Introduction

Solid-phase -peptide synthesis

-Peptides are synthesized using standard solid-phase methodology developed for -peptides²⁴, and our microwave-assisted parallel synthesis methods can be applied to both - and -peptides. Solid-phase -peptide synthesis (Fig. 1) is accomplished by (i) coupling the activated ester of a -aa residue with the free amine of a resin-bound linker or growing oligopeptide chain; (ii) removing the base-labile 9-fluorenylmethoxycarbonyl (Fmoc) group from the main chain nitrogen by treatment with piperidine (these two steps are repeated until the desired oligomer length is achieved); (iii) cleaving the peptide from the solid support with simultaneous global side-chain de-protection by treating with trifluoroacetic acid (TFA). During parallel synthesis of a peptide library, the same coupling, Fmoc-removal and cleavage reactions occur in each well of the plate, except that the addition of different protected aa to each well during the coupling steps results in each well containing a peptide with a unique sequence. For illustrative purposes, this protocol describes the synthesis of the -peptide library in Figure 2.

Figure 1: Solid-phase synthesis of -peptides.

Steps 1 and 2 are repeated to extend the -peptide chain before performing Step 3. MW, microwave irradiation.

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Figure 2: Example of a -peptide library.

Reproduced with permission from ref. 23.

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Materials

Reagents

• PEG-polystyrene (PEG-PS) rink amide resin (NovaSyn TGR, 0.25 mmol g^{- 1} loading; Novabiochem, cat. no. 01-64-0060)
• DMF (biotech grade solvent, 99.9%+) stored over Dowex ion exchange resin (Aldrich, cat. no. 494488)
  Critical Purchasing high-quality DMF and storing over ion exchange resin eliminates the traces of dimethylamine that result from the decomposition of DMF; high concentrations of dimethylamine can lead to premature Fmoc removal under microwave irradiation.
• Dichloromethane (DCM; Aldrich, cat. no. 320269)
• Piperidine (Aldrich, cat. no. 104094)
• Fmoc--aa may be synthesized^{25, 26} or bought from PepTech Corporation
• 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexaflurorophosphate (HBTU; Novabiochem, cat. no. 01-62-0010)
• 1-Hydroxybenzotriazole hydrate (HOBt; Aldrich, cat. no. 157260)
• Diisopropylethylamine (iPr₂EtN; Aldrich, cat. no. 496219)
• Triethylamine (Aldrich, cat. no. 471283)
• Acetic anhydride (Aldrich, cat. no. 242845)
• Triethylsilane (Aldrich, cat. no. 230197)
• Deionized water
• TFA (Aldrich, cat. no. T6508)
  Caution Corrosive.
• Dimethylsulfoxide (DMSO; Aldrich, cat. no. 472301)
• Acetonitrile (Aldrich, cat. no. 644536)

Equipment

• 50-ml polypropylene centrifuge tube
• 15 scintillation vials (20 ml)
• 0.1–1-ml adjustable pipette and tips
• Polypropylene pipette troughs (50 ml)
• Polypropylene 2-ml deep-well square-bottom 96-well filter plate with 25-m polyethylene frits and long drip drain ports (AWFP-F20000; Arctic White)
• Bottom sealing matte (AWSM-1003DP; Arctic White)
• Polypropylene 2-ml deep-well solid–bottom 96-well plate
• 96 Spinbar micro stir bars (PTFE, 7 2 mm; VWR, cat. no. 58948-976)
• Three plastic squirt bottles (2 500 ml and 1 250 ml)
• 12-channel multipipette (50–300 l capacity) and a case of tips
• Vacuum pump and inline 2–4-l waste trap
• Multiscreen resist vacuum manifold (Millipore through VWR, cat. no. MAVM0960R)
• CEM MARS microwave reactor with microtiter plate turntable, fiber-optic temperature probe and magnetic stirrer
• Analytical and/or semi-preparative HPLC, preferably with a fraction collector and an autosampler capable of injecting out of a 96-well plate
• Mass spectrometer (MALDI-TOF MS or LC-MS) Lyophilizer

Reagent setup

• Prepare the reagent solutions as shown in Table 1.

  Table 1: Reagent solutions for aa coupling and Fmoc–removal.

  

  
  Full table
  
  

Equipment setup

• For HPLC for analysis of crude peptide products, see Table 2.

  Table 2: Analytical HPLC equipment and conditions for analysis of crude peptide products.

  

  
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• For semi-preparative HPLC for purification of crude peptide products, see Table 3.

  Table 3: Semi-preparative HPLC equipment and conditions for purification of crude peptide products.

