Alternating Poly(ester-anhydride) by Insertion Polycondensation

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Alternating Poly(Ester-Anhydride) by Insertion Polycondensation Moran Haim Zada, ARIJIT BASU, Tal Hagigit, Ron Schlinger, Michael Grishko, Alexander Kraminsky, Ezra Hanuka, and Abraham J. Domb Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00523 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Alternating Poly(Ester-Anhydride) by Insertion Polycondensation Moran Haim-Zada,a‡ Arijit Basu,a,‡ Tal Hagigitb, Ron Schlingerb, Michael Grishkoc, Alexander Kraminskyc , Ezra Hanukac, and Abraham J Domba* a

School of Pharmacy, Institute of Drug Research, Hebrew University of Jerusalem, Israel b c

Dexcel Pharma Technologies Ltd, Or-Akiva, Israel.

TAMI- Institute for Research & Development Ltd, Haifa Bay, Israel

E-mail: [email protected] ‡

Moran Haim-Zada and Arijit Basu

contributed equally

Abstract: We report on a synthetic method where polyanhydride is used as starting material and the ester monomers are inserted through complete esterification, leading to an alternating ester-anhydride copolymer. The molar ratio of ricinoleic acid (RA) and sebacic acid (SA) was optimized until polysebacic acid is completely converted to carboxylic acid terminated RASA and RA-SA-RA ester-dicarboxylic acids. These dimers and trimers were activated with acetic anhydride, polymerized under heat and vacuum to yield alternating RA-SA copolymer. The resulting alternating poly(ester-anhydride) have the RA at regular intervals. The regular occurrences of RA side chains prevent anhydride interchange, enhancing hydrolytic stability, which allows storage of the polymer at room temperature. Keywords: Alternating copolymer, stable polyanhydride, poly(ester-anhydride), ricinoleic acid, sebacic acid.

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ACS Paragon Plus Environment

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Introduction

Biodegradable polymers have been used as temporary sutures, plates, implantable devices, as carriers for drugs, and tissue engineering.1 Anhydride based biodegradable polymers have been used as carriers for controlled drug release. 2, 3 Introduction of ester bonds along the polyanhydride chain further extends the period of release.4, 5 Poly(ε-caprolactone) alkenyl succinic anhydride poly(ester anhydrides) with improved hydrolytic stability have been reported.6, 7 Polyanhydrides degrade over a wide range of physiological conditions and via surface erosion mechanism, which enables controlled release throughout their degradation process. However, widespread use of polyanhydrides is often limited due to their limited shelf life.8, 9 Therefore, stable polyanhydrides with extended shelf life are warranted for easy fabrication and storage. Poly(ester-anhydrides) are synthesized from natural dicarboxylic acids and hydroxyalkanoic acid, where drug release and degradation profile can be tailored by changing monomer ratio. One such poly(ester-anhydride) is synthesized from sebacic (SA) and ricinoleic acid (RA), used in drug delivery.10-15 Current state of the art poly(ester-anhydride) synthesis involve one-pot melt or solvent polycondensation, resulting in only random or block copolymers.5, 1620

In a poly(ester-anhydride), ideally all the hydroxyalkanoic acid should be incorporated

with even distribution of ester bonds along the polymer chain. However, incomplete esterification of the hydroxyalkanoic acids resulted in ester and anhydride blocks along the polymer chain. This leads to rapid hydrolysis and instability of the polymer. Complete esterification is critical, and is possible only when the monomer units are arranged alternately (ie. diacid and hydroxyalkanoic acid) throughout the polymer chain. 2


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Synthetic method ensuring proper control over polyester-anhydride architecture is a key for improved biodegradable polymers.21 Thus, we report here on a strategy to synthesize esteranhydride alternating copolymers, using RA and SA as monomers.

Scheme 1 Showing the reaction between polysebacic acid (PSA) and ricinoleic acid (RA). The reaction was continued until all RA has been consumed, monitored by 1H NMR. Depending on the molar concentration of RA, four types of products may form- RA-SA dimer, RA-SA-RA trimer, PSA oligomer, and RA-PSA oligomers. The resulting polymer should have PSA repeat units, alternating SA-RA units, and random SA-RA units.

