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CS336X Automated Peptide Synthesizer Quick Reference Manual

2011 C S Bio Co.


DOC. No. 11-04-05

C S Bio Co. 20 Kelly Court Menlo Park, California, USA 94025 TEL: 650 322 1111 FAX: 650 322 2278 EMAIL: peptides@csbio.com

Reagents in Detail:
Figure 1 - CS336X

R1: DMF or NMP (main wash) - Sensor 2 (8-10 ml) Fill bottle up to 1.8 L with DMF or NMP. R2: DMF or NMP (deblock wash) - Sensor 2 Fill bottle up to 800 ml with DMF or NMP. This bottle should be used two times after piperidine wash to clean the line. R3: PIP/DMF (deblock peptide chain) - Sensor 2 Have an empty switch bottle ready at the side of the synthesizer. 2

Wear two sets of gloves. Carefully open R3 bottle. Remove lid and tubing carefully also taking up the black o-ring. Transfer to the switch bottle. Fill R3 with 20% piperidine/DMF. Replace lid and tubing onto R3 bottle. Remove upper set of gloves; dispose. R4: DCM (wash resin) - Sensor 2 Fill DCM into bottle. R5: 1.0 M DIEA/DMF (for activating the AA and HBTU) - Metered to S14, 1 ml When synthesizing 0.2 mmol of peptide you will generally use 1 mmol of amino acid (4 X excess) and so will generally use 1 mmol of DIEA. In order to get 1 mmol of DIEA to the AA tube the standard way is to make a 1 M solution of DIEA and meter to S14 (set to 1ml), this now is 1 mmol of DIEA. If you desire 2 mmol of activator the easiest way to accomplish this is to meter and deliver twice. To get a 1 M solution of DIEA: MW 129g/mol d. 0.74 g/ml 129 g/mol X 1 = 129 g/L X 0.1 L = 12.9 g/100 ml/0.74 = 17.4 ml of DIEA diluted up to 100 ml with DMF or NMP. Fill solution into the R5 bottle. R6: 0.4 M HBTU/DMF (for dissolving the AA) - Metered to S16, 2.5 ml When synthesizing 0.2 mmol of peptide you will generally use 1 mmol of amino acid (4 X excess) and so will generally use 1 mmol of HBTU. In order to get 1 mmol of HBTU to dissolve the AA the standard way is to make a 0.4 M solution of HBTU in DMF and meter to S16 (set to 2.5 ml), this will now be 1 mmol of HBTU. To get a 0.4 M solution of HBTU: MW 379 g/mol 379 g/mol X 0.4 mol/L = 156 g/L X 0.1 L = 15.7 g/100 ml. Transfer HBTU into 250 ml bottle & shake to dissolve. Transfer 0.4 M HBTU solution to bottle 6. R7: DMF or NMP (rinse needle and wash) Fill bottle up to about 800 ml with DMF or NMP. Preset: Sensor 2: 8-10 ml; Sensor 14: 1 ml; Sensor 16: 2.5 ml Recommended Volumes of Reagents Before a Synthesis Starts: R1 (DMF or NMP): 1.8 L R2 (DMF or NMP): 800ml R3 (Pip): 200 ml R4 (DCM): 300 ml R5 (DIEA): 50 ml R6 (HBTU): 50 ml R7 (DMF or NMP): 800 ml

Running the Synthesizer:


Attach RV (reaction vessel) to the synthesizer. Turn up the gases: Air - to approximately 3.1 bar, resulting in about 40 psi (that is the min) at the machine. Nitrogen - to approximately 0.3 - 0.4 bar, resulting in about 4 - 6 psi (the max is 8) at the machine. Check nitrogen in tank. Check waste container, make sure not too full and lid is tight. Make sure small cap is on and tight. Decide whether or not to change the RV filters. These are good for 4-5 syntheses. Make sure all solvents have enough to finish your synthesis. Add resin to reaction vessel, put reaction vessel on synthesizer. Add amino acids to wheel in proper sequence. Put wheel in place. Select "Go Home" Using "RV-Setup" enter the number of repeats (generally corresponding to the number of amino acids to be coupled plus 2). The extra two repeats will generally be a PreSwell protocol and a Final Deblock protocol. Tick the square to the right of the repeats to select the protocols to be used at each repeat. If you have a multiple reaction vessel system you will be prompted to enter the reaction vessel wanted. You can now save this series as an RVS file if you wish. Now select OK to return to the main screen. Enter "Run" to start synthesis at step one, or change the "Initial Queue Start Index" to begin the synthesis at a different step. CSPep Operating Screen: Figure 2 - CSPep Screen Layout

