U.S. patent application number 12/077256 was filed with the patent office on 2008-10-16 for screening of chemical compounds purified from biological sources.
Invention is credited to Peader Cremin, Gary Eldridge, Marilyn Ghanem, Chris Lee, Helene C. Vervoort, Lu Zeng.
Application Number | 20080255001 12/077256 |
Document ID | / |
Family ID | 27381716 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080255001 |
Kind Code |
A1 |
Eldridge; Gary ; et
al. |
October 16, 2008 |
Screening of chemical compounds purified from biological
sources
Abstract
A method of producing a chemical compound library comprises
extracting at least one extract from at least one species of plant;
processing at least one of the extract(s) to remove at least one
type of chemical interference to produce a processed extract;
chromatographically separating the processed extract into a
plurality of chromatographic fractions, each containing an amount
of chemical compounds; determining the amount of chemical compounds
in at least one of the chromatographic fractions; and normalizing
the chromatographic fractions in which the amounts were determined
to produce normalized chromatographic fractions, each such fraction
comprising from about 1 microgram to about 500 micrograms of each
of from one to seven chemical compounds that were present in lower
concentrations in the extract and that each have a log P of from
about -1 to about 5 and a molecular weight less than about 1000
Daltons; thereby to produce a chemical compound library from at
least one species of plant.
Inventors: |
Eldridge; Gary; (San Diego,
CA) ; Zeng; Lu; (San Diego, CA) ; Cremin;
Peader; (San Diego, CA) ; Lee; Chris; (Yorba
Linda, CA) ; Vervoort; Helene C.; (Chelmsford,
MA) ; Ghanem; Marilyn; (St. Louis, MO) |
Correspondence
Address: |
GALLOP, JOHNSON & NEUMAN, L.C.
101 S. HANLEY, SUITE 1600
ST. LOUIS
MO
63105
US
|
Family ID: |
27381716 |
Appl. No.: |
12/077256 |
Filed: |
March 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11358815 |
Feb 21, 2006 |
7367933 |
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12077256 |
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10115741 |
Apr 3, 2002 |
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11358815 |
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60280739 |
Apr 3, 2001 |
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60328788 |
Oct 15, 2001 |
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Current U.S.
Class: |
506/12 ;
506/23 |
Current CPC
Class: |
C40B 50/08 20130101;
C40B 40/00 20130101; B01J 2219/0072 20130101; B01J 2219/00759
20130101; B01J 2219/00707 20130101; C40B 40/04 20130101; B01J
2219/00315 20130101; G01N 2500/04 20130101 |
Class at
Publication: |
506/12 ;
506/23 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C40B 50/00 20060101 C40B050/00 |
Claims
1. A method of identifying biologically active chemical compounds
comprising producing a chemical compound library comprising the
steps of: (a) extracting at least one extract from at least one
species of plant; (b) processing at least one of the at least one
extract from step (a) to remove at least one type of chemical
interference to produce a processed extract; (c)
chromatographically separating the processed extract from step (b)
into a plurality of chromatographic fractions, each containing an
amount of chemical compounds; (d) determining the amount of
chemical compounds in at least one of the chromatographic fractions
from step (c); and (e) normalizing the chromatographic fractions in
which the amounts were determined in step (d) to produce normalized
chromatographic fractions, each such normalized chromatographic
fraction comprising from about 1 microgram to about 500 micrograms
of each of from about one to seven chemical compounds that were
present in lower concentrations in the extract and that each have a
log P of from about -1 to about 5 and a molecular weight less than
about 1000 Daltons; (f) thereby to produce a chemical compound
library from at least one species of plant, the chemical compound
library comprising the normalized chromatographic fractions, each
such normalized chromatographic fraction comprising from about 1
microgram to about 500 micrograms of each of the from about one to
seven chemical compounds, a majority of which have a log P of from
about -1 to about 5 and a molecular weight less than about 1000
Daltons; and screening the chemical compound library for biological
activity of said chemical compounds.
