification for the next coupling reaction.

(58) The deprotected compound, 1-hydroxy-benzotriazole hydrate (HOBt, 1.03 g, 6.9 mmol) and N-Boc-(D)-alanine (1.29 g, 6.8 mmol) were suspended in freshly distilled CH.sub.2Cl.sub.2 (50 mL), N,N-diisopropyl-N-ethylamine (DIPEA, 1.2 mL) was added dropwise and the mixture was stirred at room temperature until almost all the solids were dissolved. The mixture was cooled down to 0 C. (ice bath), and DCC (1.41 g, 6.9 mmol) was added portionwise. The reaction mixture was stirred at 0 C. for 1 hour and then allowed to slowly warm up to room temperature in 3 hours. The dicyclohexyl urea was removed by filtration and washed with ethyl acetate (220 ml). The solvent was evaporated and the desired compound was purified via column chromatography (SiO.sub.2 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2 2% MeOH) to yield 3a as a colorless to pale yellow oil (2.01 g, 5.5 mmol, 82%).

(59) .sup.1H NMR (CDCl.sub.3, 300 MHz): 6.87 (br s, 1H, CH(CH.sub.3)CONH); 5.24 (br s, 1H, OCONH); 4.59 (quint, J=7.2 Hz, 1H, CH(CH.sub.3)COO); 4.35-4.21 (m, 3H, CH(CH.sub.3)CONH, COOCH.sub.2); 3.70 (t, J=4.5 Hz, 2H, COOCH.sub.2CH.sub.2); 3.65-3.63 (m, 2H, CH.sub.2CH.sub.2OCH.sub.3); 3.56-3.54 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.45 (s, 9H, (CH.sub.3).sub.3C); 1.42 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO); 1.36 (d, J=6.9 Hz, 3H, CH(CH.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.2 (CH(CH.sub.3)COO); 171.8 (CH(CH.sub.3)CONH); 155.1 (NHCOO); 79.6 (OC(CH.sub.3).sub.3); 71.4; 70.0 (COOCH.sub.2CH.sub.2); 68.4; 63.9 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 49.4; 47.6 (CH(CH.sub.3)); 27.8 (OC(CH.sub.3).sub.3); 17.7 (2CH(CH.sub.3)) FT-IR (cm.sup.1, ATR): 3317 (br s, NH); 2977, 2932, 2881, 2828 (CH); 1739 (CO ester); 1710 (CO carbamate); 1664 (amide I); 1512 (NH carbamate/amide II); 1246 (CO carbamate), 1199 (CO ester); 1162 (CO carbamate, ester); 1129, 1109, 1055 (CO ethers). MS (ESI): m/z ([M +Na].sup.+; C.sub.16H.sub.30N.sub.2O.sub.7Na), calcd 385.20; found 385.2 [].sub.D.sup.20: +25.4 deg (c 0.81; CHCl.sub.3).

Example 4

Synthesis of 2-methyloxyethyloxyethyloxyethyl-(L)-alaninamido-(D)-alaninate (3b)

(60) This compound was synthesized according to the same procedure as described in Example 3.

(61) Compound 3b was prepared using the same procedure as described for the synthesis of 3a with the following reactants and solvents: 2b (2.10 g, 6.3 mmol), HOBt (0.965 g, 6.3 mmol), DCC (1.32 g, 6.4 mmol), N-Boc-(D)-Alanine (1.19 g, 6.3 mmol) and DIPEA (1.29 mL) in dichloromethane (20 mL). Column chromatography (SiO.sub.2 0.060-0.200 mm/CH.sub.2Cl.sub.2-2% MeOH) yielded 3b as a pale yellow oil (2.0 g, 5.0 mmol, 79%).