  

  
  Full table
  
  

Procedure

1. Points from here (point 1) up to and including point 1 are related to Plan library synthesisTiming: 15 minAfter designing the library (J.K.M., J.D. Sadowsky, S.H.G., manuscript in preparation), plan the library synthesis by preparing a map that divides the wells of the plate into different sectors for each reaction step (Fig. 3). Subdividing the plate into sectors facilitates reagent delivery using a multichannel pipette, and starting with the platemap prevents confusion during the synthesis. For convenience, it is recommended that the number of diversity elements at each position be a factor of either 8 or 12 (e.g., 1, 2, 3, 4, 6, 8 or 12). The product of the numbers of diversity elements at all positions should be equal to 96 (e.g., 1 3 2 2 4 2 = 96).

   Figure 3: Coupling protocols for stepwise synthesis of a spatially defined -peptide library (1 3 2 2 4 2 = 96 members).

   

   Note that the peptide is synthesized in the C-to-N direction by starting at the top of the figure, even though the positions are labeled according to standard peptide numbering beginning at the N-terminus. Reproduced with permission from ref. 23.

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2. Points from here (point 2) up to and including point 2 are related to Prepare reagentsTiming: 1 hCalculate and weigh the proper amount of each of the Fmoc-aa into separate 20-ml scintillation vials: [3 equivalents scale (mol) MW (g mol^{- 1}) no. of wells]/1,000 = mass of aa (mg). For example, at position 6 (standard peptide numbering beginning at the N-terminus), 316.4 mg Fmoc-(S)-³-homoGlu(t-Bu)-OH is prepared: (3 2.5 mol 439.5 g mol^{- 1} 96)/1,000 = 316.4 mg. If the same aa is incorporated at two positions of the library, then two separate vials are prepared (Table 4).

   Table 4: Masses of protected b-aa to be prepared in separate vials.

   

   
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3. Points from here (point 3) up to and including point 5 are related to Array the solid support into each well of the 96-well filter plateTiming: 30 minWeigh a slight excess of PS or PEG-PS resin [no. of wells scale (mol well^{- 1})/loading (mmol g^{- 1})/1,000 = mass of resin (g); e.g., 100 (wells) 2.5 (mol well^{- 1})/0.25 (mmol g^{- 1})/1,000 = 1.0 g] into a 50-ml polypropylene centrifuge tube. This protocol has been validated for a synthesis scale of 2.5–25 mol per well. For this example, a 2.5-mol scale has been assumed.
4. Suspend the resin in 50 ml 3:2 DCM/DMF (vol/vol, total volume with resin). Add a stir bar to the centrifuge tube and, while stirring, transfer a 500-l aliquot into each well of the sealed 2-ml deep-well polypropylene filter plate. The 3:2 DCM/DMF solvent mixture produces a relatively homogeneous resin suspension, allowing an equal amount of resin to be distributed throughout the plate by simply dispensing a constant volume of the slurry into each well.
5. Place a small (7 2 mm) magnetic stir bar inside each well.Pause Point Reagent setup and Steps 1–5 can be performed the day before the actual synthesis; the reagent solutions, dry aa and arrayed resin can be stored overnight at room temperature (20–25 °C) before proceeding to Step 6.
6. Points from here (point 6) up to and including point 11 are related to Resin washTiming: 5 minRemove the bottom sealing matte from the plate.
7. Transfer the plate quickly to the vacuum filtration manifold.
8. Drain the solution from the plate by applying vacuum.
9. Close the vacuum. Using a 500-ml squirt bottle, rapidly add DMF to each well (approximately 0.5–1 ml per well, but the actual amount does not have to be precise).
10. Repeat Steps 8 and 9 four times and then drain the solution a final time.
11. Reaffix the bottom sealing matte to the plate.
12. Points from here (point 12) up to and including point 17 are related to Fmoc-removalTiming: 10 minFill a 50-ml polypropylene trough with 25 ml of the pre-made de-protection solution of 20% piperidine in DMF (vol/vol).
13. Using a 12-channel multipipette, dispense 250 l deprotection solution into each well of the plate.
14. Place the plate on the microtiter plate turntable inside the multimode microwave cavity of the CEM MARS (Fig. 4).

    Figure 4: Picture of the experimental setup in the microwave reactor.

    

    Reproduced with permission from ref. 23.

