Chemical synthesis using solvent microdroplets

• US 20010018512A1
• Filed: 03/26/2001
• Published: 08/30/2001
• Est. Priority Date: 03/20/1997
• Status: Abandoned Application

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Blanchard, Alan P.
• Application Number:
  US09/817,491
• Publication Number:
  US 20010018512A1
• Time in Patent Office: Days
• US Class Current: 536/25.3
• 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 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/00488 by rotation of the reaction vessels
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• 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 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• 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 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00704 : integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B41J 2/01 : Ink jet
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07H 19/00 : Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/02 : in solution C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, 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

Abstract

The present invention relates to microdroplets of a solution comprising a solvent having a boiling point of 150° C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec). Such microdroplets are useful for the synthesis of chemical species, particularly biopolymers such as oligonucleotides and peptides, as well as arrays of chemical species. Preferably, the solvent has the formula (I):

wherein

A=O or S;

X, O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

Claims

1. A microdroplet of a solution, the solution comprising a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

2. The microdroplet of

claim 1

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

3. The microdroplet of

claim 2

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

4. The microdroplet of

claim 2

, wherein said solvent is propylene carbonate.

5. The microdroplet of

claim 1

, wherein the volume of said microdroplet is 100 pL or less.

6. The microdroplet of

claim 5

, wherein the volume of said microdroplet is 50 pL or less.

7. The microdroplet of

claim 1

, wherein the solution comprises a nucleoside or activated nucleoside.

8. The microdroplet of

claim 7

, wherein the solution comprises an activated nucleoside that contains an activated phosphorous-containing groups selected from the group consisting of phosphodiester, phosphotriester, phosphate triester, H-phosphonate and phosphoramidite groups.

9. A method for chemical synthesis, comprising the step of dispensing a microdroplet of a solution comprising (i) a first chemical species, and (ii) a solvent, such that the microdroplet impinges a second chemical species and the first chemical species reacts with the second chemical species to form a third chemical species, the solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

10. The method of

claim 9

, wherein said solvent has the formula (I):

wherein

A=O or S;

O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

11. The method of

claim 10

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

12. The method of

claim 10

, wherein said solvent is propylene carbonate.

13. The method of

claim 9

, wherein the volume of said microdroplet is 100 pL or less.

14. The method of

claim 13

, wherein the volume of said microdroplet is 50 pL or less.

15. The method of

claim 9

, wherein the third chemical species is formed in the presence of a catalyst.

16. The method of

claim 9

, further comprising the step of dispensing a microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the microdroplet comprising the catalyst and solvent impinges the second chemical species subsequent to impingement by the microdroplet that comprises the first chemical species and solvent.

17. The method of

claim 9

, wherein the second chemical species is attached to a substrate, directly or via a linker molecule.

18. The method of

claim 17

, wherein the substrate is selected from the group consisting of glass, silica, silicon, polypropylene, TEFLON®

, polyethylimine, nylon, fiberglass, paper and polystyrene.

19. The method of

claim 17

, wherein the second chemical species is attached to a linker that is attached to the substrate.

20. The method of

claim 9

, wherein the first chemical species and second chemical species are nucleosides or activated nucleosides.

21. The method of

claim 20

, wherein the chemical species comprises an activated nucleoside that contains activated phosphorous-containing groups selected from the group consisting of phosphodiester, phosphotriester, phosphate triester, H-phosphonate and phosphoramidite groups.

22. The method of

claim 9

, wherein the chemical being synthesized is an oligonucleotide or a peptide.

23. The method of

claim 22

, wherein the chemical being synthesized is an oligodeoxyribonucleotide or an oligoribonucleotide.

24. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having a phosphoramidite group at its 3′

position, and a protecting group at its 5′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having hydroxyl groups, and forms a microdot upon the substrate;

(b) dispensing a second microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the second microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the first nucleoside and an hydroxyl group of the linker, resulting in the conversion of the phosphoramidite group to a 3′

phosphite group, and the presence of unreacted hydroxyl groups of the linker;

(c) washing the substrate with an oxidizing agent to convert the 3′

phosphite group to a 3′

phosphate group;

(d) rinsing the substrate with a deprotecting agent which removes the protecting group from the 5′

position of the first nucleoside, and yields a 5′

hydroxyl group;

(e) dispensing a third microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group at its 3′

position, and a protecting group at its 5′

position and (ii) the solvent, such that the second microdroplet impinges the microdot; and

(f) dispensing a fourth microdroplet of a solution comprising (i) the catalyst and (ii) the solvent, such that the fourth microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 5′

hydroxyl group of the first nucleoside, resulting in the coupling of the second nucleoside to the first nucleoside.

25. The method of

claim 24

further comprising the steps of performing successive iterations of steps (c)-(f).

26. The method of

claim 24

, further comprising after step (c) and before step (d) the step of treating the substrate with a capping reagent which caps the unreacted hydroxyl groups of the linker.

27. The method of

claim 26

, wherein the capping reagent is perfluorooctanoyl chloride.

28. The method of

claim 24

wherein said solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

29. The method of

claim 28

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

30. The method of

claim 28

, wherein said solvent is propylene carbonate.

31. The method of

claim 24

, wherein the substrate is glass.

32. The method of

claim 24

, wherein the catalyst is 5-ethylthiotetrazole.

33. The method of

claim 24

, wherein the oxidizing agent is a solution comprising iodine and water.

34. An array of microdots on a substrate, said microdots comprising (i) a chemical species, and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

35. The array of

claim 34

, wherein said solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

36. The array of

claim 35

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

37. The array of

claim 35

, wherein said solvent is propylene carbonate.

38. The array of

claim 34

, wherein each of said microdots has a diameter in the range of 1 to 1000 μ

m.

39. The array of

claim 38

, wherein each of said microdots has a diameter in the range of 10 to 500 μ

m.

40. The array of

claim 39

, wherein each of said microdots has a diameter in the range of 40 to 100 μ

m.

41. The array of

claim 34

, wherein the chemical species is an oligonucleotide or a peptide.

42. The array of

claim 41

, wherein the chemical species is an oligodeoxyribonucleotide or an oligoribonucleotide.

43. The array of

claim 34

, wherein the microdots are separated from each other by hydrophobic domains.

44. An automated system comprising:

an inkjet print head for spraying a microdroplet comprising a chemical species on a substrate;

a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the microdroplet at specified sites;

a flow cell for treating the substrate on which the microdroplet is deposited by exposing the substrate to one or more selected fluids;

a treating transport for moving the substrate between the print head and the flow cell for treatment in the flow cell; and

an alignment unit for aligning the substrate correctly relative to the print head each time when the substrate is positioned adjacent to the print head for deposition.

45. The system of

claim 44

, wherein the inkjet printhead contains a solution comprising the chemical species dissolved in a solvent.

46. The system of

claim 45

, wherein the chemical species is a monomer unit of a biopolymer.

47. The system of

claim 45

, wherein the solution further comprises a catalyst.

48. The system of

claim 45

wherein the solution comprises a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

49. The system of

claim 48

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

50. The system of

claim 49

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

51. The system of

claim 48

, wherein said solvent is propylene carbonate.

52. The system of

claim 46

which is for synthesizing an oligonucleotide, and wherein the monomer is a nucleoside or nucleoside derivative.

53. The system of

claim 52

, wherein the nucleoside is a deoxyribonucleoside or a ribonucleoside.

54. The system of

claim 44

, wherein the inkjet print head comprises an array of piezoelectric pumps.

55. The system of

claim 54

, further comprising an external reservoir connected to supply the chemical species to the print head.

56. The system of

claim 55

, wherein the external reservoir contains a solution comprising the chemical species dissolved in a solvent.

