- Research article
- Open Access
Dynamic combinatorial chemistry at the phospholipid bilayer interface
© Mansfeld et al; licensee BioMed Central Ltd. 2010
- Received: 17 June 2010
- Accepted: 08 September 2010
- Published: 08 September 2010
Molecular recognition at the environment provided by the phospholipid bilayer interface plays an important role in biology and is subject of intense investigation. Dynamic combinatorial chemistry is a powerful approach for exploring molecular recognition, but has thus far not been adapted for use in this special microenvironment.
Thioester exchange was found to be a suitable reversible reaction to achieve rapid equilibration of dynamic combinatorial libraries at the egg phosphatidyl choline bilayer interface. Competing thioester hydrolysis can be minimised by judicial choice of the structure of the thioesters and the experimental conditions. Comparison of the library compositions in bulk solution with those in the presence of egg PC revealed that the latter show a bias towards the formation of library members rich in membrane-bound building blocks. This leads to a shift away from macrocyclic towards linear library members.
The methodology to perform dynamic combinatorial chemistry at the phospholipid bilayer interface has been developed. The spatial confinement of building blocks to the membrane interface can shift the ring-chain equilibrium in favour of chain-like compounds. These results imply that interfaces may be used as a platform to direct systems to the formation of (informational) polymers under conditions where small macrocycles would dominate in the absence of interfacial confinement.
- Lipid Bilayer
- Exact Mass
- Mercaptoacetic Acid
Dynamic combinatorial chemistry [1–3] is a growing field in the general area of systems chemistry [4–11] and revolves around equilibrium mixtures of molecules or supramolecules that can exchange the building blocks from which they are constituted. The resulting dynamic combinatorial libraries (DCLs) are inherently responsive to influences that alter the relative thermodynamic stabilities of the library members. For example, addition of a template (a guest molecule or a biomolecule) to a DCL will result in a stabilization of those library members that bind to the template, inducing a shift in the product distribution, ideally in favour of the best binders and at the expense of the other unwanted library members. This responsiveness makes dynamic combinatorial chemistry an important tool for the discovery of new synthetic receptors [12–21] and ligands for biomolecules [22–25]. Moreover, the technique has potential for the development of catalysts by using a transition-state analogue as a template [26, 27]. Molecular recognition in DCLs can also occur between or within library members, enabling the discovery of replicating and/or self-assembling systems [28–38], catenanes [39–41] and the exploration of folding of macromolecules [42–44].
While the vast majority of the work on dynamic combinatorial chemistry is confined to homogeneous solutions and in a few cases, two-phase systems [14, 15, 45], its application to the chemistry at interfaces is largely unexplored, apart from one example of a dynamic combinatorial approach to bilayer membrane transport . Nonetheless, molecular recognition at interfaces is of extreme importance in many different disciplines, ranging from nanotechnology to cell biology. Furthermore, molecular recognition at interfaces can differ markedly from the corresponding process in bulk solution, as a result of a different microenvironment and confinement in two dimensions [46–48]. This prompted us to develop dynamic combinatorial methodology to allow the use of this technique at interfaces. We have focussed our efforts on the phospholipid bilayers that mimic the cell membrane and now report the successful adaptation of reversible thioester chemistry [33, 44, 49–54] for making DCLs at the lipid bilayer interface. We have developed analytical protocols that enable the monitoring of product distributions of libraries of molecules that are bound to the lipid bilayer. Our results show that the kinetics of thioester exchange are comparable in bulk solution and at the bilayer interface, despite the different microenvironments of these two systems. We also show that the partitioning and two-dimensional organisation of building blocks at the membrane surface can bias the product distributions of the libraries from small mainly cyclic products in bulk solution toward larger linear products at the bilayer.
Many different reversible covalent chemistries have now been developed that allow for the construction of DCLs in aqueous solution. The most popular of these are hydrazone and disulfide chemistry . However, both processes have equilibration times that are typically in the range of 2-5 days and sometimes even several weeks . This makes these chemistries less applicable to bilayer systems, many of which have limited physical stabilities. For example, unilamellar vesicles prepared from phospholipids are often only stable for approximately 24 hours. Thus, alternative reversible chemistries are required that allow equilibrium to be reached within this timeframe and under relatively dilute conditions. Literature reports suggest that the equilibration of thioesters in bulk aqueous solution is relatively fast [33, 44, 49–54], which led us to investigate the potential of this reaction in bilayer vesicle systems. As a model system we chose unilamellar vesicles made from egg phosphatidyl choline.
Thioester chemistry in bulk aqueous solution
Rate constants defined in Figure 1 for thioester exchange and hydrolysis at different temperatures and pHs.
k 1 [M-1s-1]
k -1 [M-1s-1]
k h1 [s-1]
k 1 /k -1
0.868 × 10-2 ± 1.8 × 10-4
3.51 × 10-2 ± 1.3 × 10-3
5 × 10-5 ± 2 × 10-5
7.58 × 10-2 ± 6.8 × 10-3
18.5 × 10-2 ± 2.3 × 10-2
1.1 × 10-4 ± 1 × 10-5
2.93 × 10-2 ± 3 × 10-4
6.42 × 10-2 ± 1.1 × 10-3
6 × 10-5 ± 1 × 10-5
1.22 × 10-2 ± 1 × 10-4
2.92 × 10-2 ± 4 × 10-4
2 × 10-5 1 × 10-5
1.08 × 10-2 ± 2 × 10-4
1.00 × 10-2 ± 8 × 10-4
1.64 × 10-3 ± 3 × 10-5
8.13 × 10-2 ± 1.4 × 10-3
6.12 × 10-2 ± 2.1 × 10-3
2.29 × 10-3 ± 3 × 10-5
4.09 × 10-2 ± 1.2 × 10-3
4.01 × 10-2 ± 2.1 × 10-3
8.0 × 10-4 ± 4 × 10-5
1.54 × 10-2 ± 2 × 10-4
1.69 × 10-2 ± 6 × 10-4
3.2 × 10-4 ± 2 × 10-5
The experiments with the model thioesters 1a and 1b established that it is preferable to utilise building blocks with the acyl carbon directly attached to the aromatic core; including an aliphatic spacer leads to significant competition by hydrolysis. Therefore, in further experiments we focused on the former design.
