U.S. patent application number 14/229011 was filed with the patent office on 2014-07-31 for separation and manipulation of a chiral object.
This patent application is currently assigned to Dynamic Connections, LLC. The applicant listed for this patent is Dynamic Connections, LLC. Invention is credited to Mirianas Chachisvilis, Osman Kibar, Thomas H. Marsilje, Eugene Tu.
Application Number | 20140209464 14/229011 |
Document ID | / |
Family ID | 41434438 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140209464 |
Kind Code |
A1 |
Kibar; Osman ; et
al. |
July 31, 2014 |
Separation and Manipulation of a Chiral Object
Abstract
Among other things, for use in directional motion of chiral
objects in a mixture, a field is applied across the chamber and is
rotating relative to the chamber to cause rotation of the chiral
objects. The rotation of the objects causes them to move
directionally based on their chirality. The method applies to
sugars, proteins, and peptides, among other things, and can be used
in a wide variety of applications.
Inventors: |
Kibar; Osman; (San Diego,
CA) ; Chachisvilis; Mirianas; (San Diego, CA)
; Tu; Eugene; (San Diego, CA) ; Marsilje; Thomas
H.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dynamic Connections, LLC |
San Diego |
CA |
US |
|
|
Assignee: |
Dynamic Connections, LLC
San Diego
CA
|
Family ID: |
41434438 |
Appl. No.: |
14/229011 |
Filed: |
March 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12142545 |
Jun 19, 2008 |
8698031 |
|
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14229011 |
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|
12103281 |
Apr 15, 2008 |
7935906 |
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12142545 |
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60912309 |
Apr 17, 2007 |
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60987674 |
Nov 13, 2007 |
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Current U.S.
Class: |
204/549 ;
204/450 |
Current CPC
Class: |
C07C 303/44 20130101;
Y10T 436/143333 20150115; B03C 1/24 20130101; C07B 57/00 20130101;
Y10T 436/25375 20150115; C07C 201/16 20130101; B03C 7/026
20130101 |
Class at
Publication: |
204/549 ;
204/450 |
International
Class: |
C07C 303/44 20060101
C07C303/44; C07C 201/16 20060101 C07C201/16; C07B 57/00 20060101
C07B057/00 |
Claims
1-5. (canceled)
6. A method comprising enhancing a purity of at least one of two
enantiomers in a mixture by separating molecules of the two
enantiomers in each of a series of chambers, the purity of the
enantiomer in at least some of the successive chambers being at
increasingly higher levels, and transferring a portion of at least
one of the separated enantiomers from each of the chambers to a
previous one or a next one of the chambers in the series.
7-20. (canceled)
21. A method for use with an apparatus that comprises a chamber to
hold a mixture containing one or more enantiomers; a field source
to impose a rotating field on the mixture; the chamber having an
inlet to receive the mixture, and an outlet to remove a portion of
the mixture that contains at least one of the enantiomers in an
elevated concentration relative to its average concentration in the
mixture in the chamber; and a chemical reaction vessel connected to
the chamber to hold fluid to be delivered to the chamber; the
method comprising analyzing or processing a reaction component in
the fluid using the chamber.
22. The method of claim 21 in which the analyzing or processing
comprises analyzing purity of a reaction component.
23. The method of claim 21 in which the analyzing or processing
comprises purification of a reaction component.
24. The apparatus of claim 21 in which the analyzing or processing
comprises determining the absolute configuration of a reaction
component.
25. The method of 6 in which the chiral objects comprise achiral
molecules, such as lipids, that have chiral labels attached.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/103,281, filed Apr. 15, 2008, which claims
the benefit of priority of U.S. provisional application 60/912,309,
filed Apr. 17, 2007, and U.S. provisional application 60/987,674,
filed Nov. 13, 2007, and the entire contents of the applications
are incorporated here by reference.
BACKGROUND
[0002] This description relates to separation and manipulation of a
chiral object.
[0003] We use the term chiral object (or system) very broadly to
include, for example, any object or system that differs from its
mirror image such that its mirror image cannot be superimposed on
the original object. One kind of chiral object is a chiral
molecule, also called an enantiomer. A common feature of chiral
molecules is their "handedness" (i.e., right-handed or
left-handed). Enantiomers are a subset of chiral objects called
stereoisomers. A stereoisomer is one of a set of isomeric molecules
whose atoms have similar connectivity but differ in the way the
atoms are arranged in space. A stereoisomer includes at least one
stereocenter, which is any atom that bears groups such that an
interchanging of any two groups leads to a stereoisomer. A
stereoisomer may have one or more stereocenters. For example, for a
molecule with 3 stereocenters (e.g. S, S, S), its enantiomer would
be (R, R, R); epimers are stereoisomers that differ in only one but
not all stereocenters (e.g., S, S, R instead of S, S, S). It is
also possible for a molecule to be chiral without having a
stereocenter (the most common form of chirality in organic
compounds). In axial chirality, for example, a molecule does not
have a stereocenter but has an axis of chirality, i.e., an axis
about which a set of substituents is held in a spatial arrangement
that cannot be superimposed on its mirror image. An example is the
molecule 1,1'-bi-2-naphthol (BINOL). Although the discussion here
refers to enantiomers, it also applies to other stereoisomers, even
if they do not qualify as enantiomers. We use the term
stereoisomers broadly.
[0004] A mixture of molecules is often called racemic if it
contains equal amounts of right-handed and left-handed enantiomers,
and it is called enantiopure (or, optically pure) if only one type
of enantiomer dominates in the mixture. Here, however, we refer to
a racemic mixture more broadly to include any non-pure mixture of
stereoisomers that is not enantiopure, whether or not the amounts
of the stereoisomers are equal.
[0005] Chirality is important in chemistry, especially for
biological and drug applications. Natural biomolecules are
typically found in only one enantiomer form (e.g., proteins,
peptides, and amino acids are left-handed, and sugars are
right-handed). The fields of drug discovery, development, and
manufacturing are interested in molecules that are enantiopure,
because one form or enantiomer may work better in vivo while the
opposite form may be toxic or may cause side effects. Other
chemistry-based fields would also benefit from enantiopure
molecules including (for illustration purposes), but not limited
to: flavors and fragrances, agrichemicals, fine chemicals,
petrochemicals, and others.
[0006] Enantiopure samples are sometimes produced by asymmetric
synthesis, in which only one form of enantiomer is chemically
synthesized from the beginning. Another approach is to synthesize
both enantiomers (for example, in a racemic mixture) and then
separate the desired enantiomer from the mixture, for example,
using column chromatography, in which the mixture is run through a
chiral selector (e.g., a chemical matrix that binds preferentially
to one enantiomer and less so to its counterpart) iteratively until
a desired purity is reached.
[0007] Some molecular separation techniques are not effective for
chiral molecules, because two counterpart enantiomers generally
share physical properties, including chemical composition, charge,
size, electric and magnetic dipole moments, and energy levels.
Detection and separation of chiral molecules is typically done by
interacting the molecules with a chiral medium (e.g., a chemical
matrix). Enantiomers also can be identified by their interaction
with a chiral (e.g., circularly polarized) electromagnetic
field.
SUMMARY
[0008] In general, in an aspect, for use in directional motion of
chiral objects in a mixture, a field is applied across the chamber
and is rotating relative to the chamber to cause rotation of the
chiral objects. The rotation of the objects causes them to move
directionally based on their chirality.
[0009] Implementations may include one or more of the following
features. The chiral objects comprise sugar molecules. The sugar
molecules comprise at least one of monosaccharides, disaccharides,
oligosaccharides, or polysaccharides (glycans). The chiral objects
comprise proteins. The chiral objects comprise peptides. A purity
of at least one of two enantiomers in a mixture is enhanced by
separating molecules of the two enantiomers in each of a series of
chambers, the purity of the enantiomer in at least some of the
successive chambers being at increasingly higher levels. A portion
of at least one of the separated enantiomers is transferred from
each of the chambers to a previous one or a next one of the
chambers in the series. A result of the directional motion or
enhanced purity is used in one or more of the following: genomics,
proteomics, glycomics, chemical manufacturing, food processing,
forensics, academic research, solvent purification, asymmetric
synthesis. A product outcome of a chemical reaction is purified or
quantified, or identified. A product outcome of a chemical reaction
comprises a non-racemic product mixture or a racemic product
mixture.
[0010] In general, in an aspect, a chamber holds a mixture
containing one or more enantiomers. A field source imposes a
rotating field on the mixture. The chamber has an inlet to receive
the mixture, and an outlet to remove a portion of the mixture that
contains at least one of the enantiomers in an elevated
concentration relative to its average concentration in the mixture
in the chamber. A chemical reaction vessel is connected to the
chamber to hold fluid to be delivered to the chamber. The chamber
is used to analyze or process a reaction component in the fluid
using the chamber.
[0011] Implementations may include one or more of the following
features. The analyzing or processing comprises analyzing purity of
a reaction component, purification of a reaction component, or
determining the absolute configuration of a reaction component. The
chiral objects comprise achiral molecules, such as lipids, that
have chiral labels attached.
[0012] Implementations also may include one or more of the
following features. The field delivers insufficient energy to
damage the chiral objects. The frequency of rotation causes
directional motion at a speed that is high enough to achieve a
predetermined degree of separation of some of the chiral objects
from the mixture at a predetermined concentration in no more than a
predetermined amount of time. The field strength and the speed of
rotation of the field are selected to achieve a predetermined level
of efficiency of the directional motion. The electric field
strength is lower than 10.sup.5 V/m. The electric field has a
rotational frequency higher than 100M rotations per second. The
directional motion has a velocity of at least 0.1 angstrom per
revolution (i.e., a millimeter per second at 100 million
revolutions per second).
[0013] The chiral objects include chiral molecules. The molecules
include stereoisomers. The stereoisomers include enantiomers. The
stereoisomers include epimers. The chiral objects include
aggregates of chiral or achiral molecules or both. The chiral
objects include molecules having axial chirality.
[0014] The molecules include drug molecules. The molecules include
drug intermediate molecules. The stereoisomers have more than one
stereocenter. The chiral objects are of one type. The chiral
objects are of two types. The chiral objects are of more than two
types.
[0015] The chirality of the chiral objects is analyzed based on the
directional motion. The presence or absence of chiral objects is
detected based on the directional motion. Two or more types of
chiral objects are separated based on the directional motion. The
chiral objects are separated into two groups. The chiral objects
are separated into more than two groups. The chiral objects of the
two or more types move in opposite directions. The chiral objects
of the two or more types move in the same direction but at
different average velocities. The chiral objects of the two or more
types are separated in real-time. The chiral objects are separated
as end-point. The field includes an electric field. The field
includes a magnetic field. The field is rotated relative to the
chamber in discrete steps. The field is rotated continuously
relative to the chamber. The field is rotated around a stationary
chamber. The chamber is rotated relative to a field of fixed
orientation. The mixture includes a racemic mixture. The field is
rotated relative to the chamber in successive angular positions
around a central portion of the chamber. The field is applied from
electrodes arranged on a peripheral wall of the chamber. The
electric field is applied at successive angular orientations across
the chamber at intervals that cause the electric field to rotate
around the chamber with a selected rotational frequency profile.
The rotational frequency profile is in at least one of the ranges
of less than 1 kHz, 1 kHz to 10 kHz, 10 kHz to 100 kHz, 100 kHz to
1 MHz, 1 MHz to 10 MHz, 10 MHz to 100 MHz, 100 MHz to 1 GHz, 1 GHz
to 10 GHz, or above 10 GHz. The rotational frequency is in the RF
range. The rotational frequency is in the microwave range. The
field is applied by an electromagnetic beam that is collinear with
an axis of the chamber. The electromagnetic beam is circularly
polarized. The rotational frequency is in the RF range. The
rotational frequency is in the microwave range.
