Influence of the Backbone Structure on the Release of Bioactive

2000

Bioconjugate Chem. 2010, 21, 2000–2012

Influence of the Backbone Structure on the Release of Bioactive Volatiles from Maleic Acid-Based Polymer Conjugates Damien L. Berthier,* Nicolas Paret, Alain Trachsel, and Andreas Herrmann* Firmenich SA, Division Recherche et De´veloppement, B.P. 239, CH-1211 Gene`ve 8, Switzerland. Received May 7, 2010; Revised Manuscript Received September 22, 2010

Poly(maleic acid monoester)-based β-mercapto ketones were synthesized and investigated as potential delivery systems for the controlled release of bioactive, volatile, R,β-unsaturated enones (such as damascones and damascenones) by retro 1,4-addition. The bioconjugates were prepared in a one-pot synthesis using 2-mercaptoethanol as a linker. The thiol group of 2-mercaptoethanol adds to the double bond of the enone to form a β-mercapto ketone, which was then grafted via nucleophilic ring-opening of the remaining alcohol function onto a series of alternating copolymers of maleic anhydride and 1-octadecene, ethylene, isobutylene, and methyl vinyl ether. The influence of copolymer backbones on the release of δ-damascone was investigated in buffered aqueous solution as a function of pH and time. In the presence of a cationic surfactant, the polymer conjugates were transferred from an aqueous medium to a cotton surface. The deposition and the release of δ-damascone from the cotton surface as a function of the polymer backbone structure were measured by fluorescence spectroscopy and dynamic headspace analysis, respectively. All polymer conjugates were found to deliver higher amounts of the volatile into the headspace than the reference consisting of unmodified δ-damascone. Polymers with a hydrophobic backbone were generally efficiently deposited on the cotton surface, but released δ-damascone only moderately in solution. Conjugates with a more hydrophilic backbone release the active compound more efficiently in water, but are deposited to a lower extent onto the target surface. A good balance of the hydrophobicity and hydrophilicity of the polymer backbone is the key factor to maximize the deposition of the conjugates on the target surface and to optimize the release of the bioactive volatiles.

INTRODUCTION Semiochemicals are highly volatile, biologically active organic compounds that serve for interspecies communication via evaporation and transportation through the air by diffusion (1–3). Many of these compounds have been used as fragrances since antiquity (4, 5), and advances in organic synthesis have resulted in the introduction of new volatile compounds to the palette of the modern perfumer (6–9). As a consequence of their high volatilities (vapor pressures) (10, 11), the perception of these compounds is usually transient. Bearing in mind that the longevity of fragrance perception is an important factor in the appreciation of perfumed consumer articles (12), and that perfumes are generally complex mixtures of fragrance ingredients having different volatilities, water solubilities, and chemical functions (4, 6–9), the stability of these ingredients during storage, their efficient delivery, and their resilience in application are very important. Physical (encapsulation-based) (13, 14) or chemical (precursors) (15) delivery systems have been developed to improve substantivity, which is defined as the ability to be deposited on different substrates and the retention of volatile perfumery ingredients. Because of their particular physicochemical properties, a broad variety of polymers (12, 16–20) and polymer conjugates (15, 21–30) have been described as suitable carrier materials to control the release of fragrances. These materials can disperse hydrophobic volatile molecules in aqueous media and enhance their deposition on various substrates. Physical delivery systems are based on the preparation of polymer matrices (12, 16–19) or core-shell structures (20), * To whom correspondence should be addressed. E-mail: damien. [email protected], [email protected]. Fax: +41 22 780 33 34.

which stabilize sensitive compounds whose slow diffusion out of the encapsulating matrix or capsule improves the duration of fragrance perception. In contrast, chemical delivery systems, so-called profragrances, use mild ambient conditions to cleave the covalent bond of the volatile conjugated to a precursor substrate (15). Reaction conditions allowing the cleavage of a chemical bond between a substrate and a targeted molecule comprise changes in temperature, hydrolysis or change of pH, oxidation, and exposure to daylight or enzymes. Because of their macromolecular structure, polymer conjugates were generally found to slow down the release of volatiles compared with the corresponding monomeric, low-molecular-weight analogues (22–29). Changes in the polymer structure with respect to the hydrophilicity or hydrophobicity of the polymer backbone (28–30), as well as the presence of specific chemical functionalities in close proximity to the release unit (29), were found to strongly influence the release rates of the volatiles from their precursors. Endo and co-workers prepared a series of amphiphilic poly(methacrylate) copolymers with different ratios of hydrophobic fragrance-releasing acetal side chains and hydrophilic poly(ethylene glycol) (PEG) side chains (30). They found that the polymers aggregate to micelles, which strongly influenced the cleavage of the acid-labile acetal side chain and thus the release of the fragrance. With the polymer aggregation being temperature dependent, application of heating and cooling cycles allowed control of the rate of fragrance release from the polymeric profragrances. Recently, Fehr and Galindo (31) reported the Michael-type 1,4-addition of carboxylic acids or thiols to enones, such as damascones or damascenones, the so-called rose ketones (32, 33). The β-mercapto ketone obtained by condensation of 1-dodecanethiol and δ-damascone was especially found to be very

10.1021/bc100223s  2010 American Chemical Society Published on Web 10/11/2010

Release of Volatiles from MA-Based Conjugates Scheme 1. Fabric Softening Process in a Washing Machine

efficient as a fragrance precursor and has now been commercialized (31). From these results, we became interested in preparing polymeric fragrance delivery systems that released rose ketones by retro 1,4-addition and in investigating the influence of structural modifications on the surface deposition of the polymers from an aqueous environment and the release efficiency of the bioactive volatile. A recent study reported the pH-dependent release of δ-damascone from amphiphilic random copolymers of a β-acyloxy ketone derivative of δ-damascone and methacrylic acid (28). Poly(methacrylate)-based profragrances were synthesized via an aldol condensation of the corresponding methyl ketone with acetaldehyde, followed by esterification of the resultant β-hydroxy ketones with methacrylic acid (28, 31). Subsequent copolymerization with methacrylic acid, at different molar ratios to adjust the polarity of the polymer backbone, controlled the surface adsorption/deposition of the polymer on various substrates and its dispersion in aqueous media. Although this procedure provided access to a very efficient pH-dependent delivery system for δ-damascone by retro 1,4-addition, the preparation of the intermediate 3-hydroxy ketone of δ-damascone is costly and difficult to scale up. Because alternating copolymers of maleic anhydride can be easily modified by ring-opening of the reactive anhydride unit with alcohols or amines (34, 35), these are suitable substrates for the grafting of bioactive compounds and, at the same time, for fine-tuning the physicochemical properties of the resulting conjugate. Accordingly, various maleic acid monoesters were prepared by reaction with monomeric or polymeric alcohols (36–41) to afford a series of versatile, biocompatible delivery systems (42, 43). To be perceived, fragrance molecules have to be evaporated from a surface. In fine perfumery, surface deposition is easily achieved by spraying the volatiles together with a solvent onto the target surface (e.g., skin, fabric). However, in other types of application, surface deposition is less evident and becomes an important issue to control the release of volatiles. Scheme 1 shows a typical fabric softening process in a washing machine. A concentrated surfactant emulsion, serving as the softening agent, is rinsed into a larger volume of water corresponding to a dilution by a factor of ca. 300. The active agent is then deposited from this diluted surfactant emulsion onto the fabric (cotton) surface. After stirring for a short time, the emulsion is withdrawn and the fabric dried. To achieve a controlled release effect, it is important to understand and control the release

Bioconjugate Chem., Vol. 21, No. 11, 2010 2001 Scheme 2. Preparation of β-Mercapto Ketone 1 by 1,4-Addition of 2-Mercaptoethanol to δ-Damascone

properties of the delivery system in aqueous solution (during the washing cycle), the amount of its deposition onto the fabric, as well as the evaporation kinetics on dry fabric (after the washing cycle). In this study, we thus prepared a series of maleic anhydridebased δ-damascone conjugates in an easy one-pot, two-step process using 2-mercaptoethanol as a linker. 1,4-Addition of the thiol group to the enone double bond of δ-damascone gave β-mercapto ketone 1 (Scheme 2), which then reacted by nucleophilicring-openingwithpoly(maleicanhydride)copolymers. The release of the fragrances by retro 1,4-addition was investigated in solution as a function of pH by solvent extraction. The copolymer conjugates were labeled with pyren-1-ylmethanol (44) to quantify their deposition from an aqueous dispersion onto a cotton surface in the presence of a cationic surfactant. Finally, the influence of the polymer backbone structure on the amount of polymer deposition and release of δ-damascone was investigated by dynamic headspace analysis (45–47) on dry cotton.

