Protein Thioacylation. 1. Reagents Design and Synthesis

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Protein Thioacylation. 1. Reagents Design and Synthesis† Guy Levesque,*,‡ Philippe Arse` ne,§ Vale´ rie Fanneau-Bellenger,§ and Thi-Nha` n Pham§ Centre de Recherche, Universite´ de Bretagne-Sud, Rue de St Maude´ , F-56325 Lorient, France; and Universite´ de Caen, ISMRA, Boulevard Mare´ chal Juin, F-14050 Caen, France Received January 11, 2000; Revised Manuscript Received April 3, 2000

Thioacylation is a new way for protein chemical modification. Carboxylic dithioesters and -acids react selectively and rapidly at room temperature with aliphatic amines such as lysine -amino groups leading to thioamide formation, without any other reagent or catalyst. Various thioacylating reagents were synthesized: monofunctional dithioesters bearing on the acylating end various chemical groups such as: aliphatic chains, phenyl group, mono- and dicarboxylic acids, dialkylphosphonic ester, phosphonic acid, thiol, phenol, or quaternary ammonium group. Bifunctional dithioesters containing either a polymethylene chain or an ethylene oxide oligomer as spacer group as well as some mono- and bis(dithio acids) are described. Applications of thioacylation may be involved either in enzyme chemical modifications or in the obtention of new materials from proteins. Bifunctional reagents might be used as cross-linking or coupling reagents. Introduction Chemical modifications of proteins, and more particularly of enzymes, require them to be conducted in aqueous media and temperatures in the range 4-30 °C to avoid degradation of secondary and tertiary structures. Thus, water-soluble reagents are needed, provided that high reaction rates might be obtained for the conjugaison step. Such conditions limit the modification rates in the case of second-order kinetics, and only a few examples of protein chemical modifications are used. The -amino groups present in lysine residues are the most frequent targets for chemical modifications, mainly through acylation reactions, using mainly carboxylic acid anhydrides or so-called activated esters. Thioacylation of aliphatic primary and secondary amines by aliphatic or aromatic dithioesters R-CS2R′ is a fast and irreversible reaction we have largely applied in the field of polymer synthesis, and we report now the versatile use these reagents might find in protein modification. With such reagents, we have been able to bind proteins to various functional groups (namely, carboxylic acid, phosphonic acid or ester, phenol, ammonium ion, thiol) as well as alkyl chains or phenyl rings, through coupling with the lysine -amino groups. Dithiocarboxylic acids R-CS2H are less readily accessible reagents and we have studied their use in the chemical modification of proteins, mainly at pH near 7 or lower, as they react more rapidly than dithioesters under such conditions. The first paper is devoted to the synthesis of thioacylating reagents, and the second one reports on the hydrolytic behavior of some thiocarbonyl-containing compounds as well as their reaction kinetics, determined through thioacylation †

Dedicated to the memory of the late Professor Andre´ Thuillier. * To whom correspondence should be addressed. ‡ Universite ´ de Bretagne-Sud. § Universite ´ de Caen, ISMRA.

of gelatin as a model protein. Specific applications to enzyme modifications will be published later. Results and Discussion 1. Protein Modifications through Reactions on Lysine Residue. Protein chemical modifications may have two main purposes:2 synthesis of new materials from biologically available polymers, and enzyme modifications in order to prepare new biocatalysts. These modified enzymes may become more or less soluble in organic solvents while remaining active, and/or behave differently from the initial enzyme either in their optimum activity conditions (temperature, pH, substrate, reaction rate), or in developping new (or altered) catalytic activity. The chemical modifications may also be used either to determine which amino acid residues are involved in the active site, or to study their catalytic behavior. If the modification induces an important loss of activity, one can deduce that modified residues are located near the active site. However the persistance of such structures may also be affected by the modification conditions. Although the main enzyme research field lies now in molecular biology, the occurrence of a new general chemical modification may provide facilities for large scale reaction to introduce new chemical functions, change global net charge, and modify the hydrophilic-lipophilic balance. High reaction rates are desirable as protein denaturation may occur during the modification process, depending on residence times. Excess reagents are employed to make up for unsufficient rates. The more specific the reagents are, the best the results. Lysine residues are the most used targets in chemical modification due to the high nucleophilicity of amines, particularly under basic conditions. As our work is concerned only with reactions on lysine, we have resumed the main features of chemical modifications on the lysine amino groups, to afford comparison to the field available through thioacylation.

10.1021/bm000288k CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000

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Lysine Acylation. Lysine acylation is the more widely used modification.2-8 Large excesses (molar ratio 3-100) of carboxylic acid anhydrides are commonly used at pH 7-9 and temperature from 4 to 40 °C, for times from minutes to hours (Figure 1). A charge sign inversion is observed. Figure 4. Reaction of lysine with imidoesters and 2-iminothiolane.

Figure 1. Succinic anhydride acylation of lysine amino group in proteins.

A cosolvent (dimethyl sulfoxide) has also been added to enhance miscibility.9 Sometimes terminal amino groups must be protected.10 Substitution degrees (DS) are evaluated through reaction of remaining lysine groups with 2,4,6trinitrobenzenesulfonic acid (TNBS), the 2,4,6-trinitrophenylamino groups being determined from their UV-visible absorbance. Thiol (cysteine), phenol (tyrosine), and imidine (histidine) groups are also more or less modified. Hydroxylamine addition reverts the tyrosine acylation.11 Lysine Alkylation. Alkylations are also frequently used in protein modification, mainly as reductiVe alkylation12 using a carbonyl compound and a complex boron hydride (NaBH4 or NaCNBH3) (Figure 2). High selectivity is achieved by

Figure 2. Protein reductive alkylation.

reaction at 0 °C, pH 9, and large reagent excess.13 Improved procedures allow us to process at room temperature without opening disulfide cross-links.6,14 Carbonyl reagents may be acetone, cyclohexanone or benzaldehyde.15,16 Methanal (formaline) react twice with each lysine residue.17 Other reagents involved in alkylation reactions are prop-2-enal18 and R-lactose (R-disaccharide)19 as well as monosaccharides20 and 5-pyridoxal phosphate.21-24 Reversible alkylation is observed with an R-hydroxyaldehyde or ketone.25 As previously indicated TNBS is used to phenylate amines in lysine residues,26-29 thus grafting a large hydrophobic moiety (Figure 3). TNBS grafting results in a useful method for free amine titration.9, 27-29

Figure 3. Reaction of lysine residue with TNBS.

Lysine Amidination and the Like. Amidination (Figure 4) may be obtained by action of lysine amino groups onto

either imidoesters at pH 9 (30) or imidothiolane (Traut’s reagent) near pH 8.31 Traut’s reagent introduces a thiol group which was often used for coupling purposes in bioconjugate chemistry.1,32 Lysine carbamoylation is also readily available through reaction with isocyanates whereas cysteine and histidine residues give rise to unstable derivatives.17,33-35 Polymer Grafting, Cross-Linking, and Conjugation. Our polymer grafting interest lies in the availability of solvent-soluble or solvent-dispersible enzymes. In fact, mixtures of water and organic solvents37 are used as enzyme complete drying is generally avoided, and sometimes surfactants may be added.38 Chemically modified enzymes open new uses for enzymes in organic synthesis.39 Bifunctional reagents may induce either cross-linking of enzymes or binding to other active or inert elements.1,40 Protein grafting to polymer substrates has been often solved through actiVation of the polymer end through reactive groups such as dichloro and trichlorotriazines41 or activated esters.42,43 Cross-linking can be achieved with bifunctional reagents: lysine and cysteine have been used extensively,1 particularly in association with dialdehydes,40,44 bis(imidate)s,45 or activated diesters.46 Conjugaison of a protein to another biopolymer or chemical compound requires the use of two different reactive groups, most reagents being thus able to bind to lysine amino group at one end and to cysteine thiol group at the other.1 Numerous examples of coupling reagents associate an activated ester (for grafting on lysine) and maleimide (cysteine)40,46 or another activated ester and activated halogen,47 the reactivity of each group being selectively monitored through pH adjustments. More complex procedures involve attachment of Traut’s reagent onto lysine residues followed by coupling through the new thiol group.31,40 A pyridyldithio group linked to an activated ester containing molecule is also useful to introduce a thiol in place of an amino group.48 Our preliminary reports in enzyme modification using dithioesters concerned grafting of either amphililic PEG oligomers or hydrophobic alkyl chains or phenyl rings.49 2. Reagent Design. Reagents designed for enzyme modification must fit several criteria such as solubility and stability in aqueous media and fast and selective reactions between 4 and 30 °C at pH near neutrality (5-8, pH 9 can be used for short reaction times). Thioacylation of aliphatic primary and secondary amines by aliphatic dithioesters R-CS2R′ is a very fast, specific and irreversible reaction, the mechanism of which has been studied kinetically.50 The proposed mechanism involves a first reversible step consisting in amine nucleophilic addition

Protein Thioacylation

onto the thiocarbonyl, followed by an amine-assisted thiol elimination. According to the relative rate-constant values, the global thioacylation order can vary from 2 to 3. Some individual step rate constants could be estimated: the rate constant for amine addition to dithioester is particularly high, ca. 1 L‚mol-1‚s-1, whereas the global reaction rate depends on the relative importance of reversal to reagents from the zwitterionic intermediate to products formation. More important for protein modification is the low apparent activation energy: 10-17 kJ‚mol-1 between 20 and 40 °C, thus allowing rapid reaction, eVen near 0 °C. Dithioesters can be obtained from various carboxylic derivatives (acids, thiolesters, (thio)amides, nitriles) as well as from a Grignard reagent through addition to carbon disulfide followed by alkylation. Dithio acids are available only from Grignard reagents. Then, the choice of dithiocarboxylic reagents for enzyme (or protein) modification is governed both by their structure-dependent physicochemical properties and their synthetic availability. Water solubility is limited in simple dithioesters owing to their paraffin-like character: sulfur and carbon present nearly the same Pauling’s electro-negativity values and carbonsulfur bonds are not polarized although they are highly polarizable as a result of sulfur bonding through 3s and 3p atomic orbitals. Then, dithioester water solubility must be induced through specially designed structural features. One of the most useful structural element is present in carboxymethyl dithioesters (R-CS-S-CH2-COOH): at pH > 4.5, carboxylate ions are formed, thus allowing sufficient water solubility, eventually as micelles. However such carboxymethyl dithioesters cannot be obtained from nitriles. Carboxymethyl dithioesters are then prepared either from N,Ndialkylthioamides or from a Grignard reagent. Hydrophilic carboxylic acids were also introduced in the thioacylating side of dithioesters: specific synthesis were developed, which give rise in fair to good yields to functional reagents similar to carboxylic anhydrides. Some other ways to improve dithioesters water solubility were tested, sometimes with success: particularly efficient were the introduction of diethyl phosphonate, phosphonic acid, or quaternary ammonium groups. Stability toward water and aqueous media is not really expected in thiocarbonyl compounds as a consequence of the lower bonding enthalpy in CdS double bond as compared to CdO double bond. However aliphatic dithioesters react so rapidly in protein modification at pH 6-9 and at temperatures e30 °C that they can be considered as stable reagents in such conditions. Aromatic dithioesters are less reactive toward amine and thus hydrolysis to inactive reagents may occur before high modification yields are obtained. We have generally preferred aliphatic reagents for this specific reason (see the second paper in this series for a detailed study of the reagent stability in aqueous media). 3. Bis(dithioester)s and Bis(dithio acid)s Syntheses. We have previously synthesized several bis(dithioester)s to prepare poly(thioamide)s through a room-temperature condensation reaction with diamines.51-53 For such a purpose, these bis(dithioester)s need to be obtained in a high purity

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grade, preferentially in a crystalline form, so we have often preferred long chain alkyl or carboxymethyl bis(dithioester)s. The first bis(dithioester)s52 were prepared from dinitriles using a modification of the method of Marvel et al.54 The first step consists of the addition of a thiol on the nitrile triple bond, the presence of anhydrous hydrogen chloride being required to complete the formation of the imidoester as an imidium salt (Figure 5). Improvements lie in the use

Figure 5. Conversion of nitriles and dinitriles into dithioesters and bis(dithioesters).

of anhydrous conditions and eventually of insoluble polymeric pyridine (cross-linked polyvinylpyridine) instead of pyridine for the second step (sulfhydrolysis of the imidium salt). In the present paper, we report on the application of this synthetic way to poly(oxa)dinitriles obtained by Michael addition of diols onto acrylonitrile (Figure 6).

