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ABI 433A Peptide Synthesis

1. For PNA synthesis the HBTU solution usually does not have the additional HOBt The HBTU mw 379 3 is dissolved in NMP as shown in Table 4 6 Table 4 6 HBTU solutions using solid HBTU 0 19 M HBTU 14 4 g HBTU dissolved in NMP and diluted to 200 mL 0 38 M HBTU 28 8 g HBTU dissolved in NMP and diluted to 200 mL WARNING RESPIRATORY HAZARD Inhaling HBTU dust can cause bronchial irritation with coughing Repeated or prolonged exposure may cause allergic respiratory system sensitization Handle HBTU under a chemical fume hood The cycles designed for the 3 mL RV do not contain a loading cycle Loading Fmoc amino acids on HMP resins requires DCC in Bottle 8 Therefore use pre loaded resins for syntheses in these micromole scales Bottle 9 DCM P N 400142 Bottle 10 NMP P N 400580 Waste Container If you are using TFA pour a bottle of Ethanolamine Methanol P N 400230 into the waste container to neutralize the TFA in the waste 4 Chemistry November 2001 Applied Biosystems S Cartridge Predissolve the Fmoc amino acids Boc amino acids or Boc PNAs in NMP using five or ten equivalents of monomer for each coupling Add the mixture to the cartridge The formula for calculating monomer quantity is monomer MW X mmolesofAA mg of AA per cartridge To calculate the quantity of monomer when five equivalents is required a From Table 4 1 on page 42 find the quantity of monomer you need for your scale synthesis If
2. Module E Read Cartridge and Add Two Times HBTU and DIEA umol 5 10 20 Step Fxn Time 1 1 Wait 1 1 1 2 4 Read cartridge 10 10 10 3 6 Needle up 10 10 10 4 7 Eject cartridge 10 10 10 5 8 Advance cartridge 10 10 10 6 5 Needle down 10 10 10 7 14 Flush bottom valve block with NMP to waste 1 1 1 8 9 Flush top valve block with gas to waste 2 2 2 9 10 Flush bottom valve block with gas to waste 3 3 3 10 70 Flush bottom valve block with loop to waste 2 2 2 11 78 Pressurize manifold 5 5 5 12 98 Begin Loop UPPER 2 2 4 13 69 Deliver HBTU to measuring loop open 3 3 3 5 Cycles and Modules November 2001 Applied Biosystems 5 10 20 Step 14 10 Flush bottom valve block with gas to waste 2 2 2 15 63 Transfer measuring loop to cartridge 10 10 10 16 99 End Loop UPPER 1 1 1 17 60 Mix cartridge 2 2 2 18 98 Begin Loop UPPER 2 2 2 19 68 Deliver DIEA to measuring loop open 3 3 3 20 10 Flush bottom valve block with gas to waste 2 2 2 21 63 Transfer measuring loop to cartridge 10 10 10 22 99 End Loop UPPER 1 1 1 23 60 Mix cartridge 5 5 5 November 2001 5 Cycles and Modules 5 17 Applied Biosystems Module F Transfer Clean Cartridge and Couple Step Fxn Name Time 1 1 Wait 1 2 5 Needle down 10 3 98 Begin loop UPPER 8 4 41 Vent reaction vessel 2 5 96 Transfer cartridge to reaction vessel top closed 6 6 2 Vortex reaction vessel on 3 7 3 Vortex reaction vessel off 1 8 99 End l
3. South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Eastern As ia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 75 0 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 4202 61 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Warszawa Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 A
4. 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 1 About This Manual November 2001 Applied Biosystems November 2001 Product or Product Area Telephone Dial Fax Dial FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemiluminescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 Outside North America Region Telephone Dial Fax Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349
5. Refer to Installation Procedure on page 3 3 7 Remove the Conductivity Cell If you are going to run cycles without monitoring remove the Conductivity Cell For instructions on removing the Conductivity Cell refer to the ABI 433A User s Manual Section 8 System Description 8 Assemble the 3 mL RV Install the filter add the resin and ghten the caps on the RV see To assemble the 3 mL RV on page 2 5 9 Run a test peptide Before you use expensive monomers make a simple peptide to test the new synthesizer setup For information on running a test synthesis refer to Synthesis Setup for the 3 mL Reaction Vessel on page 4 15 and Test Synthesis Example on page 4 18 Use the normal synthesis procedure with the following two exceptions e Prepare Bottle 7 and Bottle 8 reagent solutions according to your intended scale of synthesis refer to the tables of concentrations on page 4 5 e Prepare the amino acid solutions Refer to Bulk Amino Acid Solutions on page 4 8 10 Ready to go If the test synthesis is satisfactory you may proceed to your small scale syntheses using the 3 mL RV 2 Introduction November 2001 Applied Biosystems How to Assemble the 3 mL RV WARNING CHEMICAL HAZARD To prevent serious chemical burns and eye damage make sure a plug is inserted into the bulkhead fitting ABI 431A or the sliding cover flap covers the bulkhead fitting ABI 433A Even though the 3 mL RV cycles are written without resin
6. Prepare the capping solution for Bottle 4 Note Make the capping solution fresh each week 6 Check all the other solvents and reagents Piperidine TFA if using Boc cycles DCM and NMP 7 Install or remove the conductivity cell depending on whether conductivity monitoring is used 8 Check that the 0 125 mL variable measuring loop is installed 4 Chemistry 4 15 Applied Biosystems 9 Open Flow Test 1 18 VML and send to the synthesizer Run the appropriate flow tests Check for leaks after finishing flow tests Flow Test 10 module A NMP to metering vessel Flow Test 11 module B NMP to cartridge Flow Test 2 module b TFA to metering vessel only if using Boc cycles Flow Test 1 module a Piperidine to metering vessel Flow Test 4 module d Capping solution to metering vessel Flow Test 7 module g DIEA to measuring loop check that it fills loop in 3 sec or less Flow Test 8 module h HBTU to measuring loop check that it fills loop in 3 sec or less 10 11 12 If the sequence is not already entered open New then enter and save the sequence Open the appropriate 3 mL chemistry 5 10 20 pmol a Choose Boc or Fmoc in the Chemistry Information dialog box SynthAssist User s Manual page 4 3 The possible cycles to choose from are described on pages 5 6 and 5 7 It is also possible to create your own cycles b Check Default Set and change if necessary SynthAssi
7. Resin sampling version is not available Note The bottom cap of the 3 mL Reaction Vessel has three dots for identification 2 2 2 Introduction November 2001 Applied Biosystems November 2001 3 mL RV Installation Overview This section describes the process of installing the hardware and software components of the 3 mL RV kit Each step in the installation process must be performed in the order listed in this section to ensure that the 3 mL RV System functions properly Carry out the following steps in the installation process 1 Check ABI 431A for proper vortexer bracket ignore for ABI 433A Note If you plan to use the 3 mL RV on an ABI 431A that has had the Monitoring Upgrade installed verify that the synthesizer is equipped with the vortexer bracket that accepts RTF style Reaction Vessels RTF Recessed Tab Filter If you have an ABI 433A ignore this instruction because all ABI 433A instruments have the proper vortexer bracket If the vortexer bracket does not accept RTF style Reaction Vessels do not proceed any further You need to upgrade the synthesizer s vortexer bracket before you can use the 3 mL RV For more information contact Technical Support Refer to Technical Support on page 1 3 2 Verify the contents of the 3 mL RV kit The 3 mL Reaction Vessel System kit contains a number of different parts Before you begin to install any components take a minute to inventory the kit to verify
8. The 0 125 mL Loop folder contains the new 0 125 mL cycles The 0 5 mL Loop folder contains all the old cycles previously on the SynthAssist Chemistry disk with some steps modified as described in Table 5 5 on page 5 24 gt EM NU 5 umol Folder 5 umol 3 mL RV PNA 1 MM 0 125 mL Loop Folder 10 umor Folder 10 umol 3 mL RV PNA MM 20 umol Folder 20 umol 3 mL RV E 8 1 0 5 Loop Folder ot Li Variable Measuring Loop Tw ERES 17 modified cycles Ms using the 0 5 mL Measuring Loop Flow Tests New VML see page 5 24 Tu Flow Tests Feder Flow Tests 1 18 VML Tow Flow Tests 19 23 Figure 5 1 Arrangement of folders on Variable Measuring Loop disk November 2001 5 Cycles and Modules 5 1 Applied Biosystems The Variable Loop Folder also contains four new flow tests for the Variable Measuring Loop located in the Flow Tests Folder Figure 5 1 This folder also contains the original flow tests from the ABI 433A with minor modifications made to Flow Tests 7 8 17 and 18 modules g h H and I The 3 mL RV cycles are provided with the Fmoc chemistry selected If you are using Boc resins and Boc amino acids select Boc in the information window refer to the SynthAssist 2 0 User s Manual page 4 3 5 2 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Module Descriptions for ABI 433A The modules describ
9. 1 16 42 Drain reaction vessel to waste 7 Module h Conditional Deprotection Previous Peak Step Fxn Name Time 1 137 Do module if condition not met 1 2 56 Deliver NMP to reaction vessel 2 3 79 Pressurize piperidine 10 4 51 Deliver piperidine to reaction vessel 3 5 56 Deliver NMP to reaction vessel 2 6 40 Mix reaction vessel 2 7 2 Vortex reaction vessel on 1 8 13 Flush top valve block with NMP to waste 2 9 14 Flush bottom valve block with NMP to waste 2 10 9 Flush top valve block with gas to waste 5 11 10 Flush bottom valve block with gas to waste 5 12 1 Wait 600 13 3 Vortex reaction vessel off 1 14 42 Drain reaction vessel to waste 2 15 130 Monitor previous peak 1 16 1 Wait 3 17 131 Monitoring stop 1 18 132 Read monitoring peak 1 19 42 Drain reaction vessel to waste 10 Module i Conditional Vortex 5 minutes Step Fxn Time 1 137 module if condition not met 1 2 2 Vortex reaction vessel on 300 3 3 Vortex reaction vessel off 1 November 2001 5 Cycles and Modules 5 23 Applied Biosystems 5 24 Cycles using the new 0 5 mL measuring loop Because the new 0 5 mL measuring loop is made of smaller diameter tubing the cycles using this new 0 5 mL measuring loop need to have the times in some functions changed as compared to the cycles using the original 0 5 mL measuring loop These functions are e HFxn 23 Transfer measuring loop to Act Fxn43 Transfer measuring loop to RV Fxn63 Tra
10. 112 108 216 432 Fmoc Met OH 371 5 4 503 0 113 109 218 436 Fmoc Asn Trt OH 596 7 4 729 0 118 114 228 456 Fmoc Pro OH 337 4 4 469 0 112 108 216 432 Fmoc Gln Trt OH 610 7 4 743 0 119 115 230 460 Fmoc Arg Pmc OH 662 8 4 795 0 120 116 232 464 Fmoc Ser tBu OH 383 4 4 515 0 113 109 218 436 Fmoc Thr tBu OH 397 5 4 529 0 113 109 218 436 Fmoc Val OH 339 4 4 471 0 112 108 216 432 Fmoc Trp OH 426 5 4 558 0 114 110 220 440 Fmoc Tyr tBu OH 459 5 4 591 0 115 111 222 444 Boc Ala OH 189 2 4 321 0 108 104 208 416 Boc Cys Mob OH 341 4 4 473 0 112 108 216 432 Boc Asp OBzl OH 323 4 4 455 0 111 107 214 428 Boc Glu OBzl OH 337 4 4 469 0 112 108 216 432 Boc Phe OH 265 3 4 397 0 110 106 212 424 Boc Gly OH 175 2 4 307 0 108 104 208 416 Boc His Bom OH 375 4 4 507 0 113 109 218 436 Boc His DNP OH 421 4 4 553 0 114 110 220 440 1 2 H2O 240 3 4 372 0 109 105 210 420 Boc Lys Cl Z OH 414 9 4 547 0 114 110 220 440 Boc Leu OH H20 249 3 4 381 0 110 106 212 424 Boc Met OH 249 3 4 381 0 110 106 212 424 Boc Asn Xan OH 412 4 4 544 0 114 110 220 440 Boc Pro OH 215 3 4 347 0 109 105 210 420 246 3 4 378 0 110 106 212 424 Boc Arg Mts OH 456 6 4 589 0 115 111 222 444 Boc Ser Bzl OH 295 3 4 427 0 111 107 214 428 Boc Thr Bzl OH 309 4 4 441 0 111 107 214 428 Boc Val OH 217 3 4 349 0 109 105 210 420 304 4 4 436 0 111 107 214 428 Boc Tyr Br Z OH 494 4 4 626 0 116 112 224 448 November 2001 4 Chemistry 4 9 Appli
11. 2 6 7 68 Deliver 7 to measuring loop 2 6 8 63 Transfer measuring loop to Cart 4 10 Module H Load and Cap Time sec Step Fxn Name Old New 27 69 Deliver 8 to measuring loop 3 6 28 43 Transfer measuring loop to RV 4 10 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Module a Activation amp Transfer Conditional cycles only Time sec Step Fxn Name Old New 29 70 Flush bottom valve block with loop contents to waste 2 6 31 68 Deliver 7 to measuring loop 2 6 32 63 Transfer measuring loop to Cart 4 10 FastMoc Cycles 1 0 mmol Module E Transfer Time sec Step Fxn Name Old New 4 70 Flush bottom valve block with loop contents to waste 2 6 6 68 Deliver 7 to measuring loop 2 6 7 23 Transfer measuring loop to Act 2 10 Module H Load and Cap Time sec Step Fxn Name Old New 27 69 Deliver 8 to measuring loop 3 6 28 43 Transfer measuring loop to RV 4 10 Module H Load and Cap monitoring cycles Time sec Step Fxn Name Old New 28 69 Deliver 8 to measuring loop 3 6 29 43 Transfer measuring loop to RV 4 10 Fmoc HOBt DCC 0 10 mmol Module a Activation Time sec Step Fxn Name Old New 15 68 Deliver 7 to measuring loop 3 6 17 63 Transfer measuring loop to Cart 6 10 61 69 Deliver 8 to measuring loop 3 6 62 23 Transfer measuring loop to Act 3 10 64 23 Transfer measuring loop to Act 3 5 Module h Loading Time sec Step Fxn Name Old New 45 69 Deliver 8 to measuring loop 3