EXPERIMENTAL PROCEDURES General. Commercially available reagents and solvents were used without further purification if not stated otherwise. Reactions were carried out in standard glassware under N2 or Ar, and yields were not optimized. Demineralized H2O: MilliporeSynergy-185 water purifier. IR Spectra: Perkin-Elmer-1600FTIR spectrometer; ν˜ of weak (w), medium (m), or strong (s) bands in cm-1. 1H and 13C NMR spectra: Bruker-DPX-400 spectrometer; δ in ppm downfield from Me4Si as internal standard, J in Hz. High-resolution mass spectra (HRMS) were recorded in the multimode (MM) on an Agilent 1200 RR highperformance liquid chromatograph, equipped with an Agilent Eclipse Plus C18 column (2.1 × 100 mm inner diameter (i.d.)), eluted at 0.5 mL min-1 with a gradient of water (containing 0.1% of formic acid)/acetonitrile at 50 °C and coupled to a MSD TOF HR G3250A mass spectrometer (MM source, dual mode positive) at 250 °C, with the N2 flow at 5 mL min-1, the nebulizer pressure at 40 psi, the capillary voltage at 3000 V, and the fragmentor voltage at 140 V. Analytical Size-Exclusion Chromatography (SEC). SEC analyses were carried out at room temperature (ca. 22 °C) with a system composed of a Thermo Finnigan Surveyor vacuum online degasser, quaternary LC pump, autosampler, and UV/ vis detector, combined with a Thermo Separation Products (tsp) Spectra System IR 150 refractometer and a Viscotek 270 Dual Detector viscometer. Samples were eluted from a Macherey Nagel Nucleogel GPC 104-5 column (300 × 7.7 mm i.d., particle size 5 µm) at a flow rate of 1.0 mL min-1 by using HPLC-grade tetrahydrofuran (THF) from SDS. Universal calibrations were performed with the viscometer and the RI detector using commercial poly(styrene) standards from Fluka. Ca. 40 mg of the polymer standard was accurately weighed and dissolved in 10 mL of THF; then 50 µL aliquots of those solutions were injected for the calibration. Molecular weights of poly(maleic anhydrides) were given by the manufacturer to

2002 Bioconjugate Chem., Vol. 21, No. 11, 2010

be Mn ) 30 000-50 000 g mol-1 (2), Mw ) 60 000 g mol-1 (3), Mw ) 100 000-500 000 g mol-1 (4), and Mw ) 200 000 g mol-1 (5). 3-(2-Hydroxy-ethylsulfanyl)-1-(2,6,6-trimethyl-cyclohex-3enyl)-butan-1-one (1). (E)-1-(2,6,6-Trimethylcyclohex-3-enyl)but-2-en-1-one (δ-damascone, 10.0 g, 52.1 mmol) and 1,8diazabicyclo[5.4.0]undec-7-ene (DBU, 0.78 mL, 5.21 mmol) were dissolved in THF (50 mL) prior to the dropwise addition of a solution of 2-mercaptoethanol (3.5 mL, 50 mmol) in THF (20 mL) at 45 °C; the mixture was then stirred for 12 h. Treatment with 5% aqueous HCl, extraction with Et2O, and consecutive washing of the organic phase with a saturated aqueous solution of NaHCO3 and a saturated solution of NaCl afforded an organic phase which was dried (Na2SO4), filtered, and concentrated to give a colorless oil (13.8 g, 99%). 1H NMR (400 MHz, CDCl3): δ 5.53 (m, 1H), 5.45 (m, 1H), 3.77 (m, 2H), 3.35 (sext., 6.9 Hz, 1H), 2.96 (m, 1H), 2.88 (m, 1H), 2.86-2.63 (m, 3H), 2.60-2.45 (m, 2H), 2.23 (m, 1H), 1.97 (m, 1H), 1.70 (m, 1H), 1.31 (m, 3H), 1.02 (m, 1H), 0.96 (m, 5H), 0.92 (d, 6.9 Hz, 2H), 0.88 (d, 6.9 Hz, 1H) ppm. 13C NMR (100.6 MHz, CDCl3): δ 212.76 (s), 212.61 (s), 131.81 (d), 131.68 (d), 124.23 (d), 124.13 (d), 67.96 (t), 62.89 (d), 61.15 (t), 61.13 (t), 55.13 (t), 41.75 (t), 41.70 (t), 36.48 (q), 34.38 (t), 34.27 (t), 34.05 (d), 33.98 (d), 33.20 (q), 33.13 (q), 31.75 (d), 31.69 (d), 31.44 (d), 31.38 (d), 29.85 (q), 29.75 (q), 25.60 (t), 22.32 (q), 22.14 (q), 20.75 (q), 20.73 (q), 19.92 (q), 19.88 (q) ppm. IR (neat): 3415w, 3018w, 2956m, 2928m, 2871m, 2830w, 1703s, 1667m, 1625w, 1457m, 1430w, 1386m, 1366s, 1286w, 1250w, 1212w, 1153m, 1115m, 1044s, 999s, 953w, 932w, 895w, 843w, 690s cm-1. HRMS: m/z calcd. for C15H27O2S, [M + H]+ 271.17263, found 271.17080. General Procedure to Prepare Alternating Copolymers of Maleic Acid-Based Profragrances. δ-Damascone (2.68 g, 13.94 mmol), 2-mercaptoethanol (1.08 g, 13.80 mmol), and 2,3,4,6, 7,8,9,10-octahydropyrimido[1,2-a]azepine (0.21 mL, 1.39 mmol) were dissolved in acetone (50 mL) to give a colorless solution. The solution was stirred at 45 °C for 2 h. Thin layer chromatography (TLC) in ethyl acetate showed a complete conversion; no more 2-mercaptoethanol was detected (rf ) 0.15). Pyren-1ylmethanol (31.70 mg, 0.14 mmol) was added to the solution before one of the copolymers 2-5 (13.94 mmol of maleic anhydride unit) in acetone (30 mL) was added dropwise. The reaction mixture was stirred at 45 °C for 48 h. Copolymers 6-9 were obtained by precipitation from pentane or diethyl ether, filtered, and dried under vacuum at 50 °C for 24 h. Alternating Copolymer of 1-Octadecene and Maleic Acid Mono-{2-[1-methyl-3-oxo-3-(2,6,6-trimethyl-cyclohex-3-enyl)propylsulfanyl]ethyl} Ester Labeled with Pyren-1-ylmethanol (6). Precipitation afforded a white solid (4.82 g, 70%, conversion (of 1) 51%). 1H NMR (DMSO-d6, 400 MHz): δ 8.46-8.22 (br. m, 0.08H), 7.02 (br. m, 0.01H), 5.52 (m, 1H), 5.46 (m, 1H), 4.09 (m, 1.5H), 3.51 (m, 1H), 3.28 (m, 6H), 2.96 (s, 6H), 2.74 (m, 4H), 2.59 (m, 1.5H), 2.43-2.15 (br. m, 4H), 1.93 (m, 3H), 1.63 (m, 5H), 1.18 (m, 60H), 0.96 (m, 4H), 0.82 (m, 13H) ppm. 13 C NMR (DMSO-d6, 100.6 MHz): δ 162.64 (s), 131.54 (d), 124.13 (d), 62.97 (d), 61.37 (t), 54.45 (t), 45.33 (t), 40.97 (t), 36.42 (q), 33.88 (t), 32.52 (t), 31.41 (t), 31.34 (d), 31.02 (q), 29.24 (t), 22.09 (t), 21.40 (q), 20.52 (q), 20.36 (q), 19.34 (q) ppm. IR (neat): 3634 to 2324m, 3018w, 2922s, 2852s, 2605w, 2498w, 1778w, 1726m, 1710m, 1649m, 1625w, 1567m, 1464m, 1397w, 1386m, 1366m, 1323w, 1167s cm-1. Mw ) 68 600 g mol-1. Mn ) 28 000 g mol-1. Alternating Copolymer of Isobutylene and Maleic Acid Mono-{2-[1-methyl-3-oxo-3-(2,6,6-trimethyl-cyclohex-3-enyl)propylsulfanyl]ethyl} Ester Labeled with Pyren-1-ylmethanol (7). Precipitation afforded a white solid (1.91 g, 65%, conversion (of 1) 20%). 1H NMR (DMSO-d6, 400 MHz): δ 8.63-8.19 (br.

Berthier et al.