(62) .sup.1H NMR (CDCl.sub.3, 200 MHz): 6.95 (br d, J=6.9 Hz, 1H, CH(CH.sub.3)CONH); 5.30 (br d, J=6 Hz, 1H, OCONH); 4.59 (quint, J=7.4 Hz, 1H, CH(CH.sub.3)COO); 4.32-4.20 (br m, 3H, CH(CH.sub.3)CONH, COOCH.sub.2); 3.71 (t, J=5.0 Hz, 2H, COOCH.sub.2CH.sub.2); 3.67-3.63 (m, 6H, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.58-3.55 (m, 2H, CH.sub.2OCH.sub.3);3.38 (s, 3H, OCH.sub.3); 1.45 (s, 9H, (CH.sub.3).sub.3C); 1.42 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO); 1.37 (d, J=7.0 Hz, 3H, CH(CH.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.7 (CH(CH.sub.3)COO); 172.4 (CH(CH.sub.3)CONH); 155.5 (NHCOO); 80.0 (OC(CH.sub.3).sub.3); 71.9; 70.6; 70.6; 70.5; 68.8; 64.4 (COOCH.sub.2CH.sub.2); 59.0 (OCH.sub.3); 49.9; 48.0 (CH(CH.sub.3)); 28.3 (OC(CH.sub.3).sub.3); 18.2 (CH(CH.sub.3)CONH); 18.2 (CH(CH.sub.3)COO) FT-IR (cm.sup.1, ATR): 3314 (NH); 2976, 2928, 2876 (CH); 1739 (CO ester); 1713 (CO carbamate); 1667 (amide I); 1517 (NH carbamate, amide II); 1247 (CO carbamate); 1199 (CO ester); (1162 (CO carbamate, ester); 1104, 1055 (CO ethers) MS (EST): m/z ([M+Na].sup.+: C.sub.18H.sub.34N.sub.2O.sub.8Na), calcd 429.22; found 429.2 [].sub.D.sup.20: +18.3 deg (c 1.07; CHCl.sub.3).

Example 5

Synthesis of 2-methyloxyethyloxyethyloxyethyloxyethyl-(L)-alaninamido-(D)-alaninate (3c)

(63) This compound was synthesized according to the same procedure as described in Example 3.

(64) Compound 3c was prepared using the same procedure as described for the synthesis of 3a with the following reactants and solvents: 2c (3.03 g, 8.0 mmol), HOBt (1.23 g, 8.1 mmol), DCC (1.67 g, 8.1 mmol), N-Boc-(D)-Alanine 1.53 g, 8.1 mmol) and DIPEA (1.4 mL) in dichloromethane (25 mL). Column chromatography (SiO.sub.2 0.060-0.200 mm/CH.sub.2Cl.sub.2 2% MeOH) yielded 3b as a pale yellow oil (2.88 g, 6.4 mmol, 80%).

(65) .sup.1H NMR (CDCl.sub.3, 300 MHz) : 6.87 (br s, 1HCH(CH.sub.3)CONH); 5.19 (br s, 1H, OCONH); 4.62-4.53 (m, 1H, CH(CH.sub.3)COO); 4.33-4.20 (br m, 3H, CH(CH.sub.3)CONH, COOCH.sub.2); 3.71-3.63 (m, 12H, COOCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.56-3.51 (br m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.45 (s, 9H, C(CH.sub.3).sub.3); 1.41 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO); 1.35 (d, J=7.0 Hz, 3H, CH(CH.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.2 (CH(CH.sub.3)COO); 171.8 (CH(CH.sub.3)CONH); 155.0 (NHCOO); 79.6 OC(CH.sub.3).sub.3); 71.4; 70.1; 70.0; 68.4; 63.9 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 49.5; 47.6 (CH(CH.sub.3)); 27.8 (OC(CH.sub.3).sub.3); 17.7 (2CH(CH.sub.3)) FT-IR (cm.sup.1, ATR): 3319 (NH); 2976, 2931, 2879 (CH); 1740 (CO ester); 1711 (CO carbamate); 1665 (amide I); 1513 (NH carbamate, amide II); 1248 (CO carbamate); 1200 (CO ester); 1163 (CO carbamate, ester); 1105, 1067 (CO ethers) MS (ESI): m/z ([M +Na].sup.+: C.sub.20H.sub.38N.sub.2O.sub.9Na), calcd 473.25; found 473.2 [].sub.D.sup.20: +17.4 (c 0.81; CHCl.sub.3).

