Method and device for investigating substance libraries

• US 20030165998A1
• Filed: 02/19/2003
• Published: 09/04/2003
• Est. Priority Date: 03/28/2000
• Status: Active Grant

• Current Assignee: Borstel Forschungszentrum
• Original Assignee: Laser-Und Medizin-Technologie Gmbh Berlin
• Inventors: Frey, Andreas, Helfman, Juergen, Schmidt, Marcus Alexander, Mueller, Gerhard
• Granted Patent: US 7,431,685 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00306 Reactor vessels in a multiple arrangement
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00414 using suction
• CPC Class Codes: B01J 2219/00418 using pressure
• CPC Class Codes: B01J 2219/00486 by sonication or ultrasonication
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00522 in a multiple parallel arrangement
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 : separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The aim of the invention is to investigate the bonding of substances to target molecules. This is achieved by means of a densely packed device wherein various target molecules are bonded in a large number of sample areas. Cross-contamination and evaporation need to be minimized. The active surface of the sample areas have to be maximized. The inventive solution resides in the use of carrier plates, containing densely packed capillary structures and having a very large inner surface despite small outer dimensions. 1000 times more molecules can be bonded than on a flat outer surface of a comparable size. Cross contamination is avoided by the lack of cross links between the capillaries. Evaporation is minimized by a small outer surface. After the inner surface of the capillaries has been silanized, peptides and peptidomimetics are synthesized in a locally targeted manner. The molecular interactions of components of the substance library with active substances in a solution or a suspension are investigated by means of a local resolution optical detection method. Handling, especially cleaning and covering with substances, is carried out in a simple manner by rinsing liquids through the capillary plate.

Claims

1. (amended) A device for producing peptidic or peptoid substance libraries on a planar structure that has areas which are laterally separated from each other by physical barriers, for receiving a plurality of individual peptidic or peptoid target molecules or the precursors thereof that define the substance library, the device comprising:

a synthesis table for fixing the structure in a defined position during the production method;

a pipetting robot which applies the necessary reagents for producing locally delimited sample areas from a respective group of areas and applies the necessary reagents for producing the individual target molecules at the same time or in time-displaced relationship in the respective sample areas of the structure, and

a variable-volume cavity with a discharge at an underside of the synthesis table for disposal of applied reagents.

2. (amended) The device of claim 1, wherein the structure comprises a capillary plate with a plurality of capillaries which serve to receive the individual peptidic or peptoid target molecules of the substance library or the precursors thereof.

3. (amended) The device of claim 2, wherein at least an internal surface of the capillaries is coated with an organosilane layer which has functional groups suitable for covalent bonding of the target molecules of the substance library.

4. (amended) The device of claim 3, wherein the structure has regions outside the sample areas with functional groups that are chemically deactivated.

5. (amended) The device of claim 4, wherein the planar structure is transparent or opaque and can be used for optical measurement.

26. (new) The device of claim 1, wherein the structure has regions outside the sample areas with functional groups that are chemically deactivated.

27. (new) The device of claim 4, wherein the planar structure is transparent or opaque and can be used for optical measurement.

6. (amended) A method of building up peptidic or peptoid substance libraries defined by a plurality of individual peptidic or peptoid target molecules or the precursors thereof on a planar structure having areas which are separated laterally from each other by physical parameters, the method comprising the following steps:

a) fixing the structure in a defined position on a synthesis table during the production method;

b) covering at least the laterally-separated areas of the structure with an organosilane layer which has functional groups for anchoring peptidic or peptoid target molecules;

c) defining locally delimited sample areas comprising a group of adjacent areas by selectively applying a reagent for protecting the functional groups or a protected anchor molecule for covalent bonding to the functional groups by a pipetting robot to the areas of the sample areas;

d) chemically deactivating the regions of the structure, which are outside the sample areas, with functional groups, by the application of a deactivation reagent;

e) deprotecting all protected functional groups or anchor molecules;

f) applying the amino-terminally and side chain-protected, reagents required for production of the individual target molecules to the sample areas of the structure selectively with the pipetting robot and covalently bonded to the de-protected functional groups or anchor molecules;

g) removing excess reagents by way of a variable-volume cavity with a discharge at an underside of the synthesis table for disposal thereof, and

h) repeating steps e) through g) as required.

7. (amended) The method of claim 6, wherein the structure is a capillary plate with a plurality of capillaries which serve to receive individual peptidic or peptoid target molecules of the substance library or precursors thereof, comprising the following method steps:

a) fixing the capillary plate in a defined position on a synthesis table during the production method;

b) covering at least the internal surface of the capillaries by an organosilane layer which has functional groups for anchoring peptidic or peptoid target molecules;

c) defining locally delimited sample areas comprising a group of adjacent areas by selectively applying a reagent for protecting the functional groups or a protected anchor molecule for covalent bonding to the functional groups by a pipetting robot into the capillaries of the sample areas;

d) chemically deactivating the regions of the capillary plate, which are outside the sample areas, with functional groups, by the application of a deactivation reagent;

e) deprotecting all protected functional groups or anchor molecules;

f) applying the amino-terminally and side chain-protected reagents required for production of the individual target molecules to the sample areas of the capillary plate selectively with the pipetting robot and covalently bonded to the de-protected functional groups or anchor molecules;

g) removing excess reagents by way of a variable-volume cavity with a discharge at an underside of the synthesis table for disposal thereof, and

h) repeating steps e) through g) are repeated as required.

8. (amended) The method of claim 7, wherein γ

-aminopropyltrialkoxysilane is used for silanisation in step b).

28. (new) The method of claim 7, wherein γ

-aminopropyltrialkoxysilane is used for silanisation in step b).