The above results indicate that thioester exchange proceeds smoothly in bulk solution in the required timescale. Thus, the scene was set to perform similar experiments at the lipid bilayer interface.
Thioester chemistry at the phospholipid bilayer interface
In order to study thioester chemistry at the phospholipid bilayer interface a derivative of 6 was prepared that was equipped with an alkyl chain for incorporation in lipid vesicles and a triethylene glycol spacer to facilitate access of thiols to the thioester functionality of the head group (13 in Figure 1). The synthetic route to this compound is shown in Figure 3. To set up phospholipid-based DCLs, large unilamellar vesicles (200 nm diameter) were prepared in phosphate buffer pH 8.0 by mixing egg phosphatidylcholine (egg PC) with 10 mol% 13, followed by extrusion through a polycarbonate membrane. Different thiols were then added to the resulting solutions.
Where finding suitable analytical conditions for studying DCLs in bulk solution was straightforward, identification of products in DCLs in the presence of phospholipids was considerably more challenging. Even though, lacking a chromophore, lipids do not interfere with the UV/Vis detection of compounds, they complicate the MS analysis significantly. In initial attempts we found that egg PC is not eluted completely from the column under the conditions used for analysis and leaches slowly with every gradient chromatography performed. The resulting broad MS signal of the phospholipids concealed those of any other species present in the DCLs, hampering the identification of thioester exchange products. After exploring several different approaches, the best method for removing the egg PC from the column was found to be washing with THF containing triethylamine and trifluoroacetic acid at regular intervals, usually after five to ten chromatography runs had been performed. In combination with using very low injection volumes for LC-MS analysis, these measures proved suitable for achieving MS traces that allowed the assignment of library members in a DCL containing egg PC.
Rate constants as defined in Figure 1 for thioester exchange and hydrolysis at the lipid bilayer interface in a DCL made from 13 and 14 at pH 8.0 and 40°C.
k 2 [M-1s-1]
k -2 [M-1s-1]
k 3 [M-1s-1]
k -3 [M-1s-1]
k h2 [s-1]
k h3 [s-1]
1.65 × 10-2
± 0.2 × 10-3
3.31 × 10-2
± 0.8 × 10-3
1.32 × 10-2
± 0.5 × 10-3
5.07 × 10-2
± 2.7 × 10-3
0.008 × 10-2
± 0.9 × 10-5
0.004 × 10-2
± 0.6 × 10-4
We also probed whether the thiol building blocks are able to cross the bilayer membrane and/or the membrane-bound thioesters are able to undergo flip-flop. Experiments were performed in which thiols were added after vesicle formation (single-sided addition) or where vesicles were prepared in the presence of thiol (double-sided addition). If the membrane permeation of the thiols through the bilayer and thioester flip-flop are both slow on the timescale of the experiment, single-sided and double-sided addition would result in different DCL compositions. For single-sided addition only the fraction of thioester building block present in the outer leaflet of the lipid bilayer would be exposed to the thiols and therefore be able to participate in transthioesterification. The HPLC analysis of two DCLs where thiols were added prior to, or after vesicle formation show comparable product distributions (see Additional file 1, Figure S1), indicating that diffusion of thiols across the lipid bilayer and/or flip-flop of the uncharged thioester 13 are fast on the timescale of the experiment.
Comparing DCL distributions at the bilayer interface with those in bulk solution
Although it was pointed out in the previous section that less polar thiol building blocks may favour the thioester exchange species rather than the starting material, the poor water-solubility of more hydrophobic building blocks prohibited their use in this study.
The differences in product distribution in bulk water and at the lipid bilayer observed for both aliphatic and aromatic thiol-thioester systems studied here can be attributed to differences in local concentration. Libraries at the lipid bilayer interface are biased towards species that are rich in the membrane-bound amphiphilic building block 13 which is probably caused by a local concentrating effect of the lipid vesicles.
Our results show that it is possible to use dynamic combinatorial chemistry based on reversible thioester chemistry at the phospholipid bilayer interface and achieve equilibration within 24 hours at pH 7.0 or 8.0 at millimolar building block concentrations. Conditions for LC-MS analysis of the libraries of molecules bound to the egg PC membranes have been developed. Kinetic investigations revealed that thioester hydrolysis is not competitive with thioester exchange, provided that thioesters are used that are derived from aromatic carboxylic acids. Perhaps counterintuitively, hydrolysis is less competitive at the higher pH, because the rate of hydrolysis increases less rapidly with increasing pH than the rate of exchange.
DCLs at the membrane interface facilitate access to library members featuring multiple copies of membrane-bound building blocks. Thus, at the membrane interface the composition of the DCLs is biased towards larger linear species, while smaller macrocyclic species are more abundant in solution-phase libraries. We attribute this difference to the confinement of the building blocks to two dimensions at the bilayer interface. This confinement leads to a high local concentration of the membrane-bound building blocks, shifting the ring-chain equilibrium in favour of chain-like compounds. These results imply that interfaces may be used as a platform to direct systems to the formation of (informational) polymers under conditions where small macrocycles would dominate in the absence of interfacial confinement.
Chemicals were purchased from Aldrich, Acros, Alfa Aesar, Avocado Organics, Fluka, or Lancaster Synthesis and used without further purification. Solvents used in synthesis were distilled prior to use and anhydrous solvents were distilled from a drying agent under argon. LC-MS grade solvents (acetonitrile, water, formic acid and trifluoroacetic acid) were obtained from Romil and used without further purification. Thin layer chromatography was carried out on glass or aluminium plates coated with silica gel 60 F254 (Merck). Column chromatography was performed on silica gel (0.040-0.063 mm) purchased from Breckland Scientific Systems. Silica gel (0.015-0.040 mm) from Merck was used for dry column vacuum chromatography. Dithiols 27  and 29  have been prepared following literature procedures.
2-(Benzoylthio)acetic acid 1a
Benzoic acid (10 mmol, 1.22 g) was dissolved in anhydrous CH2Cl2 (20 mL). Oxalylchloride (12 mmol, 1.02 mL) was added and the solution was stirred at room temperature overnight under a N2 atmosphere. The solvent was evaporated in vacuo and the resulting acid chloride was used for the next step without further purification.
