Synthesis and polymerization of conveniently substituted 6-amino-6-deoxy-D-galactonic acid derivatives

Methyl 6-azido-6-deoxy-2,3:4,5-di-O -isopropylidene- D -galactonate ( 1 ), prepared in two steps from D -galactono-1,4-lactone, was hydrolyzed to the corresponding acid 2 , which was esterified with phenol (PhOH) in the presence of dicyclohexylcarbodiimide (DCC) to give the phenyl ester 3 . Hydrogenolysis of the azide function of 3 yielded the hydrochloride derivative of the amino acid 4 . Compound 4 is conveniently substituted for the polycondensation; therefore, this reaction was conducted using DMF as solvent and N , N -diisopropylethylamine as a basic catalyst. The MALDI-TOF mass spectrum of the isolated product ( 5 ) indicated that this was a mixture of the cyclic trimer (M + Na + , 793.7) and linear oligomers (from the tetramer to the tetradecamer). To favor the linear polymerization the dimeric amino acid 10 was prepared starting from 2 . Thus, hydrogenation of 2 gave the amino acid 6 , and treatment of 2 with pentachlorophenol-DCC gave the pentachlorophenyl ester 7 . Coupling of 6 with 7 in the presence of DCC gave the dimer 8 , which was converted (PhOH, DCC) into 9 . Hydrogenolysis of 9 afforded the dimeric amino acid 10 , which was polymerized under the conditions employed for 4 . The resulting polyamide 11 was highly soluble in common organic solvents, and its molecular weight was established by MALDI-TOF MS and size exclusion chromatography ( M w = 2700).


Introduction
The synthesis of biodegradable polymers is a topic of current intensive research. 1Biodegradable polymers are environmentally friendly 2 and have found a wide range of practical uses, mainly as biomedical materials. 3For example, polyhydroxyalkanoates are employed as biodegradable suture strings 4 and as scaffolds for the controlled delivery of drugs. 5Similarly, the synthesis of polyamides more hydrophylic and degradable than industrial nylons has attracted considerable attention. 1,6,7Readily available, renewable natural products are the preferred substrates for the preparation of biodegradable polyamides.Amongst the natural regrowing resources, carbohydrates constitute particularly convenient starting materials, due to their abundance and configurational diversity. 6,8Thus, aldaric acids have been frequently employed for the synthesis of polyhydroxylated nylon 6,6 and related polyhydroxypolyamides.These investigations have been pioneered by Ogata et al., 9 who studied the polycondensation of ethyl galactarate with alkylenediamines.More recently it has been reported the synthesis and properties of polyhydroxypolyamides derived from D-glucaric, 10 meso-xylaric, 10,11 meso-galactaric 10,11 and Dmannaric acids. 10,12Also activated glucaric and galactaric acid derivatives were condensed with α,ω-diaminoalkyl polydimethylsiloxanes to give carbohydrate-segmented silicone polyamides, 13 and aminosaccharide-derived polymers having urea, urethane and amide linkages have been synthesized from glycosylamines. 14Stereoregular AABB type polyamides have been also prepared from 1,6-diaminoalditols and mannaric acid 15 or dioic acids. 16n contrast to the numerous reports (cited above) on the synthesis of polyhydroxylated nylon 6,6, only a few preparations of polyhydroxy derivatives of nylon 6 have been described.For the synthesis of such nylon 6 analogs, sugar amino acids have been employed as precursors.Sugar amino acids are currently exploited as peptidomimetics and as building blocks for the construction of new biomaterials. 17Thus, sugar-based polyamides have been synthesized from amino acids derived from pentoses 18 or from D-glucose or D-glucosamine, 19 and linear oligomers were obtained on polymerization of protected 6-amino-6-deoxy-D-allonate. 20We have also described the synthesis of sugar amino acids 21,22 and amino acids from other natural products 23,24 and their polymerization reactions were studied.For example, polycondensation of 6-amino-6deoxy-2,3,4,5-tetra-O-methyl-Dand L-galactonic acids 22 led to stereoregular polyamides having a relatively high molecular weight. 25We report here the attempted polymerization of 2,3:4,5-di-O-isopropylidene derivatives of 6-amino-6-deoxy-D-galactonic acid.The resulting products possess removable protecting groups that could facilitate the preparation of free polyhydroxylated analogs of nylon 6.

