Patent Application: US-201414577038-A

Abstract:
the present invention relates to a process for the preparation of oligo - functionalized polyisocyanopeptides comprising the steps of functionalizing an isocyanopeptide with oligo - side chains and subsequently polymerizing the oligo - alkylene glycol - functionalized isocyanopeptides . several isocyanopeptides may be functionalized with various linear or non - linear oligo - 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 .

Description:
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 . non - linear poly ( ethylene glycol ) ( peg ) analogs have recently attracted a great deal of attention for the development of innovative water - soluble materials . 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 . 2 so far , most non - linear peg analogs have been derived from vinyl , 3 , 4 ( meth ) acrylate , 5 , 6 , 7 , 8 , 9 , 10 and styrene 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 12 and the copper - catalyzed huisgen 1 , 3 - dipolar cycloaddition , 13 or through the direct polymerization of crown - ether appended precursors . 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 . 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 . 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 . 15 , 16 , 17 , 18 , 19 the introduction of peptide - containing side chains leads to materials with unprecedented stiffness . 20 , 21 this effect has been attributed to the development of an intramolecular hydrogen - bonding network between the peptide pendants , which adopt a β - sheet - like arrangement along the helical poly ( imine ) core . 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 . 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 . 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 , 26 whereas three ethylene glycol units were required in the case of the more hydrophobic styrene derivatives . 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 ( fig2 ). 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 %). acid - induced polymerization is the historical method for the preparation of poly ( isocyanide ) s . 27 in the case of dialananyl - isocyanides , chains with an exceptionally high degree of polymerization ( dp & gt ; 10000 ) can be obtained , but this is strongly dependent on the stereochemistry of the dipeptide fragments ; 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 + ] 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 . 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 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 4 ) 2 . 6h 2 o ], 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 . the nickel - catalyzed polymerization of isocyanides can be greatly influenced by the solvent used , both in terms of yields 17 and final polymer structure . 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 ( fig3 ). the relative polymerization rates ( kp ) according to the different solvents were found to follow the order kp [ toluene ]& gt ; kp [ dichloromethane ]& gt ; 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 . 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 . monomers 1a - c were dissolved in toluene ( 0 . 030 - 0 . 300 mol / l ) and subsequently treated with methanolic aliquots of ni ( clo 4 ) 2 . 6h 2 o 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 poly - 1c & lt ; dp poly - 1a & lt ; dp 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 . a determined 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 ; b determined from absorption measurements at 450 nm ( uv , solution at 1 mg / ml ); poly ( isocyanodialaninyl methyl ester ) possesses a very characteristic signature ( i . e ., an intense cotton effect centered at λ = 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 - π * transition of the imine groups , which are trapped in a pseudo 4 1 helical symmetry , and interact with the strong dipole that can develop along the polymer backbone ( as a result of the ideal β - sheet - like packing of the peptidic pendants ). 