2648
`
`J. Org. Chem. 1991,56,2648-2650
`Selective Reduction of Disulfides by Tris(2-carboxyethy1)phosphine
`
`John A. Burns, James C. Butler, John Moran, and George M. Whitesides*
`
`Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138
`
`Received October 15, 1990
`
`Tris(2-carboxyethy1)phosphine (TCEP) reduces disulfides rapidly and completely in water at pH 4.5. It
`preferentially reduces more strained disulfides, in accordance with the usual mechanism postulated for reduction
`of disulfides by phosphines in water. The reagent can be synthesized conveniently in large quantities by acidic
`hydrolysis of the commercially available tris(2-cyanoethy1)phosphine.
`
`RbP + RSSR + H20 - R’,P=O + 2RSH
`
`utility of TCEP as a selective reducing agent for repre-
`sentative dialkyl disulfides in aqueous solutions.
`TCEP-HC1 is a nonvolatile, water-soluble solid that is
`easily manipdated in air. It is a strong reducing agent that
`has previously been reported to cleave disulfides:8 it
`rapidly reduces even very stable alkyl disulfides such as
`truns-4,5-dihydroxy-l,2-dithiane (DTT’”) at room tem-
`perature and pH 5. Qualitative studies of the relative
`reactivities of TCEP with several disulfides also establish
`the potentially useful fact that these reductions are ki-
`netically, not thermodynamically, controlled. For example,
`1 reduces the disulfide group of lipoic acid (Lipox) to the
`corresponding dithiol (Lipd) more rapidly than it reduces
`that of 2-hydroxyethyl disulfide (MEox), although Lipd
`itself reduces MEox almost quantitatively at thermody-
`namic equilibrium.
`
`Results
`Synthesis of TCEP. TCEP.HC1 was made conven-
`iently in 50-g quantities by hydrolysis of tris(2-cyano-
`ethy1)phosphine in refluxing aqueous HC1 (eq 2). TCEP
`oxidizes rapidly in aqueous base (see below), making basic
`hydrolysis more cumbersome and less clean than acidic
`hydrolysis: separation of TCEP and TCEP oxide is dif-
`fi~ult.6*~J~ TCEP has also been made by reaction of alu-
`minum phosphide with acrylic acid.” Previous prepara-
`tions are much less convenient than the one reported here.
`Properties of TCEP. TCEPaHCl is a water-soluble
`(310 g/L), odorless, white crystalline solid that is stable
`in air for several months. The pKa of the phosphonium
`center has been estimated by titrimetry to be 7.66.9 Dilute
`solutions of TCEP oxidize only slowly in air when the pH
`is less than that pKa (Figure 1).
`Reduction of Structurally Simple Disulfides in
`Water: Competition Experiments. The reaction of
`TCEP with disulfides is so fast at pH 4.5 that it is difficult
`to measure accurately. We have, instead, measured rela-
`tive reactivities of representative disulfides toward TCEP
`by competition experiments. The competitive reductions
`were carried out by allowing 0.8 equiv of TCEP to react
`with 1.0 equiv of each of two disulfides in D20 at pH 4.5.
`At the concentrations we used (0.2-1.0 mM), the reaction
`was complete within 5 min for all disulfides.
`The NMR spectra place lower boundaries on the se-
`lectivity between each pair of disulfides. The ratio of the
`rates of reduction of Lipox and 1,2-dimethyl-3,8-dioxo-
`
`(2)
`
`Introduction
`Trialkylphosphines reduce organic disulfides to thiols
`smoothly and quantitatively in water (eq l).l The strength
`(1)
`of the phosphorus-oxygen bond renders reduction irre-
`versible. Because trialkylphosphines are kinetically stable
`in aqueous solution, selective for the reduction of the di-
`sulfide linkage, and unreactive toward many other func-
`tional groups, they are attractive as reducing agents in
`biochemical systems. Their utility has been limited by the
`low solubility of most simple trialkylphosphines in water,
`and their acceptability as reagents (especially in biochem-
`istry) by the odor and ease of autoxidation of many species
`with low molecular weights.
`Water-soluble phosphines have been used as reducing
`agents, as chelating agenta, and as ligands. Sulfonylated
`triphenylphosphines have been used extensively as ligands
`for water-soluble metal catalysts.2 They are the subject
`of several ~ a t e n t s . ~ A commercially available water-sol-
`uble triarylphosphine, tris(4-carboxyphenyl)phosphine, is
`relatively expen~ive.~ Other water-soluble phosphines
`have been made.S*6 Tris(hydroxymethyl)phosphine, a
`liquid with an unpleasant odor, can generate formaldehyde
`and other byproducts derived from it in reductions of
`proteins.’ These characteristics have prevented the gen-
`eral use of phosphines as reducing agents for water-soluble
`disulfides.
