Means for sequential solid phase organic synthesis and methods using the same

• US 4,631,211 A
• Filed: 03/25/1985
• Issued: 12/23/1986
• Est. Priority Date: 03/25/1985
• Status: Expired due to Term

• Current Assignee: NEXUS Biosystems, Inc. (Azenta, Inc.)
• Original Assignee: Scripps Clinic & Research Foundation (Scripps Health)
• Inventors: Houghten, Richard A.
• Primary Examiner(s): Kittle, John E.
• Assistant Examiner(s): Seidleck, James J.
• Application Number:
  US06/715,654
• Time in Patent Office: 638 Days
• Field of Search: 139/389, 428/35, 383/102, 525/54.11
• US Class Current: 506/30
• 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 2219/00297 "Tea bags"
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00461 Beads and reaction vessel together
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00542 Alphanumeric characters
• CPC Class Codes: B01J 2219/00558 Cuts-out
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07K 1/04 on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10T 428/1345 Single layer [continuous layer]

Abstract

A means for carrying out sequential, solid phase syntheses is disclosed as are methods of its use. The synthesis means comprises a foraminous container that encloses reactive particles. The particles are larger than any of the foraminae and have a known amount of covalently linked organic synthesis reactive functionality that is capable of reacting during the organic syntheses. The container and particles are substantially insoluble in water.

Claims

1. A means for carrying out a sequential, solid phase organic synthesis to form a reaction product containing a plurality of reacted subunits comprising an immersible mesh packet having enclosed therein a free form aliquot of reactive particles, said particles being of a size that is larger than any of the foraminae defined by said mesh packet and having a known amount of organic synthesis reactive functionality covalently linked to said particles, said reactive functionality being capable of reacting during said organic synthesis, said mesh packet and said particles being substantially insoluble in water and in organic solvents.

2. The synthesis means according to claim 1 wherein said mesh packet comprises a material selected from the group consisting of polymerized ethylene and propylene monomers.

3. The synthesis means according to claim 2 wherein said mesh of said packet is polypropylene mesh.

4. The synthesis means according to claim 3 wherein said mesh of said packet is heat sealed to enclose said particles.

5. The synthesis means according to claim 1 wherein said reactive functionality of said particles is a benzyl moiety that is a portion of a polymerized resin.

6. The synthesis means according to claim 5 wherein said resin constitutes substantially all of each of said particles.

7. The synthesis means according to claim 6 wherein the resin of said particles is comprised primarily of polymerized styrene cross-linked with divinyl benzene and includes a known amount of polymerized vinyl benzyl moiety, said polymerized vinyl benzyl moiety constituting said reactive benzyl moiety and providing a functional group through which said subunits may be covalently linked to said particles.

8. The synthesis means according to claim 7 wherein said reactive benzyl moiety is selected from the group consisting of benzyl chloride and benzylamine.

9. The synthesis means according to claim 5 wherein said particles are non-reactive with hydrogen fluoride.

10. The synthesis means according to claim 1, including a plurality of said foraminous container means each having enclosed therein said known quantity of said reactive particles, said plurality of container means being integrally formed with each other.

11. A means for carrying out a solid phase organic synthesis to form a reaction product containing a plurality of reacted subunits comprising an immersible mesh packet having enclosed therein a free form aliquot of solid support particles, said particles being of a size that is larger than any of the foraminae defined by said container and being linked to a known amount of identical organic synthesis subunits by selectively severable covalent bonds, each of said subunits containing a functional group capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable covalently linked protecting group, said packet and said particles being substantially insoluble in water and organic solvents and being non-reactive with hydrogen fluoride.

12. The synthesis means according to claim 11 wherein said mesh packet comprises a material selected from the group consisting of polymerized ethylene, propylene and mixtures thereof.

13. The synthesis means according to claim 12 wherein said mesh in said packet is made from polypropylene mesh.

14. The synthesis means according to claim 11 wherein said particles are hydrophobic resin beads.

15. The synthesis means according to claim 14 wherein the resin of said beads is comprised primarily of polymerized styrene cross-linked with divinyl benzene and includes a known amount of polymerized vinyl benzene moiety through which said subunits are covalently linked to said beads.

16. The synthesis means according to claim 15 wherein said subunits- are selected from the group consisting of (i) an alpha amino group-protected amino acid residue and (ii) an alpha amino group-protected and reactive side chain-protected amino acid residue.

