Method for producing hydrophilic monomers and uses thereof

Details Method for producing hydrophilic monomers and uses thereof Method for producing hydrophilic monomers and uses thereof

1999-07-29

2002-10-15

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S469000, C526S304000, C427S230000, C427S384000, C427S002110

Reexamination Certificate

active

06464850

ABSTRACT:

TECHNICAL FIELD
This invention relates to a method for producing hydrophilic monomers, which are particularly useful for electrophoresis, and to electrophoresis compositions, more particularly, to an electrophoresis gel composition that is hydrolytically stable and has high resolution for biological macromolecule separations. The invention further relates to a polymer composition which effectively suppresses electroendoosmosis, is hydrolytically stable and has high resolution for use in capillary electrophoresis and microchannel-based separations of macromolecules. The invention also relates to the preparation of electrophoresis compositions, electrophoresis gels, and coating compositions. The invention further relates to the use of said compositions, gels and polymer media for high resolution electrophoretic separations of proteins, nucleic acids, and other biological macromolecules.
BACKGROUND ART
The publications and other materials used herein to illuminate the background of the invention and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference.
Electrophoresis gels have been widely used for the separations of biological macromolecules such as proteins, nucleic acids, and the like. There are essentially two types of gels in use: agarose gels and polyacrylamide gels. Polyacrylamide gels, in general, have higher resolving power than agarose gels. Since gel casting is rather tedious and the quality of hand-cast gels is inconsistent, there is a need for precast, “ready to use” gels. Generally, precast gels are manufactured and supplied in buffers of pH between 8 and 9. Under these conditions, precast agarose gels are stable, and have a shelf life of one year at 4° C. However, precast polyacrylamide gels are unstable, and depending on use, have a shelf life of only three months at 4° C. As precast polyacrylamide gels age in alkaline conditions (pH above 7), the electrophoretic mobility of biological macromolecules through these gels decreases and the separation resolution deteriorates. The short shelf life of precast polyacrylamide gels is primarily attributed to the hydrolytic degradation of acrylamide moieties in the gel, while the crosslinking units, usually N,N′-methylene bisacrylamide, are relatively stable. Due to the short shelf life of precast polyacrylamide gels, it is difficult for a manufacturer to mass-produce and to store large quantities of gels, and it is inevitable that some customers have to throw away some unused but “expired” gels. Therefore, it is highly desirable to have a gel that has a similar resolution to polyacrylamide gel, but a longer shelf life. Since the manufacturing and application of precast polyacrylamide gels are well established, it is even more desirable to have a stable, high resolution gel system that can be manufactured and used in the same manner as polyacrylamide gels.
Recognizing the fact that the short shelf life of precast polyacrylamide gels is due to the hydrolytic degradation of acrylamide moieties in alkaline condition, Takeda et al. (U.S. Pat. No. 5,464,516), Engelhorn et al. (U.S. Pat. No. 5,578,180) and Bjellqvist et al. (WO 96/16724) developed neutral buffer systems to replace the conventionally used Tris-HCl buffer (pH=8.8) in sodium dodecyl sulfate (SDS) polyacrylamide gels to reportedly improve the shelf life of precast polyacrylamide gels. However, the gel running buffer has to be changed to be compatible with gel buffer, and the protein separation patterns that are obtained from these systems are different from traditional SDS polyacrylamide electrophoresis based on the Laemmli system (Laemmli,
Nature
277:680-685 (1970)).
