Biomacromolecules 2002, 3, 116-123
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Amphiphilic Block Copolymers as Bile Acid Sorbents: 1. Synthesis of Polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride)† Neil S. Cameron,‡ Adi Eisenberg,*,‡ and G. Ronald Brown§,| Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montre´ al, Que´ bec, Canada H3A 2K6, and Department of Chemistry, Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, Canada L2S 3A1 Received August 3, 2001; Revised Manuscript Received November 5, 2001
The systematic investigation of the synthesis of polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) was accomplished by employing both polystyrene-b-poly(tert-butyl acrylate) and its hydrolyzed derivative, polystyrene-b-poly(acrylic acid) (PS-b-PAA) as starting materials, and coupling them with N,N-dimethylethylenediamine (DMED). The various reactions and intermediates we examined include aluminum amides, acid chlorides, and imides derived from carbodiimides, all in a variety of solvents. We present below our investigation of several synthetic routes and conclude that the carbodiimide coupling of PS-b-PAA with DMED followed by quaternization and counterion exchange is the most effective method of achieving the target. A brief discussion of the merits of each procedure in the context of block copolymers is given, and IR spectroscopic evidence for the postpolymerization synthesis of the poly(acrylamide) block is provided. Introduction Coronary Heart Disease (CHD) and Cholesterol. As reported by Yalpani,1 “In the USA, 1.5 million people suffer heart attacks each year and 550 000 of them die. More Americans lose their lives to cardiovascular disease each year than to all other diseases combined. The associated economic impact is estimated to reach $80bn-100bn annually in medical expenses and lost output as a result of disability.” Recent surveys (1985-1990) found that more than one-third of all Canadians between the ages of 18 and 74 have two or more of the major CHD risk factors (smoking, hypertension, elevated cholesterol, physical inactivity, or obesity).2 It is now widely accepted in both the main-stream and scientific literature that one’s risk of CHD increases with age, family susceptibility (genetic predisposition), smoking, physical inactivity, obesity, hypertension, diabetes, and dislipidemias including high blood cholesterol concentrations.3-6 The National Institutes of Health (USA) very recently published the findings of the Adult Treatment Panel III (ATP III, 2001) and their recommendations to the National Cholesterol Education Program’s (NCEP) clinical guidelines for cholesterol testing and management.7,8 While ATP II guidelines (1993) identified low-density lipoprotein (LDL) cholesterol as a risk factor for CHD, ATP III places a special emphasis * To whom correspondence may be addressed: e-mail,
[email protected]; phone, (514) 398-6934; fax, (514) 398-3797. † This work was presented in part at the 73rd ACS Colloids and Surfaces Symposium (M.I.T., 1999). ‡ McGill University. § Brock University. | Deceased.
on LDL and realigns the optimal serum levels of cholesterol and triglycerides. There is substantial evidence that when the LDL transport system is overloaded, or when it does not function properly, the excess LDL is deposited on the arterial walls9,10 leading to arteriosclerosis and CHD.7,8 Treatments for Hypercholesterolemia. While careful attention to diet can result in limited serum cholesterol reductions, in many cases the recommended levels of LDL cholesterol can only be attained by administration of drugs. Broadly, these fall into two categories: (1) nonsystemic (nonabsorbable), drugs composed of orally ingested bile salt sequestrants (BSS), e.g., cholestyramine or colestipol, and (2) systemic (absorbable into the bloodstream), agents which in the main are 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors, e.g., lovastatin. In 1996, the North American annual market for cholesterol-lowering drugs exceeded $200 million and continues to show marked increases. Nonsystemic drugs have the advantage of minimal intervention in body chemistry since, by simply increasing the excretion level of bile salts with the sorbents, the liver is cued to consume endogenous cholesterol in their replacement. However, currently available sorbents suffer from poor patient compliance because of the side effects that are experienced from the large doses that are required (for example, the prescribed dose for cholestyramine can exceed 24 g/day). Of the sequestrant resins employed clinically, cholestyramine is the most prevalent, due in part to its comparative bile-binding superiority.11-18 A recent publication calls into question the use of the currently favored statin drugs (systemic HMG-reductase inhibitors) that make up more than 80% of the market of
10.1021/bm015595k CCC: $22.00 © 2002 American Chemical Society Published on Web 12/13/2001
Amphiphilic Block Copolymers as Bile Acid Sorbents
Figure 1. Chlorotrimethylammonium-bearing repeat unit for QPDA-n with an alkyl spacer where n is typically 2-12.
