pH Optimization of Amidation via Carbodiimides - Industrial

Aug 21, 2013 - Thu V. Vuong , Bing Liu , Mats Sandgren , and Emma R. Master. Biomacromolecules 2017 18 (2), 610-616. Abstract | Full Text HTML | PDF ...
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pH Optimization of Amidation via Carbodiimides Stephen A. Madison and Joseph O. Carnali* Unilever R&D, 40 Merritt Boulevard, Trumbull, Connecticut 06611, United States ABSTRACT: The use of carbodiimides to create an amine-reactive reagent is a favored means of modifying proteins, nucleic acids, and small-molecule organic compounds containing carboxylic acid groups, or vice versa. The rules for optimizing the amidation have not previously been presented quantitatively, but such optimization is critical when modifying with an expensive fluorescent dye. In this study, the reaction conditions for attaching an amine-containing dye to sodium carboxymethyl cellulose (NaCMC) via 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) are systematically examined, with an emphasis on the role played by the pKa of the coupling amine group. The reactivity is reasoned to be proportional to the product of the fractional deprotonation of NaCMC, the fractional protonation of the carbodiimide, the fractional deprotonation of the amine-containing dye, and the relative stability of the reactants and intermediates: (αCMC‑)(1 − αEDAC)(αamine)Sf. The pH dependence of this product for a given amine group pKa can be used, by comparison to experimental data, to determine the pH dependence of Sf. Substitution of the pKa for a second amine-containing dye and inverting the above procedure gives a semiquantitative prediction of the pH conditions to optimize its amidation. The results suggest that generally available pKa values and the above form of Sf can be used to optimize carbodiimide assisted amidation.

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INTRODUCTION Water-soluble cellulose derivatives are important performance enhancing additives in many commercial applications. A major subclass of these materials are the ionic derivatives such as sodium carboxy methyl cellulose (NaCMC).1 NaCMC is utilized as a thickener,2,3 a dispersing or flocculating agent,4 as a flotation aid for the benefaction of potash4 and platinum5 ores, and as a tablet excipient,6 among other applications. Cellulose is a complex polysaccharide consisting of 3000 or more D-glucose (C6H12O6) monomer units linked by β-1,4glycosidic bonds.7 In NaCMC, a fraction of the glucose hydroxyl protons are substituted by CH2COONa. NaCMC’s are characterized by the number of these substitutions per glucose unit, the degree of substitution (DS),8 and by their molecular weight. The polysaccharide charge density is directly proportional to DS, with the latter typically in the range 0.7− 1.3.9−11 Methods for the determination of NaCMC include precipitation or conductometric titration,8 spectrophotometric analysis with anthrone, gel permeation chromatography,12 NMR,13 and fluorescence quenching.7 A route potentially leading to highly sensitive detection of NaCMC is to attach/ graft some form of a spectrochemical label. A fluorescent dye can be attached to an otherwise spectroscopically invisible polymer to turn it into a spectroscopically visible species.14 Examples of such labeled polymers include cationic hydroxy ethyl cellulose and cationic polygalactomannans,14 chitosan, dextrans, and carboxy methyl cellulose.15 In general, the optimum levels of dye labeling will depend upon the fluorescent probe and solution conditions employed. A number of routes are available for fluorescently labeling polymers and biopolymers with organic probes.16 Important considerations in this regard are the absorption and emission wavelength of the fluorophore, its molar absorptivity, its quantum yield, and the reaction chemistry by which a covalent linkage may be made with the polymer. © 2013 American Chemical Society

One reactive chemistry route for incorporation of a fluorophore in NaCMC is to render the carboxylic acid group into an amine-reactive reagent via a carbodiimide.17−19 1Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) is a so-called zero-length cross-linking agent20 frequently used to couple carboxyl groups to primary amines via the scheme illustrated in Figure 1.21 This cross-linker is

Figure 1. Schematic of the reaction of EDAC with a carboxylate group to produce the O-acylisourea ester which can then react with an amine containing species to form a stable amide linkage. Received: Revised: Accepted: Published: 13547