m, 0.05H), 7.71 (m, 0.01H), 7.04 (m, 0.03H), 5.54 (m, 1H), 5.46 (m, 1H), 4.95 (m, 3H), 4.08 (m, 2H), 3.61 (m, 2H), 3.54 (m, 6H), 3.48 (m, 6H), 3.30 (m, 3H), 2.68 (m, 9H), 2.31 (m, 9H), 1.97 (m, 18H), 1.06 (br. m, 24H) ppm. 13C NMR (DMSOd6, 100.6 MHz): δ 165.31 (s), 162.25 (s), 131.56 (d), 124.13 (d), 61.45 (d), 41.77 (d), 23.40 (br. d) ppm. IR (neat): 3768 to 2495w, 2959m, 2937w, 2879m, 1786s, 1731s, 1646s, 1585w, 1469m, 1394m, 1371m, 1321m, 1175s cm-1. Alternating Copolymer of Ethylene and Maleic Acid Mono{2-[1-methyl-3-oxo-3-(2,6,6-trimethyl-cyclohex-3-enyl)propylsulfanyl]ethyl} Ester Labeled with Pyren-1-ylmethanol (8). Precipitation afforded a white solid (3.27 g, 89%, conversion (of 1) 50%). 1H NMR (DMSO-d6, 400 MHz): δ 8.52-8.16 (br. m, 0.17H), 5.54 (m, 1H), 5.47 (m, 1H), 4.09 (m, 2H), 3.52 (m, 4H), 3.25 (m, 3H), 3.01 (m, 2H), 2.87 (m, 10H), 2.71 (m, 4H), 2.32 (m, 3H), 2.12 (m, 3H), 1.93 (m, 3H), 1.63 (m, 5H), 1.43 (m, 5H), 1.24 (m, 4H), 0.95 (m, 3H), 0.84 (m, 6H) ppm. 13C NMR (DMSO-d6, 100.6 MHz): δ 211.93 (s), 176.46 (s), 131.53 (d), 124.15 (d), 63.02 (t), 61.31 (t), 60.96 (d), 54.36 (t), 54.19 (t), 53.25 (d), 49.64 (t), 42.72 (d), 37.53 (d), 34.00 (s), 32.52 (d), 31.57 (d), 31.13 (d), 31.03 (t), 28.16 (t), 25.82 (t), 23.27 (q), 21.49 (q), 21.30 (q), 20.31 (q), 19.38 (q), 18.82 (q), 9.36 (q) ppm. IR (neat): 3750 to 2162m, 2952m, 2933m, 2871m, 1786w, 1722s, 1705s, 1649m, 1562w, 1455m, 1387m, 1361m, 1156s cm-1. Alternating Copolymer of Methoxyethylene and Maleic Acid Mono-{2-[1-methyl-3-oxo-3-(2,6,6-trimethyl-cyclohex-3enyl)propylsulfanyl]ethyl} Ester Labeled with Pyren-1-ylmethanol (9). Precipitation afforded a white solid (4.89 g, 95%, conversion (of 1) 30%). 1H NMR (DMSO-d6, 400 MHz): δ 8.46-8.02 (br. m, 0.11H), 7.22 (m, 0.01H), 5.54 (m, 1H), 5.47 (m, 1H), 4.11 (m, 2H), 3.52 (m, 4H), 3.26 (m, 12H), 2.88 (m, 2H), 2.74 (m, 4H), 2.63 (m, 2H), 2.31 (m, 2H), 1.95 (m, 2H), 1.64 (m, 3H), 1.23 (m, 3H), 0.95 (m, 3H), 0.84 (m, 6H) ppm. 13 C NMR (DMSO-d6, 100.6 MHz): δ 211.70 (s), 172.67 (s), 165.30 (s), 131.46 (d), 124.67 (d), 64.80 (d), 63.23 (d), 61.30 (s), 54.16 (t), 53.32 (t), 47.78 (t), 40.91 (t), 37.49 (d), 33.99 (d), 32.54 (d), 31.65 (q), 23.20 (t), 21.47 (q), 20.47 (q), 19.36 (q) ppm. IR (neat): 3750 to 2323m, 3016w, 2956m, 2932m, 2884w, 2831w, 1855w, 1778m, 1728s, 1704s, 1648m, 1587w 1454m, 1441m, 1386m, 1366m, 1165s cm-1. Mw ) 315 000 g mol-1. Mn ) 53 000 g mol-1. Alternating Copolymer of Methoxyethylene and Maleic Acid Mono-(r-methoxy-poly(ethylene oxide)) And Mono-{2[1-methyl-3-oxo-3-(2,6,6-trimethyl-cyclohex-3-enyl)propylsulfanyl]ethyl} Esters Labeled with Pyren-1-ylmethanol (10). Copolymer 5 (10 mmol) was dissolved in acetone (40 mL) prior to the dropwise addition of a solution of 1 (8 mmol), pyren-1ylmethanol (0.01 mmol), and R-methoxy-poly(ethylene glycol) (2 mmol, Mn ) 550 g mol-1) in acetone (30 mL) and a solution of triethylamine (10 mmol) and 4-dimethylaminopyridine (DMAP, 61 mg, 0.5 mmol) in acetone (30 mL) at 0 °C. The mixture was heated at 40 °C for 24 h. The cooled mixture was then diluted with dichloromethane and extracted with aqueous HCl (5%), dried (Na2SO4), filtered, concentrated, and precipitated from pentane to afford a red solid (4.69 g, 88%, conversion (of 1) 75%). 1H NMR (DMSO-d6, 400 MHz): δ 8.46-8.02 (br. m, 0.09H), 7.22 (m, 0.02H), 5.54 (m, 1H), 5.45 (m, 1H), 4.78 (m, 0.3H), 4.57 (m, 0.3H), 4.09 (m, 1.8H), 3.51 (m, 15H), 3.33 (m, 28H), 2.88 (m, 1.3H), 2.74 (m, 3H), 2.61 (m, 2H), 2.32 (m, 2H), 1.94 (m, 2H), 1.65 (m, 3H), 1.24 (m, 3H), 0.94 (m, 3H), 0.83 (m, 6H) ppm. 13C NMR (DMSO-d6, 100.6 MHz): δ 211.71 (s), 165.31 (s), 131.51 (d), 124.10 (d), 71.16 (t), 69.66 (t), 63.18 (d), 61.30 (s), 60.99 (t), 40.99 (q), 37.54 (d), 34.02 (d), 33.85 (s), 32.57 (t), 32.51 (t), 31.64 (t), 31.14 (t), 31.02 (t), 29.04 (s), 27.81 (t), 21.45 (br. q), 20.45 (q), 19.33 (q) ppm. IR

Release of Volatiles from MA-Based Conjugates

(neat): 3675 to 2323m, 2953m, 2930m, 2884m, 2831w, 1727s, 1705s, 1648m, 1587w, 1453m, 1386w, 1365m, 1323w, 1172s cm-1. Controlled Release of δ-Damascone From Copolymers 6-10 in Buffered Solutions. According to the molecular mass of their repeat unit, copolymers 6-10 were dispersed in a buffered aqueous solution (CertiPUR from Merck, 5 mL) at pHs 4, 7, and 10, respectively. The solutions thus contained equimolar concentrations of δ-damascone (0.045 mmol mL-1 or 8.7 mg mL-1). Samples were stirred for 1 h to obtain a solution or a homogeneous dispersion. Each solution or dispersion was divided into four samples of 1 mL and mixed with 1 mL of a solution of 1,4-dibromobenzene at 150 mg L-1 (internal standard) in a mixture of isooctane/diethyl ether (9:1). The samples were stirred at 25 °C in a water bath for 1, 3, and 6 days. The δ-damascone content in the organic phase was analyzed by gas chromatography (GC) using a GC 7890A system equipped with an auto sampler 7683 series from Agilent Technologies (1 µL injected) and a HP-5 column (30 m, i.d. 0.32 mm, film thickness 0.25 µm). The volatiles were analyzed by GC at 100 °C for 1 min and then heated to 170 at 10 °C min-1, for a total run time of 8 min. Helium was the mobile gas phase; the split ratio was 50:1. A calibration curve was measured and used to quantify δ-damascone. All samples were analyzed in triplicate. Thermogravimetric Analysis (TGA). Solid contents were measured by thermogravimetry using an organic phase solution (20 µL) and placed with a precision syringe (Hamilton Bonaduz, Switzerland, 25 µL) inside an aluminum pan (MettlerToledo ME-26763 40 µL) that had been previously weighed on a precision balance (Mettler-Toledo XP204S). Measurements were carried out under a constant nitrogen flow (20 mL min-1) with a thermogravimetric analyzer (Mettler-Toledo TGA/ SDTA851e) equipped with a microbalance (accuracy: 1 µg) and an accurate oven having an internal volume of 35 mL. Temperatures were increased from 25 to 80 °C at 10 °C min-1 and kept at 80 °C for 25 min and 30 s. All measurements were performed in triplicate. The stability of copolymers 6 and 9 (data not shown) was measured by thermogravimetry. Solids were placed inside an aluminum pan that had been previously weighed on a precision balance. Temperatures were increased from 20 to 270 at 10 °C min-1 under a constant nitrogen flow (20 mL min-1). Preparation of Aqueous Surfactant Emulsions. A cationic surfactant containing emulsion with the following composition was prepared: Stepantex VK90 (Stepan) 16.5%, calcium chloride 0.2%, water 83.3%. A total of 1.0% to 5.5%, depending on the δ-damascone content of the polymers, of poly(maleic acid)based copolymers was added to the softener formulation. All values are given in % by weight. Procedure for Polymer Deposition on Cotton. The fabric softening process in a washing machine as illustrated in Scheme 1 was transferred to a laboratory scale. In a beaker (1 L), poly(maleic acid)-based copolymers 6-10 (6, 43.6 mg (2.4%) in THF (3 mL); 7, 48.3 mg (2.7%) in methanol (5 mL); 8, 23.5 mg (1.3%) in methanol (5 mL); 9, 348.7 mg (10% in dipropylene glycol (DIPG), 1.9%); 10, 312.5 mg (10% in DIPG, 1.7%), all of which can release a total amount of 8.7 mg of δ-damascone) were added to the aqueous concentrated surfactant emulsion (1.80 g, pH ca. 3.1), respectively, and the resulting mixtures stirred for 18 h. The emulsions were then placed in a beaker (1 L) and diluted with demineralized cold tap water (600 g). One cotton sheet (Eidgeno¨ssische Materialpru¨fanstalt (EMPA, Switzerland), cotton test cloth Nr. 221, cut to ca. 12 × 12 cm2 sheets (average mass ca. 3.5 g) and prewashed with an unperfumed detergent powder (perfumery application laboratories)) was added in a beaker to the diluted surfactant emulsion

