Hoppe-Seyler's Z. Physiol. Chem. Bd. 357, S. 713-719, Mai 1976
A Computer-Controlled Peptide Synthesizer Preben ViLLEMOES,ThorkildCHRiSTENSEN, and Kay BRUNFELDT The Danish Institute of Protein Chemistry, affiliated to the Danish Academy of Technical Sciences, DK-2970 Htfrsholm
(Received 6 November 1975)
Summary: A computer-controlled system for solid-phase peptide synthesis including an ana-
lytical step wise evaluation of the yields by titration with perchloric acid is described.
Ein Computer-gesteuertes System fur Peptidsynthese Zusammenfassung: Ein Computer-gesteuertes System für die Peptidsynthese an fester Phase einschließlich einer analytischen schrittweisen Aus-
wertung der Ausbeuten durch Titration mit PerChlorsäure wird beschrieben,
Key words: Solid-phase peptide synthesis, monitoring, computer control.
The experience with solid-phase peptide synthesis has shown that it often is advantageous to carry out the procedure automatically with control of the stepwise yields of both the coupling and the removal of the TV-protecting group. Such automatic systems have been developed^1'3!. The control has been achieved either by titration with perchloric acid of free -amino groups, by photometric monitoring of the concentration of the acylating reagent and the cleaved «-ammo proteciion group, or by determination of displaced
The use of a computer as control unit is, however, advantageous for several reasons. Thus changes of functions and introduction of new functions are easily carried out by minor changes in or addition of software. Computer control also allows a stepwise or continuous evaluation of analytical data, allowing a process control, ~, . , · , , , _ , · .. i_ · ·. T f %*"? descnbed bek)w ,s to be regarded as fal pment f SyStem * *"$?° ° ^ P™"0"^
P
Experimental
'
escn e
In the first two cases, the control unit is based
1) Description of reservoir and metering system
upon sequential logical. In the latter, an electromechanical system is used.
The reactor, metering vessel,and common lines are made of glass. (Fig. 1). Between the reservoirs and the solenoid
Abbreviations: Boc = f-butyloxycarbonyl-; Bzl = benzyl-; DCC = dicyclohexylcarbodiimide; EtOH = abs. ethanol; St3N = triethylamine; HOAc = acetic acid; S 2 = 2% crosslinking.
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
714
P. Villemoes, T. Chiistensen and K. Brunfeldt
Bd. 357(1976)
-Solvent inlets
HCI/Solvent
Solvents and Reagents Et, N
To waste
Fig. 1. Schematic drawing of the solid-phase peptide synthesizer. MV1 and MV2, metering vessels; SI through S9, liquid detectors; VI through V4, solenoid valves; V5 valve operated by fluid pressure in the line. -> indicates flow direction for open valve.
valves are inserted traps for protecting solvents, reagents and valves. Brown glass containers are used as reservoirs in order to minimize the destructive effect.of.light on the liquids. All the reservoirs except the one containing Et3N are connected to the metering vessel MV1, which is supplied with a side arm with 5 sensors. The levels of S2, S3 and S4 determine the volumes which can be delivered by the metering vessel. S5 is an overflow control. Sl^ must be activated in combination with S2, S3 or S4; this prevents that a single drop from a previous metering will terminate the actual metering. The principle for metering Et$N has been previously described'11 Transferring of solvents and reagents to the metering vessel, as well as draining of the reactor, is carried out by applying nitrogen pressure. The teflon tubes from the reservoirs are connected to the metering vessel via a glass manifold. The solvents, except acetic acid, are fed into the top of the manifold to ensure that the inlet system is carefully washed. A
special arrangement for metering HCI in organic solvents is used in order to minimize absorption of HCI in the teflon tubes. During measuring of the latter the valve V4 is also energized, thereby blocking the line to waste. 2) Control unit A computer, Alpha-16, from Computer Automation, U.S.A., with a 4K 16-bit core memory is used as control unit. An interface was constructed and consisted of 4 χ 4 bit input/output module, which selectively allows input/output of 4-bit information. Each 4-bit word is converted to a binary 15-bit word at the output, and input is converted from a 15-bit word to a 4-bit word. It means that it is possible to handle 15 inputs and/or 15 outputs without disturbing the other 45 inputs and/or outputs. Furthermore, all outputs are provided with a buffer flip-flop, that is set one at a time. The whole buffer can be cleared by one instruction from the computer, which makes a powerful way of terminating any