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frequently employed in situations where the reactive species are water-soluble. As indicated in Figure 1, the reaction of EDAC and a carboxylate forms an O-acylisourea intermediate. This reactive species is relatively unstable toward hydrolysis, with a half-life on the order of a few seconds in water at pH 4.75.20 If it encounters a primary amine species, it reacts to form a stable amide bond linking the two molecules and releasing a soluble substituted urea. In the absence of an available amine, the Oacylisourea can hydrolyze to regenerate the carboxyl group.22 The lifetime of the intermediate can be extended by the addition of N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfo-NHS) which converts the O-acylisourea ester to a more stable amine-reactive NHS or sulfo-NHS ester, respectively (see Figure 2). The coupling efficiency of an amine

working to functionalize alginate with biotin via EDAC activation, found higher levels of incorporation at 0.5% alginate than at 1%. These authors attributed the poorer yield at higher alginate level to difficulty in mixing the more concentrated solution. The pH of the reaction solution is critical to optimizing the amidation reaction, but the literature offers inconsistent advice in this regard. Carbodiimide activation is facilitated when the carbodiimide is protonated while the carboxylate is sufficiently ionized.30,31 The protonation of the carbodiimide nitrogen is thought to reduce the electron density at the carbodiimide central carbon favoring nucleophilic attack by the carboxylate anion.27 In the case of the carbodiimide EDAC, the protonated species is actually the dication.32 The pH conditions for carbodiimide protonation and carboxyl ionization overlap only slightly, and a pH range 4.5−4.75 has been recommended27,33,34 for the formation of O-acylisourea. The stoichiometry of the reaction shows that one proton is consumed so that the pH will increase unless the system is buffered or compensated with acid. The reaction of the O-acylisourea intermediate with an amine is an example of nucleophilic attack. Thus, the amine is best unprotonated to maintain its nucleophilicity.30 However, the primary amine will generally be protonated at the optimal pH 4.5−4.75 for O-acylisourea formation, and so will be unreactive.35 Thus, for direct, one-step amidation, a slightly higher pH is often chosen as a compromise. Some prior workers have proposed pH 5.5−8.21,36−39 The same mismatch of optimal pH conditions applies as well when NHS or sulfo-NHS is combined with EDAC to enhance amidation: the best conditions for formation of the ester are not those for amide bond formation. A compromise is usually chosen, which differs from one reported study to another. Reaction of EDAC and sulfo-NHS has been reported in the pH range 4.5−7.2.29,35 The reaction of the sulfo-NHS activated species with primary amines is again most efficient at pH > 7, when the amine is in its deprotonated form.24 Thus, a two-part approach can be taken, in which the activated species is prepared in the lower pH range where the ester is stable, and then the pH is adjusted upward for the second step of the reaction if the added amine would otherwise not be deprotonated. When attempting to achieve a given level of amine incorporation, the inherent inefficiencies of the linking reaction would suggest activating more than the theoretically required number of carboxyl groups. Typical recipes from the protein labeling literature call for a 10-fold excess of EDAC to carboxylate groups. Higher levels of activation may lead to conversion of the carboxyl groups to stable N-acylurea, and such excessive levels of activation are generally avoided on the grounds that coupling efficiency will fall off.31,40 Although individual results may vary, the overall efficiency of amine incorporation could not be improved beyond 25% by varying the level of activation.34,36,39,41 Inherent linkage inefficiencies have also led authors to employ excess amine over the level of EDAC activator.26,27,39,42 Kuo et al.27 employed amine/EDAC ratios of 3/1 to 100/1, with the idea that increased amine would favor the replacement reaction. Anjaneyulu and Staros23 demonstrated a first order dependence of amidation on amine level, but Sehgal and Vijay35 and Dulong et al.33 found that the degree of coupling increased linearly only over a narrow range of amine/EDAC.

Figure 2. Schematic of the chemical modification of the O-acylisourea ester by N-hydroxysulfosuccinimide (sulfo-NHS) to form a more stable, amine reactive sulfo-NHS ester.