Results and Discussion
Methyl 6-azido-6-deoxy-2,3:4,5-di-O-isopropylidene-D-galactonate (1) was synthesized from commercially available D-galactono-1,4-lactone in two steps, in about 80% overall yield. 22ydrolysis of the ester group of 1 with KOH in MeOH-water gave the crystalline acid 2, isolated in 97% yield (Scheme 1).The carboxylic acid group of 2 was converted into the phenyl ester by reaction with phenol in the presence of dicyclohexylcarbodiimide (DCC) and p-toluenesulfonic acid. 26Replacement of the original methyl group in 1 by phenyl was effected to increase the reactivity of the ester function in the nucleophylic attack of the amine, during the polycondensation.

Scheme 1
The hydrogenolysis of the azide group of 3 was conducted using a mixture of EtOAc-MeOH-CHCl 3 as solvent.The hydrogen chloride released by hydrogenation of chloroform led to the hydrochloride derivative 4, without affecting the acid labile isopropylidene groups. 27Compound 4 constitutes a suitable monomer for the polycondensation as it contains in the same molecule an amino and an activated carboxylic acid.However, it should be taken into account that in ωamino acids intramolecular condensation with formation of the lactam competes with the growing chain to give a linear polymer. 28Thus, attempted polymerization of active esters of tetra-O-methyl-6-amino-6-deoxy-D-gluconic 19 and D-galactonic 25 acids led to lactam formation.These monomers, compared to 4 should have a higher degree of flexibility, facilitating the lactamization, whereas the formation of the seven-member lactone ring from 4 should be more difficult because of the constrain of two dioxolane rings fused to the sugar chain.Thereore, the polymerization of 4 was attempted, using N,N-dimethylformamide (DMF) as solvent and N,N'diisopropylethylamine (DIPEA) to release the amine from its hydrochloride derivative.Analysis of the product isolated from the polycondensation reaction by MALDI-TOF mass spectrometry revealed formation of polymeric species.However, this spectrum exhibited a rather particular pattern of signals (Fig. 1), showing a strong peak at m/z = 793.7 and a distribution of less intense signals at higher m/z values.These signals were separated, as expected, by 257 mass units, which corresponds to the mass of the repeating unit of an open-chain polyamide.The MS data suggests that 5 is a mixture of oligomers having as major component (~50%) the cyclic trimer (M + Na + , calculated 794.4), and that the minor products are mainly open chain oligomers having m/z values from 1100 (M + Na + + MeOH, tetramer) to 3670 (tetradecamer).In agreement with the MS, the NMR spectra of the mixture 5 showed clearly that the highest signals maintained a good correlation with the NMR spectra reported for the cyclic trimer of the amino acid 4, which had been synthesized and isolated by Fleet et al. 29,30 Also, in agreement with our results, they have shown that polycondensation of the free amino acid 6, under conditions that favored the cyclooligomerization led to the formation of cyclooligomers, being the cyclic trimer the main product.The molecular weight for the linear polymer constituent of 5 was estimated from its MALDI-TOF MS taking into account the m/z value of each peak and its relative intensity (M w = 2028, M n = 1830, and polydispersity = 1.11).In view of the previous results, we considered that the dimeric amino acid would provide a better opportunity for the preparation of a linear polyamide, as the formation of the cyclic trimer, obtained from the monomer 4, will be prevented.For the synthesis of the dimer 8, the hydrochloride derivative of the amino acid 6 was obtained by hydrogenolysis of 2. The other precursor of the dimmer, the pentachlorophenyl ester 7, was prepared by treatment of 2 with pentachlorophenol in the presence of DCC.Reaction of the amino group of 6, using DIPEA as catalyst, with the active ester of 7 gave the expected dimer 8, which was employed crude for the esterification with phenol.The phenyl ester 9 was obtained as a crystalline product in 51% yield for the two steps.