2

Experimental

2.1 Materials Sebacic acid (SA, 99% pure; Aldrich, Milwaukee, WI), and acetic anhydride (Merck, Darmstadt, Germany) were used as received. Ricinoleic acid (RA) was prepared from the hydrolysis of castor oil as previously described.22 All solvents were analytical-grade from 3


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Sigma–Aldrich (Rosh HaAyin, Israel) or BioLab Jerusalem, and were used without further purification. HPTLC plates (high performance TLC) plates are purchased from Sigma-Aldrch (Analtech HPTLC Uniplates™: silica gel matrix coated on aluminium plates). 2.2 Spectral analysis H and 13C NMR spectra (CDCl3) were obtained on a Varian 300 MHz or 500 MHz NMR

1

spectrometer in tubes with 5 mm outside diameters. All the reaction kinetics was evaluated by 1H NMR, by calculating the relative integrals. We quantified incorporation of RA, relative ester and anhydride linkages in the reactions. CDCl3 residual solvent signals is used as used as the lock reference. The esterdicarboxylic acids were characterized by Finnigan LCQ Duo mass spectrophotometer equipped with an electrospray (ESI) interface (Thermo Quest, Tokyo, Japan) in negative ionization mode. The methanolic solutions of the compounds were directly injected into the ion source by use of a syringe pump. 2.3 Molecular weight determination The molecular weights were determined by gel permeation chromatography (GPC) system, Waters 1515. Isocratic HPLC pump with a Waters 2410 refractive in dex detector, a Waters 717 plus auto sampler, and a Rheodyne (Cotati, CA) injection valve with a 20 µL-loop. The samples were eluted with CHCl3 (HPLC grade) through linear Styragel HR5 column (Waters) at a flow rate of 1 mL/min. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA) with a molecularweight range of 500–100000.

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2.4

Synthesis

2.4.1 Poly sebacic acid (PSA) In a clean dry round bottom flask 20 g of dry sebacic acid and acetic anhydride (1:5 w/v) was refluxed, for 30 min with constant stirring. The excess acetic anhydride was evaporated to dryness under high vacuum. The clear, viscous residue was further polymerized by melt condensation at 160 °C for 4 h under vacuum (20 mbar) with constant stirring. The resulting polymer was washed with dry diethyl ether to remove excess acetic anhydride or acetic acid. 1

H NMR (300 MHz, CDCl3) δ 2.42 (m, 4H), 2.29 (m, 2H), 1.6 (m, 4H), 1.3 (m 3H). Weight

average MW, GPC = 12000 Da, polydispersity index = 1.48, Yield 85 %. 2.4.2 Synthesis of poly(RA-SA) The synthesis of the polymer was modified from the previously described method.23 Different molar ratios (RA: SA = 1:2, 2:3, 1:1, 4:3, 2:1) of RA and PSA were used. Appropriate amount of RA and PSA were melted and mixed thoroughly at 175 °C under inert nitrogen atmosphere. The resulting molten mixture was further stirred for 24 h at this temperature, under strictly inert conditions. For molar ratio RA: SA = 2: 1, the reaction was continued for 36 h, to ensure complete consumption of ricinoleic acid. Samples were collected from the reaction mixture at regular intervals to monitor free RA. The consumption of free RA was estimated by 1H NMR (integrating the peaks at δ ~3.6 ) When all the RA has been consumed, 5 equivalents of acetic anhydride were added and refluxed at 140 °C for 30 min. This resulting uniform mixture was subjected to melt condensation at 160 °C under vacuum (~10 m bar) for 6 h.

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2.5

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Purification of dimers and trimer and synthesis of the corresponding polymers