`Here we describe a convenient preparation of tris(2-
`carboxyethy1)phosphine hydrochloride (TCEP.HC1,l.HCl)
`by acidic hydrolysis of the commercially available tris(2-
`cyanoethy1)phosphine (2) (eq 2) and demonstrate the
`aq HC1
`reflux
`
`(NCCHpCH&P - (HOpCCH CH2)3PH+C1-
`
`l.HC!l
`
`(88%)
`
`2
`
`(1) Schhberg, A. Chem. Ber. 1935,68, 163-164.
`(2) Sinou, D. Bull. SOC. Chim. Fr. 1987,4J30-486. Joo, F.; Toth, Z. J.
`Mol. Catal. 1980,8, 369-383.
`(3) Varre, C.; Desbois, M.; Nouvel, J. French Patent 2,561,650, 1985.
`Sabot, J. L. French Patent 2,532,318, 1984. Kuntz, E. West German
`Patent 2,627,364, 1976.
`(4) Tria(4-carboxypheny1)phosphine costa more than $15000/mol;
`tria(2-cyanoethyl)phosphine, less than $350/mol. Prices are from Strem.
`(5) For example, P(CH2CH2CONHJ3: Jahn, W. 2. Naturforsch. 1989,
`44B, 79-82. P(pCJ34CH2N(Ac)CH2CH20H)3: Jahn, W. 2. Naturforsch.
`1989,44B, 1313-1322. Ph2PCH2(o-C6H4S03K): Paetzold, E.; Oehme, G.;
`Costisella, B. 2. Chem. 1989, 29, 447-448. P(CH2(CHzCH20).Me)3~
`Okano, T.; Morimoto, K.; Konishi, H.; Kiji, J. Nippon Kagaku Kaishr
`1985, 486-493. RCON(CH2CH2PPhz)2: Nuzzo, R. G.; Haynie, S. L.;
`Wilson, M. E.; Whitesides, G. M. J. Org. Chem. 1981, 46, 2861-2867.
`P(p-C6H4CH2NH2)$ Bartlett, P. A.; Bauer, B.; Singer, S. J. J. Am. Chem.
`SOC. 1978, 100,5085-5089.
`(6) Rauhut, M. M.; Hechenbleikner, I.; Currier, H. A.; Schaefer, F. C.;
`Wystrach, V. P. J. Am. Chem. SOC. 1959,81, 1103-1107.
`(7) Sweetman, B. J.; Maclaren, J. A. Aust. J. Chem. 1966, 19,
`2347-2354.
`
`(8) Levison, M. E.; Josephson, A. S.; Kirschenbaum, D. M. Expenen-
`tia 1969,25, 126-127.
`(9) Podlaha, J.; PodlahovH, J. Collect. Czech. Chem. Commun. 1973,
`38, 1730-1736.
`(10) Yakovenko, T. V.; Valetdinov, R. K.; Vafha, R. V. Zh. Obshch.
`Khim. 1976, 46, 278-280. Valetdinov, R. K.; Yakovenko, T. V. Soviet
`Patent 484,221,1975. Haenssle, P. West German Patent 2,902,203,1980.
`(11) Moedritzer, K. West German Patent 2,653,852, 1977.
`0022-3263/91/1956-2648$02.50/0 0 1991 American Chemical Society
`
`Page 2648
`
`

`

`Selective Reduction of Disulfides
`1,2,5,6-diazadithiocane (3)12 is >101. The corresponding
`
`s-s
`O T N F O
`
`X + A"' - Y + Ared
`
`3
`ratio for 3/DTTOx is >10:1 and for DTTOx/MEox, 2.51.
`The ratios of rates are calculated from the concentrations
`of both disulfides and both thiols by the method of Sih
`(eq 3,4; [&I = [A"'] + [AoX]).l3 The method assumes that
`rate = k~[Aox:lf([Xl,[Yl) (3)
`In ([Ared1/[Aol)/ln ([Bred1/[Bol) = k ~ / k ~
`(4)
`the reaction is first-order in disulfide, is irreversible, and
`has the same rate expression for each substrate; it does not
`depend on the participation of other species (X, Y in eq
`3). The signal-to-noise ratio in our spectrometer prevents
`us from measuring selectivities higher than -101.