Example 6

Synthesis of 2-methyloxyethyloxyethyl-(L)-alaninamido-(D)-formalaninate (4a)

(66) Compound 3a (1.26 g, 3.5 mmol) was treated with HCl (15 mL, 2 M) in ethyl acetate at room temperature. The deprotection was followed by TLC. When no protected compound remained (1 hour) the solvent was evaporated under reduced pressure. The crude material was dissolved in tertbutyl alcohol (10 mL), which was subsequently evaporated (two times). The residual tertbutyl alcohol was removed via azeotropic distillation with CH.sub.2Cl.sub.2 and the crude product was used without further purification.

(67) The deprotected compound was dissolved in ethyl formate (50 ml), sodium formate (1.89 g, 27.8 mmol) was added and the mixture was refluxed for 4 hours under argon. After cooling down to room temperature, the mixture was filtered off to remove the sodium formate, and the solvent was evaporated under reduced pressure. Column chromatography (SiO.sub.2 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2 4% MeOH) yielded 4a as a white solid (0.89 g, 3.1 mmol, 86%).

(68) .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.18 (s, 1H, CHO); 6.98 (br d, J=7.5 Hz, CH(CH.sub.3)CONH); 6.87 (br d, J=6 Hz, 1H, NHCHO); 4.69-4.45 (m, 2H, CH(CH3)CONH, CH(CH.sub.3)COO); 4.36-4.20 (m, 2H, COOCH.sub.2); 3.69-3.67 (m, 2H, COOCH.sub.2CH.sub.2); 3.63-3.61 (m, 2H, CH.sub.2CH.sub.2OCH.sub.3); 3.57-3.55 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.43 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO); 1.39 (d, J=7.0 Hz, 3H, CH(C0H.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.1 (CH(CH.sub.3)COO); 171.0 (CH(CH.sub.3)CONH); 160.9 (NHCHO; 71.4; 69.9 (COOCH.sub.2CH.sub.2); 68.5; 63.8 (COOCH.sub.2CH.sub.2); 58.4 (OCH.sub.3); 47.7; 46.6 (CH(CH.sub.3)); 17.3 (CH(CH.sub.3)CONH); 17.1 (CH(CH.sub.3)COO) FT-IR (cm.sup.1, ATR): 3287 (NH); 2980, 2941, 2881, 2816 (CH); 1740 (CO ester); 1653 (amide I, formamide I); 1524 (amide II, formamide II); 1200 (CO ester); 1160 (CO ester); 1135, 1105, 1058 (CO ethers) MS (ESI): m/z ([M +Na].sup.+: C.sub.12H.sub.22N.sub.2O.sub.6Na), calcd 313.14; found 313.3 [].sub.D.sup.20: +52.7 (c 1.4; CHCl.sub.3).

Example 7

Synthesis of 2-methyloxyethyloxyethyloxyethyl-(L)-alaninamido-(D)-formalaninate (4b)

(69) Compound 4b was prepared using the same procedure as described for the synthesis of 4a with the following reactants: 3b (0.85 g, 2.1 mmol), sodium formate (1.14 g, 16.8 mmol), ethyl formate (30 mL). Column chromatography (SiO.sub.2 0.060-0.200 mm/CH.sub.2Cl.sub.2 4% MeOH) yielded 4b as a pale yellow oil (0.63 g, 1.8 mmol, 89%).