Different molar ratios of RA and PSA are mixed at 175 °C for 24 h. The resulting molten mixture is cooled down to room temperature. The resulting mixture contains the dimer (RASA) and the trimer (RA-SA-RA), PSA oligo units and RA-SA random/block oligo units. The dimer and the trimers were purified through silica gel column chromatography (100-200 mesh). The fractions were gradually eluted with a mixture of 5-10 % ethyl acetate in hexane. The separation was monitored on TLC, using iodine vapour or by charring (10 % H2SO4 in methanol). The compounds were characterized by 1H NMR and ESI MS. The obtained RASA and RA-SA-RA subunits were further polymerized separately and together. The polymerization was aided by poly-condensation as described in the earlier section. RA-SARA trimer: 1H NMR (300 MHz, CDCl3) δ 5.34-5.44 (m, 4H), 4.90 (s, 1H), 2.29-2.37 (m, 8H), 2.03 (m, 4H), 1.25-1.61 (m, 70H), 0.87 (t, 6H). ESI MS (-ve ionization): calculated 762.6, observed 761.51. Polymer: 1H NMR (300 MHz, CDCl3) δ 5.35-5.34 (m,4H), 4.87 (t, J = 6.2 Hz, 1H), 2.44 (t, J = 7.4 Hz, 2H), 2.38 – 2.18 (m, 4H), 2.04 (t, J = 14.7 Hz, 4H), 1.72 – 1.45 (m, 10H), 1.44 – 1.14 (m, 39H), 0.99 – 0.75 (t, 6H). Weight average MW by GPC = 9800 (polydispersity-1.33). RA-SA dimer: 1H NMR (300 MHz, CDCl3 ) δ 5.45 (dd, J = 17.8, 7.1 Hz, 1H), 5.31 (dd, J = 17.8, 7.0 Hz, 1H), 5.03 – 4.72 (m, 1H), 2.30 (dt, J = 23.2, 7.3 Hz, 4H), 2.04 (dd, J = 18.1, 11.1 Hz, 4H), 1.55 (m, 5H), 1.32 (m,22H), 0.87 (t, 3H). ESI MS (-ve ionization): calculated 482.36, observed 481.26. Polymer: 1H NMR (300 MHz, CDCl3) δ 5.45 (dd, J = 18.1, 7.4 Hz, 1H), 5.32 (dd, J = 17.7, 7.9 Hz, 1H), 5.07 – 4.75 (m, 1H), 2.43 (t, J = 7.4 Hz, 1H), 2.26 (t, J = 7.4 Hz, 5H), 2.01 (d, J = 7.7 Hz, 3H), 1.74 – 1.45 (m, 10H), 1.28 (m, 25H), 0.87 (t, J = 6.4 Hz, 3H). Weight average MW by GPC = 5500 (polydispersity-1.06).

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2.5.1 Estimation of the components in the prepolymer For all the ratios of RA: SA (1:2, 2:3, 1:1, 4:3, and 2:1) three components were estimated: the RA-SA dimer, RA-SA-RA trimer, and the SA oligomers. The relative ratio of dimer and the trimer were estimated by thin layer chromatography and by purifying each component through column chromatography. The spots were identified by iodine vapor and (10% conc. H2SO4 in methanol) by charring. The relative intensities of the plots were calculated by imageJ image analysis software’s Gel analyzer module. Their relative intensities were converted to area under the curve. Dimers and trimers were further separated by column chromatography and weighed separately to cross-validate the results. The oligomers were quantified by size exclusion chromatography using the Waters 1515 system described above. The prepolymer samples were accurately weighed and dissolved in HPLC grade chloroform. Note: the solution should be made immediately before injecting. The samples were injected and the oligomers were analysed by eluting with chloroform. The areas under the peaks at threshold of MW> 1000 Da are quantified and their relative ratios were estimated. 2.5.2 Stability studies and Hydrolytic degradation These studies were carried out as reported earlier described briefly as follows:23, 24 The stability at room temperature was conducted by placing the pasty polymer samples (~50 mg) at 25 °C under a nitrogen atmosphere. Their molecular weights were recorded at regular intervals for a period of 18 months. For hydrolytic degradation studies polymer samples (~100 mg, in triplicate) were placed into 1 mL Eppendorf tubes containing 1 mL of a 0.1 M phosphate buffer saline solution (PBS, pH 7.4) at 37 °C with constant shaking (100 rpm). The buffer was replaced at regular intervals. Each time point, the polymer sample was taken out 7


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of the buffer, lyophilized, and weighed. The hydrolysis was monitored by weight loss of the sample, and molecular weight by GPC.

Table 1. Chemical shifts (NMR at 300 or 500MHz, CDCl3) for the monitored 1H and 13C nucleus.