to the more stable reactive ester can be improved 10- to 20fold.21 Further, these esters have sufficient stability to enable a two-step reaction procedure, in which the solution conditions can first be adjusted to favor ester formation and then readjusted to favor the reaction of the ester with the amine group. While these NHS or sulfo-NHS esters are more stable than O-acylisourea, they will still hydrolyze in water,20 showing general base catalysis.23,24 Reduced temperature will also slow the rate of hydrolysis, with the half-life of sulfo-NHS esters increasing by an order of magnitude at 0 °C relative to 31 °C.23,25 In the absence of a free amine, the O-acylisourea can hydrolyze to regenerate the carboxyl group as noted above. However, another rearrangement is possible, through an intramolecular acyl transfer to a stable N-acylurea via a formal 1−3 acyl shift.26−28 In certain cases, this rearrangement has even been observed to dominate over amide linkage when a primary amine was present.27 Reaction Considerations. Prior efforts at coupling to the carboxyl groups of polysaccharides via EDAC have led to some qualitative rules regarding the polysaccharide concentration, the reaction solution pH, the EDAC to carboxyl group ratio (theoretical degree of activation), the amine to EDAC ratio, and the ratio of NHS or sulfo-NHS to EDAC. Leung et al.,29 13548

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Coactivation of EDAC with either sulfo-NHS or NHS has become common in the literature,24,25,37,39,41,43−45 but again there seems to be little consensus on the key conditions. The ratio of sulfo-NHS/NHS to EDAC is observed to vary from 20/1 to 1/20 (e.g., Leung et al.29), but it seems sensible to employ a ratio exceeding unity in light of the poor stability of the O-acylisourea.25,43 Systematic studies generally support this supposition,20,22,46 but there are contrary examples.35,46 A twostep reaction scheme is used in about half of the above cited examples, with the carboxylic acid, EDAC, and NHS reacted for up to a few hours before the amine is added. Although one should presume that the prior published works represent reactions run under optimized conditions, evidence for this optimization is seldom presented. Combined with the many contradictory reaction schemes employed, it is difficult to understand the reasoning behind the conditions being chosen. In this study, a systematic examination is made of the EDAC facilitated labeling of NaCMC with a fluorescent dye. The typical fluorescent dyes which could be linked to a carboxylate group fall into two basic classes, those with the amine group attached directly to the ring system of the dye and those with the amine removed from the ring by a spacer group. The first group have a pKa of 0.5−3 and so will be unprotonated even at the lowest pH used in EDAC activation. The latter group will have a pKa of about 9−11 and so will be protonated in the pH range in which the activated O-acylisourea is stable. The major goal of this study is to highlight the key role which amine pKa plays in determining the optimal reaction conditions and to show how these conditions can be predicted. Optimization conditions for NHS coactivation will also be presented. This work will be of interest to those seeking to optimize the covalent attachment of proteins, nucleic acids, and smallmolecule organic compounds.

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Table 1. Structures and Properties of the Fluorescent Probes Considered in This Study

a

Determined in 0.1 M phosphate pH 7.0 buffer. bDetermined in ethanol. cpKa values for the primary amines are predicted using the ACD/I-Lab Web service (ACD/pKa 8.03).51

The first three entries in Table 1 have been suggested as reactive probes for carboxylates,50 but have two major disadvantages as potential fluorescent labels in the current application. First, they all have relatively low molar absorptivities (ECs) and hence are weakly fluorescent. Second, they absorb and emit in the blue region of the spectrum, a region in which a NaCMC solution may show some turbidity due to scattering. These entries are, however, relatively small molecules and so are less likely to strongly perturb the labeled molecule’s structure. The last entry in Table 1, lissamine, is a larger molecule which shows considerably brighter green fluorescence, but is prohibitively expensive. The pKa predictions listed in Table 1 refer to the equilibrium constant for the dissociation of the conjugate acid of the amine:

EXPERIMENTAL SECTION

Materials. Sodium carboxymethyl cellulose (NaCMC) was supplied as a white powder, product number C 5678, by Sigma Chemical Corporation. This NaCMC was a low viscosity material with a molecular weight of 90 kDa, degree of polymerization of 400, and degree of substitution in the range 0.65−0.90, corresponding to 6.5−9.0 carboxymethyl groups per 10 anhydroglucose units. NaCMC’s with a minimum DS of at least 0.7 are expected to be completely water-soluble,3,12 and indeed, this material gave clear aqueous solutions. The low viscosity (a 4% solution has a viscosity at 20 °C of 0.1 Pa s) facilitated the subsequent labeling reaction. The NaCMC was used as received to prepare transparent NaCMC solutions by stirring the powder in Milli-Q water overnight. The solutions were used within 48 h. 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) was purchased from Sigma-Aldrich. N-Hydroxysuccinimide (NHS) was sourced from Fluka Analytical, and Nhydroxysulfosuccinimide (sulfo-NHS) was purchased from Thermo Scientific. The fluorescent probes considered included 7-amino-1,3naphthalene-disulfonic acid monopotassium salt monohydrate (ANDSA)18,47,48 and dansylhydrazine (DH),49 both from Fluka, and N-(1-naphthyl) ethylenediamine dihydrochloride (EDAN), from Sigma-Aldrich. Lissamine (rhodamine B ethylenediamine) was purchased from Molecular Probes/ Invitrogen. Further details of the fluorescent probes are provided in Table 1.

