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Chemical Synthesis

Aldolase Antibody


"murine catalytic monoclonal antibody"

Aldrich Catalog Nos. 479950 and 481572
Request Technical Bulletin AL-207 for experimental details.

Introduction

Reactive immunization is a powerful method for the generation of efficient biocatalysts. An antibody may be tailor-made and optimized by the immune system to covalently bind to the hapten. The binding pocket is not necessarily refined for noncovalent interactions with the immunogen, allowing for the production of catalysts that are broad in scope. Antibody 38C2 was generated by using the strategy of reactive immunization, which involves a hapten with a moderately reactive functional group which induces a chemical reaction with a functionalized amino acid residue in the active site of the antibody.

Professors Lerner, Barbas and their coworkers have demonstrated that Ab 38C2 catalyzes a wide variety of crossed and self aldol reactions, including intramolecular aldol reactions. Their groups have found that Ab 38C2 catalyzes reactions involving at least 100 different substrate combinations with remarkably high rate enhancements. The biocatalyst can accept a variety of substrates with different physicochemical properties.

Mechanism

Antibody 38C2 uses an enamine mechanism known from natural class I aldolase enzymes. Natural aldolases tolerate only minor changes in the structure of the donor substrate. Ab 38C2 accepts a wide variety of ketones as substrates for aldol reactions with aldehydes. The amino group of a lysine residue in the binding pocket of the antibody reacts with the ketone substrate to form an enamine. This is followed by nucleophilic attack of the enamine on the aldehyde substrate and final hydrolytic release of the aldol product. Typical values for the Michaelis-Menten constants KM of the donors range from 1 mM to 1 M, reflecting the ability of the antibody to accept a wide variety of different ketones.

Antibody Stability

Stable at room temperature for up to 3 weeks when dissolved in different buffer solutions (pH 5.5 to 8.5) and even pure water. No detectable activity loss was found if the antibody was stored in stock solutions of 10 to 20mg/mL in PBS (phosphate buffer saline) at -78°C (stable for at least 1 year).

Scope and Applications

Antibody 38C2 catalyzes the following reactions:

  • Aldol addition and in some cases the condensation reaction
  • Crossed aldol
  • Retro aldol
  • Decarboxylation of ß-keto acids
  • Self aldol
  • Robinson annulation
  • Kinetic resolutions

An excellent teaching tool in the laboratory for:

  • HPLC characterization of the antibody catalyzed reaction
  • Titration of antibody or enzyme active sites
  • Analysis of the kinetics of the antibody or enzyme catalyzed reaction
  • Woodward UV rules
  • pKa
  • Enzyme mechanisms

Antibody QC Assay

The aldolase antibody, 38C2, can be checked for concentration and activity by incubating with acetylacetone and monitoring the formation of the stable enaminone using UV spectroscopy (lambda(max) = 318 nm, epsilon ~ 15,000; epsilon = extinction coefficient). Upon resuspension of antibody lyophilized with buffer salts, some undissolved particulate solids may be observed. These do not affect the catalysis of the antibody.

Experimental: 75 µL of a 10mg/mL solution of antibody are dissolved into 425 µL PBS (phosphate buffer saline, pH = 7.4). 100 µL of this antibody solution is placed into a cuvette and the A230 recorded for calculation of antibody concentration. Next, 10 µL of a 1 mM solution of the acetylacetone in PBS are added to the solution in the cuvette. UV scans are taken at 1, 5, and 10 minutes. The A315 of the 10 minute scan is recorded and used for calculation of activity.

Calculations:

a) Determination of antibody concentration: The OD (optical density) value at 280 nm is divided by the extinction coefficient of antibody (1.35) to give the mg/mL of antibody in the diluted sample. Dividing this number by the F.W. of the protein (F.W. ca. 150,000) gives µM of antibody in the sample. Back calculation will then give concentration of stock solution.

b) Determination of antibody activity: The OD value at 315 nm is divided by the extinction coefficient of the enaminone (15,000) to give µM of antibody binding sites. This value should be 2x the determined antibody concentration.

Instructions

Each vial contains 10mg of antibody as a lyophilized powder (F.W. ca. 150,000) plus 5-6.5 mg of phosphate buffer salts*. Prepare a stock solution by adding 1 mL of purified** water. Do not shake the stock solution - this can denature the protein. Other possible co-solvents (up to 5%) are acetonitrile and DMF, though the ketone donor may be used up to 10% v/v and will serve as cosolvent in most cases. No detectable activity loss was found if the antibody was stored in stock solutions of 10 to 20mg/mL in PBS at -78oC for up to one year. Syringes and reaction vessels do not require sterilization.

*Also available without PBS buffer.