Figure 2. Polystyrene-b-poly(N,N,N-trimethylammonium ethyleneacrylamide chloride) (PS-b-PTMEACl).
cholesterol-lowering drugs, on the basis that they may be cancer suspect agents.19 Furthermore, cerivastatin (Bayer) has recently been voluntarily withdrawn from the marketplace due to fatal rhabdomyolysis (muscle necrosis) events in some patients.20 Because the benefits of the cholesterol-lowering drugs can only be maintained with continued administration, they are used for prolonged periods, so that even limited risks are of concern. Accordingly, an urgent need remains for more effective sorbents. Scope of this paper. Previous publications by this group described alternative resins to the pharmaceutical standard, cholestyramine.21 These sorbents were composed of lightly cross-linked poly(methacrylate) beads functionalized with diamines and then quaternized to yield poly(acrylamide) (QPDA-n) with the general repeat-unit structure shown in Figure 1. Various pendant architectures and spacer lengths were investigated, and the dodecyl spacer (n ) 12) was identified as providing the most effective bile salt binding properties. Although the QPDA materials significantly outperform cholestyramine in in vitro and in vivo preclinical trials, they do not address poor patient compliance issues related to the texture of polymeric resins. There are several recent reviews of sequestrant materials in the literature,22-24 and there are new materials, some of which are now in advanced clinical trials.25,26 Following previous work with cross-linked beads,21 this paper describes the synthesis of an amphiphilic diblock copolymer with a water-insoluble polystyrene block and a polyacrylamide water-soluble block that is N-functionalized with ethylenetrimethylammonium chloride (see Figure 2). As suggested in the M.Sc. thesis of Asgari,27 self-assembled nanoarchitectures of such materials may have pharmaceutical application to the treatment of hypercholesterolemia (see the following paper).28 Nanoscale self-assemblies not only offer large surface area with the concomitant high accessibility to the bile salt binding sites but also are attractive in that these aggregates suspend completely in water to produce a slightly viscous and turbid mixture that for practical reasons can be expected to be more acceptable to patients than the clinically available polymeric sorbents. These micelles are extremely stable to loss of macromolecular chains29 and, hence, would be confined to the gastrointestinal tract. It is anticipated that the high availability of binding sites will ultimately facilitate
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substantial reduction in dosages and that the unpleasant side effects associated with resin therapy will be avoided, or at least minimized. It might be argued that if the only goal were to obtain a material that sequesters bile salts, then the complete functionalization of the parent copolymer should not be necessary. For pragmatic reasons, it would indeed be convenient to accept nonquantitative functionalization. However, there are several factors that favor total replacement of the ester (or acid) groups: (i) residual ester groups render the ostensibly hydrophilic block less water-soluble and thereby complicate the process of self-assembly; (ii) residual carboxylate groups will interact with the pendant ammonium moieties to form ionic pseudo-cross-links; (iii) residual ester or carboxylate groups represent wasted bile salt binding sites. Considerable effort was therefore invested in the functionalization of these copolymers. Experimental Section Instrumentation. Gel permeation chromatography (GPC) was performed on a Waters instrument (HR-1 and HR-4 columns) interfaced to a PC running Millenium software with a Varian RI-4 refractive index detector, calibrated against polystyrene standards. The flow rate was 0.6 mL/min and the eluant was tetrahydrofuran (THF, chromatographic grade, Aldrich). FTIR spectra were collected on a Perkin Elmer 16PC instrument interfaced to a PC running either IRDM or Spectrum software. Samples were typically prepared as KBr pellets, though in some cases films were cast on KBr windows (Wilmad). Except in rare cases, 16 scans were sufficient to obtain clean spectra. Due to the sensitivity of the carbonyl bands, FTIR offered the simplest and most revealing spectroscopic data given the limited quantity of material available. Parent Copolymer Synthesis. The parent copolymer to the postpolymerization functionalization product, polystyreneb-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) (PS-b-PTMEACl), was either polystyrene-b-poly(tertbutyl acrylate) (PS-b-PtBuA) or