57. The system of

claim 56

, wherein the solvent has a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

58. The system of

claim 57

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

59. The system of

claim 58

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

60. The system of

claim 58

, wherein said solvent is propylene carbonate.

61. An automated system for synthesizing oligonucleotides on a substrate, comprising:

an inkjet print head for spraying a solution comprising a nucleoside or activated nucleoside on a substrate;

a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the nucleoside at specified sites;

a flow cell for treating the substrate on which the monomer is deposited by exposing the substrate to one or more selected fluids;

a treating transport for moving the substrate between the print head and the flow cell for treatment in the flow cell; and

an alignment unit for aligning the substrate correctly relative to the print head each time when the substrate is positioned adjacent to the print head for deposition.

62. The system of

claim 61

, wherein the inkjet print head comprises an array of piezoelectric pumps.

63. The system of

claim 61

, further comprising:

an external reservoir connected to supply the nucleoside to the print head.

64. The system of

claim 63

, wherein the external reservoir contains a solution comprising said nucleoside or activated nucleoside dissolved in propylene carbonate.

65. The system of

claim 61

, further comprising a plurality of external reservoirs connected to the printer head, each external reservoir storing a nucleoside or activated nucleoside.

66. The system of

claim 61

, further comprising control logic configured to perform the following steps:

moving the substrate over the print head with the scanning transport;

firing the print head repeatedly to deposit the nucleoside or activated nucleoside monomer at the specified loci on the substrate; and

transferring the substrate to the flow cell with the treating transport.

67. The system of

claim 61

, wherein the scanning transport comprises:

a vacuum chuck for holding the substrate; and

a translational stage connected to move the vacuum chuck with respect to the print head.

68. The system of

claim 67

, wherein the vacuum chuck is rotatable for alignment with the print head.

69. The system of

claim 68

, wherein the vacuum chuck is engageable by a stationary element to rotate the vacuum chuck for alignment with the print head.

70. The system of

claim 67

, wherein the translational stage is driven by motorized means.

71. The system of

claim 70

, wherein the motorized means is a stepping motor.

72. The system of

claim 61

, wherein the flow cell has means for rinsing off unconnected monomers.

73. The system of

claim 61

, wherein the treating transport comprises:

a vacuum chuck for holding the substrate; and

a translational stage connected to move the second vacuum chuck and to move the substrate to and from the flow cell.

74. The system of

claim 73

, wherein the translational stage is driven by motorized means.

75. The system of

claim 74

, wherein the motorized means is a stepping motor.

76. The system of

claim 61

, wherein said alignment unit comprises a camera positioned adjacent to the substrate to positionally calibrate the substrate.

77. The system of

claim 61

, wherein said alignment unit comprises a marker that can be activated to establish one or more marks at particular loci on the substrate for positionally calibrating the substrate.

78. The system of

claim 61

, wherein said alignment unit comprises:

a marker that can be activated to establish one or more marks at particular loci on the substrate; and

a camera positioned adjacent to the substrate to located said marks relative to the printer head.

79. The system of

claim 78

, further comprising a tip that can be activated to scratch marks at particular loci on the substrate for positionally calibrating the substrate.

80. The system of

claim 61

, wherein said alignment unit comprises:

control logic connected to control the movement of the scanning transport;

a marker that can be activated to establish one or more marks at particular loci on the substrate; and

a camera positioned adjacent to the substrate to locate said marks relative to the printer head.

81. The system of

claim 80

, wherein the control logic is configured to perform the following steps:

moving the substrate over the marker to establish one or more marks on the substrate;

subsequently locating the marks with the camera;

determining the position of the substrate with respect to the print head with reference to the marks; and

calibrating the scanning transport in response to the determined position of the substrate with respect to the printer head.

82. The system of

claim 80

, further comprising a stationary element that engages the substrate chuck to rotate the substrate chuck for alignment with the print head, wherein the control logic is configured to perform the following steps:

moving the substrate over the marker to establish one or more marks on the substrate;

subsequently locating the marks with the camera;

determining misalignment of the substrate relative to the print head with reference to the marks; and

moving the translational stage to (a) engage the substrate chuck with the stationary element, and (b) rotate the substrate chuck by an angular displacement that corrects for the misalignment.