(70) .sup.1H NMR (CDCl.sub.3, 200 MHz): 8.18 (s, 1H, CHO); 7.36 (br d, J=7.4 Hz, 1H, CH(CH.sub.3)CONH); 7.15 (br d, J=7.5 Hz, 1H, NHCHO); 4.70-4.52 (m, 2H, CH(CH3)CONH, CH(CH.sub.3)COO); 4.35-4.19 (m, 2H, COOCH.sub.2); 3.73-3.60 (m, 8H, COOCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.58-3.53 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.42 (d, J=7.4 Hz, 3H, CH(CH.sub.3)COO); 1.40 (d, J=7.2 Hz, 3H, CH(CH.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.6 (CH(CH.sub.3)COO); 171.7 (CH(CH.sub.3)CONH); 161.5 (NHCHO); 71.8; 70.6; 70.5; 70.4; 68.9; 64.4 (COOCH.sub.2CH.sub.2); 58.9 (OCH.sub.3); 48.3; 47.2 (CH(CH.sub.3)); 18.00 (CH(CH.sub.3)CONH); 17.8 (CH(CH.sub.3)COO) FT-IR (cm.sup.1, ATR): 3284 (NH); 3059, 2981, 2876 (CH); 1739 (CO ester); 1654 (amide I, formamide I); 1525 (amide II, formamide II); 1201 (CO ester); 1160 (CO ester); 1135, 1099 (CO ethers) MS (ESI): m/z ([M+Na].sup.+: C.sub.14H.sub.26N.sub.2O.sub.7Na), calcd 357.17; found 357.3 [].sub.D.sup.20: +46.0 deg (c 0.81; CHCl.sub.3).

Example 8

Synthesis of 2-methyloxyethyloxyethyloxyethyloxyethyl-(L)-alaninamido-(D)-formalaninate (4c)

(71) Compound 4c was prepared using the same procedure as described for the synthesis of 4a with the following reactants: 3c (2.0 g, 4.4 mmol), sodium formate (2.40 g, 35.2 mmol), ethyl formate (50 mL). Column chromatography (SiO.sub.2 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2 4% MeOH) yielded 4c as a pale yellow oil (1.41 g, 3.7 mmol, 85%).

(72) .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.18 (s, 1H, CHO); 7.20 (d, J=7.5 Hz, 1H, CH(CH.sub.3)CONH); 7.02 (d, J=7.8 Hz, 1H, NHCHO); 4.70-4.62 (m, 1H, CH(CH.sub.3)COO); 4.57-4.50 (m, 1H, CH(CH.sub.3)CONH); 4.35-4.19 (m, 2H, COOCH.sub.2); 3.70-3.62 (m, 12H, CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.56-3.35 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.42 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO); 1.40 (d, J=7.5 Hz, 3H, CH(CH.sub.3)CONH) .sup.13C NMR (CDCl.sub.3, 75 MHz): 172.1 (CH(CH.sub.3)COO); 171.1 (CH(CH.sub.3)CONH); 161.0; 71.4; 70.1; 70.0; 69.9; 68.4; 63.9 (COOCH.sub.2CH.sub.2); 58.4 (OCH.sub.3); 47.8; 46.7 (CH(CH.sub.3)); 17.4 (CH(CH.sub.3)CONH); 17.3 (CH(CH.sub.3)CONH) FT-IR (cm.sup.1, ATR): 3300 (NH); 2982, 2875 (CH); 1738 (CO ester); 1658 (amide I, formamide I); 1530 (Amide II, formamide II); 1202 (CO ester); 1165 (CO ester); 1098 (CO ethers) MS (ESI): m/z ([M+Na].sup.+: C.sub.16H.sub.30N.sub.2O.sub.8Na), calcd 401.19; found 401.3 [].sub.D.sup.20: +42.4 (c 0.95; CHCl.sub.3).

Example 9

Synthesis of 2-methyloxyethyloxyethyl-(L)alaninamido-(D)-i socyanylalanine (1a)

(73) Formamide 3a (300 mg, 1.03 mmol) and N-methylmorpholine (570 L, 524 mg, 5.15 mmol) were dissolved in freshly distilled CH.sub.2Cl.sub.2 (50 mL) and cooled down to 30 C. (dry ice-acetone bath). A solution of diphosgene (334 L, 205 mg, 1.03 mmol) in freshly distilled CH.sub.2Cl.sub.2 (5 mL) was added dropwise to the solution over 5 minutes. After stirring the reaction mixture at 30 C. for another 10 minutes (during which the mixture turned yellow), the reaction was quenched by addition of solid sodium bicarbonate (3 g). The suspension was vigorously stirred at 30 C. for 10 minutes and then was warmed up to room temperature. The crude mixture was poured on a silica short plug (silica 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2) without further work up, and the desired compound eluted with CH.sub.2Cl.sub.2 25% acetonitrile to lead to the compound 1a as a white solid that was recrystallized from EtOH/diisopropylether (190 mg, 0.70 mmol, 68%).

