deprotection time to 22 minutes.
Modules for the Run Editor
If you want to load the C-terminal amino acid to an HMP resin and follow this with a capping step, the first cycle should be: HF.
Place the amino acid cartridges in the first three cartridge positions. After the third cartridge, place a cartridge that contains 3.0 mmol (0.60-0.70 g) benzoic anhydride.
For all subsequent cycles use: BADEF (G)
Synthesis process Modules Requires
Single coupling with resin sampling, with C-terminal amide or pre-loaded resins
BADEFG 3 amino acid cartridges Single coupling without resin sampling,
with C-terminal amide or pre-loaded resins
BADEF 3 amino acid cartridges Double coupling with resin samples BADEIADEFG 6 amino acid cartridges Double coupling without resin sampling BADEIADEF 6 amino acid cartridges
Final deprotection BIDC
Loading and capping initial resin use suggested module H modifications, shown on page 4-16, with Arg, Asn, Gln, and His derivatives
HF 3 amino acid cartridges
+ 1 cartridge containing 3 mmol benzoic anhydride (0.60-0.70 g)
FastMoc 0.25 mmol
Chemical Usage
Table 4-14 summarizes the chemical usage of the FastMoc 0.25 mmol cycles for average flow rates.
See page 4-8 for chemistry warnings.
Table 4-12. Chemical Usage of FastMoc 0.25 mmol Cycles
Table 4-13. Chemical Usage of FastMoc 1.0 mmol Cycles
Bottle Chemical mL /Bottle Cycle 1 mL / cycle Cycle 10 mL /cycle Cycle 20 mL /cycle Cycle 30 mL /cycle Cycles per bottle*** 1 Piperidine 450 5.0 5.5 6.0 6.5 78 4 DMAP† 200 5 HBTU/ HOBt 230 2.0 2.0 2.0 2.0 110 6 MeOH† 450 7 2 M DIEA 200 1 1 1 1 130 8 DCC† 200 9 DCM†† 4000 10 NMP 8000** 92 107 121 136 50 Waste — 9463 100* 115* 130* 145* 65*
† DMAP, DCC, and MeOH are only used in the loading cycle, HF
†† DCM is used only in the loading cycle (Module H) and in the final resin wash (Module C).
*This value is for non resin-sampling cycles. Resin-sampling cycles produce 15 mL more waste per cycle. ** Based on the two-bottle configuration of NMP.
***These figures are based on chemical usage with average flow rates, i.e., NMP, 2.5 mL/5 sec; DCM, 3.4 mL/5 sec; and piperidine, 1.0 mL/5 sec. If flow rates on a ABI 433A instrument are greater than these cited, the number of cycles/bottle will be proportionately lower.
Bottle Chemical mL / Bottle Cycle 1 mL / cycle Cycle 10 mL /cycle Cycle 20 mL /cycle Cycle 30 mL /cycle Cycles per bottle*** 1 Piperidine 450 9 10.5 12 14 33 4 DMAP† 200 5 HBTU/ HOBt 230 6 6 6 6 30 6 MeOH† 450 7 2 M DIEA 200 5 5 5 5 33 8 DCC† 200 9 DCM†† 4000 10 NMP** 8000** 249 269 326 394 24 Waste — 9463 270* 292* 350* 420* 26*
† DMAP, DCC, and MeOH are only used during the loading cycle
†† DCM is used only in the loading cycle (Module H) and in the final resin wash (Module C).
*This value is for non resin-sampling cycles. Resin-sampling cycles produce 15 mL more waste per cycle. ** Based on the two-bottle configuration of NMP.
***These figures are based on chemical usage with average flow rates, i.e., NMP, 2.5 mL/5 sec; DCM, 3.4 mL/ 5 sec; and piperidine, 1.0 mL/5 sec. If flow rates on a ABI 433A instrument are greater than those cited, the number of cycles/bottle will be proportionately lower.
FastMoc 0.25 mmol Modules
The FastMoc 0.25 mmol cycles are used with either the resin sampling or non-resin sampling, 41-mL reaction vessel. The Fmoc-amino acid is dissolved with 2.1 g NMP, 2.0 g of 0.45 M HBTU/HOBt in DMF, and 2 M DIEA, then transferred to the reaction vessel. For examples of FastMoc cycles and Runs you can create with these modules, see "FastMoc 0.25 mmol and 0.10 mmol Cycles" on page 7-3.
Module A - Dissolving Amino Acid Total time = 7.6 minutes At the beginning of module A, the old cartridge is ejected and the new cartridge is advanced. NMP (2.1 g) and 0.9 mmol of 0.45 M HBTU/HOBt in DMF (2.0 g) are added to the cartridge. The amino acid is dissolved by mixing for 6 minutes. The module includes flushing with NMP and gas dry with nitrogen to assure complete removal of HBTU.