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 λ = 272 nm and a smaller negative component centered at λ = 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 ( fig4 ). comparable cd signals have previously been reported for the polymerization of heptyne - functionalized ( d )- isocyanoalanyl -( l )- alanyl esters . 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 - π * 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 , n — h and c ═ o amide 1 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 n — h stretching bands are slightly less shifted than expected for the optimal β - sheet - like packing , as observed in poly -( dl )- iaa ( vn - h ˜ 3268 vs . 3252 cm − 1 , respectively ) and are very similar to the values observed for the heptyne - functionalized polymers synthesized previously . 33 it is , therefore , proposed that poly - 1a - c present the classical 4 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 . 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 ( fig5 ). 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 . 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 . ( fig6 , 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 ( fig6 , panel b ). these transitions were also associated with the formation of hydrogels . 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 . 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 ( dp ˜ 4400 ), the intensity of the cd signal slightly decreased above 40 ° c . and reached a stable value around 50 ° c . ( fig7 , δi cd360 ˜ 6 . 4 %, δi 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 . (& lt ; 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 . for poly - 1b chains with the highest degree of polymerization ( dp ˜ 7300 ), 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 fig8 , 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 ( fig8 , panel a ). on mica , thinner layers of material could be deposited , which permitted the observation of isolated fibers ( fig8 , panels b and c ). the lateral dimension of these structures is polydispersed , ranging from a few nm to several tens of nm . as shown in fig9 , 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 ( fig9 , panel a ). 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 ( fig1 ). 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 fig8 ( panel c ) and 9 ). 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 : δh = 0 . 09 nm , δw = 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 (& gt ; 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 . most rigid and rod - like polymers are able to form gels in adequate conditions ( liquid crystalline gel phase at high concentration ). 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 , 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 dp ˜ 700 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 . 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 . dichloromethane and chloroform were distilled over cacl 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 mω ). 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 254 coated glass obtained from merck and the compounds were visualized using ninhydrine or basic aqueous kmno 4 solutions . 1 h nmr and 13 c 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 büchi ® b - 545 and are reported uncorrected . mass spectrometry measurements were performed on a jeol ® accutof ® instrument ( esi ). optical rotations were measured on a p erkin e lmer ® 241 polarimeter at room temperature and are reported in 10 − 1 deg cm 2 g − 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 3 × 15 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 − 6 m in chcl 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 1024 × 1024 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 ). 