[0016] The chiral objects are loaded into the chamber at a
particular point along the chamber. The chiral objects are loaded
into the chamber without regard to their entry point along the
chamber. The field is applied to cause a concentration of the
chiral objects to reach a steady state. The field is applied and
then turned off before the concentration of the chiral objects
reaches a steady state.
[0017] The gradient of a concentration of the chiral objects in the
mixture in the chamber includes an exponential profile. The
gradient includes a linear profile. The gradient includes a
nonlinear profile. The parameters associated with the directional
motion are non-constant in the direction of the motion.
[0018] A chiral label is associated with the chiral objects.
Entities are attached to the chiral objects to increase dipole
moments of the chiral objects. Entities are attached to the chiral
objects to increase a rotational/translational coupling factor.
[0019] At least some of the chiral objects are caused to move
collectively.
[0020] The mixture includes a fluid in which the chiral objects
move. The fluid includes a gas. The fluid includes a polar
solution. The fluid includes a non-polar solution. The fluid
includes a high pressure fluid. The fluid is in a supercritical
phase. The fluid's composition or properties are controlled.
[0021] The chiral objects exhibit a smaller dipole moment than
molecules of the polar solution. The directional motion is achieved
by rotating molecules of the fluid to impart angular momentum on
the objects to cause them to rotate. The directional motion occurs
within a flow of the mixture (e.g., along the chamber). The mixture
is caused to flow in a manner that counters the directional motion
of the chiral objects. The applied field has a profile other than
constant along a direction of the chamber. The direction is
orthogonal to a length of the chamber.
[0022] The directional motion is controlled using feedback. An
outcome of the directional motion is monitored.
[0023] The enhancing occurs during processing periods and the
transferring occurs in intervals that are at least partly
non-overlapping with the processing periods. The portion of the
separated enantiomer in each chamber that is transferred is a
higher concentration portion or a lower concentration portion than
the average concentration in the chamber. The average concentration
of each of the enantiomers in each of the chambers remains
essentially unchanged over time. There is more than one such series
of chambers operated in parallel. The purities of both of the
enantiomers are enhanced. The chambers are of the same size. The
enantiomers are in a mixture and the mixture is transferred from
chamber to chamber. The enantiomers are in a mixture and the
enantiomers are extracted from the mixture before being transferred
from chamber to chamber. The portion that is transferred is
one-quarter. The portion that is transferred is less than
one-quarter. The portion that is transferred to the next chamber is
different from the portion transferred to the previous chamber. The
additional series of chambers have different lengths from the
primary series. The output of the additional series of chambers is
transferred to the first or last chamber of the primary series.
Initial purity levels of the enantiomers are established in each of
the chambers. The process is run continuously for real-time
purification of one or both enantiomers. The portions of the chiral
objects are transferred to and from the chamber using a pump. The
pump is mechanical. The pump is not mechanical.
[0024] An outcome is monitored chemically. An outcome is monitored
optically. An outcome is monitored electronically. Software is used
to control, manage, or compliment an outcome. The software
calculates or predicts expected performance or performance limits.
The software calculates or predicts an average velocity of the
chiral objects. The software calculates or predicts a direction of
motion of one or more of the chiral objects. The system is fully
automated. The system is modular.
[0025] Environmental parameters are controlled or optimized,
including temperature or pressure. One of the environmental
parameters is controlled remotely. Control parameters are
optimized, calibrated, or monitored. The control parameter includes
applied voltage, rotation frequency, duration of the applied field,
or selection of a fluid medium. Performance parameters are also
optimized or monitored. The performance parameter includes
reliability, repeatability, or reproducibility.
[0026] Multiple runs are performed in parallel. Performance runs
are allowed or managed in series. The environment for the chiral
objects is isotropic. The environment for the chiral objects is
anisotropic or asymmetric in at least one dimension of the chamber.
Diffusion inside the chamber is reduced. Convection inside the
chamber is reduced.
[0027] The chamber holds one enantiomer. The chamber holds two
enantiomers. The chamber holds more than two enantiomers. A
diameter of the chamber is in the millimeter scale. A diameter of
the chamber is in the micrometer scale. A diameter of the chamber
is in the nanometer scale. The chamber has a cross-section that is
circular. The chamber has a cross-section that is non-circular. The
electrodes are on an inner wall of the chamber to be in contact
with the mixture. The electrodes are on an inner wall of the
chamber and not arranged to be in contact with the mixture. The
electrodes are on an outer wall of the chamber, but not in contact
with the mixture. The electrodes include metal or a semiconductor.
The electrodes have a cross-section that is circular. The
electrodes have a cross-section that is non-circular. The
electrodes have a cross-section that is asymmetric. There are two
electrodes. There are three electrodes. There are more than three
electrodes. The electric field is applied to only two of the
electrodes at a time. The electric field is applied to more than
two of the electrodes at a time.
[0028] The chamber includes or is part of a disposable. The
disposable includes a cartridge. The cartridge includes a chamber
to hold a sample. The cartridge includes multiple chambers to hold
a sample. The chamber cross-section is circular. The chamber
cross-section is non-circular. The chamber includes a capillary.
The capillary includes glass. The capillary includes quartz. The
capillary includes polymer. The capillary includes material that is
not glass, quartz or polymer. The capillary is coated on the inside
surface. The chamber includes a microfluidic channel on a chip. The
channel is formed by photolithography. The channel is formed by
laminating layers of a material. The channel is formed by both
photolithography and laminating layers of a material.
[0029] The cartridge includes electrodes arranged about the chamber
to produce a rotating electric field. The electrodes are placed
about the chamber in an axial geometry. The electrodes are placed
about the chamber in an orthogonal geometry. The electrodes are
placed about the sample chamber at an angle that is not axial or
orthogonal. The electrode includes a coil. The electrodes include a
continuous set of electrodes per sample chamber. The electrodes
include discrete sets of electrodes per sample chamber. The
electrodes include a waveguide for circularly polarizing microwave
field. The electrodes generate a rotating field with an axis of
rotation orthogonal to direction of flow. The rotating field is
generated at a T-junction and/or a Y-junction.
[0030] The cartridge includes one or more ports to inject and/or
extract the sample. The ports include fluidic interconnects. The
interconnects include luer fittings. The interconnects include
screw fittings. The cartridge includes a detection zone to monitor
the concentration of a sample. The cartridge includes a controller
to control and/or monitor environmental parameters. The
environmental parameter includes pressure. The environmental
parameter includes temperature. The cartridge includes a connector
to make electrical contact with the electrodes. The connector
include electrical interconnects. The capillary is surrounded by
electrodes that generate a rotating field whose axis is co-linear
with the length of the capillary. The capillary and electrodes are
held by a larger tubing. The tubing includes metal. The tubing
includes dielectric material.
[0031] The electrodes and/or the electrical connections are
integrated onto a board. The board includes a printed circuit
board. The board includes a through hole for a capillary. The
cartridge includes a microfluidic device. The device includes
glass. The device includes quartz. The device includes polymer. The
polymer includes epoxy. The device includes elastomer.
[0032] The electrodes are deposited on or near vertices of the
channel. The electrodes are arranged to be in contact with a medium
in the sample chamber. The electrodes are arranged to be insulated
from a medium in the sample chamber. The channel cross-section is a
square. The sample chamber includes a channel. The sample chamber
includes multiple channels. The sample chamber includes one or more
T- or Y-junctions. The chamber contains a chemical matrix. The
matrix includes glass. The matrix includes silica. The matrix
includes diatomaceous earth. The matrix includes polymer. A
direction of the chiral object's motion is monitored to determine
its absolute configuration.
[0033] The mixture is non-pure. The mixture is enantiopure. The
chiral objects have only one stereocenter. The chiral objects have
more than one stereocenter. The direction of the field is reversed
for the same chiral objects to confirm the absolute configuration.
Software is used to calculate or predict the speed or direction of
motion for a chiral object. The apparatus is used as a stand-alone
system. The apparatus is used as an add-on to another chiral
separation instrument. The field is applied to a chiral HPLC
column. The apparatus is used as an add-on to a standard HPLC
column. A result is used in analytical chemistry. A result is used
in drug discovery. A result is used in drug development. A result
is used in drug manufacture. A result is used in medical
diagnostics. A result is used in fine chemical or synthetic
intermediate manufacture. A result is used in agrochemicals. A
result is used in petrochemicals. A result is used in flavors and
fragrances. A result is used in process monitoring.
[0034] The chiral objects include achiral objects with chiral
labels attached. The chiral objects include unknown molecules. The
chiral objects include known molecules.
[0035] A result is used to quantify a specific property of the
chiral object. The specific property of the chiral object includes
its propeller propulsion efficiency. The specific property of the
chiral object includes its absolute configuration. The specific
property of the chiral object includes its presence or absence in
the solution. The specific property of the chiral object includes
the magnitude or the orientation of its dipole moment. The system
or the resulting separations are using in electro-rotary chemistry.
The electro-rotary chemistry includes chiral synthesis. The
electro-rotary chemistry includes reactions or applications
involving catalysis. The electro-rotary chemistry includes the
study or the detection of molecular interactions. The spatial
concentration profile of the chiral molecules is changed to
manipulate the chemical reactions involving them. The system is
used to separate or purify the chiral molecules from achiral
impurities in the solution. The chiral labels are self-assembled.
The chiral labels are self-activated. The chiral labels are
pre-activated. The achiral objects include molecules, such as DNA,
RNA, peptides, proteins or amino acids. The achiral objects include
living organisms, such as viruses, bacteria or cells. More than one
type of chiral label is used for multiplex assays. The chiral
labels are used for debulking or enriching sample matrices.
[0036] The chiral labels comprise propeller entities. The propeller
entities are conjugated to antibodies or nucleic acids. The
propeller entities comprise at least two components. The chiral
labels comprise aptamers. The aptamers become chiral or reverse
their chirality upon binding to achiral objects. Intermolecular
interactions are induced to convert achiral objects into chiral
objects. Intermolecular interactions are induced to change the
propeller efficiency of chiral objects.
[0037] The technique enables stereoseparation of previously
unseparated racemic drugs, such as metaxalone. Accordingly another
aspect of the invention features compositions that are
enantomerically enriched in + metaxalone or in - metaxalone.
Enantiomerically enriched means that there is a substantial excess
of the designated stereoisomer. Preferably the other stereoisomer
is not present in physiologically significant amounts, or is not
present in any detectable amount.
[0038] These and other features, aspects, and implementations, and
combinations of them, may be expressed as methods, apparatus,
systems, program products, means for performing functions,
compositions, purified entities, molecules, and in other ways.
[0039] Other aspects, features, and advantages will become apparent
from the following description and from the claims.
DESCRIPTION
[0040] FIG. 1 is a perspective side view and an end view of a
cylinder.
[0041] FIG. 2 shows schematic high voltage ((a), at the top) and
low voltage ((b), at the bottom) profiles over time (left to
right).
[0042] FIG. 3 is a graph of gradients.
[0043] FIG. 4 is a schematic diagram of multiple separation
chambers.
[0044] FIG. 5 is a table representing a separation sequence.
[0045] FIG. 6 shows schematic linear (left) and trigonometric
(right) profiles using high voltage ((a), at the top) and low
voltage ((b), at the bottom).
[0046] FIG. 7 is a schematic diagram of multiple separation
chambers.
[0047] FIG. 8 is a block diagram.
[0048] FIG. 9 is a cross-sectional view of a chamber.
[0049] FIGS. 10 and 11 are a cross-sectional view and a side view
of a bundle of chambers.
[0050] FIG. 12 is a top view of a wire bundle.
