Step-by-Step Guide for KitAlysis™ High-Throughput Medium (5,6,7) Ring Closing Metathesis Reaction Screening Kit

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Medium (5,6,7) Ring-Closing Metathesis Procedure

6 pre-weighed catalysts (x4)

4 degassed solvents: Chlorobenzene (ClPh), Toluene (PhMe), Isopropyl Acetate (i-PrOAc), and Methyl-THF (Me-THF)

1 (optional) acid additive (degassed neat TFA)

Materials Required – Set Up

  • 1 mylar (foil) bag from the KitAlysis Grubbs Metathesis Reaction Screening Kit and you will use the following components:
    • 6 x 4 pre-weighed catalysts in glass vials loaded with stir bars and topped with cap mat
    • Four empty 4 mL substrate vials
  • 1 NEW KitAlysis 24-Well Reaction Block Replacement Film
  • 1 (2mL) ampule each of PhCl, PhMe, i-PrOAc, Me-THF, and TFA (optional) from provided ampule boxes
  • Four NEW stir bars
  • KitAlysis 24-Well Reaction Block (sold separately)
  • KitAlysis Benchtop Inertion Box (sold separately) or glove box/glove bag
  • KitAlysis Torque Screwdriver Set (sold separately)

Additional (not included) items needed:

  • Pipette (0-100 µL) & tips
  • 4 (1 mL) syringes with long needles
  • Your olefin metathesis substrate
  • Nitrogen (or Argon): from hood line or tank
  • 1 hot plate with stir capabilities
  • 1 stir plate (or hot plate to be used without heat)
  • HPLC vials, 96-well HPLC auto sampler block, or TLC plates.

Solutions to make: Click here to download the excel file to enter in molecular weights for recipe

Set Up Procedure

(See KitAlysis video for useful visuals)

Note on Acid (TFA) additive: Follow the instructions as outlined below, adding the acid additive (TFA) as the last step of the procedure directly to each individual reaction vial. DO NOT ADD TFA DIRECTLY TO THE SUBSTATE STOCK SOLUTIONS as this may cause your substrates to crash out of solution. It is advised to run a second screen in parallel without acid in order to compare the results.

When to use TFA: See the “TFA Additive Guidelines” below

  • Preheat a hot plate to 60 °C (temperatures range but do not exceed 80 °C).  It is recommended to use an oil bath or second reaction block to hold temperature and avoid spiking. Lower temperatures may be better but 60 °C is a great place to start.
  • Place a NEW KitAlysis 24-Well Reaction Block Replacement Film on reaction block lid and verify all holes, including the temperature probe hole, line up with the corresponding holes on the film.
  • Check all screws to ensure they are not stripped. Replace any stripped screws with provided replacements.
  • Place the KitAlysis Benchtop Inertion Box, with tubing connected to inert gas onto the second, non-heated stir plate (see KitAlysis Benchtop Inertion Box set-up for details)
  • Place KitAlysis 24-Well Reaction Block with lid into KitAlysis Benchtop Inertion Box. Start nitrogen flow and purge 5 minutes. Leave nitrogen flowing for remainder of set up.
  • Prepare empty 4 mL substrate vials to make substrate mixtures. Label as:
    • “ClPh Substrate Mixture A,”
    • “Toluene Substrate Mixture B,”
    • i-PrOAc Substrate Mixture C”
    • “Me-THF Substrate Mixture D.”
  • Add one stir bar to each of the 4 labeled substrate vials.
  • Weigh your olefin metathesis substrate into each of the 4 substrate vials according to the substrate mixture recipe (provided in the downloadable Excel file). If your cross metathesis partners is volatile, add it last (after solvent mixtures have been added) directly to requisite reaction vials via pipette from a separate vial that has been quickly purged and then capped before placing into Inertion Box.
  • Partially open the lid on the KitAlysis Benchtop Inertion Box. Place the “ClPh Substrate Mixture A,” and the “Toluene Substrate Mixture B,” in two of the holes located on the left hand side of the Inertion Box diffuser tray. Place the “i-PrOAc Substrate Mixture C” and “Me-THF Substrate Mixture D.” in two holes on the right hand side of the plate. Ensure that one vial is placed in the center hole on both the left and right hand side of the diffuser tray. This vial placement allows for the best flow of inert gas (remove lids from the solid mixtures before placing them into the recommended holes, keeping the lids in the Inertion Box for later use if needed).
  • Transfer the capped, 24-vial, preloaded catalysts into the reaction block making sure to load it according to the matching diagram on the packaging and the Reaction Block (A1 in upper left ). Leave the vial mat on.
  • Place 1 ampule each of of ClPh, PhMe, i-PrOAc, and Me-THF into ampule holes located along the bottom of Inertion Box, below the Reaction Block.
  • Once all components are in the KitAlysis Benchtop Inertion Box, close the lid and purge for an additional 5 minutes. Leave nitrogen flowing for remainder of set up.
  • Using the ampule cracker, open all ampules of solvent located in the inertion box.
  • Purge needle and syringe in a nitrogen diffuser hole 2x by pulling and then pushing plunger. Using purged needle and syringe, add required solvent amounts to corresponding substrate mixture vials.  
  • Stir mixtures until in solution (1-2 min). For slurries, see “additional tips” below.
  • While mixtures are stirring, carefully remove the cap mat from the 24-vial, preloaded catalysts in the reaction block.  
  • Dose stock solutions, using a new pipette tip for each substrate mixture. You may have a very small amount of excess solution remaining. Save it as a reaction standard for HPLC/TLC later.
    • Dose 100 µL of “ClPh Substrate Mixture A” to vials A1-A6 according to scheme below.
    • Dose 100 µL “Toluene Substrate Mixture B” to vials B1-B6 according to scheme below.
    • Dose 100 µL “i-PrOAc Mixture C” to vials C1-C6 according to scheme below.
    • Dose 100 µL “Me-THF Substrate Mixture D” to vials D1-D6 according to scheme below.