Several vinyl-based monomers were proposed to replace acrylamide in the standard polyacrylamide gel system in order to improve gel stability. Shorr and Jain (U.S. Pat.No. 5,055,517) disclosed the use of N-mono- or di-substituted acrylamide monomers, such as N,N′-dimethylacrylamide (DMA), in electrophoresis gels. Although DMA is more stable than acrylamide, DMA is very hydrophobic and is useful in only a limited number of electrophoretic applications, such as for certain types of nucleic acid analyses.
Kozulic and Mosbach (U.S. Pat. No. 5,319,046) disclosed the use of N-acryloyl-tris-(hydroxymethyl)aminomethane (NAT), and Kozulic (U.S. Pat. No. 5,202,007) disclosed the use of sugar-based acrylamide derivatives in electrophoresis gels. Because of the presence of several hydroxyl groups in the monomers, these monomers are extremely hydrophilic. However, Chiari et al (
Electrophoresis
15:177-186 (1994)) reported that NAT is less stable than acrylamide. On the basis of molecular modeling, Miertus et al (
Electrophoresis
15:1104-1111 (1994)) concluded that, when there are two atoms between the amide linkage and the hydroxyl group (as is the case for NAT, sugar-based acrylamide derivatives, and N-(2-hydroxyethyl)acrylamide), the hydroxyl group facilitates the hydrolysis of amide linkages.
In a series of articles and a U.S. patent, Righetti et al. (U.S. Pat. No. 5,470,916
; Electrophoresis
15:177-186 (1994);
Electrophoresis
16:1815-1829 (1995)) disclosed the use of N-mono- and di-substituted hydroxyethoxyethyl-(meth)acrylamides and their analogs in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these
N-(Hydroxyethoxyethyl)acrylamide (HEEAA) was identified as the preferred monomer, because of its extreme hydrophilicity and resistance to alkaline hydrolysis.
However, Righetti et al. (WO 97/16462
; Electrophoresis
17:723-731 (1996);
Electrophoresis
17:732-737 (1996);
Electrophoresis
17:738-743 (1996)) subsequently reported that the HEEAA monomer had a peculiar tendency to auto-polymerize during storage as a 50% aqueous solution at 4° C., even in the presence of free radical inhibitor. In view of this auto-polymerization tendency of HEEAA, Righetti et al. disclosed in these references the use of N-mono- and di-substituted hydroxyalkyl-(meth)acrylamides as an alternative in electrophoresis gels. The formula of the monomers disclosed by Righetti et al. in these references is:
N-(Hydroxypropyl)acrylamide (HPAA) was claimed by Righetti et al. to be extremely hydrophilic and resistant to alkaline hydrolysis. However, to applicants' knowledge there have been no further reports on HPAA-based gels by Righetti's group or other groups, and there have been no HPAA-based commercial products.
For capillary electrophoresis (CE) of biological molecules, linear non-crosslinked polymers are commonly used rather than crosslinked gels due to easy replacement of media between runs. Many water-soluble, non-ionic polymers were shown to have utility as sieving media for CE. These include polyacrylamide (
J Chromatogr
, 516:33-48 (1990)), substituted celluloses (U.S. Pat. No. 5,534,123), polyethylene oxide (
J Chromatogr
781:315-25 (1997)), and polyacrylamide derivatives (U.S. Pat. Nos. 5,552,028 and 5,567,292) for capillary-based DNA sequencing. For CE of double-stranded DNA fragments, agarose (
Electrophoresis
12:1059-1061 (1991)), polymers made from N-substituted acrylamide monomers (
Electrophoresis
15:177-186 (1994);
Electrophoresis
17:723-731 (1996); U.S. Pat. No. 5,470,916), polyvinyl alcohol, and polyvinylpyrrolidone (U.S. Pat. No. 5,089,111) are known. These suggest that any hydrophilic, non-charged polymer will have some utility in electrophoretic separation of biological molecules in capillaries (
Electrophoresis
19:3114-3127 (1998)).
A problem in capillary electrophoresis (CE) is electroendoosmosis (EEO) which must be lo suppressed in order to obtain good resolution of analytes. Typically, the capillaries used in CE are fused silica glass. The silanol groups on the inner surface of capillaries will become negatively charged under the alkaline pH of separation buffers, and because these are fixed charges, when a high voltage is applied (>100 V/cm) the mobile solution in the capillary is pulled toward the cathode. This phenomenon, termed EEO, retards the migration of negatively charg

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