[0051] FIG. 13 is a top view of a connector.
[0052] FIGS. 14 and 15 are a perspective view and a side view of a
capillary and electrodes.
[0053] FIG. 16 is a block diagram
[0054] FIG. 17 is a perspective view of a disposable chamber.
[0055] FIGS. 18 and 19 are a schematic sectional end view and a
sectional top view of a set of chambers.
[0056] FIGS. 20 and 21 are a schematic perspective view and a
schematic sectional side view of a chamber.
[0057] FIGS. 22 and 23 are schematic views of a T-junction chamber
and a tree of T-junction chambers.
[0058] FIGS. 24 and 25 are a side view and a top view of fluidic
interconnections to a chamber.
[0059] FIGS. 26 through 28 are graphs of experimental results.
[0060] FIG. 29 is a theoretical graph.
[0061] As we discuss here, separation and manipulation of chiral
objects can be achieved without using chiral media, by relying on
the susceptibility of the objects to external forces and the
handedness of the objects. For example, the dipole moment that
characterizes chiral molecules is susceptible to being rotated by a
rotating external electromagnetic field. And the left handedness or
right handedness of counterpart enantiomers, for example, can be
used to transform the rotational motion of the enantiomers into
translational (i.e., directional) motion of the two counterpart
enantiomers in opposite directions. The helical handedness of some
molecules, for example, is similar to the opposite handedness of
left-handed and right-handed macroscopic propellers, which (when
rotated) can propel themselves and objects to which they are
attached in respectively opposite directions through a medium in
which they are held.
[0062] The propeller of each molecule is characterized by the
spatial configuration of its chiral features. As the propeller
rotates, these spatial features act against the fluid resistance of
the mixture that holds the molecules to force the propeller and the
molecule to move in a direction. We sometimes refer to this
transformation of rotational motion of the propeller into
directional motion depending on the handedness of the propeller as
the propeller effect.
[0063] In some examples, an external rotating electrical field is
applied to a sample of chiral molecules. An electric dipole of each
chiral molecule lines up with the external electric field and
rotates with it causing rotation of the chiral molecule. The
handedness (i.e., chirality or chiral features) of the molecule
(which can be viewed as a tiny propeller) transforms this rotation
into a linear (i.e., directional or translational) motion (E. M.
Purcell, "The efficiency of propulsion by a rotating flagellum,"
Proc. Natl. Acad. Sci. USA, Biophysics, v 94, pp 11307-11311,
October 1997).
[0064] At a molecular level and in a fluid (characterized by very
low Reynolds numbers), inertial forces on the chiral molecules are
negligible. The motion of the molecule that results from its
rotation is similar to a left-handed or right-handed corkscrew
motion. For a particular force applied by the rotation of the
molecules in the mixture, the S and R enantiomers will acquire the
same velocities but in opposite directions. Concentration gradients
for both enantiomers will be established based on the magnitude of
these velocities, compared to the inherent diffusive flux of the
molecules in the mixture, which is characterized by the diffusion
constant.
[0065] The magnitude and profile of the concentration gradient for
each enantiomer, and thus, the enrichment achieved, will depend on
the efficiency of the enantiomers' propeller (that is, the
efficiency with which its spatial configuration is converted to a
linear force on the molecule, which relates to the size, shape and
orientation of the propeller, among other things), the effective
length of the container that holds the mixture, how long the field
is applied, the electric field strength, the frequency of rotation,
and properties of the fluid in which the enantiomers are held in
the mixture, among other things. We sometimes refer to the
propeller efficiency as the propeller propulsion efficiency.
[0066] We describe how to use a propeller effect to separate or
manipulate chiral objects (such as chiral molecules) that is
relatively inexpensive (because it uses simple equipment), achieves
relatively high throughput (i.e., reaches a higher purity in a
shorter period of time), applies to a broad range of types of
objects and molecules, and produces (arbitrarily) high purity
levels.
[0067] We also describe how to amplify (if there is a need) the
separation to reach the arbitrarily high purity level for one or
both of the enantiomers, for example, without sacrificing other
performance parameters. Note that, although we use enantiomers in
describing some implementations here, the principles apply broadly
to any chiral molecules or chiral objects.
[0068] In some implementations, to separate the counterpart
enantiomers, a cylindrical (e.g., glass) container 10 (FIG. 1) is
filled with a racemic mixture 12 (held in a fluid, for example, in
solution in a solvent). Parallel stripes of longitudinal electrodes
14 are spaced at regular angular intervals 15 around the outer
surface 16 of the container and are parallel to the longitudinal
cylindrical axis 18. Each of the molecules 20 in the racemic
mixture has a permanent electric dipole moment. We sometimes refer
to the space within the container as a chamber. Although we often
describe the chiral objects as being in a solution having a
solvent, we mean to include by such references any kind of fluid or
medium in which the molecules or other chiral objects may move.
[0069] A voltage profile over time 21 is applied between pairs of
the electrodes in sequence. First, two of the electrodes that are
on opposite sides of the container (for example, electrodes 22,
24), which establishes an electric field (26) inside the container
that causes the electric dipole moments of both enantiomers to
align themselves with the electric field all along the length of
the cylinder. The voltage is applied to a succession of pairs of
electrodes on opposite sides of the container (FIG. 2). For
example, electrodes 32, 34 receive the voltage at the middle times
in FIG. 2 and electrodes 36, 38 at the later times in FIG. 2. Thus,
in evenly spaced timed steps, the applied voltage (and the
direction of the field induced by the voltage) rotates 28, 30
around the cylinder at a fixed rotational frequency (e.g., a number
of rotations around the cylinder per second). The stepping of the
voltage can be controlled to cause the rotational frequency to be
in the kHz-GHz range or in specific sub-ranges of that broader
range. In some examples, the rotational frequency could be varied
over time.
[0070] The rotation of the external field causes a continuous
torque to be applied to the dipole moment of each of the molecules,
causing it to rotate with the field. In this example, both
enantiomers rotate in the same angular direction. During rotation
their chiral features (that is, the spatial features that represent
their handedness) will act as tiny propellers. Of course, the
right-handed and the left-handed chiral features of the molecules
of the two counterpart enantiomers will cause them to behave
respectively as oppositely handed propellers.
[0071] Rotation of the tiny propellers by the field-applied torque
is transformed into translational motion (i.e., linear motion) of
the two enantiomers in opposite directions. For example, if the
right-handed molecules are propelled to the right end of the
container, then the torque will cause the left-handed molecules to
be propelled to the left end.
[0072] Based on the strength and rotational frequency of the
electric field, the molecule's dipole moment and its angle relative
to the molecule's propeller axis, the solution properties, and
other parameters, the two enantiomers will both move at a net
velocity (v) along the cylindrical axis, but in opposite directions
(i.e., one at +v, the other at -v). A concentration gradient 40, 42
(FIG. 3) will be established along the length L of the container
(i.e., along the cylindrical axis) for each enantiomer.
[0073] The gradients for the respective enantiomers will be in
opposite directions. If the right handed molecules have a velocity
+v (traveling to the right), their concentration will increase
towards the right; and vice versa for the left handed molecules.
The magnitude of the concentration gradient that can be established
for a given set of parameters will be limited by the diffusion
constant for the molecule (given the properties of the setup, such
as temperature and the choice of fluid in which the molecules are
moving). After a long enough time (i.e., enough for diffusion or
the enrichment to reach a steady state), the ratio of the
concentrations of the enantiomers at the two ends of the container
depends on a factor exp(+vL/D), where exp stands for exponential, v
is the linear velocity of the molecule that is caused by the
externally applied field, L is the effective length of the
container, and D is the diffusion constant of the molecule in the
particular solvent (although we sometimes use the word solvent, the
principles apply to any fluid in which the molecules are held) in
which the racemic mixture is dissolved. The formula for the
steady-state concentration at an arbitrary position x is given
by:
C ( x ) = C Ave v L D ( v L D - 1 ) v D x , ##EQU00001##
where C.sub.Ave is average concentration within the chamber, and x
is distance from one edge of the chamber.
[0074] To understand how the propeller effect depends functionally
on molecular parameters and experimental configurations, a
rotational diffusive flux theory can be applied to calculate the
efficiency of chiral separation using propeller motion. The
efficiency of separation of enantiomer molecules depends on (1) the
average distance traveled per one full revolution (L.sub.rev), (2)
the frequency of rotation (f), and (3) the magnitude of rotating
electric field (E).
[0075] The factor L.sub.rev is determined by properties of the
molecule, including its geometry, conformational state(s),
orientation of its dipole moment with respect to its propeller
axis, solvent, temperature, and viscosity, among others. According
to our molecular dynamic simulations, L.sub.rev is typically in the
range of 0.1 to 4 .ANG./per one revolution for most chiral
molecules. L.sub.rev is basically a fixed parameter for a given
choice of a chiral molecule and solvent combination (assuming
solubility related issues are negligible).
[0076] Consequently, optimization of enantiomer separation (or
manipulation) using the propeller effect depends on knowing (and
using) the relationship of separation efficiency to the other two
parameters (f and E). Because the molecular rotational relaxation
time of a small molecule (the time it takes the molecule to rotate
fully in response to the rotating electric field) is typically on
the order of 1-100 ps in a solution (mixture) at room temperature,
most of the small molecules should be able to follow rotation of
the electric field at frequencies up to .about.10 GHz. Therefore,
the dependence of the average directional velocity resulting from
the propeller effect on the rotational frequency of the field is
expected to be linear up to .about.10 GHz and (assuming f remains
below .about.10 GHz) can be expressed as
<v>=LrevfF(E), (Eq. 1), where F(E) is a function that
incorporates the dependence of the propeller effect on electric
field strength.
[0077] It has been argued empirically that the dependence of the
propeller effect on the electric field should be quadratic at low
field magnitudes (Baranova, N. B. & Zeldovich, B. Y. Separation
of Mirror Isomeric Molecules by Radio-Frequency Electric-Field of
Rotating Polarization. Chemical Physics Letters 57, 435-437
(1978)), i.e., F(E).about.E.sup.2 for small E (where "small" refers
to the ratio of a potential energy difference that the field can
impose on the molecule (.about..mu.E, where .mu. is the electric
dipole moment of the molecule) compared to k.sub.BT (the unit
thermal energy). Baranova assumed (without proving) that the
translational velocity of a molecule due to the propeller effect in
a rotating electric field should be proportional to the product of
the electric field and a time derivative of the electric field
(equation 2a of Baranova).
[0078] Other later studies (Gelmukhanov, F. K. & Ilichov, L. V.
Orientation of Stereoisomers by Electromagnetic-Field. Optics
Communications 53, 381-384 (1985); and Evans, M. W. & Evans, G.
J. The Effect of External Electric-Fields on Molecular Liquids and
Induced Translational Motion. Journal of Molecular Liquids 29,
11-35 (1984). did not question Baranova's equation 2a. The Evans
study interpreted the propeller effect as described by Baranova, as
being due to "magnetic orientation of the particles in the field of
circular polarization" even though Baranova only considered
interaction at the electrical dipole level. The theoretical
analysis in Baranova relied on a two-dimensional (2D) stochastic
Langevin equation to describe a chiral molecule having electric
dipole moment and exposed to the rotating electric field. The
average translational velocity was found by perturbative solution
of Langevin equation to the second order in the electric field
which resulted in quadratic dependence on the electric field
magnitude (equation 6 of Baranova).
[0079] We have now shown that the behavior of propeller motion in a
rotating electric field can be solved exactly, without empirical
assumptions, and that the result of the exact derivation is in
direct contrast with Baranova's reasoning. We infer that the
original empirical assumption (equation 2a of Baranova) is
unjustified and therefore the conclusions of Baranova are incorrect
and misleading. We have found complete analytical solutions for
this problem both in 2D and in 3D cases, which are valid at any
electric field strength.