1. Available Functions: This section lists all of the standard functions that the CS Bio Synthesizer can accomplish. Functions can also be customized by modifying the .map file. 2. Functions Queue: This section is where synthesis protocols (a series of functions) and a single or multiple functions will be put to be run. From this area a series of functions can be saved as a .CFN (Custom Function file) and/or recalled if previously saved. While the synthesizer is running the protocol being run will be shown and the step operating will be highlighted in this section. 3. RV Setup: This section is where the number of repeats of the function queue synthesis protocols can be set, in other words, .CFN file could be put in sequence and create an RVS file. In this section the desired synthesis protocols to be run are selected in the order they are required. This section also allows the user to quickly activate the steps that are currently set-up in the function queue. This section also allows the user to select a previously saved series of steps. More in-depth explanation of this function is described in the manual. 4. Function Variables: This section shows the parameters of selected function in the function queue section. Certain parameters of the function in this section will be shown: the function name, the function type, the number of phases, the number of repeats and the time of any time dependent phases. This section also allows editing of certain parameters, for instance, 25 seconds could be edited to 30 seconds in a Mix_30sec function. The repeatability of a function could also be edited. For example, to accomplish a mix of 10 minutes, the repeat line would be set to 20 for a Mix_30sec function. 5

5. Initial Queue Start Index: This section allows a saved synthesis protocol (.CFN) to be started at user inputted step number. 6. Run RV Setup File/Pause: Here, a synthesis protocol (.CFN or .RVS file) can be started, stopped, paused or resumed. 7. Logbook: This section shows a log of every step that was run. Logbook can be set to autosave after a specific numbers of text line shown, and also the details of the logbook can also be modified in the administration section. 8. Sensor I/O (input/output): This section shows the real time on-off position of every sensor. A sensor is on when a concave button with the sensor button text displayed in red and is triggered by a complete circuit. A sensor is off when a convex button with the sensor button text is displayed in blue and is triggered by a broken circuit. Sensor 1: Lower level of MV. This sensor is used to detect the solvent transfer from the MV to the TV. It is also used to detect the amino acid solvent at the end of the step "AA_To_MV", if this sensor does not detect the AA solvent the synthesizer will stop. Sensor 2: This sensor is used to measure the volume of the solvents from bottles 1-4. This sensor is set to a volume of 8 to 10 ml. Sensor 3: Top level of the MV. This sensor is a solvent overflow sensor. Sensor 4: Located on the 1/8 Teflon tubing between the TV and the RV. This sensor is used to detect liquid in the solvent line during the transfer of solvent from the TV. This function uses three phases, sensor on (solvent in line), sensor off (no solvent in line), and 5-10 seconds of time to blow the line clear. Minimum of 6 ml required. If less than 6 ml of solvent is to be transferred a timed transfer should be used. Sensor 5: This sensor is located on the 1/8 Teflon tubing between the RV and waste. This sensor is used to detect liquid in the solvent line during the drain step. This function uses three phases, sensor on (solvent in line), sensor off (no solvent in line), and 5-10 seconds of time to blow the line clear. Minimum of 6 ml required. If less than 6 ml is to be drained a timed drain should be used. Sensor 7: This sensor is the sensor that detects the tab on the wheel. This detects the "Home Position" (position1) on the wheel. Sensor 8: This sensor detects the needle in the inner position on the wheel. When the needle is in the inner position (pos. 25-36) this sensor is red. 6

Sensor 9: This sensor detects the needle in the outer position on the wheel. When the needle is in the outer position (pos. 1-24) this sensor is red. Sensor 10: This sensor detects when the needle is up. (Retract Needle) Sensor 11: This sensor detects when the needle is in the down position. (Inject Needle) The software will not allow the metering of solvents from bottle 5-7 to the MVAA or the delivery of solvent from the MVAA to the AA tube (Deliver_AA_Solvent) if this sensor is not on (red). Sensor 12: This sensor detects the slot in the outer edge of the wheel and allows the software to keep track of the wheel position. Sensor 13: This sensor detects the wheel (carousel) in the tower. Sensor 14: This sensor is at the lower level of the MVAA and is set to 1ml. The activator is generally measured with this sensor. Sensor 16: This sensor is at the upper level of the MVAA and is set to 2.5ml. The AA disolution solvent (R6) and the needle wash solvent (R7) is generally measured with this solvent. 9. Permanent Log File: By using permanent log file, the user can create a log file that logs and save data on every step of a protocol. Therefore, even in cases of unexpected crashes or power failure, the user will know what step the protocol stopped at. A Standard Synthesis Protocol is as Follows: RB: Solution/Duration 3 X R1: DMF 30 sec 1 X R3: 20% Piperdine 5 min 1 X R3: 20% Piperdine 20 min 2 X R2: DMF 30 sec 2 X R4: DCM 30 sec 3 X R1: DMF 30 sec

Activate AA/HBTU solution with base (DIEA or NMM) approximately 1 - 3 min. Add AA soln. to resin, couple 30 mins - 1hr. Repeat.