R3NH+ ⇔ R3N: + H+ K a = [R3N][H+]/[R3NH+]

Generally speaking, alkyl and nonaromatic heterocyclic amines are slightly stronger bases than ammonia (whose pKa is 9.3 in water), while aryl amines are much weaker bases than ammonia. In terms of the kinds of fluorescent molecules considered here as labeling species, two classes can be distinguished in terms of their pKa. Those which do not contain an aryl amine will be largely protonated at the pH used in the amidation, while those with an aryl amine (the only example considered here being ANDSA) will be largely deprotonated. Procedures. A 1% w/w solution of NaCMC was prepared and allowed to become homogeneous by stirring overnight prior to use. Assuming an average degree of substitution of 7.5 13549

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wavelength region including the spectral peak of the label species. Further dilutions were required in many cases to keep the measured absorbance in the range of 0−1 absorbance units. The measured absorbance at the spectral peak was converted to a bound-label content by reference to a calibration curve of the label in water, the assumption being made that the molar absorptivity of the bound label was the same as that of the free species.42 Additional Considerations. The pH conditions for optimized reactivity can be estimated by considering in turn the ionization of the CMC, the deprotonation of the amine label, the activation of the carbodiimide, and the stability of the active species. The ionization of CMC has been modeled by Feng et al.53 using the modified Henderson−Hasselbalch equation fitted to experimental data. These authors give the degree of ionization α as

carboxymethyl groups per 10 anhydroglucose units, the molecular weight per carboxylate group in the unlabeled material is estimated at 296. Thus, the 1% NaCMC solution represents 0.034 M in carboxylic acid groups. In the majority of labeling runs reported here, it was planned to label one out of every five of these carboxylic acid groups. Typically, 20 g of 1% NaCMC solution was diluted with an equal volume of Milli-Q water for a total of 0.68 mmol of acid. For experiments without NHS/sulfo-NHS stabilizer, the label species was then added as a solid and dissolved by stirring for several minutes. The solution pH was then adjusted to the desired level via dropwise addition of 0.1 M NaOH solution. The desired level of EDAC powder was next dissolved in 2 mL of water and added to the NaCMC solution with stirring and continual pH monitoring. An increase in pH was often observed upon EDAC addition and immediately corrected by addition of 0.01 M HCl so that the pH was maintained at the desired level for 12 h. The halflife of the reaction of carbodiimides and carboxylic acids has been measured to be on the order of seconds,52 so that a reaction period of a few hours is expected to be more than sufficient. At the end of the reaction period, the transparent solution was split into 10 mL aliquots and transferred to plastic 50 mL centrifuge tubes. To cause precipitation of the labeled polysaccharide, the aliquots were mixed with 2 mL of 1 M NaCl solution and diluted to the 50 mL mark with reagent ethanol. Centrifugation on an Eppendorf 5804 centrifuge for 10 min at 3000 rpm served to sediment the polysaccharide. The supernatant was poured off, and the labeled NaCMC was redissolved in 10 mL of water. Spectrophotometric analysis of the supernatant indicated that it contained labeling species not associated with the polysaccharide. The redissolved polysaccharide solution was again diluted with ethanol to precipitate the polymer. This dissolution/precipitation regime was repeated27,31 until the supernatant gave an absorbance at the spectral peak of the labeling species which was within the instrument background. The labeled polysaccharide was airdried and then dried in a vacuum-oven at ambient temperature and stored over Drierite. On several occasions, the redissolved polymer after the first precipitation was dialyzed29 against MilliQ water using a Spectra/Por 7 dialysis membrane with a 1000 Da cutoff. The dialysate was changed twice daily over a period of three days, and then the polymer solids were isolated by freeze-drying (FTS Systems Flexi-Dry MP). No difference in the mass of recovered solids or degree of conversion (see below) was observed between the two isolation techniques. For runs with the EDAC/sulfo-NHS (or NHS) activation system, a two-step procedure was explored. The solution of 0.68 mmol in carboxylic acid groups from the NaCMC was adjusted to the desired pH and then the sulfo-NHS (or NHS) added and dissolved with stirring, followed25 by the EDAC. The pH was maintained as described above for a period of 5− 15 min and then the labeling species added with continued stirring. As the label dissolved, the solution pH was adjusted to whatever new level was desired and maintained for a period of 12 h. The labeled material was then isolated as above. As detailed below, most of the reported work made use of NHS at a favorable pH of 4.75 for the first step of the procedure (activation). In the amidation step, the pH was raised to 7 to favor deprotonation of the amine-containing dye, which was chosen to be EDAN. For routine analysis of the degree of conversion of the labeling reaction, the labeled NaCMC was dissolved at a level of 0.2% in water and its absorption spectrum collected over a