(74) .sup.1H NMR (CDCl.sub.3, 300 MHz): 6.96 (br s, 1H, NH); 4.59 (q, J=7.2 Hz, 1H, CH(CH.sub.3)COO); 4.34 (br t, J=4.5 Hz, 2H, COOCH.sub.2); 4.27 (q, J=6.9 Hz, CH(CH.sub.3)CONH); 3.73 (br t, J=4.5 Hz, 2H, COOCH.sub.2CH.sub.2); 3.67-3.63 (m, 2H, CH.sub.2CH.sub.2OCH.sub.3); 3.56-3.51 (m, 2H, CH.sub.2OCH.sub.3); 3.39 (s, 3H, OCA; 1.66 (d, J=6.6 Hz, 3H, CNCH(CH.sub.3)); 1.49 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO) .sup.13C NMR (CDCl.sub.3, 75 MHz): 171.5 (CH(CH.sub.3)COO); 165.2 (CH(CH.sub.3)CONH); 161.0 (NC); 76.8; 71.4; 70.1; 68.4; 64.2 (COOCH.sub.2CH.sub.2); 58.6 (OCH.sub.3); 52.9; 49.1 (CH(CH.sub.3)); 19.2 (CH(CH.sub.3)CONH); 17.6 (CH(CH.sub.3)CONH) FT-IR (cm.sup.1, ATR): 3313 (NH); 2887, 2880 (CH); 2140 (NC isocyanide); 1740 (CO ester); 1678 (CO, amide I); 1537 (NH, amide II); 1200 (CO, ester); 1106, 1024 (CO ethers) HRMS (ESI): m/z ([M +Na].sup.+: C.sub.12H.sub.201\1.sub.2O.sub.5Na), calcd 295.12617; found 295.12699 [].sub.D.sup.20: 2 (c 0.29; CH.sub.2Cl.sub.2) mp: 53 C.

Example 10

Synthesis of 2-methyloxyethyloxyethyloxyethyl-(L)alaninamido-(D)-isocyanylalanine (1b)

(75) Compound 1b was prepared using the same procedure as described for the synthesis of 1a with the following reactants: 4b (1.5 g, 4.49 mmol), N-methylmorpholine (1.23 mL, 1.13 g, 11.1 mmol), diphosgene (461 L, 755 mg, 3.83 mmol) in dichloromethane (200 mL+10 mL). Column chromatography (SiO.sub.2 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2- 25% acetonitrile) yielded 1b as a pale yellow gel (1.15 g, 3.65 mmol, 81%).

(76) .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.06 (br s, 1H, NH); 4.60 (quint, J=7.2 Hz, 1H, CH(CH.sub.3)COO); 4.35-4.26 (br m, 3H, COOCH.sub.2, CH(CH.sub.3)CONH); 3.72 (t, J=4.5 Hz, 2H, COOCH.sub.2CH.sub.2); 3.66 (br s, 6H, OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.59-3.54 (m, 2H, CH.sub.2OCH.sub.3); 3.38 (s, 3H, OCH.sub.3); 1.66 (d, J=6.6 Hz, 3H, CH(CH.sub.3)CONH); 1.47 (d, J=6.9 Hz, 3H, CH(CH.sub.3)COO) .sup.13C NMR (CDCl.sub.3, 75 MHz): 171.5 (CH(CH.sub.3)COO); 165.3 (CH(CH.sub.3)CONH); 160.8 (NC); 78.8; 71.4; 70.2; 70.1; 70.0; 68.5; 64.2 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 52.8; 48.0 (CH(CH.sub.3)); 19.2 (CH(CH.sub.3)CONH); 17.6 (CH(CH.sub.3)CONH) FT-IR (cm.sup.1, ATR): 3312 (NH); 2885, 2878 (CH); 2140 (NC isocyanide); 1740 (CO ester); 1681 (CO, amide I); 1536 (NH, amide II); 1200 (CO, ester); 1099, 1025 (CO ethers) HRMS (ESI): m/z ([M +Na].sup.+: C.sub.14H.sub.24N.sub.2O.sub.6Na), calc 339.15285; found 339.15321 [].sub.D.sup.20: 10 deg (c 0.70; CH.sub.2Cl.sub.2).