Figure 6. Synthesis of polyoxabis(dithioesters) 4a-c.

In an other attempt to develop rapid and efficient synthesis of bis(dithioester)s, we have applied the synthetic way first described by Leon55 to bis(thioamides) particularly those derived from diacylbenzenes via the Willgerodt-Kindler reaction (this way allows the direct one-pot synthesis of ω-phenyl-N,N-dialkylthiocarboxamides from acetophenone or propiophenone). Finally, some of the previously unknown bis(dithio acid)s as well as several bis(dithioester)s were prepared through the addition of carbon disulfide to double Grignard reagents derived from R,ω-dihalogenoalcanes. Such a synthesis is well-known in the case of monohalogenoalcanes either in a direct manner56 or via the cuprates.57 Although the yields remain fair or even low, this straightforward way is really attractive (one-pot synthesis), even for obtaining aliphatic carboxymethyl bis(dithioesters).

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Figure 7. Synthesis of dithioesters bearing ω-carboxylic acids 7a-d.

Synthesis of Functional Dithioesters from Nitriles. Nitriles are useful starting materials for dithioesters synthesis via the Marvel modification of Pinner’s reaction (Figure 5). Moreover it appeared during the course of this work that some functional groups can support the reaction conditions (i.e., the use of anhydrous hydrogen choride in the first step) without any protection: this was particularly useful for the synthesis of ω-functionalized dithioesters. Starting from readily available products, we were able to prepare various interesting dithioesters. Synthesis of Polyoxabis(dithioesters) 4a-c. Dinitriles containing the required number of ether groups 3a-c were obtained through a Michael base-catalyzed addition of the corresponding diols (poly(ethylene oxide) oligomers) to excess acrylonitrile according to an adaptation of a known procedure.58 The usual conversion of nitriles into dithioesters affords good to high yields of polyoxabis(dithioester)s 4a-c (x ) 1, 2, 3) (Figure 6). Application to higher poly(ethylene oxide)s offered products in which the final functionality was lying between 1.0 and 1.6 in respect to purification problems occurring at each step. Then polycondensation polymers prepared from R,ω-diamino-polyoxyethylene and bis(dithioester)s were preferred for enzyme modification and grafting.49 Synthesis of Dithioesters Bearing ω-Carboxylic Acids 7a-d. As will be seen in the next few papers, the chain length between the functional groups in reactive dithioesters is not the most important feature for the chemical modification of enzymes, provided that the two groups are far enough to avoid cyclization reactions during the synthetic path (six atoms or more). As several mercapto- mono- or dicarboxylic acids are readily available, we have prepared some carboxylic acidbearing dithioesters through addition of mercapto acids on acrylonitrile according to Hurdand and Gershbein,59 followed by the conversion of the nitrile into dithioester in the usual way (Figure 7). The ω-carboxy dithioester 7c was obtained from the ω-cyano acid 6c. These mono- and dicarboxy dithioesters in which functional groups lie in the acylating moiety are interesting reagents as they mimic quite well dicarboxylic acid anhydrides usually used in enzyme acylation. Several acid and diacid derivatives 7a-d could be readily obtained and used later to investigate reagent structure influence onto modified enzymes properties.

Synthesis of a Dithioester Bearing an ω-Quaternary Ammonium Ion, 11. As the primary and secondary aliphatic amines react very rapidly with dithioesters, the existence of ω-amino-dithioesters could be expected only as their salts with strong acids or for N,N-dialkyl derivatives. Moreover, such compounds would not affect the protein external charges after modification of lysine residuessat least under acid conditions. Then, it seemed more interesting to use a reagent able to replace primary amines by quaternary ammonium ions, which are nearly indifferent to pH variations. Compound 11 was obtained in a few high-yield steps (Figure 8).

Figure 8. Synthesis of dithioester 11 bearing an ω-quaternary ammonium ion.

Starting from 2-(N,N-dimethylamino)ethanol 8, reaction with acrylonitrile affords the nitrile 9, then the usual sequence transforms the nitrile group into dithioester. The aminodithioester 10 is isolated as an hydrochloride, which in turn liberates the parent amine, and then methyl iodide alkylation yields 11 (R ) C2H5, soluble in water), each step with good to excellent yields (ca. 50% overall). Synthesis of ω-Phosphonoester Dithioester 13 and ω-Phosphonoacid Dithioester 14. Phosphonic esters and acids are interesting functional groups, inducing high water affinity. Phosphonic acids are stronger than carboxylic ones and therefore grafting of such acids in place of -amino lysine groups would produce important behavior modifications in the modified proteins. Although such synthesis have been first realized starting from diethyl cyanomethyl-phosphonate NC-CH2-PO(OEt)2, we have obtained better yields from diethyl 4-cyanobutyl phosphonate NC-(CH2)4-PO(OEt)2. From these cyanophosphonates (Figure 9), dithioesters 13a-b and acids 14a-b were prepared through some modification of the

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carboxymethyl benzenebis(dithiocarboxylic ester)s according to a procedure first described by N. H. Leon55 and also to unprotected acylphenols which provides a simple synthesis of hydroxyphenyl-bearing dithioesters. Synthesis from Carboxylic Acids via Amides and Thioamides. The synthesis described in Figure 11 gives fair to high yields in each step. In particular, conversion of amides into thioamides by Lawesson’s reagent71-73,80,84 is nearly quantitative.

Figure 9. Synthesis of ω-dithioester phosphonates 13a,b and ω-dithioester phosphoacids 14a,b.

procedure described by Masson et al.60 Some other β-phosphonodithioesters were described from methylphosphonic esters by lithiation, reaction of CuI, and then coupling with alkyl chlorodithioformate.61 Dithioesters bearing a phosphonic acid such as 14a-b are obtained from the corresponding phosphonic esters by action of halotrimethylsilane followed by alcoolysis according to a known method.62,63 Synthesis of Dithioesters from Carboxylic Acids and Amides. Various dithioesters, such as 15 were obtained directly from carboxylic acids using Davy’s reagent,64-69 through the methyl thiolesters (Figure 10). However such a

Figure 10. Synthesis of dithioesters from carboxylic acids using the Davy reagent.

multistep reaction afford generally low yields, and its main interest lies in the one-pot synthesis of complex molecules such as dimethyl 2,6-naphthalenebis(dithiocarboxylate).70 More conveniently we have used an indirect way which includes preparation of N,N-dialkylthioamides, which are in turn alkylated by alkyl halides (simple halides or haloacetic acid) thus leading to the formation of imidothiocarboxylate salts which undergo hydrogen sulfide splitting to dithioesters, as in the synthesis from nitriles and thiols. However water exclusion is stringently needed in the last step. Thioamides are prepared by a variety of different ways: • Aminolysis of dithioesters (quantitative yield). This synthesis transform a water insoluble alkyl dithioester into a soluble carboxymethyl dithioester. • Thionation of N,N-dialkylcarboxamides using, for example, Lawesson’s reagent (direct synthesis of N,N-dimethylamides from carboxylic acids by heating in hexamethylphosphoramide).71-73,80,84 • Wilgerodt-Kindler reaction applied to acyl benzenes. This way88 was successfully applied to the synthesis of

Figure 11. Synthesis of dithioester 19 from carboxylic acid via amide 16 and thioamide 17: (a) heating to reflux with (Me2N)3PdO; (b) thionation by Lawesson’s reagent; (c) alkylation with R′X; (d) reaction with anhydrous H2S.

Synthesis from Acylbenzenes via Thioamides. A ready access to arylalkylthioamides is given by the sulfonic acidcatalyzed Willgerodt-Kindler reaction:74-77 heating of an alkylaryl ketone with sulfur and excess secondary amine (usually morpholine) induces a migration of the functional group toward the free aliphatic chain end with thioamide formation (Figure 12). This reaction was successfully applied particularly to p-bis(acetyl)benzene, offering the complex bis(thioamide) 21d in good yield, as well as to 4-hydroxyacylbenzenes without protecting the phenolic hydroxyl group. From these mono- and bis(thioamides), dithioesters 22a-c (n ) 0, 1; R′ ) CH3, CH2CO2H, CH2CO2Me) and bis(dithioesters) 22d (n ) 0; R′ ) CH2CO2H) were prepared through thioamide alkylation into imidothioester halides followed by reaction with anhydrous hydrogen sulfide. The presence of phenolic hydroxyl group in the starting acylbenzene is allowed and thus dithioesters 22a-c (n ) 0, 1; R1 ) CH3, CH2CO2H, CH2CO2Me) were prepared without any phenol group protection. Synthesis of Dithio Acids, Bis(dithio acid)s and Bis(dithioester)s from Grignard Reagents. Dithio acids are not well-known compounds for two major reasons: lowmolecular weight derivatives present very unpleasant smells and only one useful synthetic path is known. Grignard reagents are the unique source for dithiocarboxylic acids.56 As these reagents are difficult to prepare in the presence of unprotected functional groups, only hydrocarbon chains and phenyl rings can be introduced easily into dithio acid containing molecules. We have synthesized (Figure 13) according to Beiner and Thuillier56 monofunctional derivatives and bis(dithioester) 24 and bis(dithio acid) 25 and its quaternary ammonium salt 26 from 1,6-dibromohexane through bis(organometallic) intermediates.85,86 Quaternary ammonium dithiocarboxylic salts are commun storage forms which limit oxidation and oligomerization and generate immediately free acids at pH g 2 (approximate value of the dithio acids’ pKas). Although bis(dithio acid)s yields are generally fair or low (and vary significantly without identified reasons), the use of bis-Grignard reagents represents a very simple one-pot synthesis which allow one to obtain test samples of bis(dithio acid)s 25 and bis(dithioesters) 26.

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Figure 12. Synthesis of dithioesters 22 a-d by the Willgerodt-Kindler reaction.

Figure 13. Synthesis of bis(dithioesters)s 24 and bis(dithio acid)s 25 using salts 23 derived from Grignard reagents: (a) magnesium, THF, and then CS2 addition; (b) alkylation by R-X; (c) aqueous HCl; (d) tetramethylammonium hydroxide.