9. (amended) The method of 8, wherein a protected α

-aminopoly(ethyleneglycol)-ω

-carboxylic acid active ester is used as the protected anchor molecule in step c).

10. (amended) The method of claim 9, wherein fluorenylmethoxycarbonyl (FMOC)-protected α

-aminopoly(ethyleneglycol)-ω

-propionic acid-N-hydroxybenzotriazole ester is used as the protected anchor molecule in step c).

30. (new) The method of claim 9, wherein fluorenylmethoxycarbonyl (FMOC)-protected α

-aminopoly(ethyleneglycol)-ω

-propionic acid-N-hydroxybenzotriazole ester is used as the protected anchor molecule in step c).

11. (amended) A device for binding soluble or suspendable active substances to solid-phase bonded peptidic or peptoid target molecules, wherein the target molecules are bonded on substance libraries in the form of a planar structure with areas which are separated laterally from each other by physical barriers, the device comprising:

an incubation table for fixing the structure in a defined position during binding of the active substances; and

a closed variable volume cavity which is provided with a valve and into which liquids with the soluble or suspendable active substances and other liquids and gases can be introduced and removed, the variable volume cavity positioned on at least one side of the structure.

12. (amended) The device of claim 11, wherein the structure comprises a capillary plate with a plurality of capillaries which serve to receive individual peptidic or peptoid target molecules of the substance library or the precursors thereof.

13. (amended) The device of claim 12, further comprising:

an ultrasonic transmitter which is in contact with the capillary plate.

14. (amended) A device for investigating the interaction of soluble and suspendable active substances with solid-phase bonded peptidic or peptoid target molecules on substance libraries in the form of a planar structure which is freely accessible to optical measurement and having areas which are laterally separated from each other by physical barriers, the device comprising:

an incubation table for fixing the structure in a defined position during the investigation in a defined position, and

an optical measuring device having a radiation source for an excitation light and having a detection optical means.

15. (amended) The device of claim 14, wherein the structure comprises a capillary plate with a plurality of capillaries which serve to receive individual peptidic or peptoid target molecules of the substance library or the precursors thereof.

16. (amended) The device of claim 15, further comprising:

an ultrasonic transmitter which is in contact with the capillary plate.

17. (amended) The device of claim 16, wherein the optical measuring device comprises a fluorescence spectrometer.

18. (amended) The device of claim 17, wherein the radiation source comprises a laser.

31. (new) The device of claim 14, wherein the optical measuring device comprises a fluorescence spectrometer.

32. (new) The device of claim 17, wherein the radiation source comprises a laser.

19. (amended) A method of investigating the interaction of soluble and suspendable active substances with solid-phase bonded peptidic or peptoid target molecules on substance libraries in the form of a planar structure with areas which are laterally separated from each other by physical barriers, the method comprising the step of:

providing an excitation light by way of an optical measuring device having a radiation source, which light scans the areas and is collected by way of a detection optical means.

20. (amended) The method of claim 19, wherein the structure comprises a capillary plate having a plurality of capillaries which serve to receive individual peptidic or peptoid target molecules of the substance library or the precursors thereof.

21. (amended) The method of claim 20, wherein the optical measuring device comprises a fluorescence spectrometer and the soluble or suspendable active substances and/or solid-phase bonded target molecules are fluorescence-marked.

22. (amended) The method of claim 21 wherein the fluorescence marking is excited and detected with light in the UV, in the visible range or near infrared.

23. (amended) The method of claim 22, wherein binding or non-binding of the active substance to the target molecule or the reaction of the active substance with the target molecule is detected by fluorescence or fluorescence extinction.

24. (amended) The method of claim 23, wherein fluorescence excitation is effected with a pulsed diode laser with a pulse duration of less than 10 ns.

25. (amended) The method of claim 23, wherein detection of the fluorescence is effected for the suppression of stray light in a time gate after decay of the excitation pulse.

33. (new) The method of claim 20, wherein the optical measuring device comprises a fluorescence spectrometer and the soluble or suspendable active substances and/or solid-phase bonded target molecules are fluorescence-marked.

34. (new) The method of claim 33 wherein the fluorescence marking is excited and detected with light in the UV, in the visible range or near infrared.

35. (new) The method of claim 19, wherein binding or non-binding of the active substance to the target molecule or the reaction of the active substance with the target molecule is detected by fluorescence or fluorescence extinction.

36. (new) The method of claim 34, wherein binding or non-binding of the active substance to the target molecule or the reaction of the active substance with the target molecule is detected by fluorescence or fluorescence extinction.

37. (new) The method of claim 35, wherein fluorescence excitation is effected with a pulsed diode laser with a pulse duration of less than 10 ns.

38. (new) The method of claim 36, wherein fluorescence excitation is effected with a pulsed diode laser with a pulse duration of less than 10 ns.

39. (new) The method of claim 22, wherein fluorescence excitation is effected with a pulsed diode laser with a pulse duration of less than 10 ns.

40. (new) The method of claim 34, wherein fluorescence excitation is effected with a pulsed diode laser with a pulse duration of less than 10 ns.

41. (new) The method of claim 35, wherein detection of the fluorescence is effected for the suppression of stray light in a time gate after decay of the excitation pulse.

42. (new) The method of claim 36, wherein detection of the fluorescence is effected for the suppression of stray light in a time gate after decay of the excitation pulse.

43. (new) The method of claim 22, wherein detection of the fluorescence is effected for the suppression of stray light in a time gate after decay of the excitation pulse.

44. (new) The method of claim 34, wherein detection of the fluorescence is effected for the suppression of stray light in a time gate after decay of the excitation pulse.

29. (new) The method of 8, wherein a protected α

-aminopoly(ethyleneglycol)-ω

-carboxylic acid active ester is used as the protected anchor molecule in step c).