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
Bd. 357 (1976)
A Computer-Controlled Peptide Synthesizer
function. By splitting the 60 inputs and outputs into 4 words of 15 bits, it was possible to get satisfactory flexibility. 32 of the outputs are provided with a power amplifier to drive the solenoid valves. The rest of the outputs are for control of analytical equipment, such as titiation equipment and eventual future equipment. The inputs are connected to the liquid detectors (sensors) and to data and control inputs from the titration equipments, etc. Thus 60 input and 60 output lines are achieved. The coding of a synthesis is performed on punched tape in ASCII code and read into the computer via the tape reader of an ASR 33 teletype. The syntax is: a letter, a number, and a space. The letter denotes the basic function, the number attributes to the basic function the desired parameters, and the space separates the complete functions. As an example: A2 X4 Yl. A2 means metering methylene chloride (A) to the level of sensor S3 (2) on the side arm of the metering vessel, followed by draining of the liquid into the reactor. X4 means stirring (X) for 3 minutes (4). Yl means draining of reactor (Y) in less than five minutes (1). Programming for removal of the Boc-group and two titration cycles is shown in the upper part of Fig. 2; the
REMOVAL OF THE BOC-GROUP AND TWO B2 X4 Yl B2 X4 Yl V2 X4 Y l W2 X6 A2 X4 Y l A2 X4 Yl A2 X4 Y l Al W0 A I X4 X2 Yl A2 X4 Y l A2 X4 B2 X4 Y l B2 A2 V0 Y l A2 X4 Y l A2 Al W0 A I X4 X2 Yl A2 X4 Y l A2 X4 B2 X4 Y l B2 A2 V0 Y l A2 X4 Y l A2 B2 00165 00002 X4 00166 00001 Yl 00169 00003 00000 B2 00169 B2 00208 X4 00170 00001 X4 00209 Yl 00173 00003 Yl 00212 W 2 00173 00000 i B2 00212 X4 00174 00001 X4 00213 Yl 00177 00003 Yl 00216 W2 00177 00000 B2 00216 X6 00178 00001 X4 00217 Yl 00208 00030 Jf Yl 00220
/// /
715
lower part shows the print-out during the execution of the program. All functions are printed out by the teletype together with elapsed time and duration of the previously performed function in minutes. The alarm system is based on the same principles as described in ref J41. If an alarm occurs, the cause is printed out and the synthesizer is immediately paralyzed. It is possible to stop the process manually by making a console interrupt at the computer, whereby all outputs from the computer are turned off. Reactivating is done via the full stop of the teletype keyboard. After the interruption, notes may be written by the teletype. It is furthermore possible manually to let the synthesizer go on stand-by, when the function under execution is terminated. 3) Software The software package contains three sections: Section 1 When a code has been read, the software checks for valid letter, number and syntax errors, incorrect operation of the tape reader or tape out. If an error is detected, the teletype will print out a message, and the synthesizer will be paralyzed.
TITRATIONS Y l B2 X4 Y l B2 X4 Y l B2 X4 Y l Yl X4 Yl X4
A2 Yl A2 Yl
X4 A2 X4 A2
Yl X4 Yl X4
B2 X4 Y l B2 X 4 Y l Yl B2 X4 Y l B2 X4 Y l Yl .
00000 / A2 00001 /X4 00003 / Y1 00000 / A2 00001 / X4 00003 / Y1 00000 1' A2 00001 / X4 00003 f Yl
00220 00221 00224 00224 00225 00228 00228 00229 00232
CODE T I M E
00000 00001 00003 00000 00001 00003 00000 00001 00003 DURATION
Fig. 2. Programming for removal of the Boc-group and two titration cycles. The upper part shows the codes at the punch tape. See Table 1. Yl means draining of reactor. The lower part shows a print-out during the synthesis. The three columns indicate the code, elapsed time and duration of the single functions. It is to be noted that the figures in the third column refer to the duration of the previous functions. Thus 0001 refers to the duration of function B2 starting after an elapsed time of 165 minutes. The time is only printed in full minutes.