Bioconjugate Chem., Vol. 21, No. 11, 2010 2003

(pH ca. 4.1) and manually stirred for 3 min, left standing for 2 min, and then wrung out by hand and weighed (average mass ca. 7 g) to estimate the quantity of residual water and to ensure that each cotton sheet was loaded with the same amount of diluted surfactant emulsion. As a reference sample, a solution (1 mL) containing an equimolar amount of unmodified δ-damascone (87.2 mg in 10 mL of acetone) was added to another 1.80 g of the original fabric softener formulation, which was treated as described above. All cotton sheets were line dried for 3 days. Procedure for Fluorescence Measurements. Poly(maleic acid)-based copolymers 6-10 were added to the aqueous surfactant emulsion and diluted with demineralized cold tap water as described above. Several aliquots of the emulsions were pipetted off (1.0, 0.5, and 0.1 mL) and adjusted to 1.0 mL with the diluted aqueous surfactant emulsion. For the verification of the linearity of the fluorescence intensity with respect to the copolymer concentration, additional points using 0.75 and 0.25 mL of the emulsion were recorded. All samples were then further diluted with demineralized water by a factor of 100 and analyzed by fluorescence spectroscopy. Measurements were carried out on a Jobin Yvon Fluorolog-3 spectrometer equipped with DataMax software. The average spectra of three scans were recorded between 370 and 400 nm, with an excitation wavelength of 334 nm, increment 0.5 nm, integration time 0.5 s, slit excitation 1.5 nm, and slit emission 1.5 nm. Data were analyzed at 378, 384, 389, and 398 nm. The fluorescence intensity measured for the 1.00 mL aliquot served as the reference corresponding to 0% of deposition and the other aliquots as external standard calibration of the corresponding polymer. Then, one cotton sheet (see above) was added to each beaker containing the diluted emulsions with the copolymer, and manually stirred for 3 min, left standing for 2 min, and then wrung out by hand, and line-dried for 3 days (see above). The remaining emulsion (1.00 mL) was diluted by a factor of 100 and analyzed by fluorescence spectroscopy as described above to determine the amount of remaining nondeposited copolymer. The missing amount with respect to the first fluorescence measurement was assumed as being deposited on the fabric surface. All measurements were carried out in duplicate. Procedure for Dynamic Headspace Sampling. One of the dry cotton sheets was put into a headspace sampling cell (internal volume ca. 160 mL), thermostatted at 25 °C, and exposed to a constant airflow (200 mL min-1), respectively (48). The air was filtered through active charcoal and aspirated through a saturated solution of NaCl, corresponding to a constant humidity of ca. 75% (49). For 15 min, the volatiles were adsorbed onto a waste Tenax cartridge and then for 15 min onto a clean Tenax cartridge. The sampling was repeated seven times every 60 min (45 min trapping on the waste cartridge and 15 min on a clean cartridge) (48); the waste cartridges were discarded. The cartridges with the volatiles were desorbed on a Perkin-Elmer TurboMatrix ATD 350 desorber coupled to a Perkin-Elmer Autosystem XL gas chromatograph equipped with a J&W Scientific DB1 capillary column (30 m, i.d. 0.25 mm, film thickness 0.25 µm) and a Perkin-Elmer Turbomass Upgrade mass spectrometer. The volatiles were analyzed by GC using a two-step temperature gradient starting from 70 to 130 °C at 3 °C min-1 and then heating to 260 at 25 °C min-1. The injection temperature was at 240 °C and the detector temperature at 260 °C. External standard calibrations were carried out using six different concentrations of δ-damascone in acetone (varying between 1.96 × 10-6 and 9.26 × 10-4 mol L-1). Each calibration solution (2 µL) was injected with a microliter syringe onto three clean Tenax cartridges, respectively. All the cartridges were desorbed immediately under the same conditions as those resulting from the headspace sampling (see above). Because


Berthier et al.

Scheme 3. Preparation of Poly(maleic acid monoester)-Based δ-Damascone Conjugates 6 (x ) 55, y ) 0.1, z ) 50), 7 (x ) 65, y ) 0.3, z ) 260), 8 (x ) 640, y ) 1, z ) 640), and 9 (x ) 320, y ) 1, z ) 750)

the quantities of released compounds were monitored at very low concentrations, the calibration curve was forced through the origin of the coordinate system. Plotting the concentrations (in ng L-1) against the peak areas gave a straight line with a correlation coefficient of r 2 > 0.9990. All experiments were carried out in duplicate.

RESULTS AND DISCUSSION Preparation of Poly(maleic acid)-Based Profragrances. Poly(maleic acid)-based conjugates of δ-damascone were prepared in a one-pot procedure consisting of two reaction steps. In the first step, 2-mercaptoethanol, serving as a linker (50), was reacted with a slight excess of δ-damascone to give 1,4-addition product 1 in very good yields (Scheme 2). This reaction step was carried out in the presence of catalytic amounts of DBU as a base (31). β-Mercapto ketone 1 was isolated for full characterization. 1H NMR analysis showed the disappearance of the enone double bond and the appearance of two signals corresponding to the formation of the thioether (a sextuplet at 3.3-3.4 ppm for the CH group of the rose ketone moiety and a multiplet at 2.5-2.8 for the CH2 group of the ethylene linker), whereas the signals of the other double bonds remained unchanged. After complete reaction of the mercaptoethanol, as shown by TLC in ethyl acetate, one of the commercially available poly(maleic anhydride) copolymers 2-5, respectively, was added as the second reaction step to react with the primary alcohol function of 1 upon ring-opening of the maleic anhydride function (Scheme 3). Copolymers 2-5, obtained by copolymerization of maleic anhydride with 1-octadecene, isobutylene, ethylene, or methyl vinylether, respectively, are commercially available. Alternating copolymer 5, for example, is supplied by ISP Technologies under the trade name Gantrez and used as an ingredient in toothpaste and various pharmaceutical products (43). Depending on the solubility of the copolymers, different solvents (DMF, THF, or acetone) were used for the reaction with β-mercapto ketone 1. Four different bases, namely, pyridine, tributylamine, triisobutylamine, and triethylamine (Et3N), with a catalytic amount of DMAP, were tested to open the maleic anhydride moiety of 2-5 with 1 to give polymer conjugates 6-10 (Scheme 3); triethylamine led to the best conversions (36–41). In this work, the one-pot two-step preparation of copolymers 6-10 was carried out in acetone, which is a convenient solvent

for an industrial scaleup. Intermediate 1 was synthesized in situ by adding 2-mercaptoethanol to δ-damascone in the presence of DBU as catalyst. When the conversion was completed, the acetone solutions of copolymers 2-5 were added to 1. We observed that the presence of DBU as a base was sufficient to reach acceptable conversion (see Experimental Procedures). Grafting a fluorescent marker onto the polymer backbone (44) is convenient to measure the deposition on various surfaces. We labeled our polymer conjugates by adding pyren-1-ylmethanol to the reaction mixture (51). Its concentration was limited to 0.1 mol % to avoid saturation of the fluorescent signal and to keep the physical properties of copolymers 6-10. Amphiphilic polymer conjugates are known to aggregate in surfactant emulsions (52). However, this aspect was not studied in the context of this work because it requires higher grafting of the fluorescence moiety. The conversion of the grafting reaction was analyzed by 1H NMR spectroscopy by integrating the signals of the cyclohexene double bonds (between 5.45 and 5.85 ppm) of alcohol 1 and comparing them with characteristic resonances from the different copolymers 2-5, such as the signal of the methyl group of the alkyl chain of 1-octadecene in 2 (at 0.87 ppm), the signal of the dimethyl group of the isobutylene unit in 3 (at 1.07 ppm), the broad signal of the poly(ethylene) backbone in 4 (1.40 to 2.15 ppm), and the signal of the methyl ether (at 3.36 ppm) of copolymer 5. Despite the fact that ring-opening of maleic anhydride creates carboxylic acid functions that increase the polarity of the polymer backbone, this effect was counterbalanced by the introduction of the rather hydrophobic rose ketone release unit. In contrast to copolymer 9, conjugates 6-8 were found to be almost insoluble in aqueous media and gave only dispersions. This solubility profile indicates that minor structural modifications of the copolymer structure have an important influence on the physicochemical properties of the resulting conjugate (28, 29). The release of δ-damascone from the thioether conjugate is pH dependent. Although relatively stable under acidic conditions, the precursors slowly release the bioactive compound at pH values above 6 (31). Furthermore, for an efficient release, the polymer must be soluble, or at least welldispersed, in water (28). To fine-tune the water solubility, we grafted poly(ethylene glycol) side chains onto the polymer backbone by using the same one-pot synthesis described above. PEG is a water-soluble and biocompatible polymer that has already been used to modify



Scheme 4. Preparation of Poly(maleic acid monoester)-Based δ-Damascone Conjugate 10 (a ) 800, b ) 215, c ) 1, d ) 59)

Scheme 5. Mechanism of Release of δ-Damascone from Copolymers 6 (x ) 55, y ) 0.1, z ) 50), 7 (x ) 65, y ) 0.3, z ) 260), 8 (x ) 640, y ) 1, z ) 640), 9 (x ) 320, y ) 1, z ) 750), and 10 (x ) 1015 (x ) a + b, see Scheme 4), y ) 1 () c), z ) 59 () d))

different poly(maleic anhydride) copolymers of styrene (53), 1-hexene (54), and methyl vinyl ether (55). We thus prepared a PEG-modified analogue of copolymer 9 by adding a mixture of a poly(ethylene glycol) monomethyl ether (20 mol % with respect to the maleic anhydride unit) and 1 (80 mol %) to polymer 5 to afford conjugate 10 (Scheme 4). The grafting of the PEG chains on 5 was complete, and conversion of 1 was estimated at 75 mol %. Measurement of the Controlled Release of δ-Damascone from Poly(Maleic Acid)-Based Conjugates in Buffered Solutions at pH 4, 7, and 10 as a Function of Time. In technical applications or under physiological conditions, Michael-type 1,4addition of thiols to R,β-unsaturated ketones can be reversible, as described for the reaction of prostaglandins with various thiols (56). In the present study, the release of δ-damascone from copolymers 6-10 is presumably also controlled by this mechanism (Scheme 5). The influence of the copolymer structure on the release of δ-damascone from conjugates 6-10 was first studied in buffered aqueous solution at different pH values. The pH influences the kinetics of the retro 1,4-addition. Furthermore, the solubility of the different delivery systems in aqueous media is impacted by the pH because the carboxylic acid functions can be deprotonated under neutral or alkaline conditions (28). The amount of each polymer was chosen to release the same concentration of δ-damascone in all samples, that is to say, 8.7 mg mL-1 or

8700 ng µL-1. In order to prevent equilibration, a nonmiscible organic solvent, a mixture of isooctane/diethyl ether (9:1), was added to the system to continuously extract the released δ-damascone (30). Organic and water phases were gently stirred so as not to mix with each other at pH 4 (the pH of fabric softener applications), 7, and 10 (the pH of liquid detergent applications) for different times. The quantity of δ-damascone released into the organic phase was analyzed by GC. Results were plotted as a function of pH for copolymers 6-10 after 1, 3, and 6 days (Figures 1a-d). In solution, the release of δ-damascone is strongly influenced by the polymer backbone. When the polymer contained methyl groups or hydrogen atoms, as in copolymers 7 and 8, respectively, the solubility in the buffered solutions was very limited, especially at low pH. Both copolymers 7 and 8 gave stable dispersions at pH 4 and 7, and were soluble only at pH 10. They released only 253.9 and 325.2 ng µL-1 of δ-damascone at pH 10 after 6 days, which corresponded to 2.9% and 3.7% of δ-damascone grafted on the copolymers, respectively. When the comonomer of the backbone contained a methoxy group, the resulting copolymer 9 was soluble at all pH values. A faster release was observed with a δ-damascone concentration of 999.5 ng µL-1 measured at pH 10 after 6 days. This trend suggests that the higher solubility of a copolymer facilitates the release by retro 1,4-addition. This result was confirmed at the example of copolymer 10, which was modified with PEG side


Berthier et al.