12. 5 Press fit the ferrule and fitting together leaving approximately 1 inch of tube protruding from the ferrule 3 8 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems November 2001 fitting ferrule Figure 3 5 Trimming the long red tube 6 Holding the fitting ferrule assembly between your fingers press the trimmed end of the red tube down onto a hard surface to bring the ferrule flush with the end of the tube Figure 3 6 Figure 3 6 Pressing long red tube into fitting and ferrule 7 Reinstall the long red tube between the flange coupling and cross fitting 8 Rerun flow test a to determine whether the measuring loop is within specification Table 3 1 If further calibration is required repeat the calculation procedure starting on page 3 6 To calibrating the 0 5 mL Measuring Loop 1 Connect the new blue tube labeled ASSY TUBE 0 5 ML LOOP P N 604127 between the two pieces of red tube P N 604129 and 604130 using two flange couplings P N 110070 Figure 3 7 2 Place an empty tared cartridge with a septum into the guideway under the needle assembly and place the pusher block against the cartridge 3 Installing the Variable Measuring Loop 3 9 Applied Biosystems set 3 Run flow test b located in the file Flow Tests New VML The steps in this module are listed on page 5 28 4 Check all the tube connections you have made to ensure that there are no
13. Assemble the 3 mL RV 3 Installing the Variable Measuring Loop Equipment Required Installation Procedure 4 Chemistry Cycle Times Solvent Consumption Reagent and Bottle Positions Bulk Amino Acid Solutions Peptide Nucleic Acids Recommended Modifications Lowering Resin Substitution PNA Monomer Solutions Synthesis Setup for the 3 mL Reaction Vessel Test Synthesis Example 5 Cycles and Modules Cycles in SynthAssist 2 0 Software Module Descriptions for ABI 433A Fmoc and Boc Cycles Cycles grouped by coupling Cycle Modifications Deprotection Monitoring 1 1 1 1 1 9 1 3 1 3 1 3 1 3 1 4 1 7 2 1 2 1 2 2 2 5 2 5 3 1 3 1 4 1 4 2 4 3 4 4 4 8 4 12 4 13 4 14 4 15 4 18 5 1 5 1 5 3 5 6 5 6 5 8 5 9 iii Applied Biosystems Example of a Deprotection Calculation 5 11 SynthAssist Dictionary 5 12 Modules for 433A Peptide Synthesizer 5 13 Cycles using the new 0 5 mL measuring loop 5 24 Flow Tests Folder 5 27 Appendix 1 Plumbing Schematics A 1 iv November 2001 Applied Biosystems November 2001 1 About This Manual This manual describes how to install and use the 3 mL Reaction Vessel System on the ABI 433A Peptide Synthesizer This manual contains information that you might need to refer to from time to time so it is recommended that you insert this manual at the back of the ABI 433A Peptide Synthesizer User s Manual for future reference Contents of the Ma
14. Caution Reaction vessels are designed to be tightened by hand Use only your hands or the ABI supplied black wrench to tighten the 3 mL RV caps Figure 2 2 Use the open ended wrench to tighten the 3 mL RV caps 2 6 2 Introduction November 2001 Applied Biosystems 3 Add the appropriate amount of resin to the 3 mL RV Figure 2 3 Refer to Section 4 Chemistry for resin quantity specifications Figure 2 3 Filling the 3 mL RV with resin 4 Place a filter on the knife edge of the open end of the 3 mL RV Tightly screw on the cap using the procedure described in step 2 Place the RV in the RV holder on the synthesizer Use the black open ended wrench to tighten the 3 mL RV caps November 2001 2 Introduction 2 7 Applied Biosystems November 2001 Installing the Variable Measuring Loop The ABI 433A is built using a 0 5 mL fixed measuring loop to deliver the solutions from Bottles 7 and 8 The Variable Measuring Loop kit contains the tubes required to change the fixed measuring loop to one that can deliver either 0 5 mL for 0 10 0 25 0 50 and 1 0 mmol cycles or 0 125 mL for 5 10 or 20 umol cycles Perform the entire installation procedure to calibrate both measuring loops IMPORTANT Once you install the Variable Measuring Loop you must always use the new cycles included on the floppy disk The old cycles will not function properly with the Variable Measuring Loop hardware Equipment Required Wre
15. Module information 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems November 2001 4 Chemistry The cycles provided for the 5 10 and 20 pmol scales use the HBTU activation strategy for both Fmoc and Boc chemistries This approach is equivalent to Applied Biosystems FastMoc chemistry for the higher scales However we refer to these cycles as either Fmoc or Boc cycles because modules from both chemistries are present within a single chemistry file This is useful when both Fmoc and Boc deprotections are required for one synthesis The 3 mL Reaction Vessel System uses five equivalents of amino acid monomer which is predissolved in NMP before being added to an empty amino acid cartridge Activation occurs through the use of HBTU located in Bottle 8 and DIEA located in Bottle 7 The measuring loop for Bottles 7 and 8 is 0 125 mL and the concentration of the DIEA solution in Bottle 7 and the HBTU solution in Bottle 8 varies according to the scale of synthesis The amounts of the various reagents are summarized in Table 4 1 on page 4 2 In the Boc cycle neutralization and coupling are performed simultaneously This process is often called in situ neutralization and is possible because the HBTU activation uses excess DIEA The only different Boc derivative that should be used with this approach is Boc Asn Xan or Boc Asn Trt instead of Boc Asn 1 Schn lzer M Alewood P Jones A Alewood D and
16. for 2 minutes The conductivity of the deprotection solution is measured and the resin is drained This process is continued for at least three deprotections At the end of the third deprotection the value of the second and third deprotections are compared to see if they are within the limits defined by step 54 End loop monitoring A value of 25 in step 54 means that if the third deprotection is within 2 5 of the second deprotection then no more deprotections will occur Three to four deprotections will occur Module I Vortex Total time 5 0 minutes The reaction vessel is vortexed for 5 minutes Use this module to extend the coupling or capping Module a Read Cartridge Total time 0 9 minutes The cartridge name is read the old cartridge is ejected and the new cartridge is advanced Use this module when the material is already in its activated form such as when using fluorescein isothiocyanate The material must be dissolved in at least 0 36 mL of solvent The number of equivalents is user determined and may depend on the expense and availability of the material as well as the efficiency of the reaction 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Module b Piperidine Deprotection no monitoring Total time 8 7 minutes The resin is washed one time with NMP A 20 piperidine NMP solution is introduced and allowed to deprotect for 2 minutes The RV is drained anda second treatment with 20 pip
17. leaks If you find a leak tighten the connection with your fingers 5 Weigh the cartridge containing the NMP from the 11 step module Consult Table 3 2 to determine whether the weight falls within the specified range Table 3 2 0 5 mL measuring loop weight specification Weight of NUP Resulting Volume 4 loops 1 loop 2 037 2 095 g 0 493 0 507 mL 6 Run flow test b repeatedly until you achieve three successive results that are consistent in their weight measurements After the results are consistent then go on to step 7 7 Determine how much the NMP is over the desired weight and use this information to determine approximately how much of the blue tube to trim The example below shows how to calculate the amount of tube to cut The linear volume of the blue tube is about 0 005 mL per DAS UR centimeter J 25 gi ao middle S valve B o block B e 284 N yellow D o tube short red sil 5 _ 604128 tube 4 604130 1 K 3 ie gt E cross flange A lt fitting coupling 110070 Note all tube long red lengths not blue tube tube necessarily not to scale 604129 drawn to scale 604127 Figure 3 7 New 0 5 mL measuring loop configuration 3 10 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems WARNING Wear protective gloves when handling tubes that may contain even small amounts
18. other capping solution often used in Peptide Nucleic Acid PNA synthesis is a 1 25 25 mixture of acetic anhydride pyridine NMP Note Make both capping solutions fresh each week Bottle 7 DIEA solution The concentrations of DIEA for different scales of synthesis and their preparation using 2 M DIEA P N 401517 are shown in Table 4 4 Table 4 4 DIEA solutions Scale DIEA Preparation 5 umol 0 40 M 40 mL 2 M DIEA diluted to 200 mL in NMP 10 0 80 M 80 mL 2 M DIEA diluted to 200 mL in NMP 20 umol 1 60 M 160 mL 2 M DIEA diluted to 200 mL in NMP 4 Chemistry 4 5 Applied Biosystems 4 6 Bottle 8 HBTU solution Note Before running any synthesis verify that the tube for Bottle 8 has the HBTU filter installed The HBTU solution can be made with or without the additional HOBt For peptide synthesis it is usually made with the additional HOBt according to the directions on the HBTU Activation kit P N 401132 This gives a 0 45 M HBTU 0 45 M HOBt solution which should be diluted with NMP to give the desired solution as shown in Table 4 5 on page 4 6 Table 4 5 HBTU solutions using 0 45 M HBTU HOBt Scale HBTU HBTU HOBt Preparation Volume 5 umol 0 19M Dilute 84 mL 0 45 M HBTU HOBt 1 X 0 125 mL 0 125 mL to 200 mL with NMP 10 umol 0 38 M Dilute 169 mL 0 45 MHBTU HOBt 1 X 0 125 mL 0 125 mL to 200 mL with NMP 20 umol 0 38 M Dilute 169 mL 0 45 MHBTU HOBt 2 X 0 125 mL 0 250 mL to 200 mL with NMP
19. sampling functions hazardous solvents such as DCM NMP or DMF may squirt out of the resin sampler bulkhead fitting AT EYE LEVEL Always wear protective lab coat chemical resistant gloves and safety goggles To assemble the 3 mL RV 1 Hold the RV in a vertical position and place an RV filter on the protruding knife edge found just inside the openings at either end of the RV Figure 2 1 The filter forms a seal with the knife edge when the RV cap is screwed in place reaction vessel RV cap filter knife edge reaction vessel RV Figure 2 1 Placing RV filter on inner knife edge of reaction vessel 2 Screw on the RV cap making sure to hold the RV in a vertical position at all times November 2001 2 Introduction 2 5 Applied Biosystems Caution Hold the RV in a vertical position when screwing on the RV cap If you turn the RV on its side while tightening its cap the filter may become crooked and form an imperfect seal As a result resin may escape and clog the in line filter Tighten the cap until you feel a firm resistance This resistance indicates that the primary seal is forming between the filter and the recessed knife edge Visually check the filter placement by looking through the open end of the RV The surface of the filter should be flat and smooth with no protrusions beyond the knife edge Use the black open ended wrench to tighten and loosen both caps on 3 mL RV see Figure 2 2
20. that it is complete Check the packing list s in the 3 mL Reaction Vessel System kit to verify that all the pieces are included before you begin any installation procedure If your kit is missing any parts contact Applied Biosystems Technical Support Refer to Technical Support on page 1 3 3 Inspect the RV Caution The pieces of the 3 mL 8 mL Reaction Vessels are not interchangeable Do not mix parts from the two Reaction Vessels Because the 3 mL Reaction Vessel RV is similar in shape to the 8 mL RV the top and bottom caps of the 3 mL RV both have distinctive markings for easy recognition the top cap of the 3 mL RV has 3 rings and the bottom cap has 3 dots 4 Copy the new software modules to your hard disk To copy the new software modules drag the Variable Loop Folder from the disk in the kit onto your hard drive Put the new cycles in the SynthAssist chemistry folder 2 Introduction 2 3 Applied Biosystems 2 4 IMPORTANT Once you install the Variable Measuring Loop you must always use the new cycles included on the disk in the kit The old cycles will not function properly with the Variable Measuring Loop hardware 5 Remove the old cycles from the Macintosh hard drive The old cycles will not function properly with the new Measuring Loop hardware Always use the new cycles from this point on 6 Install the Variable Measuring Loop For the procedure describing how to install the Variable Measuring Loop