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
716
P. Villemoes, T. Christensen and K. Brunfeldt
Bd. 357(19776)
If a valid code has been read, the teletype prints the code and the elapsed time. After execution of the function, the duration is printed out. Section 2 Section 2 comprises three types of functions, namely: a) metering solvents and reagents, b) stirring and reaction time, and c) drain reactor. Furthermore special functions are included, such as metering of HC1 in organic solvents and meterin a) Metering solvents and reagents. Before the function is initiated all liquid sensors are tested for sensing light, i.e. no presence of liquid or malfunction, and furthermore the maximum allowed time for the mete r ing is set, normally five minutes. The master valve and the valve corresponding to the code are now energized, and sensors SI through S5 are watched. For completion of metering, the liquid sensors must have detected liquid in the proper sequence. Thus the sequence for metering to level two has to be: 1) SI no liquid, S3 no liquid; 2) SI liquid, S3 no liquid; 3) SI liquid, S3 liquid. Only if this sequence is accomplished are the abovementioned valves disenergized and solenoid valve VI energized, causing the liquid to be transferred into the reactor. Now, sensor SI and S6 have to go through a similar sequence as SI and S3. 20 s after the last drop has passed the liquid sensor S6, the computer terminates the function. Special metering functions are carried out in a way similar to the normal metering. b) Agitation. As seen in Fig. 1, a reactor with stirring is used. Five standard times, 1, 2, 3, 30 and 120 min, are available with or without stirring. Other agitation times can be obtained by a combination of the standard times. During the function, the computer compares elapsed time with the preset time, and when equalized, the function is terminated. c) Drain reactor. This function is carried out in a similar way as the draining of the metering vessel, but supplied with an extended drain time. Section 3 This section takes care of the analytical equipment, in the present system the titration equipment. The titrations can be performed with or without a time limit. In the first case mentioned the computer terminates the titration after 60 min, and executes the next seven functions sufficiently for two succeeding washings and for addition of methylene chloride. Finally, the synthesizer is paralyzed. As a software example, the drain metering vessel part is shown in flow chart form, Fig. 3.
]—
A Computer-Controlled Peptide Synthesizer Preben ViLLEMOES,ThorkildCHRiSTENSEN, and Kay BRUNFELDT The Danish Institute of Protein Chemistry, affiliated to the Danish Academy of Technical Sciences, DK-2970 Htfrsholm
(Received 6 November 1975)
Summary: A computer-controlled system for solid-phase peptide synthesis including an ana-
lytical step wise evaluation of the yields by titration with perchloric acid is described.
Ein Computer-gesteuertes System fur Peptidsynthese Zusammenfassung: Ein Computer-gesteuertes System für die Peptidsynthese an fester Phase einschließlich einer analytischen schrittweisen Aus-
wertung der Ausbeuten durch Titration mit PerChlorsäure wird beschrieben,
Key words: Solid-phase peptide synthesis, monitoring, computer control.
The experience with solid-phase peptide synthesis has shown that it often is advantageous to carry out the procedure automatically with control of the stepwise yields of both the coupling and the removal of the TV-protecting group. Such automatic systems have been developed^1'3!. The control has been achieved either by titration with perchloric acid of free -amino groups, by photometric monitoring of the concentration of the acylating reagent and the cleaved «-ammo proteciion group, or by determination of displaced
The use of a computer as control unit is, however, advantageous for several reasons. Thus changes of functions and introduction of new functions are easily carried out by minor changes in or addition of software. Computer control also allows a stepwise or continuous evaluation of analytical data, allowing a process control, ~, . , · , , , _ , · .. i_ · ·. T f %*"? descnbed bek)w ,s to be regarded as fal pment f SyStem * *"$?° ° ^ P™"0"^
P
Experimental
'
escn e
In the first two cases, the control unit is based
1) Description of reservoir and metering system
upon sequential logical. In the latter, an electromechanical system is used.
The reactor, metering vessel,and common lines are made of glass. (Fig. 1). Between the reservoirs and the solenoid
Abbreviations: Boc = f-butyloxycarbonyl-; Bzl = benzyl-; DCC = dicyclohexylcarbodiimide; EtOH = abs. ethanol; St3N = triethylamine; HOAc = acetic acid; S 2 = 2% crosslinking.