pHCMC = pKCMC + n log[α /(1 − α)]

(1)

where the factor n depends on the ionic strength I via the empirical expression n = 1.1−0.23 log(I) and pKCMC = 3.22− 0.37 log(I).54 Equation 1 expresses the fact that the ionization of CMC shifts to lower pH with increasing ionic strength as a result of the polyelectrolyte effect. In the ionic strength range of interest, CMC is almost completely ionized over the range pH 4−6. In practice, it is known that all NaCMCs become insoluble in water below pH 3.1 The deprotonation of the primary amine group of the label can be represented by the unmodified form of eq 1 with n = 1 and the pKa of the amine as in Table 1 replacing pKCMC. Equation 1 is used along with Microsoft Office Excel 2007 to plot the fractional protonations and deprotonations which appear in Figures 7−9. For the sake of comparison, the level of activation of CMC carboxylate groups is expressed in terms of the level of EDAC. Since [EDAC] < [−COOH], it is assumed that all the added EDAC is converted to amine reactive ester when calculating conversion fractions.43

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RESULTS

Amine/EDAC Ratio. As a preliminary step, the effect of variation in the ratio of amine-to-activated carboxylate was studied for the NaCMC/ANDSA system. The NaCMC level was maintained at 0.5% (0.017 M in −COOH) in the reaction system, and the EDAC was dosed so as to activate one in every five carboxy methyl groups. The pH was maintained at 5.5 for the duration of the reaction. The molar ratio of ANDSA to EDAC was varied over the range 1−5, and the fractional conversion of carboxylate to amide linkage was monitored. Figure 3 indicates that the extent of amidation increases smoothly with the excess amine, linearly over the range ANDSA/EDAC = 0−3, and then a little less strongly. Clearly the intuitive notion of more amine favoring the replacement reaction holds for this system. An obvious disadvantage of this approach is that the amine is in this case a fluorescent dye which could be extremely expensive. At prices as high as $100/ mg, employing large excesses of dye, which will largely have to be removed from the reaction medium and discarded, is a poor means of increasing the degree of conversion. Dependence of Fractional Conversion on pH. As noted above, the pKa’s of the dyes ANDSA and EDAN differ by some 6.5 units. To study the role of reaction pH on the extent of amidation, the EDAC facilitated reaction of NaCMC and each of the dyes was run at a series of pH’s. The other reaction 13550

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Figure 3. Fractional amidation of NaCMC with the fluorescent dye ANDSA for a range of dye levels at a fixed level of activation via EDAC. Reaction conditions as in the text.