[0080] The starting point for our analysis is a rotational
diffusion equation for a molecule with a dipole moment exposed to a
rotating electric field. We assume that the electric field
rotational frequency is much lower than the rotational relaxation
time (i.e., that the molecule can easily follow the field
rotation). This enables us to use a stationary diffusion equation
to find an equilibrium angular distribution of molecular dipole
moments as a function of orientation and magnitude of the electric
field. The solutions for function F(E) describing the dependence of
the propeller effect on the electric field can be found using two
approaches that yield virtually identical results.
[0081] The first approach relies on calculating the fraction of the
molecules that are following the rotation of the electric field.
This fraction depends on the temperature. At higher temperatures,
this mobile or correlated fraction is smaller because molecules
have more kinetic energy to escape from alignment with the electric
field.
[0082] Another approach relies on calculation of a net rotational
diffusive flux resulting from an infinitesimally small change in
the orientation of electric field.
[0083] Our expressions for 2D and 3D cases are, respectively:
F ( E ) .apprxeq. 1 - 1 .mu. E k B T I 0 ( .mu. E k B T ) and ( Eq
. 2 ) F ( E ) .apprxeq. 1 - 2 .mu. E k B T ( 2 .mu. E k B T - 1 ) ,
( Eq . 3 ) ##EQU00002##
where .mu. is the electric dipole moment of the enantiomer, k.sub.B
is the Boltzman constant, T is temperature, and I.sub.0 is a
modified Bessel function of the first kind.
[0084] The solution of the 3D case should include the contribution
to the propeller effect from preferential orientation of dipole
moments in the plane parallel to electric field rotation plane;
however, we have shown that this contribution is small (at most a
factor of .about.1.274 at weak fields) and therefore is not
included in Equation 3. When interaction energy of the electric
dipole moment with the electric field is small compared with
k.sub.BT (i.e. .mu.E<<k.sub.BT), equations 2 and 3 yield
following asymptotic solutions:
F ( E ) .apprxeq. .mu. E k B T 1 + .mu. E k B T , and ( Eq . 4 ) F
( E ) .apprxeq. 2 .mu. E k B T 1 + 2 .mu. E k B T , ( Eq . 5 )
##EQU00003##
for the 2D and 3D cases, respectively.
[0085] These expressions indicate that, for small electric fields,
the propeller effect varies linearly with the electric field
magnitude. Thus, at low electric fields, the propeller effect is
linearly proportional both to the frequency of rotation and to the
magnitude of the electric field. In other words, in terms of
optimization of the propeller effect, the rotational frequency and
strength of the field are interchangeable variables
(<v>E.times.f). This result is in direct contrast to
Baranova's conclusion that <v>E.sup.2.times.f. Furthermore,
this result indicates that the magnitude of the propeller effect at
any practical electric field strength is much larger than what is
predicted by Baranova's conclusion. A theoretical comparison of the
two alternative expressions for F(E) as a function of E is shown in
FIG. 29.
[0086] Our conclusion demonstrates, contrary to the implication of
Baranova's reasoning, that the efficiency of the propeller effect
can be improved by increasing the frequency of electric field up to
the GHz range, even though the magnitudes of achievable electric
field are low at such high frequencies. In other words, we have
discovered that high propeller efficiencies (for example, propeller
efficiencies at levels that can be used to produce economically
practical separations) can be achieved in spite of the lower field
magnitudes that are associated with producing high frequencies, as
long as the decrease in electric field magnitude is compensated by
an equivalent (e.g., the same amount of) increase in the rotational
frequency factor.
[0087] This combining of low field magnitude and high frequency to
achieve practical levels of efficiency would appear not to be
possible based on Baranova's (incorrect) quadratic dependence of
efficiency on electric field amplitude. From the quadratic
dependence, it can be inferred that higher efficiency could be
better achieved by a higher strength of the electric fields than by
increasing the rotation frequency (which, in practical terms, is
typically accompanied by lower field strength). Increasing the
power of a microwave field, for example, would not be practical,
because the power (.about.1 GW) needed to achieve the needed
electric field magnitude (10.sup.6-10.sup.8 V/m) implied by the
quadratic dependence would far exceed the amount of energy that
could be released due to dielectric loss in the solvent (which
would lead to unacceptable heating and even boiling of the
solvent).
[0088] According to our expressions (Eqs. 4 and 5), however,
electric fields as low as 10.sup.4 V/m can lead to efficient
propeller motion, if the frequency of electric field rotation is
increased proportionately e.g., to the 100 MHz-10 GHz range for
such low fields, which are attainable using available electronic
components and circuits. Then the voltage applied across the
electrodes may be reduced while the rotation frequency of the
electric field can be increased by the same factor to produce an
equivalent linear velocity of the chiral molecules (i.e., similar
or even higher <v> for the enantiomers). For example, a
rotating field at 10 kHz generated by +/-5,000V is expected to be
as efficient as +/-50V at 1 MHz.
[0089] Our theoretical and experimental analysis indicates that the
propeller effect works even at low field magnitudes, so that the
molecules can be rotated by a rotating electric field or a rotating
electromagnetic field (i.e., a circularly polarized), within a
broad variety of frequency ranges (e.g., RF, microwave, infrared,
visible, UV, or other frequencies). If the applied field is
electromagnetic, practical power levels (e.g., for which sourcing
or heating is not an issue) that provide low electric fields, can
be used, and the relatively lower molecule velocity resulting from
these practical power levels can be more than compensated by an
increase in the rotation frequency of the field (e.g., in the MHz
range for RF up to GHz range for microwave). By using a rotating
electric field at relatively low rotational frequencies (<1 GHz)
and low power levels using, for example, a multiple electrode setup
(FIG. 1), the absorption of energy from the externally applied
field by the racemic solution (which can produce undesirable
heating of the solution) is reduced or eliminated. This approach
also simplifies the overall setup and decreases its cost, because
the electric fields can be generated purely electronically.
[0090] For example, suppose a 2% composite enrichment of two
enantiomer concentrations could be achieved over a 1 mm long
container (i.e., a 51-49 ratio of the concentration levels of the
two enantiomers at opposite ends of the container, and L=1 mm). The
upper limit for the time period required for the enrichment to
reach a steady state (in other words, the time after which further
enrichment will no longer occur) is proportional to L.sup.2 (i.e.,
t.sub.steady-state L.sup.2/D), so for L=1 mm and D.about.10.sup.-5
cm.sup.2/s, t.sub.steady-state.about.10.sup.3s.about.half an hour.
And if the characteristic length of the propeller effect (D/v) is
significantly smaller than the length of the container (L) (i.e.,
D/v <<L), the time required for separation is given by
.about.L/v. The container length (L) could be increased to achieve
any desired purity level. The time required for diffusion to reach
steady-state increases quadratically with L. However, the
displacement of the molecules due to the propeller effect is linear
in time, while diffusive spread scales as the square root of time.
Given sufficient time, linear motion always overtakes diffusion.
Therefore high purity levels (e.g., 99%-1%) can be achieved in a
sufficiently long container, without establishing concentration
equilibrium.
[0091] To achieve higher purities in less time, multi-step
amplification can be used. One way to amplify an arbitrarily low
purity level in one container to achieve an arbitrarily high
desired purity level (within a reasonable amount of time) is shown
in FIG. 4. In this example, we assume a linear concentration
gradient (instead of an exponential one) for simplicity, but the
amplification algorithm that we discuss may be modified for any
type of gradient profile.
[0092] In this example, we have .about.2N chambers, N=10, lined up
consecutively and numbered from N.sub.-10 to N.sub.+10 (only some
of them are shown). A strictly racemic mixture 60, of 50%-50%
concentration, is in the middle chamber, N.sub.0. Neighboring
chambers are connected to each other using, e.g., pumps and
barriers (not shown), and reservoirs (not shown) are operated for
temporary storage of solutions obtained in a time succession of
purification steps. During each purification step, the contents of
each chamber are kept separate from the contents of its neighbors
(i.e., the contents are not allowed to interact). In the times
between successive purification steps, all or portions of each
chamber's contents may be transferred to the next and/or to the
previous neighboring chamber in the sequence.
[0093] Neighboring chambers are separated by walls 62. For purposes
of describing concentrations of the enantiomers, each chamber has a
middle point 64, a left point 66, and a right point 68. The numbers
shown across the tops of the chambers in FIG. 1 represent
concentrations of the right-handed enantiomer, for example, and
numbers shown across the bottoms of the chambers represent
concentrations of the left-handed enantiomer.
[0094] Initially each chamber contains a pre-determined average
concentration of each enantiomer, e.g., an average concentration of
100 in N.sub.0, 105 in N.sub.1, 110 in N.sub.2, (using arbitrary
units for concentration levels). For example, in FIG. 4, in chamber
N.sub.2, the average concentration level of the right-handed
enantiomer is 100-110-120, for an average of 110. We discuss the
concentration levels of only one type of enantiomer, e.g.,
right-handed molecules, as their concentration levels are amplified
along the chambers going from chamber N.sub.0 to chamber N.sub.+10.
The concentration of left-handed molecules is being purified at the
same time along the chambers towards the left (going from N.sub.0
to N.sub.-10). We will describe later how the initial
concentrations in each chamber may be established in a brief setup
time.
[0095] During each of a time series of separation steps, a rotating
field is applied separately to each of the chambers as explained
earlier. Assume that the applied field on each chamber creates a
net force acting on the right-handed molecules (corresponding to a
directional velocity of the molecules) that yields a concentration
gradient of 20 (again, in arbitrary units) at the end the time
period of a given step. So, at the end of the step, inside the
N.sub.0 chamber (with average concentration 100), the concentration
of the right-handed molecules at the far left end of the chamber
will be 90, in the middle will be 100, and at the far right end
will be 110. For the next chamber (N.sub.1), the average is assumed
to be 105, and the gradient would go from 95 to 115; for N.sub.2,
the gradient has an average of 110 at the middle point, with 100
and 120 at either end, and so on.
[0096] During the purification time step that lasts for the period
t.sub.1, equal magnitude concentration gradients (of 20
concentration points in this example) would be produced in each
chamber, centered at the average level for that chamber.
[0097] At the end of t.sub.1, the volume of the solution that is in
the left half (e.g., half 67) of each chamber is transferred 67 to
the right half of the previous (next to the left) chamber. For
example, the solution in the left half of chamber N.sub.1, from the
left end to the middle-point, including all the molecules in it, in
which the concentration ranges from 95 to 105 for right-handed
molecules and 105 to 95 for left-handed molecules, respectively, is
transferred to the previous chamber, N.sub.0. Similarly, the volume
of the solution that is in the right half of chamber N.sub.1 (from
the middle-point to the right end, with levels ranging from 105 to
115 for right-handed and 95 to 85 for left-handed molecules,
respectively), is transferred 69 to the left half of chamber
N.sub.2, and so forth. The average concentration of the half-volume
transferred to N.sub.2 both from N.sub.1 and from N.sub.3 is 110
and 90 for right- and left-handed molecules, respectively, the same
as the average of the whole N.sub.2 chamber before t.sub.1.
Similarly, the average of N.sub.0 was 100 for both enantiomers,
which is exactly the average of the right half-volume of N.sub.-1,
as well as the average of the left half-volume of N.sub.1. Thus,
the average concentration and the total volume in each chamber
remain constant from one time step to the next.