1

H NMR (δ ppm) before after polymerization polymerization 1. RA 2. Vinyl 3. -COOH 4. ester carbonyl 5. anhydride carbonyl 6. α-proton ester 7. α-proton anhydride

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C NMR (δ ppm) before after polymerization polymerization

3.62 4.8 5.4

4.87 4.7 5.4

71.7 127 130

73.9 124 132.5

-

-

180

Not observed

-

-

-

-

-

-

Not relevant

2.45

Not relevant

2.25

173.6 169.6

-

-

-

-

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Unreacted RA δ ~ 3.6 ppm, RA -acetate δ ~ 4.1 ppm, and δ ~ 4.8 ppm RA-ester. These protons were used monitored the reaction progress between RA and PSA. The methylene (-CH2) protons (δ ~ 2.25 for anhydride and 2.45 for ester) enabled quantification of ester: anhydride linkages. 13C NMR (500 MHz, CDCl3) reveals incorporation of -C=C-, anhydride, and complete consumption of RA in the final polymer. 3 areas are focussed: 160-180 ppm - area of carboxyl, 135-120 ppm - area of double bonds and 75-70 ppm – area of free hydroxylic groups.

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Results and discussion

A synthetic method for RA-SA alternating copolymer formation was investigated with the emphasis on the reaction conditions. The focus was particularly on the degree of conversion of ricinoleic acid when reacted with PSA to form the diacid oligomers. The reaction progress was monitored by 1H and 13C NMR for RA consumption, and formation of new ester bonds. To synthesize an ideal alternating poly(ester-anhydride), we focused on two aspects: 1. Complete consumption of RA prior to polycondensation 2. Optimizing the appropriate molar ratio of RA: PSA to ensure complete esterification of RA-SA. On reacting ricinoleic acid with PSA at 120 ºC for 2 h, about 80% of ricinoleic acid is reacted with PSA while about 20% remain intact. When moving to the next step, the unreacted RA reacts with acetic anhydride to form RA acetyl ester on the RA hydroxyl, blocking the -OH to take part in further polymerization. The acetyl ester RA acetate anhydride units serve as chain terminators as they react only through the carboxylate residue or form RA oligomers (MW~ 2500) which are incorporated as blocks in the final polymer.

Poly(ester-anhydride)

synthesized by this method therefore contain blocks of RA-RA and SA-SA, along with randomized RA-SA units. This uneven distribution of anhydride and ester bonds made this polymer unstable at room temperature and sensitive to hydrolysis.25 We modified this reported method and optimized the conditions until all RA is consumed. In this work, RA and PSA reaction mixture was heated at 175 °C to ensure RA full esterification in PSA as 9


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monitored by 1H NMR (proton at C12, δ ~ 3.6 ppm). Scheme1 shows different products that may form during the polymerization of SA-RA. Chemical shift value for the proton at position 12 of unreacted RA, acetylated/self-condensed RA, and esterified polymer was observed at ~3.6 ppm, ~4.1 ppm and ~4.8 ppm respectively.26

The relative ratio of the protons in the NMR spectra revealed that the RA was consumed after 24 h at 175 °C, for all ratios of RA: PSA (Figure1). The reaction was continued for 36 h for molar ratio of SA: RA = 1: 2 to ensure complete consumption of RA (after 24 h ~2 % of the RA remained unreacted). The prepolymer (before polycondensation) and final polymer were sampled and their 13C NMR was recorded, for further confirmation. In 13 C NMR, three areas were monitored: δ 160-180 ppm - areas for carbonyls, δ 135-120 ppm - areas for double bonds, and 75-70 ppm – area for free hydroxyl groups. The carbonyl group of carboxylic acid, ester, and anhydride showed peaks at δ ~ 180 ppm, δ 173.6 ppm, and δ 169.6 ppm, respectively. We observed the two alkenyl carbons at δ 125 and 132.6 ppm, respectively in both the prepolymer and the final polymer. This observation confirmed the full incorporation of RA in the final polymer. The –CHOH carbon in RA showed a peak at δ 71.7 ppm, the esterified RA showed a peak at δ 73.9 ppm. In both pre- and final polymer we observed peaks at δ 73.9 ppm and no peaks at δ 71.7 ppm. This observation suggests no residual unreacted RA in the prepolymer. It also confirms the incorporation of RA esters in the RA-SA polymeric framework (spectra presented in Supporting Information). The chemical shifts values in δ ppm for the possible bonds are shown in Table 1. Integration of these proton signals (α to the