Figure 5. Fractional amidation of NaCMC with the fluorescent dye EDAN over a range of reaction pH. Reaction conditions as in the text. Solid line is the predicted fractional amidation according to eq 3 (see Discussion below).

conditions were again a NaCMC level maintained at 0.5% (0.017 M in −COOH), EDAC dosed so as to activate one in every five carboxy methyl groups, and the amine (dye)-toEDAC ratio maintained at 3-to-1 for the sake of comparison. The dye DH proved to be too insoluble under the reaction conditions and Lissamine was too costly for this study. ANDSA. For this dye, the pH was varied incrementally over the range 4.75−7. The lower limit was set by the assumption that pH 4.75 was the optimum for formation of the Oacylisourea ester. The fractional conversion is seen in Figure 4

pH 4.75 for the formation of the O-acylisourea and pH 7 for the amidation . The reaction conditions were again a NaCMC level maintained at 0.5% (0.017 M in −COOH), with EDAC dosed so as to activate one in every five carboxy methyl groups. For this part of the study, the amine (dye)-to-EDAC ratio was reduced to 1-to-1, and the ratio of (sulfo-)NHS-to EDAC was varied from 0 to 20. Unlike the reactions run in the absence of (sulfo-)NHS above, the reaction temperature was here maintained at 4 °C throughout.35 As above, it is assumed that the level of EDAC expresses the degree of activation. The level of amine is kept just sufficient to react with all of the amine reactive ester under idealized conditions. The expected yield is much less than 100%, allowing ample opportunity to explore the enhancement by sulfo-NHS or NHS. The effect of varying the NHS/EDAC molar ratio on the fractional conversion of intended carboxylate groups is shown in Figure 6. The fractional conversion is indeed very small in the absence

Figure 4. Fractional amidation of NaCMC with the fluorescent dye ANDSA over a range of reaction pH. Reaction conditions as in the text. Solid line is a linear fit to the data according to: conversion fraction = −0.1575(pH) + 1.1706.

to be highest at this pH as well, with a conversion of about 0.42. The fractional conversion then drops off linearly with increasing pH and was not readily detectable beyond pH 7. EDAN. For this dye, the reaction pH was varied incrementally from 4.75 to 9.0. Although pH 4.75 is presumably the optimum pH for the formation of the O-acylisourea ester, the fractional conversion of the CMC is very low at this pH, as shown in Figure 5. The conversion increases markedly to a broad maximum of about 0.23 in the pH range 6−7, and then falls off with higher pH. The distinct difference observed between the pH dependence of the reaction with ANDSA and that with EDAN, under identical experimental conditions, can reasonably be attributed to some difference in the nature of the dyes. Benefit of Coactivation with (Sulfo-)NHS. The pHdependence of the amidation reaction displayed in Figure 5 suggests that the reaction with EDAN would be a good candidate for a two-step reaction using NHS or sulfo-NHS to coactivate the NaCMC. The two-step approach provides an opportunity to utilize both of the optimal pH’s identified above:

Figure 6. Fractional amidation of NaCMC with the fluorescent dye EDAN using a two-step route consisting of activation with EDAC in the presence of the coactivator NHS. The mole ratio of NHS to EDAC is varied at a constant level of activation at 4 °C (□) and at room temperature (■). Sulfo-NHS replaces NHS in one instance (△). Other reaction conditions as in the text.

of NHS, on the order of 0.02. However, this quantity increases very strongly with addition of NHS, showing a peak fractional conversion of about 0.42, a 20-fold improvement over that in the absence of NHS. The improvement peaks by a NHS/ EDAC ratio of 3 and then falls off in a linear fashion to a fractional conversion of 0.2 (a 10-fold enhancement) at an NHS/EDAC ratio of 20. A few comparative reactions were run 13551

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at room temperature instead of 4 °C and yielded a lower fractional conversion as shown in Figure 6. Likewise, sulfo-NHS was found to be inferior to NHS for this system, though only a single trial was performed. The period during which NHS and EDAC are allowed to react with NaCMC at pH 4.75, before addition of EDAN and readjustment of the pH, was also varied, over a range 5−15 min. No effect of this small variation is observed, though larger variations might be expected to have a more significant effect. In the classic work of Staros et al.,22 a 15-fold enhancement of EDAC mediated coupling was reported but at a very low sulfo-NHS/EDAC ratio of only 0.05. Higher ratios gave a reduced benefit. It is unclear why the optimum coactivator/EDAC ratio should occur in that or the present study.