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 . unless mentioned , cd spectra were taken from 1 mg / ml samples in freshly distilled dichloromethane or mq water ( 18 . 2 mω ) on a jasco ® j - 810 spectrometer . 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 . nmr spectra were taken on bruker ® avance ® 200 or 300 mhz . when recorded in deuterated chloroform , the chemical shifts were calibrated on tms signal . 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 . 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 .). the measurements were done on mica samples prepared by spin coating solution of poly1a - d in chloroform ( 0 . 5 mg / ml ; 25 μl ) on freshly cleaved mica support . nsg - 10 golden coated tips ( nt - mdt , moscov , russia ) were used to take the micrograph . 2 -( 2 - methoxyethoxyl ) 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 2 cl 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 ( 2 × 20 ml ) and the solvents evaporated . column chromatography ( sio 2 , 0 . 060 mm to 0 . 200 mm / 1 % meoh in ch 2 cl 2 ) yielded compound 2a as a colorless to pale yellow oil ( 2 . 40 g , 8 . 3 mmol , 79 %). 1 h nmr ( cdcl 3 , 300 mhz ): 5 . 14 ( br s , 1h , — n h —); 4 . 32 - 4 . 28 ( br m , 3h , — c h ( ch 3 )—, — cooc h 2 —); 3 . 71 ( t , j = 4 . 5 hz , — cooch 2 c h 2 —); 3 . 66 - 3 . 63 ( m , 2h , — c h 2 ch 2 och 3 ); 3 . 56 - 3 . 53 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 45 ( s , 9h , ( c h 3 ) 3 c —); 1 . 39 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 )—) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 8 (— ch ( ch 3 ) c oo —); 154 . 5 (— nh c oo —); 79 . 2 (— o c ( ch 3 ) 3 ); 71 . 4 ; 70 . 0 (— cooch 2 c h 2 —); 68 . 4 (— o c h 2 c h 2 o —); 63 . 7 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 48 . 7 (— c h ( ch 3 )—); 27 . 8 (— oc ( c h 3 ) 3 ); 18 . 1 (— ch ( c h 3 )—) ft - ir ( cm − 1 , atr ): 3352 ( br s , n — h ); 2977 , 2932 , 2881 , 2825 ( c — h ); 1742 ( c ═ o ester ); 1712 ( c ═ o carbamate ); 1518 ( n — h carbamate ); 1248 ( c — o carbamate ), 1164 ( c — o carbamate , ester ); 1109 , 1067 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 13 h 25 no 6 na ), calcd 291 . 17 ; found 291 . 1 [ α ] d 20 : − 7 . 7 ( c 0 . 81 ; chcl 3 ). this compound was synthesized according to the same procedure as described in example 1 . compound 2b was prepared using the same procedure as described for the synthesis of 2a with the following reactants and solvents : 2 -( 2 -( 2 - methoxyethoxyl ) 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 2 , 0 . 060 mm to 0 . 200 mm / 1 % meoh in ch 2 cl 2 ) yielded compound 2b as a colorless to pale yellow oil ( 2 . 35 g , 7 . 1 mmol , 86 %). 1 h nmr ( cdcl 3 , 300 mhz ): 5 . 17 ( br s , 1h , — n h —); 4 . 35 - 4 . 27 ( br m , 3h , — c h ( ch 3 )—, — cooc h 2 —); 3 . 71 ( t , j = 4 . 5 hz , 2h , — cooch 2 c h 2 —); 3 . 67 - 3 . 63 ( br s , 6h , — oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 57 - 3 . 53 ( m , 2h , ch 2 c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 45 ( s , 9h , ( c h 3 ) 3 c —); 1 . 39 ( d , j = 7 . 2 hz , — ch ( c h 3 )—) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 8 (— ch ( ch 3 ) c oo —); 154 . 6 (— nh c oo —); 79 . 2 (— o c ( ch 3 ) 3 ); 71 . 4 ; 70 . 1 ; 70 . 0 ; 68 . 4 ; 63 . 7 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 48 . 7 (— c h ( ch 3 )—); 27 . 8 (— oc ( c h 3 ) 3 ); 18 . 1 (— ch ( c h 3 )—) ft - ir ( cm − 1 , atr ): 3341 ( n — h ); 2976 , 2876 ( c — h ); 1744 ( c ═ o ester ); 1714 ( c ═ o carbamate ); 1556 ( n — h carbamate ); 1249 ( c — o carbamate ); 1164 ( c — o carbamate , ester ); 1108 , 1068 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 15 h 9 no 7 na ), calcd 358 . 18 ; found 358 . 1 [ α ] d 20 : − 0 . 97 ( c 0 . 30 ; chcl 3 ). compound 2c was prepared using the same procedure as described for the synthesis of 2a with the following reactants and solvents : 2 -( 2 -( 2 - methoxyethoxyl ) 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 2 , 0 . 060 mm to 0 . 