1. Method of assembling peptide fragments for manufacturing a polypeptide comprising n peptide fragments and at least n\u22121 amino acids bearing a thiol function, represented by the formula:
A1-C1-A2-C2-A3- . . . -Ci\u22121-Ai- . . . -Cn\u22121-An\u2003\u2003(I)
in which A1, A2, A3, . . . Ai . . . , An are peptide fragments,
C1, C2, C3 . . . Ci\u22121 . . . Cn\u22121 are amino acid residues bearing a thiol function,
n is comprised between 3 and 50,
and
i is any integer between 2 and n, said method involving:
(a1) at least one step of preparing a fragment Y-A1-SEAoff (II1) in which A1 represents a peptide fragment the C-terminal of which bears a cyclic bis(2-sulphanylethyl)amino group
called SEAoff, and
Y is a fragment capable of reacting with a function of a solid support so as to form a bond between A1 and a solid support,
(b) at least one step of reaction of Y-A1-SEAoff (II1) with a solid support designated \u25a1-Y\u2032, \u25a1 representing the solid support itself and Y\u2032 representing a reactive function capable of reacting with Y to form a group Z according to the diagram:
(a2) at least one step of preparing a fragment H-C1(PG1)-A2-SEAoff (II2) in which C1, A2 and SEAoff are as defined above and (PG1) represents H or a protective group of the thiol of the amino acid C1,
(c1) at least one step of preparing a thioester peptide of formula (III1\u2032) starting from bis(2-sulphanylethyl)amino peptide \u25a1-Z-A1-SEAoff (III1) according to the diagram:
by the action of a thiol R-SH, optionally in the presence of a reducing agent of the cyclic disulphides, where R can be an alkyl or aryl radical, optionally substituted,
(d1) at least one step of condensation of (III\u20321) with the peptide fragment (II2) in the presence of an aromatic thiol ArSH under conditions in which PG1 is eliminated when it is different from H:
(an\u22121) at least one step of preparing a fragment Cn\u22121(PG n\u22121)-An where (PGn\u22121) represents H or a protective group of the thiol of the amino acid Cn\u22121,
(dn\u22121) at least one step of condensation of Cn\u22121(PG n\u22121)-An with
\u25a1-Z-A1-C1-A2- . . . Ci\u22121Ai- . . . Cn\u22122An\u22121SEAoff\u2003\u2003(IIIn\u22121)
in the presence of an aromatic thiol ArSH under conditions in which PGn\u22121 is eliminated when it is different from H in order to give:
\u25a1-Z-A1-C1-A2- . . . Ci\u22121Ai- . . . Cn\u22121An\u2003\u2003(IVn).
2. Method according to claim 1, which further comprises:
(e) a step of detaching the peptide A1-C1-A2- . . . Ci\u22121Ai- . . . Cn\u22121An (I) from the solid support.
3. Method according to claim 1 or claim 2, in which the solid support \u25a1 is selected from resins, in particular from resins based on polystyrene, polyacrylamide, polyethylene glycol, cellulose, polyethylene, polyester, latex, polyamide, polydimethylacrylamide, synthetic or natural hydrophilic polymers, glass beads, silica gels.
4. Method according to any one of claims 1 to 3, in which are C1, . . . Ci . . . Cn are cysteines.
5. Method according to any one of claims 1 to 4, in which PG1, . . . PGi . . . PGn are tert-butyl sulphenyl groups.
6. Method according to any one of claims 1 to 5, in which:
Y\u2032 comprises a function selected from an azide and Y is selected from the groups comprising an alkyne function, or
Y\u2032 comprises an alkyne function and Y is selected from the groups comprising an azide function or
Y\u2032 comprises an aldehyde function, Y is H and the N-terminal amino acid of A1 is selected from a cysteine, a serine or a threonine, or
Y\u2032 comprises an aldehyde function, Y comprises an NH2 group capable of forming a Schiff base.
7. Method according to any one of claims 1 to 6, in which R is selected from an alkyl radical comprising 1 to 12 carbon atoms, linear or branched, optionally substituted, or C6-C12 aryl, optionally substituted.
8. Method according to any one of claims 1 to 7, in which, for every i=2, . . . n\u22122, the method comprises
(ci) at least one step of conversion of
\u25a1-Z-A1-C1- . . . Ci\u22121AiSEAoff\u2003\u2003(IIIi)
to
\u25a1-Z-A1-C1- . . . Ci\u22121AiSR\u2003\u2003(III\u2032i)
by the action of a thiol R-SH, optionally in the presence of a reducing agent of cyclic disulphides,
(di) at least one step of condensation of Ci(PGi)Ai+1SEAoff where (PGi) represents H or a protective group of the thiol of the amino acid Ci, with
\u25a1-Z-A1-C1- . . . Ci\u22121Ai\u2014SR\u2003\u2003(III\u2032i)
in order to give
\u25a1-Z-A1-C1- . . . CiAi+1SEAoff\u2003\u2003(IIIi+1).