Figure 1. a-d. Concentrations of δ-damascone released from copolymers 7 (a, R1 ) R2 ) Me), 8 (b, R1 ) R2 ) H), 9 (c, R1 ) MeO), and 10 (d, R1 ) MeO + PEG chain ester) as a function of pH after 1, 3, and 6 days as determined by GC analysis.

Figure 2. Concentrations of δ-damascone released from copolymer 6 as a function of pH after 1, 3, and 6 days as determined by GC analysis.

chains. Its solubility was significantly improved, and the amount of δ-damascone released from copolymer 10 increased faster than in the case of the unmodified copolymer 9, even at pH 4 (Figure 1d). The measured δ-damascone concentrations varied between 836.2 ng µL-1 at pH 4 and 1689.9 ng µL-1 at pH 10, corresponding to a release of 19.4%. Nonetheless, high water solubility might prevent an efficient deposition of the copolymers on a fabric surface. Therefore, we were also interested in understanding the release from more hydrophobic structures, such as copolymer 6 with a hexadecyl side chain. As observed with copolymers 7-10, the release of δ-damascone from 6 increased with time at different pH values (Figure 2). However, after 1 and 3 days, we observed a more important δ-damascone release at pH 4 than at pH 10. These results are different from those obtained with copolymers 7-10 and in contradiction with the mechanism of release (Scheme 5). In addition, high variability of the data was observed, indicating an uncontrolled process. In comparison with copolymers 7-10, the presence of the hexadecyl side chain should change the solubility profile of copolymer 6, and we expected that a large part of the copolymer was dissolved in the organic phase. The solubility of all copolymers was thus estimated by thermogravimetry. We placed 20 µL of the organic phase (isooctane/diethylether 9:1) inside an aluminum pan, which was dried in a thermogravimeter at

Figure 3. Concentrations (in mg mL-1) of copolymers 6-10 extracted from buffered aqueous solutions into the organic phase as a function of pH after 1 day as determined by thermogravimetric analysis.

80 °C for 30 min. A plateau was reached, indicating that the samples were dried. The experiment was repeated with the pure extraction solvent, which served as the reference. The difference between both plateaus corresponds to the concentration of polymer in the organic phase. Data obtained from measurements performed after one day are reported in Figure 3. Concentrations of copolymers were measured at different pH values as a function of time (Table 1). As expected, the concentrations of copolymers 7-10 in the organic phase are relatively low. The concentration of copolymer 7 in the organic phase was at maximum 1-2%. The concentration of copolymer 8 leveled off at around 6-7%, indicating a saturation of the organic phase, even after 1 day. Solubilization of copolymers 9 and 10 in the organic phase increased slowly with time but did not exceed 8% and 7%, respectively. In contrast, 45% and 58% of copolymer 6 were detected in the organic phase at pH 4 and 7, respectively (Table 1). These concentrations increased with time to reach a maximum at about 78% at pH 7 after 6 days and decreased to 12% at pH 10. At this pH, the carboxylic acid functions of the copolymer are mostly deprotonated, which makes the polymer less soluble in the organic phase. Furthermore, the release of δ-damascone increases the polarity of the polymer and thus decreases its solubility in the organic phase.



Table 1. Concentrations (in µg µL-1 and %) of Copolymers 6-10 in the Organic Phase as a Function of pH and Time copolymers dissolved in organic phase [µg µL-1] ([%]) pH 4 7 10

day #

6

7

8

9

10

1 3 6 1 3 6 1 3 6

19.63 (45.2) 22.08 (50.9) 24.00 (55.3) 25.13 (57.6) 31.75 (73.2) 33.70 (77.7) 5.25 (12.1) 1.00 (2.3) 0.91 (2.1)

0.50 (1.0) 0.14 (0.3) 0.29 (0.6) 0.52 (1.1) 0.48 (1.0) 0.67 (1.4) 0.35 (0.7) 0.19 (0.4) 0.31 (0.7)

1.62 (6.9) 1.51 (6.4) 1.35 (5.7) 1.79 (7.5) 1.69 (7.2) 1.35 (5.7) 1.41 (6.0) 1.44 (6.1) 1.48 (6.3)

0.22 (0.6) 0.44 (1.3) 2.71 (7.8) 0.55 (1.6) 0.73 (2.1) 1.43 (4.1) 1.87 (5.4) 0.68 (2.0) 1.84 (5.3)

0.97 (2.2) 2.67 (6.1) 1.78 (6.7) 0.90 (2.1) 1.35 (3.1) 1.27 (4.0) 1.20 (2.8) 1.42 (3.3) 2.22 (7.0)

These results suggest that a large part of copolymer 6 was dissolved in the organic phase at pH 4 and 7. Its concentration in buffered aqueous solution must thus have decreased, which then slowed down the release of δ-damascone (Figure 2). The concentration of δ-damascone extracted from the aqueous phase was determined by GC analysis using a calibrated flame ionization detector (FID). The presence of copolymer 6 in the organic phase suggests that, at an injection temperature of 250 °C, additional amounts of δ-damascone and other volatiles might be generated by thermal degradation of the polymer conjugate. Therefore, we measured the stability of the copolymer at this temperature by using TGA. Pure copolymer 6 was placed inside an aluminum pan, which was transferred into the TGA. The temperature was increased from 25 to 270 at 10 °C min-1 and the mass loss measured as a function of time (Figure 4). A significant mass loss was detected in the range from 150 to 270 °C, corresponding to 38% of the total polymer mass. The same experiment was carried out with the starting copolymer 2, and a mass loss of only 8% was measured in this case. This result demonstrated that copolymers of maleic acid were not stable at 250 °C (injector temperature represented by a dotted line in Figure 4) and that they degraded (57). We assume that this degradation first releases precursor 1, which then further degrades (Scheme 6). This was confirmed by TGA analysis of 1 for which a complete mass loss was measured at 270 °C, suggesting either evaporation or degradation of 1. With the boiling point of δ-damascone being 253 °C, we thus assume that copolymer 6 degrades, in fact, in two steps, as illustrated

Figure 4. Thermogravimetric analysis of β-mercapto ketone 1 and copolymers 2 and 6 from 25 to 270 °C at 10 °C min-1.

in Scheme 6. The thermal degradation of copolymer 6 extracted into the organic phase thus explains the large amount of δ-damascone detected in our GC measurements. With the ester bond being much more stable than the β-mercapto ketone moiety (15), we did not expect the release of 2-mercaptoethanol under the mild reaction conditions found in the targeted application. This was confirmed by the fact that no smell arising from sulfur compounds was detected in the applications. However, even if we were not able to accurately measure the release of δ-damascone from 6 at pH 4 and 7, the low concentration of this copolymer in the organic phase at pH 10 (Table 1), combined with the high concentration of released δ-damascone, suggested that copolymer 6 might nevertheless be a suitable δ-damascone delivery system in practical applications. The efficient release of δ-damascone at pH 10 emphasizes the importance of the polarity of the backbone on the release rate. The tendency observed at pH 10 was confirmed (at lower extent) at pH 4, which is the pH of our model application. Deposition of the Copolymers from an Aqueous Environment onto Cotton. To test the potential of the different δ-damascone conjugates for practical applications in functional perfumery, we investigated the release of the active volatile in a fabric softening composition. Fabric softeners are particularly interesting in the context of this project, as they are usually acidic (pH ca. 3.1), which means that the polymer conjugates are expected to be stable during storage. In use (e.g., during a normal washing cycle), the fabric softener is diluted with water, resulting in an increase of pH to ca. 4.1, which should trigger the release of the damascone. Fabric softener formulations typically consist of about 16% by weight of a cationic surfactant in water, such as a quaternized triethanolamine ester of a fatty acid (TEA-esterquat) (58, 59). Cationic surfactants are efficiently deposited onto cotton surfaces (58–61) and therefore facilitate the transport of apolar organic molecules such as fragrances from an aqueous environment onto textiles (62–65). It can be expected that the polymer conjugates are also deposited with the surfactant onto cotton surfaces. To ensure that the quantity of polymer deposited onto the cotton depends on the polymer structure and not on the deposition process, the amount of the diluted surfactant emulsion deposited on the cotton sheet was controlled (by weighing a constant amount of remaining water). As outlined above, the polymer deposition should depend on the copolymer structure and should thus be influenced by the nature of the comonomer (28). The polarity of the copolymer is expected to be further influenced by the pH of the system. Besides the choice of the linker (66) and efficient deposition on the target surface, the amount of δ-damascone released into the headspace also depends on the release kinetics of the active compound from the corresponding polymer conjugate; these are dependent on the water solubility of the polymer, as previously shown for a series of poly(methacrylate) derivatives (28). At acidic pH, as in the softener formulation, the carboxylic acid functions of polymers 6-10 are protonated. The pKa of the maleic acid units is a function of the degree of dissociation and


Berthier et al.