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15. Position the fiber-optic temperature probe in the center of the plate (e.g., well D6) using the arm attached to the turntable, ensuring that the tip of the probe is in contact with the bottom of the well.
    Critical step If the fiber-optic probe is not positioned inside the well and in the solution, the microwave reactor will not accurately detect the increasing temperature of the solution. As the air temperature being sensed by the probe does not increase, the reactor will continue to irradiate at full power throughout the experiment, overheating the samples.
16. Irradiate the sample (600 W maximum power, 80 °C, ramp 2 min, hold 2 min).
17. Remove the plate from the microwave reactor, and wash the resin (5 DMF) as in Steps 6–11.
18. Points from here (point 18) up to and including point 28 are related to Amino acid couplingTiming: 20 minTo a vial containing pre-weighed solid Fmoc-aa from Step 2, add the pre-made solutions of HBTU, DMF, HOBt and iPr₂EtN sequentially (volumes according to Table 5). For example, at position 6 of our library, there is only one diversity element (i.e., the same aa is coupled to every member of the library). So, to the 316.4 mg solid Fmoc-(S)-³-hGlu(t-Bu)-OH are added 1,440 ml 0.5 M HBTU in DMF, followed by 10.56 ml DMF, 1,440 ml 0.5 M HOBt in DMF and finally 1,440 ml 1.0 M iPr₂EtN in DMF.

    Table 5: Volumes of coupling reagents to be added depending on the number of different aa residues to be coupled at a given position (i.e., diversity elements) for a 2.5-mol-scale synthesis.

    

    
    Full table
    
    
19. Vortex the mixture until the solid aa is dissolved, and then allow the solution to stand for 60–90 s.
20. Pour the mixture into a polypropylene trough. Using a multichannel pipette, transfer 150 l of the solution to each well in the appropriate sector(s) of the plate according to the platemap prepared in Step 1.
21. If there is more than one diversity element at the given position of the library, then the different aa will be coupled sequentially (one at a time). After adding the coupling solution to the appropriate wells, transfer 150 l DMF to all the wells in the sector(s) of the plate not containing aa coupling solution. For example, at position 5 of our library, each of the three different aa will be incorporated into a different third of the library members in three sequential coupling reactions. First, Fmoc-(S)-³-hPhe-OH is activated (Steps 20–21) and delivered to each well in columns 1–4 (32 wells total). DMF is added to the wells in columns 5–12 (64 remaining wells).
22. Place the plate on the microtiter plate turntable inside the multimode microwave cavity of the CEM MARS.
    Critical step Position the fiber-optic temperature probe in the center of the sector containing aa coupling solution (e.g., well D3 for the coupling of Fmoc-(S)-³-hPhe-OH at position 5) using the arm attached to the turntable, ensuring that the tip of the probe is in contact with the bottom of the well. If the fiber-optic probe is not positioned inside the well and in the coupling solution, the microwave reactor will continue to irradiate at full power throughout the experiment, overheating the samples.
23. Irradiate the sample (600 W maximum power, 70 °C, ramp 2 min, hold 4 min).
24. Remove the plate from the microwave reactor, and wash the resin (5 DMF) as in Steps 6–11.
25. Repeat Steps 18–24 until all the diversity elements have been incorporated for that position. For example, the coupling of Fmoc-³-hGlu(t-Bu)-OH at position 6 is performed once in all 96 wells before proceeding to Step 26. At position 5, Fmoc-³-hPhe-OH is coupled to the resin in the 32 wells of columns 1–4, followed by the coupling of Fmoc-³-hTrp(Boc)-OH to the resin in the 32 wells of columns 5–8, and finally Fmoc-³-hLeu-OH is coupled to the resin in the 32 wells of columns 9–12. After the three sequential coupling reactions are complete, proceed to Step 26.Pause Point After a coupling reaction, the washed resin can be stored at room temperature for several hours or overnight.
26. Repeat Steps 12–25, adding one monomer per cycle until the growing peptide chains reach the desired oligomer length.
27. Remove the Fmoc protecting group from the N-terminal residue by repeating Steps 12–17.
28. Wash the resin five times as in Steps 6–11, except use DCM instead of DMF.
29. Points from here (point 29) up to and including point 32 are related to AcetylationTiming: 20 minIn a separate vial, prepare the acetylation cocktail by mixing 14 ml DCM, 0.1 ml triethylamine and 0.5 ml acetic anhydride. This reagent should be prepared fresh at this step.
30. Pour the solution into a polypropylene trough. Using a multichannel pipette, transfer the acetylation cocktail to each well in the plate (150 l per well).
31. Allow the plate to stand for 5–15 min inside a hood. (The plate may be gently agitated on an orbital shaker if desired.)
32. Wash the resin five times as in Steps 6–11, except use DCM instead of DMF.Pause Point After acetylation, the washed resin can be stored at room temperature for several hours or overnight.
33. Points from here (point 33) up to and including point 37 are related to CleavageTiming: 4 hIn the hood, use a polypropylene trough and a multichannel pipette to transfer 50 l triethylsilane to each well of the plate (approximately 5 ml triethylsilane total).
34. In the hood, use a trough and a multichannel pipette to transfer 50 l water to each well of the plate (approximately 5 ml water total).
35. In a well-ventilated hood, use a small polypropylene squirt bottle to add TFA slowly to each well of the plate (approximately 500 l per well). Cover the plate with aluminum foil, and allow the plate to stand for 2 h inside the hood. (The plate may be gently agitated on an orbital shaker if desired.)
    Caution TFA is corrosive. Make sure to wear eye protection, a lab coat and gloves.
36. Remove the aluminum foil from the plate. Remove the bottom sealing matte carefully and quickly and place the synthesis plate into a 2-ml deep-well 96-well solid-bottom plate. Allow the cleavage solution to be transferred to the solid-bottom plate by gravity filtration.
    Caution TFA is corrosive. TFA is much less viscous than the DMF used in earlier steps of the synthesis. The removal of the bottom sealing matte and transfer to the solid–bottom plate must be performed quickly to avoid drips and carefully to prevent spills.
37. Concentrate the cleavage solution by rotary evaporation in a rotary concentrator at 35 °C for 2 h (Genevac). Alternatively, concentrate the samples with a stream of nitrogen by positioning the plate under an inverted funnel attached to a nitrogen line.
    Caution TFA is corrosive. Take care that the nitrogen flow rate is sufficiently low not to cause spattering.
38. Points from here (point 38) up to and including point 40 are related to Library analysis and screeningTiming: 4 h–5 dOnce TFA and triethylsilane have been removed (some water may remain), add DMSO to dissolve the peptide product mixtures in each well (250 l DMSO per well to obtain approximately 10 mM stock solution, assuming a 100% yield).
39. Assess the purity of the library members by analyzing the peptide product mixtures on an analytical RP-HPLC (see EQUIPMENT SETUP) and collecting fractions based on a UV absorbance threshold. Measure peptide masses of the collected fractions using MALDI-TOF MS (-cyano-4-hydroxycinnamic acid matrix). If an LC-MS is available, then determine peptide masses of the eluting peaks in real time. Initial purity of the library members is calculated from the integrated trace of UV absorbance at 220 nm as follows: initial purity (%) = (peak area of desired product/sum of all peak areas) 100. The actual number obtained for initial purity depends on many factors, including the wavelength monitored, injection amount and elution gradient. We have found it useful to report the actual HPLC traces of library members (see Supporting Information of ref. 23) to allow the reader to assess the quality of the library.
40. Purify each of the crude peptides using a semi-preparative RP-HPLC, collecting the desired product using a UV absorbance or mass detection threshold (see EQUIPMENT SETUP)²⁷ followed by concentration by lyophilization, or screen the crude products in a relevant biological assay, purifying only the hits for re-testing and validation (J.K.M., J.D. Sadowsky, S.H.G., manuscript in preparation).Troubleshooting