17. The synthesis means according to claim 16 wherein said alpha amino group protecting group is an N-tert-butoxycarboxyl group.

18. The synthesis means according to claim 17 wherein said amino acid residue is linked to said resin through an ester bond between the carboxyl group of said amino acid and said polymerized vinyl benzyl moiety.

19. The synthesis means according to claim 17 wherein said amino acid residue is linked to said resin through an amide bond between the carboxyl group of said amino acid residue and the amino group of a disulfide-containing linking group that is bonded to said polymerized vinyl benzyl moiety, said linking group, written in the direction from left to right and from said amino acid toward said benzyl moiety, being represented by the formula:

space="preserve" listing-type="equation">--NHCH.sub.2 CH.sub.2 SSCH.sub.2 (CH.sub.2).sub.x CONH--

wherein x is a numeral having the value of zero or one.

20. A method of carrying out an organic solid phase synthesis to form a reaction product containing a plurality of desired reacted subunits comprising the steps of:

(a) providing a solid phase organic synthesis means comprising an immersible mesh packet having enclosed therein a free form aliquot of reactive particles, said particles being of a size that is larger than any of the foraminae defined by the mesh packet and having a known amount of organic synthesis reactive functionality covalently linked to said particles, said reactive functionality being capable of reacting during said organic synthesis, said packet and said particles being substantially insoluble in water and organic solvents;

(b) immersing said mesh packet in a liquid reaction medium containing first subunits and reacting the first subunits with said reactive functionalities to form within the immersed packet a selectively severable covalent bond between said first subunits and said particles, and to form a synthesis means containing particle-linked subunits, said first subunits including a first functional group that reacts with said reactive functionality and a second reactive functional group capable of reacting during said reaction, but that is protected from reaction by a selectively removable, covalently linked protection group;

(c) removing said protecting group from said second reactive functional group while said packet is immersed in a liquid medium to form a synthesis means containing particle-linked subunits having free reactive functional groups;

(d) admixing with the synthesis means containing particle-linked subunits having free reactive functional groups, while said packet is immersed in a liquid medium, an excess of identical, known other subunits that contain (i) a functional group capable of reacting with the free reactive groups of said particle-linked subunits and (ii) a second reactive functional group capable of reacting during the organic synthesis, but that is protected from so reacting by a selectively removable covalently linked protecting group;

(e) reacting said free reactive groups and said admixed functional groups, while said packet is immersed in a liquid medium, to form covalent bonds, couple the subunit to the particle and form particle-linked subunit reaction products;

(f) separating unreacted subunits from said particle-linked reaction products;

(g) removing the protecting groups of the coupled subunits of step (d), while said packet is immersed in a liquid medium, to form particle-linked reaction products containing free reactive functional groups;

(h) thereafter, serially repeating steps (d), (e), (f) and (g) until particle-linked reaction products containing the desired numer and identity of reacted subunits are synthesized;

(i) severing said selectively severable bond between said first subunit and said particle to form a mixture of severed, free reaction products and reacted particles;

(j) separating said severed, free reaction products from said reacted particles; and

(k) recovering said severed reaction products.

21. The method according to claim 20 wherein said particles are hydrophobic resin beads.

22. The method according to claim 21 wherein the resin of said beads is comprised primarily of polymerized styrene cross-linked with divinyl benzene and includes a known amount of polymerized vinyl benzene moiety through which said subunits are covalently linked to said beads.

23. The method according to claim 22 wherein said subunits are selected from the group consisting of (i) an alpha amino group-protected amino acid residue and (ii) an alpha amino group-protected and reactive side chain-protected amino acid residue.

24. A method of carrying out an organic solid phase synthesis to form a reaction product containing a plurality of desired reacted subunits comprising the steps of:

(a) providing a solid phase organic synthesis means comprising an immersible mesh packet having enclosed therein a known quantity of resin particles having a known amount of a selectively severable, covalently linked identical organic synthesis subunits that include a selectively removable protecting group covalently bonded to a reactive functional group, said resin particles being of a size that is larger than any of the foraminae defined by said mesh packet, said packet and said particles being substantially insoluble in water and organic solvents and non-reactive with hydrofluoric acid;

(b) immersing said mesh packet in a liquid reaction medium for removal of said protecting group and selectively removing said protecting group to form within the immersed packet a synthesis means containing resin-linked subunits having a free reactive group;