  • If using TFA, remove one empty solvent ampule from the inertion box. Replace it with an ampule of TFA.
  • Using an ampule cracker, open the TFA.
  • Using a pipette, dose 5 µL TFA to each reaction vial. (It helps to say the name of the vial (A1, A2, etc.) as you go along to ensure you stay on track as you dose.)
  • After all substrate mixtures (and acid additive if using)  have been dosed according to recipe, take the KitAlysis 24-Well Reaction Block lid and line up the screws with the holes in the plate. Ensure the temperature probe holes line up on both the lid and the block.
  • Screw on lid according to directions and pattern shown in the “additional tips” section below. Before removing KitAlysis 24-Well Reaction Block from the Inertion Box, ensure that the lid is evenly sealed onto the base. Do this visual check to avoid having to unscrew the lid and screw again.
  • Once completely sealed, remove the KitAlysis 24-Well Reaction Block from the Inertion Box. Place on preheated hot plate with probe through the lid and inserted into the block. Heat at 60 °C (and to no more than 80 °C) overnight stirring at or near 300 rpm.
  • Turn off nitrogen flow to box and dispose of any unused chemicals.
  • At reaction completion, follow the Work-Up Procedure provided below that will quench reactions, make them more suitable for analysis, and add the internal standard.

Quench Solution/Internal Standard Recipe

  • 49 mL CH3CN
  • 1 mL AcOH
  • 15.4 mg Biphenyl (KitAlysis Internal Standard provided in the kit) There is excess in the bottle so be sure to weigh it out.

Note: This recipe makes 50 mL which is enough stock solution for four screens. The amount of internal standard is 10 mol% per reaction (1 µmol) . So a big product peak to small internal standard indicates a good reaction. Integrate to compare reactions against one another (product/internal standard).