Module B - Piperidine Deprotection Total time = 8.8 minutes This module begins with one NMP wash of the resin. A solution of
18% piperidine/NMP is introduced and allowed to deprotect for 3 minutes. The RV is drained and a second treatment with 20% piperidine is
performed. The valve blocks are then rinsed thoroughly. The resin continues to deprotect for an additional 7.6 minutes during module A.
Module C - DCM Washes Total time = 7.6 minutes The resin is washed 6 times with DCM. After the final wash, the DCM in the resin sample line is removed. This module is used only at the end of the synthesis.
Module D - NMP Washes Total time = 4.6 minutes The RV is drained and the resin is washed 5 times with NMP. During each NMP wash the resin sample line is rinsed with NMP. There is an add time of 1 in the loop function, so after cycle 4, the number of washes is 6, and after cycle 14, the number of washes is 7, and so forth.
Module E - Add DIEA and Transfer to RV Total time = 2.2 minutes At the beginning of the module, 1 mL of 2 M DIEA in NMP is added to the cartridge, to initiate the activation of the amino acid. The activated amino acid is then transferred from the cartridge to the RV.
Module F - Clean cartridge, Couple, Total time = 22.2 minutes
Drain and NMP Washes
During this module, the amino acid cartridge is washed 3 times with NMP. This NMP is transferred to the Activator Vessel and used later in the module to wash the resin in the RV. Coupling occurs during this cartridge washing, and the coupling is then continued for another 15 minutes. The RV is drained and the resin is washed with the NMP from the activator vessel.
Module G - Resin Sample Total time = 1.7 minutes This module takes a resin sample. Place a blank tube between each resin sample tube. The tared resin sample test tube should contain MeOH and a few drops of acetic acid.
Module H - Loading and Capping Total time = 54 minutes This module is used to load the first amino acid onto the HMP resin. The 1.00 mmol Fmoc amino acid is dissolved in an NMP/DCM mixture and transferred to the RV, followed with 1 mL of 1.0 M DCC/NMP and 0.1 equivalent of DMAP. The resin is mixed for 30 minutes. The RV is drained and the resin is washed 2 times with 50% MeOH and 50% DCM and 5 times with DCM. The resin is then capped with 3 mmol benzoic anhydride (0.60- 0.70 g) which has been placed in one cartridge.
The loading and capping is done with modules HF. It requires 1 Fmoc- amino acid cartridge and 1 cartridge containing 3.0 mmol (0.60-0.70 g) of benzoic anhydride.
Module H is modified for Arg, Asn, Gln and His the following way:
IMPORTANT Do not load with unprotected Fmoc-Asn or Fmoc-Gln. Loading
with His(Trt) can cause racemization.
Although it is possible to load Fmoc-Pro onto the HMP resin, the formation of a diketopiperazine during synthesis drastically reduces the yield of the final peptide-resin (see "References" on page 3-22, reference 23).
Module I - 15 Min Wait Total time = 15 minutes Module I (same as Module i) is used in the last cycle to extend the total deprotection time to 22 minutes.
Fmoc-His(Bum) Fmoc-Arg(Mtr) Fmoc-Arg(Pmc)
Fmoc-Gln(Trt) Fmoc-Asn(Trt)
Step 14 (#9 CART) 0 sec 0 sec 4 sec
FastMoc 0.10 mmol
Chemical Usage
FastMoc 0.10 mml cycles are usually used with the non–resin-sampling, 8-mL
reaction vessel. The Fmoc-amino acid is dissolved and activated in the cartridge in a mixture of 2.0 g of 0.45 M HBTU/HOBt in DMF, 2 M DIEA, and 0.8 mL NMP.
See page 4-8 for chemistry warnings.
Note To take resin samples, use a resin-sampling, 8 mL reaction
vessel.
Table 4-14. Chemical Usage of 0.10 mmol FastMoc Cycles
WARNING CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak hazardous chemicals. Secure each four-liter bottle in a low-density polyethylene safety container. Fasten the cover on the safety container and lock the handles in an upright position.
FastMoc 0.10 mmol Modules
For examples of FastMoc cycles and Runs you can create with these modules, see "FastMoc 0.25 mmol and 0.10 mmol Cycles" on page 7-3.