Timing

Step 1: Library planning should be given careful consideration, but preparing the platemap requires only 15 min.
Steps 2–11: Preparation for the synthesis requires 2–3 h, including REAGENT SETUP.
Steps 12–28: Each deprotection/coupling cycle requires 30 min; the overall synthesis time depends on the length of the peptide.
Steps 29–37: The cleavage reaction requires 2 h; concentrating the cleavage solution is highly dependent on the flow rate of the nitrogen, often requiring an overnight period.
Steps 38–40: Required time is highly dependent on the HPLC and MS equipment available to the researcher and the degree to which the library is characterized, ranging from a few hours to 1 week.

Troubleshooting

If analysis of the crude library members using HPLC and MS reveals that the product mixtures contain substantial amounts of deletion peptide side products resulting from incomplete coupling reactions, then in the second synthesis consider doing Steps 18–24 two times (i.e., double coupling) for the difficult aa coupling steps. Alternatively, modify Step 23 of the difficult coupling steps by subjecting the reaction mixture to multiple cycles (e.g., three cycles) of microwave irradiation, waiting for approximately 10 min between cycles for the plate to cool to room temperature⁷. Overall, the reaction solvent can also be modified to facilitate difficult couplings².

Anticipated results

With the microwave-assisted parallel peptide synthesis protocol, 96 different hexapeptides can be prepared in less than 24 h (excluding characterization). The peptide product mixtures should contain one major species corresponding to the desired product. The initial purity of the peptide products is typically approximately 60% (Fig. 5) and 50% yield (using UV peak area for a given injection volume and a calibration curve to correlate with peptide concentration and hence amount^{23, 28}) for a hexamer, but this decreases with increasing length of the peptide. This protocol has been applied to peptides containing both - and -aa with equivalent results.

Figure 5: HPLC chromatogram (UV absorbance at 220 nm) of a -peptide (H-ACHC-³-hTrp-³-hOrn-ACHC-³-hPhe-³-hGlu-OH) prepared as a member of a library.

Reproduced with permission from ref. 23. See the Supporting Information of the original reference for HPLC traces of all library members.

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