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DISCUSSION The effect of variation in the amine to EDAC ratio on the extent of amidation was determined for the NaCMC/ANDSA system in Figure 3. The trend shown in the figure is one of approximately linear increase in product formation with amine concentration at a fixed level of activation. This linear trend is in keeping with first order reaction kinetics, as was found by Anjaneyulu and Staros.23 As noted above, the use of excess amine to drive the amidation reaction to higher yield is uneconomical for an expensive amine reagent, unless it can be readily recovered. The major impetus for this work was to identify more economical conditions for linking NaCMC with a fluorescent, amine-containing dye costing over $50 per milligram. The use of a coactivation agent proved to be a viable means of improving the efficiency of the labeling procedure. The method still hinges on identifying the ideal pH at which to run the second step of the reaction. It has been shown that this pH is highly dependent on the nature of the amine. While this dependence can be determined by trial-and-error, it is obviously preferable to be able to predict the likely range, again especially with expensive labeling materials. A possible scheme for such a prediction, as well as a better understanding of the amidation process with carbodiimides, is presented below. There is a marked and drastically different effect of pH on the fractional amidation seen in Figures 4 and 5. As indicated in the Introduction, pH is expected to play a large role in the formation of the O-acylisourea ester, in the stability of this intermediate, and in the subsequent reaction with the amine to be incorporated. The influence of pH on the formation of the O-acylisourea ester can be examined in terms of the pH range for favorable speciation of EDAC and the carboxyl groups of NaCMC. Carbodiimide activation should be favored when the carbodiimide nitrogen (pKa = 3.132,55) is protonated while the CMC carboxylate is sufficiently ionized. The likelihood of these two conditions occurring simultaneously is proportional to (αCMC‑)(1 − αEDAC). The speciation of NaCMC can be approximated as a function of pH according to eq 1, with the ionic strength I taken as 0.05 M. The deprotonation of EDAC can be also be approximated by eq 1, with n = 1. Figure 7 shows the fractional speciation according to these approximations. EDAC remains largely protonated only up to a pH of about 3, while NaCMC begins to become significantly deprotonated beyond pH 4. As a result, the product of the two speciations displayed in Figure 7 should be the controlling factor in terms of the ester formation. This product shows a peak centered at about pH 3 and decaying at higher pH. A calculation by Chan and Cox55 for an analogous system with acetic acid as the

Figure 7. Fractional ionization of the carboxyl groups of CMC (---) and protonation of EDAC () calculated as described in the text as a function of pH. Square symbols (□) are the product of the two fractions (αCMC‑)(1 − αEDAC), representing the occurrence of oppositely charged species.

carboxylate gave a similar speciation curve. Thus speciation considerations for this reactant pair predict a useful pH range for EDAC activation in the vicinity of the pH 4.5−4.75 range noted in the Introduction.27,33,34 Note that stability considerations regarding EDAC and the O-acylisourea ester have not yet been taken into account in Figure 7 but will be considered below. Attempts to directly study the stability of the O-acylisourea ester proved unsuccessful. Working with propionic acid in place of NaCMC, a species with the mass/charge ratio expected for the intermediate was isolated by mass spectroscopy but found to be stable over a period of many days. It was concluded that the corresponding N-acylurea was forming in the absence of a free amine. A less direct way to follow the stability of the reactants and intermediate products is to extend the above speciation product idea and apply it to the fractional amidation of NaCMC with ANDSA (Figure 4). Optimal conditions for amidation include protonation of EDAC, ionization of the CMC carboxylate, and deprotonation of the amine being incorporated. With these assumptions, the fractional amidation would be expected to be proportional to fractional amidation ∝ (αCMC −)(1 − αEDAC)(αamine)

(2)

For ANDSA (pKa = 2.7), the fractional deprotonation is calculated (again using eq 1 with n = 1) as a function of pH and shown in Figure 8. The deprotonation is essentially complete by pH 4. The speciation product is then calculated as in eq 2, with Figure 8 showing a peak centered at around pH 3.5 and clearly controlled by the product (αCMC‑) (1 − αEDAC) in Figure 7. This peak would suggest that amidation at pH 4.5 should be favored by a factor of over 100 versus that at pH 7. In light of the far more modest declining trend seen in Figure 4, it is postulated that the discrepancy is due to the pH-dependent stability of the carbodiimide and the O-acylisourea ester intermediate. The contribution of instability to the fractional amidation could be expressed by adding to the product in eq 2 an empirical factor which describes how the relative stability affects the amidation. 13552

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Figure 9. Fractional deprotonation of EDAN () and speciation product according to eq 2 (scaled by 106, ---), both plotted against the left-hand ordinate. The relative stability of the reactants and the Oacylisourea intermediate (Sf, □) plotted against the right-hand ordinate. The predicted fractional amidation according to eq 3 (multiplied by a scaling factor of 103, ···) is also plotted against the lefthand ordinate.