2. A method as set forth in claim 1 wherein the at least one
extract in step (a) is extracted from a plurality of species of
plant.
3. A method as set forth in claim 2, wherein the at least one
extract in step (a) is extracted from the plurality of species of
plant in an extraction with an organic solvent.
4. A method as set forth in claim 2, wherein the at least one
extract in step (a) is extracted from the plurality of species of
plant in a multiple step extraction comprising a first extraction
step carried out with a first organic solvent and thereafter a
second extraction step whereby insoluble material is extracted by
use of a mixture of a second organic solvent, which may or may not
be the same as the first organic solvent, and water.
5. A method as set forth in claim 4, wherein the first organic
solvent is about 50% by weight ethanol and about 50% weight ethyl
acetate and wherein the mixture of the second organic solvent and
water is about 70% by weight methanol and about 30% by weight
water.
6. A method as set forth in claim 1, wherein in step (b), the at
least one of the at least one extract from step (a) is processed to
remove a tetramer or greater of a polyphenolic compound.
7. A method as set forth in claim 1, wherein in step (b), the at
least one of the at least one extract from step (a) is processed to
remove a compound with a molecular weight of greater than 1000
daltons.
8. A method as set forth in claim 1, wherein the at least one
extract is processed in step (b) by flash chromatography to remove
the at least one type of chemical interference.
9. A method as set forth in claim 1, wherein the at least one
extract is processed in step (b) by use of a size exclusion filter
to remove the at least one type of chemical interference.
10. A method as set forth in claim 1, wherein the at least one
extract is processed in step (b) by use of a polyamide column to
remove the at least one type of chemical interference.
11. A method as set forth in claim 1, wherein in step (b), the at
least one of the at least one extract from step (a) is processed to
remove a compound with a with a log P of greater than about 5 or
less than -1.
12. A method as set forth in claim 1, further comprising the step
of providing pre-determined analytical data comprising molecular
ions and chromatographic elution conditions for the at least one
chromatographic fraction.
13. A method as set forth in claim 1, further comprising the step
of providing NMR spectra for the at least one chromatographic
fraction.
14. A method as set forth in claim 1, further comprising the step
of providing MS/MS fragmentation patterns for the at least one
chromatographic fraction.
15. A method as set forth in claim 1, further comprising the step
of providing LC-ELSD-MS data sufficient to identify the amounts of
each of said chemical compounds for the at least one
chromatographic fraction.
16. A method as set forth in claim 1, wherein said chromatographic
fractions are produced by eluting one to five chemical compounds
with related physical properties from a reverse phase
chromatography column with a flow rate of more than 0.48 and less
than 0.80 column volumes per minute of a solvent system and where
an acetonitrile concentration in the solvent system increases by
more than 0.008% per second and less than 0.035% per second.
17. A method as set forth in claim 1, wherein each of the
normalized chromatographic fractions of step (e) has associated
therewith a retention time, molecular weight, mass and number of
compounds which are recorded in a database.
18. A method as set forth in claim 1, further comprising the step
of preparing a sublibrary of chemical compounds in the library in
which the chemical compounds in the sublibrary have similar
predetermined molecular ions.
19. A method as set forth in claim 1, further comprising the step
of preparing a sublibrary of chemical compounds in the library in
which the chemical compounds in the sublibrary have similar
predetermined log Ps.
20. A method as set forth in claim 1, wherein step (c) of said
method is carried out with a reverse phase chromatography column
having a flow rate of more than 0.48 and less than 0.80 column
volumes per minute of a solvent system, wherein an acetonitrile
concentration in said solvent system increases at more than 0.008%
per second and less than 0.035% per second; and wherein at least
fifteen chromatographic fractions are collected.
21. A method as set forth in claim 1 wherein the chemical compound
library comprises at least about 100 compounds from one.
22. A method as set forth in claim 1 wherein the chemical compound
library comprises at least two or more isomeric forms of a chemical
compound.