`
`Discussion
`TCEP reduces disulfides rapidly and cleanly in water
`at pH 5 at room temperature. Because it is easy to make
`and convenient to use, it should find wide applicability as
`a reducing agent for water-soluble disulfides, particularly
`in biochemical applications. Our determinations of the
`selectivity of the reaction proceeded with minimal diffi-
`culty.
`The observed order of rates of reduction of disulfides
`by TCEP (Lipox > 3 > DTT" > Meox) is kinetically de-
`termined: the order of thermodynamic reactivity, and the
`order expected if rates of reduction correlated with the free
`energy of reduction of the disulfides under acidic condi-
`tions, is MEox > 3 > Lip" > DTT0x.14 The order of rates
`observed for TCEP reduction of disulfides does, however,
`correlate qualitatively with the order of rates of thiolate-
`disulfide self-exchange (eq 5);15 the rate constant for ex-
`SRS + -SRS- F! -SRS- + SRS
`n
`n
`(5)
`change between 1,2-dithiolane and 1,3-propanedithiolate
`is 650 times larger than that between 1,2-dithiane and
`1,4-butanedithiolatea We have suggested that rate con-
`stants for thiolate-disulfide interchange are determined
`in large part by the ground-state strain in the CSSC
`we infer that the susceptibility of these disulfides
`to reduction by TCEP is largely determined by the same
`factor.
`This kinetic selectivity offers the opportunity to ma-
`nipulate the distribution of species in a mixture of di-
`sulfides and thiols in a way that would be difficult to
`achieve by other means. The reduction of disulfides by
`TCEP proceeds readily at low pH. At these values of pH,
`thiolate-disulfide interchange is effectively prevented
`because only low levels of thiolate are present.
`It is
`therefore possible to generate thiol from disulfide with
`kinetic selectivity under conditions in which thiolate-di-
`sulfide interchange does not occur. For example, we were
`able to reduce Lipox with good selectivity in the presence
`
`(12) Singh, R.; Whitesides, G. M. J. Org. Chem., in press.
`(13) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
`Sot. 1982.104.7294-7299.
`(14) Houk, .J.; Whitesides, G. M. J. Am. Chem. SOC. 1987, 109,
`6825-6836.
`(15) Singh, R.; Whitesides, G. M. J. Am. Chem. SOC. 1990, 112,
`6304-6309.
`
`J. Org. Chem., Vol. 56, No. 8, 1991 2649
`
`4- 6.6 2ov
`I
`
`I ,
`,",
`30 40
`20
`50
`10
`time (hours)
`Figure 1. Solutions of TCEP (5 mM) in acetate or phosphate
`buffers (0.4 M) autoxidized slowly at pH <7. The solutions were
`stirred vigorously under air at room temperature and analyzed
`periodically by 'H NMR spectroscopy.
`
`0
`
`0
`
`5.0,
`70
`
`60
`
`80
`
`Scheme I. Mechanism of Reduction of Disulfides by
`Phosphines in Water
`R , P : - S C S e R',P'SRS e r R,P'SRSH % R',P=O + HSRSH
`y RDS
`-r
`
`5
`
`4
`of MEox using TCEP at pH 4.5.
`The selectivity of this reaction supports the mechanism
`usually postulated for reduction of disulfides by phos-
`phines: cleavage of the disulfide bond appears to be the
`rate-determining step (RDS) (Scheme I).16J7 The order
`of kinetic reactivities is the same as the order of reactivity
`of disulfides as measured in thiolate-disulfide exchange
`between dithiolates and their corresponding disulfide^.'^
`We infer from that correspondence that the S-S bond is
`partially broken in the transition state, as it is in the
`transition state for thiolate-disulfide interchange.18 If the
`RDS were to occur after the cleavage of the S-S bond, the
`kinetic selectivity would be different. For this type of
`mechanism, assuming little interaction between the thio-
`phosphonium moiety and the thiolate moiety (intermediate
`4) or the thiol group (intermediate 5), the transition states
`derived from those intermediates have no S-S bond and
`resemble the thiol products of the thiolate-disulfide in-
`terchange. As a result, the rates of reduction by phos-
`phines should follow the thermodynamic order of disulfide
`reactivity, not the kinetic order.