Scheme 6. Proposed Mechanism of Degradation of Copolymer 6 (x ) 55, y ) 0.1, z ) 50) at 250°C in the GC Injector

Table 2. Amount of Deposition of Pyrene-Labeled Poly(maleic acid)-Based δ-Damascone Conjugates 6-10 on Cotton as Determined by Fluorescence Spectroscopy (data recorded at 389 nm, excitation at 334 nm)

copolymer

deposition determined with respect to reference [%]

deposition determined by external standard calibration [%]

average deposition [%]

amount of conjugated δ-damascone deposited onto the cotton surface [%]

6 7 8 9 10

44 28 40 29 23

43 41 50 32 25

44 35 45 31 24

3.83 3.05 3.92 2.70 2.09

has been reported to be between 4 and 8 (67). Therefore, upon dilution during the application, the pH increases and the polymers become partially deprotonated, which results in an increased solubility of the conjugates in the aqueous medium. With the release being pH dependent (Figure 1) and triggered at a pH of ca. 6.5 and above (31), additional carboxylic acid functions are generated on the polymer backbone (Scheme 5). The water solubility of the polymer still increases, which should then accelerate the release of the active volatile. The amount of polymer deposition on the cotton surface was determined with the help of the fluorescence label of copolymers 6-10. In a typical experiment, equimolar amounts of copolymers with respect to the total amount of active compound to be released were solubilized in an appropriate solvent and pipetted into an aqueous emulsion of a TEA-esterquat, corresponding to a total of ca. 1.0-5.5% by weight of polymer in the surfactant emulsion. After the emulsion was stirred for several hours, it was diluted with tap water and a small aliquot pipetted off. The measured fluorescence intensity served as the reference, corresponding to 0% of deposition. Then, one cotton sheet was added and stirred manually for 5 min (to allow for the deposition of the surfactant and the polymer conjugate onto the cotton surface). The cotton sheet was removed before another aliquot of the surfactant emulsion was pipetted off, and then the cotton sheet was wrung out by hand and line-dried for 3 days. The measured fluorescence intensity reflected the quantity of the remaining nondeposited copolymer in the surfactant emulsion. The missing amount with respect to the reference measurement was assumed as being deposited on the fabric surface. The release of δ-damascone from the dry cotton surface was then analyzed by dynamic headspace analysis as described below. Despite the fact that the fluorescence label corresponded to only about 0.1 mol % by weight of the total mass of the copolymer, its concentration in the surfactant emulsion was by far too high for accurate fluorescence measurements. A nonlinear fluorescence response was obtained by diluting the sample by a factor varying between 2 and 10. The surfactant emulsions were thus diluted by a factor of 100 and the fluorescence spectra recorded between 370 and 400 nm with an excitation at 334 nm. Additionally, the difference in fluorescence intensity measured at different wavelengths before and after addition of

the cotton sheet was compared with data obtained by external standard calibration. Data were analyzed at 378, 384, 389, and 398 nm. The dependence of the fluorescence intensity at the different wavelengths with respect to the amount of polymer in the emulsion, after dilution by a factor 100, was verified with the example of δ-damascone conjugate 9. At all wavelengths, a linear relationship was observed when varying the polymer concentration over 1 order of magnitude, thus covering a range corresponding to 0-90% of deposition. Extrapolation of the fluorescence intensities to zero polymer concentration revealed some remaining fluorescence at 378 nm, whereas at the other wavelengths, the fluorescence decreased to a fluorescence intensity value close to zero. With the curve recorded at 389 nm having the steepest slope, we selected this wavelength for further analysis. Table 2 lists the data obtained for the deposition of poly(maleic acid)-based δ-damascone conjugates 6-10 on cotton and indicates the total amount of conjugated δ-damascone transferred to the cotton surface. Our measurements confirmed the expected dependence of the polymer structure on the amount of surface deposition. According to the copolymer structures, different amounts of the conjugates were deposited on the cotton surface, varying between 24% for the more hydrophilic polymer 10 and 45% for the more apolar structures 6 and 8, thus confirming an inverse correlation of the deposition with the ease of dispersion of the materials in an aqueous environment, which was also reported previously for a series of poly(methacrylate) derivatives (28, 30). Nevertheless, for practical application, the amount of δ-damascone to be delivered from the target surface is the most important factor to be considered for the efficiency of a delivery system. Increasing the deposition on the target substrate is the first step of optimization; controlling the rate of the covalent bond cleavage reaction is another parameter to be considered. The latter strongly depends on the local environment of the release site (28–30). As the next step, we thus determined the release of δ-damascone from the different poly(maleic acid) conjugates by dynamic headspace analysis after deposition of the polymers from an aqueous environment onto a cotton



Figure 5. Headspace concentrations (in ng L-1 of air) measured for the evaporation of δ-damascone from poly(maleic acid)-based conjugates 6 (-[-), 7 (-×-), 8 (-9-), 9 (-•-), and 10 (-2-) on dry cotton after 3 days with respect to unmodified δ-damascone as the reference (--O---).

Figure 6. Correlation of δ-damascone concentrations measured in buffered aqueous solution (pH 4, after 3 days, black bars), in the headspace above the cotton surface (after 3 days of line-drying, gray bars) with the amount of polymer deposited onto the surface (dotted line) for copolymers 7-10.

surface, and we tried to correlate the amount of deposition with the release efficiency. Dynamic Headspace Analysis of the Controlled Release of δ-Damascone from Poly(maleic acid)-Based Conjugates on Dry Cotton. δ-Damascone conjugates 6-10 were deposited from a TEA-esterquat emulsion onto the cotton surface as described above. After line-drying for 3 days, one of the dry towels was put into a headspace sampling cell that was controlled for temperature and relative humidity of the air (48). A constant flow of air of ca. 200 mL min-1 was continuously pumped across the cell. After equilibrating the system, the volatiles were adsorbed onto a clean Tenax cartridge for 15 min and then onto a waste cartridge for 45 min. The sampling was repeated seven times at constant time intervals every 60 min. At the end of the experiment, the sample cartridges were thermally desorbed and the volatiles quantified by GC analysis (48). The absolute headspace concentrations of δ-damascone were determined by external standard calibration. All experiments were carried out in duplicate and compared with a reference sample composed of unmodified δ-damascone, which was prepared and analyzed under the same conditions as the polymer conjugates. The measured headspace concentrations of δ-damascone released from polymers 6-10 or from the reference sample on dry cotton after 3 days are illustrated in Figure 5. The data show that the headspace concentrations were higher when δ-damascone was released from either one of the polymeric precursors 6-10 as compared with the unmodified δ-damascone used as the reference, thus confirming the expected resiliency of fragrance release. The release of δ-damascone was found to be particularly efficient from polymer conjugates 6, 9, and 10, with increased headspace concentrations (measured at the end of the experiment after 450 min, Figure 5) by a factor of 30, 75, or 50, respectively, with respect to the δ-damascone reference. With a human group olfactory threshold for δ-damascone of 0.021 ng L-1 air (32, 33), all detected concentrations lie above the human detection limit and are therefore easily perceived by humans (Figure 5). The observed constant increase of some of the concentration curves of δ-damascone released from the precursors at the beginning of the experiment may result from the changing conditions at the cotton surface from the line-drying to the headspace sampling cell (48). A constant airflow at ca. 75% of humidity was aspirated through the headspace sampling cell. Although the ambient humidity during the line-drying of the fabric was estimated as being quite high, the cotton sheets may still take up some humidity during the experiment, which in return may trigger some fragrance release. After ca. 200 min, a constant or slightly decreasing amount of δ-damascone was released from the samples. Due to the fact that the headspace

above the cotton surface, and thus the δ-damascone evaporated from the surface, was constantly removed, the system is not at equilibrium. The observed constant release at the end of the measurements thus corresponds to a constant release rate over time. Comparison of the release rates observed by dynamic headspace analysis (Figure 5) with those from solution measurements (Figure 1) generally showed a good correlation. With the exception of copolymer 6 (which was extracted into the organic phase and thermally degraded in the GC injector), the relative amount of δ-damascone released was found to low for copolymer 7 as compared to copolymers 8-10. This was the case for both headspace analysis and solution experiments. Despite its better deposition, copolymer 8 released less δ-damascone than copolymers 9 and 10, which correlates to a less efficient release in solution. In contrast, copolymer 10 with a faster release in solution released less δ-damascone into the headspace than copolymer 9 due to its lower deposition on the cotton surface. Considering the amount of deposition of the different polymers (Table 2) and the efficiency of release observed in solution (Figure 1), one can see that these two parameters have an opposite influence on the amount of δ-damascone released into the headspace. In solution, polymer conjugate 10 with hydrophilic PEG chains near the releasing unit was found to release the highest amount of δ-damascone of the series of compounds tested in this work. However, as a result of a limited deposition on the cotton surface, its final performance was found to be comparable to copolymer 6 with hydrophobic alkyl side chains. The latter is suspected to release the volatile less efficiently in solution, at pH between 4 and 7, but was better deposited onto cotton. Our measurements carried out in aqueous-buffered solution have shown that the structure of the polymer influences the rates of release, independently of other parameters such as the surface deposition. We therefore expected that copolymer 10 should give rise to a higher release of δ-damascone than copolymers 7-9. The headspace concentrations of δ-damascone released from the polymers on the cotton surface were then measured after line-drying for 3 days. These concentrations correspond to the momentaneous release at the given time. The total amount released over time was influenced by the polymer structures (release efficiency) and depended on the amount of polymer deposited onto the surface (maximum amount to be released). The maximum amount of δ-damascone that can be released into the air (as required for the practical application) was estimated from measurement of the deposition of the polymers on the target surface (Table 2). Figure 6 correlates the concentrations of δ-damascone released in solution (at pH 4 after 3 days, Figure 1) and the momentaneous headspace concentration measured


above the cotton surface (constant value measured after 3 days of line-drying and 450 min of sampling, Figure 5) together with the deposition (data determined by fluorescence measurements, Table 2) for polymers 7-10 (horizontal axis). Accordingly, a well-adjusted balance between hydrophobicity (deposition) and hydrophilicity (solubility, release kinetics) is presumably the most important criterion for the choice of the appropriate delivery system with respect to the targeted application. The presence of PEG side chains favors the release but not the deposition. Therefore, polymer conjugate 9 seems to represent the best structural compromise of the delivery systems investigated in this work by giving rise to the highest headspace concentrations of δ-damascone after 3 days. The ease of structural modifications, allowing fine-tuning of the physicochemical properties of bioconjugates, makes polymeric delivery systems particularly interesting for the controlled release of bioactive compounds in various applications.