Example 11

Synthesis of 2-methyloxyethyloxyethyloxyethyloxyethyl-(L)alaninamido-(D)-isocyanylalanine (1c)

(77) Compound 1c was prepared using the same procedure as described for the synthesis of 1a with the following reactants: 4c (526 mg, 1.39 mmol), N-methylmorpholine (384 L, 354 mg, 3.5 mmol), diphosgene (143 L, 235 mg, 1.19 mmol) in dichloromethane (200 mL+10 mL). Column chromatography (SiO.sub.2 0.060 mm to 0.200 mm/CH.sub.2Cl.sub.2 25% acetonitrile) yielded 1c as a pale yellow viscous oil (402 mg, 1.11 mmol, 80%).

(78) .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.00 (d, J=6.3 Hz, 1H, CH(CH.sub.3)CONH); 4.62-4.52 (m, 1H, CH(CH.sub.3)COO); 4.32-4.21 (m, 3H, COOCH.sub.2, CH(CH.sub.3)CONH); 3.70 (t, J=4.8 Hz, 2H, COOCH.sub.2CH.sub.2); 3.63.sup.-3.61 (m, 10H, CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3); 3.54-3.51 (m, 2H, CH.sub.2OCH.sub.3); 3.36 (s, 3H, OCH.sub.3); 1.63 (d, J=6.9 Hz, 3H, CH(CH.sub.3)CONH); 1.46 (d, J=7.2 Hz, 3H, CH(CH.sub.3)COO) .sup.13C NMR (CDCl.sub.3, 75 MHz) : 171.5 (CH(CH.sub.3)COO); 165.4 (CH(CH.sub.3)CONH); 160.5 (NC); 78.1; 71.4; 70.1; 70.0; 68.3; 64.2 (COOCH.sub.2CH.sub.2); 58.5 (OCH.sub.3); 52.9; 52.8; 48.1 (CH(CH.sub.3)); 19.2 (CH(CH.sub.3)CONH); 17.5 (CH(CH.sub.3)CONH) FT-IR (cm.sup.1, ATR): 3313 (NH); 2880, 2875 (CH); 2140 (NC isocyanide); 1740 (CO ester); 1680 (CO amide I); 1531 (NH amide II); 1201 (CO ester); 1103, 1026 (CO ethers) HRMS (ESI): m/z ([M +Na].sup.+: C.sub.16H.sub.28N.sub.2O.sub.6Na), calc 383.17889; found 383.17942 [].sub.D.sup.20: 5 deg (c 0.51; CH.sub.2Cl.sub.2).

Example 12

Synthesis of poly(2-methyoxywthyloxyethyloxyethloxy-(L)-alaninamido-(D)-aisocyanylalanine)

(79) To a solution of monomer (40 mg, 15 mmol) in freshly distilled toluene (3 mL) was added a solution of Ni(ClO4)2.Math.H2O (0.147 mol/L, 10 L) in toluene-30% methanol. The reaction mixture was vigorously stirred under air in a sealed flask, for two hours. The solvent was evaporated and the crude polymer redissolved in CHCl13 (3 mL). It was precipitated against diethyl ether (10 mL) and collected by centrifugation (3500 rpm, 7 min). The pale yellow precipitate was redissolved in CHC13 (3 mL) and precipitated against diethyl ether (10 mL) and collected by centrifugation two other times before being dried under vacuum to yield polymer (29.2 mg, 10.9 mmol, 73%) as a yellow-brown glassy solid.