All the described compounds are reasonably stable in usual laboratory conditions (temperature, hygrometry). Dithioesters disappear to form thioamides by reacting with any N-Hcontaining compound. Storage in a dry, cold environment is possible for months or even years without significant alterations. Free dithio acids are rapidly oxidized by air, and the ammonium salts are their most valuable form, usually stored at -18 °C to avoid slow internal SN2 substitution reaction between the ammonium alkyl substituents and the highly nucleophilic dithiocarboxylate; halogen-containing solvents must be avoided with these salts for the same reason. Conclusion Many types of thiocarboxylic compounds have been synthesized to provide interesting reagents able to graft selectively onto the lysine -amino group in protein; the thioacylating part can bear quite different substituents such as alkyl chains, phenyl groups, mono- and dicarboxylic acids, dialkylphosphonates and phosphonic acids, thiol, phenols, and a quaternary ammonium group. Then they are able to replace selectively the lysine amino group by each of these structures. Moreover bis(thioacylating) agents are also available, opening ways to protein cross-linking or conjugaison of protein to amino-bearing compound as was already demonstrated in the case of R,ω-diaminopolyoxyethylene.49

Reagent structures were selected to allow synthesis in a few steps from readily available compounds. A great deal of effort was devoted to promote reagent solubility in buffered aqueous media. Most of these sulfur compounds are stable for months at 4-20 °C. Experimental Part THF was distilled over sodium benzophenone ketyl. Monomers and the model compound were purified by flash chromatography (silica gel or alumina). 1H NMR 60 MHz and 250 MHz spectra were run on Varian EM 360 and Bruker AC 250 spectrometers with TMS as an internal reference. The products were dissolved in the mentioned solvent. Data are given in the following order: chemical shift in ppm, multiplicity (s, singlet; d doublet; t, triplet; q, quartet; hept, heptuplet; m, multiplet), coupling constant in hertz, assignment. 13C NMR spectra were determined at 20.15 MHz with a Bruker WP 80 spectrometer operating with broad band 1H decoupling. The solvent used is indicated. IR absorption spectra were recorded as liquid thin films between NaCl plates or as solids in KBr pellets, or dissolved in CDCl3 or CCl4 on a Perkin-Elmer 257IR spectrophotometer and a Pye-Unicam SP 3-100 or a Perkin-Elmer 16 PC Fourier transform spectrometer. The mentioned IR absorptions were observed as strong bands in cm-1. UV spectra were executed on a Perkin-Elmer λ 15 or a Beckman DU-7. The products were dissolved in CHCl3, CH2Cl2, H2O, or THF.

Protein Thioacylation Elemental analyses were performed by “Service Central d’Analyze” of the CNRS in Vernaison. The results were described as percentages. Mass spectra were recorded with a Nermag R 10 10 H spectrometer in electronic impact at 70 eV (the molecular ion and the most abundant ions are reported). 1. Synthesis of Functional Dithioesters from Nitriles. Synthesis of Imidothioester Chlorhydrate. In a 500 mL round-bottom, three-necked flask equipped with a gas inlet and a magnetic stirrer was placed the nitrile (1 mol; 1 equiv) or dinitrile (0.5 mol) in 250 mL of dry dichloromethane. A solution of dry ethanethiol or dodecanethiol (2 mol; 2 equiv) diluted in CH2Cl2 was added to the stirred nitrile solution previously cooled to 0 °C. The mixture was saturated with hydrogen chloride; gas was passed in rather rapidly at first and then slowly for 1-2 h at 0 °C. The stirred saturated mixture was allowed to react for 15 h or more at 0 °C and concentrated in vacuo. No nitrile signal, from any remaining nitrile, could be observed near ν ) 2250 cm-1. Synthesis of Dithioesters. The dry crude mono imidothioester chlorhydrate (1 equiv) or bis(imidothioester) chlorhydrate (0.5 equiv) was dissolved in 150 mL of dichloromethane. A suspension of dry polyvinylpyridine (Reillex 402, 8.8 mequiv g-1) (PVPr) (113 g; 2 equiv) in CH2Cl2 (150 mL) was added to the solution of chlorhydrate and the mixture, cooled in an ice-salt bath, was saturated with dry hydrogen sulfide. Gas was passed rapidly for 1 h and then slowly for 6-8 h, and the saturated solution was allowed to react about 15 h at 0 °C. Polyvinylpyridine was filtered off, and then the reaction mixture was concentrated under Vacuum. Dithioesters was purified by distillation under reduced pressure or by flash chromatography on silica gel with the eluting solvent: cyclohexane/CH2Cl2 mixture 95/5, or cyclohexane. Ethyl Hexanedithioate (1a), CH3(CH2)4CSSC2H5 (C8H16S2 ) 176.345 g). Yield: 76%, orange liquid. Eb0.1-0.5 ) 40-45 °C. 1H NMR (CDCl3): 0.89 (3H, t, CH3), 1.32 (3H, t, SCH2 CH3), 1.83 (6H, m, CH2), 3.02 (2H, t, CH2CdS), 3.15 (2H, t, SCH2. 13C NMR (CDCl3): 12.30 (CH3), 13.90 (SCH2CH3, SCH2CH3), 22.41, 30.41, 30.91, 31.02 (CH2), 52.16 (CH2CS2), 238.79 (CdS). UV-vis (CDCl3): 308 (log  ) 4.12), 453 (log  ) 1.43). Ethyl Heptanedithioate (1b), CH3 (CH2)5CSSC2H5 (C9H18S2 ) 190.370 g). Yield: 81%, orange liquid, Eb3 ) 101 °C. 1H NMR (CDCl3): 0.90 (3H, t J ) 8 Hz, CH3), 1.20 (3H, t J ) 8 Hz, SCH2CH3), 1.30-1.80 (8H, m, CH2), 2.95 (2H, t J ) 8 Hz, CH2Cd S), 3.20 (2H, q J ) 8 Hz, SCH2). 13C NMR (CDCl3): 12.24 (CH3), 14.57 (SCH2CH3), 23.19, 29.56, 32.39, 33.14 (CH2), 52.03 (CH2CS2), 238.45 (CdS). UV-vis (CHCl3): 308 (log  ) 401). Anal. Calcd for C4H18S2: C, 56.78; H, 9,53; S, 33.68. Found: C, 57.12; H, 9.62; S, 33.32. Dodecyl Decanebis(dithioate) (1c), C12H25SSC(CH2)8CSSC12H25 (C34H66S4 ) 603.161 g). Yield: 60%, yellow powder. Mp: 34 °C. 1H NMR (CDCl ): 0.85 (6H, t J ) 8 Hz, CH ), 1.25 (20H, m, 3 3 CH2), 3.00 (4H, t, CH2CdS), 3.20 (4H, t, CH2S). Dodecyl Dodecanebis(dithioate) (1d), (C12H25SSC(CH2)10CSSC12H25. Yield: 80%, yellow powder. Mp: 40 °C. 1H NMR (CDCl3): 0.85 (6H, t, J ) 8 Hz, CH3), 1.25 (56H, m, CH2), 2.98 (4H, t, J ) 8 Hz, CH2CdS), 3.18 (4H, t, J ) 8 Hz, CH2S). 13C NMR (CDCl3): 14.05 (CH3), 22.69, 27.40, 28.76, 29.21, 29.36, 31.27, 31.94, 36.58 (CH2), 52.32 (CH2CS2), 239.43 (CdS). UVvis (CHCl3): 309 (log  ) 4.41). UV-vis (hexane): 305 (log  ) 4.38). Anal. Calcd for C36H70S4 (631.215 g): C, 68.50; H, 11.19; S, 20.32. Found: C, 68.48; H, 11.34; S, 19.60. Ethyl (p-Bromophenyl)ethanedithioate (1f). The reaction was performed from (p-bromophenyl)acetonitrile (10 g, 51 mmol) in chloroform (50 mL, ethanethiol (7.4 mL, 102 mmol) and pyridine (15.5 mL). The H2S-saturated reaction mixture was concentrated

Biomacromolecules, Vol. 1, No. 3, 2000 393 and washed with 5 N sodium hydroxide (7.3 mL) (solution pH must be basic) than extracted with chloroform. After the reaction was dried over MgSO4, the solvent was removed in vacuo to give a red oil. This was purified by flash chromatography on silica gel (eluant: cyclohexane/ethyl acetate 95/5). Yield: 69%. 1H NMR (CDCl3): 1.54 (3H, t ) 7.5 Hz, SCH2CH3), 3.18 (2H, q J ) 7.5 Hz, SCH2CH3), 4.23 (2H, s, CH2CdS), 7.23, 7.44 (4H, 2d J ) 8 Hz, C6H4). 13C NMR (CDCl3): 12.10 (CH3), 31.30 (SCH2), 57.30 (CH2CdS), 121.40, 130.90, 131.70, 136.10 (C6H4), 234.70 (Cd S). Anal. Calcd for C10H11BrS2 (275.21 g): C, 43.64; H, 4.03; S, 23.30. Found: C, 43.61; H, 3.98; S, 23.09. 2. Synthesis of Polyoxabis(dithioesters) 4a-c. Dinitriles Containing Ether Groups. In a round-bottom, three-necked flask equipped with a condenser, a nitrogen gas inlet, and a magnetic stirrer were placed the diol (1a-c) (0.167 mol) and 40% potassium hydroxide in water (1 mL). The mixture was stirred under nitrogen, and hydroquinone (0.095 g; 8 × 10-4 mol) was dissolved into it. Acrylonitrile (18 g; 0.34 mol) was added dropwise, and the resulting solution was stirred over 20 h at room temperature. After quenching by an ice-cooled solution of HCl 20%, extraction with CHCl3, and washing with NaCl solution, the organic layer was dried over MgSO4, evaporated, and distilled under reduced pressure. 4,7-Dioxadecane-1,10-dinitrile (3a), NC-(CH2)2O-(CH2)2O(CH2)2CN (C8H12N2O2 ) 168.197 g). Yield: 65%, colorless liquid, Eb0,1 ) 118 °C. 1H NMR (CDCl3): 2.65 (4H, t, CH2CN), 3.70 (8H, m, CH2O). 13C NMR (CDCl3): 18.87 (CH2 CN), 65.95 (CH2CH2CN), 70.51 (CH2O), 118.21 (CN). IR (film NaCl plates): 2240 (CN), 1100 (C-O-C). 4,7,10-Trioxatridecane-1,13-dinitrile (3b), NC-(CH2)2-O(CH2)2O-(CH2)O-(CH2)2CN (C10H16N2O3 ) 212.251 g). Yield: 49%, colorless liquid, Eb0,1 ) 145 °C. 1H NMR (CDCl3): 2.60 (4H, t, CH2CN), 3.65 (12H, m, CH2O). 13C NMR (CDCl3): 18.87 (CH2CN), 65.95 (CH2CH2CN), 70.59, 70.65 (CH2O), 118.09 (CN). IR (film NaCl plates): 2250 (CN), 1115 (C-O-C). 4,7,10,13-Tetraoxahexadecane-1,16-dinitrile (3c), NC(CH2)2O(CH2)2O(CH2)2O(CH2)2O(CH2)2CN (C12H20N2O4 ) 256.304 g). Yield: 41%, colorless liquid, Eb0,1 ) 195-205 °C. 1H NMR (CDCl3): 2.62 (4H, t, CH2CN), 3.65 (16H, m, CH2O). 13C NMR (CDCl3): 18.31, 18.73 (CH2 CN), 65.88 (CH2CH2CN), 70.41, 70.51 (CH2O), 118.35 (CN). IR (film NaCl plates): 2245 (CN), 1110 (CO-C). Polyoxabis(dithioester). Dodecyl 4,7-dioxadecanebis(dithioate) (4a), C12H25SSC(CH2)2O-(CH2)2O(CH2)2CSSC12H25. Yield: 70%, yellow powder. Mp: 36 °C. 1H NMR (CDCl3): 0.85 (6H, t J ) 8 Hz, CH3), 1.25 (40H, m, CH2), 3.32 (8H, t, J ) 8 Hz, CH2CdS, CH2S), 3.57 (4H, s, OCH2O), 3.88 (4H, t, OCH2). 13C NMR (CDCl3): 14.09 (CH3), 22.72, 27.33, 29.18, 29.38, 29.52, 29.67; 31.97; 36.72 (CH2), 51.86, 71.09 (CH2CS2,CH2O), 234.82 (CdS). Anal. Calcd for C32H62O2S4 (607.106 g): C, 63.31; H, 10,29; O, 5.27; S, 21.13. Found: C, 63.03; H, 10.16; O, 5.37; S, 20.76. 3. Synthesis of Dithioesters Bearing ω-Carboxylic Acids 7ad. Preparation of ω-Cyanocarboxylic Acids. To a stirred solution of mercaptocarboxylic acids 5a,b,d (0.1 mol) and 5 N aqueous sodium hydroxide solution (20 mL) was added freshly distilled acrylonitrile (0.12 mol) dropwise. The temperature was raised, and the resulting mixture was stirred and refluxed for 5 h at 60 °C and then cooled. After quenching by 30% aqueous HCl (or 30% H2SO4, the mixture was extracted with diethyl ether. The organic layer was dried over magnesium sulfate and concentrated in vacuo. The residue was purified by chromatography on silica gel with the eluting solvent: chloroform. 2-(2-cyanoethyl)thioethanoic acid (6a). HOOCCH2 SCH2CH2CN. Yield: 70%, white powder. 1H NMR (CDCl3): 2.83 (4H, m,