To a stirred solution of mercaptoacetic acid (1.87 mmol, 0.13 mL) and triethylamine (3.82 mmol, 0.53 mL) in a 1:1 mixture of water and acetonitrile (10 mL), the acid chloride (2.01 mmol, 0.31 g) was added. After 4 hours the mixture was washed three times with hexane. The aqueous phase was acidified and extracted three times with CH2Cl2. The combined organic phases were dried over sodium sulphate, filtered and the solvent was evaporated. Column chromatography (silica, DCM/acetone/formic acid 96:4:0.5) yielded 1.37 g (71% over two steps) of a white solid. Characterisation matched that described in ref. .
2-(2-Phenylacetylthio)acetic acid 1b
Prepared analogous to the procedure for the synthesis of 1a. Yield: 1.32 g (63% over two steps) of an oil that crystallized upon scratching. 1H-NMR (CDCl3, 400 MHz): δ = 7.37 - 7.27 (m, 5 H, a-c), 3.89 (s, 2 H, e), 3.70 (s, 2 H, d). 13C-NMR (CDCl3, 400 MHz): δ = 195.8, 173.9, 132.7, 129.7, 128.8, 127.7, 49.9, 31.3. Exact mass calculated: 209.0278, found: 209.0277 [M-H+]. Melting point: 90 - 92°C.
S,S-Diacetic acid benzene-1,3-bis(carbothioate) 6
Mercaptoacetic acid (2.02 mmol, 0.14 mL) and triethylamine (3.97 mmol, 0.55 mL) were dissolved in a 1:1 mixture of water and acetonitrile (10 mL). After addition of isophthaloyl dichloride (0.99 mmol, 0.20 g) the solution was stirred at room temperature for 5 hours. The mixture was washed with hexane three times and then lyophilised. The residue was redissolved in water and the product precipitated by acidifying with 3N HCl. Recrystallisation from methanol yielded a white powder (33 mg, 11%).
1H-NMR (CD3OD, 400 MHz): δ = 8.49 (t, 1 H, 4J = 1.5, d), 8.23 (dd, 2 H, 4J = 1.7, 3J = 7.8, e), 7.69 (t, 1 H, J = 7.8, f), 3.94 (s, 4 H, b). 13C-NMR (CD3OD, 400 MHz): δ = 190.9, 171.9, 138.3, 133.1, 131.0, 126.5, 32.3. Exact mass calculated: 336.9817, found: 336.9825 [M+Na+]. Melting point: 199 - 201°C.
2-(2-(2-(Dodecyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 20
Anhydrous pyridine (10.01 mmol, 0.81 mL) was added to a solution of triethyleneglycol monododecyl ether (2.01 mmol, 0.69 mL) in anhydrous CH2Cl2 (20 mL) stirring at 0°C under a N2 atmosphere. After 10 minutes p-toluenesulfonyl chloride (4.98 mmol, 0.95 g) was added. The solution was allowed to warm up slowly to room temperature and left to stir for a further 2 days before washing it with 3 N HCl, saturated sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate and filtered. After the solvent had been evaporated the residue was purified using dry column vacuum chromatography (0-35% EtOAc in hexane (v/v) in 2.5% increments) to yield a clear oil (0.69 g, 73%).
1H-NMR (CDCl3, 400 MHz): δ = 7.77 (d, 2 H, J = 8.3, c), 7.31 (d, 2 H, J = 8.4, b), 4.13 (t, 2 H, J = 4.7, d), 3.66 (t, 2 H, J = 4.7, e) 3.59-3.51 (m, 8H), 3.41 (t, 2 H, J = 6.9, g), 2.42 (s, 3 H, a), 1.56 (m, 2 H, h), 1.29-1.23 (m, 18 H, i), 0.85 (t, 3 H, j). 13C-NMR (CDCl3, 400 MHz): δ = 144.6, 133.0, 129.7, 127.9, 71.4, 70.7, 70.6, 70.4, 69.9, 69.1., 68.6, 31.8, 29.6 - 29.4, 29.4, 29.2, 26.0, 22.6, 21.5, 14.0. Exact mass calculated: 473.2937, found: 473.2949 [M+H+].
Dimethyl 5-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)isophthalate 21
2-(2-(2-(Dodecyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (20; 4.99 mmol, 2.36 g) and dimethyl-5-hydroxyisophthalic acid (5.00 mmol, 1.05 g) were dissolved in anhydrous DMF (100 mL) and potassium carbonate (19.97 mmol, 2.76 g) was added. The mixture was stirred at 100°C for 4 hours. After cooling down, CH2Cl2 (100 mL) was added and the mixture was stirred for 10 minutes at room temperature, then washed with 3 N HCl and brine, dried over sodium sulfate and filtered. The solvent was evaporated to yield 2.23 g (87%) of a clear oil.
1H-NMR (CDCl3, 400 MHz): δ = 8.27 (t, 1 H, J = 1.5, b), 7.77 (d, 2 H, J = 1.5, c), 4.21 (t, 2 H, J = 4.8, d), 3.93 (s, 6 H, a), 3.89 (t, 2 H, J = 4.8, e), 3.75 - 3.56 (m, 8 H, f), 3.44 (t, 2 H, J = 6.9, g), 1.56 (m, 2 H, h), 1.31 - 1.25 (m, 18 H, i), 0.87 (t, 3 H, j). 13C-NMR (CDCl3, 400 MHz): δ = 166.1, 158.9, 131.8, 123.1, 120.0, 71.6, 70.9, 70.7, 70.7, 70.1, 69.5, 68.1, 52.4, 31.9, 29.7 - 29.6, 29.5, 29.3, 26.1, 22.7, 14.1. Exact mass calculated: 511.3271, found: 511.3292 [M+H+].
5-(2-(2-(2-(Dodecyloxy)ethoxy)ethoxy)ethoxy)isophthalic acid 22
Dimethyl 5-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)isophthalate (21; 4.36 mmol, 2.20 g) was dissolved in THF (60 mL). 1 M KOH (20 mL) was added and the solution was stirred for 15 hours. The THF was evaporated and the remaining aqueous solution was acidified and extracted with CH2Cl2 three times. The organic phase was washed with 3 N HCl and brine, dried over sodium sulfate. Evaporation of solvent gave 2.06 g (98%) of an off-white solid.