9. Method according to any one of claims 1 to 8, in which, for every i=2, . . . n\u22122, the steps di) of the method are carried out under conditions in which PGi is removed selectively in situ, without affecting SEAoff and the solvent of the reaction is an aqueous buffer, with pH between 4 and 9, containing an aromatic thiol.
10. Method of manufacturing a medicinal product comprising at least:
manufacturing at least one polypeptide by the method according to any one of claims 1 to 9, and
combining it with a pharmaceutically acceptable support.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. An audio system testing arrangement, comprising:
a controller configured to:
be coupled to a communication bus of a motor vehicle;
run a plurality of test scripts via the communication bus, the test scripts testing features of an audio system within the vehicle; and
determine a fault of the audio system dependent upon results of the test scripts; and
a display andor speaker configured to annunciate the determined fault of the audio system.
2. The arrangement of claim 1 further comprising a microphone configured to sense audible sounds emitted by a speaker of the vehicle, the controller being configured to determine the fault of the audio system dependent upon a signal generated by the microphone.
3. The arrangement of claim 1 further comprising a temperature sensor configured to sense a temperature of the audio system, the controller being configured to determine the fault of the audio system dependent upon a signal generated by the temperature sensor.
4. The arrangement of claim 3 wherein the signal generated by the temperature sensor includes a temperature and a date and time stamp associated with the temperature.
5. The arrangement of claim 1 wherein the test scripts include a plurality of first test scripts that store information about an audio system of the vehicle and a plurality of second test scripts that diagnose the audio system.
6. The arrangement of claim 1 further comprising a first switch connected to the controller and a second switch connected to the controller, the first switch being configured to cause the controller to run a next said test script in response to the first switch being actuated, the second switch being configured to cause the controller to run a previous said test script in response to the second switch being actuated.
7. The arrangement of claim 1 wherein the controller is disposed within a portable module that is configured to be temporarily plugged into the motor vehicle.
8. An audio system testing method, comprising the steps of
coupling a controller to a signal bus of a motor vehicle;
using the controller to run a plurality of test scripts via the signal bus, the test scripts testing features of an audio system within the vehicle;
determining a fault of the audio system dependent upon results of the test scripts; and
annunciating the determined fault of the audio system.
9. The method of claim 8 wherein the determined fault is annunciated by a display screen andor a loudspeaker.
10. The method of claim 8 further comprising:
sensing audible sounds emitted by a speaker of the vehicle; and
determining the fault of the audio system dependent upon the sensed audible sounds.
11. The method of claim 8 further comprising:
sensing a temperature of the audio system; and
determining the fault of the audio system dependent upon the sensed temperature.
12. The method of claim 11 further comprising generating and storing in memory a date and time stamp in association with the temperature.
13. The method of claim 8 wherein the test scripts include a plurality of first test scripts that store information about an audio system of the vehicle and a plurality of second test scripts that diagnose the audio system.
14. The method of claim 8 further comprising:
causing the controller to run a next said test script by actuating a first switch; and
causing the controller to run a previous said test script by actuating a second switch.
15. The method of claim 8 wherein the controller is disposed within a portable module, the method further comprising temporarily plugging the portable module into the motor vehicle.
16. An audio system testing arrangement, comprising:
a controller configured to:
run a plurality of test scripts via a communication bus of a motor vehicle, the test scripts including a plurality of first test scripts that store information about an audio system of the vehicle and a plurality of second test scripts that diagnose the audio system; and
determine a fault of the audio system dependent upon results of the test scripts; and
a display andor speaker configured to annunciate the determined fault of the audio system.
17. The arrangement of claim 16 further comprising a microphone configured to sense audible sounds emitted by a speaker of the vehicle, the controller being configured to determine the fault of the audio system dependent upon a signal generated by the microphone.
18. The arrangement of claim 16 further comprising a temperature sensor configured to sense a temperature of the audio system, the controller being configured to determine the fault of the audio system dependent upon a signal generated by the temperature sensor.
19. The arrangement of claim 16 further comprising a first switch connected to the controller and a second switch connected to the controller, the first switch being configured to cause the controller to run a next said test script in response to the first switch being actuated, the second switch being configured to cause the controller to run a previous said test script in response to the second switch being actuated.
20. The arrangement of claim 16 wherein the controller is disposed within a portable module that is configured to be temporarily plugged into the motor vehicle.