[0098] In summary, each chamber generates a concentration gradient
of 20 in each time step, and by moving portions of the solution
(more generally the mixture) between neighboring chambers, a
desired purity level can be achieved for volumes in chambers that
are away from the center chamber. While each chamber imparts a
relatively small amount of enrichment, operating multiple chambers
in sequence can amplify the overall purification level
significantly. An advantage of using a multi-step amplification
setup (as opposed to a single, long container) is that the total
time to reach a certain throughput will be reduced by a factor of
.about.N (i.e., the number of steps). The time is reduced by a
factor of 10, because even though the number of steps increases by
N, the time it takes for a single container to reach steady-state
decreases by a factor of N.sup.2 (because this time period is
quadratic with container length). As a result, for an enrichment
factor of X per chamber, each enantiomer will be enriched by a
factor of (1+X).sup.N, so the overall purity level will be
(1+X).sup.2N (because the overall purity is equal to the product of
both enantiomers' enrichment). As an example, for X=1%, a 99%-1%
purification can be achieved with .about.200 steps.
[0099] The enriched sample can be collected from the final chamber
(i.e., N.sub.10 in this example). At the end of each time step,
there is no N.sub.11 to which to transfer the right half volume of
N.sub.10. Instead, a small fraction of that N.sub.10 volume is
collected 70, and the same volume of racemic mixture is injected 72
into N.sub.10. The partial amount of solution (with enriched
enantiomers) that can be extracted from the last chamber is
.about.V/2N (i.e., throughput is V/2N per time step), where V is
the volume of a single chamber (so V/2 is the volume of the upper
half), and N is the number of amplification steps (10, in this
example). Extracting this volume of solution and substituting the
same volume of racemic solution keeps both the volume and the
average concentration in the last chamber constant from one time
step to the next. This scheme enables continuous purification
(enrichment) of enantiomers.
[0100] The above description assumed that increasing average
concentrations in the respective consecutive chambers are
pre-established at the start. These concentrations can be set up
initially within a short period of time as shown in FIG. 5. We
first fill N.sub.0 with a racemic mixture (the first row in the
table), i.e., with concentrations of 100-100 for both the
left-handed and right-handed molecules. We assume 10 (arbitrary)
units of volume in each chamber. Then we run chamber N.sub.0 for
one time step (during t.sub.1), transfer the upper half volume
(which has average concentrations of 105 and 95 of right-handed and
left-handed molecules, respectively) to N.sub.1, and transfer the
lower half volume (which has average concentrations of 95 and 105
of right-handed and left-handed molecules, respectively) to
N.sub.-1. After these transfers, N.sub.0 is completely empty, and
N.sub.1 and N.sub.4 are each half full with their respective
correct average concentrations (i.e., V.sub.1=V.sub.-1=5).
[0101] We then fill N.sub.0 with a racemic mixture again (i.e.,
V.sub.0=10) and repeat the process during t.sub.2 (line 2 of the
table, without any transfers from N.sub.1 and N.sub.-1). At the end
of t.sub.2, N.sub.1 and N.sub.-1 are filled (i.e.
V.sub.1=V.sub.-1=10) with their operational average concentrations
of the two enantiomers and N.sub.0 is empty. We repeat the initial
process (of filling N.sub.0 with a racemic mixture), and at each
time step transfer half of the volume of the outermost chambers
(that are full) to their subsequent neighbors until all the
chambers are filled. Note that the total time it takes to fill the
whole setup is not on the order of 2.sup.N, since we can start
doing multiple half-volume transfers in parallel as more and more
chambers are being filled. Instead, the initial set-up time is
linear with number of steps, on the order of .about.4N, where 2N
comes from the fact that it takes two time steps to fill the
outermost chamber, another N because we need to wait a third time
step before we can make a transfer out of the outermost chamber,
and another final N to fill up all the half-filled chambers at the
end of the initial setup. In FIG. 5, we can see that it takes three
time steps to bring each additional chamber into operation in the
system (so setup time is on the order of .about.3N). We need a
final stage t.sub.13 for cleanup on the order of another
.about.N.
[0102] As shown in FIG. 16, a system 220 for receiving and
processing chiral objects 228 and delivering, for example, enriched
chiral objects 230 after processing, can include a set 222 of
chambers 224, a fluid subsystem 226 and an electrical subsystem 250
both coupled to each of the chambers, and controls 268 that provide
control signals 234, 264 to and receive feedback signals 232, 266
from the subsystems to cause the set of chambers to safely,
efficiently, effectively, and quickly produce the enriched chiral
objects.
[0103] In the fluid subsystem, reservoirs 244 temporarily hold
supplies 246 of fluids, chiral objects, enriched chiral objects,
and volumes of fluids that are passed from chamber to chamber
during processing. The fluids are moved in and out of reservoirs
and along conduits 240 and through valves 240 by electrically
controlled pumps 236 based on the control signals from the
controls. Sensors 248 can detect the presence, movement, velocity,
volume, and composition of any of the fluids, chiral objects, or
other materials that are present in or moving through the system.
Corresponding feedback signals can be sent back the controls.
[0104] In the electrical subsystem, voltages to drive electrodes in
the chambers are provided by power supplies 260 (which also power
other components in the electrical subsystem, the fluid subsystem,
and the controls. Other kinds of fields can be produced by field
sources 258. Power is routed along electrical conductors 252
through controlled switches 254 and can be filtered, enhanced, and
otherwise processed by electronics 256. Sensors can detect the
voltages, currents, field strengths, rotational velocities, timing,
and other characteristics of all electrical parameters and provide
corresponding feedback signals back to the controls. The control
signals control the operation of the power supplies, switches,
field sources, electronics, and sensors.
[0105] The controls 268 may include processors 270, memory 272,
network connections 275, input/output interfaces 274, and software
276, among other things. A user interface 287 enables a user to
observe and control the operation of the system. The software 276
includes an operating system 278, database and other applications
280, field rotation algorithms, fluid flow algorithms 284, and
process algorithms 286. The controls can communicate with other
devices and users through a network 289.
[0106] In other parts of this description, references to software
include, for example, portions of software 276 in FIG. 16.
[0107] A wide variety of modifications and enhancements to features
described above are possible, including, for example, the
following:
[0108] The chiral objects may be, for example, chiral molecules,
stereoisomers, enantiomers, epimers, or aggregates of a number of
chiral or achiral molecules. These chiral molecules may be, for
example, drug molecules, drug intermediate molecules, sugars (such
as monosaccharides, disaccharides, oligo- and poly-saccharides
(glycans)), proteins, peptides, glycoproteins, glycolipids, or
other macromolecules. The chiral objects may have one stereocenter
or more than one stereocenter.
[0109] There may be only one type of chiral object in the mixture,
or two, or more than two.
[0110] The molecules may belong to different categories, classified
based on their various features, e.g., side groups, number of
stereocenters, molecular size, 3D shape or structure, or
interaction with the solvent or other fluid.
[0111] The proposed method may be used to analyze the chirality of
the objects, to detect their presence and/or absence, or to
separate two or more types of chiral objects.
[0112] Molecules may have more than one stereocenter. Molecules
with more than one stereocenter may be separated into two groups or
they may be separated into multiple groups (one for each
stereocenter combination). These multiple peaks may be monitored
(i.e., detected and/or analyzed) in real-time or as an
end-point.
[0113] Epimers may be separated based on different rotation
frequencies and/or different effective velocities they experience
in response to the same applied field, even if their velocities
have the same sign (i.e. even if their linear motion is in the same
direction).
[0114] The applied field may be an electric field or a magnetic
field.
[0115] The applied field may be rotated around a stationary
chamber, or conversely, the orientation of the field may be fixed
(i.e., static electric field or linearly polarized electromagnetic
field) and the chamber may be rotated.
[0116] The mixture may be racemic, non-pure, or enantiopure.
[0117] The applied field may be generated by electrodes arranged
around the chamber or it may be a field component of an
electromagnetic beam (e.g., that is collinear with the chamber).
The rotation frequency of the field may be less than 1 kHz, between
1-10 kHz, 10-100 kHz, 100 kHz-1 MHz, 1-10 MHz, 10-100 MHz, 100
MHz-1 GHz, 1-10 GHz, or above 10 GHz. It may be in the RF range or
in the microwave range.
[0118] The separation of the two chiral objects may continue until
the concentration gradient reaches steady-state (the longer we
continue, the more stable the gradient will be). Or a time-gated
cutoff scheme may be used, in which the molecules are released from
a particular point in the chamber (e.g. one end of it, or from the
mid-point). Then the chiral objects diffuse across the length of
the container (with different velocities depending on their
handedness) for a selected period of time, at the end of which the
diffusion is stopped (either by terminating the applied field or
mechanically) and the molecules are collected from a region where
the desired purity is reached (e.g., from the tails of the
distribution profiles). Note that the higher the desired purity,
the lower the yield.
[0119] The time period applied may be adjusted to trade off
enrichment consistency and throughput. That is, the longer the
processing time, the more stable the concentration gradient will be
(i.e., lower statistical noise in concentration levels), but the
throughput will also be lower because the same quantity of material
will be enriched over a longer period of time.
[0120] The concentration gradient profile may be exponential,
linear, or non-linear.
[0121] The non-exponential profiles may be obtained by using
separation parameters that are not constant along the length of the
chamber (e.g. field intensity, rotation frequency, solution
properties such as viscosity, pH, or co-solvent concentration).
[0122] The v/D ratio (i.e., velocity of a molecule divided by its
diffusion constant) may be improved (i.e., increased) by attaching
a chemical molecule (e.g., a chiral label) to the molecules of
interest to enhance the propeller effect (e.g., by increasing the
molecule's dipole moment and/or its rotational-translational
coupling factor). Such chiral labels may also be attached to
improve other system properties (e.g., noise, yield, purity,
throughput).
[0123] The v/D ratio may also be improved (i.e., increased) by
causing the molecules to experience a collective motion, in which
they "sense" each other and act in unison (so that the net force
acting on the respective molecules is additive, and yet, the
diffusion constant acting on the aggregate is relatively smaller).
This collective behavior may be accomplished using chemical (e.g.,
liquid crystal-like) techniques or physical techniques (e.g.,
ferromagnetism), or combinations of them.
[0124] The molecules may be in solution, in gas, or in high
pressure fluid (e.g., supercritical CO.sub.2, N.sub.2, or
Argon).
[0125] Different solvents may be used inside the container. These
include polar solutions, e.g. water, polar organics (e.g.,
ethylacetate, acetonitrile, dimethylformamide (DMF),
dimethylsulfoxide (DMSO)), alcohols (R--OH, with R.dbd.C.sub.n),
dichloromethane (DCM), methoxymethylether, tetrahydrofuran (THF),
and the like, and nonpolar solutions, e.g. carbontetrachloride
(CCl.sub.4), alkanes such as cyclohexane, hexane or pentane, and
the like. Solvents may have different thermodynamic properties
(e.g., temperature, pressure) or physiochemical properties (e.g.,
different buffers, concentrations, pH, molecule solubility, ionic
strength, viscosity, osmolarity).
[0126] The solvent composition may be adjusted (e.g., pH level) to
optimize the propeller technique (e.g., neutralize the molecules to
ensure a zero net charge per molecule).
[0127] If the solvent is polar (e.g., the solvent molecule has a
permanent or an induced electric dipole), the solvent molecules may
be rotated with an applied electric field, which in turn will
impart angular momentum on the molecules of interest (polar or
non-polar). And with this angular momentum, the molecules of
interest may rotate and experience linear motion based on their
chirality, as described previously.
[0128] The separation may be accomplished in a solution that is
flowing in the chamber along its length or in a steady
(non-flowing) solution. For a particular application, a
counter-flow may be set up inside the container to oppose the
propeller force, to set an opposing threshold for the molecules to
overcome.