Work-up Procedure and Analysis

  • Cool Reaction Block. Remove lid using small, non-torque KitAlysis screwdriver.
  • Check each vial for solvent loss, and record.
  • Aliquot 500 µL of prepared quench solution to each reaction vial, using a pipette for accuracy.
  • Replace reaction block lid, tighten middle screw and stir on stir plate (NO HEAT) for 2-3 minutes. DO NOT INVERT BLOCK.
  • After 2-3 minutes of stirring, let plate rest (without stirring) for 5 minutes to allow insoluble material to settle out of solution to the bottom of the vials.
  • While plate is resting, add 700 µL of HPLC Grade Acetonitrile to each 24 individually labeled (A1, A2 etc,) HPLC vial or to each of 24 wells of a 96-well HPLC/UPLC auto sampler block (use the upper left quadrant as it corresponds to the reaction block vial array). See “additional recommended materials” below for suggestions on the auto sampler block and cap mat.
  • Remove lid on KitAlysis 24-Well Reaction Block carefully.
  • Using a clean pipette each time, remove a 25 µL aliquot from each vial into corresponding HPLC vials or HPLC block. Be careful to pull material from the top of the vials to avoid any precipitate.   
  • Run on HPLC auto sampler. You may need to adjust the amount of acetonitrile from the suggested 700 µL to accommodate your unique HPLC system.
  • See “troubleshooting” below for additional help

Note on catalyst quenching: The internal standard quench should be sufficient in most cases, however an additional catalyst quench may be desired prior to the HPLC analysis step. Choice of quenching agent may depend on substrate compatibility. For options see:

  1. Blacquiere, J. M.; Jurca, T.; Weiss, J.; Fogg, D. E. Adv. Synth. Catal. 2008, 350, 2849 – 2855.
  2. Yee, N. K.; Farina, V.; Houpis, I. N.; Haddad, N.; Frutos, R. P.; Gallou, F.; Wang, X.-j.; Wei, W.; Simpson, R. D.; Feng, X.; Fuchs, V.; Xu, J.; Tan, J.; Zhang, L.; Xu, J.; Smith-Kennan, L. L.; Vitous, J.; Ridges, M. D.; Spinelli, E. M. Johnson, M. J. Org. Chem. 2006, 71, 7133-7145.

TFA Additive Guidelines

Two essential scenarios where an acid additive should be considered:

  1. Strongly coordinating group anywhere in the molecule (primary/secondary amine, thiol, urea, isonitrile etc). Additive should be matched to the pKa or Lewis basicity of the coordinating group (amines – HCl/TFA, ureas – boron or aluminum Lewis acids)

    Example 1:

  1. Weakly coordinating groups that are near the catalyst initiation site (amides, esters, ketones, alcohols that are 5 or 6 bonds away from the olefin).

    Example 2:

Additional Tips

Sealing the Plate-screwing down the cover

Sealing the plate properly is critical to success. The key is light, even pressure to the lid of the block to keep the cover flat while sealing.

  1. Line-up screws making sure that the base and lid temperature probe holes line-up in the KitAlysis 24-Well Reaction Block.
  2. Initially flush: Using your thumb and forefinger, press the lid of the box until it becomes flush with the vials. Then, using the KitAlysis Torque Screwdriver, insert screws until flush, but not tight, with the top of the box, following the cross pattern provided below. Check to see that the lid is evenly sealed onto the base on all sides. Do this visual check to avoid having to unscrew the lid and screw again.
  3. Tighten: Repeat the same pattern until the KitAlysis Torque Screwdriver “clicks” indicating complete tightness. Go around the block once more for a final check to ensure all screws are tight.

Probe for hot plate does not fit into reaction block hole

Use a small oil bath or other metal block (for best results) in the back of the hot plate and place the probe in there. Place reaction block as close to the center of the hot plate as possible for more even stirring.

Slurry Additions

Due to narrow opening of pipette tips, slurry additions will result in blockages. To aid in uniform dosing, simply snip off the end of the tip at the first marker (~10 mm). To ensure an evenly dispersed aliquot, it is critical that the mixture is stirring well while you draw aliquots for dosing into the corresponding reaction vial.

Reaction Troubleshooting

If no conversion is observed (starting material is not consumed), try one or more of the following:

  1. Increase reaction temperature
  2. Assess starting material for potential coordinating groups and screen acid additives (see above)
  3. Assess starting material purity – could it contain a peroxide or impurity that is highly coordinating?

If the starting material is consumed but the reaction mixture is messy, double bond isomerization or other unwanted side reactions may have occurred. Try one or more of the following:

  1. Reduce reaction temperature
  2. Reduce reaction time
  3. Reduce catalyst loading
  4. Add isomerization inhibitors (see Metathesis Guide for more detail)