(80) .sup.1H NMR (CDCl3, 300 MHz): 3.74-3.36 (br m, 13H); 1.60 (br s, 6H); FT-IR (cm.sup.1, ATR): 3263 (NH); 2927, 2880 (CH); 1740 (CO); 1656 (CO amide I); 1531 (NH amide II); 1214 (CO ester); 1108 (CO ethers) []D20: +75 (c 0.03; CH2Cl2) Mn: 478 KDa Mw: 716 KDa.

(81) Compound 1b (40 mg, XX) was dissolved in freshly distilled toluene (2 ml) and a solution of Ni(ClO.sub.4).sub.2.Math.H.sub.2O previously dissolved in a mixture of toluene and methanol (2-1) was added. The reaction mixture was stirred vigorously for 4 hours. The viscous solution was diluted with tetrahydrofurane (4 ml) and precipitated against diethyl ether (15 ml). The precipitate was collected via centrifugation (6 minutes, 4000 rpm) and the supernatant discarded. The gel like material was redissolved with tetrahydrofuran (6 ml) and precipitated against diethylether (15 ml). The cycle was repeated three times to yield a colorless to pale yellow glassy solid.

Example 13

Polymerization of Compound 1c

(82) Compound 1c was polymerized following the same procedure as compound 1b.

Example 14

Co-polymerization of Compounds 1b and 1c

(83) A mixture of both compounds 1b and 1c was treated with Ni9ClO4)2 following the same procedure as for compound 1b.

Example 15

Co-polymerization of Compounds 1c and 1d

(84) The copolymerization of 1c and 1d was conducted following the same procedure as in Example 14.

Example 16

Influence of the Solvent Used in the Polymerization Process

(85) The same monomers were used as in the examples described above.

(86) TABLE-US-00003 [Ni(ClO.sub.4).sub.2H.sub.2O] (mol %) 1.sup.a 0.1.sup.a 0.01.sup.b yield Mw.sup.c yield Mw.sup.c yield Mw.sup.c (%) (Da) PDI.sup.c (%) (Da) PDI.sup.c (%) (Da) PDI.sup.c toluene 90 505000 3.01 88 nd.sup.d nd.sup.d 78 nd.sup.d nd.sup.d tetrahydrofuran 83 554000 2.79 38 652000 2.30 0.4 nd.sup.e nd.sup.e dichloromethane 57 145000 1.88 28 144000 1.86 5 122000 1.88 methanol 51 248000 2.75 4 72000 1.42 0 reaction conditions: .sup.aisocyanide 20 mg/ml, catalyst dissolved in methanol-toluene 1-2 mixtures, 4 hours, RT .sup.bisocyanide 20 mg/ml, catalyst dissolved in methanol-toluene 1-2 mixtures, 12 hours, RT .sup.cResults obtained by analytical gel permeation chromatography, ReproGel column (300 8 mm, 5 m, linear, Dr Maisch Gmbh, Ammerbuch-Entrigen, Germany) run at 35 C. with THF-0.25% tetrabutyl ammonium bromide. Calibration used: poly (ethylene glycol) standards. .sup.dnot determined: substantial fraction to all polymer above the column resolution limit (2,000,000 Da). .sup.enot determined: to low material quantity collected to run a proper analysis.

Example 17

Synthesis of Polymer poly-1a

(87) To a solution of 1a (40 mg, 15 mmol) in freshly distilled toluene (3 mL) was added a solution of Ni(ClO.sub.4).sub.2.Math.H.sub.2O (0.147 mol/L, 10 L) in toluene-30% methanol. The reaction mixture was vigorously stirred under air in a sealed flask for two hours. The solvent was evaporated and the crude polymer redissolved in CHCl.sub.3 (3 mL). It was precipitated against diethyl ether (10 mL) and collected by centrifugation (3500 rpm, 7 minutes). The pale yellow precipitate was redissolved in CHCl.sub.3 (3 mL) and precipitated against diethyl ether (10 mL) and collected by centrifugation two other times before being dried under vacuum to yield poly-1a (29.2 mg, 10.9 mmol, 73%) as a yellow-brown glassy solid.