23. A method as set forth in claim 1, wherein the chemical compound
library is screened by testing the chromatographic fractions in
parallel using at least one biological assay.
24. A method as set forth in claim 23, wherein the at least one
biological assay detects efficacy in treatment of a disease or in
control of biological pests.
25. A method as set forth in claim 1, wherein the concentration of
each of the chemical compounds in the chromatographic fractions
tested is at least one micromolar.
26. A method as set forth in claim 21 wherein the chemical compound
library contains at least one triterpene compound, one lignan
compound, one flavonoid compound and one alkaloid compound.
27. A method as set forth in claim 21 wherein the chemical compound
library comprises at least about 200 compounds from one plant
species.
28. A method as set forth in claim 1 wherein the chemical compound
library comprises at least about 80 normalized chromatographic
fractions.
29. A method as set forth in claim 28 wherein the chemical compound
library comprises at least about 100 normalized chromatographic
fractions.
30. A method as set forth in claim 29 wherein the chemical compound
library comprises at least about 150 normalized chromatographic
fractions.
31. A method as set forth in claim 1 wherein each of the compounds
has a log P of from about -1 to about 5 and a molecular weight less
than about 1000 Daltons.
32. A method of identifying biologically active chemical compounds
comprising screening a chemical compound library for biological
activity of said chemical compounds, the chemical compound library
having been produced by a process comprising the steps of: (a)
extracting at least one extract from at least one species of plant;
(b) processing at least one of the at least one extract from step
(a) to remove at least one type of chemical interference to produce
a processed extract; (c) chromatographically separating the
processed extract from step (b) into a plurality of chromatographic
fractions, each containing an amount of chemical compounds; (d)
determining the amount of chemical compounds in at least one of the
chromatographic fractions from step (c); and (e) normalizing the
chromatographic fractions in which the amounts were determined in
step (d) to produce normalized chromatographic fractions, each such
normalized chromatographic fraction comprising from about 1
microgram to about 500 micrograms of each of from about one to
seven chemical compounds that were present in lower concentrations
in the extract and that each have a log P of from about -1 to about
5 and a molecular weight less than about 1000 Daltons; (f) thereby
to produce a chemical compound library from at least one species of
plant, the chemical compound library comprising the normalized
chromatographic fractions, each such normalized chromatographic
fraction comprising from about 1 microgram to about 500 micrograms
of each of the from about one to seven chemical compounds, a
majority of which have a log P of from about -1 to about 5 and a
molecular weight less than about 1000 Daltons.
33. A method as set forth in claim 32 wherein the at least one
extract in step (a) is extracted from a plurality of species of
plant.
34. A method as set forth in claim 33, wherein the at least one
extract in step (a) is extracted from the plurality of species of
plant in an extraction with an organic solvent.
35. A method as set forth in claim 33, wherein the at least one
extract in step (a) is extracted from the plurality of species of
plant in a multiple step extraction comprising a first extraction
step carried out with a first organic solvent and thereafter a
second extraction step whereby insoluble material is extracted by
use of a mixture of a second organic solvent, which may or may not
be the same as the first organic solvent, and water.
36. A method as set forth in claim 35, wherein the first organic
solvent is about 50% by weight ethanol and about 50% weight ethyl
acetate and wherein the mixture of the second organic solvent and
water is about 70% by weight methanol and about 30% by weight
water.
37. A method as set forth in claim 32, wherein in step (b), the at
least one of the at least one extract from step (a) is processed to
remove a tetramer or greater of a polyphenolic compound.
38. A method as set forth in claim 32, wherein in step (b), the at
least one of the at least one extract from step (a) is processed to
remove a compound with a molecular weight of greater than 1000
daltons.
39. A method as set forth in claim 32, wherein the at least one
extract is processed in step (b) by flash chromatography to remove
the at least one type of chemical interference.
40. A method as set forth in claim 32, wherein the at least one
extract is processed in step (b) by use of a size exclusion filter
to remove the at least one type of chemical interference.