200 mm / 1 % meoh in ch 2 cl 2 ) yielded compound 2c as a pale yellow oil ( 6 . 88 g , 18 . 1 mmol , 93 %). 1 h nmr ( cdcl 3 , 300 mhz ): 5 . 18 ( br s , 1h , — n h —); 4 . 33 - 4 . 27 ( br m , 3h , — c h ( ch 3 )—, cooc h 2 —); 3 . 71 ( t , j = 4 . 8 hz , 2h , — cooch 2 c h 2 —); 3 . 66 - 3 . 63 ( m , 10h , — oc h 2 c h 2 oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 56 - 3 . 53 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 44 ( s , 9h , — c ( c h 3 ) 3 ); 1 . 39 ( d , j = 7 . 0 hz , 3h , — ch ( c h 3 )—) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 8 (— ch ( ch 3 ) c oo —); 154 . 6 (— nh c oo —); 79 . 2 (— o c ( ch 3 ) 3 ); 71 . 4 ; 70 . 1 ; 70 . 0 ; 68 . 4 ; 63 . 7 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 48 . 7 (— c h ( ch 3 )—); 27 . 8 (— oc ( c h 3 ) 3 ); 18 . 1 (— ch ( c h 3 )—) ft - ir ( cm − 1 , atr ): 3337 ( n — h ); 2975 , 2935 , 2879 ( c — h ); 1743 ( c ═ o ester ); 1712 ( c ═ o carbamate ); 1520 ( n — h carbamate ); 1249 ( c — o carbamate ); 1165 ( c — o carbamate , ester ); 1107 , 1069 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 17 h 33 no 8 na ), calcd 402 . 21 ; found 402 . 1 [ α ] d 20 : − 5 . 4 deg ( c 1 . 04 ; chcl 3 ). 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 2 cl 2 and the crude product was used without further purification for the next coupling reaction . 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 2 cl 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 ( 2 × 20 ml ). the solvent was evaporated and the desired compound was purified via column chromatography ( sio 2 0 . 060 mm to 0 . 200 mm / ch 2 cl 2 - 2 % meoh ) to yield 3a as a colorless to pale yellow oil ( 2 . 01 g , 5 . 5 mmol , 82 %). 1 h nmr ( cdcl 3 , 300 mhz ): 6 . 87 ( br s , 1h , — ch ( ch 3 ) con h —); 5 . 24 ( br s , 1h , — ocon h —); 4 . 59 ( quint , j = 7 . 2 hz , 1h , — c h ( ch 3 ) coo —); 4 . 35 - 4 . 21 ( m , 3h , — c h ( ch 3 ) conh —, — cooc h 2 —); 3 . 70 ( t , j = 4 . 5 hz , 2h , — cooch 2 c h 2 —); 3 . 65 - 3 . 63 ( m , 2h , — c h 2 ch 2 och 3 ); 3 . 56 - 3 . 54 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 45 ( s , 9h , ( c h 3 ) 3 c —); 1 . 42 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —); 1 . 36 ( d , j = 6 . 9 hz , 3h , — ch ( c h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 2 (— ch ( ch 3 ) c oo —); 171 . 8 (— ch ( ch 3 ) c onh —); 155 . 1 (— nh c oo —); 79 . 6 (— o c ( ch 3 ) 3 ); 71 . 4 ; 70 . 0 (— cooch 2 c h 2 —); 68 . 4 ; 63 . 9 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 49 . 4 ; 47 . 6 (— c h ( ch 3 )—); 27 . 8 (— oc ( c h 3 ) 3 ); 17 . 7 ( 2 ×— ch ( c h 3 )—) ft - ir ( cm − 1 , atr ): 3317 ( br s , n — h ); 2977 , 2932 , 2881 , 2828 ( c — h ); 1739 ( c ═ o ester ); 1710 ( c ═ o carbamate ); 1664 ( amide i ); 1512 ( n — h carbamate / amide ii ); 1246 ( c — o carbamate ), 1199 ( c — o ester ); 1162 ( c — o carbamate , ester ); 1129 , 1109 , 1055 ( c — o ethers ). ms ( esi ): m / z ([ m + na ] + : c 16 h 30 n 2 o 7 na ), calcd 385 . 20 ; found 385 . 2 [ α ] d 20 : + 25 . 4 deg ( c 0 . 81 ; chcl 3 ). this compound was synthesized according to the same procedure as described in example 3 . 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 2 0 . 060 - 0 . 200 mm / ch 2 cl 2 - 2 % meoh ) yielded 3b as a pale yellow oil ( 2 . 0 g , 5 . 0 mmol , 79 %). 1 h nmr ( cdcl 3 , 200 mhz ): 6 . 95 ( br d , j = 6 . 9 hz , 1h , — ch ( ch 3 ) con h —); 5 . 30 ( br d , j = 6 hz , 1h , — ocon h —); 4 . 59 ( quint , j = 7 . 4 hz , 1h , — c h ( ch 3 ) coo —); 4 . 32 - 4 . 20 ( br m , 3h , — c h ( ch 3 ) conh —, — cooc h 2 —); 3 . 71 ( t , j = 5 . 0 hz , 2h , — cooch 2 c h 2 —); 3 . 67 - 3 . 63 ( m , 6h , — oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 58 - 3 . 55 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 45 ( s , 9h , ( c h 3 ) 3 c —); 1 . 42 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —); 1 . 37 ( d , j = 7 . 