83. The system of

claim 61

, further comprising a transfer station that supports the substrate for transfer between the treating transport and the scanning transport.

84. A method of controlling a system synthesizing a biopolymer on a substrate using a computer having a memory for storing a control program and data, wherein the system has an inkjet print head for spraying a microdroplet on the substrate, a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the microdroplet, an alignment unit for detecting misalignment of the substrate with respect to the print head at each deposition step, a flow cell for treating the substrate, and a treating transport for moving the substrate between the printer head and the flow cell, the method comprising the steps of:

(a) aligning the substrate relative to the print head by processing data from the alignment unit and by sending a signal to the scanning transport to move the substrate so as to correct misalignment of the substrate;

(b) selectively depositing a microdroplet on the substrate by sending a signal to the print head to spray the microdroplet and by sending a signal to the scanning transport to move the substrate adjacent to the print head so that the microdroplet can be deposited at specified loci on the substrate; and

(c) controlling treatment of the substrate by sending a signal to the treating transport to move the substrate to the flow cell and by sending a signal to the flow cell to control operation of the flow cell.

85. The method of

claim 84

, wherein the microdroplet comprises a monomer unit of a biopolymer.

86. The method of

claim 85

, wherein the microdroplet further comprises a catalyst.

87. The method of

claim 84

, wherein the microdroplet comprises a solvent.

88. The method of

claim 87

, wherein the solvent has a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

89. The method of

claim 88

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

90. The method of

claim 89

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

91. The method of

claim 87

, wherein said solvent is propylene carbonate.

92. The method of

claim 84

, further comprising the steps of repeating said steps (a)-(c).

93. The method of

claim 85

, further comprising the steps of repeating said steps (a)-(c) to form a two-dimensional biopolymer array.

94. The method of

claim 84

, wherein the biopolymer is an oligonucleotide.

95. The method of

claim 94

, wherein the oligonucleotide is an oligodeoxyribonucleotide or an oligoribonucleotide.

96. The method of

claim 85

, further comprising repeating said steps (a)-(c) to form a two-dimensional oligonucleotide array.

97. The method of

claim 84

, wherein the step of aligning the substrate comprises the steps of:

(a) moving the substrate over a marker to establish one or marks on the substrate;

(b) subsequently locating the marks with a camera;

(c) determining misalignment of the substrate relative to the print head with reference to the marks; and

(d) moving the substrate to correct the misalignment.

98. The method of

claim 97

, wherein the step of moving the substrate to correct the misalignment is done by moving the substrate in a linear motion in X and Y directions and by rotating the substrate.

99. A method of controlling a system synthesizing oligonucleotides on a substrate using a computer having a memory for storing a control program and data, wherein the system has an inkjet print head for spraying a solution comprising a nucleoside or activated nucleoside, on the substrate, a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the solution, an alignment unit for detecting misalignment of the substrate with respect to the print head at each deposition step, a flow cell for treating the substrate, and a treating transport for moving the substrate between the printer head and the flow cell, the method comprising the steps of:

(a) placing the substrate with respect to the print head and establishing marks on the substrate;

(b) selectively depositing the solution on the substrate by sending a signal to the print head to spray the solution and by sending a signal to the scanning transport to move the substrate adjacent to the print head so that the solution can be deposited at specified loci on the substrate;

(c) controlling treatment of the substrate by sending a signal to the treating transport to move the substrate to the flow cell and by sending a signal to the flow cell to control operation of the flow cell; and

(d) placing the substrate adjacent to the print head and aligning the substrate with respect to the print head by processing data from the alignment unit and by sending a signal to the scanning transport to move the substrate so as to correct misalignment of the substrate.

100. The method of

claim 99

, further comprising repeating said steps (a)-(d) to form a two-dimensional oligonucleotide array.

101. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having an activated O-succinate group at its 3′

position, and a protecting group at its 5′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having an amine group, and forms a microdot upon the substrate;

(b) rinsing the substrate with a deprotecting agent which removes the protecting group from the 5′

position of the first nucleoside, and exposes a 5′

hydroxyl group;

(c) dispensing a second microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group, containing a cyanoethyl group, at its 3′

position, and a protecting group at its 5′

position and (ii) the solvent, such that the second microdroplet impinges the microdot;

(d) dispensing a third microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the third microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 5′

hydroxyl group of the first nucleoside, resulting in the conversion of the phosphoramidite group to a phosphite group;

(e) washing the substrate with an oxidizing agent to convert the 3′

phosphite group to a 3′

phosphate group;

(f) performing successive iterations of steps (b)-(e);

(g) treating the product of step (f) with a second deprotecting agent that converts the cyanoethylphosphate groups, of the product of step (f), to phosphate groups; and

(h) treating the product of step (g) with a hydrolyzing agent which cleaves the oligonucleotide from the linker.

102. The method of

claim 101

wherein said solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

103. The method of

claim 101

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

104. The method of

claim 101

, wherein said solvent is propylene carbonate.

105. The method of

claim 101

, wherein the substrate is glass.

106. The method of

claim 101

, wherein the catalyst is 5-ethylthiotetrazole.

107. The method of

claim 101

, wherein the oxidizing agent is a solution comprising iodine and water.

108. The method of

claim 101

, wherein the hydrolyzing agent is selected from the group consisting of hydroxide ion, CH₃NH₂ and concentrated aqueous NH₄OH.

109. An automated system for synthesizing oligonucleotides on a substrate, comprising:

an inkjet print head having an array of pumps for depositing a nucleoside or activated nucleoside at specified loci on the substrate;

a first translational stage having at least two axes of movement;

a first substrate chuck mounted for movement by the translational stage, the first substrate chuck being adapted to hold the substrate and move adjacent to the print head;

a flow cell that receives the substrate and that exposes the substrate to one or more selected fluids;

a second translational stage that has at least two axes of movement;

a second substrate chuck mounted for movement by the second translation stage, the second substrate chuck being adapted to hold the substrate and move between the print head and the flow cell;

control logic connected to control movement of the first and second translational stages;

a marker positioned adjacent to the print head that can be activated to mark particular loci on the substrate for positionally calibrating the substrate with respect to the print head; and

a camera positioned adjacent to the print head to positionally calibrate the substrate with respect to the print head, wherein the camera is connected to provide images to the control logic.

110. The system of

claim 109

, wherein the first and the second translational stages are driven by stepping motors.

111. The system of

claim 109

, wherein the first and second substrate chucks are vacuum chucks.

112. The system of

claim 109

, wherein the first substrate chuck is rotatable for alignment with the print head.

113. The system of

claim 109

, further comprising a tip that can be activated to scratch marks at particular loci on the substrate for positionally calibrating the substrate.

114. The system of

claim 109

, further comprising:

a reservoir connected to the print head to supply the nucleoside or activated nucleoside to the print head, wherein

the reservoir contains said nucleoside or activated nucleoside dissolved in a solvent.

115. The system of

claim 114

, wherein the solvent has a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

116. The system of

claim 115

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₂-C₂₀ straight or branched chain alkyl.

117. The system of

claim 116

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

118. The system of

claim 115

, wherein said solvent is propylene carbonate.

119. A solution comprising a solvent and a nucleoside or activated nucleoside, said solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

120. The solution of

claim 119

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₂-C₂₀ straight or branched chain alkyl.

121. The solution of

claim 120

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

122. The solution of

claim 119

, wherein said solvent is propylene carbonate.

123. The solution of

claim 119

, wherein the solution comprises an activated nucleoside that contains an activated phosphorous-containing group selected from the group consisting of phosphodiester, phosphotriester, phosphate triester, H-phosphonate and phosphoramidite group.