(c) admixing, while said packet is immersed in a liquid medium, an excess of identical subunits that contain (i) a second reactive functional group capable of reacting with the free reactive groups of said resin-linked subunits, and (ii) another reactive functional group that is capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable, covalently bonded protecting group;

(d) reacting said free reactive groups and said second reactive groups, while said packet is immersed in a liquid medium, to form covalent bonds, couple said admixed subunit to the resin and form resin-linked reaction products;

(e) separating unreacted subunits from said resin-linked reaction products;

(f) selectively removing the protecting groups said coupled subunits of step (c), while said packet is immersed in a liquid medium, to form resin-linked reaction products containing free reactive functional groups;

(g) thereafter, serially repeating steps (c), (d), (e) and (f) until resin-linked reaction products containing the desired number of reacted subunits are synthesized;

(h) severing said selectively severable bond between said first-named subunits and said resin to form free reaction products and reacted particles;

(i) separating said free reaction products from said reacted particles; and

(j) recovering said severed reaction products.

25. The method according to claim 24 wherein the resin of said particles is comprised primarily of polymerized styrene cross-linked with divinyl benzene and includes a known amount of polymerized vinyl benzyl moiety through which said subunits are covalently linked to said particles.

26. The method according to claim 25 wherein said subunits that contain a selectively removable protected reactive functional group are N-tert-butoxycarbonyl-protected amino acid residues.

27. The method according to claim 26 wherein said amino acid residue is linked to said resin through an ester bond between the carboxyl group of said amino acid and said polyerized vinyl benzyl moiety.

28. The method according to claim 25 wherein said amino acid residue is linked to said resin through an amide bond between the carboxyl group of said amino acid and the amino group of a disulfide-containing linking group that is bonded to said polymerized vinyl benzyl moiety, said linking group, written in the direction from left to right and from said amino acid toward said benzyl moiety, being represented by the formula:

space="preserve" listing-type="equation">--NHCH.sub.2 CH.sub.2 SSCH.sub.2 (CH.sub.2).sub.x CONH--

wherein x is a numeral having the value of zero or one.

29. A method for simultaneously adding a subunit to each of a pluraltiy of resin particle-linked subunits in an organic solid phase synthesis comprising the steps of:

(a) providing a pluraltiy of synthesis means each of which comprises an immersible mesh packet having enclosed therein a known quantity of particles, said particles being of a size that is larger than any of the foraminae defined by said mesh packet and having a known amount of an organic synthesis subunit covalently linked to said particles by selectively severable bonds, the subunit distal to the particle/subunit link containing a reaction functional group capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable covalently bonded protecting group, said packet and said particles being substantially insoluble in water and organic solvents;

(b) immersing each said mesh packet in a liquid reaction medium for removal of said protecting group and selectively removing said protecting group from said particle-linked subunits in each of said synthesis means to form an admixture containing a plurality of synthesis means containing particle-linked subunits having free reactive groups;

(c) immersing said synthesis means in a liquid medium containing an excess of identical subunits each of which contains (i) a second reactive functional group capable of reacting with the free reactive group of another reactive functional group that is capable of reacting said particle-linked subunits and (ii) another reactive functional group that is capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable, covalently bonded protecting group;

(d) froming covalent bonds between said free reactive groups and said second reactive group, while said packet is immersed, to couple said admixed subunits to said particles and form a plurality of synthesis means that contain particle-linked reaction products whose distal subunits contain selectively removable, protected reactive functional groups;

(e) separating said admixed, unreacted subunits from said particle-linked reaction products; and

(f) thereafter separating said plurality of synthesis means from each other.

30. The method according to claim 29 wherein said particles include a resin and said subunits are linked to said particles through said resin.

31. The method according to claim 30 wherein the resin of said particles is comprised primarily of polymerized styrene cross-linked with divinyl benzene and includes a known amount of polymerized vinyl benzyl moiety through which said subunits are covalently linked to said particle.

32. The method according to claim 31 wherein said subunits that contain a removably protected reactive functional group are N-tert-butoxycarbonyl-protected amino acid residues.

33. The method according to claim 32 wherein said amino acid residue is linked to said resin through an ester bond between the carboxyl group of said amino acid and said polymerized vinyl benzyl moiety.