21. 0 NMP Washes 3 times 1 4 Cycle Time for Boc chemistry Approximately 50 minutes Solvent Consumption One cycle consumes a total volume of approximately 60 mL of solvent Each of the three scales of synthesis use the same quantity of solvent One reason for this is that the quantity of solvent needed to wash the 3 mL RV and amino acid cartridge are the same for each scale In addition the quantities of resin used in the 5 10 and 20 umol scale syntheses are so small that the solvent used to wash the RV and cartridge is more than enough to wash the resin November 2001 4 Chemistry 4 3 Applied Biosystems Reagent and Bottle Positions WARNING CHEMICAL HAZARDS Chemicals used on the ABI 433A can be hazardous and cause injury illness or death Become completely familiar with the Material Safety Data Sheet MSDS for each hazardous chemical before attempting to operate the instrument or use the reagents MSDSs are provided in the Safety Supplement of the ABI 433A User s Manual When working with hazardous chemicals wear all appropriate safety attire listed in the MSDSs To minimize inhalation of the chemicals do not leave any chemical bottles uncapped IMPORTANT Cartridges swell after extended contact with solvents such as NMP and DCM After only a single synthesis cycle a cartridge can swell enough to exceed the recommended cartridge size Reusing a cartridge can result in the cartridge becoming stuck in the autosampler an
22. 2 6 3 Vortex reaction vessel off 1 7 42 Drain reaction vessel to waste 5 8 41 Vent reaction vessel 2 9 50 Flow NMP through reaction vessel to waste 5 10 42 Drain reaction vessel to waste 10 11 56 Deliver NMP to reaction vessel 2 12 79 Pressurize piperidine 10 13 51 Deliver piperidine to reaction vessel 3 14 56 Deliver NMP to reaction vessel 2 15 40 Mix reaction vessel 2 16 2 Vortex reaction vessel on 120 17 3 Vortex reaction vessel off 1 18 42 Drain reaction vessel to waste 10 19 56 Deliver NMP to reaction vessel 2 20 79 Pressurize piperidine 5 21 51 Deliver piperidine to reaction vessel 3 22 56 Deliver NMP to reaction vessel 2 23 40 Mix reaction vessel 2 24 2 Vortex reaction vessel on 300 25 3 Vortex reaction vessel off 1 26 42 Drain reaction vessel to waste 5 27 41 Vent reaction vessel 2 28 50 Flow NMP through reaction vessel to waste 5 29 42 Drain reaction vessel to waste 10 5 Cycles and Modules 5 21 Applied Biosystems Module c Final DCM Washes Step Fxn Name Time 1 1 Wait 1 2 12 Flush bottom valve block with DCM to waste 1 3 9 Flush top valve block with gas to waste 2 4 10 Flush bottom valve block with gas to waste 2 5 98 Begin loop UPPER 6 6 55 Deliver DCM to reaction vessel 5 7 40 Mix reaction vessel 2 8 2 Vortex reaction vessel on 1 9 40 Mix reaction vessel 2 10 1 Wait 5 11 3 Vortex reaction vessel off 1 12 42 Drain reaction vessel to waste 5 13 41 Vent reaction vessel 2 14 49 Flow DCM through reaction ve
23. 6 46 23 Transfer measuring loop to Act 3 10 48 23 Transfer measuring loop to Act 3 5 5 Cycles and Modules 5 25 Applied Biosystems Fmoc HOBt DCC 0 25 mmol Module a Activation Time sec Step Fxn Name Old New 16 68 Deliver 7 to measuring loop 3 6 19 63 Transfer measuring loop to Cart 6 10 21 63 Transfer measuring loop to Cart 6 5 54 69 Deliver 8 to measuring loop 3 6 55 23 Transfer measuring loop to Act 3 10 57 23 Transfer measuring loop to Act 3 5 Module h Loading Time sec Step Fxn Name Old New 45 69 Deliver 8 to measuring loop 3 6 46 23 Transfer measuring loop to Act 3 10 48 23 Transfer measuring loop to Act 3 5 Boc HOBt DCC 0 10 mmol Module a Activation Time sec Step Fxn Name Old New 21 68 Deliver 7 to measuring loop 3 6 23 63 Transfer measuring loop to Cart 6 10 61 69 Deliver 8 to measuring loop 3 6 62 23 Transfer measuring loop to Act 3 10 64 23 Transfer measuring loop to Act 3 5 Boc HOBt DCC 0 50 mmol Module a Activation Time sec Step Fxn Name Old New 34 70 Flush bottom valve block with loop contents to waste 2 6 37 68 Deliver 7 to measuring loop 3 6 40 63 Transfer measuring loop to Cart 5 10 73 70 Flush bottom valve block to waste 2 6 76 69 Deliver 8 to measuring loop 3 6 78 23 Transfer measuring loop to Act 2 10 80 23 Transfer measuring loop to Act 2 5 82 23 Transfer measuring loop to Act 2 5 5 26 5 Cycles and Modules November 2001 Applied Biosystems Flow Tests Fol
24. 9 115 230 460 Boc Tyr Br Z OH 494 4 2 560 0 128 123 246 492 November 2001 4 Chemistry 4 11 Applied Biosystems 4 12 Peptide Nucleic Acids Recommended Modifications Peptide Nucleic Acids PNAs are DNA analogues with a polyamide backbone consisting of an uncharged 2 aminoethylglycine aeg unit instead of the charged ribose phosphate backbone of DNA The first publication on PNAs was in 19911 At the time of writing of this manual February 1996 there have been over 75 papers on the synthesis and use of PNAs The disk contained in the 3 mL Reaction Vessel kit contains special cycles designed specifically for synthesizing PNAs These cycles are based on modifications and improvements provided by PNA Diagnostics Copenhagen who have been synthesizing PNAs on the ABI 433A with the 3 mL RV since November 1994 As new improvements are made in the PNA cycles you should include these improvements in your syntheses PNA Diagnostics has recommended the following changes which are included in the 5 umol 10 umol and 20 pmol PNA cycle files l After you add the DIEA allow for a 60 second activation period of the PNA monomer To accomplished this extend the time in module A step 23 from 5 seconds to 60 seconds It may be that the activation of the PNA takes more time than amino acids 2 Additional DCM washes are necessary between the TFA deprotection and the NMP washes To make this change a module G was added
25. A acetic anhydride in capping solution 45 activation 4 1 amino acid adding to cartridge 47 solutions using five equivalents 4 9 solutions using ten equivalents 4 11 B Boc cycle times 43 5 8 in situ neutralization 41 bottle position capping solution 4 5 DCM 46 DIEA 45 HBTU 46 NMP 46 piperidine 44 TFA 44 waste container 4 6 bracket vortexer 2 3 C calibration 3 6 capping solutions 4 5 cartridge adding amino acids 4 7 reusing 4 4 conductivity cell 2 4 monitoring 5 9 coupling concentration 42 volume 4 2 customer support See technical support 1 3 cycle times Boc 43 5 8 Fmoc 42 5 8 November 2001 cycles see also modules Boc HOBt DCC 0 10 mmol 5 26 Boc HOBt DCC 0 5 mmol 5 26 combination of Boc and Fmoc 5 10 FastMoc 0 10 amp 0 25 mmol 5 24 FastMoc 1 0 mmol 5 25 final deprotection 5 7 final deprotection with acetylation 5 7 Fmoc amp Boc 5 6 Fmoc HOBt DCC 0 10 mmol 5 25 Fmoc HOBt DCC 0 25 mmol 5 26 grouped by coupling 5 6 in SynthAssist 5 1 installing new software 2 3 modifications 5 8 on floppy disk 5 1 single couple 5 6 single couple with capping 5 6 5 7 using activated material 5 7 using new 0 5 meas loop 5 24 D DCM bottle position 4 6 deprotection calculation 5 11 monitoring 5 9 diagram plumbing A 1 dictionary 5 12 DIFA bottle position 4 5 concentration 42 4 5 in capping solution 45 Documents on Demand 1 7 downloading see resin Index
26. ABI 433A Peptide Synthesis 3 mL Reaction Vessel User s Manual E Copyright 2001 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures ABI PRISM the ABI PRISM design Aquapore Applied Biosystems Brownlee GeneScan INHERIT Masterpiece MicroCoat MPLC NEWGUARD POLYPORE Precipitette ProBlott ProSort ProSpin SeqEd SPHERI10 SPHERIS SynthAssist and VeloSep are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries Amplicover Anitron AutoAssembler BaseSprinter Biobytes CATALYST FastPhoramidite GeneAssist Genotyper HLP Hot Start ONESTEP PCR MATE PDQ Phosphalink PROCISE ProFocus Sequence Navigator StockMarks Stretch and Synergy are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries All other trademarks are the sole property of their respective owners Printed 11 2001 Applied Biosystems November 2001 Contents 1 About This Manual Contents of the Manual User Attention Words Technical Support Contacting Technical Support To Contact Technical Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax To Reach Technical Support Through the Internet To Obtain Documents on Demand 2 Introduction About the 3 mL Reaction Vessel System 3 mL Reaction Vessel 3 mL RV Installation Overview How to
27. E e mail address for technical support 1 3 equivalents calculating 4 7 F filter HBTU 46 Fmoc cycle times 42 5 8 on HMP resins 4 6 folders on floppy disk 5 1 G gas assisted tubing removal 3 3 glycosylated amino acids 4 10 H HBTU activation 4 1 bottle position 4 6 concentration 42 4 6 filter 4 6 solutions 4 6 with HOBt 46 help See technical support 1 3 HOBt in capping solution 4 5 I in situ neutralization 4 1 Internet address Documents on Demand 1 7 K kit feedback monitoring 2 1 M manual content descriptions 1 1 User Attention Words 1 2 modifying cycles 5 8 Applied Biosystems modules see also cycles A Read Cart amp Add HBTU DIEA 5 3 5 13 a Read Cartridge 5 4 5 21 b Piperidine Deprotection 5 5 5 21 B TFA Deprotection 5 3 5 14 C Capping 5 3 5 16 c Final DCM Washes 5 5 5 22 descriptions 5 3 D NMP Wash 5 3 5 16 d NMP Wash from Activator 5 5 5 22 E Read Cartridge amp Add Double HBTU DIEA 5 3 5 16 DIEA Neutralization 5 5 5 23 F Transfer Clean amp Couple 5 4 5 18 G DCM Washes 5 4 5 19 h Cond Deprotec Prev Peak 5 5 5 23 H Piperidine Deprotection 5 4 5 19 i Conditional Vortex 5 5 5 23 installing new software 2 3 5 4 5 20 monitoring 2 4 5 9 monomer concentration 4 2 dissolving 4 7 PNA 2 1 5 12 solutions 4 14 storage 4 7 table of volumes 4 9 N neutralization and coupling 4 1 NMP bottle position 4 6 density 3 6 in capping solut
28. Figure 3 3 Measuring loop tube with hex nut installed 2 Attach this short red tube to Valve 13 on the 11 port Valve Block Figure 3 4 Use only your fingers to tighten the fitting Caution Do not over tighten the measuring loop fitting Damage to the ferrule from over tightening can cause leaks in the measuring loop 3 After the fitting is finger tight use the 5 16 inch wrench to tighten the fitting a maximum of one quarter turn more Tighten the metal hex nut finger tight 4 Attach the new long red tube labeled ASSY TUBE CPLG TO X FTG P N 604129 to the cross fitting Figure 3 4 again using your fingers Note You will trim the long red piece of tube to calibrate the 0 125 mL measuring loop in step 3 below A spare piece of tube is provided in the Tubing Kit 5 Connect the two red tubes P N 604129 and 604130 with a 1 4 28 flange coupling P N 110070 Figure 3 4 using only your fingers until the connections are tight 6 Remove the existing tube that connects the cross fitting to Angar Valve 29 and replace it with the new yellow tube labeled ASSY TUBE X FTG TO VALVE 29 P N 604128 Figure 3 4 Be sure the fitting is straight as you screw it into the Angar valve to avoid cross threading 7 After the fitting is finger tight use the 5 16 inch wrench to tighten the fitting one quarter turn more 3 Installing the Variable Measuring Loop 3 5 Applied Biosystems i middle valve i bl
29. Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time 1 About This Manual 1 3 Applied Biosystems To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Dial Fax Dial ABI PRISM 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM9877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32
30. Kent S B H 1992 n situ neutralization in Boc chemistry solid phase peptide synthesis nternational Journal of Peptide amp Protein Research 40 180 193 4 Chemistry 4 1 Applied Biosystems Table 4 1 Concentration and amounts of reagents Resin 5 pmol Monomer 25 umol 0 110 mL HBTU 019 M HBTU X 0 125 mL 23 0 umol DIEA 0 40 M DIEA X 0 125 mL 50 Coupling Volume 0 35 mL Coupling Concent 0 0 68 10 umol 50 0 220 mL 0 38 M HBTU X 0 125 mL 47 5 umol 0 80 M DIEA X 0 125 mL 100 umol 0 47 mL 0 100 M X X 20 0 1 mmol 100 0 440 mL 0 38 M HBTU 0 250 mL 95 umol 1 60 M DIEA 0 125 mL 200 0 69 mL comparison 0 138 M 0 167 M Cycle Times The cycle time for both the Fmoc and Boc cycles is approximately 50 minutes The cycles and their durations are listed in Table 4 2 and Table 4 3 Table 4 2 Fmoc Cycle with 3 mL RV Module Total Time min Piperidine Deprotection 2 times NMP Washes 6 times Activate monomer Coupling Capping NMP Washes 3 times Cycle Time for Fmoc chemistry 7 0 2 9 0 3 35 1 0 1 4 Approximately 50 minutes 4 Chemistry November 2001 Applied Biosystems Table 4 3 Boc Cycle with 3 mL RV Module Total Time min DCM Wash 1 time 0 9 TFA Deprotection 2 times 6 0 DCM Wash 1 time 0 5 NMP Washes 6 times 2 9 Activate monomer 0 3 Coupling 35 Capping 1
31. L bottle and place the bottle in the Bottle 7 position Turn on Fxn 68 and let the ABI 433A run until all the NMP has been removed from Bottle 7 Turn off Fxn 68 Turn on Fxn 70 for about 10 seconds 4 Add 25 mL NMP to Bottle 7 and 8 then run Flow Test 17 and 18 Expected Found Flow Test 17 amp 18 0 515 to 0 554 g to cartridge 5 Empty both Bottles 7 and 8 by turning on Fxn 68 and 69 until the Bottles are empty 6 Turn on Fxn 70 for about 10 seconds 3 2 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems November 2001 Installation Procedure Removing the existing 0 5 mL measuring loop tube The existing 0 5 mL tube is located between Valve 13 the 11 port Valve Block and the Teflon cross fitting Figure 3 2 To remove the 0 5 mL measuring loop 1 Ifyou have not already done so remove the right side panel from the instrument Refer to Figure 3 1 to identify the correct panel to remove d eet 200 a 250065 AG 055 right side panel user accessible Figure 3 1 ABI 431 433 showing location of right side user accessible panel 2 Remove the end of the 0 5 mL loop from the cross fitting Figure 3 2 To find which tube to remove locate valve 13 on the middle valve block trace that tube back to the cross fitting then remove the tube end from the cross fitting 3 At the middle valve block loosen the metal h