*Important* If your synthesis stops for any reason during a run the screen will freeze
and Fig.3 will be displayed. Until the "OK" is pushed the synthesis screen will retain the information needed to restart your synthesis at the proper place. You will need two pieces of information, first the queue repetition; the example shown in Fig. 3 is Queue repetition 3/9. Second, you will need to know what step (Queue Index) the run stopped at. In the example shown, the synthesis stopped at step 15. Now when you have fixed the problem and you want start the synthesis again you will first go to "RV Setup" (see Fig. 4) uncheck the "Run all Files", set the "Start File" to the queue repetition you wish to start at. Then select "OK". Now set the "Initial Queue Start Index" to the step you wish to start at. You can now press Run RV Setup File.

Figure 3.

Figure 4.

Abbreviations:
NMP = n-Methylpyrrolidone HBTU = N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-Nmethylmethanaminium hexafluorophosphate N-oxide DIEA = N,N-diisopropylethylamine DCM = Dichlormethane Fmoc = 9-Fluorenylmethoxycarbonyl DMF = Dimethylformamide HATU = 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate Methanaminium HCTU = 2-(6-Chloro-1H-benzotriazole-1-yl) - 1,1,3,3 - tetramethylaminium hexafluorophosphate BOP = Benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate PyBOP = Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate TBTU = O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate DMAP = 4-Dimethylaminopyridine DIC = N,N'-diisopropylcarbodiimide DCC = N,N'-dicyclohexylcarbodiimide 9

HOBT = N-Hydroxybenzotriazole Ac2O = Acetic Anhydride NOTE: Although it says 'DMF' on the bottles, you can use 'NMP' instead.

Necessary Solutions and Gases: General Remarks


All solutions are relatively stable EXCEPT that of the activator (normally HBTU). It is always possible to replace/top-up solutions that are running low. Possible quick pause points: During incubation times or longer wash steps; 1) press PAUSE button, 2) turn off gases, 3) change/top up, 4) press RESUME button. Regarding the gases, have in mind that not much N2 is used during a run. However, a run can consume up to half of an air bottle! As with the solutions, it is easy to switch over a bottle during a pause. It is, however, important that none of the gases run out during unsupervised hours (e.g., overnight)! The solvent that is mainly used is either DMF or NMP.

Tables:
Table 1 - Fmoc Amino Acids: MW & mM (g) No. Description MW 0.4 mM (g) 0.125 0.260 0.239 0.165 0.234 0.244 0.5 mM (g) 0.156 0.324 0.298 0.206 0.293 0.305 1.0 mM (g) 0.311 0.649 0.597 0.412 0.586 0.611 1.6 mM (g) 0.498 1.038 0.955 0.658 0.937 0.977

1A 2R 3N 4D 5C 6Q

Fmoc-L-Ala Fmoc-L-Arg(Pbf) Fmoc-L-Asn(Trt) Fmoc-L-Asp(OtBu) Fmoc-L-Cys(Trt) Fmoc-L-Gln(Trt)

311.3 648.8 596.7 411.5 585.7 610.7

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No.

Description

MW

0.4 mM (g) 0.170 0.119 0.248 0.141 0.141 0.187 0.149 0.155 0.135 0.153 0.159 0.211 0.184 0.136

0.5 mM (g) 0.213 0.149 0.310 0.177 0.177 0.234 0.186 0.194 0.169 0.192 0.199 0.263 0.230 0.170

1.0 mM (g) 0.426 0.297 0.620 0.353 0.353 0.469 0.372 0.387 0.337 0.383 0.398 0.527 0.460 0.339

1.6 mM (g) 0.681 0.476 0.992 0.565 0.565 0.750 0.594 0.620 0.540 0.613 0.636 0.843 0.735 0.543

7E 8G 9H 10 I 11 L 12 K 13 M 14 F 15 P 16 S 17 T 18 W 19 Y 20 V

Fmoc-L-Glu(OtBu) Fmoc-L-Gly Fmoc-L-His(Trt) Fmoc-L-Ile Fmoc-L-Leu Fmoc-L-Lys(Boc) Fmoc-L-Met Fmoc-L-Phe Fmoc-L-Pro Fmoc-L-Ser(tBu) Fmoc-L-Thr(tBu) Fmoc-L-Trp(Boc) Fmoc-L-Tyr(tBu) Fmoc-L-Val

425.5 297.3 619.7 353.4 353.4 468.5 371.5 387.4 337.4 383.4 397.5 526.6 459.6 339.4

Table 2 - Reagents, MW, & 1 M Conversion Description Fmoc Rink Linker HMPS Linker BOP HATU HBTU MW 539.6 182.2 442.3 380.3 379.3 11 1M -

Description HCTU PyBOP TBTU DMAP DIC DCC DIEA HOBT Acetic Anhydride

MW 413.7 520.3 321.1 122.2 126.2 206.3 129.3 135.1 102.1

1M 156 ml/L 174.2 ml/L 94.4 ml/L

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