34. The method according to claim 31 wherein said amino acid residue is linked to said resin through an amide bond between the carboxyl group of said amino acid and the amino group of a disulfide-containing linking group that is bonded to said polymerized vinyl benzyl moiety, said linking group, written in the direction from left to right and from said amino acid toward said benzyl moiety, being represented by the formula:

space="preserve" listing-type="equation">--NHCH.sub.2 CH.sub.2 SSCH.sub.2 (CH.sub.2).sub.x CONH--

wherein x is a numeral having the value of zero or one.

35. The method according to claim 29 wherein said linked subunits are amino acid residues polypeptide bonded in a reaction product sequence.

36. The method according to claim 35 wherein said amino acid residue reaction product sequences correspond to the amino acid sequence of a single proteinaceous material except for the identify of one amino acid residue in each of said subunit reaction product sequences.

37. The method according to claim 29 further including serially repeating strps (c), (d) and (e) prior to step (f) until plural synthesis means are produced in which each of said synthesis means contains a particle-linked subunit reaction product having a desired number and identity of reacted subunits.

38. The method according to claim 29 further including the steps of

(g) immersing said separated synthesis means in a liquid reaction medium for removal of said distal protecting group and selectively removing the distal protecting group from the subunit reaction products of at least one of the separated the subunit reaction products of at least one of the separated synthesis means of step (f) to form a synthesis means containing particle-linked subunits having free reactive groups;

(h) immersing said synthesis means formed in step (g) in a liquid medium containing an excess of identical subunits each of which contains (i) a second reactive functional group capable of reacting with said free reactive groups of the particle-linked subunits, and (ii) another reactive functional group that is capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable, covalently bonded protecting group;

(i) forming covalent bonds between said free reactive groups and said second reactive group, while the synthesis in step (g) is immersed in a liquid medium to couple said admixed subunits to said particles and form a synthesis means containing particle-linked reaction products whose distal subunits contain selectively removable, protected reactive functional groups;

(j) separating unreacted subunits from the particle-linked reaction products contained in synthesis means produced in step (i);

(k) therqafter combining said synthesis means produced in step (i) with another synthesis means to form a new plurality of synthesis means, each comprising an immersible mesh packet having enclosed therein a known quantity of particles, said particles being of a size that is larger than any of the foraminae defined by said mesh packet and having a known amount of organic synthesis subunit covalently linked to said particles by selectively severable bonds, the subunit distal to the particle/subunit link containing a reactive functional group capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable, covalently bonded protecting group, said packet and said particles being substantially insoluble in water and organic solvents;

(l) selectively removing said distal protecting groups from said particle-linked subunits in each of said new plurality of synthesis means to form a further plurality of synthesis means containing particle-linked subunits having free reactive groups;

(m) immersing said further pluraltiy of synthesis means in a liquid medium containing an excess of identical subunits each of which contains (i) a second functional group capable of reacting with the free reactive functional group of said particle-linked subunits, and (ii) another reactive functional group that is capable of reacting during said organic synthesis, but that is protected from so reacting by a selectively removable, covalently linked protecting group;

(n) forming covalent bonds between said free reactive groups and said second reactive groups, while said further plurality of synthesis means is immersed in a liquid medium to couple said admixed subunits to said particles and form an additional plurality of synthesis means that contain particle-linked reaction prodcuts whose distal subunits contain selectively removable, protected reactive functional groups;

(o) separating said admixed, unreacted subunits from said particle-linked reaction products; and

(p) thereafter separating individual synthesis means constituting said additional pluraltiy of synthesis means from each other.

39. The method according to claim 38 wherein individual synthesis means of the new pluraltiy of synthesis means contain different subunits.

40. The method according to claim 29 wherein a portion of the synthesis means constituting the plurality of synthesis means in step (a) is enclosed in a resealingly closable, foraminous envelope means, said envelope means comprising first and second opposing webs at least one of which is foraminous, said webs being partially secured together at their peripheries;

said first web being operably connected to a male portion of a resealable clsoure means, and said second web being operably connected to a female defines a mouth which communicates with the interior of said envelope means, said male and female portions of said closure means matingly engaging to resealably close said envelope means when pressed together in face-to-face relation, the foraminae of said envelope means being sized to permit drainage of a solvent used in said synthesis within a time period of at least 5 minutes at synthesis temperature while retaining said synthesis means within said envelope means, said envelope means being substantially insoluble in water and organic solvents.