32. a 0 125 mL 0 50 mL Variable Measuring Loop e SynthAssist disk containing the Variable Measuring Loop Cycles e boxes of empty amino acid cartridges e this manual The cycles use HBTU activation strategy for both Fmoc and Boc chemistries This approach is equivalent to Applied Biosystems FastMoc cycles when using Fmoc amino acids However we refer to these cycles as either Fmoc or Boc cycles because modules from both chemistries are present within a single chemistry file This is useful when both Fmoc and Boc deprotections are required for one synthesis The 3 mL Reaction Vessel System is designed primarily for installation in the ABI 433A Peptide Synthesizer The 3 mL Reaction Vessel System will also function on the ABI 431A Peptide Synthesizer if equipped as follows e The ABI 431A has the Feedback Monitoring kit e The ABI 431A has the vortexer bracket that accepts RTF style Recessed Tab Filter reaction vessels For information about whether your instrument accepts RTF style reaction vessels see 3 mL RV Installation Overview on page 2 3 Note For information about upgrading or retrofitting an ABI 431A contact Applied Biosystems Technical Support Refer to Technical Support on page 1 3 2 Introduction 2 1 Applied Biosystems 3 mL Reaction Vessel 3 mL Reaction Vessel Part Number 402776 lt gt Filter Box of 30 Part Number 401524 Use with 5 umol 10 umol and ae 20 pmol scale syntheses 2
33. a 5 pmol scale synthesis is being performed you need 25 pmol of monomer 5 equivalents b Ifyou use Fmoc Ala MW 311 3 then the calculation using the formula above is as follows 311 3 X 025 78mg c According to the calculation 7 8 mg of Fmoc Ala is needed for a 5 junol scale synthesis The formula for calculating the quantity of NMP required to dissolve the monomer is monomer quantity X Vol of solvent per mmol Volume of NMP in mmol To calculate the quantity of NMP required a Usean amount of NMP that equals 4 mL 1 mmol of monomer b Ifyou use Fmoc Ala the quantity of NMP required is determined as follows 0 025 mmol 0 10 According to the calculation 0 10 mL of NMP is required for a 5 nmol scale synthesis using Fmoc Ala If the same monomer is used several times then a larger amount of material can be dissolved The resultant monomer solution can be refrigerated and stored for a week Details for this are given in the next section November 2001 4 Chemistry 4 7 Applied Biosystems 4 8 Bulk Amino Acid Solutions When the same monomer is used several times per week a larger amount of solution can be prepared Two calculations are required one for millimoles of amino acid and one for quantity of solvent To calculate the number of mmoles of amino acid in 0 25 g Fmoc Ala grams of monomer MW 0 25 g 75 1009 1000 no of mmol 0 803 mmol To calcu
34. able Measuring Loop 3 11 Applied Biosystems 7 Reinstall the long red tube between the flange coupling and cross fitting 8 Rerun flow test b to determine whether the measuring loop is within specification Table 3 2 If further calibration is required follow this procedure again 9 Check one more time all the tube connections you have made to ensure that there are no leaks 10 Reinstall the inline filter you removed earlier step 2 on page 3 6 11 Run flow tests and d to check the variable measuring loop These tests will let you verify that the measuring loop fills within the correct time The 0 125 mL loop should fill within three seconds and the 0 50 mL measuring loop should fill within five seconds Module c is for Bottle 7 and module d is for Bottle 8 see page 5 28 for details The Variable Measuring Loop is now installed and calibrated Table 3 3 shows which tubes to use for each scale synthesis Table 3 3 Measuring Loop Configurations synthesis scale 5 umol 10 umol 20 umol 0 1 mmol 0 25 mmol 0 5 mmol 1 0 mmol short red tube e e e e e e e long red tube e e e e e e e blue tube removed removed removed e e e e use this tube for synthesis Note When you remove the blue tube for the 5 10 and 20 umol scales also remove one of the flange couplings then connect the two red tubes together Refer to Section 4 for Chemistry information or Section 5 for Cycle and
35. artridge 5 60 5 60 5 60 For PNA cycles step 23 Mix Cartridge is 60 seconds 5 Cycles and Modules 5 13 Applied Biosystems Module B TFA Deprotection Step Fxn Name Time 1 1 Wait 1 2 12 Flush bottom valve block with DCM to waste 1 3 55 Deliver DCM to reaction vessel 4 4 40 Mix reaction vessel 2 5 2 Vortex reaction vessel on 1 6 10 Flush bottom valve block with gas to waste 6 7 3 Vortex reaction vessel off 1 8 73 Vent TFA without gas 2 9 75 Vent TFA with gas 2 10 76 Pressurize TFA 15 11 11 Flush top valve block with DCM to waste 2 12 9 Flush top valve block with gas to waste 3 13 42 Drain reaction vessel to waste 10 14 49 Flow DCM through reaction vessel to waste 10 15 42 Drain reaction vessel to waste 10 16 41 Vent reaction vessel 2 17 72 Deliver TFA to reaction vessel 15 18 40 Mix reaction vessel 2 19 1 Wait 20 41 Vent reaction vessel 21 12 Flush bottom valve block with DCM to waste 22 55 Deliver DCM to reaction vessel 23 41 Vent reaction vessel 24 11 Flush top valve block with DCM to waste 25 45 Deliver DCM to reaction vessel top 26 2 Vortex reaction vessel on 27 9 Flush top valve block with gas to waste 28 10 Flush bottom valve block with gas to waste 29 73 Vent TFA without gas 30 74 Back flush TFA 31 73 Vent TFA without gas 32 13 Flush top valve block with NMP to waste 33 14 Flush bottom valve block with NMP to waste 34 12 Flush bottom valve block with DCM to waste 35 11 Flush top valve block with DCM to
36. at Internet for fax or http www appliedbiosystems com techsupp e mail delivery b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery 1 8 1 About This Manual November 2001 Applied Biosystems November 2001 2 Introduction About the 3 mL Reaction Vessel System The 3 mL Reaction Vessel System makes it possible for the ABI 433A to synthesize peptides and peptide analogues on the 5 10 and 20 umol scale These scales of synthesis are very useful when using expensive monomers such as glycosylated amino acids isotopically labeled amino acids and peptide nucleic acid PNA monomers The 3 mL Reaction Vessel System is established by installing the 3 mL Reaction Vessel kit P N 402067 which contains the following components 3 mL Reaction Vessel 3 mL RV e tubing kit containing tubing required to make
37. bDAFd 4 Tyr Fmoc Depro Single bDAFd 5Asp Fmoc Depro Single bDAFd 6 lle Fmoc Depro Single bDAFd 7 Ala Fmoc Depro Single bDAFd 8 Ala Fmoc Depro Single bDAFd 9 Gin Fmoc Depro Single bDAFd 10 Val Fmoc Depro Single bDAFd 11 Fmoc Final Depro bDc 4 18 4 Chemistry November 2001 Applied Biosystems Cleavage 6 8 mg of resin was treated with 200 pL of 90 TFA 5 EDT 2 5 thioanisole and 2 5 20 for 2 hours Filter resin precipitate peptide in 10 mL methyl t butyl ether centrifuge decant redissolve peptide 0 5 mL TFA reprecipitate in 10 mL ether centrifuge decant dissolve peptide in 10 acetic acid and lyophilize Weight 1 8 mg theory 2 5 mg HPLC Performed on an ABI 130A Micro Separation System equipped with an Aquapore OD 300 C18 300 A pore size 7 particle size reverse phase column Flow rate of 250 pL min with a gradient of 5 60 B in 45 minutes where buffer is 0 10 TEA H20O and buffer B is 0 08 acetonitrile Detection was at 214 nm 19 79 LELLEL GL GG Gg SL Gg GL gZ FEL LB BL BLBLo pp Lp 1 bgggrglgrgrlgrgrgrgrgrgrgrrgrgrrrgrgragg wn N ev lt Figure 4 1 HPLC results November 2001 4 Chemistry 4 19 Applied Biosystems 5 Cycles and Modules Cycles in SynthAssist 2 0 Software The disk provided with the 3 mL Reaction Vessel Kit contains new cycles written in SynthAssist software that use the Variable Measuring Loop Figure 5 1 shows the folder structure
38. bout This Manual 1 5 Applied Biosystems Region Telephone Dial Fax Dial South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 United Kingdom Warrington 44 0 1925 825650 44 0 1925 282502 Cheshire All other countries not listed 44 0 1925 282481 44 0 1925 282509 Warrington UK Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 1 About This Manual November 2001 Applied Biosystems To Reach Technical Support Through the Internet We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products You can also order technical documents or an index of available documents and have them faxed or e mailed to you through our site The Applied Biosystems Web site address is http www appliedbiosystems com techsupp To submit technical questions from North America or Europe Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting
39. d Boc Cycles PrPk previous peak monitoring Boc Depro Single Boc Depro Single cap Boc Depro Single 10 eq Boc Depro Single 10 eq cap Boc Depro Single no 7 amp 8 Boc Final Depro Boc Final Depro amp Acetylation Fmoc Depro Single Fmoc Depro Single cap Fmoc Depro Single 10 eq Fmoc Depro Single 10 eq cap Fmoc Depro Single no 7 amp 8 Fmoc Final Depro Fmoc Final Depro amp Acetylation PrPk Fmoc Depro Single cap PrPk Fmoc Depro Single 10 eq cap PrPk Fmoc Depro Single no 7 amp 8 PrPk Fmoc Final Depro PrPk Fmoc Final Depro amp Acetylation NMP Wash Final Wash Cycles grouped by coupling BDAFd BDAFCd BDEFd BDEFCd BDfDaFIllld BDc BDCCIDc bDAFd bDAFCd bDEFd bDEFCd bDaFllId bDc bDCCIDc HhDAFiiiCidD HhDEFiiiCidD HhDaFilliiidD HhDc HhDCCIiDc D following cycles single coupling with five equivalents of monomer Boc Depro Single Fmoc Depro Single BDAFd bDAFd The following cycles are single coupling with five equivalents of monomer followed by capping with a pre mixed solution of acetic anhydride Boc Depro Single cap Fmoc Depro Single cap PrPk Fmoc Depro Single cap BDAFCd bDAFCd HhDAFiiiCidD The following cycles are single coupling with 10 equivalents of monomer Boc Depro Single 10 eq Fmoc Depro Single 10 eq 5 6 5 Cycles and Modules BDEFd bDEFd November 2001 Applied Biosystems The following cycles are single coup
40. d shutting down your synthesis Bottle 1 Piperidine P N 401750 Piperidine is used for Fmoc removal Even if only Boc cycles are used Piperidine or DIEA is needed in Bottle 1 because Flow Test 2 contains a step that uses Bottle 1 to neutralize the metering vessel Bottle 2 TFA P N 400137 When synthesizing peptides with Boc protected amino acids use 100 TFA in Bottle 2 When synthesizing PNAs use 95 TFA 5 m cresol in Bottle 2 Use Bottle 2 bottle seal P N 400789 when using TFA in Bottle 2 Avoid leaving TFA installed on an unused synthesizer for an extended period of time Because an unused synthesizer does not backflush the TFA tubing with nitrogen TFA fumes are in long term contact with synthesizer valves If you plan to not use TFA for an extended period of time for example two weeks or more remove the TFA bottle from the instrument See Waste Container on page 4 6 for information about neutralizing TFA in the waste container 4 4 4 Chemistry November 2001 Applied Biosystems November 2001 Bottle 4 Capping solution Two capping solutions have been used The typical capping solution for peptides is 0 5 M acetic anhydride 0 125 M DIEA and 0 015 M HOBt in NMP This is made by combining the following components 19 mL of acetic anhydride P N 400660 9mL of 100 DIEA P N 400136 or 26 mL of 2 M DIEA P N 401517 6mLof 1M HOBt NMP P N 400662 and diluting to 400 mL with NMP P N 400580 The
41. der The Flow Test folder contains three flow test files Flow Tests New VML Flow Tests 1 18 VML Flow Tests 19 23 Flow Tests New VML The Flow Tests New VML file contains 4 flow tests that are needed to install and check the variable measuring loop VML system Flow Test a Calibrate 0 125 mL VML This flow test is used when calibrating the 0 125 mL measuring loop as described on page 3 6 When running this flow test Bottle 8 is filled with NMP and the in line filter to the cartridge is replaced with a flange coupling P N 110070 After the calibration of the 0 125 mL measuring loop is completed calibrate the new 0 50 mL measuring loop However before starting a synthesis remove the flange coupling and re attach the in line filter This flow test uses an empty tared cartridge with the septum installed The flow test fills the 0 125 mL measuring loop with NMP and delivers this NMP to the cartridge 10 times Step Fxn Name Time 1 5 Needle down 10 2 78 Pressurize manifold 15 3 98 Begin Loop UPPER 10 4 69 Deliver 8 to measuring loop open 5 5 10 Flush bottom valve block with gas to waste 2 6 63 Transfer measuring loop to cartridge 10 7 99 End Loop UPPER 1 8 6 Needle up 10 9 7 Eject cartridge 10 10 70 Flush bottom valve block with loop to waste 5 11 10 Flush bottom valve block to waste 5 November 2001 5 Cycles and Modules 5 27 Applied Biosystems 5 28 Flow Test b Calibrate 0 50 mL VML This flo