polystyrene-b-poly(acrylic acid) (PS-b-PAA). The parent block copolymer samples were synthesized via anionic polymerization under an ultrapure nitrogen atmosphere (Matheson) in flame-dried glassware.30-32 Solvent and monomer transfers were accomplished via cannula and septa. Freshly distilled THF was collected from sodium/benzophenone under nitrogen. The PAA-containing polymer was derived by acidcatalyzed elimination of isobutene by well-established means.30-32 While it is true that the modification of PS-bPtBuA is a postpolymerization functionalization of the polymer, we draw a distinction between the preparation of the parent PtBuA or PAA blocks and their more challenging PTMEACl sibling in the following section. PS-b-PtBuA. In a typical polymerization, 500 µL of R-methylstyrene (RMS) was added to 200 mL of THF containing LiCl to provide gegenions in 5-10-fold excess as compared with the number of polymer chains to be initiated. The reaction was titrated dropwise with a suspen-
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sion of sec-butyllithium (1.3 M in THF, Aldrich) until a dark red color persisted indicating stable RMS anionic monomers and/or nascent oligomers. The reaction was then initiated with sec-butyllithium in 10% excess and the temperature was reduced to approximately 240 K in a dry ice/acetone bath. Cryo-distilled styrene monomer that had been stirred over CaH2 and further dried with fluorenyllithium immediately prior to distillation was then added to the reaction, and the deep red color temporarily turned orange, the color associated with the styryl anion, until all the styrene monomer was consumed. The styrene block was then auto-end-capped with traces of remaining RMS, and the deep red RMS color associated with the living RMS anion was regenerated. An aliquot of PS was removed for GPC analysis, and based on the confirmed degree of polymerization, the quantity of tert-butylacrylate (tBuA) to be added for the desired architecture was determined. The acrylate monomer was prepurified over (and cryo-distilled from) CaH2. The reaction temperature was reduced to 195 K in a dry ice/acetone bath, and on addition of tBuA, the deep red color of the RMS anion was replaced by the nearly colorless acrylate anion. An aliquot ranging in volume from several drops to a large fraction of this reaction mixture was extracted and “killed” in degassed, cold methanol to avoid coupling. Since the residual reaction mixture still contained living polymer chains, further addition of tBuA was possible thereby generating a family of polymers based on a fixed PS block length with a series of different tBuA block lengths. PS-b-PAA. Acid-catalyzed elimination of the tert-butyl pendants was employed to prepare PS-b-PAA from PS-bPtBuA. The acrylate-containing polymer was dissolved in toluene, and 2-5% toluenesulfonic acid (TsOH) was added relative to the number of ester groups to be converted (i.e., mole percent). The reaction was left at reflux for several hours whereupon the toluene was removed by rotary evaporation and the residue was redissolved in THF. The polymer was precipitated in water and infrared spectroscopy of cast films or KBr pellets confirmed the quasi-complete reaction where the characteristic carboxylic acid bands were present (O-H at 3000-2500 cm-1 and carbonyl at 17251700 cm-1).33 Furthermore, there was little or no evidence for residual ester carbonyls (∼1730 cm-1) or tert-butyl groups (a doublet at 1395-1385 and 1365 cm-1) (see Figure 3). Postpolymerization Functionalization. Amidation of these block copolymers was accomplished with varying degrees of success using several different synthetic pathways (see Figure 4). The pendant ester groups were reacted directly either with the corresponding amine or with an aluminum amide. Alternatively, the pendant ester groups were eliminated to yield carboxylic acids, which either were converted to the acid chloride and reacted with or were carbodiimide coupled to an amine. Pellets (KBr) were prepared containing analyte, and the solid-state IR amide I (1680-1630 cm-1) and amide II (1570-1515 cm-1) diagnostic bands were monitored to follow the reactions.34 Discussion of pertinent spectra will be found following the Experimental Section where appropriate. Parent copolymers, polydispersities, and degrees
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Figure 3. FTIR spectra of one of the parent PS-b-PtBuA samples and a pair of derivative hydrolyzed polymers based on the same polystyrene block. Note the displacement of the carbonyl band (17301710 cm-1) and the virtual disappearance of the tert-butyl bands (∼1390, 1370 cm-1).