1H-NMR (CDCl3, 400 MHz): δ = 8.02 (t, 1 H, J = 1.2, a), 7.58 (d, 2 H, J = 1.1, b), 4.15 (t, 2 H, J = 4.2, c), 3.92 (t, 2 H, J = 4.0, d), 3.86 - 3.63 (m, 8 H, e), 3.45 (t, 2 H, J = 6.8, f), 1.56 (m, 2 H, g), 1.30 - 1.21 (m, 18 H, h), 0.86 (t, 3 H, i). 13C-NMR (CDCl3, 400 MHz): δ = 169.2, 158.5, 130.9, 123.8, 120.1, 71.6, 70.7, 70.5,70.4, 69.9, 69.6, 67.7, 31.9, 29.7 - 29.6, 29.5, 29.5, 29.3, 26.0, 22.7, 14.1. Exact mass calculated: 483.2958, found: 483.2968 [M+H+]. Melting point: 80 - 82°C.
S,S-Bis(furan-2-ylmethyl) 5-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)benzene-1,3-bis(carbothioate) 13
5-(2-(2-(2-(Dodecyloxy)ethoxy)ethoxy)ethoxy)isophthalic acid (22; 0.40 mmol, 193 mg), 2-furanmethanethiol (0.99 mmol, 0.1 mL) and DMAP (0.10 mmol, 12 mg) were dissolved in anhydrous CH2Cl2 (10 mL) and cooled to 4°C on an ice bath. Dicyclohexylcarbodiimide (1.00 mmol, 206 mg) was added and the solution was stirred over night, allowing it to warm up to room temperature slowly. The mixture was extracted with 3 N HCl and brine. The organic layer was dried over sodium sulfate and filtered. After solvent evaporation the residue was purified using dry column vacuum chromatography (0-25% EtOAc in hexane (v/v) in 2% increments) to yield a clear oil (226 mg, 83%).
1H-NMR (CDCl3, 400 MHz): δ = 8.13 (t, 1 H, J = 1.5, e), 7.68 (d, 2 H, J = 1.5, f), 7.35 (d, 2 H, a), 6.32 - 6.29 (m, 4 H, b, c), 4.36 (s, 4 H, d), 4.21 (t, 2 H, J = 4.8, g), 3.88 (t, 2 H, J = 4.8, h), 3.74 - 3.56 (m, 8 H, i), 3.44 (t, 2 H, j), 1.56 (m, 2 H, k), 1.31 - 1.25 (m, 18 H, l), 0.88 (t, 3 H, m). 13C-NMR (CDCl3, 400 MHz): δ = 189.7, 159.3, 150.0, 142.4, 138.3, 118.7, 117.8, 110.7, 108.3, 71.6, 71.0, 70.7, 70.7, 70.0, 69.5, 68.2, 31.9, 29.7, 29.6, 29.5, 29.3, 26.1, 22.7, 14.1. Exact mass calculated: 675.3025, found: 675.3043 [M+H+].
Dimethyl 5-(dimethylcarbamothioyloxy)isophthalate 23
Dimethyl-5-hydroxyisophthalic acid (9.99 mmol, 2.10 g) was dissolved in anhydrous DMF (10 mL) and cooled to 4°C. DABCO (19.97 mmol, 2.24 g) was added in portions, followed by dropwise addition of a solution of dimethylthiocarbamoyl chloride (19.98 mmol, 2.47 g) in anhydrous DMF (10 mL). The resulting suspension was allowed to warm up to room temperature slowly. After 24 hours the reaction mixture was poured into water (100 mL) and the precipitate was filtered off and washed with water. Recrystallisation from methanol yielded a white powder (2.32 g, 78%).
1H-NMR (CDCl3, 400 MHz): δ = 8.57 (t, 1 H, J = 1.5, b), 7.92 (d, 2 H, J = 1.5, c), 3.93 (s, 6 H, a), 3.45 (s, 3 H, d), 3.36 (s, 3 H, d). 13C-NMR (CDCl3, 400 MHz): δ = 187.0, 165.4, 153.9, 131.6, 128.4, 128.0, 52.5, 43.4, 38.8. Exact mass calculated: 298.0749, found: 297.0749 [M+H+]. Melting point: 105 - 106°C.
Dimethyl 5-(dimethylcarbamoylthio)isophthalate 24
Dimethyl 5-(dimethylcarbamothioyloxy)isophthalate (23; 2.99 mmol, 890 mg) was heated at 215°C under a N2 atmosphere for 1 hour. The mixture was cooled to about 70°C and ethanol (20 mL) was added. The pale brown crystals that appeared upon cooling down to room temperature were filtered off to give the product (802 mg, 90%).
1H-NMR (CDCl3, 400 MHz): δ = 8.69 (t, 1 H, J = 1.5, b), 8.34 (d, 2 H, J = 1.5, c), 3.94 (s, 6 H, a), 3.10 (s, 3 H, d), 3.04 (s, 3 H, d). 13C-NMR (CDCl3, 400 MHz): δ = 165.5, 140.7, 131.2, 130.5, 52.5, 37.0. Exact mass calculated: 298.0749, found: 297.0746 [M+H+]. Melting point: 122 - 124°C.
5-Mercaptoisophthalic acid 25
A 1.2 M solution of potassium hydroxide in a 1:1 mixture of ethanol and water (20 mL) was degassed by purging with N2 for at least 2 hours. Dimethyl 5-(dimethylcarbamoyl-thio)isophthalate (24; 2.02 mmol, 0.60 g) was added and the mixture was stirred at 80°C for 30 minutes under nitrogen, and then for another 1.5 hours, allowing it to cool down to room temperature slowly. Addition of concentrated HCl (10 mL) led to the precipitation of the product which was filtered off, washed with dilute HCl (0.3%) and dried under vacuum (yield: 0.33 g, 83%).
1H-NMR (CD3OD, 400 MHz): δ = 8.36 (t, 1 H, J = 1.5, a), 8.11 (d, 2 H, J = 1.5, b). 13C-NMR (CD3OD, 400 MHz): δ = 168.2, 135.4, 134.5, 133.3, 128.4. Exact mass calculated: 196.9911, found: 196.9914[M-H+]. Melting point: 280 - 282°C.