42. e measuring loop and other tubes contain N methylpyrrolidone NMP Always wear protective lab coat gloves and safety goggles when handling tubes that may contain even small amounts of reagents such as NMP 3 Installing the Variable Measuring Loop 3 7 Applied Biosystems Example for calculating how much to cut 0 125 mL measuring loop weight of 10 loops of NMP delivery 1 35 g weight of 1 loop of NMP delivery s 0 1359 _NMP weight volume of 1 loop of NMP delivery NMP density 1 033 g mL difference between the volume of NMP delivery and the upper volume specification Table 3 1 0 131mL 0 127mL 0 004 mL excess amount of tube to be cut _ volumeinexcess _ 0 004 2 cm to cut linear vol of tube 0 002 mL cm To cut measuring loop to correct length 1 Disconnect the long red tube from the flange coupling and cross fitting Remove the long red tube to a secure work surface 2 Separate the ferrule white from the fitting black then slide both the ferrule and fitting several inches farther onto the tube Figure 3 5 Note When estimating how much tube to trim it is better to cut too little than to cut too much 3 Using a new single edged razor blade trim the appropriate amount of tube determined in step 9 above using a perfectly straight cut Figure 3 5 4 Slide the ferrule and fitting to within 1 inch of the trimmed end of the tube
43. e to fill the loop has been increased from 4 to 6 seconds In modules H and I the time to fill the loop has been changed from 3 to 6 seconds and the time to deliver the loop contents to the cartridge has been increased from 5 to 10 seconds Once the variable measuring loop has been installed Flow Tests 1 18 VML should be used instead of Flow Test 1 18 When Flow test 17 and 18 are used with the 0 125 mL measuring loop only one quarter of the amount of solution shown in the ABI 433A User s Manual pages 6 31 through 6 32 will be obtained Flow Tests 19 23 The Flow Tests 19 23 file is identical to the Flow Test 19 23 file that is described in the ABI 433A User s Manual This file is included on the disk for your convenience November 2001 5 Cycles and Modules 5 29 Applied Biosystems A Appendix Plumbing Schematics Installing the Measuring Loop for the 3 mL RV involves changing some of the tubes also known as plumbing The schematics on pages A 2 and A 3 show what the plumbing should look like after modifications are made November 2001 A Appendix 4 Applied Biosystems onset 602977 602977 w TO VALVE 7 eman L BLOCKS 430048 3 Figure A 1 Plumbing diagram for 0 125 mL configuration A 2 A Appendix November 2001 Applied Biosystems November 2001 Figure A 2 Plumbing diagram for 0 500 mL configuration A Appendix A 3 Applied Biosystems Index
44. ed Biosystems 4 10 If you want to perform a synthesis in which some of the monomers are very expensive such as glycosylated amino acids and the remaining monomers relatively inexpensive run the synthesis using five equivalents of the expensive monomer and ten equivalents of the inexpensive monomer When you select the activation to use five equivalents of the expensive monomer choose cycles that contain module A for example BDAFd When you select the activation to use ten equivalents of the inexpensive monomer choose cycles that contain module E for example BDEFd When you use ten equivalents of monomer you can make the monomer solution more concentrated for example 1 mmol dissolved in 2 mL NMP Table 4 8 gives the volumes for the three scales when using ten equivalents of monomers 4 Chemistry November 2001 Applied Biosystems w T r Table 4 8 Amino acid solutions when using ten equivalents 1 mmol in 2 mL NMP weight g weight volume mL of solution for of mmol 9 of So monomer 50 pmol 100 pmol 200 pmol AAand lution for amino acid MW 2mLNMP 50 pmol resin 5 10 umol 20 pmol Fmoc Ala OH 311 3 2 377 0 119 115 230 460 Fmoc Cys Trt OH 585 7 2 652 0 133 128 256 512 Fmoc Asp OtBu OH 411 4 2 477 0 124 119 238 476 Fmoc Glu OtBu OH 425 5 2 492 0 125 120 240 480 Fmoc Phe OH 387 4 2 453 0 123 118 236 474 Fmoc Gly OH 297 3 2 363 0 118 114 228 456 Fmoc His Trt OH 619 7 2 686 0 134 129 258 516 Fmoc
45. ed in this section are used for both Boc and Fmoc syntheses The TFA deprotection for the Boc removal is written in module B The piperidine deprotection for the Fmoc removal is written in module b when there is no monitoring and in modules H and h when there is conductivity monitoring Module A Read Cartridge and add HBTU and DIEA Total time 1 2 minutes The amino acid cartridge name is read the old cartridge is ejected and the new cartridge is advanced HBTU 0 95 equiv is added to the cartridge For the 5 pmol cycles 0 125 mL of 0 19 HBTU is added for the 10 umol cycles 0 125 mL of 0 38 M HBTU is added for the 20 umol cycles 0 250 mL 2 loops of 0 38 M HBTU is added Next the DIEA solution is added to the cartridge to initiate activation of the carboxylic acid group The concentration of the DIEA is 0 40 M for the 5 jumol scale synthesis 0 80 M for the 10 j1mol scale synthesis and 1 6 M for the 20 pmol scale synthesis When synthesizing PNAs the 5 second activation period on step 23 is extended to 60 seconds Module B TFA Deprotection Total time 8 6 minutes The resin is washed one time with DCM then treated for 1 minute with TFA After draining the resin is treated again with TFA this time for 4 1 minutes At the end of the module there is a single DCM wash Module C Capping Total time 1 6 minutes The resin is drained the capping solution from Bottle 4 is added and the reaction vess
46. el is vortexed for 1 minute Module D NMP Washes Total time 2 9 minutes The resin is drained and washed six times with NMP Module E Read Cartridge and Add Double the Amount of HBTU and DIEA Total time 2 0 minutes This module is identical to module A except twice the amount of HBTU and DIEA is added This module is used with the less expensive amino acids Module A uses five equivalents of amino acids and module E uses 10 equivalents of amino acids 5 Cycles and Modules 5 8 Applied Biosystems 5 4 Module F Transfer Clean Cartridge and Couple Total time 35 1 minutes At the beginning of this module the activated monomer is transferred to the reaction vessel and the cartridge is washed two times with NMP This NMP is transferred to the Activator Vessel and is used later in module d After the cartridge is washed the coupling is continued You can modify the coupling time by changing the number of loops in step 29 Module G DCM Washes Total time 2 4 minutes The resin is drained and washed five times with DCM This is an optional module that you might use in the Boc cycles after the TFA deprotections In the PNA cycles the resin is drained and washed only twice see page 5 19 Module H Piperidine Deprotection Prev Peak Total time if 3 loops 12 4 minutes if 4 loops 16 3 minutes The resin is washed three times with NMP A 20 piperidine NMP solution is introduced and allowed to deprotect
47. ep 3 Fxn 1 Wait which lets you check the waste line to make sure the liquid has completely filled the measuring loop It does not matter what liquid is in Bottle 7 When the 0 125 mL measuring loop is attached the loop should fill in 3 seconds When the 0 50 mL measuring loop is attached the loop should fill in 5 seconds Step Fxn Name Time 1 78 Pressurize manifold 15 2 68 Deliver 7 to measuring loop open 6 3 1 Wait 5 4 70 Flush bottom valve block with loop to waste 10 5 10 Flush bottom valve block with gas to waste 2 6 14 Flush bottom valve block with 10 to waste 2 7 10 Flush bottom valve block with gas to waste 10 5 Cycles and Modules November 2001 Applied Biosystems Flow Test d Test Bottle 8 VML This flow test is identical to flow test c except it is to check the measuring loop when using Bottle 8 Step Fxn Name Time 1 78 Pressurize manifold 15 2 69 Deliver 8 to measuring loop open 6 3 1 Wait 5 4 70 Flush bottom valve block with loop to waste 10 5 10 Flush bottom valve block with gas to waste 2 6 14 Flush bottom valve block with 10 to waste 2 7 10 Flush bottom valve block with gas to waste 10 Flow Tests 1 18 VML The Flow Tests 1 18 VML file contains the 18 flow tests described in the 433A User s Manual These flow tests are the identical except for Module g Flow Test 7 Module h Flow Test 8 Module H Flow Test 17 Module I Flow Test 18 In modules g and h the tim
48. eridine NMP is performed for 5 minutes You can change the time in step 24 to extend the second treatment At the end of the module the resin is drained Module c Final DCM Washes Total time 5 1 minutes The resin is drained and washed six times with DCM At the end of the cycle the resin is drained for 30 seconds and the Activator Vessel is washed with DCM and drained Use this module at the end of a synthesis Module d NMP Wash from Activator Total time 1 4 minutes The NMP that was used in Module F to wash the cartridge is used in this module to wash the resin after the coupling is completed Module f DIEA Neutralization Total time 1 5 minutes The resin is washed two times with NMP and 0 125 mL of the DIEA solution Module h Conditional Piperidine Deprotection Total time 10 8 minutes This module is used only when the maximum deprotection loops have been used in module H When this module is active a 20 piperidine NMP solution is introduced and allowed to deprotect for 10 minutes The conductivity of the solution is measured at the end of the deprotection Module i Conditional Vortex Total time 5 0 minutes This module is used only when the maximum deprotection loops have been used in module H When this module is active the reaction vessel is vortexed for 5 minutes Use this module to conditionally extend the coupling or the capping 5 Cycles and Modules 5 5 Applied Biosystems Fmoc an
49. ex nut then loosen the black bushing that secures the other end of the 0 5 mL loop 4 Pull the 0 5 mL tube out of the valve block very carefully making sure that the white ferrule does not pull loose from the tube If it is difficult to pull out the tube and ferrule use the following procedure for gas assisted removal of the tube a Switch the ABI 433A to manual control use the manual control menu b Turn on Fxn 10 Gas B VB for about 10 seconds c Turn off Fxn 10 3 Installing the Variable Measuring Loop 3 3 Applied Biosystems angar valve 29 valve 13 0 5 mL measuring loop gt removed in OU Cross step 2 and 4 fitting Note all tube lengths not necessarily drawn to scale Figure 3 2 Original 0 5 mL measuring loop configuration d Open valves 17 16 and 13 to send gas through the tube e Place your fingertip over the cross fitting end of the tube to cause the gas pressure inside the tube to build up f Carefully remove the tube which should now be easier to remove because of the gas pressure assistance g Turn off the valves 3 4 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems November 2001 To connect the variable measuring loop tube 1 Locate the short red tube labeled ASSY VALVE 13 TO CLPG P N 604130 and install the metal hex nut removed in step 3 above measuring loop fitting hex nut
50. extending filling times for the measuring loop The correct cycles are in the 0 50 mL Loop Folder and they have VML in the Information Box Note If you haven t already removed the original cycles in the Chemistry folder of SynthAssist remove them from the computer now drag them into the trash then empty the trash November 2001 4 Chemistry 4 17 Applied Biosystems Test Synthesis Example Before performing a synthesis using expensive monomers make a simple peptide to verify that all the changes made to the measuring loop cycles and reagents are correct Table 4 11 shows an example of a 5 mol 65 74 synthesis using Fmoc cycles Table 4 11 5 pimol ACP synthesis using Fmoc cycles ACP 65 74 Val GiIn Ala Ala lle Asp Tyr lle Asn Gly Fmoc Gly HMP resin 0 0082 g 0 65 mmol g 0 0053 mmol Bottle 7 0 40M DIEA Bottle 8 0 19M HBTU Fmoc amino acids Use the following volumes of amino acids solutions 1 mmol amino acid 4 mL NMP Fmoc Asn Trt 114 uL Fmoc Tyr tBu 111 uL Fmoc Ala 107 uL Fmoc Val 108 uL Fmoc lle 108 uL Fmoc Asp OtBu 110 uL Fmoc Gln Trt 115 uL Final weight of resin 0 0142 g Theory 0 0155 g Cycle information Cycle 5 Type Fmoc see SynthAssist User s Manual p 4 3 Default set Default Fmoc Depro Single bDAFd Preload NMP Wash D Load None End Final Fmoc Depro bDc Cycles Amino Acid Cycle Modules 1 Gly NMP Wash D 2 Asn Fmoc Depro Single bDAFd 3 lle Fmoc Depro Single
51. g Cycle Modules Boc Depro Single BDAFd Final DCM Wash determine the substitution follow procedure located page 3 18 of the ABI 433A User s Manual PNA Monomer Solutions The Boc PNA monomers are predissolved in NMP and the appropriate amount of the solution is pipeted into a cartridge Table 4 10 gives the volume of each dissolved monomer for the 3 scales of syntheses when using 4 mL of NMP to dissolve 1 mmol of monomer All the monomers dissolve at this concentration at room temperature except Boc Gaeg Z OH which requires sonication or heating to 50 C to dissolve the solid but it will stay in solution once it has dissolved In Table 4 10 the actual density of 1 06 g mL was used to calculate the volumes of the four PNA monomers The density of NMP is 1 033 g mL The reason 5 3 equivalents of PNA monomer is used instead of the 5 equivalents used with the amino acids is to use a greater excess of monomer over the uronium activator Table 4 10 PNA Monomer solutions in NMP when using 5 3 equivalents weight g weight g volume pL of solution for of mmol of solu monomer 26 5 53 pmol 106 pmol AA and tion for amino acid MW 4mLNMP 26 5 umol resin 5 10 umol 20 pmol Boc Aaeg Z OH 527 5 4 660 0 123 116 232 464 Boc Caeg Z OH 503 5 4 636 0 122 115 230 460 Boc Gaeg Z OH 543 5 4 676 0 123 116 232 464 Boc Taeg OH 348 4 4 480 0 118 112 224 448 4 14 4 Chemistry Nove
52. heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not already done so then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts within 24 to 48 hours To Obtain Documents on Demand Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail or by download from our Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http www appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number c Use the index number when requesting documents following the procedures below by phone for fax delivery a From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request November 2001 1 About This Manual Applied Biosystems To order documents Then through the a Access the Applied Biosystems Technical Support Web site
53. ide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AS Applied NP Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 11 2001 Part Number 904323B an Applera business
54. ion 4 5 P peptide nucleic acid PNA background 4 12 dictionary 5 12 monomer solutions 4 14 monomers 2 1 peptide test 2 4 piperidine bottle position 4 4 plumbing schematic 1 PNA see peptide nucleic acid pyridine in capping solution 4 5 R reagents concentrations of 4 2 recessed tab filter 2 3 resin concentration 42 lowering substitution 4 13 RTF see recessed tab filter 5 schematic plumbing A 1 solvent consumption 4 3 SynthAssist 5 1 dictionary 5 12 synthesis example 4 18 setup checklist 4 15 test 2 4 418 T technical support 1 3 1 8 e mail address 1 3 Internet address 1 7 telephone fax 1 4 1 6 test synthesis 2 4 TFA bottle position 4 4 3mL RV assembly 2 5 caps marks on 2 3 closing and tightening 2 5 filter 2 2 2 5 installation checklist 2 3 non interchangeability 2 3 system requirements 2 1 tubing gas assisted removal 3 3 Index U User s Manual see manual V variable measuring loop 0 125 mL calibra ng 3 6 configuration 3 6 0 5 mL calibra ng 3 9 configuration 3 10 equipment required 3 1 folder 5 2 installation procedure 3 3 tubing connecting 3 5 gas assisted removal 3 3 removing 3 3 vortexer bracket 2 3 w waste container 4 6 WWW address Applied Biosystems 1 7 Documents on Demand 1 7 November 2001 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldw
55. ions are not implemented 1 About This Manual November 2001 Applied Biosystems November 2001 Technical Support Contacting Technical Support You can contact Applied Biosystems for technical support by telephone or fax by e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below To Contact Technical Support by E Mail Contact technical support by e mail for help in the following product areas Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing corelab appliedbiosystems com Peptide and DNA Synthesis Biochromatography PerSeptive DNA tsupport appliedbiosystems com PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers LC MS apisupport sciex com or Applied Biosystems MDS Sciex api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com Hours for Telephone Technical Support In the United States and Canada technical support is available at the following times Product
56. late the quantity of NMP required for 0 25 g Fmoc Ala no of mmol X vol of solvent per mmol solvent required 0 803 mmol 3 21 The amount of this solution to pipet into the cartridge has been calculated for each Fmoc and Boc amino acid using a density of 1 04 g mL for each solution This information is shown in Table 4 7 for the three different scales of syntheses when five equivalents of monomer is used For example in Table 4 7 107 pL of the Fmoc Ala solution is used for the 5 umol cycle Note The solution volumes given in Table 4 7 and Table 4 8 represent the minimum amount of amino acid to use 4 Chemistry November 2001 Applied Biosystems spe d Table 4 7 Amino acid solutions when using five equivalents 1 mmol in 4 mL NMP weight g weight volume mL of solution for of mmol 9 ofso monomer 25 pmol 50pmol 100 pmol AAand lution for amino acid MW 4mLNMP 25 pmol resin 5 10 umol 20 pmol Fmoc Ala OH 311 3 4 443 0 111 107 214 428 Fmoc Cys Trt OH 585 7 4 718 0 118 114 228 456 Fmoc Asp OtBu OH 411 4 4 543 0 114 110 220 440 Fmoc Glu OtBu OH 425 5 4 557 0 114 110 220 440 Fmoc Phe OH 387 4 4 519 0 113 109 218 436 Fmoc Gly OH 297 3 4 429 0 111 107 214 428 Fmoc His Trt OH 619 7 4 752 0 119 115 230 460 Fmoc lle OH 353 4 4 485 0 112 108 216 432 Fmoc Lys Boc OH 468 6 4 600 0 115 111 222 444 Fmoc Leu OH 353 4 4 485 0
57. ling with five equivalents of monomer followed by capping with a pre mixed solution of acetic anhydride Boc Depro Single 10 eq cap BDEFCd Fmoc Depro Single 10 eq cap bDEFCd PrPk Fmoc Depro Single 10 eq cap HhDEFiiiCidD The following cycles are single treatment with a material that is already in an activated form for example a solution containing fluorescein isothiocyanate The cycle with the TFA deprotection requires a neutralization step Boc Depro Single no 7 amp 8 BDfDaFllld Fmoc Depro Single no 7 amp 8 bDaFllld PrPk Fmoc Depro Single no 7 amp 8 HhDaFllliiidD The previous cycles do not include capping The following cycles may be written if capping is desired Boc Depro Single no 7 amp 8 cap BDfDaFIllCd Fmoc Depro Single no 7 amp 8 cap bDaFIllCd PrPk Fmoc Depro Single no 7 amp 8 cap HhDaFIlliiiCdD The following cycles are final deprotection cycles The final deprotection is usually not performed when using the Boc protecting group The Boc group is left on the resin and is removed during the cleavage If one does not want to remove the final Boc group then the syntheses is finished with a final DCM wash module c instead of modules BDc Boc Final Depro BDc Fmoc Final Depro bDc PrPk Fmoc Final Depro HhDc The following cycles are final deprotection and acetylation cycles After the deprotection there are two treatments with the capping solution After the second treatment the acetylation is c
58. lle OH 353 4 2 419 0 121 116 232 464 Fmoc Lys Boc OH 468 6 2 535 0 127 122 244 488 Fmoc Leu OH 353 4 2 419 0 121 116 232 464 Fmoc Met OH 371 5 2 438 0 122 117 234 468 Fmoc Asn Trt OH 596 7 2 663 0 133 128 256 512 Fmoc Pro OH 337 4 2 403 0 120 116 232 464 Fmoc Gln Trt OH 610 7 2 677 0 134 129 258 516 Fmoc Arg Pmc OH 662 8 2 729 0 136 131 262 524 Fmoc Ser tBu OH 383 4 2 449 0 123 118 236 472 Fmoc Thr tBu OH 397 5 2 464 0 123 118 236 472 Fmoc Val OH 339 4 2 405 0 120 116 232 464 Fmoc Trp OH 426 5 2 493 0 125 120 240 480 Fmoc Tyr tBu OH 459 5 2 526 0 126 121 242 484 Boc Ala OH 189 2 2 255 0 113 109 218 436 Boc Cys Mob OH 341 4 2 407 0 120 116 232 464 Boc Asp OBzl OH 323 4 2 389 0 120 116 232 464 Boc Glu OBzl OH 337 4 2 403 0 120 116 232 464 Boc Phe OH 265 3 2 331 0 117 113 226 452 Boc Gly OH 175 2 2 241 0 112 108 216 432 375 4 2 441 0 122 117 234 468 Boc His DNP OH 421 4 2 487 0 124 119 238 476 1 2 H2O 240 3 2 306 0 115 111 222 444 Boc Lys Cl Z OH 414 9 2 481 0 124 119 238 476 Boc Leu OH H20 249 3 2 315 0 116 112 224 448 Boc Met OH 249 3 2 315 0 116 112 224 448 Boc Asn Xan OH 412 4 2 478 0 124 119 238 476 Boc Pro OH 215 3 2 281 0 114 110 220 440 Boc Gln OH 246 3 2 312 0 116 112 224 448 Boc Arg Mts OH 456 6 2 523 0 126 121 242 484 Boc Ser Bzl OH 295 3 2 361 0 118 114 228 456 Boc Thr Bzl OH 309 4 2 375 0 119 115 230 460 Boc Val OH 217 3 2 283 0 114 110 220 440 Boc Trp OH 304 4 2 370 0 11
59. lt to detect changes to the point that the system may be effective at detecting only very poor deprotections at the small scales Because the signal to background ratio is small for the 5 10 and 20 umol cycles the Previous Peak Deprotection monitoring cycles have capping and extra NMP washes Module D The capping is included because uncoupled amino groups seem to cause an ion exchange effect The extra washing is included to make sure most of the conductive species from the coupling is removed 5 Cycles and Modules 5 9 Applied Biosystems When you use the Previous Peak PrPk Fmoc Deprotection cycles connect the conductivity cell between the bottom in line filter and valve 10 If you use only the non monitoring cycles Boc cycles and non monitoring Fmoc Cycles remove the conductivity cell and the small connecting tube to help minimize the volume between the valve block and the reaction vessel When using a combination of Boc and Fmoc cycles we recommend that you use non monitoring Fmoc cycles This is because the long term effect of TFA on the conductivity cell is not known Only the Previous Peak algorithm cycles are included on the disk contained in the 3 mL Reaction Vessel Kit Before you use the 1st Peak X algorithm you must perform enough syntheses to obtain a confident estimate of the X value to use For example if the baseline is steady at 700 the X value to use could be 68 or 69 If the base line is not steady then do n
60. mber 2001 Applied Biosystems November 2001 Synthesis Setup for the 3 mL Reaction Vessel You can use the following checklist with the ABI 433A FastMoc Quick Start Card to help set up your synthesis Much of the information on the Quick Start Card however is incorrect when you are doing a synthesis using the 3 mL RV cycles For example predissolved monomers bottle position HBTU concentration DIEA concentration and measuring loop volume are all different WARNING Always wear gloves protective clothing and eye protection when working with chemicals and bottles under pressure l Check the barcode reader nitrogen tank and waste container for proper connection and operation 2 Ifnecessary change the in line filters top RV bottom RV and cartridge Note For the next step weigh the HBTU in a fume hood 3 Prepare DIEA and HBTU solutions according to your synthesis scale then place the solutions in Bottles 7 and 8 Install an HBTU line filter on the end of the tube in Bottle 8 Reagent Scale 5 10 umol 20 umol DIEA Bottle 7 0 40 M 0 80 M 1 60 M HBTU Bottle 8 0 19 M 0 38 M 0 38 M 4 Prepare the monomer solutions using 1 mmol 4 mL NMP when using five equivalents and 1 mmol 2 mL NMP when using ten equivalents These monomer solutions can be stored for up to one week when refrigerated Add the appropriate amount of solution to the cartridges according to Table 4 7 Table 4 8 and Table 4 10 5
61. measuring loop 10 times from Bottle 8 and delivers the contents to the cartridge position The steps in this module are listed on page 5 27 As flow test a runs watch the flow at the waste port to verify that reagents are flowing quickly enough You should see the NMP fill the tube within three seconds of the start of delivery If the NMP does not fill the tube within the proper time a tube restriction or fitting leak is the probable cause Check all the tube connections you have made to ensure that there are no leaks If you find a leak on the valve block tighten the fitting no more than one quarter turn If the leak is on a fitting tighten the fitting with your fingers Weigh the cartridge containing the NMP from the 11 step module Consult Table 3 1 to determine whether the weight falls within the specified range Table 3 1 0 125 mL measuring loop weight specification Weight of NUP Resulting Volume 10 loops 1 loop 1 27 1 319 0 123 0 127mL Repeat steps 3 through 7 repeatedly until you achieve three successive results that are consistent in their weight measurements After the results are consistent then go on to step 9 Determine how much the NMP is over the desired weight and use this information to determine approximately how much tube to trim The example below shows how to calculate the amount of tube to cut The linear volume of the red tube is about 0 002 mL per centimeter WARNING CHEMICAL HAZARD Th
62. nches 5 16 inch 3 8 inch and 7 16 inch New single edged razor blade To prepare for installation WARNING CHEMICAL HAZARD Chemicals in bottles under pressure can discharge dangerous liquids into eyes and onto skin Always wear protective lab coat gloves and safety goggles when working with chemicals and bottles under pressure 1 Run Flow Tests 10 NMP to the metering vessel and 11 NMP to cartridge to verify proper reagent flow before doing any hardware installation See section on Flow Tests in 433A User s Manual Expected Found Flow Test 10 2 45 to 2 55 mL to RV position Flow Test 11 1 95 to 2 35 g to cartridge If the flow test values are not correct replace the inline filters then repeat the tests If the flow test values are still not correct adjust the bottom regulator then repeat the tests 3 Installing the Variable Measuring Loop 3 1 Applied Biosystems 2 Clean the existing 0 50 mL measuring loop with NMP by using the following procedure a d Add about 25 mL NMP to an empty 200 mL bottle and place the bottle in the Bottle 8 position Switch the ABI 433A to manual control use the manual control menu Turn on Fxn 69 and let the ABI 433A run until all the NMP has been removed from Bottle 8 Turn off Fxn 69 Turn on Fxn 70 for about 10 seconds 3 Clean the Bottle 7 tube with NMP by using the following procedure a Add about 25 mL NMP to an empty 200 m