Figure 4. A functionalization summary for PS-b-PtBuA. The R and R′ reflect the different substituents for the 1-[3-(dimethylamino)propyl]3-ethylcarbodiimide hydrochloride (EDC) and dicyclohexylcarbodiimide (DCC) adducts.
of functionalization as determined by FT-IR as a function of coupling agent are presented in Table 1. Direct Ester to Amide Transformation. The direct functional group conversion from ester to amide was attempted by simply refluxing a solution of ester-containing polymer in an excess of monodentate diamine (neat or in dimethylformamide, DMF). In a typical reaction, 1 mmol of tert-butyl acrylate units from PS175-b-PtBuA65 was refluxed with 4 mmol of N,N-dimethylethylenediamine (DMED, Aldrich, redistilled) in dioxane. Reaction times were on the order of 1 week at elevated temperature approaching 374 K. The polymer was then precipitated and washed with methanol/water (MilliQ). The conversion, as evaluated by FTIR, was determined to be >90% according to the relative intensities of the ester carbonyl and amide I bands.
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Table 1. Reaction Summary DPa PS-b-PTMEAClm
n
m
PIa
175 175 25 25 175 175 385 385 80 80 95 80 80
65 35 280 280 35 65 80 80 330 330 65 130 330
1.04 1.06 1.07 1.07 1.06 1.04 1.12 1.12 1.25 1.25 1.24 1.19 1.25
coupling agent with DMEDb
conversionc (%, best case)
AlEt3 AlMe3 AlMe3 AlMe3 SOCl2 C2O2Cl2 C2O2Cl2 DCC DCC EDC EDC
>95 >95 95d ∼50d >80d ∼50 >95 ∼60 >95e >95e >95 >95
a Degree of polymerization (DP) and polydispersity index (PI) of parent copolymer. b DMED ) N,N-dimethylaminoethylenediamine. c Measured by FT-IR (amideI/ester). d Geling always observed. e Trace byproducts/side reactions.
Ester to Amide Transformation via Aluminum Amide. The aluminum amide mediated functional group conversion of an ester to an amide was attempted starting with either triethyl- or trimethylaluminum (AlEt3 or AlMe3).35 In a typical AlEt3 reaction, 2 mmol of AlEt3 was stirred with 4 mmol of DMED in 5 mL of freshly distilled CH2Cl2 for at least 15 min. Then 270 mg of PS25-b-PtBuA280 containing 2 mmol of ester groups was added in 5 mL of CH2Cl2, and the reaction was left at reflux overnight. The reaction was quenched with acidic water yielding a gel-like white precipitate. Spectroscopy indicated incomplete conversion due to the presence of a strong IR band at 1730 cm-1. Therefore, to increase reactivity, AlMe3 was employed under similar conditions. A sample of PS25-b-PtBuA280 containing 1 mmol of ester groups was added to the reaction product of 3 mmol of Me3Al and 4 mmol of DMED in 5 mL of freshly distilled CH2Cl2. The reaction was allowed to proceed at reflux for 72 h and was then quenched with acidic water. The aqueous phase was then rendered basic, and the organic phase was collected, the solvent was removed by evaporation, and the residue was redissolved in dioxane. The dioxane solution was dialyzed against distilled water and then lyophilized, yielding a faintly yellow powder. FTIR spectroscopy indicated virtually quantitative conversion since quaternization led to no methyl ester carbonyl band. Thionyl Chloride-Mediated Transformation. To increase susceptibility of the acrylate block to nucleophilic addition/elimination, the ester was converted to the corresponding carboxylic acid, and the acid chloride intermediate was synthesized. In a typical reaction, SOCl2 was added in slight excess to a PS385-b-PAA80 sample dissolved in freshly distilled THF. The reaction solution was allowed to stir at 316 K for 14 h whereupon the reaction was cooled to 273 K and excess DMED was then added. The reaction was allowed to proceed for 4 h and was quenched with 25 mL water (MilliQ). Oxalyl Chloride Mediated Transformation. Water was added dropwise to a solution of PS80-b-PAA330 (100 mg) in
Figure 5. FTIR spectra for the DCC coupling of PS95-b-PAA65 at t ) 0, 4, and 30 h, isolated product and quaternized product (from top to bottom). Note the imide band disappearance (2117 cm-1) and the new amide band(s) ∼1600 cm-1.