S,S-(5-Mercaptoisophthalic acid) benzene-1,3-bis(carbothioate) 28
5-Mercaptoisophthalic acid (25; 2.02 mmol, 0.40 g) and triethylamine (11.97 mmol, 1.66 mL) were dissolved in a 1:1 mixture of water and acetonitrile (50 mL). Isophthaloyl dichloride (0.99 mmol, 0.20 g) was added and the solution was stirred at room temperature for 4 hours. The mixture was extracted with hexane three times and after evaporation of the solvent, the residue was redissolved in water. Fractional precipitation by careful addition of 3 N HCl gave a white powder which was recrystallised from methanol to yield the product (47 mg, 9%).
1H-NMR (DMSO-d 6 , 400 MHz): δ = 8.55 (t, 2 H, J = 1.6, a), 8.46 (t, 1 H, J = 1.8, c), 8.34 dd, 2 H, J = 7.8, J = 1.8, d), 8.28 (m, 4 H, b), 7.75 (t, 1 H, J = 7.8, e). 13C-NMR (DMSO-d 6 , 400 MHz): δ = 187.9, 165.7, 139.2, 136.3, 132.6, 131.2, 131.1, 130.7, 130.0, 127.9, 127.7, 125.4. Exact mass calculated: 548.9926, found: 548.9929[M+Na+]. Melting point: 263 - 265°C.
Methyl 4-mercaptobenzoate 26
4-Mercaptobenzoic acid (8.56 mmol, 1.32 g) was dissolved in methanol (40 mL). After addition of concentrated sulfuric acid (1 mL), the solution was refluxed for 24 hours. The solvent was evaporated and the residue was purified using dry column vacuum chromatography (0-25% EtOAc in hexane (v/v) containing 0.1% formic acid, in 2% increments) to yield a white solid (1.43 g, 99%).
1H-NMR (CDCl3, 400 MHz): δ = 7.89 (dt, 2 H, J = 8.5, J = 2.0, b), 7.29 (dt, 2 H, J = 8.5, J = 2.0, c), 3.90 (s, 3 H, a), 3.60 (s, 1 H, d). 13C-NMR (CDCl3, 400 MHz): δ = 166.6, 138.3, 130.2, 128.1, 127.2, 52.1. Exact mass calculated: 191.0140, found: 191.0137 [M+Na+]. Melting point: 57 - 58°C.
S,S-Bis(methyl-4 benzoate) 5-(2-(2-(2-(dodecyloxy)ethoxy)ethoxy)ethoxy)benzene-1,3-bis(carbothioate) 30
5-(2-(2-(2-(Dodecyloxy)ethoxy)ethoxy)ethoxy)isophthalic acid (22, 0.40 mmol, 193 mg), methyl 4-mercaptobenzoate (26; 1.00 mmol, 168 mg) and DMAP (0.10 mmol, 12 mg) were dissolved in anhydrous CH2Cl2 (10 mL) and cooled to 4°C on an ice bath. Dicyclohexylcarbodiimide (1.00 mmol, 206 mg) was added and the solution was stirred overnight, allowing it to warm up to room temperature slowly. The mixture was extracted with 3 N HCl and brine, and the organic layer was dried over sodium sulfate and filtered. After solvent evaporation the residue was purified using dry column vacuum chromatography (0-25% EtOAc in hexane (v/v) in 2% increments) to yield a clear oil (180 mg, 35%).
1H-NMR (CDCl3, 400 MHz): δ = 8.26 (t, 1 H, J = 1.4, d), 8.13 (dt, 2 H, J = 8.5, J = 1.9, b), 7.77 (d, 2 H, J = 1.5, e), 7.62 (dt, 2 H, J = 8.5, J = 1.9, c), 4.25 (t, 2 H, J = 4.6, f), 3.95 (s, 6 H, a), 3.92 (t, 2 H, J = 4.6, g), 3.76 - 3.56 (m, 8 H, h), 3.44 (t, 2 H, i), 1.60 - 1.55 (m, 2 H, j), 1.32 - 1.24 (m, 18 H, k), 0.87 (t, 3 H, l). 13C-NMR (CDCl3, 400 MHz): δ = 188.0, 166.3, 159.6, 138.2, 134.6, 132.5, 131.2, 130.2, 118.9, 118.3, 71.5, 70.9, 70.7, 70.7, 70.0, 69.5, 68.3, 52.3, 31.9, 29.6, 29.5, 29.3, 26.1, 22.7, 14.1. Exact mass calculated: 783.3237, found: 783.3263 [M+H+].
Preparation of vesicles
A solution of 25 mg egg PC in chloroform (1 mL) was placed in a pyrex glass test tube and, where appropriate, a stock solution of one of the amphiphilic thioester building blocks in chloroform was added to give the desired ratio of lipid to thioester. The chloroform was evaporated under a stream of nitrogen to leave a lipid film on the wall of the test tube, which was dried under vacuum over night. Then, 100 mM phosphate buffer (1.65 mL) was added to give a concentration of 20 mM egg PC and the solution was vortexed for 30 seconds or until the lipid film was completely dispersed. The mixture was allowed to rest, vortexed again for 30 seconds and allowed to rest for 20 minutes. Five freeze-thaw cycles were followed by extrusion through polycarbonate membranes, ten times through 400 nm pore size and ten times through 200 nm pore size. The resulting vesicle solution was allowed to rest for one hour before libraries were set up.
Preparation of DCLs
DCLs in bulk aqueous solution were prepared by dissolving building blocks in 100 mM phosphate buffer to the appropriate concentrations (library volume: 0.5 mL) and adjusting the pH to 8 with 1 M KOH. These stock solutions were mixed to give the final concentrations given in the text.
The libraries were then stirred at 40°C for 5 hours, unless otherwise stated, and analysed by HPLC and LC-MS.
DCLs at the bilayer interface were prepared by dissolving thiol building blocks in 100 mM phosphate buffer to the appropriate concentrations and adjusting the pH with 1 M KOH. These stock solutions were then mixed with the freshly prepared vesicle solutions. In some cases, when the thiols to be used were not soluble in water, a highly concentrated stock solution in 2-propanol was prepared and a few microlitres of this were injected into the vesicle solution to give the appropriate final concentration. Libraries were equilibrated at 40°C for 24 hours and analysed by HPLC and LC-MS.