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hydroxyl group of ricinoleic acid) were used to determine bond formation. 100

% of unconsumed RA

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SA: RA 2:1 3:2 1:1 3:4 1:2

80

60

40

20

0 0

1

2

4

6

8

10

24

36

hour

Figure1. Consumption of RA under the described reaction conditions (175 °C). The consumption was monitored by recording the 1H-NMR and integrating the proton 'α' to the hydroxyl group of ricinoleic acid; δ ~ 3.6 PPM. The reaction was followed for 24 hour, but for SA: RA 1:2 the reaction was carried out for 36 hour.

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Optimizing the ratio of RA

Scheme 2 Top: The kinetics of the reaction may be shifted by increasing the molar concentration of RA from oligomers to ester dicarboxylic acids (RA-SA and RA-SA-RA). Bottom: A segment of alternating poly(RA-SA) is shown. The molar ratio of 4:3 RA: SA seems to favour the synthesis of alternating polymer.

At the prepolymer stage, primarily four types of products are formed depending on the ratio PSA: RA (Scheme1), provided all RA has been reacted. We observed the formation of ester dicarboxylic acids, RA-SA-RA (trimer) and RA-SA (dimer), oligo units of both PSA and RA-SA. These dicarboxylic acid dimers and trimers polymerized in the next step into poly(ester-anhydride) with alternating architecture, RA-SA-RA-SA. The kinetic of the reaction may be favoured towards complete conversion of the oligo units to ester dicarboxylic acids, by increasing the ratio of RA in the reaction mixture (Scheme2). We 12


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isolated these proposed dimers and trimers by preparative column chromatography (through gradual elution with 5-10% ethyl acetate: hexane). Their structures were confirmed by 1H NMR and mass spectrometry.

Figure 2. 1H NMR (CDCl3, 300 MHz) spectra of poly(RA-SA).

As shown in Scheme 2, an alternating SA-RA copolymer section consists of one trimer and two dimer units along the polymer chain. The molar ratio of SA: RA should be ideally 3: 4. Moreover, polymerizing the trimer: dimer in 1: 2 molar ratio should yield perfectly alternating polymer. As demonstrated by polymerizing the isolated the trimer (RA-SA-RA) and the dimer (RA-SA). 1H NMR spectra were used to calculate the ratio of ester to anhydride bonds. Ester linkages consistently increased with the amount of RA. The two protons adjacent to the ester bonds (---CH2-COO-CH---, δ = 2.43 ppm) and anhydride bonds 13


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(---CH2-COO-CO-CH2---, δ = 2.25 ppm) were monitored to study these reactions. Figure2 shows NMR of RA-SA polymer. Five different molar ratios of RA and PSA (RA: PSA – 1:2; 2:3, 1:1, 4:3, 2:1) were evaluated. The RA-OH cleaves PSA to form the ester dicarboxylic acids RA-SA or RA-SARA. The formation of the dimer RA-SA is always predominant due to steric factors. The trimer can form when two units of RA cleaves the adjacent anhydride bond of PSA concomitantly. This is prominent if RA is in molar excess. The enhanced amount of RA drives the formation of RA-SA-RA trimer. Our aim in this section is find the ratio of PSA: RA that provides the ideal ratio of trimer: dimer (approximately 1:2). We started with mole excess of SA and gradually to twice mole excess of RA.

relative % of ester-dicarboxylic acids

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100 90 80 70 60 50 40 30 20 10 0

Trimer Dimer

2:1

3:2

1:1

3:4

1:2

Molar ratio SA: RA

Figure 3. Ratio of esterdicariboxylic acids-dimer (RA-SA) and trimer (RA-SA-RA) formed during mixing PSA and RA at 175 °C. Different ratios of ricinoleic acid (RA) and sebacic acid (SA) yields different ratio of dimer and trimer. The ratio is calculated by separating the mixture through column chromatography followed by weighing the samples, and through estimating their relative intensities on TLC plates.

