Intramolecular thiolysis of 4-mercaptobutyrate esters: developing a “traceless” linker for alcohol release from self-assembled monolayers on gold

A series of esters of dithiobutyric acid was prepared using the carbodiimide coupling system. These esters were used to examine the kinetic feasibility of release of alcohols by reductive cleavage of the disulfide. Release of p-nitrophenol was rapid following reduction with dithiothreitol at pH ~10.5. Intramolecular thiolysis is at least one hundred-fold faster then base hydrolysis at this pH. NMR experiments established rapid alcohol release for phenolates and ethanolamine derivatives but alkyl substrates were found to release slowly. Self-assembled monolayers (SAMs) formed from nitrophenol or ethanolamine derivatives produce the expected quantity of alcohol following reductive release from gold powder .


Introduction
Ion channels in planar bilayers offer a way to observe single molecular events.Typically a vast excess of putative channel forming compound is added in the hope of haphazardly inserting a single channel into a planar bilayer.We seek a method to reliably embed a few molecules into a lipid bilayer.To provide a high level of control over channel deposition we envisage release from a gold electrode via reductive cleavage of a gold-sulfur bond.Conventional microelectrodes and circuitry would be capable of delivering as few as 10 2 electrons.Figure 1 introduces the concept of a "traceless" linker, providing a method of selectively delivering a few molecules at a defined point and time via a thiolactonization release of the channel compound as a bound alcohol.The adsorption of dialkyl disulphides has been established 1,2 with the formation of goldsulfur bonds at the expense of the sulfur-sulfur bond.Spontaneous formation of self-assembled monolayers (SAMs) based on the formation of gold-sulfur bonds was pioneered by Nuzzo and Allara in 1983. 3[6][7][8][9][10] In order for the "traceless" linker to be effective, a process for reductively cleaving the goldsulfur bond and simultaneously releasing the alcohol is required.As suggested in Figure 1, thiolactone was evaluated for its potential to release the alcohol after reductive cleavage of the gold-sulfur bond.Results by Schjånberg 11 show that the rate of lactonization for 4mercaptobutanoic acid is comparable to the rate of hydrolysis of this ester, and that at equilibrium the ratio of γ-thiolactone: 4-mercaptobutanoic acid is nearly 1:1 at pH 1.The rate for hydrolysis of the δ-lactone of 5-mercaptopentanoic acid results primarily in the mercaptocarboxylate form at equilibrium at the same pH. 11This indicates that 4mercaptobutanoic acid linker (n=3 in Figure 1) would be favoured for an intramolecular nucleophilic catalysis of alcohol release.A conformationally restricted example of such a nucleophilic catalysis was reported by Fife 12 for the release of nitrophenolate from a 2mercaptophenyl carbamate, also via γ-thiolactone intermediate.
The goal of this study was to establish alcohol release occurs as required and that release occurs at a competent rate for the application envisaged.Working with phenol esters we established that phenolate release, following reduction of the disulfide bond by dithiothreitol (DTT), occurs and is not rate determining.The ability to release other alcohols was shown by NMR scale thiolactonization reactions initiated by DTT.With release of alcohols by thiolactonization established, monolayers were formed on gold surfaces, reductively cleaved, and the expected alcohols were detected both by UV/VIS spectrometry and by HPLC analysis.

Synthesis of dithiobutyrate diesters
Five esters were prepared as shown in scheme 1.The yields ranged from 35% to 56% and represent the isolation of the di-substituted esters except in the case of Fmoc-ethanolamine in which the 62% yield is the combined yield for the synthesis and isolation of both mono and di-substituted esters.All products were fully characterised by conventional techniques.

Kinetics of phenolate release in solution
The initial focus of this study was the rate of p-nitrophenolate release from reductive cleavage of 1 as this release provided a simple spectroscopic probe.Chemical reductive cleavage of the disulphide bond was envisaged, followed by thiolysis of the ester as indicated in scheme 2. No simple method of reduction was found near neutral pH and reagents required basic conditions to be effective.Sodium borohydride, sodium cyanoborohydride, and triphenylphosphine were evaluated and were shown to be ineffective or kinetically incompetent as reducing agents for the disulphide bond at pH 7. Initial testing showed that dithiothreitol (DTT) in 6:4 water: acetonitrile at a pH of 10.4, was an effective reducing agent for the disulphide bond.

Scheme 1 Scheme 2
The observed pseudo first order rate of base hydrolysis in an acetonitrile/water buffer solution at pH 10.4 was established to be 21.9 ± 0.7 s -1 .This result gave a half-life for base hydrolysis of 3200 s in the absence of DTT.
By varying the concentrations of disulphide and DTT, the kinetic competence of the thiolysis was established (Table 1).This experiment was conducted by adding 6 parts of aqueous solution containing 1eq.DTT, and 1.5 eq.sodium hydroxide, to 4 parts acetonitrile containing 1 eq. of disulphide.DTT induced release of phenolates was shown to proceed with a half-life below 600 s.As reported by Whitesides for DTT reduction of other disulfides, 13 the overall reaction was shown to be first order in both disulfide and DTT (Table 1).Under equivalent disulfide concentrations there is an apparent 100-fold decrease in the half-life for phenolate release (22s vs. 3000s) compared to the direct hydrolysis.No precise estimate of kthio (as defined in scheme 2) was possible, but these experiments establish the kinetic competence of scheme 2 as a means to release phenolate into solution.