394 Biomacromolecules, Vol. 1, No. 3, 2000 CH2CH2), 3.33 (2H, s, CH2COOH), 9.3 (1H, s, COOH). IR (KBr pellets): 1740 (CdO), 2260 (CN), 3080 (OH). 3-(2-cyanoethyl)thiopropanoic acid 6b. HOOCCH2CH2SCH2CH2CN. Yield: 57%, white solid. 1H NMR (CDCl3): 2.73 (8H, m, CH2), 9.9 (1H, s, COOH). IR (KBr pellets): 1717 (CdO), 2250 (CN), 3200 (OH). 2-(2-cyanoethyl)thiobutanedioic acid 6d. HOOCCH2CH2CH2SCH2CH2CN. Yield: 95%, white powder. The reaction was also realized with 2-mercaptobutanedioic acid (5d) (34.2 mL) (18.6 g; 0.3 mol), 5 N aqueous NaOH solution (50 mL) and freshly distilled acrylonitrile (23.7 mL) (19.1 g; 0.36 mol). After refluxing the mixture for 5 h at 60 °C and quenching by 30% aqueous HCl, water was evaporated under reduced pressure (the crude product is very soluble). The residue was diluted in acetone, dried over MgSO4 and the solvent was removed in vacuo. The crude product was crystallized from acetone to give pure nitrile 6d. 1H NMR (D2O): 2.80 (6H, m, CH2), 3.70, (1H, t J ) 8 Hz, CH), 8.60 (2H, s, COOH). 13C NMR (D2O); 18.11 (CH2CN), 26.66 (SCH2), 36.21 (CH2COOH), 41.94 (CH), 120.12 (CN), 174.35, 175.23 (CdO). IR (KBr pellets): 1700 (CdO), 2240 (CN), 3000 (OH). Anal. Calcd for C7 H9NO4S (203.22): C, 41.37; H, 4.47; N, 6.89; S, 15.78. Found: C, 41.35; H 4.52; N, 6.55; S, 15.47. ω-Dithioester Carboxylic Acids 7a-d. According to the procedure described above, the reaction was carried out in a 250 mL round-bottom flask, with ω-cyanocarboxylic acids 6a-d (0.1 mol) dissolved in CH2Cl2 or for 7d in CH2Cl2/DMF (2/1) 150 mL, ethanethiol (0.1 mol) 1 equiv, and 6 equiv of polyvinylpyridine (70 g). After hydrogen sulfide was passed over the reaction for 2 h at 0 °C, the saturated solution was allowed to react overnight. The resulting mixture was filtered, the solvent was removed under Vacuum and poured in 10% dilute HCl and then extracted with chloroform, dried over MgSO4, and concentrated under reduced pressure. ω-Carboxydithioesters 7a-c were purified by chromatography on silica gel using dichloromethane as the eluent. 2-[2-(Ethylthiothiocarbonyl)ethylthio]ethanoic Acid (7a), HOOCCH2SCH2CSSEt. Yield: 25%, orange liquid. 1H NMR (CDCl3): 1.30 (3H, t J ) 7 Hz, CH3), 3.23 (8H, m, 4 CH2), 11.03 (1H, s, COOH). 13C NMR (CDCl3): 11.58 (CH3), 30.24, 32.35, 33.25 (CH2), 49.93 (CH2COOH), 175.44 (COOH), 234.13 (CdS). IR (film NaCl plates): 1710 (CdO), 3050 (OH). UV-vis (EtOH): 308 (log  ) 4.05). 3-[2-(Ethylthiothiocarbonyl)ethylthio]propanoic Acid (7b), HOOCCH2CH2SCH2CH2CSSEt. Yield: 35%, orange liquid. 1H NMR (CDCl3): 1.30 (3H, t J ) 7 Hz, CH3), 2.71 (4H, m, CH2Cd O, CH2-CdS), 3.11 (6H, m, CH2SCH2, SCH2), 10.16 (1H, s, COOH). 13C NMR (CDCl3): 12.07 (CH3), 26.81, 30.67, 32,33, 34.33, 34.64 (4 CH2), 51.13 (CH2COOH), 177.58 (COOH), 235.00 (CdS). IR (film NaCl plates): 1710 (CdO), 3050 (CH). UV-vis (EtOH): 308.2 (log  ) 4.05). 11-(Ethylthiothiocarbonyl)undecanoic Acid (7c), HOOC(CH2)10CSSEt. Yield: 70%. Mp: 50 °C. 1H NMR (CD3 OD): 1.30 (19 H, m, CH3, CH2), 2.26 (2H, m, CH2COOH), 3.13 (4H, m, CH2CdS, SCH2), 11.06 (1H, s, COOH). 13C NMR (CDCl3): 12.23 (CH3), 24.71, 28.74, 29.23, 29.33, 30.63, 31.22, 34.11 (7 CH2), 52.21 (CH2COOH), 180.12 (COOH), 239.28 (CdS). IR (KBr pellets): 1710 (CdO), 3050 (OH). UV-vis (EtOH): 306.3 (log  ) 4.04), 308.5 (log  ) 4.08). 2-[2-(Ethylthiothiocarbonyl)ethylthio]butanedioic Acid (7d), HOOCCH2CH(COOH)S(CH2)2CSSEt. Yield: ) 95%, yellow powder. Mp: ) 150 °C. 7d was purified by precipitation in 10% dilute hydrochloric acid and drying under Vacuum. 1H NMR (CD3OD): 0,90 (3H, t, J ) 6 Hz, CH3), 2.10-3.30 (9H, m, CH2, CH), 10.25 (2H, s, COOH). 13C NMR (D2O): 12,49 (CH3), 31.48, 32.84, 37.51 (CH2), 43.17 (CH), 52.22 (CH2CS2), 173.96, 175.06 (Cd

Levesque et al. O), 237.08 (CdS). UV-vis (D2O): 307 (log  ) 4.02). IR (KBr pellets): 1690, 1710 (CdO), 3100 (OH). Anal. Calcd for: C9H14O4S3 (282.37) C, 38.28; H, 5.01; S, 34.06. Found: C, 38.18; H, 5.09; S, 34.00. Mass: 282 (M+• 0.3); 264 (2); 235 (2); 204 (2); 165 (7); 132 (20); 104 (32); 71 (100); 60 (34); 59 (36); 45 (95). 4. Synthesis of a Dithioester Bearing an ω-Quaternary Ammonium. ω-Aminonitrile 9. Freshly distilled acrylonitrile 33.25 mL (26.5 g, 0.5 mol) was slowly added (3 h) to a stirred solution of 2-(N,N-dimethyl)aminoethanol 8 (44.6 g, 0.5 mol), 40% aqueous potassium hydroxide (5 mL), and hydroquinone (0.5 g). The temperature was maintained at 20 °C, and the mixture was stirred for 20 h. The black solution (pH ≈ 9) was poured dropwise into 20% aqueous hydrochloric acid (7 mL). The nitrile was isolated by extracting the water layer three times with chloroform (the ω-aminonitrile is very soluble in water), drying the combined extracts with magnesium sulfate, and removing the solvent under reduced pressure. The black crude product was distilled under Vacuum. 6-(N,N-Dimethylamino)-4-oxahexanenitrile (9).Yield: 70%, colorless liquid. Eb12 ) 106 °C. 1H NMR (CDCl3): 2.20 [6H, s, N(CH3)2], 2.50 (4H, 2t, J ) 6 Hz, NCH2 and CH2CN), 3.60 (4H, 2t, J ) 6 Hz, CH2-O-CH2). 13C NMR (CDCl3): 18.27 (CH2CN), 45.40 (NCH3), 59.10 (NCH2), 65.32, 70.21 (CH2-O-CH2), 121.07 (CN) IR (film NaCl pellets): 1140 (CH2OCH2), 1455 [(CH3)2N], 2240 (CN), 2760, 2980 (CH). Anal. Calcd for C7H14N2O (142.20): C, 59.12; H, 9.92; N, 19.70; 0,11.25. Found: C, 58.84; H, 9.80; N, 19.43; O 11.45. Mass: 142 (M+•, 0.3); 72 (3); 58 (100); 54 (2); 42 (8). ω-(Dimethylamino)dithioester 10. After reaction of excess hydrogen chloride on a mixture of 0.1 mol of nitrile 9 with 0.15 mol of ethanethiol in 200 mL of chloroform according to the usual conversion procedure and after concentration to remove excess HCl, the resulting salt was dissolved in 200 mL of chloroform and added to 70 g of PVPr. After the usual saturation with anhydrous H2S and overnight reaction, the filtered solution was washed with 5% aqueous NaOH and dried over anhydrous MgSO4. Evaporation of the solvent in vacuo furnished the product 10 as a orange oil. The crude ω-aminodithioester 10 was pure enough to use in the next step. Yield: 80%. Ethyl [6-(N,N-Dimethylamino)-4-oxahexane]dithioate (10). 1H NMR (CDCl3), 1.30 (3H, t J ) 6 Hz, SCH2CH3), 2.10 (6H, s, NCH3), 2.45 (2H, t J ) 6 Hz, NCH2); 3.30 (4H, m, SCH2, CH2CS2), 3.85 (4H, t, J ) 6 Hz, CH2OCH2). 13C NMR (CDCl3): 12.56 (CH3), 30.92 (SCH2), 45.64 (NCH3), 51.07 (CH2CdS), 57.80 (NCH2), 65.19 (CH2CH2 CS2), 69.88 (NCH2CH2O), 233; 26 (Cd S). Anal. Calcd for C9H19NOS2 (221.38): C, 48.83; H, 8.65; N, 6.33; O, 7.23; S, 28.97. Found: C, 48.99; H, 8.75; N, 6.37; O, 7.42; S, 28.52. Dithioester ω-Quaternary Ammonium Salt 11. To a stirred solution of the aminodithioester 10 (22.1 g, 0.1 mol) in freshly distilled, dry THF (40 mL) cooled at -10 °C, 1.5 equiv of methyl iodide (9 mL) (21.2 g, 0.15 mol) was added dropwise under nitrogen atmosphere. A yellow precipitate was formed, and the resulting solid was filtered and washed with diethyl ether and then dried under Vacuum. The resulting crude salt 11 was purified by recrystallization from methanol. Yield: 95%, yellow powder. Mp: 94 °C. Ethyl [6-(N,N,N-trimethyliodoammonio)-4-oxahexane]dithioate (11). 1H NMR (CDCl3): 1;30 (3H, t J ) 6 Hz, SCH2CH3), 3.20 (4H, m, SCH2, CH2CdS), 3.50 (9H, s, NCH3), 3,90 (6H, m, CH2OCH2, NCH2). 13C NMR (CCl4): 12.26 (SCH2CH3); 30.87 (SCH2), 50.98 (CH2CS2), 54.86, 55.04, 55.21 (NCH3), 65.05 (NCH2CH2O), 70.81 (OCH2CH2CS2), 65.95 (NCH2), 234.79 (Cd S). UV-vis (CH2Cl2); 309 (log  ) 4.14). Anal. Calcd for C10H22-