0 hz , 3h , ch ( c h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 7 (— ch ( ch 3 ) c oo —); 172 . 4 (— ch ( ch 3 ) c onh —); 155 . 5 (— nh c oo —); 80 . 0 (— o c ( ch 3 ) 3 ); 71 . 9 ; 70 . 6 ; 70 . 6 ; 70 . 5 ; 68 . 8 ; 64 . 4 (— coo c h 2 ch 2 —); 59 . 0 (— o c h 3 ); 49 . 9 ; 48 . 0 (— c h ( ch 3 )—); 28 . 3 (— oc ( c h 3 ) 3 ); 18 . 2 (— ch ( c h 3 ) conh —); 18 . 2 (— ch ( c h 3 ) coo —) ft - ir ( cm − 1 , atr ): 3314 ( n — h ); 2976 , 2928 , 2876 ( c — h ); 1739 ( c ═ o ester ); 1713 ( c ═ o carbamate ); 1667 ( amide i ); 1517 ( n — h carbamate , amide ii ); 1247 ( c — o carbamate ); 1199 ( c — o ester ); ( 1162 ( c — o carbamate , ester ); 1104 , 1055 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 18 h 34 n 2 o 8 na ), calcd 429 . 22 ; found 429 . 2 [ α ] d 20 : + 18 . 3 deg ( c 1 . 07 ; chcl 3 ). this compound was synthesized according to the same procedure as described in example 3 . 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 2 0 . 060 - 0 . 200 mm / ch 2 cl 2 - 2 % meoh ) yielded 3b as a pale yellow oil ( 2 . 88 g , 6 . 4 mmol , 80 %). 1 h nmr ( cdcl 3 , 300 mhz ): 6 . 87 ( br s , 1h , — ch ( ch 3 ) con h —); 5 . 19 ( br s , 1h , — ocon h —); 4 . 62 - 4 . 53 ( m , 1h , — c h ( ch 3 ) coo —); 4 . 33 - 4 . 20 ( br m , 3h , — c h ( ch 3 ) conh —, — cooc h 2 —); 3 . 71 - 3 . 63 ( m , 12h , — cooch 2 c h 2 oc h 2 c h 2 oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 56 - 3 . 51 ( br m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 45 ( s , 9h , — c ( c h 3 ) 3 ); 1 . 41 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —); 1 . 35 ( d , j = 7 . 0 hz , 3h , ch ( c h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 2 (— ch ( ch 3 ) c oo —); 171 . 8 (— ch ( ch 3 ) c onh —); 155 . 0 (— nh c oo —); 79 . 6 (— o c ( ch 3 ) 3 ); 71 . 4 ; 70 . 1 ; 70 . 0 ; 68 . 4 ; 63 . 9 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 49 . 5 ; 47 . 6 (— c h ( ch 3 )—); 27 . 8 (— oc ( c h 3 ) 3 ); 17 . 7 ( 2 ×— ch ( c h 3 )—) ft - ir ( cm − 1 , atr ): 3319 ( n — h ); 2976 , 2931 , 2879 ( c — h ); 1740 ( c ═ o ester ); 1711 ( c ═ o carbamate ); 1665 ( amide i ); 1513 ( n — h carbamate , amide ii ); 1248 ( c — o carbamate ); 1200 ( c — o ester ); 1163 ( c — o carbamate , ester ); 1105 , 1067 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 20 h 38 n 2 o 9 na ), calcd 473 . 25 ; found 473 . 2 [ α ] d 20 : + 17 . 4 ( c 0 . 81 ; chcl 3 ). 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 2 cl 2 and the crude product was used without further purification . 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 2 0 . 060 mm to 0 . 200 mm / ch 2 cl 2 - 4 % meoh ) yielded 4a as a white solid ( 0 . 89 g , 3 . 1 mmol , 86 %). 1 h nmr ( cdcl 3 , 300 mhz ): 8 . 18 ( s , 1h , — c h o ); 6 . 98 ( br d , j = 7 . 5 hz , — ch ( ch 3 ) con h —); 6 . 87 ( br d , j = 6 hz , 1h , — n h cho ); 4 . 69 - 4 . 45 ( m , 2h , — c h ( ch3 ) conh —, — c h ( ch 3 ) coo —); 4 . 36 - 4 . 20 ( m , 2h , — cooc h 2 —); 3 . 69 - 3 . 67 ( m , 2h , — cooch 2 c h 2 —); 3 . 63 - 3 . 61 ( m , 2h , — c h 2 ch 2 och 3 ); 3 . 57 - 3 . 55 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — och 3 ); 1 . 43 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —); 1 . 39 ( d , j = 7 . 0 hz , 3h , ch ( c0 h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 1 (— ch ( ch 3 ) c oo —); 171 . 0 (— ch ( ch 3 ) c onh —); 160 . 9 (— nh c ho ; 71 . 4 ; 69 . 9 (— cooch 2 c h 2 —); 68 . 5 ; 63 . 8 (— coo c h 2 ch 2 —); 58 . 4 (— o c h 3 ); 47 . 7 ; 46 . 6 (— c h ( ch 3 )—); 17 . 3 (— ch ( c h 3 ) conh —); 17 . 1 (— ch ( c h 3 ) coo —) ft - ir ( cm − 1 , atr ): 3287 ( n — h ); 2980 , 2941 , 2881 , 2816 ( c — h ); 1740 ( c ═ o ester ); 1653 ( amide i , formamide i ); 1524 ( amide ii , formamide ii ); 1200 ( c — o ester ); 1160 ( c — o ester ); 1135 , 1105 , 1058 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 12 h 22 n 2 o 6 na ), calcd 313 . 14 ; found 313 . 3 [ α ] d 20 : + 52 . 7 ( c 1 . 4 ; chcl 3 ). 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 2 0 . 