(88) .sup.1H NMR (CDCl.sub.3, 300 MHz): 3.74-3.36 (br m, 13H); 1.60 (br s, 6H); FT-IR (cm.sup.1, ATR): 3263 (NH); 2927, 2880 (CH); 1740 (CO); 1656 (C0 amide I); 1531 (NH amide II); 1214 (CO ester); 1108 (CO ethers) [].sub.D.sup.20: +75 (c 0.03; CH.sub.2Cl.sub.2) Mn: 478 KDa Mw: 716 KDa.

Synthesis of Polymer poly-1b

(89) Poly-1b was prepared using the same procedure as described for poly-1a, except that tetrahydrofuran was used to redissolve the polymer during the purification. The following reactants were used: 1b (40 mg, 12.6 mmol), Ni(ClO.sub.4).sub.2.H.sub.2O solution (0.126 mol/L, 10 L), in toluene (2 mL). Purification of the crude mixture yielded poly-1b (33.7 mmol, 10.7 mmol, 85%) as a deep yellow glassy solid.

(90) .sup.1H NMR (CDCl.sub.3, 300 MHz): 3.68-3.34 (b rm, 17H); 1.58 (br s, 6H); FT-IR (cm.sup.1, ATR): 3260 (NH); 2917, 2875 (CH); 1740 (CO); 1657 (CO amide I); 1530 (NH amide II); 1210 (CO ester); 1105 (CO ethers) [].sub.D.sup.20: +105 (c 0.03; CH.sub.2Cl.sub.2) Mn: 830 KDa Mw: 1327 KDa.

(91) 1b (41 mg, 12.9 mmol), Ni(ClO.sub.4).sub.2.H.sub.2O solution (1.29 mmol/L, 10 L), in toluene (2 mL). Purification of the crude mixture yielded poly-1b (32.0 mmol, 10.1 mmol, 78%) as a pale yellow glassy solid.

(92) .sup.1H NMR (CDCl.sub.3, 300 MHz): 3.68-3.34 (br m, 17H); 1.58 (br s, 6H); FT-IR (cm.sup.1, ATR): 3260 (NH); 2917, 2875 (CH); 1740 (CO); 1657 (CO amide I); 1530 (NH amide II); 1210 (CO ester); 1105 (CO ethers) Mn: 2306 KDa Mw: 3458 KDa.

Synthesis of Polymer poly-1c

(93) Poly-1c was prepared using the same procedure as described for poly-1b. The following reactants were used: 1c (50.1 mg, 13.8 mmol), Ni(ClO.sub.4).sub.2.H.sub.2O solution (0.138 mol/L, 10 L), in toluene (2.5 mL). Purification of the crude mixture yielded poly-1c (45.2 mg, 12.5 mmol, 90%) as a deep yellow glassy solid.

(94) .sup.1H NMR (CDCl.sub.3, 300 MHz): 3.71-3.35 (br m, 21H); 1.63 (br s, 6H); FT-IR (cm.sup.1, ATR): 3261 (NH); 2917, 2879 (CH); 1740 (CO); 1656 (CO amide I); 1529 (NH amide II); 1213 (CO ester); 1109 (CO ethers) [].sub.D.sup.20: +175 (c 0.01; CH.sub.2Cl.sub.2); [].sub.D.sup.20: +169 (c 0.03; H.sub.2O) Mn: 249 KDa Mw: 323 KDa.

(95) 1c (56.3 mg, 15.6 mmol), Ni(ClO.sub.4).sub.2.H.sub.2O solution (1.56 mmol/L, 10 L), in toluene (2.8 mL). Purification of the crude mixture yielded poly-1b (43.9 mmol, 12.2 mmol, 78%) as a pale yellow glassy solid.

(96) .sup.1H NMR (CDCl.sub.3, 300 MHz): 3.71-3.35 (brm, 14H); 1.63 (brs, 6H); FT-IR (cm.sup.1, ATR): 3260 (NH); 2917, 2875 (CH); 1740 (CO); 1657 (CO amide I); 1530 (NH amide II); 1210 (CO ester); 1105 (CO ethers) Mn: 1589 KDa Mw: 2702 KDa.

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