63. ne to reaction vessel 45 56 Deliver NMP to reaction vessel 46 40 Mix reaction vessel 47 2 Vortex reaction vessel on 48 3 Vortex reaction vessel off 49 42 Drain reaction vessel to waste 50 130 Monitor previous peak 51 1 Wait 52 131 Monitoring stop 53 132 Read monitoring peak Q N I I0 Q N S N s N 54 134 End loop monitoring 25 55 10 Flush bottom valve block with gas to waste 3 56 40 Mix reaction vessel 2 57 2 Vortex reaction vessel on 5 58 3 Vortex reaction vessel off 1 59 42 Drain reaction vessel to waste 5 60 41 Vent reaction vessel 2 61 50 Flow NMP through reaction vessel to waste 5 62 42 Drain reaction vessel to waste 10 Module I Vortex 5 minutes Step Fxn Name Time 1 1 Wait 1 2 2 Vortex reaction vessel on 300 3 3 Vortex reaction vessel off 1 5 20 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Module a Read Cartridge Step Fxn Name Time 1 1 Wait 1 2 4 Read cartridge 10 3 6 Needle up 10 4 7 Eject cartridge 10 5 8 Advance cartridge 10 6 5 Needle down 10 7 14 Flush bottom valve block with NMP to waste 1 8 9 Flush top valve block with gas to waste 2 9 10 Flush bottom valve block with gas to waste 5 10 60 Mix cartridge 5 Module b Piperidine Deprotection no monitoring Step Fxn Time 1 1 Wait 1 2 56 Deliver NMP to reaction vessel 3 3 40 Mix reaction vessel 2 4 2 Vortex reaction vessel on 5 5 40 Mix reaction vessel
64. nsfer measuring loop to Cart e 68 Deliver 7 to measuring loop e 69 Deliver 8 to measuring loop FExn 70 Flush bottom valve block to waste Table 5 5 shows the modifications that are made to the cycles that use the 0 5 mL Measuring Loop These new cycles are included in the Variable Measuring Loop disk The cycles that have been modified are from the SynthAssist Chemistry disk version 2 0 3 Check to see if your 0 5 mL measuring loop fills in 6 seconds with Bottle 7 or 8 Flow Tests VML modules and d This test should be done with the solution that is being used in the synthesis because the viscosity of the solutions affect the flow rate If it is difficult to see the liquid as it fills the measuring loop check for the appearance of the liquid at the waste tube valve position 6 top of the 11 port valve block If the time is less than 6 seconds you might want to reduce the time of delivery for Fxn 68 and or Fxn 69 Table 5 5 will provide the information on where Fxn 68 and 69 appear In addition if you have already modified the cycles previously provided in the SynthAssist Chemistry disk then you need to modify the cycles to match the specifications shown in Table 5 5 Table 5 5 Changes to cycles on ABI 433A chemistry disk version 2 03 FastMoc Cycles 0 10 amp 0 25 mmol Module E Transfer Time sec Step Fxn Name Old New 5 70 Flush bottom valve block with loop contents to waste
65. nual Section 1 About this Manual Briefly describes each section of this manual explains the User Attention Words and tells how to get help Section 2 Introduction Describes the purpose of the 3 mL Reaction Vessel System and provides brief chemistry information Section 3 Measuring Loop Installation Gives the procedure for changing and calibrating the new Variable Measuring Loop required for use with the 3 mL Reaction Vessel Section 4 Chemistry Provides information about reagents bottle positions preparing solutions synthesis setup and example Section 5 Cycles and Modules Describes the new cycles and modules designed to be used specifically with the 3mL Reaction Vessel Appendix Contains plumbing schematics for the new 0 125 mL and 0 500 mL measuring loop configurations 1 About This Manual 1 1 Applied Biosystems User Attention Words Throughout the 3 mL Reaction Vessel User s Manual four kinds of information are set off from the regular text Each User Attention Word requires a particular level of observation or action that is significant to the user s safety or to proper instrument operation Note Used to call attention to information IMPORTANT Indicates information that is necessary for proper instrument operation Caution Damage to the instrument could result if you do not comply with this information WARNING Physical injury to the user or other persons could result if these precaut
66. ock JH i 2 Angar valve 29 25 a llow valve 13 Ge ye h n I tube 21 1 604128 29 short red tube 22 604130 M prd gt gt n ose _ cross 5 fitting 9 P x Va long red flange tube Note all tube coupling 604129 lengths not 110070 necessarily drawn to scale Figure 3 4 New 0 125 mL measuring loop configuration To calculate the 0 125 mL Measuring Loop calibration The long red tube labeled ASSY CPLG TO X FTG P N 604129 must be trimmed so it will deliver between 0 123 mL and 0 127 mL 1 Install a bottle of NMP at the Bottle 8 position NMP has a density of 1 033 g mL which will be used for calculating the calibration later Table 3 1 and Table 3 2 2 Replace the cartridge inline filter with a flange coupling This is only for calibrating the measuring loop You will replace the inline filter later in this section 3 Place empty tared cartridge with a septum into the guideway under the needle assembly and place the pusher block against the cartridge 4 Ifyou have not already done so load the new cycles provided on the Variable Measuring Loop Cycles disk In the Flow Tests folder open Flow Tests New VML and send to the ABI 433A 3 6 3 Installing the Variable Measuring Loop November 2001 Applied Biosystems November 2001 Run flow test a module a This module fills the
67. of reagents such as N methylpyrrolidone NMP Example for calculating how much to cut 0 5 mL measuring loop weight of 4 loops of NMP delivery 2 373 g _ 2 373g _ weight of 1 loop of NMP delivery loops 0 593 g _NMP weight amp volume of 1 loop of NMP delivery NMP density 05939 1 033 g mL difference between the volume of NMP delivery and the upper volume specification Table 3 2 0 574mL 0 507mL 0 067 mL excess amount of tube to be cut volume of NMP 0 067mL 13 4 cm to cut linear vol of tube 0 005 mL cm To cut measuring loop to correct length 1 Disconnect the blue tube from the flange couplings Remove the blue tube to a secure work surface 2 Separate the ferrule white from the fitting black then slide both the ferrule and fitting several inches farther onto the tube Figure 3 5 on page 3 9 3 Using the single edged razor blade trim the appropriate amount of tube determined in step 7 above using a perfectly straight cut 4 Slide the ferrule and fitting to within 1 inch of the trimmed end of the tube 5 Press fit the ferrule and fitting together leaving approximately t inch of tube protruding from the ferrule 6 Holding the fitting ferrule assembly between your fingers press the trimmed end of the red tube down onto a hard surface to bring the ferrule flush with the end of the tube Figure 3 6 on page 3 9 November 2001 3 Installing the Vari
68. ontinued for an additional 5 minutes Boc Final Depro amp Acetylation BDCCIDc Fmoc Final Depro amp Acetylation bDCCIDc PrPk Fmoc Final Depro amp Acetylation HhDCCliDc November 2001 5 Cycles and Modules 5 7 Applied Biosystems 5 8 Fmoc Cycle no monitoring Table 5 1 Fmoc Cycle described by modules Module b Piperidine Deprotection 8 7 min 1st treatment 2 min 2nd treatment 5 min Module D NMP Wash 2 9 min Module A Read Cart Add 7 amp 8 1 2 min Module F Transfer amp Coupling 35 1 min Module C Capping 1 6 min 1 min of capping Module d NMP Wash from Act 1 4 min Total Fmoc Cycle Time 50 9 min Boc Cycle Table 5 2 Boc Cycle described by modules Module B TFA Deprotection 8 6 min 1st treatment 1 min 2nd treatment 4 1 min Module D NMP Wash 2 9 min Module A Read Cart Add 7 amp 8 1 2 min Module F Transfer amp Coupling 35 1 min Module C Capping 1 6 min 1 min of capping Module d NMP Wash from Act 1 4 Total Boc Cycle Time 50 8 min Cycle Modifications Cycles often need to be modified to change times number of washes or other cycles parameters Refer to Table 5 3 for a list of common cycle modifications Table 5 3 Common Cycle Modifications If you want this Then do this Longer or shorter TFA deprotections Change the time in module B step 71 for the Boc deprotections page 5 14 Longer or shorter coupling times Change the loop count in Module F step 29 page 5 4 Longer o
69. oop UPPER 1 9 40 Mix reaction vessel 1 10 2 Vortex reaction vessel on 1 11 62 Drain cartridge to waste 10 12 98 Begin loop UPPER 3 13 67 Deliver NMP to cartridge small needle 2 14 62 Drain cartridge to waste 5 15 99 End loop UPPER 1 16 98 Begin loop UPPER 2 17 65 Deliver NMP to cartridge 22 18 60 Mix cartridge 10 19 24 Transfer cartridge to activator 20 20 62 Drain cartridge to waste 10 21 99 End loop UPPER 1 22 98 Begin loop UPPER 2 23 67 Deliver NMP to cartridge small needle 2 24 62 Drain cartridge to waste 10 25 99 End loop UPPER 1 26 62 Drain cartridge to waste 10 27 60 Mix cartridge 5 28 61 Vent cartridge 2 29 98 Begin loop UPPER 60 30 2 Vortex reaction vessel on 15 31 3 Vortex reaction vessel off 13 32 41 Vent reaction vessel 2 33 99 End loop UPPER 1 5 18 5 Cycles and Modules November 2001 Applied Biosystems Module G DCM Washes Step Fxn Name Time 1 1 Wait 1 2 3 Vortex reaction vessel off 1 3 98 Begin loop UPPER 5 PNA 2 4 41 Vent reaction vessel 2 5 49 Flow DCM through reaction vessel to waste 2 6 42 Drain reaction vessel to waste 5 7 55 Deliver DCM to reaction vessel 4 8 40 Mix reaction vessel 2 9 2 Vortex reaction vessel on 3 10 40 Mix reaction vessel 2 11 3 Vortex reaction vessel off 1 12 42 Drain reaction vessel to waste 7 13 99 End loop UPPER 1 14 42 Drain reaction vessel to waste 5 Module H Piperidine Deprotection Previous Peak Step Fxn Name Time 1 1 Wait 1 2 135 Monitoring re
70. ot use the 1st peak X algorithm To change Module H from Previous Peak to 1st Peak X make the following changes Step Function Description Change step 3 Fxn 110 Begin lower loop change time 2 to 1 step 24 Fxn 130 Monitor previous peak time 1 change to Fxn 128 Monitoring first peak X time X value step 50 Fxn 130 Monitor previous peak time 1 change to Fxn 128 Monitoring first peak X time X value step 57 Fxn 2 Vortex reaction vessel time 5 to 120 To change Module h from Previous Peak to 1st Peak X make the following change Step Function Description Change step 15 Fxn 130 Monitor previous peak time 1 change to Fxn 128 Monitoring first peak X time X value 5 Cycles and Modules November 2001 Applied Biosystems Example of a Deprotection Calculation Module H Piperidine Deprotection Previous Peak determines the number of deprotections based on the measured conductivity monitoring values At the end of the third deprotection the value of the second and third deprotections are compared to see if they are within the limits defined by step 54 End loop monitoring SynthAssist uses the following formula to calculate whether three or four deprotections are used Value of 2nd deprotect Value of 3rd deprotect percentage Value of 2nd deprotect If the percentage calculated is lower than 2 5 then no more deprotections will occur For example if the values of the th
71. ottom valve block with gas to waste 6 71 2 Vortex reaction vessel on 180 72 3 Vortex reaction vessel off 1 73 41 Vent reaction vessel 3 74 42 Drain reaction vessel to waste 5 75 12 Flush bottom valve block with DCM to waste 1 76 55 Deliver DCM to reaction vessel 4 77 40 Mix reaction vessel 2 78 2 Vortex reaction vessel on 5 79 3 Vortex reaction vessel off 1 70 41 Vent reaction vessel 2 81 42 Drain reaction vessel to waste 10 82 49 Flow DCM through reaction vessel to waste 10 83 42 Drain reaction vessel to waste 10 November 2001 5 Cycles and Modules 5 15 Applied Biosystems Module C Capping 1 minute Step Fxn Name Time 1 1 Wait 1 2 77 Pressurize Cap Solution 10 3 42 Drain reaction vessel to waste 10 4 17 Flush bottom valve block with Cap Sol to waste 2 5 10 Flush bottom valve block with gas to waste 2 6 52 Deliver Cap Solution to reaction vessel 8 7 40 Mix reaction vessel 2 8 2 Vortex reaction vessel on 60 9 3 Vortex reaction vessel off 1 Module D NMP Washes Step Fxn Name Time 1 1 Wait 1 2 3 Vortex reaction vessel off 1 3 98 Begin loop UPPER 6 4 41 Vent reaction vessel 2 5 50 Flow NMP through reaction vessel to waste 2 6 42 Drain reaction vessel to waste 5 7 56 Deliver NMP to reaction vessel 4 8 40 Mix reaction vessel 2 9 2 Vortex reaction vessel on 3 10 40 Mix reaction vessel 2 11 3 Vortex reaction vessel off 1 12 42 Drain reaction vessel to waste 7 13 99 End loop UPPER 1 14 42 Drain reaction vessel to waste 5
72. r shorter capping times To change the number of NMP washes To change the amount of NMP in each wash Change the time in Module C step 8 Change the loop count in Module D step 3 Change the time listed in Module D step 7 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Deprotection Monitoring Difficulties arise when using monitoring at the small scales made possible by the 3 mL RV Synthesis reagents and system electronics combine to generate a background conductivity value which is referred to as a noise baseline For example a noise baseline of 700 units is typical for the 5 jumol scale The initial Fmoc deprotection of such a small quantity of resin will generate only slight additional conductivity for a total conductivity of perhaps 1000 units during the initial deprotection Figure 5 2 The conductivity from the initial deprotection which in this example is less than 50 above the background of 700 is difficult to discriminate from the background conductivity 1200 1000 800 600 400 200 Start Finish 6 26 PM Leu Leu Ala Thr Ser Lys Lys 5 33 1 10 96 1 11 96 Figure 5 2 Background and initial deprotection conductivity using 3 mL RV In larger syntheses such as those using the 0 1 mmol scale the signal to noise ratio is much higher As deprotection continues the conductivity above the noise baseline becomes progressively smaller and it becomes more difficu