NMR Scale thiolactonization experiments
The DTT cleavage of the disulphide bond of 1 was conducted in deuterated acetonitrile.The DTT was added with 1.5 equivalents of NaOD in D 2 O.This cleavage resulted in the release of pnitrophenolate in less than 5 minutes.At this stoichiometric ratio the reaction proceeds to an equilibrium mixture of disulfide esters and mercaptobutyrates.If intramolecular thiolysis were slow, then the direct observation of a thiolate ester would be possible.If thiolysis proceeded quickly, then the only the mercaptobutyrate would be evident.
Figure 2(a) shows the 1 H-nmr spectrum of 1 in CD 3 CN.The aromatic protons are at 8.25 ppm and 7.34 ppm.Three methylene signals were present: two protons α to ester (2.74 ppm), two protons β to the ester (2.11 ppm), and the two protons α to the disulfide (2.83 ppm).Figure 2(c) shows the 1 H-nmr of p-nitrophenolate under the same conditions with two aromatic signals at 7.92 ppm and 6.36 ppm.The appearance of three new signals (2.65 ppm, 2.41 ppm, and 2.21 ppm) represents the appearance of the mercaptobutyrate ion.The methylene positions from the disulfide ester remain (2.83ppm, 2.74 ppm, and 2.10 ppm).The presence of thiolactone is not observed as the mercaptobutyrate form dominates the equilibrium under the conditions of the reaction (pH =10.3).The dithiothreitol reagent also contributes resonances to Figure 2(b), these are found around 3.5 ppm.This reaction was also conducted in deuterated methanol with a similar result.This NMR experiment confirms the kinetic result above, namely that phenolate is rapidly released, presumably via intramolecular thiolysis.
In contrast, the DTT cleavage of the hexyl ester 3 in deuterated methanol resulted in cleavage of the disulphide bond with no release of the deprotonated hexanol.This was clearly observable as the resonance for the methylene protons next to the disulphide shifted from 2.73 ppm to 2.50 ppm, while the hexyl methylene protons next to the ester remain close to 4.08 ppm.This is a direct observation of a stable thiolate ester that indicates the expected thiolysis/ hydrolysis does not occur rapidly in this instance.
Other esters show a range of behaviors between these two limits.p-Methoxyphenol is released quickly from 2(under 10 minutes), and Fmoc-ethanolamine is released at a feasible rate from 4(under 30 minutes).The long chain alcohol from 5 bola was shown to release very slowly, and showed complete conversion after 3 days, a rate consistent with direct hydrolysis.

Reductive cleavage and release from gold powders
Experimental detection of reductive release from gold requires a sufficient surface area to produce a detectable amount of product.A convenient form is the use of spherical gold powder (5.5-9.0 micron) with a surface area of 345 to 565 cm 2 / g.At a surface concentration of 10 14 molecules/ cm 2 this corresponds to release of 57 nanomoles of material per gram of gold powder.Electrolysis of the suspended powder in a ml or less of electrolyte solution will then give a concentration of approximately 60 µM per gram of gold powder.
Spherical gold powder was cleaned, and absorption was conducted overnight in a dichloromethane solution of 1 (approx.0.1 M).The gold powder was rinsed until the presence of 1 was undetectable by UV spectrometry.The release was conducted in 1M KCL solution, at a potential of -2.0 V (vs.AgCl/Ag reference) applied for 30 sec.The release solution turned visibly yellow, and UV/VIS spectrometry confirmed the presence of pnitrophenolate.Although the result was gratifying, the amount of nitrophenolate was much higher then expected, probably due to carryover from the absorption step.
Fluorescence detection was then used to improve the detection of the residual disulfide to confirm negligible carryover.Absorption of 4 was conducted in ethanol, and reductive cleavage was conducted in acetonitrile solution.Analysis by LC showed release of a quantity of Fmocethanolamine corresponding to approximately 10% of the expected release and represents the overall effectiveness of the process outlined in Figure 1.Since our goal is to limit the amount of material released, this efficiency is acceptable.
The overall fitness of Figure 1 is thus established.The next stage will be to prepare a suitable mercaptobutyrate ester of a channel-forming compound and proceed to a controlled embedding in a planar bilayer.Our efforts in this are will be reported in due course.

Experimental Section
General Procedures.All NMR spectra were recorded on a Bruker AC300 FT NMR spectrometer. 1 H-NMR and 13 C-NMR were referenced to the solvent residual peaks as follows: CDCl 3 δ H 7.26, δC 77.16, CD 3 CN δ H 1.94, and CD 3 OD δ H 3.31.UV/VIS spectra were recorded on a Cary50 UV/VIS spectrometer.

Figure 1 .
Figure 1.Proposed Pathway for Release of Channel Compounds.
Figure 2(b) shows the 1 H-nmr 5 minutes after the addition of 1.1 equivalents of basic DTT to the deuterated acetonitrile solution of the disulphide.Two new resonances (8.01 ppm and 6.63ppm) indicate the appearance of pnitrophenolate signals, while two resonances (8.24 ppm and 7.33 ppm) show the presence of unreleased p-nitrophenolate ester.