ISSN 1424-6376
Page 80 © ARKAT USA, Inc Catalytic hydrogenation of 9 in the presence of chloroform afforded the crystalline hydrochloride derivative 10, the desired amino acid dimer suitable for the homopolycondensation.This reaction was conducted in solution of DMF containing DIPEA for the activation of the amine.The mixture was purified by dilution with CH 2 Cl 2 and washing with water.The syrup obtained became solid upon standing, and this product (11) was analyzed by MALDI-TOF MS.The mass spectrum showed a distribution of open-chain oligomers having m/z values from 1069 (M + Na + , tetramer) to 4153 (M + Na + , hexadecamer).As expected, the m/z difference between vicinal peaks was 514, which corresponded to the mass of the dimer 10, once incorporated into the polyamide chain.On the basis of m/z values of each peak and its respective intensity the molecular weight of 11 was calculated (M w = 2288, M n = 2067, and polydispersity = 1.11).The 1 H-NMR spectrum of 11 showed a pattern of signals similar to that of 5.However, the 13 C-NMR spectrum of 5 showed a resonance at 74.9 ppm, signal which was absent in the spectrum of 11.Interestingly, similar high field resonances (assigned to C-2) were observed for the cyclic trimer 29 (74.95 ppm) and cyclic tetramer 30 (74.4 ppm) derivatives of 6; whereas in the respective spectra of the linear oligomers, the highest field resonances for carbons bonded to oxygen appeared at 76.5-77.0ppm. 30This result seems to bring additional support to the structure assigned for the polycondensation products 5 and 11.
Finally, the molecular weight of 5 and 11 was determined using size exclusion chromatography (SEC).Interestingly, compound 5 showed a main peak corresponding to M w = 2546 and a shoulder of lower M w , in agreement with the composition of the open chain and cyclic oligomers detected by MS.As expected, SEC of polyamide 11 gave a single peak, from which a M w = 2700 was calculated, this value was coincident with that determined by MS.
In contrast to regular nylons, polyamide 11 was highly soluble in practically all organic solvents, including chloroform.The enhanced solubility in this solvent is a rather common phenomenon for polyamides derived from carbohydrates. 18,19,25n summary, we described here convenient procedures for the synthesis of monomeric (4) and dimeric (10) sugar amino acids as precursors of polyamides.Polymerization of 4 led to a cyclic trimer and linear oligomers, whereas, polymerization of 10 led to the linear polyamide 11.
In both cases the linear polymer constituent of 5 and 11 showed relatively low molecular weights.We can conclude that the substitution pattern in the precursors exerts a strong influence in the molecular weight of the polymer obtained as, under the same conditions, polycondensation of the per-O-methyl analog of 10 led to a linear polyamide having a high M w . 25Polyamides of the type of 11 possessing readily removable O-protecting groups are convenient precursors of analogs of nylon 6 having free hydroxyl groups in their chains.

Experimental Section
General Procedures.Melting points (mp) were determined with a Fisher-Johns apparatus and are uncorrected.Analytical thin-layer chromatography (TLC) was performed on 0.2 mm silica gel 60 F 254 (Merck) aluminum supported plates.Visualization of the spots was effected by charring with a solution of 5% sulfuric acid in EtOH, containing 0.5% p-anisaldehyde.Column chromatography was performed with silica gel 60 (230-400 mesh, Merck).Optical rotations were measured with a Perkin-Elmer Model 343 digital polarimeter at 25 o C in the solvent indicated in each individual case.Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC ISSN 1424-6376 Page 82 © ARKAT USA, Inc 200 or Bruker AMX-500 spectrometers.TMS was employed as an internal standard for solutions in CDCl 3 .Size exclusion chromatography (SEC) was performed at room temperature with a Waters apparatus equipped with a Waters 2414 refractive index detector and Styragel HR (7.8 x 300 mm) Waters columns, using THF as mobile phase.The flow rate was 1mL min -1 .Calibration was based on polystyrene standards.