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
714
P. Villemoes, T. Chiistensen and K. Brunfeldt
Bd. 357(1976)
-Solvent inlets
HCI/Solvent
Solvents and Reagents Et, N
To waste
Fig. 1. Schematic drawing of the solid-phase peptide synthesizer. MV1 and MV2, metering vessels; SI through S9, liquid detectors; VI through V4, solenoid valves; V5 valve operated by fluid pressure in the line. -> indicates flow direction for open valve.
valves are inserted traps for protecting solvents, reagents and valves. Brown glass containers are used as reservoirs in order to minimize the destructive effect.of.light on the liquids. All the reservoirs except the one containing Et3N are connected to the metering vessel MV1, which is supplied with a side arm with 5 sensors. The levels of S2, S3 and S4 determine the volumes which can be delivered by the metering vessel. S5 is an overflow control. Sl^ must be activated in combination with S2, S3 or S4; this prevents that a single drop from a previous metering will terminate the actual metering. The principle for metering Et$N has been previously described'11 Transferring of solvents and reagents to the metering vessel, as well as draining of the reactor, is carried out by applying nitrogen pressure. The teflon tubes from the reservoirs are connected to the metering vessel via a glass manifold. The solvents, except acetic acid, are fed into the top of the manifold to ensure that the inlet system is carefully washed. A
special arrangement for metering HCI in organic solvents is used in order to minimize absorption of HCI in the teflon tubes. During measuring of the latter the valve V4 is also energized, thereby blocking the line to waste. 2) Control unit A computer, Alpha-16, from Computer Automation, U.S.A., with a 4K 16-bit core memory is used as control unit. An interface was constructed and consisted of 4 χ 4 bit input/output module, which selectively allows input/output of 4-bit information. Each 4-bit word is converted to a binary 15-bit word at the output, and input is converted from a 15-bit word to a 4-bit word. It means that it is possible to handle 15 inputs and/or 15 outputs without disturbing the other 45 inputs and/or outputs. Furthermore, all outputs are provided with a buffer flip-flop, that is set one at a time. The whole buffer can be cleared by one instruction from the computer, which makes a powerful way of terminating any
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
Bd. 357 (1976)
A Computer-Controlled Peptide Synthesizer
function. By splitting the 60 inputs and outputs into 4 words of 15 bits, it was possible to get satisfactory flexibility. 32 of the outputs are provided with a power amplifier to drive the solenoid valves. The rest of the outputs are for control of analytical equipment, such as titiation equipment and eventual future equipment. The inputs are connected to the liquid detectors (sensors) and to data and control inputs from the titration equipments, etc. Thus 60 input and 60 output lines are achieved. The coding of a synthesis is performed on punched tape in ASCII code and read into the computer via the tape reader of an ASR 33 teletype. The syntax is: a letter, a number, and a space. The letter denotes the basic function, the number attributes to the basic function the desired parameters, and the space separates the complete functions. As an example: A2 X4 Yl. A2 means metering methylene chloride (A) to the level of sensor S3 (2) on the side arm of the metering vessel, followed by draining of the liquid into the reactor. X4 means stirring (X) for 3 minutes (4). Yl means draining of reactor (Y) in less than five minutes (1). Programming for removal of the Boc-group and two titration cycles is shown in the upper part of Fig. 2; the
REMOVAL OF THE BOC-GROUP AND TWO B2 X4 Yl B2 X4 Yl V2 X4 Y l W2 X6 A2 X4 Y l A2 X4 Yl A2 X4 Y l Al W0 A I X4 X2 Yl A2 X4 Y l A2 X4 B2 X4 Y l B2 A2 V0 Y l A2 X4 Y l A2 Al W0 A I X4 X2 Yl A2 X4 Y l A2 X4 B2 X4 Y l B2 A2 V0 Y l A2 X4 Y l A2 B2 00165 00002 X4 00166 00001 Yl 00169 00003 00000 B2 00169 B2 00208 X4 00170 00001 X4 00209 Yl 00173 00003 Yl 00212 W 2 00173 00000 i B2 00212 X4 00174 00001 X4 00213 Yl 00177 00003 Yl 00216 W2 00177 00000 B2 00216 X6 00178 00001 X4 00217 Yl 00208 00030 Jf Yl 00220
/// /
715
lower part shows the print-out during the execution of the program. All functions are printed out by the teletype together with elapsed time and duration of the previously performed function in minutes. The alarm system is based on the same principles as described in ref J41. If an alarm occurs, the cause is printed out and the synthesizer is immediately paralyzed. It is possible to stop the process manually by making a console interrupt at the computer, whereby all outputs from the computer are turned off. Reactivating is done via the full stop of the teletype keyboard. After the interruption, notes may be written by the teletype. It is furthermore possible manually to let the synthesizer go on stand-by, when the function under execution is terminated. 3) Software The software package contains three sections: Section 1 When a code has been read, the software checks for valid letter, number and syntax errors, incorrect operation of the tape reader or tape out. If an error is detected, the teletype will print out a message, and the synthesizer will be paralyzed.