[0129] The effective electric field profile inside the container
may be modified and controlled. This may be accomplished by a fixed
parameter (e.g., shape of the tube, inner and outer diameter of the
tube, material type, solvent conductivity, or pH) or by an
adjustable parameter (e.g., by adjusting the applied field
frequency along the z-axis, or apply a particular voltage to each
electrode around the ring, rather than applying only the peak
voltage to only the two opposing electrodes). Two example voltage
profiles (linear and trigonometric on the left and right for high
voltage on the top and low voltage on the bottom) are shown in FIG.
6.
[0130] The purification may be carried out in a predefined
unchanging mode (e.g., automated, with no feedback), or it may be
controlled and/or calibrated using real-time detection of the
concentrations (or other parameters) of the output (or other
portions of the mixture) for feedback.
[0131] The setup may have only one chamber or many chambers. It may
have one sequence of chambers or many sequences running in
parallel. One goal is to achieve a particular purity level in the
shortest time possible (i.e., maximize throughput).
[0132] The enrichment may be carried out in only one direction
(i.e., concentration of only one enantiomer is increased) or in
both directions (i.e., both enantiomers are purified).
[0133] All chambers in a sequence may be of the same size or of
different sizes (e.g., to account for concentration
non-uniformities).
[0134] From one chamber to the next, only the molecules may be
transferred or the whole volume of solution (fluid mixture) may be
transferred.
[0135] Exactly half of each chamber's volume may be transferred to
the next chamber or a volume that is less than half of the chamber
may be transferred (i.e., a portion of a particular chamber's
volume stays in that chamber).
[0136] The same volume may be transferred in both forward and
backward directions (i.e., symmetric transfer) or the volume
transferred forward may be different from that of the backward
transfer (i.e., asymmetric transfer).
[0137] Depending on the concentration gradient profiles, different
algorithms may be employed to improve a particular parameter of the
system (e.g., purity level, purity consistency, shortest initial
setup time, overall throughput, throughput within a fixed amount of
time, for example).
[0138] For a given sequence of chambers, additional chambers may be
operated in parallel to increase the overall throughput. In the
example of FIG. 7, the first three chambers 202 are replicated 204
(in addition to the original set of chambers; in this case four
replications of N.sub.0, two of N.sub.1, and one of N.sub.2), and a
portion of the replicated N.sub.2's output is directly injected 80
into the final collection chamber (instead of a racemic mixture).
Then three times the volume can be extracted from the last chamber
for each time step (i.e., throughput is increased by a factor of
3), while keeping the average concentration of the last chamber
constant after each time step.
[0139] The replicated chambers could supplement the purification of
the chambers at the beginning, at the end, and/or in the middle of
the sequence. These replicated chambers may be provided for only
one chamber or multiple chambers in the original setup.
[0140] The concentration gradients in each chamber may be initially
established by incremental and sequential processing (as in FIG.
5), or simultaneously for many or all chambers. The required
average concentrations also may be established externally and then
loaded into multiple chambers simultaneously.
[0141] This initial establishing of the gradients may be automated
or it may be calibrated by feedback using real-time detection and
monitoring of the output (or other portions of the solutions).
[0142] The solution and/or the molecules may be input to each
container and/or collected from each container (after enrichment)
using various pumping mechanisms (e.g., mechanical, pressure, or
membrane).
[0143] The detection and monitoring of the output may be done
optically (e.g., by circular dichroism, optical index
birefringence, optical absorption, or fluorescence), chemically
(e.g., by chemical sensors or chiral columns or selectors or mass
spectroscopy) or electronically (e.g., by chirally selective
electrodes). It can be done in real-time or at an end-point of the
processing.
[0144] The system (for example, the software 276 of FIG. 16) may
include software to control, manage, or compliment the results. It
may also include software to calculate and/or predict expected
performance or performance limits, or average velocity of the
chiral objects, or the direction of motion for each absolute
configuration of a chiral object.
[0145] The system may be a fully automated, turnkey system. It may
include features or modules such as sample preparation,
auto-sampling, microfluidics, and fraction collection.
[0146] The system may control and/or optimize the environmental
parameters (such as temperature or pressure). These parameters may
be adjusted by hardware or software, and by online/remote
access.
[0147] The system may automatically optimize or calibrate or
monitor the various control parameters (e.g., applied voltage,
frequency of rotation, duration of the applied field, which solvent
to be used) to optimize the performance parameters (e.g., purity,
throughput, yield, total run-time, multi-step amplification).
[0148] The system may optimize or monitor the reliability,
repeatability, and reproducibility of the various runs.
[0149] The system may allow and/or manage multiple runs in parallel
or in series, with different samples, solvents, or function and
application.
[0150] The environment (the fluid, solution, or mixture) for the
molecules may be asymmetric or anisotropic in at least one
dimension (e.g., an anisotropic gel).
[0151] Various diffusion and convection reduction techniques may be
used in the solvent, fluid, medium, or mixture (e.g., gel
technology, bead technology, orientation of the chamber).
Properties of the medium may be changed (e.g., packing with C18
bonded silica and/or porous versus nonporous media).
[0152] The propeller effect may be used at a small scale (e.g.,
microscale) or in a large scale (e.g., batch size). The chamber may
be in macroscale, microscale, or nanoscale.
[0153] The container cross-section may be circular, rectangular,
triangular or some other geometry, to enable practical electrode
deposition and to achieve an optimum electric field profile
inside.
[0154] If the applied field is a rotating electric field, the
electrodes may be placed on the outer surface or on the inner
surface, and, for the latter, they may be in contact with the
solution or they may be laminated so as not to be in contact with
the solution.
[0155] The electrodes may be of various materials (e.g., metal,
semiconductor), types, or shapes (e.g., rectangular, spherical,
etched radially outward from the center of the cylinder), and their
characteristics may be selected to optimize the electric field
profile and properties (e.g., improve the breakdown voltage).
[0156] The number of electrodes may be reduced to lower the cost of
the electronics or it may be increased to achieve a more uniform
electric field profile inside the container.
[0157] As shown schematically in FIG. 8, chambers 92 can be
implemented by providing electrodes 96 (and electrical connections
102), fluid connections 94, environmental monitors and controls
100, and detection zones 98. Depending on the application, these
components can be manufactured in very high volumes, assembled in
combinations and sub-combinations, and delivered as complete
systems, or as modular subsystems that can be combined in a wide
variety of combinations by the user. The sizes, configurations,
materials, costs, and scale of the components, and the
sub-combinations and combinations of them can vary depending on the
application. In particular, individual components,
sub-combinations, and combinations can be produced in inexpensive,
disposable forms, and we sometimes call these disposable
components, or simply disposables. We also sometimes refer to
components or sub-combinations of them (for example, a chamber with
its electrodes and fluid connections, or a set of chambers with
their electrodes and fluid connections) as cartridges 99.
Cartridges can be disposables. In some examples, a cartridge,
therefore, may include (a) a chamber or chambers 92 to hold the
sample (we sometimes use the word sample to refer to a volume of
fluid or solution that contains chiral objects), (b) electrodes 96
arranged about the chamber or chambers to produce a rotating
electric field, (c) fluid interconnections 94 to inject and extract
the sample or portions of it from the chamber or chambers, (d) a
detection zone 98 to monitor the concentration of the sample or
portions of it, (e) a device or devices (e.g., local sensors) 100
to control/monitor environmental parameters (e.g., pressure,
temperature), and (e) electrical interconnections 102 to make
electrical contact with the electrodes.
[0158] High volume manufacturing techniques could be used to
produce cartridges. For example, the chamber cross-section may be
circular, rectangular, triangular, or some other geometry, to
enable practical electrode deposition and to achieve an optimum
electric field profile inside. As shown in FIG. 9, the sample
chamber(s) 110 may be a capillary or capillaries. The capillaries
may be of various materials (e.g., glass, polymer, quartz), and
they may be coated on all or part of the inside surfaces. The
sample chamber may be a microfluidic channel on a chip, formed by
photolithography or by laminating multiple layers of material or a
combination of both.
[0159] The electrodes 112 may be placed around the sample chamber
and oriented axially (that is, with the long dimension of each
electrode parallel to the longitudinal axis of the chamber) so that
the enantiomers move (and the fluid in which they are held flows)
longitudinally (along the axis). In some examples, the electrodes
may be oriented orthogonally to the longitudinal axis of the
chamber so that the enantiomers move transversely to a direction of
flow of the solution or fluid during separation (e.g., across a
Y-junction). The electrodes may be placed about the sample chamber
at an angle that is neither parallel nor orthogonal. One or more of
the electrodes may be an alternating current driven coil that
causes the enantiomers to move longitudinally to the direction of
flow of the fluid when the wavelength of the AC field approximates
the coil diameter. A tubular ground shield 114 can be placed to
surround the electrodes.
[0160] The sample chamber may bear a continuous set of electrodes
that extend along the full length of the chamber and generate a
uniform field across the chamber all along its length. As shown in
FIGS. 20, 21, in some examples, the sample chamber may bear
discrete sets 302, 304, 306, 308 of electrodes (each set occupying
only a particular sub-length along the length of the chamber).
Different sets of the electrodes can generate rotating electric
fields 310, 312 having the same or different parameters.
[0161] Instead of using electrodes, a rotating electric field may
be generated by circularly polarized microwaves that are propagated
axially along the sample chamber, which acts as a waveguide.
[0162] As shown in FIG. 22, in some ways of manufacturing
cartridges, a chip can bear multiple chambers 120, each ending in a
T- or Y-junction 122. Electrodes 124 on each chamber generate a
transverse rotating field 126 relative to a direction of flow 128
of the solution (or fluid). In such an arrangement, the enantiomers
are enriched along the edges of each sample chamber by the
propeller effect and then are physically separated 130, 132 at the
junction and drawn away in two opposite directions along a conduit
133. As shown in FIG. 23, this configuration may be repeated 134 on
a single (or multiple chips) and organized in a tree to achieve a
desired enrichment factor.
[0163] The disposable cartridge may be compatible with available
liquid chromatography instruments that have appropriate fluidic
interconnects. Typical connections may be luer or screw
fittings.
[0164] For example, returning to FIG. 9, a capillary 110 may be
surrounded by a set of electrodes 112. The assembly may be held
together by larger tubing 114. For high frequency operation, the
tubing may be metal to serve as a ground shield. For high voltage
operation, the larger tubing may be a dielectric material. As shown
in FIG. 10, two or more of the assemblies of FIG. 9 may be grouped
to form a bundle 113 of sample chambers with electrodes 115, 117
(we sometimes call the bundle a column). Hexagonal close packing is
a possible geometry for the column.
[0165] As shown in FIG. 11, the capillaries could be bundled
together in a column 119 and terminated in luer fluidic fittings
121, 123, for example, to form a disposable cartridge that could be
plugged into and removed from a larger system easily.
[0166] Electrical connections between the electrodes of the
cartridge and a source (not shown) may be made by a set of bundled
wires 130 (FIG. 12) that are permanently connected to the
electrodes at the column (and in that case part of the disposable
cartridge) and have a connector 133 at the source end. As shown in
FIG. 13, in some examples, the connections can be made through
rigid or flexible printed circuit boards 134, or elastomeric
connectors that are part of the disposable cartridge.
[0167] The conductors can be terminated at one or both ends by
adapters for easy connection or disconnection (plug-n-play). Such a
column 119 containing one or more capillary assemblies, fluidic
connections, and electrical connections could be made in large
quantities as a disposable cartridge and multiple copies of the
disposable cartridge could be assembled to form a complete
separation device.
[0168] As shown in FIGS. 14 and 15, in some implementations, the
electrodes 136 and electrical connections 138 may be integrated on
a printed circuit board 140 (PCB) to reduce the cost of the
disposable cartridge. A rigid PCB can have ten circuit layers, for
example, and be 1/8'' thick. A through hole 142 in the PCB would
allow a capillary chamber 110 to pass through and an electrode
pattern would be generated in each layer of the PCB. Multiple PCBs
can be stacked to create a sample chamber of a selected length.