Protein Thioacylation INOS2 (363.32): C, 33.06; H, 6,10; I, 34.93; N, 3.85; 0, 4.40; S, 17.65. Found: C, 33.01; H, 6,02; I, 34.85; N, 3.91; O,4.71; S, 17.39. Mass: 363 (M+•, 0.3), 334 (2), 305 (3), 273 (1), 165 (39), 132 (21); 127 (6), 71 (84), 58 (100), 45 (65). 5. Synthesis of ω-Phosphonodithioesters. Diethyl (4-Cyano)butylphosphonate 12b. In a 250 mL round-bottom, three-necked flask equipped with a Claisen, a condenser, and a magnetic stirrer were placed 5-bromovaleronitrile (5-bromopentanenitrile) (80.6 g, 0.5 mol) and freshly distilled triethyl phosphite (126.2 g, 1 mol). The mixture was refluxed for 5 h at 160 °C. Then the bromoethane formed (about 37 mL, 0.5 mol) was distilled off (Eb760 ) 37-40 °C). The pure cyanophosphonate was obtained by fractional distillation under Vacuum. Yield: 80%, colorless liquid. Eb0,01 ) 120 °C. 1H NMR (CDCl3): 1.30 (6H, t, J ) 7 Hz, CH3); 1.60 (2H, dt JHH ) 7 Hz, JHP ) 20 Hz, PCH2), 1.00-2.00 (4H, m, CH2), 2.80 (2H, t J ) 7 Hz, CH2CN), 4.05 (dq JHOP ) JHH ) 7 Hz, OCH2). 13C NMR (CCl ): 16.35 (d J 4 CCOP ) 6 Hz, CH3CH2O), 18.09 (CH2CN), 21.45 (CH2CH2CN), 26.48 (d JCP ) 186 Hz, PCH2), 27.06 (d JCCP ) 12 Hz, PCH2CH), 62.27 (d JCOP ) 6.5 Hz, OCH2), 119.34 (CN); 31P NMR (CDCl3): 30.24. IR (CHCl3): 1160 (P-O), 1230 (PdO), 2220 (CN). Anal. Calcd for C9H18NO3P (219.22): C, 49.31; H, 8.27; N, 6.39; O, 21.89; P, 14.13. Found: C, 49.44; H, 8.28; N, 6.45; O, 22.11; P, 13,57. Mass: 219 (M+•, 10), 204 (18), 191 (30), 178 (77), 163 (71), 151 (100), 137 (42), 124 (99), 110 (44), 55 (83), 41 (70). ω-Phosphonodithioesters 13a,b. To a stirred solution of cyanomethylphosphonate 12a (MERCK or ACROS) (17.7 g, 0.1 mol) or ω-cyanophosphonate 12b (21.9 g, 0.1 mol) in chloroform (100 mL) cooled at 0 °C, ethanethiol, 2 equiv (12.4 g, 0.2 mol), was added. The conversion nitrile-dithioester was realized according to the procedures described previously for 1a-d. After confirmation that the reaction was complete by IR (no nitrile signal near 2250 cm-1), hydrogen sulfide was passed through the suspension of imidothioester chlorhydrate in CHCl3 (100 mL) using 2 equiv PVPr (23 g). The orange dithioesters 13a and 13b were purified by distillation under reduced pressure. Diethyl (Ethylthiothiocarbonyl)methylphosphonate (13a). Yield: 50%, orange liquid. Eb0.01 ) 110 °C. 1H NMR (CCl4): 1.25 (9H, t J ) 6 Hz, CH3), 3.15 (2H, q J ) 6 Hz, SCH2), 3.70 (2H, d JHP ) 23 Hz, PCH2), 4.05 (2H, dq JHOP ) JHH ) 7 Hz, OCH2). 13C NMR (CCl4): 12.12 (CH3), 16.37 (d JCCOP ) 5.8 Hz, CH3CH2O), 32.61 (s, SCH2), 51.09 (d JCP ) 186 Hz, PCH2), 63.85 (d JCOP ) 6.6 Hz, OCH2), 228.52 (d JCCP ) 7.7 Hz, CdS). IR (CCl4): 1160 (P-O), 1240 (PdO). Anal. Calcd for C8H17O3PS2 (256.32): C, 37.49; H, 6.68; O, 18.20; P, 12.08; S, 25.02. Found: C, 37.64; H, 6.88; O, 18.13; P, 11.89; S, 24.86. Diethyl [4-(Ethylthiothiocarbonyl)]butylphosphonate (13b). Yield: 90%, orange liquid. Eb7×10-4 ) 70 °C. 1H NMR (CDCl3): 1.30 (9H, t J ) 7 Hz, CH3), 1.60 (2H, dt JHH ) 7 Hz, CH3), 1.60 (2H, dt JHH ) 7 Hz, JHP ) 20 Hz, PCH2), 1.00-2.00 (4H, m, CH2), 3.00 (2H, t, J ) 7 Hz 2H, CH2CS2), 3.15 (2H, q J ) 6 Hz, SCH2), 4.10 (4H, dq JHOP ) JHH ) 7 Hz, OCH2). 13C NMR (CCl4): 12.14 (s, CH2CH3), 16.43 (d JCCOP ) 6 Hz, CH3CH2O), 26.03 (d JCP ) 183 Hz, PCH2), 30.85 (d JCCP ) 12 Hz, PCH2CH2), 31.17 (CH2); 32.04 (SCH2), 51.24 (CH2CS2), 61.34 (d JCOP ) 6.5 Hz, OCH2), 237.95 (CdS). 31P NMR (CDCl3): 31.46. UV-vis (CH2Cl2): 307.5 (log  ) 4.01). IR (CHCl3): 1160 (P-O), 1230 (PdO). Anal. Calcd for C11H23O3PS2 (298.40): C, 44.27; H, 7.77; O, 16.08; P, 10.38; S, 21.49. Found: C, 44.10; H, 7.81; O, 16.33; P, 11,07; S, 21.15. Mass: 298 (M+•, 4), 282 (9), 269 (15), 253 (24), 237 (54), 193 (12), 179 (56), 165 (25), 152 (81), 137 (48), 81 (52), 71 (35), 55 (100), 41 (30). ω-Dithioester Phosphonic Acid 14a,b. According to the procedure described earlier,62,63 under nitrogen, a solution of

Biomacromolecules, Vol. 1, No. 3, 2000 395 dithioester 13a or 13b (0.1 mol) in CH2Cl2 was cooled at 0 °C; bromotrimethylsilane (30 mL, 2.2 equiv) (33.65 g, 0.22 mol) was added dropwise, and the reaction mixture was stirred at room temperature for 24 h. The solvent was evaporated at 12 mmHg and then at 10-3 mmHg to eliminate excess BrSiMe3. Ethanol (100 mL) was added directly into the solution, and the mixture was stirred for 1 h. The solvent was removed under Vacuum. The crude product was diluted in CHCl3 and washed with 10% hydrochloric acid. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. Dithioesters 14a, 14b were purified by precipitation in petroleum ether then recrystallized from chloroform/ethyl acetate (1/1) (for 14a) and from diethyl ether (for 14b). (Ethylthiothiocarbonyl)methylphosphonic Acid (14a). Yield: 90%. Mp: 125 °C. 1H NMR (D2O): 1.30 (3H, t J ) 6 Hz, CH3), 3.20 (2H, q J ) 6 Hz, SCH2), 3.80 (2H, d JHP ) 23 Hz, PCH2), 10.90 (1H, s, POH). 13C NMR (D2O): 11.77 (CH3), 32.53 (SCH2), 49.96 (d JCP ) 184 Hz, PCH2), 228.58 (d JCCP ) 7.7 Hz, CdS). 31P NMR (D O): 15.8. UV-vis (D O): 310 (log  ) 4.08). IR (KBr 2 2 pellets): 1150 (P-O), 1240 (PdO), 3000 (OH). Anal. Calcd for C4H9O3PS2 (200.21): C, 23.99; H, 4.53; O, 23.97; P, 15.46; S, 32.03. Found: C, 24.21; H, 4.62; O, 23.80; P, 15.20; S, 31.77. Mass: 200 (M+•; 1), 120 (12), 92 (9), 59 (100), 47 (25), 45 (21). 4-(Ethylthiothiocarbonyl)butylphosphonic Acid (14b). Yield: 80%. Mp: 61 °C. 1H NMR (CDCl3): 1.20 (3H, t J ) 7 Hz, CH3), 1.50 (2H, dt, JHH ) 7 Hz JHP ) 20 Hz, PCH2), 1.00-2.00 (4H, m, CH2), 2.75 (2H, t J ) 7 Hz, CH2CS2), 2.90 (2H, q J ) 6 Hz, SCH2), 10.90 (1H, s, P-OH). 13C NMR (CDCl3): 12.38 (SCH2CH3), 26.16 (d JCP ) 185 Hz, PCH2), 31.00 (d JCCP ) 10 Hz, PCH2CH2); 31.31 (CH2), 32.16 (SCH2), 51.30 (CH2CS2), 237.99 (CdS). 31P NMR (CDCl3): 34.36. UV-vis (CH2Cl2): 308.5 (log  ) 4.02). IR (KBr pellets): 1160 (P-O), 1230 (PdO), 3000 (OH). Anal. Calcd for C7 H15O3PS2 (242.29): C, 34.70; H, 6.24; O, 19.81; P, 12.78; S, 26.46. Found: C, 34.73; H, 6.33; O, 20.32; P, 12.48; S, 26.10. Mass: 242 (M+•; 3), 213 (6), 181 (29), 137 (11), 123 (28), 96 (100), 82 (22), 71 (20), 62 (58), 55 (40), 47 (59), 41 (29). 6. Synthesis of Dithioesters from Carboxylic Acids. By Davy’s Reagent, via Thiolester. Methyl (p-Bromophenyl)ethanedithioate (15a). According to the known procedure,64,67 a stirred suspension of P4S10 (2 equiv) (21.49 g, 48.4 mmol) and dry methanol (2.5 mL, 60 mmol) in 1,2-dichlorobenzene (8 mL) was heated at 40 °C to initiate the reaction and then to 120 °C to dissolve phosphorus pentasulfide P4S10 and finally at the reflux temperature for 15 min. The reaction mixture was cooled at 100 °C before addition of (pbromophenyl)ethanoic acid (5.2 g, 24.2 mmol). The solution was stirred and heated under reflux for 10 min and then allowed to cool gradually to 130 °C. P4S10 (24.2 mmol) was introduced into the red mixture containing the thiolester. The reaction mixture was stirred and heated again at 160 °C for 20-30 min. The inorganic phosphorus compounds were precipitated by pouring the reaction mixture into an excess of cyclohexane (about three volumes) and removed by filtration. After solvent evaporation under Vacuum, 6.4 g of crude product was separated. Dithioester 15a was purified by flash chromatography (silica gel) in cyclohexane/ethyl acetate (95/5). A total of 1.9 g of 15a was isolated. Yield: 30%. 1H NMR (CDCl3): 2.55 (3H, s, SCH3), 4.24 (2H, s, CH2CS2), 7.19, 7.40 (4H, 2d J ) 8 Hz, C6H4). 13C NMR (CDCl3): 20.45 (SCH3), 57.09 (CH2CS2), 121.50, 130.89, 131.76, 136.05 (C6H4), 235.40 (CdS). Anal. Calcd for C9H9BrS2 (261.18): C, 41.39; H, 3.47; S, 24.55. Found: C, 41.62; H, 3.53; S, 24.33. Mass: 262 (38), 260 (42), 171 (67), 169 (100), 45 (87). Methyl 6-Bromohexanedithioate (15b). 15b was obtained by the reaction of P4S10 (0.25 mol) methanol (24 g, 0.75 mol), in 1,2dichlorobenzene (150 mL) and 6-bromohexanoic acid (0.2 mol) in 1,2-dichlorobenzene (50 mL). After flash chromatography on silica