`Conclusions
`Although the reaction of phosphines with disulfides in
`the presence of water to form thiols (eq 1) has been known
`since 1935,' little use has been made of the reaction be-
`cause the commonly available phosphines are malodorous
`or insoluble in water. TCEP is a convenient phosphine
`for reduction of disulfides in water: TCEP.HC1, an
`odorless, crystalline, air-stable solid, is soluble in water and
`reacts rapidly (<5 min) with disulfides at room tempera-
`ture in dilute (1 mM) solutions! Related phosphines with
`different charges (e.g., P(CH2CH2CONH2)J are also
`available from 2 by similar synthetic routes.4~~ A test of
`
`(16) Grayson, M.; Farley, C. E. Chimie Organique du Phosphore,
`Colloquee Internationaux du Centre National de la Recherche Scienti-
`fique No. 182, CNRS, Paris, 1970, pp 275-284.
`(17) Overman, L. E.; O'Connor, E. M. J. Am. Chem. SOC. 1976, 98,
`771-775. Overman, L. E.; Petty, S. T. J. Org. Chem. 1975,40,2779-W82.
`Overman, L. E.; Matzinger, D.; O'Connor, E. M.; Overman, J. D. J. Am.
`Chem. SOC. 1974,96,6081-6089.
`(18) Rosenfield, R. E.; Parthasarthy, R.; Dunitz, J. D. J. Am. Chem.
`SOC. 1977,99,4860-4862.
`
`Page 2649
`
`

`

`J. Org. Chem. 1991,56, 2650-2655
`2650
`the mechanism of the reaction suggests that it is an SN2
`10.9 Hz, 51.2 = 8.0 Hz), 2.20 (dt, 6 H, J2,p = 11.5 Hz).
`mechanism, with the RDS being attack of the phosphine
`J1,p
`Competitive Reductions. The competitive reductions were
`carried out by diluting from stock solutions 1 equiv of each of
`nucleophile on the S-S bond.
`two disulfides and 1 equiv of 2-butyne-1,4-diol (as an internal
`standard for 'H NMR spectroscopy) in buffer (20 mM acetate-d3
`Experimental Section
`In mild acid, neither autoxidation nor
`in DzO, pD = 4.5).
`General. Tris(2-cyanoethy1)phosphine was obtained from
`thiol-disulfide interchange occurs at an appreciable rate. An
`American Cyanamid.le Deuterated materials were obtained from
`aliquot of TCEP-HC1 stock solution in DzO (0.8 equiv) was added
`Cambridge Isotope Laboratories. Other chemicals were obtained
`to the reaction mixture. The reaction mixture was transferred
`from Aldrich.
`to an NMR tube which was flushed with argon, stoppered, and
`Tris(2-carboxyet hy1)phosphine Hydrochloride. A slurry
`closed with Parafilm. At the concentrations (0.2-1.0 mM) and
`of tris(2-cyanoethyl)phosphine (44.6 g, 0.231 mol) in a n a l aqueous
`temperature (22-25 "C) we used, the reaction waa complete within
`HCl(l50 mL) was heated at reflux under an argon atmosphere
`5 min for all disulfides.
`for 2 h. A clear white precipitate formed when the hot clear
`Air Oxidation of Dilute Solutions of TCEP. Deuterated
`solution was cooled to 0 "C. The precipitate was isolated by
`buffer solutions were made by neutralizing solutions of acetic
`filtration. Recrystallization from water (200 mL), filtration, and
`acid-d4 (0.40 M) or phosphoric acid-d3 (0.35 M) with 40% NaOD
`drying in vacuo afforded 28.5 g of white crystals (99.4 mmol,43%).
`in D20 to pD 5.0 (acetate), 6.6,7.4, and 11.6 (phosphate). To 4.5
`The combined supernatants were concentrated to 110 mL by
`mL of each solution was added 500 pL (25 mmol) of a solution
`boiling. More white crystalline precipitate formed when the
`of TCEPeHCl (49 mM in DzO). The reaction mixtures were
`mixture waa cooled to 0 "C. Filtration, rinsing with 20 mL of water
`vigorously stirred under air. Aliquots were examined by 'H NMR
`at 0 OC, and drying in vacuo gave 29.5 g of white crystals (88%
`spectroscopy after 30 min, 23 h, and 72 h.