060 - 0 . 200 mm / ch 2 cl 2 - 4 % meoh ) yielded 4b as a pale yellow oil ( 0 . 63 g , 1 . 8 mmol , 89 %). 1 h nmr ( cdcl 3 , 200 mhz ): 8 . 18 ( s , 1h , — c h o ); 7 . 36 ( br d , j = 7 . 4 hz , 1h , — ch ( ch 3 ) con h —); 7 . 15 ( br d , j = 7 . 5 hz , 1h , — n h cho ); 4 . 70 - 4 . 52 ( m , 2h , — c h ( ch3 ) conh —, — c h ( ch 3 ) coo —); 4 . 35 - 4 . 19 ( m , 2h , — cooc h 2 —); 3 . 73 - 3 . 60 ( m , 8h , — cooch 2 c h 2 oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 58 - 3 . 53 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 42 ( d , j = 7 . 4 hz , 3h , — ch ( c h 3 ) coo —); 1 . 40 ( d , j = 7 . 2 hz , 3h , ch ( c h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 6 (— ch ( ch 3 ) c oo —); 171 . 7 (— ch ( ch 3 ) c onh —); 161 . 5 (— nh c ho ); 71 . 8 ; 70 . 6 ; 70 . 5 ; 70 . 4 ; 68 . 9 ; 64 . 4 (— coo c h 2 ch 2 —); 58 . 9 (— o c h 3 ); 48 . 3 ; 47 . 2 (— c h ( ch 3 )—); 18 . 00 (— ch ( c h 3 ) conh —); 17 . 8 (— ch ( c h 3 ) coo —) ft - ir ( cm − 1 , atr ): 3284 ( n — h ); 3059 , 2981 , 2876 ( c — h ); 1739 ( c ═ o ester ); 1654 ( amide i , formamide i ); 1525 ( amide ii , formamide ii ); 1201 ( c — o ester ); 1160 ( c — o ester ); 1135 , 1099 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 14 h 26 n 2 o 7 na ), calcd 357 . 17 ; found 357 . 3 [ α ] d 20 : + 46 . 0 deg ( c 0 . 81 ; chcl 3 ). 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 2 0 . 060 mm to 0 . 200 mm / ch 2 cl 2 - 4 % meoh ) yielded 4c as a pale yellow oil ( 1 . 41 g , 3 . 7 mmol , 85 %). 1 h nmr ( cdcl 3 , 300 mhz ): 8 . 18 ( s , 1h , — cho ); 7 . 20 ( d , j = 7 . 5 hz , 1h , — ch ( ch 3 ) con h —); 7 . 02 ( d , j = 7 . 8 hz , 1h , — n h cho ); 4 . 70 - 4 . 62 ( m , 1h , — c h ( ch 3 ) coo —); 4 . 57 - 4 . 50 ( m , 1h , — c h ( ch 3 ) conh —); 4 . 35 - 4 . 19 ( m , 2h , — cooc h 2 —); 3 . 70 - 3 . 62 ( m , 12h , — c h 2 oc h 2 c h 2 oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 56 - 3 . 35 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 42 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —); 1 . 40 ( d , j = 7 . 5 hz , 3h , ch ( c h 3 ) conh —) 13 c nmr ( cdcl 3 , 75 mhz ): 172 . 1 (— ch ( ch 3 ) c oo —); 171 . 1 (— ch ( ch 3 ) c onh —); 161 . 0 ; 71 . 4 ; 70 . 1 ; 70 . 0 ; 69 . 9 ; 68 . 4 ; 63 . 9 (— coo c h 2 ch 2 —); 58 . 4 (— o c h 3 ); 47 . 8 ; 46 . 7 (— c h ( ch 3 )—); 17 . 4 (— ch ( c h 3 ) conh —); 17 . 3 (— ch ( c h 3 ) conh —) ft - ir ( cm − 1 , atr ): 3300 ( n — h ); 2982 , 2875 ( c — h ); 1738 ( c ═ o ester ); 1658 ( amide i , formamide i ); 1530 ( amide ii , formamide ii ); 1202 ( c — o ester ); 1165 ( c — o ester ); 1098 ( c — o ethers ) ms ( esi ): m / z ([ m + na ] + : c 16 h 30 n 2 o 8 na ), calcd 401 . 19 ; found 401 . 3 [ α ] d 20 : + 42 . 4 ( c 0 . 95 ; chcl 3 ). formamide 3a ( 300 mg , 1 . 03 mmol ) and n - methylmorpholine ( 570 μl , 524 mg , 5 . 15 mmol ) were dissolved in freshly distilled ch 2 cl 2 ( 50 ml ) and cooled down to − 30 ° c . ( dry ice - acetone bath ). a solution of diphosgene ( 334 μl , 205 mg , 1 . 03 mmol ) in freshly distilled ch 2 cl 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 2 cl 2 ) without further work up , and the desired compound eluted with ch 2 cl 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 %). 1 h nmr ( cdcl 3 , 300 mhz ): 6 . 96 ( br s , 1h , — n h —); 4 . 59 ( q , j = 7 . 2 hz , 1h , — c h ( ch 3 ) coo —); 4 . 34 ( br t , j = 4 . 5 hz , 2h , — cooc h 2 —); 4 . 27 ( q , j = 6 . 9 hz , — c h ( ch 3 ) conh —); 3 . 73 ( br t , j = 4 . 5 hz , 2h , — cooch 2 c h 2 —); 3 . 67 - 3 . 63 ( m , 2h , — c h 2 ch 2 och 3 ); 3 . 56 - 3 . 51 ( m , 2h , — c h 2 och 3 ); 3 . 39 ( s , 3h , — oc h 3 ); 1 . 66 ( d , j = 6 . 6 hz , 3h , c ≡ nch ( c h 3 )—); 1 . 49 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —) 13 c nmr ( cdcl 3 , 75 mhz ): 171 . 5 (— ch ( ch 3 ) c oo —); 165 . 2 (— ch ( ch 3 ) c onh —); 161 . 0 (— n c ); 76 . 8 ; 71 . 4 ; 70 . 1 ; 68 . 4 ; 64 . 2 (— coo c h 2 ch 2 —); 58 . 