396 Biomacromolecules, Vol. 1, No. 3, 2000 gel with hexane as eluting solvent pure bromodithioester 15b was isolated as an orange liquid. Yield: 60%. 1H NMR (CDCl3): 1.8 (6H, m, CH2), 2.62 (3H, s, SCH3), 3.08 (2H, t, CH2CdS), 3.43 (2H, t, CH2Br). 6-Mercaptohexanedithioate 15c. A solution of sodium (2.76 g, 0.12 mol) in dry methanol (100 mL) was saturated with hydrogen sulfide; then methyl 6-bromo-hexanedithioate 15b (24.12 g, 0.10 mol) was added. A slow stream of dry hydrogen sulfide was passed for 2 h, and the solution was allowed to react for about 48 h. The reaction mixture was poured into dilute HCl (100 mL) and extracted with chloroform. The organic layer was dried over MgSO4 and concentrated under Vacuum. 15c was purified by flash chromatography on silica gel with the eluting solvent: hexane. Yield: 34%, orange liquid. 1H NMR (CDCl3): 1.55 (9H, m, CH2, SH,), 2.60 (3H, s, SCH3), 3.02 (2H, t, CH2CdS). 13C NMR (CDCl3): 19.88 (SCH3), 24.43, 27.33, 30.44, 35.54 (CH2), 51.53 (CH2CdS), 239.10 (CdS). UV-vis (EtOH): 303.8 (log  ) 4.08). By Lawesson’s Reagent via Amides and Thioamides. N,NDimethylthioacetamide (15a). N,N-Dimethylacetamide (2.61 g, 30 mmol) was added to a suspension of freshly prepared Lawesson’s reagent (6.74 g, 16.5 mmol) in anhydrous toluene (25 mL) under nitrogen atmosphere. The solution was refluxed for 3 h and then cooled at room temperature. After decantation and filtration through aluminum oxide to remove inorganic phosphorus compound the solvent was removed under Vacuum. The crude product was purified by distillation under reduced pressure. Yield: 74%. Eb13 ) 110 °C. Mp ) 72-6 °C. 1H NMR (CDCl3): 3.10-3.25 [6H, 2s, (CH3)2N], 2.40 (3H, s, CH3CdS). 13C NMR (CDCl3): 32.80 (CH3CS), 44.30, 42.30 [(CH3)2N], 199.4 (CdS). Carboxymethyl Ethanedithioate (17a). A stirred yellow solution of N,N-dimethylthioacetamide (4 g, 38.8 mmol) and bromoacetic acid (5.39 g, 38.8 mmol dissolved in N,N-dimethylformamide (60 mL) was heated for 2 h at 75 °C and then cooled at room temperature. After H2S gas was passed over the reaction for a period of 2 h, the saturated mixture was allowed to react overnight. DMF was removed under reduced pressure. The N,N-dimethylammonium bromide salt was precipitated in ethyl acetate and eliminated by filtration. After evaporation of the combined filtrates under Vacuum, the dithioester 19a was purified by recrystallization from petroleum ether. 19a (4.22 g) was obtained as an orange powder. Yield: 72%. Mp: 81.6 °C. 1H NMR (CDCl3): 2.87 (3H, s, CH3), 4.12 (2H, s, SCH2), 10.22 (1H, m, COOH). 13C NMR (CDCl3): 38.50, 38.90 (CH3, CH2), 172.60 (CdO), 230.50 (CdS). 7. Synthesis of Dithioesters from Acyl- and Diacylbenzene. Preparation of Thioamides. Thioamide 21a. A mixture of 4-hydroxyacetophenone (1.36 g,10 mmol), sulfur (2.5 equiv, 0.8 g, 24.74 mmol), dry morpholine (2 equiv, 1.8 mL, 20 mmol), and p-toluenesulfonic acid (0.026 equiv, 48 mg, 0.26 mmol) were heated under reflux (130 °C) for 6 h 30 min. After cooling, the resulting solution was poured into methanol (20 mL) and stirred overnight. The mixture was filtered. The combined filtrates was concentrated under reduce pressure. Crude thioamide 21a was obtained as a brown oil. The purification by flash chromatography on silica gel with cyclohexane/ethyl acetate 60/40 as eluting solvent, furnished thioamide 21a (1.36 g). Yield: 57%, yellow powder. Mp: 128 °C. 1H NMR (CDCl ): 3.41 (2H, t J ) 5 Hz, NCH morpholine), 3.64 3 2 (2H, t J ) 5 Hz, NCH2), 3.76 (2H, t J ) 5 Hz, OCH2), 4.27 (2H, s, CH2CdS), 4.35 (2H, t, OCH2), 4.35 (2H, t, OCH2), 6.79-7.26 (4H, 2 d AB syst, J1 ) 9 Hz, J2 ) 2 Hz, C6H4). 13C NMR (CDCl3): 49.80 (CH2CdS), 50.33, 50.86 (CH2NCH2), 66.26, 66.46 (CH2OCH2), 115.95, 127.80, 129.12, 154.79 (C6H4), 200.56 (CdS). Thioamide 21d. The reaction was realized with 4-hydroxypropiophenone (20 g, 0.13 mol), sulfur (2.5 equiv, 10.67 g, 0.33 mol), morpholine (2 equiv, 23 mL, 0.26 mol), and p-toluenesulfonic acid

Levesque et al. (0.025 equiv, 0.573 g, 3.37 mmol). The product was purified as described previously. Pure thioamide 21d (13.95 g) was isolated as a yellow powder. Yield: 42% Mp: 131 °C. 1H NMR (CDCl3): 3.05 (4H, m, NCH2 and C6H4CH2), 3.41 (2H, q, NCH2), 3.50 (2H, q, OCH2), 3.70 (2H, q, OCH2), 4.35 (2H, t, CH2CSN), 5.08 (1H, s, OH), 6.78, 7.10 (4H, 2 d, AB syst J1 ) 9 Hz, J2 ) 2 Hz, C6H4). 13C NMR (CDCl ): 35.01 (CH C H ), 44.61 (CH CdS), 50.09 3 2 6 4 2 (CH2NCH2), 50.09, 50.27 (CH2NCH2), 66.21, 66.52 (CH2OCH2), 115.56, 129.85, 132.22, 154.54 (C6H4), 202.96 (CdS). Thioamide 21f. The reaction was performed with 1,4-diacetylbenzene (0.05 mol), sulfur (3.21 g, 0.1 mol), morpholine (18.9 mL, 0.21 mol), and para-toluenesulfonic acid (0.480 g, 2.6 × 10-3mol). After purification by recrystallization from CH 2Cl2 pure, thioamide 21f was isolated as a pale yellow powder. Yield: 66%. Mp: 206 °C. 1H NMR (CDCl3): 3.50 (4H, m, CH2CdS), 3.75 (8H, m, CH2NCH2), 4.40 (8H, m, CH2OCH2), 6.80, 7.40 (4H, 2d AB syst, C6H4). Preparation of Dithioesters 22a-e. Carboxymethyl (p-Hydroxyphenyl)ethanedithioate (22a). A mixture of dry thioamide 21a (2.37 g; 9.99 mmol) and bromoacetic acid (1.1 equiv, 1.54 g, 11.08 mmol) in dry freshly distilled THF (20 mL) was stirred under nitrogen for 48 h at room temperature; a white precipitate formed and the resulting mixture was cooled to 0 °C and saturated with hydrogen sulfide for 2 h. The solvent was removed under Vacuum, and the resulting solution was washed with 10% dilute hydrochloric acid and extracted with CHCl3. The organic layer was dried over MgSO4 and filtered, and the solvent was removed from the extract in vacuo. Crude dithioester 22a (3.6 g) was obtained as an orange oil. The purification by flash chromatography on silica gel with ethyl acetate/methanol 70/30 or dichloromethane/methanol 90/10 gives the dithioester 22a. Yield: 24%. 1H HMR (CDCl3): 4.06 (2H, s, CH2CS2), 4.24 (2H,s, CH2CO2H), 6.72, 7.16 (4H, 2d, J1 ) 9 Hz, J2 ) 2 Hz. 13C NMR (CD3OD): 39.70 (CH2CdS), 57.60 (CH2CO2H), 116.40, 130.70, 158.00 (C6H4), 170.00 (CO2H), 237.00 (Cd S). Silylation of Dithioester 22a. The dithioester 22a was also purified by reaction with excess chlorotrimethylsilane. Under nitrogen atmosphere, chlorotrimethylsilane (3.6 mL, 28 mmol) was added to a solution of crude dithioester 22a (2 g, 8.26 mmol) in dry pyridine (20 mL). The mixture was stirred for 4 h at room temperature and filtered off (to eliminate pyridinium chlorhydrate). The filtrates were concentrated under Vacuum, and the crude silylated dithioester (2.3 g) was purified by distillation under reduced pressure (Eb0,005 ) 95 °C). 1H NMR (CDCl3): 0.19 (3H, s, SiCH3), 3.92 (2H, s, CH2CdS), 3.98 (2H, s, CH2CdO), 6.72, 7.10 (4H, 2d AB syst, J1 ) 9 Hz, J2 ) 2 Hz, C6H4). 13C NMR (CDCl3): 0.30 (SiCH3), 40.40 (CH2CdS), 59.50 (CH2CdO), 115.60, 120.20, 130.40, 155.10 (C6H4, 174.40 (CdO), 223.20 (CdS). Methoxycarboxymethyl (p-Hydroxyphenyl)ethanedithioate (22b). The methyl ester of dithioester 22a was obtained according to the procedure described above for the preparation of compound 22a from thioamide 21a (1.18 g, 5 mmol), methyl bromoacetate (0.52 mL, 5.5 mmol), and dry THF (10 mL). Then the reaction mixture was concentrated, washed with dilute acid, and extracted with dichloromethane. After the usual workup, crude dithioester 22b was obtained as yellow orange oil (1.4 g). Dithioester 22b was purified by flash chromatography (silica gel), eluant: CH2Cl2. Yield: 50% (640 mg). 1H NMR (CDCl3): 3.70 (3H, s, OCH3), 4.00 (2H, s, CH2CdS), 4.21 (2H, s, CH2CdO), 6.77, 7.14 (4H, 2d, J1 ) 9 Hz, J2 ) 2 Hz, C6H4), 6.79 (1H, s, OH). 13C NMR (CDCl3): 38.80 (OCH3), 53.30 (CH2CdS), 56.70 (CH2CdO), 115.70, 128.40, 130.60, 155.40 (C6H4), 168.90 (CdO), 235.10 (CdS). Anal. Calcd for C11H12O3S2 (256.20): S, 25.02. Found: S, 25.10. Methyl (p-Hydroxyphenyl)ethanedithioate (22c). The reaction was performed on thioamide 21a (2 g; 8.43 mmol) in chloroform