The dimers and the trimers were separated by chromatography, characterized, and estimated. Their relative percentages were calculated. Their intensities were quantified on TLC plates

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and identified by iodine followed by (20% H2SO4 in methanol) charring. The oligomers left behind were quantified by gel permeation chromatography. For SA: RA 2:1 molar ratio, trimer was not observed. For molar ratio SA: RA 3: 4 and 1: 2 significant amount of trimer is observed (28 and 33 % respectively). In all the reactions the dimer is dominating. The oligomers were not observed in SA: RA 3: 4. Increased amount of RA drives the reaction towards formation of ester dicarboxylic acids (RA-SA and RA-SARA). At SA: RA ratio of 3: 4 the prepolymer consisted solely with these dimers and trimers. When the ratio of RA is increased further, it takes longer time to consume the RA due to ester formation between carboxylic acid and the RA hydroxyl group. As PSA is exhausted, the excess RA ends up in forming excess trimers (33%) and RA-RA esters. Figure3 shows the relative amount of ester di-carboxylic acids, dimers (RA-SA) and trimers (RA-SA-RA). Molar ratio of SA: RA 3: 4 is forming RA-SA dimers and RA-SA-RA trimers as major products, the amount of oligomers are also negligible, and no self-condensation with RA is observed. Making it the ideal workable ratio of RA: SA to synthesize alternating poly(esteranhydride) of RA and SA. However, to obtain ideal alternating polymer the trimer and the dimer fragments should be polymerized in a 1: 2 molar ratio, respectively (Scheme2, bottom). Effect on stability Polyanhydrides/ester-anhydrides are unstable at room temperature, and decrease in molecular weight with time. In ricinoleic acid (RA) sebacic acid (SA) alternating copolymer, each anhydride bond is shielded by the RA -(CH2)6-CH3 fatty side chain, to provide a stable polyanhydride. We previously reported the alternating RA-SA copolymer with enhanced stability at room temperature for more than 18 months.24 The random copolymer and PSA 15


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undergoes > 80 % drop in molecular weight within 1 month.23 Therefore, the alternating copolymer may be used in similar delivery vehicles, but may be stored and handled easily.

4

Conclusions

Reproducible synthetic method for alternating poly(ester-anhydride) was developed. The two key points to synthesis of alternating poly(ester-anhydride) are: i) monitor complete consumption of the hydroxyalkanoic acid (RA in this case). Polycondensation is applied only when all RA hydroxyl groups are incorporated as esters. ii) To use appropriate molar ratio of RA so the prepolymer consists solely of ester dicarboxylic acids trimers (RA-SA-RA) and dimers (RA-SA) in the ratio of 1: 2. Alternating RA-SA polymer has regular and more condensed units of RA alkyl side chains. This pendant alkyl groups cause steric hindrance between the polymer chains, reducing the anhydride interchange. Therefore, this new polymer of RA-SA prolongs the stability, hydrolytic degradation, and shelf-life. Moreover, this method can be applied to prepare alternating poly(ester-anhydride) from any pair of diacid and hydroxyl acid starting materials. Acknowledgements Arijit Basu would like to thank planning and budget commission of Israel for providing postdoctoral fellowships. We acknowledge Mohammed Alyan for helping us to synthesize additional compounds. References 1. Doppalapudi, S.; Jain, A.; Khan, W.; Domb, A. J., Biodegradable polymers—an overview. Polym. Adv. Technol. 2014, 25, (5), 427-435. 2. Kumar, N.; Langer, R. S.; Domb, A. J., Polyanhydrides: an overview. Adv. Drug Del. Rev. 2002, 54, (7), 889-910. 16


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Graphical Abstract Alternating Poly(Ester-Anhydride) by Insertion Polycondensation Moran Haim-Zada,a‡ Arijit Basu,a,‡ Tal Hagigitb, Ron Schlingerb, Michael Grishkoc, Alexander Kraminskyc , Ezra Hanukac, and Abraham J Domba* a

School of Pharmacy, Institute of Drug Research, Hebrew University of Jerusalem, Israel b c

Dexcel Pharma Technologies Ltd, Or-Akiva, Israel.

TAMI- Institute for Research & Development Ltd, Haifa Bay, Israel

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Alternating Poly(ester-anhydride) by Insertion Polycondensation

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