6 (— o c h 3 ); 52 . 9 ; 49 . 1 (— c h ( ch 3 )—); 19 . 2 (— c h ( c h 3 ) conh —); 17 . 6 (— ch ( c h 3 ) conh —) ft - ir ( cm − 1 , atr ): 3313 ( n — h ); 2887 , 2880 ( c — h ); 2140 ( n ≡ c isocyanide ); 1740 ( c ═ o ester ); 1678 ( c ═ o , amide i ); 1537 ( n — h , amide ii ); 1200 ( c — o , ester ); 1106 , 1024 ( c — o ethers ) hrms ( esi ): m / z ([ m + na ] + : c 12 h 20 n 2 o 5 na ), calcd 295 . 12617 ; found 295 . 12699 [ α ] d 20 : − 2 ( c 0 . 29 ; ch 2 cl 2 ) mp : 53 ° c . 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 μl , 755 mg , 3 . 83 mmol ) in dichloromethane ( 200 ml + 10 ml ). column chromatography ( sio 2 0 . 060 mm to 0 . 200 mm / ch 2 cl 2 - 25 % acetonitrile ) yielded 1b as a pale yellow gel ( 1 . 15 g , 3 . 65 mmol , 81 %). 1 h nmr ( cdcl 3 , 300 mhz ): 7 . 06 ( br s , 1h , — n h —); 4 . 60 ( quint , j = 7 . 2 hz , 1h , — c h ( ch 3 ) coo —); 4 . 35 - 4 . 26 ( br m , 3h , — cooc h 2 —, — c h ( ch 3 ) conh —); 3 . 72 ( t , j = 4 . 5 hz , 2h , — cooch 2 c h 2 ); 3 . 66 ( br s , 6h , — oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 59 - 3 . 54 ( m , 2h , — c h 2 och 3 ); 3 . 38 ( s , 3h , — oc h 3 ); 1 . 66 ( d , j = 6 . 6 hz , 3h , ch ( c h 3 ) conh —); 1 . 47 ( d , j = 6 . 9 hz , 3h , — ch ( c h 3 ) coo —) 13 c nmr ( cdcl 3 , 75 mhz ): 171 . 5 (— ch ( ch 3 ) c oo —); 165 . 3 (— ch ( ch 3 ) c onh —); 160 . 8 (— n c ); 78 . 8 ; 71 . 4 ; 70 . 2 ; 70 . 1 ; 70 . 0 ; 68 . 5 ; 64 . 2 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 52 . 8 ; 48 . 0 (— c h ( ch 3 )—); 19 . 2 (— ch ( c h 3 ) conh —); 17 . 6 (— ch ( c h 3 ) conh —) ft - ir ( cm − 1 , atr ): 3312 ( n — h ); 2885 , 2878 ( c — h ); 2140 ( n ≡ c isocyanide ); 1740 ( c ═ o ester ); 1681 ( c ═ o , amide i ); 1536 ( n — h , amide ii ); 1200 ( c — o , ester ); 1099 , 1025 ( c — o ethers ) hrms ( esi ): m / z ([ m + na ] + : c 14 h 24 n 2 o 6 na ), calc 339 . 15285 ; found 339 . 15321 [ α ] d 20 : − 10 deg ( c 0 . 70 ; ch 2 cl 2 ). 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 μl , 354 mg , 3 . 5 mmol ), diphosgene ( 143 μl , 235 mg , 1 . 19 mmol ) in dichloromethane ( 200 ml + 10 ml ). column chromatography ( sio 2 0 . 060 mm to 0 . 200 mm / ch 2 cl 2 - 25 % acetonitrile ) yielded 1c as a pale yellow viscous oil ( 402 mg , 1 . 11 mmol , 80 %). 1 h nmr ( cdcl 3 , 300 mhz ): 7 . 00 ( d , j = 6 . 3 hz , 1h , — ch ( ch 3 ) con h —); 4 . 62 - 4 . 52 ( m , 1h , — c h ( ch 3 ) coo —); 4 . 32 - 4 . 21 ( m , 3h , — cooc h 2 —, — c h ( ch 3 ) conh —); 3 . 70 ( t , j = 4 . 8 hz , 2h , — cooch 2 c h 2 ); 3 . 63 - 3 . 61 ( m , 10h , — c h 2 c h 2 oc h 2 c h 2 oc h 2 ch 2 och 3 ); 3 . 54 - 3 . 51 ( m , 2h , — c h 2 och 3 ); 3 . 36 ( s , 3h , — oc h 3 ); 1 . 63 ( d , j = 6 . 9 hz , 3h , ch ( c h 3 ) conh —); 1 . 46 ( d , j = 7 . 2 hz , 3h , — ch ( c h 3 ) coo —) 13 c nmr ( cdcl 3 , 75 mhz ): 171 . 5 (— ch ( ch 3 ) c oo —); 165 . 4 (— ch ( ch 3 ) c onh —); 160 . 5 (— n c ); 78 . 1 ; 71 . 4 ; 70 . 1 ; 70 . 0 ; 68 . 3 ; 64 . 2 (— coo c h 2 ch 2 —); 58 . 5 (— o c h 3 ); 52 . 9 ; 52 . 8 ; 48 . 1 (— c h ( ch 3 )—); 19 . 2 (— ch ( c h 3 ) conh —); 17 . 5 (— ch ( c h 3 ) conh —) ft - ir ( cm − 1 , atr ): 3313 ( n — h ); 2880 , 2875 ( c — h ); 2140 ( n ≡ c isocyanide ); 1740 ( c ═ o ester ); 1680 ( c ═ o amide i ); 1531 ( n — h amide ii ); 1201 ( c — o ester ); 1103 , 1026 ( c — o ethers ) hrms ( esi ): m / z ([ m + na ] + : c 16 h 28 n 2 o 6 na ), calc 383 . 17889 ; found 383 . 17942 [ α ] d 20 : − 5 deg ( c 0 . 51 ; ch 2 cl 2 ). to a solution of monomer ( 40 mg , 15 mmol ) in freshly distilled toluene ( 3 ml ) was added a solution of ni ( clo4 ) 2 . h2o ( 0 . 147 mol / l , 10 μl ) 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 chcl3 ( 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 chcl3 ( 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 . 1h nmr ( cdcl3 , 300 mhz ): 3 . 74 - 3 . 36 ( br m , 13h ); 1 . 