Protein Thioacylation (90 mL) with methyl iodide (2.5 equiv, 1.35 mL, 21.10 mmol). After being stirred at 50 °C over 24 h, the solution was cooled at 0 °C and saturated with anhydrous H2S during 3 h and then allowed to react overnight. The mixture was filtered and concentrated under reduced pressure. Crude dithioester 22c (orange oil) was purified by flash chromatography on silica gel. Eluent: cyclohexane/ethyl acetate 70/30. Yield: 5%, orange liquid. 1H NMR (CDCl ): 2.59 (3H, s, SCH ), 4.26 (2H, s, CH CdS), 3 3 2 4.83 (1H, s, CH), 6.80, 7.25 (4H, 2d J1 ) 9 Hz J2 ) 2 Hz, C6H4). 13C NMR (CDCl ): 20.41 (SCH ), 57.14 (CH CdS), 115.50, 3 3 2 129.48, 130.51, 154.94 (C6H4), 237.49 (CdS). Mass: 198 (51), 150 (27), 108 (39), 107 (100), 91 (30). Carboxymethyl (p-Hydroxyphenyl)propanedithioate (22d). According to the procedure described for the synthesis of 22a, the reaction was realized with thioamide 21d (0.50 g, 1.99 mmol) and dry bromoacetic acid (1.1 equiv, 0.305 g, 2.19 mmol), and then THF was replaced by chloroform (10 mL) before reaction with H2S. After the usual workup, the product was purified by flash chromatography (silica gel) with the eluting solvent: cyclohexane/ ethyl acetate 70/30. Byproducts identified as the ester 22g and thiolester 22h were isolated with 22d. 1H NMR (CD3OD): 2.28 (2H, t, PhCH2), 3.10 (2H, t, CH2CdS), 3.65 (2H, s, SCH2COOH), 6.80, 7.09 (4H, 2d. AB syst J1 ) 9 Hz J2 ) 2 Hz, C6H4). 13C NMR (CD3OD): 36.00 (CH2Ph), 45.63 (CH2-CdS), 67.28 (CH2COOH), 116.31, 130.62, 132.32, 157.18 (C6H4), 177.50 (COOH), 234.07 (CdS). Methyl (p-Hydroxyphenyl)propanedithioate (22e). The reaction was performed with thioamide 21d (2 g, 7.96 mmol) in chloroform (20 mL), with ICH3 (2.5 equiv, 1.25 mL, 19.9 mmol). After purification by flash chromatography on silica gel (eluent: cyclohexane/ethyl acetate 70/30), 22e was given as a yellow-orange solid; Yield: 10%. Mp: 46 °C. 1H NMR (CDCl3): 2.61 (3H, s, SCH3), 3.07 (2H, m, CH2Ph), 3.25 (2H, m, CH2CS2), 4.62 (1H, s, OH), 6.77, 7.07 (4H, 2d AB syst J1 ) 9 Hz J2 ) 2 Hz, C6H4). 13C NMR (CDCl3): 20.15 (SCH3), 36.51 (CH2Ph), 53.64 (CH2CS2), 115.37, 129.75, 132.53, 154.05 (C6H4), 238.51 (CdS). Mass: 212 (M+•, 19) 107 (100), 91 (48), 77 (33), 43 (39). 1,4-Benzenebis[(carboxymethyl) ethanedithioate] (22f). The reaction was realized with bis(thioamide) 21f (1.983 g, 5 mmol) and bromoacetic acid (1.417 g, 10.2 mmol), in dry THF (20 mL). After the usual workup, the bis(dithioester) was purified by recrystallization from toluene/chloroform. Yield: 8%, pale yellow powder. Mp: 202 °C. 1H NMR (DMSO): 3.15 (4H, s, PhCH2Cd S), 4.35 (4H, s, SCH2COOH), 7.28, 8.15 (4H, 2d, C6H4). 8. Synthesis from Grignard Reagents. Dithioesters and Bis(dithioesters). Bis(dithioester)s and dithioesters were prepared by reaction of carbon disulfide according to the following procedures, described earlier.56,78-83 A Grignard solution was prepared under nitrogen from magnesium (1.1 equiv, 0.55 mmol) and alkyl halides (amounts: see below) (1.1 equiv, 0.5 mol) in freshly distilled dry THF (100 mL). The mixture was then diluted with THF (200 mL) and cooled to 0 °C, and a solution of dry carbon disulfide (1.5 equiv, 0.75 mol) in dry THF (50 mL) was added slowly. The yellow-red mixture was stirred over a period of 2 h at room temperature and then cooled, and iodomethane (1.1 equiv, 0.55 mol) or sodiun chloroacetate (1.1 equiv, 0.55 mol, dissolved in the smallest amount of water needed) was added. The reaction mixture was stirred for the time indicated below. After quenching by aqueous ammonium chloride for 24a and 24c or by an ice-cooled solution of 6 N HCl (400 mL) for 24b and 24d, they are extracted with ethyl ethersa saturated NaCl solution was added if necessary. The organic layer was dried over magnesium sulfate and concentrated in Vacuo. The residue was chromatographed on silica gel

Biomacromolecules, Vol. 1, No. 3, 2000 397 with the eluting solvent indicated below or distilled or recrystallized to afford pure products. Methyl octanebis(dithioate) (24a). 24a was purified by distillation. Yield: 41%. Eb7×10-3 ) 144 °C, orange liquid. 1H NMR (CDCl3): 1;00-2.00 (8H, m, CH2), 2.65 (6H, s, CH3), 3.05 (4H, t J ) 6 Hz, CH2CdS). 13C NMR (CDCl3): 19.88 (CH3), 28.28, 30.86 (CH2), 51.66 (CH2CdS, 239.11 (CdS). UV-vis (CHCl3): 304 (log  ) 4.34). Anal. Calcd for C10H18S4 (266.51): C, 45.05; H, 6.80; S, 48.10. Found: C, 45.32; H, 7.03; S, 47.67. Mass: 266 (M+•, 23), 251 (65), 219 (43), 106 (52), 71 (100), 59 (79). Carboxymethyl octanebis(dithioate) (24b). The purification was performed by crystallization from chloroform. Yield: 37%, yellow-orange solid. Mp: 140 °C. 1H NMR [(CD3)2CdO]: 1.002.00 (8H, m, CH2), 3.05 (4H, t J ) 7 Hz, CH2CdS), 4.25 (4H, s, SCH2), 8.05 (2H, s, COOH). 13C NMR [(CD3)2CdO)]: 28.78, 31.54 (CH2), 38.09 (SCH2), 51.87 (CH2CS), 168.10 (CdO), 238.55 (Cd S). UV-vis (CH2Cl2): 307 (log  ) 4.36). IR (KBr pellets): 1700 (CdO), 3000 (OH). Anal. Calcd for C12H18 O4S4 (345.53): C, 40.65; H, 5.11; O, 18.05; S, 36.17. Found: C, 40.82; H, 5.23; O, 18.27; S, 35.87. Mass: 354 (M+•; 0.2), 202 (2), 129 (3), 111 (6), 97 (8), 92 (27), 71 (35), 56 (61), 43 (100). Methyl Undecanebis(dithioate) (24c). 24c was purified by crystallization from isooctane. Yield: 48%, yellow powder. Mp: 25 °C. 1H NMR (CDCl3): 1.00-2.00 (16H, m, CH2), 2.60 (6H, s, CH3), 3.05 (4H, t J ) 6 Hz, CH2CdS). 13C NMR (CDCl3): 19.92 (CH3), 28.65, 29.31, 30.49, 31.06 (CH2), 52.03 (CH2CdS), 238.45 (CdS). UV-vis (CHCl3): 305.6 (log  ) 4.32. Anal. Calcd for C14H26S4 (322.59): C, 52.12; H, 8.12; S, 39.75. Found: C, 53.30; H, 8.38, S, 39.39. Mass: 323 (19), 322 (M+•, 23), 307 (65), 275 (43), 127 (23), 106 (52), 71 (81), 59 (79), 55 (100). Carboxymethyl Undecanebis(dithioate) (24d). The crude product was recrystallized from chloroform to furnish pure bis(dithioester) 24d. Yield: 43%, yellow solid. Mp: 139 °C. 1H NMR [(CD3)2CO]: 1.00-2.00 (16 H, m, CH2), 3.10 (4H, t J ) 7 Hz, CH2CdS), 4.20 (4H, s, SCH2), 7.80 (2H, s, COOH). 13C NMR [(CD3)2CO]: 28.80-31.72 (CH2), 38.95 (SCH2), 52.00 (CH2Cd S), 168.24 (CdO), 238.72 (CdS). UV-vis (CH2Cl2): 306.5 (log  ) 4.33). IR (KBr pellets): 1700 (CdO), 3000 (OH). Anal. Calcd for C16H26O4S4 (410.64): C, 46.80; H, 6.38; O, 15.58; S, 31.23. Found: C, 46.95; H, 6.41; O, 15.74; S, 30.81. Carboxymethyl Ethanedithioate (24e). 24e was purified by recrystallization from petroleum ether. Yield: 65%, yellow powder. Mp: 81 °C. See 17a. Anal. Calcd for C4H6O2 S2 (150.17): C, 31.99; H, 3.99; O, 21.31; S, 42.70. Found: C, 32.05; H, 3.72; O, 21.47; S, 42.35. Mass: 150 (M+•; 21), 59 (100), 58 (25), 45 (53). Carboxymethyl Octanedithioate (24 g). The crude product was recrystallized from petroleum ether. Yield: 62%, yellow powder. Mp: 58 °C. 1H NMR (CDCl3): 0.80 (3H, t, J ) 6 Hz, CH3), 1.001.50 (10H, m, CH2), 3.10 (2H, t, J ) 8 Hz, CH2CdS), 4.20 (2H, s, SCH2), 7.90 (1H, s, COOH). 13C NMR (CDCl3): 13.68 (CH), 22.00, 32.50 (CH2), 38.10 (CH2CH2CdS), 62.75 (CH2CdS), 174.10 (CdO), 236.27 (CdS). UV-vis (CHCl3): 304 (log  ) 4.08). IR (CHCl3): 1700 (CdO), 3000 (OH). Anal. Calcd. for C10H18O2S2 (234.38): C, 51.24; H, 7.68; O, 13.65; S, 27.36. Found: C, 51.32; H, 7.65; O, 13.58; S, 27.15. Mass: 234 (M+•, 2), 175 (91), 150 (12), 143 (32), 109 (15), 87 (12), 77 (16), 67 (89), 58 (100). Carboxymethyl Dodecanedithioate (24i). 24i was purified by recrystallization from petroleum ether. Yield: 57%, yellow powder. Mp: 68 °C. 1H NMR (CDCl3): 0.85 (3H, t J ) 6 Hz, CH3), 1.001.50 (18 H, m, CH2), 3.05 (2H, t J ) 8 Hz, CH2CdS), 4.15 (2H, s, SCH2), 7.60 (1H, s, COOH). 13C NMR (CDCl3): 14.11 (CH3), 22.00-32.00 (CH2), 38.15 (CH2CH2CdS), 51.45 (CH2CdS), 173.56 (CdO); 236.61 (CdS). UV-vis (CHCl3): 304 (log  )