5 g of DMF in order to induce micellization at about 10% water content (w/w). The micelles were then dialyzed against an aqueous solution of NaOH at pH 12, and the sample was lyophilized. The resulting PS-b-PANa was resuspended in 100 mL of THF to which 5.5 mmol of oxalyl chloride was added. The reaction was stirred overnight under an argon atmosphere whereupon a large excess of DMED was added. The reaction mixture was stirred for 24 h, dialyzed against water, lyophilized, and, finally, quaternized. DCC-Mediated Transformation. Dicyclohexylcarbodiimide (DCC) was added to a solution of PS-b-PAA in THF in 50% excess. After 3-4 h the solution became cloudy and the reaction was allowed to continue overnight. Excess DMED was then added, and the reaction solution was stirred for 1 day. The polymer was precipitated, collected, and redissolved in DMF whereupon MeI was added and left for up to 12 h. Excess MeI was removed by rotary evaporation, and then aggregation was induced by addition of deionized water (MilliQ). The suspension was dialyzed against distilled water for 4 days. One reaction was followed spectroscopically, and the resulting FTIR spectra may be found in Figure 5. EDC-Mediated Transformation. To a solution of 94 mg of PS80-b-PAA330 in 5 mL of CH2Cl2, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (395 mg) was added and the solution was stirred for 5-10 min. Excess DMED (500 µL, or 4.5-fold excess over all acid groups) was then added and was stirred for 24 h, whereupon the polymer was precipitated into MeOH/H2O and dialyzed against MilliQ H2O. The functionalized copolymer was then reprecipitated in saturated NaClaq and was dialyzed against MilliQ water to remove impurities. The polymer was
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lyophilized to yield 140 mg of product and was quaternized in MeOH with MeI in 20-fold excess. The iodide counterion was exchanged for chloride by precipitation in NaCl(aq), followed by dialysis against 0.1 M NaCl(aq) and then against MilliQ H2O. Quaternization. Over the course of these syntheses, two different quaternization conditions were employed.36 Either a huge excess of neat MeI swamped the system or MeI was used in methanol with a weak base (20 mL of MeOH, 1 g of KHCO3, 1 mL of MeI). Results and Discussion Given the relative simplicity of transforming an ester to an amide in the absence of other chemically sensitive groups, we would have had reason to be disappointed had we not met with success. However, the development or modification of the amphiphilic nature of the diblock copolymers that accompanies the conversion presents special problems.41 Additionally, the stringent requirements for preparation of a material that would be successful in nanoscale self-assembly also provided unanticipated challenges. Inherent Challenges to Diblock Copolymer Functionalization. Cross-Linking. Previous work has demonstrated conclusively that the amidation of poly(methyl methacrylate) (PMMA) resins using diamines is accompanied by crosslinking that consumes potential binding sites and adversely affects the binding characteristics of the resulting sorbents.37 This complication can be avoided by use of selective protecting groups on the diamine.38 However, the extensive workup required to obtain product of the required purity compounded with the additional synthetic steps required for deprotection made this approach unattractive for the functionalization of the diblock copolymer. Instead, we chose to use a commercially available monodentate diamine: N,Ndimethylethylenediamine, where one amine is primary (a good nucleophile) and the other is tertiary (a relatively poor nucleophile, but a reasonably good base). The presence of the tertiary amine not only precludes cross-linking but also simplifies the quaternization step. Solubility. The preferred reaction medium for postpolymerization functionalization of amphiphilic diblock copolymers is one that keeps the reacting polymer in intimate contact with the reagent(s) at all times, regardless of the degree of functionalization. Furthermore, the reaction medium choice must take into account the static and, in our case, generally insoluble block. In the event of aggregation (self-assembly) during the reaction, it is important that the nonreactive block not shield the reacting species. Polyacrylamide is insoluble in nearly all of the common solvents for polystyrene, poly(acrylic acid), and poly(tert-butylacrylate); morpholine and water are the only solvents listed for this polymer.39 It is known that the amidation of PMMA with mono-tert-butoxycarbonyl (mono-BOC)-protected 1,6-hexanediamine works well in DMF at high temperatures (∼150 °C) over several days,38 suggesting that the alkylated polyacrylamide at least swells in DMF. The solvents of choice were therefore either the diamine itself or hot DMF, while others including dichloromethane, THF, 1,4-dioxane, and dimethyl sulfoxide (DMSO) were found to be unsuitable.