Fluidic and electrical interconnects could be as described above.
In this example, the disposable cartridge could be the capillary
and the fluidic interconnects to the capillary.
[0169] As shown in FIGS. 17 and 18, in a more comprehensive
integration approach, microfluidic separation devices 150 could be
fabricated in glass, quartz, polymers, epoxy, or elastomers, for
example. Electrodes 152, 154, 156, 158 would be deposited at or
near the corners of each channel 160 to impose a rotational
electric field E to achieve longitudinal separation 161 along the
length of the chamber. The electrodes may be in contact with or
insulated from the medium (fluid) that is held in the sample
chamber. In some implementations, the channel cross-section is
square as shown in FIG. 17. The side walls 164, 166 of the sample
chamber may be of the same or different material as the top or
bottom substrate 168, 170. The top and bottom substrates 168, 170
may be the same or different materials. As shown in FIGS. 18 and
19, an array 170 of such sample chambers with electrodes may be
fabricated using lithography, LIGA (x-ray lithography), molding,
stamping, and/or printing methods. The interconnected sample
chambers may define a straight or serpentine (as in FIG. 19) or any
other path. The sample chamber may be a single channel or may have
one or more T- or Y-junctions. A series of these channels and
junctions on a device operated using a transverse propeller effect
can iteratively separate enantiomers with high purity.
[0170] As shown in FIG. 24, fluidic connections 180, 182 to the
chamber 184 may include tubing ports such as Upchurch Nanoports. A
plastic housing 183 can be attached to the microchannel device to
provide molded fittings to attach tubing.
[0171] As shown in FIG. 25, electrical connections can be
end-connectors, elastomeric connectors, pogo pins, or other devices
190 to make contact with the contact pads 192 of the patterned
electrodes. Both fluidic (and electrical) connections may be made
using a clamshell device that mechanically forms a seal around the
fluidic ports and makes good contact with the bond pads. The
disposable in such cases could be the microchannel device without
the clamshell.
[0172] The separation techniques described here have an extremely
broad range of possible applications.
[0173] In some applications, the system can determine the absolute
configuration of a molecule by measuring the direction in which the
molecule moves in response to the applied electric field. In such
applications, the mixture may be non-pure or it may be
enantiopure.
[0174] The rotation of the electric field may be reversed (using
software or hardware), to confirm the absolute configuration
determination, because, when the electric field is reversed, the
chiral molecules of interest should travel in the opposite
direction.
[0175] The separation techniques described here (which we sometimes
refer to broadly as simply "the technique") may be used as a
stand-alone separation system or as an add-on to another chiral
separation technique (e.g., high-performance liquid chromatography,
HPLC). For example, the electric field may be applied to a chiral
HPLC column (for additional enrichment).
[0176] The technique may be used as an add-on to a standard HPLC
column (to achieve separation of size and chirality in one
step).
[0177] The technique or the resulting separations may be used for
the detection, determination of composition, quantification,
separation and/or purification of various objects in various fields
(e.g., analytical chemistry, drug discovery, drug development, drug
manufacturing, genomics, proteomics and/or glycomics) For example,
the technique or the resulting separations can be applied to sugars
in glycomics applications or to peptides or proteins in proteomics
applications. The technique can also be applied to the purification
of modified carbohydrates or protein-carbohydrate interactions. The
techniques may also be used for diagnostic applications, e.g.,
detection and/or quantification of various post-translational
modifications (e.g., glycosylation, phosphorylation) on
proteins/peptides, or similar modifications on DNA (e.g.,
methylation) and other epi-genetic applications, or of biomarkers
in disease states. The technique or the resulting separations may
also be used in chemical manufacturing applications, e.g., custom
synthesis, batch enrichment, in fine chemical or synthetic
intermediate manufacture, or applied to agrichemicals,
petrochemicals, flavors and fragrances, or used in food processing,
forensic applications, and/or in academic research.
[0178] The technique or the resulting separations may also be used
for the detection, determination of composition, quantification,
separation, and/or removal of chiral impurities from solvents or
solutions.
[0179] The technique or the resulting separations may also be used
in asymmetric synthesis. Currently in asymmetric synthesis and
methodology development, reactions are run sequentially or in
parallel, with each reaction having different parameters (e.g.,
reaction conditions, catalysts) to determine the optimized reaction
conditions to achieve high enantiopurity and/or a desired single
enantiomer of a particular absolute (e.g., left- or right-handed)
configuration. Upon completion of the chemical reaction, the
reaction is worked up, purified of achiral impurities and starting
materials and then, e.g., injected onto a chiral chromatography
column for enantiomeric excess ratio (% ee) analysis and/or
separation into pure optical isomers. The determination of the most
suitable chiral chromatography column and condition choices can
require significant development time. This is eventually followed
by very low throughput analysis (e.g., x-ray crystallography) to
determine the absolute configuration of the enantiomers. Overall,
this process is laborious and time intensive, significantly adding
to, for example, the development time for new asymmetric synthetic
routes (e.g., in process chemistry) or for asymmetric synthesis
methodology development (e.g., analysis of exploratory reactions
utilizing novel chiral catalysts).
[0180] The techniques described here use a separation/analysis
chamber into which a chemical reaction mixture can be directly
injected without prior processing or refinement. At a given time,
the reaction mixture can be transferred from a reaction vessel into
the separation chamber using standard fluidic technology. A field
can then be applied across the chamber that is rotating relative to
the chamber to cause chiral objects present in the reaction mixture
to rotate and the rotation of the objects causing them to move in a
direction based on their chirality. Any achiral materials present
would not move. The chiral product(s) would move at different
speeds and/or directions inside of the separation chamber, allowing
simultaneous: A) isolation/purification of optical isomers both
from each other as well as from achiral impurities; B)
determination of % ee of the reaction mixture, e.g., by comparing
the area under the curve (AUC) of the separated peaks as measured
by a detector; and/or C) determination of the absolute
configuration of the objects based upon the direction/manner of an
optical isomer's movement in the separation chamber. The reactions
and analyses could be run in parallel if desired.
[0181] Similarly, at a given time, a reaction mixture from a
chemical reaction that is believed or known to produce a racemic
mixture of products can be transferred from a reaction vessel into
the separation chamber using standard fluidic technology. When the
rotating field is applied across the chamber, achiral materials
would not move. The chiral product(s) would move at different
speeds and/or directions allowing the processing described above.
The reactions and analyses could be run in parallel if desired.
[0182] The objects may be achiral objects with chiral labels
attached.
[0183] The technique can be applied to known molecules or to
previously unknown molecules or to known molecules that have
unknown properties as they relate to the separation techniques
described here.
[0184] For molecules that are known, the system can be modeled a
priori to predict, adjust, and/or optimize performance, or to
detect their presence or absence in the solution.
[0185] For molecules that are unknown or have properties that are
unknown, the system can be used for post-analysis, to detect,
identify, and/or quantify the composition and/or the specific
characteristics of the chiral objects.
[0186] The analysis may include determining the absolute
configuration of a molecule, the molecule's absence or presence in
the mixture, the molecule's propeller efficiency, the number of its
stereocenters and their contributions to the molecule's propeller
properties, or the quantitative measurement of the mixture's
enantiomeric excess. The chirality of a sample may be quantified,
e.g., by applying a force for translational motion in a time-gated
mode, and then measuring the drag force acting on the molecule for
or against the linear motion.
[0187] The formed gradient may be measured to deduce
characteristics of a chiral object's electric dipole moment, its
propeller properties, and/or its interaction with the solvent or
with other molecules.
[0188] The technique and/or a similar setup may be used for
electrorotary chemistry (e.g., chemistry, in the presence of and/or
due to, a rotating electric field), e.g., chiral synthesis,
catalysis and other catalytic reactions and applications, shifting
of a reaction's equilibrium by separating and/or transporting the
intermediate or product of the reaction, or manipulating the
reaction probability of specific reactants by matching their
rotations.
[0189] The technique and/or a similar setup may also be used in
electrorotary chemistry to study molecular interactions, or to
detect specific molecules and/or their reactions (e.g., by
attaching a propeller object to single molecule and then study its
properties during a reaction).
[0190] The system and the technique may be used to separate, sort,
transport, purify, and/or manipulate chiral molecules and
objects.
[0191] Furthermore, certain chemical reactions involving chiral
molecules may be manipulated (e.g., by changing the concentration
of a chiral molecule in a certain region). Or a continuous
concentration gradient may be used to apply different
concentrations of molecules onto an array (e.g., of surface
receptors) in parallel and obtain multiple results simultaneously
(rather than having to run an experiment with a different
concentration of the chiral molecule each time).
[0192] In some applications, the method can be used to separate
and/or purify chiral molecules from achiral impurities, using the
propeller effect to remove chiral molecule(s) of interest from an
initial mixture containing contaminants.
[0193] A similar approach or technique may be applied to other
molecules or objects besides enantiomers, whether chiral or achiral
(in which case chiral labels may be attached to make them chiral),
e.g., DNA, RNA, proteins, protein post-translational modifications
(PTMs), peptides, lipids, amino acids, virus, bacteria, or cells.
These chiral labels may be molecules or objects that are
self-assembled, self-activated, or pre-activated.
[0194] Multiple chiral labels having different, discrete propeller
efficiencies may be used for multiplex assays.
[0195] Chiral labels can used for debulking and enriching sample
matrices for cells and molecules of interest.
[0196] Propeller molecules can be conjugated to antibodies or to
nucleic acids to provide analyte selectivity. Additional
specificity can be imparted by a propeller sandwich configuration
whereby two binding events must occur before a functional propeller
is formed. For example, achiral conjugates can target discrete
epitopes on a ligand. Presence of target molecules results in
binding of both achiral conjugates in proximity, which produces a
chiral propeller.
[0197] Aptamer structures can be designed as target-activated
chiral labels. The unbound aptamer can be achiral or chiral and
becomes chiral or oppositely chiral, respectively, in the presence
of target molecules.
[0198] The propeller effect may also be induced in biomolecules
without the need for chiral labels. Intermolecular interactions
(antibody-antigen, protein-protein, protein-nucleic acid) can
induce conformational changes. Some of these changes may change
apparent propeller efficiency of the complex and can be exploited.
Physicochemical conditions can be adjusted to enhance the effect
between bound and unbound target molecules. For example, high
pressure can reduce molecular fluctuations and rigidify the
complex.
[0199] The technique will enable discovery of therapeutic
compositions, in a variety of ways. Some examples are set forth
below.
[0200] Skelaxin.TM. (Metaxalone,
(.+-.)-5-[(3,5-dimethylphenoxy)methyl]oxazolidin-2-one, CAS
Registry Number=[1665-48-1]) (Appendix B) has been previously
isolated and identified as a racemic mixture of its two
enantiomers. The method/apparatus described here could separate the
previously known racemic mixture (Metaxalone) into its purified
stereoisomeric constituents which will have distinct biological
and/or toxicological attributes, and to an enantiomer with
properties that are preferred for therapeutic usage. An "active
agent" that incorporates the preferred enantiomer of Metaxalone,
when administered to a patient, alone or in combination with
another compound, element, or mixture, would confer, directly or
indirectly, a desired physiological effect on the patient. The
indirect physiological effect may occur through a metabolite or
other indirect mechanism. When the active agent is a compound, then
salts, solvates (including hydrates) of the free compound or salt,
crystalline forms, non-crystalline forms, and any polymorphs of the
compound are included. All forms are contemplated regardless of the
methods used to obtain them. All forms (for example solvates,
optical isomers, enantiomeric forms, polymorphs, free compound and
salts of an active agent) of the preferred stereoisomer of
metaxalone or other active agent may be employed either alone or in
combination.