MALDI-TOF-MS Analysis.
Measurements were performed using a Shimadzu Kratos, Kompact MALDI 4 (pulsed extraction) laser desorption time-of-flight mass spectrometer (Shimadzu, Kyoto, Japan) equipped with a pulsed nitrogen laser (λ em = 337 nm; pulse width = 3 ns), tunable PDE and PSD (MS/MS device) modes.Experiments were performed using firstly the full range setting for laser firing position in order to select the optimal position for data collection, and secondly fixing the laser firing position in the sample sweet spots.The samples were irradiated just above the threshold laser power for obtaining molecular ions and with higher laser power for studying cluster formation.Thus, the irradiation used for producing a mass spectrum was analyte-dependent.Usually 100 spectra were accumulated.All samples were measured in the linear and the reflectron modes, and as routine in both the positive-and the negative-ion mode were detected, although no signals corresponding to the oligomers in the negative-ion mode were detected.As the signals of oligomer ions were not enough intense in the reflectron positive-ion mode, PSD experiments were not performed.2,5-Dihydroxybenzoic acid (DHB, gentisic acid) and 9H-pyrido [3,4-b]indole (nor-harmane) were employed as matrices.Matrix stock solutions were made by dissolving 2 mg of the selected compound in 0.2 mL of 1:1 (v/v) MeOH-H 2 O or in 0.2 mL of 2:3 (v/v) MeCN-H 2 O. Analyte solutions were freshly prepared by dissolving the samples (1 mg) in MeOH (0.5 mL).To prepare the analyte-matrix deposit the two methods reported by us were used: method A (thin layer deposit) and method B (mixture method). 31,32he resulting solid partially crystalline layers were found to be relative homogeneous in both cases.Nor-harmane and gentisic acid as matrices showed signals of higher quality by using Method A. The spectra were calibrated by using as external calibration reagents: (a) commercial proteins (neurotensin; insulin) with SA as matrix in positive-or negative-ion mode and (b) α-, βand γ-cyclodextrins with nor-harmane as matrix in positive-or in negative-ion mode.The Kratos Kompact calibration program was used.
The residue was diluted with water (80 mL) and extracted with CH 2 Cl 2 (3 x 50 mL).The aqueous phase was acidified with aqueous 5 % HCl to pH 3 and extracted again with CH 2 Cl 2 (3 x 50 mL).The organic phase was dried (MgSO 4 ) and concentrated to afford a solid, which was recrystallized from EtOAc-hexane to give 2 (0.47 g, 97%); mp 76 ºC; [α] D +40.7 (c 1.1, CHCl 3 ); To a solution of 2 (0.50 g, 1.66 mmol) and phenol (0.18 g, 1.90 mmol) in pyridine (5 mL), p-toluenesulfonic acid monohydrate (p-TSA, 0.017 g, 0.09 mmol) was added 26 and the mixture was stirred at room temperature for 15 min.Upon addition of dicyclohexylcarbodiimide (DCC, 0.48 g, 2.30 mmol) the stirring was continued for additional 24 h, when TLC (hexane-EtOAc 4:1) showed the complete conversion of the starting material (R f 0.12) into a main spot having R f 0.57.The mixture was diluted with ethyl ether (80 mL) and filtered.The filtrate was extracted with water (3 x 50 mL) and the organic phase was dried (MgSO 4 ), filtered and concentrated.The residue crystallized from EtOAc-hexane to give pure 3 (0.46 g, 74%); mp 60 ºC; [α] D +8.9 (c 1.The mixture was concentrated and the residue was dissolved in sat.aq.NaHCO 3 (100 mL).The solution was extracted with CH 2 Cl 2 (3 x 100 mL) and the aqueous phase was acidified with HCl (10%) to pH 3, and extracted with CH 2 Cl 2 (3 x 100 mL).The organic extract was dried (MgSO 4 ), filtered and concentrated to afford 8 as a colorless syrup.This product was used for the next step without further purification.
To a solution of crude 8 (0.56 g, 1.00 mmol) and phenol (0.14 g, 1.49 mmol) in anhydrous pyridine (7 mL) p-TSA monohydrate (0.01 g, 0.05 mmol) was added 26 and the solution was stirred at room temperature for 15 min.Upon addition of DCC (0.39 g, 1.80 mmol) the stirring was continued overnight.Monitoring of the reaction mixture by TLC (EtOAc) showed the conversion of 8 (R f 0.10) into a main spot (R f 0.80).The mixture was diluted with water