139. An inkjet print head containing the solution of

claim 119

.

140. An inkjet print head containing the solution of

claim 120

.

141. An inkjet print head containing the solution of

claim 121

.

142. An inkjet print head containing the solution of

claim 122

.

124. An apparatus programmed for controlling a system synthesizing a biopolymer on a substrate, wherein the system has an inkjet print head for spraying a microdroplet on the substrate, a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the microdroplet, an alignment unit for detecting misalignment of the substrate with respect to the print head at each deposition step, a flow cell for treating the substrate, and a treating transport for moving the substrate between the printer head and the flow cell, the controller comprising:

(a) means for controlling alignment of the substrate relative to the print head by processing data from the alignment unit and by sending a signal to the scanning transport to move the substrate so as to correct misalignment of the substrate;

(b) means for controlling selective deposition of a microdroplet on the substrate by sending a signal to the print head to spray the microdroplet and by sending a signal to the scanning transport to move the substrate adjacent to the print head so that the microdroplet can be deposited at specified loci on the substrate; and

(c) means for controlling treatment of the substrate by sending a signal to the treating transport to move the substrate to the flow cell and by sending a signal to the flow cell to control operation of the flow cell.

125. The apparatus of

claim 124

, wherein the inkjet print head contains a solution comprising a monomer unit of a biopolymer.

126. The apparatus of

claim 125

, wherein the solution further comprises a catalyst.

127. The apparatus of

claim 125

, wherein the solution comprises a solvent that has a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

128. The apparatus of

claim 127

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

129. The apparatus of

claim 124

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

130. The apparatus of

claim 127

, wherein said solvent is propylene carbonate.

131. An apparatus programmed for controlling a system synthesizing a biopolymer on a substrate, wherein the system has an inkjet print head for spraying a microdroplet on the substrate, a scanning transport for scanning the substrate adjacent to the print head to selectively deposit the microdroplet, an alignment unit for detecting misalignment of the substrate with respect to the print head at each deposition step, a flow cell for treating the substrate, and a treating transport for moving the substrate between the printer head and the flow cell, said apparatus comprising one or more computer systems programmed for:

(a) controlling alignment of the substrate relative to the print head by processing data from the alignment unit and by sending a signal to the scanning transport to move the substrate so as to correct misalignment of the substrate;

(b) controlling selective deposition of a microdroplet on the substrate by sending a signal to the print head to spray the microdroplet and by sending a signal to the scanning transport to move the substrate adjacent to the print head so that the microdroplet can be deposited at specified loci on the substrate; and

(c) controlling treatment of the substrate by sending a signal to the treating transport to move the substrate to the flow cell and by sending a signal to the flow cell to control operation of the flow cell.

132. The apparatus of

claim 131

, further comprising a reservoir connected to the print head to suppy a nucleoside or activated nucleoside to the print head, wherein the reservoir contains said nucleoside or activated nucleoside dissolved in a solvent.

133. The apparatus of

claim 131

, wherein the inkjet print head contains a solution comprising a monomer unit of a biopolymer.

134. The apparatus of

claim 133

, wherein the solution further comprises a catalyst.

135. The apparatus of

claim 133

, wherein the solution comprises a solvent that has a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above.

136. The apparatus of

claim 135

, wherein the solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

137. The apparatus of

claim 136

, wherein the solvent is selected from the group consisting of:

N-methyl-2-pyrrolidone;

2-pyrrolidone;

propylene carbonate;

γ

-valerolactone;

6-caprolactam;

ethylene carbonate;

γ

-butyrolactone;

δ

-valerolactone;

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone;

ethylene trithiocarbonate; and

1,3-dimethyl-2-imidazolidinone.

138. The apparatus of

claim 135

, wherein said solvent is propylene carbonate.

143. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having a phosphoramidite group at its 5′

position, and a protecting group at its 3′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having hydroxyl groups, and forms a microdot upon the substrate;

(b) dispensing a second microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the second microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the first nucleoside and an hydroxyl group of the linker, resulting in the conversion of the phosphoramidite group to a 5′

phosphite group, and the presence of unreacted hydroxyl groups of the linker;

(c) washing the substrate with an oxidizing agent to convert the 5′

phosphite group to a 5′

phosphate group;

(d) rinsing the substrate with a deprotecting agent which removes the protecting group from the 3′

position of the first nucleoside, and yields a 3′

hydroxyl group;

(e) dispensing a third microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group at its 5′

position, and a protecting group at its 3′

position and (ii) the solvent, such that the second microdroplet impinges the microdot; and

(f) dispensing a fourth microdroplet of a solution comprising (i) the catalyst and (ii) the solvent, such that the fourth microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 3′

hydroxyl group of the first nucleoside, resulting in the coupling of the second nucleoside to the first nucleoside.

144. The method of

claim 143

, further comprising after step (c) and before step (d) the step of treating the substrate with a capping reagent which caps the unreacted hydroxyl groups of the linker.

145. The method of

claim 144

, wherein the capping reagent is perfluorooctanoyl chloride.

155. The method of any one of claims 143, 146, 147, 150, 153, or 154, wherein said solvent has the formula (I):

wherein

A=O or S;

X=O, S or N(C₁-C₄ alkyl);

Y=O, S, N(C₁-C₄ alkyl) or CH₂; and

R=C₁-C₂₀ straight or branched chain alkyl.

146. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having an activated O-succinate group at its 5′

position, and a protecting group at its 3′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having an amine group, and forms a microdot upon the substrate;

(b) rinsing the substrate with a deprotecting agent which removes the protecting group from the 3′

position of the first nucleoside, and exposes a 3′

hydroxyl group;

(c) dispensing a second microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group containing a cyanoethyl group, at its 5′

position, and a protecting group at its 3′

position and (ii) the solvent, such that the second microdroplet impinges the microdot;

(d) dispensing a third microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the third microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 3′

hydroxyl group of the first nucleoside, resulting in the conversion of the phosphoramidite group to a phosphite group;

(e) washing the substrate with an oxidizing agent to convert the 5′

phosphite group to a 5′

phosphate group;

(f) performing successive iterations of steps (b)-(d);

(g) treating the product of step (f) with a second deprotecting agent that converts the cyanoethyl groups, of the product of step (f), to phosphate groups; and

(h) treating the product of step (g) with a hydrolyzing agent which cleaves the oligonucleotide from the linker.

147. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having a phosphoramidite group at its 3′

position, and a protecting group at its 5′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having hydroxyl groups, and forms a microdot upon the substrate;

(b) dispensing a second microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the second microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the first nucleoside and an hydroxyl group of the linker, resulting in the conversion of the phosphoramidite group to a 3′

phosphite group, and the presence of unreacted hydroxyl groups of the linker;

(c) washing the substrate with an oxidizing agent to convert the 3′

phosphite group to a 3′

phosphate group;

(d) rinsing the substrate with a deprotecting agent which removes the protecting group from the 5′

position of the first nucleoside, and yields a 5′

hydroxyl group;

(e) dispensing a third microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group at its 5′

position, and a protecting group at its 3′

position and (ii) the solvent, such that the second microdroplet impinges the microdot; and

(f) dispensing a fourth microdroplet of a solution comprising (i) the catalyst and (ii) the solvent, such that the fourth microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 5′

hydroxyl group of the first nucleoside, resulting in the coupling of the second nucleoside to the first nucleoside.

148. The method of

claim 147

, further comprising after step (c) and before step (d) the step of treating the substrate with a capping reagent which caps the unreacted hydroxyl groups of the linker.

149. The method of

claim 148

, wherein the capping reagent is perfluorooctanoyl chloride.

150. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having a phosphoramidite group at its 5′

position, and a protecting group at its 3′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having hydroxyl groups, and forms a microdot upon the substrate;

(b) dispensing a second microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the second microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the first nucleoside and an hydroxyl group of the linker, resulting in the conversion of the phosphoramidite group to a 5′

phosphite group, and the presence of unreacted hydroxyl groups of the linker;

(c) washing the substrate with an oxidizing agent to convert the 5′

phosphite group to a 5′

phosphate group;

(d) rinsing the substrate with a deprotecting agent which removes the protecting group from the 3′

position of the first nucleoside, and yields a 3′

hydroxyl group;

(e) dispensing a third microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group at its 3′

position, and a protecting group at its 5′

position and (ii) the solvent, such that the second microdroplet impinges the microdot; and

(f) dispensing a fourth microdroplet of a solution comprising (i) the catalyst and (ii) the solvent, such that the fourth microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 3′

hydroxyl group of the first nucleoside, resulting in the coupling of the second nucleoside to the first nucleoside.

151. The method of

claim 150

, further comprising after step (c) and before step (d) the step of treating the substrate with a capping reagent which caps the unreacted hydroxyl groups of the linker.

152. The method of

claim 151

, wherein the capping reagent is perfluorooctanoyl chloride.

153. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having an activated O-succinate group at its 5′

position, and a protecting group at its 3′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having an amine group, and forms a microdot upon the substrate;

(b) rinsing the substrate with a deprotecting agent which removes the protecting group from the 3′

position of the first nucleoside, and exposes a 3′

hydroxyl group;

(c) dispensing a second microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group containing a cyanoethyl group, at its 3′

position, and a protecting group at its 5′

position and (ii) the solvent, such that the second microdroplet impinges the microdot;

(d) dispensing a third microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the third microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 3′

hydroxyl group of the first nucleoside, resulting in the conversion of the phosphoramidite group to a phosphite group;

(e) washing the substrate with an oxidizing agent to convert the 5′

phosphite group to a 5′

phosphate group;

(f) performing successive iterations of steps (b)-(d);

(g) treating the product of step (f) with a second deprotecting agent that converts the cyanoethyl groups, of the product of step (f), to phosphate groups; and

(h) treating the product of step (g) with a hydrolyzing agent which cleaves the oligonucleotide from the linker.

154. A method for synthesizing an oligonucleotide, comprising the steps of:

(a) dispensing a first microdroplet of a solution comprising (i) a first nucleoside having an activated O-succinate group at its 3′

position, and a protecting group at its 5′

position and (ii) a solvent having a boiling point of 150°

C. or above, a surface tension of 30 dynes/cm or above, and a viscosity of 0.015 g/(cm)(sec) or above, such that the microdroplet impinges a substrate with a linker attached thereto having an amine group, and forms a microdot upon the substrate;

(b) rinsing the substrate with a deprotecting agent which removes the protecting group from the 5′

position of the first nucleoside, and exposes a 5′

hydroxyl group;

(c) dispensing a second microdroplet of a solution comprising (i) a second nucleoside having a phosphoramidite group, having a cyanoethyl group, at its 5′

position, and a protecting group at its 3′

position and (ii) the solvent, such that the second microdroplet impinges the microdot;

(d) dispensing a third microdroplet of a solution comprising (i) a catalyst and (ii) the solvent, such that the third microdroplet impinges the microdot and facilitates a reaction between the phosphoramidite group of the second nucleoside and the 5′

hydroxyl group of the first nucleoside, resulting in the conversion of the phosphoramidite group to a phosphite group;

(e) washing the substrate with an oxidizing agent to convert the 3′

phosphite group to a 3′

phosphate group;

(f) performing successive iterations of steps (b)-(d);

(g) treating the product of step (f) with a second deprotecting agent that converts the cyanoethylphosphate groups, of the product of step (f), to phosphate groups; and

(h) treating the product of step (g) with a hydrolyzing agent which cleaves the oligonucleotide from the linker.