HPLC analysis was performed on an Agilent HP 1100 system fitted with an online degasser, quaternary pump, autosampler, heated column compartment and diode array detector set to a wavelength of 260 nm.
HPLC gradient A
Acetonitrile (0.1% FA)
Water (0.1% FA)
HPLC gradient B
Acetonitrile (0.1% FA)
Water (0.1% FA)
HPLC gradient C
Acetonitrile (0.1% FA)
Water (0.1% FA)
LC-MS analysis was performed using an Agilent HP 1100 system coupled to an Agilent XCT iontrap MSD mass spectrometer. For DCLs in bulk water an injection volume of 2 μL was used, while for DCLs at the bilayer interface an injection volume of 0.5 μL was used. Mass spectra were acquired in standard-enhanced scan mode using a drying temperature of 335°C, a nebuliser pressure of 20.0 psi, drying gas flow of 6.0 L/min and an ICC target of 50,000 ions. The instrument was tuned for the target mass of 1200. Agilent Chemstation software (Rev A.10.02) and Bruker Daltonik LC/MSD Trap software 5.2 (Build 374) was used to operate the LC-MS and analyse the data produced.
We are grateful for financial support from the EPSRC and the COST CM0703 action on Systems Chemistry. We thank Dr. Ana Belenguer for help with the HPLC and LC-MS analyses.
- Corbett PT, Leclaire J, Vial L, West KR, Wietor J-L, Sanders JKM, Otto S: Dynamic combinatorial chemistry. Chem Rev 2006, 106: 3652–3711. 10.1021/cr020452pView ArticleGoogle Scholar
- Lehn JM: From supramolecular chemistry towards constitutional dynamic chemistry and adaptive chemistry. Chem Soc Rev 2007, 36: 151–160. 10.1039/b616752gView ArticleGoogle Scholar
- Ladame S: Dynamic combinatorial chemistry: on the road to fulfilling the promise. Org Biomol Chem 2008, 6: 219–226. 10.1039/b714599cView ArticleGoogle Scholar
- Stankiewicz J, Eckardt LH: Chembiogenesis 2005 and systems chemistry workshop. Angew Chem Int Ed 2006, 45: 342–344. 10.1002/anie.200504139View ArticleGoogle Scholar
- Whitesides GM, Ismagilov RF: Complexity in chemistry. Science 1999, 284: 89–92. 10.1126/science.284.5411.89View ArticleGoogle Scholar
- Newth D, Finnigan J: Emergence and self-organization in chemistry and biology. Aust J Chem 2006, 59: 841–848. 10.1071/CH06292View ArticleGoogle Scholar
- Ludlow RF, Otto S: Systems chemistry. Chem Soc Rev 2008, 37: 101–108. 10.1039/b611921mView ArticleGoogle Scholar
- Peyralans JJP, Otto S: Recent highlights in systems chemistry. Curr Opin Chem Biol 2009, 13: 705–713. 10.1016/j.cbpa.2009.08.006View ArticleGoogle Scholar
- Nitschke JR: Systems chemistry: Molecular networks come of age. Nature 2009, 462: 736–738. 10.1038/462736aView ArticleGoogle Scholar
- Gibb BC: Teetering towards chaos and complexity. Nat Chem 2009, 1: 17–18. 10.1038/nchem.148View ArticleGoogle Scholar
- Balazs AC, Epstein IR: Emergent or just complex? Science 2009, 325: 1632–1634. 10.1126/science.1178323View ArticleGoogle Scholar
- Au-Yeung HY, Pengo P, Pantos GD, Otto S, Sanders JKM: Templated amplification of a naphthalenediimide-based receptor from a donor-acceptor dynamic combinatorial library in water. Chem Commun 2009, 419–421. 10.1039/b816979aGoogle Scholar
- Lippert AR, Keleshian VL, Bode JW: Dynamic supramolecular complexation by shapeshifting organic molecules. Org Biomol Chem 2009, 7: 1529–1532. 10.1039/b822585kView ArticleGoogle Scholar
- Perez-Fernandez R, Pittelkow M, Belenguer AM, Lane LA, Robinson CV, Sanders JKM: Two-phase dynamic combinatorial discovery of a spermine transporter. Chem Commun 2009, 3708–3710. 10.1039/b902842kGoogle Scholar
- Saggiomo V, Lüning U: Transport of calcium ions through a bulk membrane by use of a dynamic combinatorial library. Chem Commun 2009, 3711–3713. 10.1039/b902847aGoogle Scholar
- Sun JY, Patrick BO, Sherman JC: A new carceplex, and a crystal structure and dynamic combinatorial chemistry of a carceplex. Tetrahedron 2009, 65: 7296–7302. 10.1016/j.tet.2008.11.110View ArticleGoogle Scholar
- Ulrich S, Lehn JM: Adaptation and optical signal generation in a constitutional dynamic network. Chem Eur J 2009, 15: 5640–5645. 10.1002/chem.200900238View ArticleGoogle Scholar
- Ulrich S, Lehn JM: Adaptation to shape switching by component selection in a constitutional dynamic system. J Am Chem Soc 2009, 131: 5546–5559. 10.1021/ja809828gView ArticleGoogle Scholar
- Xu XN, Wang L, Wang GT, Lin JB, Li GY, Jiang XK, Li ZT: Hydrogen-bonding-mediated dynamic covalent synthesis of macrocycles and capsules: new receptors for aliphatic ammonium ions and the formation of pseudorotaxanes. Chem Eur J 2009, 15: 5763–5774. 10.1002/chem.200900309View ArticleGoogle Scholar
- Leclaire J, Husson G, Devaux N, Delorme V, Charles L, Ziarelli F, Desbois P, Chaumonnot A, Jacquin M, Fotiadu F, Buono G: CO 2 binding by dynamic combinatorial chemistry: an environmental selection. J Am Chem Soc 2010, 132: 3582–3593. 10.1021/ja909975qView ArticleGoogle Scholar
- Saggiomo V, Goeschen C, Herges R, Quesada R, Lüning U: Ion transport across membranes facilitated by a dynamic combinatorial library. Eur J Org Chem 2010, 2337–2343.Google Scholar