Module A - Dissolving Amino Acid Total time = 7.6 minutes
Bottle Chemical mL per Bottle Cycle 1 mL / cycle Cycle 10 mL /cycle Cycle 20 mL /cycle Cycle 30 mL /cycle Cycles per bottle*** 1 Piperidine 450 2.0 2.0 2.0 2.0 225 4 DMAP† 2 5 HBTU/ HOBt 230 2.0 2.0 2.0 2.0 110 6 MeOH† 450 7 2 M DIEA 200 1 1 1 1 130 8 DCC† 200 9 DCM†† 4000 10 NMP 8000** 45 53 61 69 120 Waste — 9463 50 58 66 74 120
† DMAP, DCC, and MeOH are only used in the loading cycle.
†† DCM is used only twice during a peptide synthesis with 0.10 mmol cycles: once when the first amino acid is loaded and again, 15 mL, in the final washes.
*This value is for non resin-sampling cycles. Resin-sampling cycles produce 15 mL more waste per cycle. ** Based on the two-bottle configuration of NMP.
***These figures are based on chemical usage with average flow rates, i.e., NMP, 2.5 mL/5 sec; DCM, 3.4 mL/5 sec; and piperidine, 1.0 mL/5 sec. If flow rates on a ABI 433A instrument are greater than these cited, the number of cycles/bottle will be proportionately lower.
At the beginning of module A, the old cartridge is ejected and the new cartridge is advanced. NMP (2.1g) and 0.9 mmol of 0.45 M HBTU/HOBt in DMF (2.0 g) are added to the cartridge. The amino acid is dissolved by mixing for 6 minutes. The module includes flushing with NMP and gas dry with nitrogen to assure complete removal of HBTU.
Module B - Piperidine Deprotection Total time = 2.9 minutes This module begins with one NMP wash of the resin. A 22% piperidine/ NMP solution is introduced and allowed to deprotect for 2 minutes. The RV is drained and a second treatment with 22% piperidine is performed. The valve blocks are then rinsed thoroughly. The resin will continue to deprotect for an additional 7.6 minutes during module A.
Module C - DCM Washes Total time = 6.3 minutes The resin is washed 6 times with DCM. After the final wash, the DCM in the resin sample line is removed. This module is used only at the end of the synthesis.
Module D - NMP Washes Total time = 2.5 minutes The RV is drained and the resin is washed 4 times with NMP. During each NMP wash the resin sample line is rinsed with NMP. There is an add time of 1 in the loop function, so after cycle 4, the number of washes is 5, and after cycle 14, the number of washes is 6, and so forth.
Module E - Add DIEA and Transfer to RV Total time = 2.1 minutes At the beginning of the module, 1 mL of 2 M DIEA in NMP is added to the cartridge, to initiate the activation of the amino acid. The activated amino acid is then transferred from the cartridge to the RV.
Module F - Clean Cartridge, Couple, Total time = 9.3 minutes
Drain and NMP Washes
During this module, the amino acid cartridge is washed 2 times with NMP. This NMP is transferred to the Activator Vessel and used later in the module to wash the resin in the RV. Coupling occurs during this cartridge washing, and the coupling is then continued for another 4.5 minutes. The RV is drained and the resin is washed with the NMP from the Activator Vessel.
Module G -Resin Sample Total time = 1.4 minutes This module takes a resin sample. Place a blank tube between each resin sample tube. The tared resin sample test tube should contain MeOH and a few drops of acetic acid.
Module H - Loading and Capping Total time = 51 minutes This module is used to load the first amino acid onto the HMP resin. The 1.00 mmol Fmoc amino acid is dissolved in an NMP/DCM mixture and transferred to the RV, followed with 1 mL of 1.0 M DCC/NMP and 0.1
equivalent of DMAP. The resin is mixed for 30 minutes. The RV is drained and the resin is washed 2 times with 50% MeOH and 50% DCM and 5 times with DCM. The resin is then capped with 3 mmol benzoic anhydride (0.60- 0.70 g) which has been placed in one cartridge.
The loading and capping is done with modules HF. It requires 1 Fmoc- amino acid cartridge and 1 cartridge containing 3.0 mmol (0.60-0.70 g) of benzoic anhydride.
Module H is modified for Arg, Asn, Gln and His the following way:
IMPORTANT Do not load with unprotected Fmoc-Asn or Fmoc-Gln. Loading
with His(Trt) can cause racemization.
Although it is possible to load Fmoc-Pro onto the HMP resin, the formation of a diketopiperazine during synthesis drastically reduces the yield of the final peptide-resin (see "References" on page 3-22, reference 23).
Module I - 15 Min Wait Total time = 15 minutes Module I (same as Module i) is used in the last cycle to extend the total deprotection time to 17 minutes.
Fmoc- His(Bum) Fmoc-Arg(Mtr) Fmoc -Arg(Pmc) Fmoc-Gln(Trt) Fmoc-Asn(Trt)
Step 14 (#9 CART) 0 sec 0 sec 4 sec