TITRATIONS Y l B2 X4 Y l B2 X4 Y l B2 X4 Y l Yl X4 Yl X4
A2 Yl A2 Yl
X4 A2 X4 A2
Yl X4 Yl X4
B2 X4 Y l B2 X 4 Y l Yl B2 X4 Y l B2 X4 Y l Yl .
00000 / A2 00001 /X4 00003 / Y1 00000 / A2 00001 / X4 00003 / Y1 00000 1' A2 00001 / X4 00003 f Yl
00220 00221 00224 00224 00225 00228 00228 00229 00232
CODE T I M E
00000 00001 00003 00000 00001 00003 00000 00001 00003 DURATION
Fig. 2. Programming for removal of the Boc-group and two titration cycles. The upper part shows the codes at the punch tape. See Table 1. Yl means draining of reactor. The lower part shows a print-out during the synthesis. The three columns indicate the code, elapsed time and duration of the single functions. It is to be noted that the figures in the third column refer to the duration of the previous functions. Thus 0001 refers to the duration of function B2 starting after an elapsed time of 165 minutes. The time is only printed in full minutes.
Brought to you by | University of Authenticated Download Date | 6/10/15 5:56
716
P. Villemoes, T. Christensen and K. Brunfeldt
Bd. 357(19776)
If a valid code has been read, the teletype prints the code and the elapsed time. After execution of the function, the duration is printed out. Section 2 Section 2 comprises three types of functions, namely: a) metering solvents and reagents, b) stirring and reaction time, and c) drain reactor. Furthermore special functions are included, such as metering of HC1 in organic solvents and meterin a) Metering solvents and reagents. Before the function is initiated all liquid sensors are tested for sensing light, i.e. no presence of liquid or malfunction, and furthermore the maximum allowed time for the mete r ing is set, normally five minutes. The master valve and the valve corresponding to the code are now energized, and sensors SI through S5 are watched. For completion of metering, the liquid sensors must have detected liquid in the proper sequence. Thus the sequence for metering to level two has to be: 1) SI no liquid, S3 no liquid; 2) SI liquid, S3 no liquid; 3) SI liquid, S3 liquid. Only if this sequence is accomplished are the abovementioned valves disenergized and solenoid valve VI energized, causing the liquid to be transferred into the reactor. Now, sensor SI and S6 have to go through a similar sequence as SI and S3. 20 s after the last drop has passed the liquid sensor S6, the computer terminates the function. Special metering functions are carried out in a way similar to the normal metering. b) Agitation. As seen in Fig. 1, a reactor with stirring is used. Five standard times, 1, 2, 3, 30 and 120 min, are available with or without stirring. Other agitation times can be obtained by a combination of the standard times. During the function, the computer compares elapsed time with the preset time, and when equalized, the function is terminated. c) Drain reactor. This function is carried out in a similar way as the draining of the metering vessel, but supplied with an extended drain time. Section 3 This section takes care of the analytical equipment, in the present system the titration equipment. The titrations can be performed with or without a time limit. In the first case mentioned the computer terminates the titration after 60 min, and executes the next seven functions sufficiently for two succeeding washings and for addition of methylene chloride. Finally, the synthesizer is paralyzed. As a software example, the drain metering vessel part is shown in flow chart form, Fig. 3.
]—