- Nasr G, Petit E, Vullo D, Winum JY, Supuran CT, Barboiu M: Carbonic anhydrase-encoded dynamic constitutional libraries: toward the discovery of isozyme-specific inhibitors. J Med Chem 2009, 52: 4853–4859. 10.1021/jm900449vView ArticleGoogle Scholar
- Nasr G, Petit E, Supuran CT, Winum JY, Barboiu M: Carbonic anhydrase II-induced selection of inhibitors from a dynamic combinatorial library of Schiff's bases. Bioorg Med Chem Lett 2009, 19: 6014–6017. 10.1016/j.bmcl.2009.09.047View ArticleGoogle Scholar
- Scott DE, Dawes GJ, Ando M, Abell C, Ciulli A: A fragment-based approach to probing adenosine recognition sites by using dynamic combinatorial chemistry. ChemBioChem 2009, 10: 2772–2779. 10.1002/cbic.200900537View ArticleGoogle Scholar
- Vongvilai P, Ramström O: Dynamic asymmetric multicomponent resolution: lipase-mediated amidation of a double dynamic covalent system. J Am Chem Soc 2009, 131: 14419–14425. 10.1021/ja9052015View ArticleGoogle Scholar
- Brisig B, Sanders JKM, Otto S: Selection and amplification of a catalyst from a dynamic combinatorial library. Angew Chem Int Ed 2003, 42: 1270–1273. 10.1002/anie.200390326View ArticleGoogle Scholar
- Vial L, Sanders JKM, Otto S: A catalyst for an acetal hydrolysis reaction from a dynamic combinatorial library. New J Chem 2005, 29: 1001–1003. 10.1039/b505316aView ArticleGoogle Scholar
- Sadownik JW, Philp D: A simple synthetic replicator amplifies itself from a dynamic reagent pool. Angew Chem Int Ed 2008, 47: 9965–9970. 10.1002/anie.200804223View ArticleGoogle Scholar
- Das AK, Hirst AR, Ulijn RV: Evolving nanomaterials using enzyme-driven dynamic peptide libraries (eDPL). Faraday Discuss 2009, 143: 293–303. 10.1039/b902065aView ArticleGoogle Scholar
- Hunt RAR, Ludlow RF, Otto S: Estimating equilibrium constants for aggregation from the product distribution of a dynamic combinatorial library. Org Lett 2009, 11: 5110–5113. 10.1021/ol901656xView ArticleGoogle Scholar
- Minkenberg CB, Florusse L, Eelkema R, Koper GJM, van Esch JH: Triggered self-assembly of simple dynamic covalent surfactants. J Am Chem Soc 2009, 131: 11274–11275. 10.1021/ja902808qView ArticleGoogle Scholar
- Nguyen R, Allouche L, Buhler E, Giuseppone N: Dynamic combinatorial evolution within self-replicating supramolecular assemblies. Angew Chem Int Ed 2009, 48: 1093–1096. 10.1002/anie.200804602View ArticleGoogle Scholar
- Ura Y, Beierle JM, Leman LJ, Orgel LE, Ghadiri MR: Self-assembling sequence-adaptive peptide nucleic acids. Science 2009, 325: 73–77. 10.1126/science.1174577View ArticleGoogle Scholar
- Wang GT, Lin JB, Jiang XK, Li ZT: Cholesterol-appended aromatic imine organogelators: a case study of gelation-driven component selection. Langmuir 2009, 25: 8414–8418. 10.1021/la804188zView ArticleGoogle Scholar
- Williams RJ, Smith AM, Collins R, Hodson N, Das AK, Ulijn RV: Enzyme-assisted self-assembly under thermodynamic control. Nature Nanotech 2009, 4: 19–24. 10.1038/nnano.2008.378View ArticleGoogle Scholar
- Otto S: Molecular self-assembly. Helping themselves. Nature Nanotech 2009, 4: 13–14. 10.1038/nnano.2008.387View ArticleGoogle Scholar
- Orrillo AG, Furlan RLE: Supramolecular interactions between library members modulate the behavior of dynamic combinatorial libraries. J Org Chem 2010, 75: 211–214. 10.1021/jo902296hView ArticleGoogle Scholar
- Carnall JMA, Waudby CA, Belenguer AM, Stuart MCA, Peyralans JJP, Otto S: Mechanosensitive self-replication driven by self-organization. Science 2010, 327: 1502–1506. 10.1126/science.1182767View ArticleGoogle Scholar
- Au-Yeung HY, Pantos GD, Sanders JKM: Amplifying different catenanes in an aqueous donor-acceptor dynamic combinatorial library. J Am Chem Soc 2009, 131: 16030–16032. 10.1021/ja906634hView ArticleGoogle Scholar
- Chung MK, White PS, Lee SJ, Gagne MR: Synthesis of interlocked 56-membered rings by dynamic self-templating. Angew Chem Int Ed 2009, 48: 8683–8686. 10.1002/anie.200903478View ArticleGoogle Scholar
- Au-Yeung HY, Pantos GD, Sanders JKM: Dynamic combinatorial synthesis of a catenane based on donor-acceptor interactions in water. Proc Natl Acad Sci USA 2009, 106: 10466–10470. 10.1073/pnas.0809934106View ArticleGoogle Scholar
- Price JL, Hadley EB, Steinkruger JD, Gellman SH: Detection and analysis of chimeric tertiary structures by backbone thioester exchange: packing of an alpha helix against an alpha/beta-peptide helix. Angew Chem Int Ed 2010, 49: 368–371.View ArticleGoogle Scholar
- Bromley EHC, Sessions RB, Thomson AR, Woolfson DN: Designed alpha-helical tectons for constructing multicomponent synthetic biological systems. J Am Chem Soc 2009, 131: 928–930. 10.1021/ja804231aView ArticleGoogle Scholar
- Pomerantz WC, Hadley EB, Fry CG, Gellman SH: In situ monitoring of backbone thioester exchange by F-19 NMR. ChemBioChem 2009, 10: 2177–2181. 10.1002/cbic.200900380View ArticleGoogle Scholar
- Choudhary S, Morrow JR: Dynamic acylhydrazone metal ion complex libraries: A mixed-ligand approach to increased selectivity in extraction. Angew Chem Int Ed 2002, 41: 4096–4098. Publisher Full Text 10.1002/1521-3773(20021104)41:21<4096::AID-ANIE4096>3.0.CO;2-0View ArticleGoogle Scholar