[0201] In addition to purification and isolation of the unknown
optically pure enantiomers of Metaxalone, the described technique
can be used to purify or isolate other known, optically active
molecules.
[0202] The technique separates stereoisomeric mixtures of
biologically active substances into their purified stereoisomeric
constituents, which will have distinct biological and/or
toxicological attributes, enabling one purified stereoisomer to be
designated as "preferred" for therapeutic usage. An "active agent"
that incorporates this preferred stereoisomer for therapeutic
usage, when administered to a patient, alone or in combination with
another compound, element, or mixture, may confer, directly or
indirectly, a desired physiological effect on the patient. The
indirect physiological effect may occur through a metabolite or
other indirect mechanism. When the active agent is a compound, then
salts, solvates (including hydrates) of the free compound or salt,
crystalline forms, non-crystalline forms, and any polymorphs of the
compound are included. All forms are contemplated regardless of the
methods used to obtain them. All forms (for example solvates,
polymorphs, free compound and salts of an active agent) of the
purified preferred stereoisomer of the biologically active
substance may be employed either alone or in combination.
[0203] The technique can be used in the purification of chiral
chemical intermediates in addition to the purification of chiral
biologically-active substances. Examples of chiral substances that
can be purified into their optically pure form by a process
employing the described method/apparatus are contained in the books
"Chiral Drugs", Cynthia A. Challener ed., Ashgate Publishing Ltd.,
2001, and references cited there, and "Chiral Intermediates",
Cynthia A. Challener ed., Ashgate Publishing Ltd., 2001, and
references cited there. Additional known chiral substances that can
be purified into their optically pure form by a process employing
the described method/apparatus are described in the books
"Chirality in Drug Research", Eric Francotte and Wolfgang Lindner
eds., Wiley-VCH, 2007 and references cited there, "Fine Chemicals",
Peter Polak, Wiley, 2007 and references cited there, and "The Merck
Index (14.sup.th edition)", Merck Research Laboratories, 2006, and
references cited there. All of the cited books and the references
cited in them are incorporated by reference here.
[0204] Additional example chiral molecules that can be purified
into their optically pure form by the technique are listed in
Tables 1 and 2 (Appendix D). The five above cited references, Table
1, Table 2, and chiral molecule examples contained there are
offered by way of illustration but are not meant to limit the scope
of the molecules that may be subjected to the technique, both in
terms of known and unknown chiral molecules that can be purified
into optically pure form by the process employing the described
technique.
[0205] Other implementations are also within the scope of the
following claims.
APPENDIX A
Examples
[0206] Six copper wire electrodes were arranged around a fused
quartz capillary (FIG. 1). The electrodes and capillary were fixed
in place by G10 high dielectric material to prevent arcing between
the electrodes. The capillary (Polymicro, O.D. 665 um, I.D. 150 um)
was connected to a pump (KDS-210) and an injection valve for sample
introduction. The resulting enantiomer enrichment was detected by
splitting the sample volume in half and running the leading and
trailing fractions into a combination CD/UV detector (Jasco
CD-2095).
[0207] A racemic mixture of
1,1'-bi-2-naphthol-bis(trifluoromethanesulfonate), sigma-aldrich
#514292, was injected into the capillary surrounded by electrodes
(200 nl @ .about.0.5 mg/ml, cyclohexane). The sample was subjected
to a rotating field at 10,000 volts peak-peak and 40 KHz for 17
hrs, at room temperature. Absorbance and CD measurements, both at
290 nm, were recorded for the leading and trailing fractions of the
sample (approx. 100 nl each). Measurements (CD:Abs)--control
mixture was +0.022:2.038, leading fraction was -0.004: 0.446,
trailing fraction was +0.010: 0.326. Post separation measurements
were non-quantitative due to Taylor dispersion and sample dilution.
The results indicated that separation did occur and that the
expected propeller stereoisomer moved in the correct direction.
[0208] The experiment was repeated with 52-hour separation. Sample
flow rate was reduced from 4 ul/min to 2 ul/min. Recorded
measurements were -0.014:1.905 and +0.008:1.584 for leading and
trailing fractions. Control was +0.022:2.038. The results were
consistent with the first experiment.
[0209] In another experiment, custom compounds were used which had
been synthesized according to methods described in Van Es, J. J. G.
S. et al. Synthesis and characterization of optically active cyclic
6,6'-dinitro-1,1'-binaphthyl-2,2'-diethers (Tetrahedron Asymmetry
8, 1825-1831 (1997)). Racemic mixture of
2,2'-(1,3-propylenedioxy)-6,6'-dinitro-1,1'-binaphthlene (1b) and
optically pure isomers of
2,2'-(1,4-butylenedioxy)-6,6'-dinitro-1,1'-binaphthlene (1a+,
1a-).
[0210] In an additional experiment, compound 1b was dissolved in
cyclohexane at approximately 0.5 mg/ml. Two hundred nanoliters was
injected into the capillary area surrounded by electrodes and a
rotating electric field was applied for 70 hrs at room temperature.
Leading and trailing fractions were measured at 303 nm and compared
to starting material. Results are shown in the graph in FIG. 26,
which illustrated that CD values inversely correlate with
absorbance during elution of the first peak and then show positive
correlation during the second peak elution. The starting material
had a CD value of 0.001 and absorbance of 1.577.
[0211] The prior experiment was repeated for 110 hrs and 250 nl
fractions were collected and serially injected into the detector.
The peak measurements were CD=-0.009, Abs=1.30 for the first peak
(graph 2) which is consistent with results observed (FIG. 27) in
the prior experiment. The second peak was not observed due to
insufficient sample collection tubes.
[0212] A demonstration of separation at low voltage and high
frequency is shown in FIG. 28. A 10-cm long capillary (100 um ID,
360 um OD) was used as sample chamber. Four electrodes (32g magnet
wire) were fixed around the capillary using adhesive. A signal
generator provided a sinusoidal signal at 1V that was passed
through a 4-way splitter and amplified to 36V peak-to-peak at the
sample chamber. The 90.degree. phase shift was accomplished by
adjusting the cable length from the splitter to the amplifiers
(Minicircuits ZHL-03-5WF). A sample of amino binap at 0.15 mgs/ml
in cyclohexane was injected into the capillary through a Rheodyne
sample injector valve and introduced into the electrode region. The
rotating electric field was turned on for 40 hrs and then eluted as
described previously. The results show that the leading fraction
(peak eluting .about.1600) was enriched for the (-) enantiomer
relative to the racemic starting material (peak eluting
.about.500). The trailing fraction (peak eluting .about.2500) was
enriched for the (+) enantiomer. The elution times are not
meaningful because each sample was injected serially while the
detector was collecting data continuously. Absorbance and CD values
were monitored at 250 nm using Jasco CD-2095 detector.
[0213] The same experimental setup was repeated at 300 MHz and
resulted in no enantiomeric enrichment, as expected (data not
shown). This was because the electric field was rotating out of
phase and not creating the propeller motion.
APPENDIX B
The Chiral Molecule Metaxalone
##STR00001##
[0214] Metaxalone
(Skelaxin.RTM., King Pharmaceuticals)
[0215] 2006 US sales=$480 Million Commercially available from
chemical suppliers
APPENDIX C
Example Chiral Molecules
TABLE-US-00001 [0216] TABLE 1 Drugs Sold Racemic (Example Trade
Name) Generic Name Racemate CAS Prevacid Lansoprazole 103577-45-3
Effexor XR Venlafaxine 93413-69-5 Norvasc Amlodipine 88150-42-9
Protonix Pantoprazole 102625-70-7 Wellbutrin XL Bupropion
34841-39-9 Toprol XL Metoprolol 37350-58-6 Zyrtec (3 forms
Cetirizine 83881-51-0 in Top 200) Coreg Carvedilol 72956-09-3
Adderall XR Amphetamine 300-62-9 Aciphex Rabeprazole 117976-89-3
Concerta (Ritalin) Methylphenidate 113-45-1 Aricept Donepezil
120014-06-4 Zofran Ondansetron 99614-02-5 Provigil Modafinil
68693-11-8 Skelaxin Metaxalone 1665-48-1 Allegra Fexofenadine
83799-24-0 Ditropan XL Oxybutynin 5633-20-5 Astelin Azelastine
58581-89-8 Prilosec Omeprazole 73590-58-6 Coumadin Warfarin
81-81-2
TABLE-US-00002 TABLE 2 Drugs Sold Optically Pure (Example Optically
Trade Name) Generic Name Pure CAS Lipitor Atorvastatin 134523-00-5
Nexium Esomeprazole 161796-78-7 Singulair Montelukast 158966-92-8
Plavix Clopidogrel 113665-84-2 Zocor Simvastatin 79902-63-9 Lexapro
Escitalopram 128196-01-0 Zoloft Sertraline 79617-96-2 Topamax
Topiramate 97240-79-4 Levaquin Levofloxacin 100986-85-4 Valtrex
Valacyclovir 124832-27-5 Zetia Ezetimibe 163222-33-1 Cymbalta
Duloxetine 136434-34-9 Crestor Rosuvastatin 287714-41-4 Diovan
Valsartan 137862-53-4 Nasonex Mometasone 105102-22-5 Flomax
Tamsulosin 106133-20-4 Omnicef Cefdinir 91832-40-5 Altace Ramipril
87333-19-5 Oxycontin Oxycodone 76-42-6 Lyrica Pregabalin
148553-50-8 Spiriva Tiotropium 186691-13-4 Detrol Tolterodine
124937-51-5 Lunesta Eszopiclone 138729-47-2 Synthroid Levothyroxine
51-48-9 Strattera Atomoxetine 83015-26-3 Pravachol Pravastatin
81093-37-0 Pulmicort Budesonide 51333-22-3 Yasmin Drospirenone
67392-87-4 Keppra Levetiracetam 102767-28-2 Flovent Fluticasone
80474-14-2 Prograf Tacrolimus; 104987-11-3 FK-506; Fujimycin
Xalatan Latanoprost 130209-82-4 Cialis Tadalafil 171596-29-5
Reyataz Atazanavir 198904-31-3 Kaletra Lopinavir 192725-17-0 Avelox
Moxifloxacin 354812-41-2 Paxil Paroxetine 61869-08-7 Xopenex
Levalbuterol 18559-94-9 Sustiva Efavirenz 154598-52-4 Nasacort
Triamcinolone 124-94-7 Norvir Ritonavir 155213-67-5 Viread
Tenofovir 147127-20-6 Zyvox Linezolid 165800-03-3 Relpax Eletriptan
143322-58-1 Lumigan Bimatoprost 155206-00-1 Zithromax Azithromycin
83905-01-5 Mirapex Pramipexole 104632-26-0 Avodart Dutasteride
164656-23-9 Casodex Bicalutamide 90357-06-5 Vigamox Moxifloxacin
354812-41-2 Lescol Fluvastatin 93957-54-1 Tussionex Hydrocodone
125-29-1 Sensipar Cinacalcet 226256-56-0 Inderal Propranolol
525-66-6 Prilosec Omeprazole 73590-58-6 Biaxin Clarithromycin
81103-11-9 Nebcin Tobramycin 32986-56-4 Proscar Finasteride
98319-26-7 Kadian Morphine 57-27-2 Codeine Codeine 76-57-3 Travatan
Travoprost 157283-68-6 Dovonex Calcipotriol 112965-21-6 Zomig
Zolmitriptan 139264-17-8 Suboxone Buprenorphine 52485-79-7 Taxol
Paclitaxel 33069-62-4 Dexamethasone Dexamethasone 50-02-2
* * * * *