`yield for the combined crops). 'H NMR (DzO): 6 2.76 (dt, 6 H,
`JlP = 18.2 Hz, J1,2 = 7.0 Hz), 2.47 (dt, 6 H, Jzp = 13.9 Hz). Mp:
`Acknowledgment. We thank our colleague R. H. Holm
`176 OC (lit! mp 175-177 OC). W: A- = 218 nm, c = 180 L mol-'
`for the use of a UV spectrometer. This research was
`cm-'; A,,
`= 192 nm, c = 150 L mol-' cm-'. Anal. Calcd for
`C9H1606PCk c, 37.71; H, 5.63; P, 10.81; c1, 12.37. Found: c,
`supported by the NIH through Grant GM39589 and by
`the National Science Foundation under the Engineering
`37.61; H, 5.65; P, 10.99; C1, 12.38.
`Research Center Initiative Biotechnology Process Engi-
`Tris(2-carboxyethy1)phosphine Oxide. A crystal of iodine
`neering Center (Cooperative Agreement CDR-88-03014).
`was allowed to react with an aliquot (0.5 mL) of a solution of
`NMR facilities were provided by the National Science
`TCEP.HCl(10 mM) in D20. *H NMR (DzO): 6 2.66 (dt, 6 H,
`Foundation under grant CHE-84-10774. J.A.B. was a
`National Science Foundation predoctoral fellow,
`1986-1989.
`
`(19) American Cyanamid no longer sells tris(2-cyanoethy1)phoephine.
`It is available from Strem.
`
`Structure Determination of Natural Epoxycyclopentanes by X-ray
`Crystallography and NMR Spectroscopy'
`
`Elin S. Olafsdottir, Alex M. Starensen,* Claus Cornett, and Jerzy W. Jaroszewski*
`Department of Organic Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2,
`DK-2100 Copenhagen, Denmark
`Received September 18,1990
`
`Two stereoisomeric epoxycyclopentane cyanohydrin glucosides were isolated, along with several previously
`described cyclopentene cyanohydrin glucosides, from Passiflora suberosa L. (Passifloraceae) and Kiggelaria afncana
`L. (Flacourtiaceae); they represent new members of a rare class of natural nonannellated cyclopentane derivatives.
`The new glucosides were shown, by NMR spectroscopy (including NOE measurements), X-ray crystallography,
`and enzymatic hydrolysis to the corresponding cyanohydrins, to be (lR,2R,3R,4R)- and (ls,2s,3S,4s)-l-(gD-
`glucopyranosyloxy)-2,3-epoxy-4-hydroxycyclopentane-l-carbonitrile and named suberin A and B, respectively.
`The crystal structure of suberin A waa determined at 110 K and refined to R = 0.036 for 2198 unique reflections;
`the cyclopentane ring forms an envelope with C5 placed 0.41 A away from the plane of the remaining four carbon
`atoms, and to the same side as the three oxygen substituents. In addition to the glucosides, two amides,
`and (1S,4R)-1,4-dihydroxy-2-cyclopentene-
`(1S,2S,3R,4R)-2,3-epoxy-1,4-dihydroxycyclopentane-l-carboxamide
`1-carboxamide, were isolated from P. suberosa and characterized; the amides are probably artefacts, and their
`formation represents a novel enzymatic transformation of plant cyanohydrins.
`
`Introduction
`In contrast to the vast abundance of natural products
`having a cyclopentane ring as a part of a polycyclic system,
`relatively few naturally occurring nonannellated cyclo-
`pentane derivatives are known. Such natural products can
`
`(1) Part 13 of the series on natural cyclopentanoid cyanohydrin gly-
`cosides. For part 12, see: Olafsdottir, E. S.; Jaroszewski, J. W.; Seigler,
`D. S. Phytochemistry 1991,30, 867.
`0022-3263/91/1956-2650$02.50/0
`0 1991 American Chemical Society
`
`be divided into four major groups. These are the prosta-
`glandins? the antibiotics pentenomycins and related mold
`and bacterial metabolites,36 a few monoterpenes6 including
`
`(2) Roberta, S. M.; Newton, R. F. Rostaglondim and Thromboxanes;
`Butterworth: London, 1982.
`(3) Smith, A. S.; Branca, S. J.; Pilla, N. N.; Guaciaro, M. A. J. Org.
`Chem. 1982,47,1855. Verlaak, J. M. J.; Klunder, A. J. H.; Zwanenburg,
`B. Tetrahedron Lett. 1982, 23, 5463. Hetmanski, M.; Purcell, N.;
`Stoodley, R. J. J . Chem. SOC., Perkin Tram. 1 1984, 2089. Achab, S.;
`Cosson, J.-P.; Das, B. C. J. Chem. SOC., Chem. Commun. 1984, 1040.
`
`Page 2650
`
`