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4.07). IR (KBr pellets): 1700 (CdO), 3000 (OH). Anal. Calcd. for C14H26O2S2 (290.49): C, 57.88; H, 8.95; O, 11.01; S, 22.07. Found: C, 58.06; H, 9.05; O, 11.11; S, 21.66. Mass: 290 (M+•, 0.4), 231 (17), 199 (6), 150 (6), 95 (7), 87 (6), 71 (35), 69 (20), 59 (46), 58 (63), 55 (77), 43 (84), 41 (100). Carboxymethyl Tridecanedithioate (24j). The purification by recrystallization from petroleum ether furnished 24j as a yellow powder. Yield: 62%. Mp: 69 °C. 1H NMR (CDCl3): 0.85 (3H, t J ) 6 Hz, CH3), 1.10-1.40 (20H, m, CH2), 3.00 (2H, t J ) 8 Hz, CH2CdS), 4.10 (2H, s, SCH2), 8.10 (1H, s, COOH). 13C NMR (CDCl3): 14.05 (CH3), 21.70-32.00 (CH2), 38.17 (CH2CH2CdS), 51.54 (CH2CdS), 173.26 (CdO), 236.54 (CdS). UV-vis (CHCl3): 305 (log  ) 4.08). IR (KBr pellets): 1700 (CdO), 3000 (OH). Anal. Calcd for C15H28O2 S2 (304.50): C, 58.97; H, 9.24; O, 10.47; S, 20.99. Found: C, 59.16; H, 9.27; O, 10.71; S, 20.76. Mass: 304 (M+•, 1), 271 (1), 245 (25), 213 (18), 150 (6), 95 (11), 71 (38), 69 (34), 59 (44), 58 (62), 55 (75), 43 (79), 41 (100). Bis(dithio acid)s 25 a,c. Bis (dithio acid)s were prepared according to the described method for 24a-d. After CS2 addition, the mixture was stirred for 2 h at room temperature and then cooled. Ice-cooled 6 N HCl solution (200 mL) was added, then the mixture was extracted with diethyl ether and dried over MgSO4, and the solvent was removed under reduced pressure. Crude bis(dithio acid)s 25a-c were obtained as dark red oils and used without further purification. Quaternary Ammonium Salts of Bis(dithio acid)s 26a,c. 2 N tetramethylammonium hydroxide solution (Acros) was standardized before used by 0.1 N HCl (phenolphthalein as indicator). Crude bis(dithio acid)s 25a,c (0.2 g) prepared above were dissolved in methanol, and then an excess of titrated 2 N tetramethylammonium hydroxide solution was added. The excess amount of tetramethylammonium hydroxide was titrated by 0.1 N HCl solution to deduce the quantity of bis(dithio acid)s 26a,c present in the crude products. Quaternary ammonium salts of bis(dithio acid)s 26a,c were obtained by the reaction of crude bis(dithio acid)s 25a,c and stoichiometric amounts of tetramethylammonium hydroxide. Crude salts were precipitated in petroleum ether and purified by recrystallization from 2-ethoxyethanol. Chloroform and dichloromethane must be avoided at this stage as dithiocarboxylate ions are strong nucleophilic reagents. Tetramethylammonium Octanebis(dithioate) (26a). Yield: 21%, orange solid. Mp: 190 °C. 1H NMR (D2O): 1.00-2.00 (8H, m, CH2), 3.10 (4H, t J ) 7 Hz, CH2CdS), 3.15 (24H, s, N-CH3). 13C NMR (D O): 28.08, 32.02 (CH ), 38.95 (SCH ), 55.24, 55.44, 2 2 2 55.63 (NCH3), 61.58 (CH2CdS), 270.68 (CdS). UV-vis (D2 O): 334 (log  ) 4.45). Anal. Calcd. for C16H36N2S4 (384.70): C, 49.95; H, 9.43; N, 7.28; S, 33.33. Found: C, 49.99; H, 9,48; N, 7.04; S, 32.91. Mass: 384 (M+•, 0.6), 342 (0.3), 326 (0.2), 279 (0.7) 243 (4.3), 201 (5.3), 149 (6.1), 129 (4.9), 97 (10.3), 69 (74.5), 56 (100). Tetramethylammonium Dodecanebis(dithioate) (26c). Yield: 17%, orange solid. Mp: 195 °C. 1H NMR (D2 O): 1.00-2.00 (16H, m, CH2), 3.05 (4h, t J ) 7 Hz, CH2CdS), 3.15 (24H, s, NCH3). 13C NMR (D O): 28.60, 29.30, 29.67, 30.65 (CH , 38.95 (SCH ), 2 2) 2 55.13, 55.32, 55.51 (NCH3), 62.13 (CH2CdS), 269.84 (CdS). UV-vis (H2O): 335 (log  ) 4.42). Anal. Calcd for C20H44N2S4 (440.84): C, 54.49; H, 10.06; N, 6.35; S, 29.09. Found: C, 54.38; H, 10.27; S, 28.76. Tetramethylammonium Pentanedithioate (26f). Yield: 40%, orange powder. Mp: 65 °C. 1H NMR (CDCl3): 0.90 (3H, t J ) 6 Hz, CH3), 1.10 (2H, q J ) 6 Hz, CH2CH2CdS), 1.60 (2H, q J ) 6 Hz, CH2CH3), 3.10 (2H, t J ) 8 Hz, CH2CdS), 3.20 (12H, s, NCH3). 13C NMR (CDCl3: 13.72 (CH3), 22.35 (CH2CH3), 34.75 (CH2CH2CdS), 55.98, 56.17, 56.35 (NCH3) 62.77 (CH2CdS), 271.56 (CdS). UV-vis (H2O): 33.45 (log  ) 4.02). Anal. Calcd

Levesque et al. for C9H21NS2 (207.40): C, 52.12; H, 10,12; N, 6.75; S, 30.92. Found C, 55.22; H, 10.19; N, 6.78; S, 30,72. Mass: 207 (M+•, 0.4), 134 (1), 101 (12), 92 (31), 77 (35), 71 (45), 59 (76), 58 (64), 41 (100). Tetramethylammonium Octanedithioate (26g). Yield: 7%, orange solid. Mp: 66 °C. 1H NMR (CD3OD): 0.9 (3H, t, J ) 6 Hz, CH3), 1.00-1.82 (10H, m, CH2), 3.10 (2H, t, CH2CdS), 3.20 (12H, s, NCH3). 13C NMR (D2O): 14.56 (CH3), 23.19, 29.56, 29.69, 32.39, 33.13 (CH2), 56.09, 56.30, 56.48 (NCH3), 62.53 (CH2Cd S), 270.26 (CdS). UV-vis (H2O): 334.3 (log  ) 4.00). Tetramethylammonium Nonanedithioate (26h). Yield: 31%, orange powder. Mp: 67 °C. 1H NMR (CDCl3): 0.90 (3H, t J ) 6 Hz, CH3), 1.10-1.80 (12H, m, CH2), 3.10 (2H, t J ) 8 Hz, CH2Cd S), 3.20 (12H, s, NCH3), 13C NMR (CDCl3): 14.56 (CH3), 23.2033.10 (CH2), 56.09, 56.30, 56.49 (NCH3), 62.53 (CH2CdS), 270.26 (CdS). UV-vis (H2 O): 335 (log  ) 4.02). Anal. Calcd for C13H29NS2(263.51): C, 59.25; H, 11.09; N, 5.31; S, 24.33. Found: C, 59.36; H, 11.31; N, 0.25; S, 24.07. Tetramethylammonium Tridecanedithioate (26j). Yield: 28%, orange powder. Mp: 68 °C. 1H NMR (CDCl3): 0.90 (3H, t J ) 6 Hz, CH3), 1.10-1.80 (20H, m, CH2), 3.10 (2H, t, J ) 8 Hz, CH2Cd S), 3.20 (12H, s, NCH3). 13C NMR (CDCl3): 14.02 (CH3), 22.6032.80 (CH2), 55.17, 55.36, 55.99 (NCH3), 63.14 (CH2CdS), 267.37 (CdS). UV-vis (H2O): 332 (log  ) 4.01). Anal. Calcd for C17H37NS2 (319.62): C, 63.88; H, 11.67; N, 4.38; S, 20.06. Found: C, 63.99; H, 11.72; N, 4.44; S, 19.81. Mass: 319 (M+•, 1), 246 (6), 213 (14), 106 (34), 95 (24), 71 (38), 59 (71), 43 (100). Tetramethylammonium Pent-4-enedithioate (26k). Yield: 15%, orange powder. Mp: 132 °C. 1H NMR (CD3OD): 2.6 (2H, m, CH2), 3.2 (14H, m, NCH3 and CH2CdS), 5.10 (3H, m, CHdCH2). 13C NMR (D2O): 36.62 (CH2), 55.87, 56.07, 56.27 (NCH3), 61.12 (CH2CdS), 115.43 (dCH2), 138.82 (dCH), 269.34 (CdS). UVvis (H2O): 335.5 (log  ) 4.04).

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