- Ariga K, Kunitake T: Molecular recognition at air-water and related interfaces: Complementary hydrogen bonding and multisite interaction. Acc Chem Res 1998, 31: 371–378. 10.1021/ar970014iView ArticleGoogle Scholar
- Kikuchi JI, Ogata T, Inada M, Murakami Y: Circular dichroism of an aromatic guest induced by a chiral steroid cyclophane in aqueous solution and synthetic bilayer membrane. Chem Lett 1996, 771–772. 10.1246/cl.1996.771Google Scholar
- Onda M, Yoshihara K, Koyano H, Ariga K, Kunitake T: Molecular recognition of nucleotides by the guanidinium unit at the surface of aqueous micelles and bilayers. A comparison of microscopic and macroscopic interfaces. J Am Chem Soc 1996, 118: 8524–8530. 10.1021/ja960991+View ArticleGoogle Scholar
- Larsson R, Pei ZC, Ramström O: Catalytic self-screening of cholinesterase substrates from a dynamic combinatorial thioester library. Angew Chem Int Ed 2004, 43: 3716–3718. 10.1002/anie.200454165View ArticleGoogle Scholar
- Woll MG, Gellman SH: Backbone thioester exchange: a new approach to evaluating higher order structural stability in polypeptides. J Am Chem Soc 2004, 126: 11172–11173. 10.1021/ja046891iView ArticleGoogle Scholar
- Larsson R, Ramström O: Dynamic combinatorial thiolester libraries for efficient catalytic self-screening of hydrolase substrates. Eur J Org Chem 2005, 285–291.Google Scholar
- Hadley EB, Gellman SH: An antiparallel alpha-helical coiled-coil model system for rapid assessment of side-chain recognition at the hydrophobic interface. J Am Chem Soc 2006, 128: 16444–16445. 10.1021/ja067178rView ArticleGoogle Scholar
- Woll MG, Hadley EB, Mecozzi S, Gellman SH: Stabilizing and destabilizing effects of phenylalanine -> F-5-phenylalanine mutations on the folding of a small protein. J Am Chem Soc 2006, 128: 15932–15933. 10.1021/ja0634573View ArticleGoogle Scholar
- Hadley EB, Witek AM, Freire F, Peoples AJ, Gellman SH: Thermodynamic analysis of beta-sheet secondary structure by backbone thioester exchange. Angew Chem Int Ed 2007, 46: 7056–7059. 10.1002/anie.200702449View ArticleGoogle Scholar
- Lam RTS, Belenguer A, Roberts SL, Naumann C, Jarrosson T, Otto S, Sanders JKM: Amplification of acetylcholine-binding catenanes from dynamic combinatorial libraries. Science 2005, 308: 667–669. 10.1126/science.1109999View ArticleGoogle Scholar
- Podhradsky D, Drobnica L, Kristian P: Reactions of cysteine, its derivatives, glutathione, coenzyme A, and dihydrolipoic acid with isothiocyanates. Cell Mol Life Sci 1979, 35: 154–155. 10.1007/BF01920581View ArticleGoogle Scholar
- Danehy JP, Noel CJ: The relative nucleophilic character of several mercaptans toward ethylene oxide. J Am Chem Soc 1960, 82: 2511–2515. 10.1021/ja01495a028View ArticleGoogle Scholar
- Kuzmic P: Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase. Anal Biochem 1996, 237: 260–273. 10.1006/abio.1996.0238View ArticleGoogle Scholar
- Connors KA, Bender ML: Kinetics of alkaline hydrolysis and n-butylaminolysis of ethyl p-nitrobenzoate and ethyl p-nitrothiolbenzoate. J Org Chem 1961, 26: 2498–2504. 10.1021/jo01351a086View ArticleGoogle Scholar
- Brown RS, Aman A: Intramolecular catalysis of thiol ester hydrolysis by a tertiary amine and a carboxylate. J Org Chem 1997, 62: 4816–4820. 10.1021/jo9623954View ArticleGoogle Scholar
- Hupe DJ, Jencks WP: Nonlinear structure-reactivity correlations - acyl transfer between sulfur and oxygen nucleophiles. J Am Chem Soc 1977, 99: 451–464. 10.1021/ja00444a023View ArticleGoogle Scholar
- Pohl ER, Wu D, Hupe DJ: Effect of solvation on beta-values for formyl, acetyl, and pivaloyl transfer between sulfur and oxygen nucleophiles. J Am Chem Soc 1980, 102: 2759–2763. 10.1021/ja00528a040View ArticleGoogle Scholar
- Epand RM, Kraayenhof R: Fluorescent probes used to monitor membrane interfacial polarity. Chem Phys Lipids 1999, 101: 57–64. 10.1016/S0009-3084(99)00055-9View ArticleGoogle Scholar
- Mazeres S, Schram V, Tocanne JF, Lopez A: 7-Nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior. Biophys J 1996, 71: 327–335. 10.1016/S0006-3495(96)79228-7View ArticleGoogle Scholar
- Doyle EL, Hunter CA, Phillips HC, Webb SJ, Williams NH: Cooperative binding at lipid bilayer membrane surfaces. J Am Chem Soc 2003, 125: 4593–4599. 10.1021/ja021048aView ArticleGoogle Scholar
- Au-Yeung HY, Cougnon FBL, Otto S, Pantos GD, Sanders JKM: Exploiting donor-acceptor interactions in aqueous dynamic combinatorial libraries: exploratory studies of simple systems. Chem Sci 2010., 1:Google Scholar
- Corbett PT, Sanders JKM, Otto S: Exploring the relation between amplification and binding in dynamic combinatorial libraries of macrocyclic synthetic receptors in water. Chem Eur J 2008, 14: 2153–2166. 10.1002/chem.200701413View ArticleGoogle Scholar
- Tecilla P, Jubian V, Hamilton AD: Synthetic hydrogen bonding receptors as models of transacylase anezymes. Tetrahedron 1995, 51: 435–448. 10.1016/0040-4020(94)00907-CView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.