CONCLUSION Besides prolonging the duration of evaporation, polymeric bioconjugates have additional advantages as delivery systems for bioactive volatiles such as fragrances. Labile covalent bonds to be cleaved in the conjugate to release bioactive compounds can be stabilized by the encapsulating polymer matrix, and thus, degradation can be (partially) prevented during storage in harsh media. Furthermore, the ease of structural modification by choosing suitable co-monomers allows preparation of copolymers with enhanced surface deposition and/or better dispersion in aqueous media. Damascones or damascenones, the so-called rose ketones, are important fragrance raw materials frequently used in perfumery. With 2-mercaptoethanol as a linker, R,β-unsaturated enones such as δ-damascone form the corresponding β-mercapto ketone, which then reacts under mild conditions with poly(maleic anhydride) copolymers to afford the corresponding conjugates in a two-step, one-pot procedure with very good yields and high conversions. The release of δ-damascone from the polymer conjugates by retro 1,4-Michael-type addition was investigated in buffered aqueous solution as a function of pH and time, as well as after deposition onto a cotton surface in the presence of a cationic surfactant. Grafting a fluorescence label to the polymer backbone allowed quantification of polymer deposition from an aqueous environment on a cotton surface. The most efficient release of δ-damascone was achieved for a polymer conjugate representing a well-balanced compromise of hydrophilicity (giving rise to high release rates) and hydrophobicity (increased surface deposition) of the polymer backbone. Our data thus show that the detailed understanding of the influence of the polymer structure on the deposition and the reactivity of the conjugates is the most important prerequisite toward the design of new polymer-based bioconjugates. Poly(maleic acid)-based copolymers were found to be particularly suitable as profragrances because they can be easily modified to cover a broad range of polarity and structural functionality within the polymer backbone. A series of these compounds with different backbone functionalities are widely available and thus allow easy access to a broad variety of structures. Therefore, the polymer conjugates described in this work (68) might be easily adapted for the release of bioactive compounds in various biomedical applications.

ACKNOWLEDGMENT The authors thank Philippe Chevalier for his assistance with the polymer synthesis, Daniel Grenno for recording the highresolution mass spectra, and Dr. Roger Snowden for constructive comments on the manuscript.

Berthier et al.

Supporting Information Available: Numerical data for Figures 1 and 5, and fluorescence spectra of the deposition measurements listed in Table 2. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Pichersky, E., Noel, J. P., and Dudareva, N. (2006) Biosynthesis of plant volatiles: nature’s diversity and ingenuity. Science 311, 808–811. (2) Baldwin, I. T., Halitschke, R., Paschold, A., von Dahl, C. C., and Preston, C. A. (2006) Volatile signaling in plant-plant interactions: “talking trees” in the genomics era. Science 311, 812–815. (3) Bergstro¨m, L. G. W. (2008) Chemical communication by behaviour-guiding olfactory signals. Chem. Commun. 3959–3979. (4) Arctander, S. (1969) Perfume and FlaVor Chemicals, Vol. 1 (A-J) and 2 (K-Z), published by the author, Montclair, NJ. (5) Surburg, H., and Panten, J. (2006) Common Fragrance and FlaVor Materials, 5th ed. (Surburg, H., and Panten, J., Eds.) pp 1-318, Wiley-VCH, Weinheim. (6) Kraft, P., Bajgrowicz, J. A., Denis, C., and Fra´ter, G. (2000) Odds and trends: recent developments in the chemistry of odorants. Angew. Chem., Int. Ed. 39, 2980–3010. (7) Brenna, E. (2003) Enzyme-mediated syntheses of chiral communication substances: fragrances for perfumery applications. Curr. Org. Chem. 7, 1347–1367. (8) Chapuis, C., and Jacoby, D. (2001) Catalysis in the preparation of fragrances and flavours. Appl. Catal., A 221, 93–117. (9) Saudan, L. (2007) Hydrogenation processes in the synthesis of perfumery ingredients. Acc. Chem. Res. 40, 1309–1319. (10) Friberg, S. E. (1998) Fragrance compounds and amphiphilic association structures. AdV. Colloid Interface Sci. 75, 181–214. (11) Zhang, Z., Friberg, S. E., and Aikens, P. A. (2000) Change of amphiphilic association structures during evaporation from emulsions in surfactant-fragrance-water systems. Int. J. Cosmet. Sci. 22, 181–199. (12) Milotic, D. (2003) The impact of fragrance on consumer choice. J. Consum. BehaV. 3, 179–191. (13) Ness, J., Simonsen, O., and Symes, K. (2003) Microcapsules for household products. Microspheres, Microcapsules Liposomes 6, 199–234. (14) Park, S.-J., and Arshady, R. (2003) Microcapsules for fragrances and cosmetics. Microspheres, Microcapsules Liposomes 6, 157–198. (15) Herrmann, A. (2007) Controlled release of volatiles under mild reaction conditions: from nature to everyday products. Angew. Chem., Int. Ed. 46, 5836–5863. (16) Saito, Y., Miura, K., Tokuoka, Y., Kondo, Y., Abe, M., and Sato, T. (1996) Volatility and solubilization of synthetic fragrances by Pluronic P-85. J. Dispersion Sci. Technol. 17, 567– 576. (17) Suzuki, K., Saito, Y., Tokuoka, Y., Abe, M., and Sato, T. (1997) Poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) triblock copolymer as a sustained-release carrier for perfume compounds. J. Am. Oil Chem. Soc. 74, 55–59. (18) Kayali, I. (2003) Solubilization of fragrance compounds in block copolymer/water system. Jordan. J. Appl. Sci. 5, 42–49. (19) Berthier, D. L., Schmidt, I., Fieber, W., Schatz, C., Furrer, A., Wong, K., and Lecommandoux, S. (2010) Controlled release of volatile fragrance molecules from PEO-b-PPO-b-PEO block copolymer micelles in ethanol-water mixtures. Langmuir 26, 7953–7961. (20) Ternat, C., Ouali, L., Sommer, H., Fieber, W., Velazco, M. I., Plummer, C. J. G., Kreutzer, G., Klok, H.-A., Månson, J.-A. E., and Herrmann, A. (2008) Investigation of the release of bioactive volatiles from amphiphilic multiarm star-block copolymers by thermogravimetry and dynamic headspace analysis. Macromolecules 41, 7079–7089. (21) Perry, R. J. (2005) “Pro-fragrant” silicone delivery polymers. DeliVery System Handbook for Personal Care and Cosmetic