**Ultrafiltered and autoclaved water (Catalog #95289) - the antibody does not contain any preservatives.

Typical experimental

Analytical Scale

To a 6.25 mM solution of the aldehyde in 160 µL of PBS were added 10 µL of acetone and 30 µL of a 270 µM solution of antibody (regarding binding sites) in PBS. The final concentrations were 5 mM of aldehyde and 40 µM of antibody in a total volume of 200 µL containing 5% (v/v) of acetone. After 18h, 12mL of CH2Cl2 were added, the organic extract was dried (MgSO4)and evaporated. The residue was redissolved in ca. 1 mL of i-PrOH and the ee was determined by normal phase HPLC using an appropriate chiral column for enantiomer separation.

Preparative Scale

Example 1

Cyclopentanone (1 mol, 88 mL) was dissolved in 912 mL of MOPS buffer (100 mM, pH = 7.4). Next, Ab 38C2 (0.67 mmol, 0.1 ga) was added and the first addition of 4-(2-methylpropionamido)benzaldehyde (1 mmol, 0.2 g) was made. The reaction was allowed to stir for 48 h followed by the next addition of aldehyde (1 mmol, 0.2 g) was added. After another 48 h, the third and fourth additions of aldehyde (2.35 mmol, 0.45 g) were made for a total of 0.85 g in the reaction. The reaction progress was monitored by high pressure liquid chromatography [Hitachi HPLC system (pump L-7100, UV detector L-7400 and integrator D-7500)] using a Rainin column (Microsorb-MV, C18, 300 Å, 5mm; 250 x 4.6 mm) and acetonitrile/water mixtureb (containing 0.1 % trifluoroacetic acid) with eluents at a flow rate 1.0 mL/min. When equilibrium was reached (ca. 21 days) the reaction mixture was saturated with NaCl and divided into two 500 mL portions for workup. Each portion was extracted with 3 x 250 mL ethyl acetate, dried (MgSO4), rotovapped to yield 1.46 g of crude product. Purification by FC (60:40, ethyl acetate/hexane) gave 0.9 g (yield = 72%) of pure product, de = 95%.

a 5 mL of a 20 mg/mL solution

b 20% CH3CN / 80% water

Example 2

To a solution of 4-nitrocinnamaldehyde (110 mg, 0.61 mmol) in 15 mL of DMF and 31 mL of acetone, PBS buffer (571 mL, degassed and kept under argon) was added slowly. Antibody 38C2 (8.0 mL of a 240 µM solution regarding binding sites) was added. The final concentrations of 4-nitrocinnamaldehyde and Ab38C2 were 1.0 mM and 3.0 µM, respectively in a total volume of 625 mL containing 5% (v/v) of acetone. The reaction mixture was kept in a dark place at room temperature for seven (7) days under argon. The reaction mixture was saturated with sodium chloride and extracted with 3 x 150 mL of ethyl acetate. The extracts were dried over MgSO4 and rotavapped to yield 140 mg of crude product. Purification by FC (1:2, ethyl acetate/hexane) gave 96 mg (yield = 67%) of pure aldol product, ee = 91 %.

Kinetic Resolution

In a typical procedure a 5 µL of 100 mM stock solution of aldol in acetonitrile was added to 95 µL of a 40 µM solution of antibody in 50 µM PBS. After 50% conversion (time varied depended on the catalytic rate of the antibody corresponding to the specific aldol) the remaining aldol was purified on analytical reverse phase HPLC column, the solvent was removed by freeze-dryer affording enantiomerically pure aldol. The product was redissolved in ca. 100 µL of i-PrOH and the ee was determined by normal phase HPLC using an appropriate chiral column for enantiomer separation.

Packaging

Lyophilized antibody 38C2 is packaged in sterilized (autoclaved) clear low extractable borosilicate glass vials with gray butyl 2 leg lyophilization stoppers and open top aluminum crimped seals.

Handling Precautions

Please refer to the Safety Data Sheet. This product is not for use in humans. This antibody is believed to be non-hazardous and non-toxic.

References

  • Wagner, J.; Lerner, R.A.; Barbas, C.F., III Science 1995, 270, 1797.
  • Wirshing, P.; Ashley, J.A.; Lo, C.-H.L.; Janda, K.D.; Lerner, R.A. Science 1995, 270, 1775.
  • Björnestedt, R.; Zhong, G.; Lerner, R.A.; Barbas, C.F., III J. Am. Chem. Soc. 1996, 118, 11720.
  • Zhong, G.; Hoffmann, T.; Lerner, R.A.; Danishefsky, S.J.; Barbas, C.F., III J. Am. Chem. Soc. 1997, 119, 8131.
  • Shulman, A.; Shabat, D.; Barbas, C.F., III; Keinan, F. J. Chem.Ed. 1997, submitted for publication.
  • "Coming soon: Off-the-shelf catalytic antibody" Chemical and Engineering News 1997, September 1, p.10.
  • Barbas, C.F., III. et al. Science 1997, 278, 2085.