Cameron et al.
Macrosurfactants. For purification of these amphiphilic block copolymers, solvent extraction workup techniques have proven largely ineffective since the macromolecules are also macrosurfactants. Even when extracted or “salted out” with saturated aqueous NaCl, the amphiphilic nature of the polymers in question precludes clean phase separation. Instead, the laborious cyclical process of dissolution, micellization, dialysis, lyophilization, and redissolution was employed to remove reaction byproducts and impurities. The possibility of cross-linking and the inaccessibility of the coronal units near the core-corona interface rendered reactions on micellized copolymers unattractive.40 Functional Group Conversion. For PMMA cross-linked resins near-quantitative conversion of the pendant esters to amides has been achieved by reaction with diamines, using high temperatures for protracted reaction times.21,41 The corresponding amidation of tert-butyl ester containing copolymers, at elevated temperature over several days, resulted in a final product showing no residual ester (∼1730 cm-1). However, it was insoluble in all common solvents and only swelled in water suggesting that the rigorous conditions involved resulted in concomitant cross-linking side-reactions possibly mediated by N-methyl migration. Two functionalized and soluble PS175 samples were obtained, albeit in small quantities, that exhibited a minor ester carbonyl peak (∼1730 cm-1) in the IR spectra. However, the amide peaks dominated suggesting nearly quantitative conversion. At reduced temperatures, even over very long reaction times, there was no appreciable conversion. Dialkylaluminum Amides. Not only reactivity and solubility but also steric hindrance plays a significant role in the successful functionalization of the coronal block. In the case of the aluminum amide mediated reaction (see Figure 4ii, iii), the intermediate is a four-center cycle. Crowding from the tertiary carbons of the tert-butyl acrylate groups as well as the polymer coil itself slowed the kinetics of reaction significantly as compared with the small-molecule analogue reaction, such that even switching from the diethyl to the dimethyl aluminum amide had profound effects on the kinetics and yield.35 The reaction involving Et3Al-mediated transamidation was so slow as to be essentially unsuccessful. Although trace residual ester carbonyl groups were indicated by the peaks around 1730 cm-1 in the IR spectra for the Me3Al-mediated functionalization of PS175-b-PtBuA35 and PS175-b-PtBuA65, the functionalization and quaternization of PS25-b-PtBuA280 was spectroscopically complete. In some measure, these reactions were therefore successful. A perennial beˆ te noire of block copolymer synthesis and functionalization, especially where nanoscale self-assembly is concerned, is purity due to the sensitivity of the polyelectrolytic block to its microenvironment. While infrared spectroscopy indicated few, if any, residual ester groups for the aluminum amide-mediated functionalizations, the separation of copolymer from the aluminum oxide required an aqueous acid/organic extraction. Because the pendant amide has a proton acceptor, both the product and the byproduct were soluble in an aqueous phase. Despite attempts at centrifugation, dialysis, and a variety of extraction techniques,
Amphiphilic Block Copolymers as Bile Acid Sorbents
Figure 6. N-Acyl rearrangement mechanism in carbodiimide coupling.
reliably clean, pure copolymer in workable quantities using this coupling agent was ultimately elusive. Acid Chlorides via Thionyl Chloride or Oxalyl Chloride. Due to the relatively nonreactive ester moiety, alternatives that activate carbonyl groups to nucleophilic attack were explored. The pendant ester was therefore hydrolyzed to the corresponding carboxylic acid and was then predisposed to nucleophilic addition-elimination by the formation of the acid chloride. Reactions with thionyl chloride required a large excess of amine, due to the generation of hydrochloric acid at both the chlorination and amidation steps (see Figure 4iv), and IR spectroscopy indicated substantial, but incomplete, carbonyl conversion. An alternative route to the desired product using oxalyl chloride (see Figure 4v) as the chlorinating agent was attempted since the byproduct of this reaction is CO2, with no deactivation of the nucleophile. However, the complete initial ionization of the pendant carboxylate groups rendered the reaction complicated. Reproducibility was an issue due to the insolubility of the reaction intermediate. Indeed, complete amidation of all pendant groups was not accomplished. The hydrolysis of the initial ester followed by the deprotonation of the carboxylic acid groups was paralleled by a shift in the FTIR carbonyl band from ∼1730 to ∼1600 cm-1. The amide bands (