73. ree deprotections are 995 775 and 766 775 766 9 then 002 1 296 775 775 And only three deprotections will occur However if the values of the three deprotections are 903 792 and 771 799 771 91 then 0 026 26 799 792 And four deprotections will occur in Module H If four deprotections occur in Module H then Module h will become active Note You can modify the value in step 54 to change the sensitivity of the monitoring A lower value will provide greater sensitivity November 2001 5 Cycles and Modules 5 11 Applied Biosystems SynthAssist Dictionary You may use compounds with these cycles that are not in the SynthAssist Dictionary For information on creating new amino acids or protecting groups in the Dictionary refer to Chapter 5 of the SynthAssist 2 0 User s Manual For peptide nucleic acid monomers enter into the Dictionary the formula and suggested code shown in Table 5 4 Table 5 4 Peptide Nucleic Acid Monomers Monomer Formula Code Aaeg C44 His N7 Os One Letter Code 1 Caeg C10 His One Letter Code 2 Gaeg C44 His N7 O4 One Letter Code 3 Taeg C44 His N4 O5 One Letter Code 4 If you use benzyloxycarbonyl protection for monomers A and remember to choose Z side chain protection Using the one letter codes shown in Table 5 4 puts the four PNA monomers at the start of the palette rather than mixing
74. s aggregation of the PNA Lowering the Resin Substitution The following procedure for lowering the substitution for MBHA resin is adapted from a procedure developed by PNA Diagnostics To lower the substitution of MBHA resin 1 Wash 3 0 g MBHA resin 0 45 mmol g 1 35 mmol twice in DCM 2 Wash the resin in 5 DIEA in DCM for 3 minutes 3 Wash the resin twice in DCM The resin is now neutralized 4 Dissolve 0 60 mmol PNA monomer in 7 5 mL NMP 5 Add 1 2 mmol DIEA to the monomer solution 6 Dissolve 224 mg 0 59 mmol HBTU in 7 5 mL NMP and add this to the monomer solution 7 Activate the monomer for 2 minutes 8 Add the activated monomer solution to the neutralized resin 9 Allow the reaction to proceed for 1 hour 10 Filter the resin 11 Wash the resin with 1x NMP 12 Make 50 mL of capping solution using a 1 2 2 ratio of acetic anhydride pyridine and NMP 13 Addthe capping solution to the resin and allow the reaction to proceed for 1 hour Successful capping will produce a negative Ninhydrin test 14 Wash the resin with DMF 15 Wash the resin with 4x DCM 16 Wash the resin with 5 DIEA in DCM 17 Wash the resin with 4x DCM 18 Dry the resin in a vacuum 4 Chemistry 4 13 Applied Biosystems To determine the new loading of the resin couple Fmoc Gly to about 20 mg of the resin Follow the procedure for a 5 pmol Boc synthesis using the following cycles Table 4 9 Cycles for determining resin loadin
75. set 1 3 110 Begin loop lower 2 4 42 Drain reaction vessel to waste 7 5 98 Begin loop UPPER 3 6 56 Deliver NMP to reaction vessel 3 7 40 Mix reaction vessel 2 8 2 Vortex reaction vessel on 2 9 40 Mix reaction vessel 2 10 3 Vortex reaction vessel off 1 11 42 Drain reaction vessel to waste 7 12 41 Vent reaction vessel 2 13 50 Flow NMP through reaction vessel to waste 3 14 42 Drain reaction vessel to waste 7 15 99 End loop UPPER 1 16 56 Deliver NMP to reaction vessel 2 17 79 Pressurize piperidine 10 18 51 Deliver piperidine to reaction vessel 3 19 56 Deliver NMP to reaction vessel 2 20 40 Mix reaction vessel 2 21 2 Vortex reaction vessel on 120 22 3 Vortex reaction vessel off 1 23 42 Drain reaction vessel to waste 2 24 130 Monitor previous peak 1 25 1 Wait 3 26 131 Monitoring stop 1 27 132 Read monitoring peak 1 November 2001 5 Cycles and Modules 5 19 Applied Biosystems a 3 D Step Fxn 28 111 End loop lower 29 133 Begin loop monitoring 30 42 Drain reaction vessel to waste 31 98 Begin loop UPPER 32 56 Deliver NMP to reaction vessel 33 40 Mix reaction vessel 34 2 Vortex reaction vessel on 35 40 Mix reaction vessel 36 3 Vortex reaction vessel off 37 42 Drain reaction vessel to waste 38 41 Vent reaction vessel 39 50 Flow NMP through reaction vessel to waste 40 42 Drain reaction vessel to waste 41 99 End loop UPPER 42 56 Deliver NMP to reaction vessel 43 79 Pressurize piperidine 44 51 Deliver piperidi
76. ssel to waste 3 15 42 Drain reaction vessel to waste 10 16 99 End loop UPPER 1 17 42 Drain reaction vessel to waste 30 18 29 Flow DCM through activator to waste 5 19 22 Drain activator to waste 30 20 11 Flush top valve block with DCM to waste 1 21 12 Flush bottom valve block with DCM to waste 1 22 10 Flush bottom valve block with gas to waste 10 23 9 Flush top valve block with gas to waste 10 Module d NMP Wash from Activator Step Fxn Name Time 1 1 Wait 1 2 98 Begin loop UPPER 3 3 3 Vortex reaction vessel off 1 4 28 Pressurize activator 4 5 42 Drain reaction vessel to waste 7 6 38 Transfer activator to reaction vessel top open 6 7 40 Mix reaction vessel 1 8 2 Vortex reaction vessel on 3 9 40 Mix reaction vessel 2 10 99 End loop UPPER 1 11 22 Drain activator to waste 5 12 3 Vortex reaction vessel off 1 13 42 Drain reaction vessel to waste 7 5 22 5 Cycles and Modules November 2001 Applied Biosystems Module f DIEA Neutralization Step Fxn Time 1 1 Wait 1 2 3 Vortex reaction vessel off 1 3 98 Begin loop UPPER 2 4 42 Drain reaction vessel to waste 7 5 56 Deliver NMP to reaction vessel 4 6 40 Mix reaction vessel 2 7 78 Pressurize manifold 5 8 68 Deliver DIEA to measuring loop open 3 9 43 Transfer measuring loop to reaction vessel 10 10 56 Deliver NMP to reaction vessel 2 11 40 Mix reaction vessel 2 12 2 Vortex reaction vessel on 3 13 40 Mix reaction vessel 2 14 3 Vortex reaction vessel off 1 15 99 End loop UPPER
77. st User s Manual page 4 14 c Save and send chemistry to synthesizer Add resin to the 3 mL RV The 3 mL RV must be closed and tightened completely before any synthesis is started You will feel resistance when you tighten the caps of the RV This resistance is the result of O rings beginning to become compressed Keep tightening the RV caps until the RV is completely closed To completely tighten the RV caps you may need to use the wrench included in the 3 mL RV kit If the RV caps are not completely tight resin will escape from the body of the vessel during the synthesis For more information about assembling and closing the 3 mL RV refer to page 2 5 13 Open New Run using the following steps a Choose sequence b Choose resin c Enter the resin substitution 4 16 4 Chemistry November 2001 Applied Biosystems d Enter the weight of resin e Check cycles to make sure the correct cycles are entered You may want some cycles to be different from the default f Check amino acids pop up menu g Save Run 14 Send Run File to synthesizer 15 Load the cartridges that contain the monomer solutions into the guideway 16 Place RV on synthesizer and begin synthesis Reminder when using the 8 mL 40 mL and 55 mL Reaction Vessels Always check that the 0 50 mL variable measuring loop is installed when you use the cycles written for the 8 mL 40 mL and 55 mL RV In addition you must use the cycles containing the
78. them with the amino acids 5 Cycles and Modules November 2001 Applied Biosystems November 2001 Modules for 433A Peptide Synthesizer The modules described in this section are used for both Boc and Fmoc syntheses The TFA deprotection for the Boc removal is written in module B The piperidine deprotection for the Fmoc removal is written in module b when there is no monitoring and in modules H and h when there is conductivity monitoring Module A Read Cartridge and Add HBTU and DIEA 5 10 20 1 1 Wait 1 1 1 2 4 Read cartridge 10 10 10 3 6 Needle up 10 10 10 4 7 Eject cartridge 10 10 10 5 8 Advance cartridge 10 10 10 6 5 Needle down 10 10 10 7 14 Flush bottom valve block with NMP to waste 1 1 1 8 9 Flush top valve block with gas to waste 2 2 2 9 10 Flush bottom valve block with gas to waste 3 3 3 10 70 Flush bottom valve block with loop to waste 2 2 2 11 78 Pressurize manifold 5 5 5 12 98 Begin Loop UPPER 1 1 2 13 69 Deliver HBTU to measuring loop open 3 3 3 14 10 Flush bottom valve block with gas to waste 2 2 2 15 63 Transfer measuring loop to cartridge 10 10 10 16 99 End Loop UPPER 1 1 1 17 60 Mix cartridge 2 2 2 18 98 Begin Loop UPPER 1 1 1 19 68 Deliver DIEA to measuring loop open 3 3 3 20 10 Flush bottom valve block with gas to waste 2 2 2 21 63 Transfer measuring loop to cartridge 10 10 10 22 99 End Loop UPPER 1 1 1 23 60 Mix c
79. to the cycle BDAFCd was changed to BGDAFCd You can change the number of loops in module G from five to two step 3 3 For capping use a 1 25 25 solution of acetic anhydride pyridine and NMP 4 Use a resin with a loading of 0 20 mmol g or less If the resin you use has a higher substitution you can lower the resin substitution For more information on lowering the resin subs tution see Lowering the Resin Substitution on page 4 13 5 Use 9596 5 m cresol instead of 10096 TFA in Bottle 2 6 Usea greater excess of PNA monomer over the uronium activator than when synthesizing peptides For PNA synthesis use 0 9 equivalent of uronium activator per 1 0 equivalent of PNA monomer For peptide synthesis use 0 95 equivalent of uronium activator per 1 0 equivalent of amino acids 7 Ifyou are using HBTU for activation do not add any additional HOBt Nielsen PE Egholm M Berg R F and Buchardt O 1991 Sequence Selective Recognition of DNA by Strand Displacement with a Thymine Substituted Polyamide Science 254 1497 1500 4 Chemistry November 2001 Applied Biosystems November 2001 Synthesizing PNAs can be more challenging and more expensive than peptides or DNA For the first PNA synthesis you perform choose a relatively simple oligomer such as Taeg g Lys NHo PNAs are often synthesized with an amino acid at the C or N terminus The C terminus is often started with a lysine amide to suppres
80. w test is used when calibrating the new 0 50 mL measuring loop as described on page 3 9 of this manual Perform this calibration immediately after calibrating the 0 125 mL loop When running this flow test Bottle 8 is filled with NMP and the in line filter to the cartridge is replace with a flange coupling P N 110070 After the calibration of the measuring loop is complete and before a synthesis is started remove the flange coupling and re attach the in line filter This flow test uses an empty tared cartridge with the septum installed The flow test fills the 0 50 mL measuring loop with NMP and delivers this NMP to the cartridge 4 times Step Fxn Name Time 1 5 Needle down 10 2 78 Pressurize manifold 15 3 98 Begin Loop UPPER 4 4 69 Deliver 8 to measuring loop open 8 5 10 Flush bottom valve block with gas to waste 2 6 63 Transfer measuring loop to cartridge 10 7 99 End Loop UPPER 1 8 6 Needle up 10 9 7 Eject cartridge 10 10 70 Flush bottom valve block with loop to waste 5 11 10 Flush bottom valve block to waste 5 Flow Test c Test Bottle 7 VML Use this flow test after the 0 125 mL and 0 50 mL measuring loops are calibrated They can be used with either of the two measuring loops The objective of the flow test is to insure that the measuring loop can be filled when Bottle 7 is used The flow test is very similar to Flow Test 7 which is described in the Model 433A User s manual except there is an additional step added st
81. waste 36 9 Flush top valve block with gas to waste 37 10 Flush bottom valve block with gas to waste 38 76 Pressurize TFA 5 39 3 Vortex reaction vessel off 40 41 Vent reaction vessel 41 42 Drain reaction vessel to waste 0 42 41 Vent reaction vessel 43 72 Deliver to reaction vessel 44 40 Mix reaction vessel 45 1 Wait N N GQ q O O N N N Q N N N N N 5 14 5 Cycles and Modules November 2001 Applied Biosystems 3 Step Fxn 46 41 Vent reaction vessel 2 47 12 Flush bottom valve block with DCM to waste 2 48 55 Deliver DCM to reaction vessel 1 49 41 Vent reaction vessel 2 50 11 Flush top valve block with DCM to waste 2 51 45 Deliver DCM to reaction vessel top 1 52 2 Vortex reaction vessel on 1 53 9 Flush top valve block with gas to waste 3 54 10 Flush bottom valve block with gas to waste 10 55 73 Vent TFA without gas 5 56 74 Back flush TFA 6 57 73 Vent TFA without gas 2 58 75 Vent TFA with gas 4 59 74 Back flush TFA 4 60 73 Vent TFA without gas 3 61 13 Flush top valve block with NMP to waste 2 62 14 Flush bottom valve block with NMP to waste 2 63 98 Begin loop UPPER 3 64 12 Flush bottom valve block with DCM to waste 2 65 11 bFlush top valve block with DCM to waste 2 66 10 bFlush bottom valve block with gas to waste 2 67 9 bFlush top valve block with gas to waste 2 68 99 End loop UPPER 1 69 9 Flush top valve block with gas to waste 6 70 10 Flush b

ABI 433A Peptide Synthesis

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