Release of Volatiles from MA-Based Conjugates Products: Technology, Applications, and Formulations (Rosen, M. R., Ed.) pp 667-682, William Andrew Publishing, Norwich. (22) Kamogawa, H., Okabe, S., and Nanasawa, M. (1976) Synthesis of polymerizable acetals. I. Vinylbenzaldehyde acetals with perfume alcohols. Bull. Chem. Soc. Jpn. 49, 1917–1919. (23) Kamogawa, H., Haramoto, Y., and Nanasawa, M. (1979) Synthesis of polymerizable acetals. II. Readily hydrolyzable acetals from citronellol and vitamins. Bull. Chem. Soc. Jpn. 52, 846–848. (24) Kamogawa, H., Mukai, H., Nakajima, Y., and Nanasawa, M. (1982) Chemical release control - Schiff bases of perfume aldehydes and aminostyrenes. J. Polym. Sci., Polym. Chem. Ed. 20, 3121–3129. (25) Kamogawa, H., Haramoto, Y., Misaka, Y., Asada, Y., Ohno, Y., and Nanasawa, M. (1985) Chemical release control: sulfonate esters from perfume and herbicide alcohols and p-styrenesulfonyl chloride. J. Polym. Sci., Polym. Chem. Ed. 23, 1517–1526. (26) Galiogˇlu, O., and Akar, A. (1988) Perfume alcohols supported to sulfochlorinated poly(styrene-co-divinyl benzene) beads. J. Polym. Sci., Part A: Polym. Chem. 26, 2355–2357. (27) Kamogawa, H., Kohno, H., and Kitagawa, R. (1989) Chemical release control: carbamates of 3-vinylphenyl and 2-methacryloyloxyethyl isocyanates and perfume and herbicide alcohols. J. Polym. Sci., Part A: Polym. Chem. 27, 487–495. (28) Berthier, D., Trachsel, A., Fehr, C., Ouali, L., and Herrmann, A. (2005) Amphiphilic polymethacrylate- and polystyrene-based chemical delivery systems for damascones. HelV. Chim. Acta 88, 3089–3108. (29) Trachsel, A., de Saint Laumer, J.-Y., Haefliger, O. P., and Herrmann, A. (2009) Parameters influencing the release of tertiary alcohols from the surface of “spherical” dendrimers and “linear” stylomers by neighbouring-group-assisted hydrolysis of 2-carbamoylbenzoates. Chem.sEur. J. 15, 2846–2860. (30) Morinaga, H., Morikawa, H., Wang, Y., Sudo, A., and Endo, T. (2009) Amphiphilic copolymer having acid-labeile acetal in the side chain as a hydrotrope: controlled release of aldehyde by thermoresponsive aggregation-dissociation of polymer micelles. Macromolecules 42, 2229–2235. (31) Fehr, C., and Galindo, J. (2005) Aldols by Michael addition: application of the retro-Michael addition to the slow release of enones. HelV. Chim. Acta 88, 3128–3136. (32) Kastner, D. (1994) 25 Jahre Rosenketone - Thema mit Variationen. Parfuem. Kosmet. 75 (3), 170–81. (33) Williams, A. (2002) Rose ketones: celebrating 30 years of success. Perfum. FlaVor. 27 (2), 18–32. (34) Shulkin, A., and Sto¨ver, H. D. H. (2002) Polymer microcapsules by interfacial polyaddition between styrene-maleic anhydride copolymers and amines. J. Membr. Sci. 209, 421–432. (35) Donati, I., Gamini, A., Vetere, A., Campa, C., and Paoletti, S. (2002) Synthesis, characterization, and preliminary biological study of glyconjugates of poly(styrene-co-maleic acid). Biomacromolecules 3, 805–812. (36) Boztugˇ, A., and Basan, S. (2003) Synthesis and characterization of maleic anhydride-styrene-allyl propionate terpolymer ester derivatives. J. Appl. Polym. Sci. 89, 296–299. (37) Xi, F., Basset, Jr, W., and Vogl, O. (1983) Esters of alternating copolymers of maleic or 2,3-dimethylmaleic anhydride with alkyl vinyl ethers. J. Polym. Sci., Part A: Polym. Chem. 21, 891–911. (38) Chiellini, E. M., Leonardi, G., Giannasi, D., and Solaro, R. (1992) Partial esters of alternating copolymers of maleic anhydride and alkyl vinyl ethers for pharmaceutical applications. J. Bioact. Compat. Polym. 7, 161–174. (39) Zatz, J. L., Weiner, N. D., and Gibaldi, M. (1968) Monomolecular film properties of protective and enteric film formers I. Effect of polymer side chain on surface pressure-area isotherms. J. Pharm. Sci. 57, 1440–1442. (40) Zatz, J. L., Weiner, N. D., and Gibaldi, M. (1970) Orientation of monolayers of half-esters of poly(methyl vinyl ether maleic anhydride). J. Colloid Interface Sci. 33, 1–7.

Bioconjugate Chem., Vol. 21, No. 11, 2010 2011 (41) Finne, U., Kyyro¨nen, K., and Urtti, A. (1989) Drug release from monoisopropyl ester of poly(vinyl methyl ether-maleic anhydride) can be modified by basic salts in the polymer matrix. J. Controlled Release 10, 189–194. (42) Uglea, C. V., Panaitescu, L., Spridon, D., Ursu, D., Popa, I., and Ottenbrite, M. (1996) Biological activity of maleic anhydride copolymers. I. Biocompatibility and antitumoural effects of maleic anhydride-vinyl acetate, maleic anhydride-methyl methacrylate and maleic anhydride-styrene copolymers. J. Biomater. Sci., Polym. Ed. 8, 269–280. (43) Plochocka, K. (2001) Polymeric delivery and release systems for oral care actives (to ISP Investments Inc.), US Patent 6,315,987, Chem. Abstract 135, 362395. (44) Winnik, F. M., and Regismond, S. T. A. (1996) Fluorescence methods in the study of the interactions of surfactants with polymers. Colloids Surf., A 118, 1–39. (45) Vitenberg, A. G. (2003) Equilibrium model in the description of gas extraction and headspace analysis. J. Anal. Chem. 58, 2– 15. (46) Snow, N. H., and Slack, G. C. (2002) Head-space analysis in modern gas chromatography. Trends Anal. Chem. 21, 608–617. (47) Penton, Z. E. (2002) Headspace gas chromatography. Compr. Anal. Chem. 37, 279–296. (48) Levrand, B., Fieber, W., Lehn, J.-M., and Herrmann, A. (2007) Controlled release of volatile aldehydes and ketones from dynamic mixtures generated by reversible hydrazone formation. HelV. Chim. Acta 90, 2281–2314. (49) Nyqvist, H. (1983) Saturated salt solutions for maintaining specified relative humidities. Int. J. Pharm. Technol. Prod. Manuf. 4, 47–48. (50) Davaran, S., Rashidi, M. R., and Hashemi, M. (2003) Synthesis and hydrolytic behaviour of 2-mercaptoethyl ibuprofenatepolyethylene glycol conjugate as a novel transdermal prodrug. J. Pharm. Pharmacol. 55, 513–517. (51) Burattini, S., Colquhoun, H. M., Greenland, B. W., Hayes, W., and Wade, M. (2009) Pyrene-functionalised, alternating copolymimide for sensing nitroaromatic compounds. Macromol. Rapid Commun. 30, 459–463. (52) Yamamoto, H., Mizusaki, M., Yoda, K., and Morishima, Y. (1998) Fluorescence studies of hydrophobic association of random copolymers of sodium 2-(acrylamido)-2-methylpropanesulfonate and N-dodecylmethacrylamide in water. Macromolecules 31, 3588–3594. (53) Zhu, L.-P., Yi, Z., Liu, F., Wei, X.-Z., Zhu, B.-K., and Xu, Y.-Y. (2008) Amphiphilic graft copolymers based on ultrahigh molecular weight poly(styrene-alt-maleic anhydride) with poly(ethylene glycol) side chains for surface modification of polyethersulfone membranes. Eur. Polym. J. 44, 1907–1914. (54) Mazi, H., Kibarer, G., Emregu¨l, E., and Rzaev, Z. M. O. (2006) Bioengineering functional copolymers. IX. Poly[(maleic anhydride-co-hexene-1)-g-poly(ethylene glycol)]. Macromol. Biosci. 6, 311–321. (55) Ding, L. M., Shi, J., and Yang, C. Z. (1997) Ion-conducting polymers based on modified alternating maleic anhydride copolymer with oligo(oxyethylene) side chains. Synth. Met. 87, 157–163. (56) Suzuki, M., Mori, M., Niwa, T., Hirata, R., Furuta, K., Ishikawa, T., and Noyori, R. (1997) Chemical implications for antitumor and antiviral prostaglandins: reaction of ∆7-prostaglandin A1 and prostaglandin A1 methyl esters with thiols. J. Am. Chem. Soc. 119, 2376–2385. (57) Ladavie`re, C., Delair, T., Domard, A., Pichot, C., and Mandrand, B. (1999) Studies of the thermal stability of maleic anhydride co-polymers in aqueous solution. Polym. Degrad. Stab. 65, 231–241. (58) Levinson, M. I. (1999) Rinse-added fabric softener technology at the close of the twentieth century. J. Surfactants Deterg. 2, 223–235. (59) Mishra, S., and Tyagi, V. K. (2007) Esterquats: the novel class of cationic fabric softeners. J. Oleo Sci. 56, 269–276.

2012 Bioconjugate Chem., Vol. 21, No. 11, 2010 (60) Berg, J. C. (1985) The role of surfactants. Absorbency (Chatterjee, P. K., Ed.) pp 149-195, Elsevier, New York. (61) Laughlin, R. G. (1991) Fabric softening. Cationic Surfactants - Physical Chemistry, Surfactant Sci. Ser., Vol. 37 (Rubingh, D. N., Holland, P. M., Eds.), pp 449-467, Dekker, New York. (62) Escher, S., and Oliveros, E. (1994) A quantitative study of factors that influence the substantivity of fragrance chemicals on laundered and dried fabrics. J. Am. Oil Chem. Soc. 71, 31– 40. (63) Stora, T., Escher, S., and Morris, A. (2001) The physicochemical basis of perfume performance in consumer products. Chimia 55, 406–412. (64) Liu, H., Obendorf, S. K., Leonard, M. J., Young, T. J., and Incorvia, M. J. (2005) Adsorption of aroma chemicals on cotton fabric from aqueous systems. J. Surfactants Deterg. 8, 311–317.

Berthier et al. (65) Obendorf, S. K., Liu, H., Tan, K., Leonard, M. J., Young, T. J., and Incorvia, M. J. (2009) Adsorption of aroma chemicals on cotton fabric in different aqueous environments. J. Surfactants Deterg. 12, 43–58. (66) Schoenmakers, R. G., van de Wetering, P., Elbert, D. L., and Hubbell, J. A. (2004) The effect of the linker on the hydrolysis rate of drug-linked ester bonds. J. Controlled Release 95, 291– 300. (67) Delben, F., Paoletti, S., Porasso, R. D., and Benegas, J. C. (2006) Potentiometric titrations of maleic acid copolymers in dilute aqueous solution: experimental results and theoretical interpretation. Macromol. Chem. Phys. 207, 2299–2310. (68) Berthier, D., and Herrmann, A. (2008) Polymer conjugates for a controlled release of active molecules (to Firmenich SA), WO 2008/044178, Chem. Abstract 148, 472574. BC100223S

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