Figure 8. Fractional deprotonation of ANDSA () and speciation product according to eq 2 (---), both plotted on the left-hand ordinate. Relative stability of the reactants and the O-acylisourea ester intermediate (Sf, □) plotted on the right-hand ordinate, all presented as a function of pH.

amidation fraction ∝ (αCMC −)(1 − αEDAC)(αamine)Sf

(3)

Sf is expected to have a dependence on pH and be determined by the nature of the reactants and the O-acylisourea ester intermediate. Other factors, including the inherent reactivity of the deprotonated amine being incorporated, would be reflected in the proportionality constant implicit in eq 3. Applying eq 3 to the amidation fraction of ANDSA in Figure 4, the factor Sf(pH) can be determined. In this determination, the amidation fraction in Figure 4 is fitted to a linear function of pH as indicated in the figure. It should be noted that Sf, as shown in Figure 8, contains the proportionality constant which makes eq 3 an equality rather than a proportionality. The determined Sf is a sharply peaked function of pH with a maximum at around pH 7. The prediction for Sf can only be made in the pH range of the data in Figure 4. The decline in Sf below pH 5 is in keeping with the instability of EDAC itself below this pH as reported by Williams and Ibrahim,32 Nakajima and Ikada,30 Lewis et al.,25 and Liu et al.,56 presumably due to hydrolysis. The decline past pH 7 can be attributed to the susceptibility of the O-acylisourea intermediate to hydrolysis which was observed by Liu et al.56 to become significant above pH 6.5. This susceptibility could be linked to the concentration of water which far exceeds that of the amine.30 Thus, the form of Sf determined in this study is not inconsistent with the limited stability data available for EDAC and the O-acylisourea ester intermediate. With the relative stability function Sf in hand, it is possible to test the strategy of eq 3 by applying it to the amidation fraction of EDAN (Figure 5). The factors (αCMC‑)(1 − αEDAC) are the same as in Figure 7, and αamine for EDAN (pKa = 9.5) is shown in Figure 9. The speciation product (equivalent to eq 2) thus has factors which peak at widely separate pH’s so that the product itself is small and spans the gap from pH 4 to pH 9. Taking the same Sf as above, the complete product (equivalent of eq 3) is shown in Figures 9 and 5, where a factor of 1000 has been applied to bring the product on the same scale as the amidation fraction. It is clear that the reaction product of eq 3 gives a semiquantitative description of the pH dependent amidation with EDAN in Figure 5. Unlike the trend of the speciation product of eq 2 for ANDSA (Figure 8), that for EDAN (Figure 9) is increasing rather than decreasing as the pH

increases past pH 4. Thus, the contribution of the αamine factor acts to shift the amidation with EDAN to a higher pH than was observed with ANDSA, as shown in Figure 5. However, the pH peak of the amidation with EDAN is seen to be controlled by the stability factor Sf. The two factors speciation product and stability serve to predict the observed pH optimum for the amidation reaction. A similar pH sweet spot was observed for EDAC assisted amidation of a carboxylated hydrogel with ethylenediamine.30 The treatment via eq 3 is obviously not quantitative, as the reactivity extends roughly one pH unit higher than that predicted. Also, the proportionality constant has changed considerably between ANDSA and EDAN, with EDAN being much more reactive on an absolute basis than would be expected in light of its partial protonation and the reduced stability of the intermediate at the higher pH. As noted with the introduction of Sf, a number of unidentified factors could be rolled up into the proportionality constant which could be different between EDAN and ANDSA and which could account for the difference in absolute reactivity. It would be speculation to discuss these factors further, but it is clear that these differences do not appear to be significantly pH dependent. The utility of eq 3 in predicting the pH optimum suggests that the pKa of the amine to be incorporated plays a major role in this optimization and that the determined stability factor Sf has semiquantitative significance. The present results imply that generally available pKa values and the above form of Sf can be used to optimize EDAC assisted amidation.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the assistance of Dr. Anil Patel with the mass spectroscopy measurements and thank Unilever R&D for permission to publish this manuscript. 13553

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