60 ( br s , 6h ); ft - ir ( cm − 1 , atr ): 3263 ( n — h ); 2927 , 2880 ( c — h ); 1740 ( c ═ o ); 1656 ( c ═ o amide i ); 1531 ( n — h amide ii ); 1214 ( c — o ester ); 1108 ( c — o ethers ) [ α ] d20 : + 75 ( c 0 . 03 ; ch2cl2 ) mn : 478 kda mw : 716 kda . compound 1b ( 40 mg , xx ) was dissolved in freshly distilled toluene ( 2 ml ) and a solution of ni ( clo 4 ) 2 . h 2 o 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 . compound 1c was polymerized following the same procedure as compound 1b . a mixture of both compounds 1b and 1c was treated with ni9clo4 ) 2 following the same procedure as for compound 1b . the copolymerization of 1c and 1d was conducted following the same procedure as in example 14 . the same monomers were used as in the examples described above . a isocyanide 20 mg / ml , catalyst dissolved in methanol - toluene 1 - 2 mixtures , 4 hours , rt b isocyanide 20 mg / ml , catalyst dissolved in methanol - toluene 1 - 2 mixtures , 12 hours , rt c results obtained by analytical gel permeation chromatography , reprogel column ( 300 × 8 mm , 5 μm , linear , dr maisch gmbh , ammerbuch - entrigen , germany ) run at 35 ° c . with thf - 0 . 25 % tetrabutyl ammonium bromide . calibration used : poly ( ethylene glycol ) standards . d not determined : substantial fraction to all polymer above the column resolution limit ( 2 , 000 , 000 da ). e not determined : to low material quantity collected to run a proper analysis . to a solution of 1a ( 40 mg , 15 mmol ) in freshly distilled toluene ( 3 ml ) was added a solution of ni ( clo 4 ) 2 . h 2 o ( 0 . 147 mol / l , 10 μl ) 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 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 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 . 1 h nmr ( cdcl 3 , 300 mhz ): 3 . 74 - 3 . 36 ( br m , 13h ); 1 . 60 ( br s , 6h ); ft - ir ( cm − 1 , atr ): 3263 ( n — h ); 2927 , 2880 ( c — h ); 1740 ( c ═ o ); 1656 ( c ═ o amide i ); 1531 ( n — h amide ii ); 1214 ( c — o ester ); 1108 ( c — o ethers ) [ α ] d 20 : + 75 ( c 0 . 03 ; ch 2 cl 2 ) mn : 478 kda mw : 716 kda . 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 4 ) 2 . h 2 o solution ( 0 . 126 mol / l , 10 μl ), 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 . 1 h nmr ( cdcl 3 , 300 mhz ): 3 . 68 - 3 . 34 ( b rm , 17h ); 1 . 58 ( br s , 6h ); ft - ir ( cm − 1 , atr ): 3260 ( n — h ); 2917 , 2875 ( c — h ); 1740 ( c ═ o ); 1657 ( c ═ o amide i ); 1530 ( n — h amide ii ); 1210 ( c — o ester ); 1105 ( c — o ethers ) [ α ] d 20 : + 105 ( c 0 . 03 ; ch 2 cl 2 ) mn : 830 kda mw : 1327 kda . 1b ( 41 mg , 12 . 9 mmol ), ni ( clo 4 ) 2 . h 2 o solution ( 1 . 29 mmol / l , 10 μl ), 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 . 1 h nmr ( cdcl 3 , 300 mhz ): 3 . 68 - 3 . 34 ( br m , 17h ); 1 . 58 ( br s , 6h ); ft - ir ( cm − 1 , atr ): 3260 ( n — h ); 2917 , 2875 ( c — h ); 1740 ( c ═ o ); 1657 ( c ═ o amide i ); 1530 ( n — h amide ii ); 1210 ( c — o ester ); 1105 ( c — o ethers ) mn : 2306 kda mw : 3458 kda . 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 4 ) 2 . h 2 o solution ( 0 . 138 mol / l , 10 μl ), 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 . 1 h nmr ( cdcl 3 , 300 mhz ): 3 . 71 - 3 . 35 ( br m , 21h ); 1 . 63 ( br s , 6h ); ft - ir ( cm − 1 , atr ): 3261 ( n — h ); 2917 , 2879 ( c — h ); 1740 ( c ═ o ); 1656 ( c ═ o amide i ); 1529 ( n — h amide ii ); 1213 ( c — o ester ); 1109 ( c — o ethers ) [ α ] d 20 : + 175 ( c 0 . 01 ; ch 2 cl 2 ); [ α ] d 20 : + 169 ( c 0 . 03 ; h 2 o ) mn : 249 kda mw : 323 kda . 1c ( 56 . 3 mg , 15 . 6 mmol ), ni ( clo 4 ) 2 . h 2 o solution ( 1 . 56 mmol / l , 10 μl ), 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 . 1 h nmr ( cdcl 3 , 300 mhz ): 3 . 71 - 3 . 35 ( brm , 14h ); 1 . 63 ( brs , 6h ); ft - ir ( cm − 1 , atr ): 3260 ( n — h ); 2917 , 2875 ( c — h ); 1740 ( c ═ o ); 1657 ( c ═ o amide i ); 1530 ( n — h amide ii ); 1210 ( c — o ester ); 1105 ( c — o ethers ) mn : 1589 kda mw : 2702 kda . 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