41. A method as set forth in claim 32, wherein the at least one
extract is processed in step (b) by use of a polyamide column to
remove the at least one type of chemical interference.
42. A method as set forth in claim 32, wherein in step (b) the at
least one of the at least one extract from step (a) is processed to
remove a compound with a with a log P of greater than about 5 or
less than -1.
43. A method as set forth in claim 32, wherein the chemical
compound library comprises at least about 100 compounds from one
plant species.
44. A method as set forth in claim 32 wherein the chemical compound
library comprises at least two or more isomeric forms of a chemical
compound.
45. A method as set forth in claim 32, wherein the chemical
compound library is screened by testing the chromatographic
fractions in parallel using at least one biological assay.
46. A method as set forth in claim 45, wherein the at least one
biological assay detects efficacy in treatment of a disease or in
control of biological pests.
47. A method as set forth in claim 32, wherein the concentration of
each of the chemical compounds in the chromatographic fractions
tested is at least one micromolar.
48. A method as set forth in claim 43 wherein the chemical compound
library contains at least one triterpene compound, one lignan
compound, one flavonoid compound and one alkaloid compound.
49. A method as set forth in claim 43 wherein the chemical compound
library comprises at least about 200 compounds from one plant
species.
50. A method as set forth in claim 32 wherein the chemical compound
library comprises at least about 80 normalized chromatographic
fractions.
51. A method as set forth in claim 50 wherein the chemical compound
library comprises at least about 100 normalized chromatographic
fractions.
52. A method as set forth in claim 51 wherein the chemical compound
library comprises at least about 150 normalized chromatographic
fractions.
53. A method as set forth in claim 32 wherein each of the compounds
has a log P of from about -1 to about 5 and a molecular weight less
than about 1000 Daltons.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This is a divisional of pending U.S. Ser. No. 10/115,741,
filed Apr. 3, 2002, which in turn claims the benefit of U.S.
provisional application Ser. Nos. 60/280,739, filed Apr. 3, 2001,
and 60/328,788, filed Oct. 15, 2001; and claims priority to PCT
application Serial No. WO 99/20291, filed Oct. 23, 1998, which in
turn claims priority to U.S. application Ser. No. 08/956,600, filed
Oct. 23, 1997; and is a continuation-in-part of PCT application,
Serial No. WO 01/33193, filed Nov. 2, 2000, which in turn claims
the benefits of U.S. provisional application Ser. Nos. 60/163,070,
filed Nov. 2, 1999, 60/189,872, filed Mar. 16, 2000, and
60/209,636, filed Jun. 6, 2000.
FIELD OF THE INVENTION
[0002] The present invention is directed to screening of chemical
compounds purified from plants or other biological materials for
testing in biological assays, and more particularly to such
screening for high throughput testing of chemical compounds present
in biological sources in low concentrations.
BACKGROUND
[0003] It is well known in the art of discovering novel chemical
compounds with therapeutic effects that plants have yielded some of
the most important molecules in history. Civilizations around the
globe have exploited the medicinal benefits of plants for
millennia. Today, private, public, and government institutions
devote extensive resources searching for molecules in plants that
may have a potential economic and humanitarian impact.
Technological advances in laboratory automation, biochemistry, and
molecular biology enable us to currently screen hundreds of
thousands of molecules for biological activity every day.
[0004] Cragg, et al., in "The search for new pharmaceutical crops:
Drug discovery and development at the National Cancer Institute,"
161-167, describe the extensive natural library testing program of
the National Cancer Institute (NCI) and the methods used to prepare
plant extracts for screening. Cardellina II, et al., in J. Nat.
Prod., 56(7), 1123-1129 (1993), describe the screening program of
the NCI and also specifically discuss the chemical interferences
ubiquitous within plants and current techniques used to remove
these chemicals before screening or after screening. Turner, in J.
Ethnopharm., 51, 39-44 (1996), describes screening plants at a
large pharmaceutical company. Borris, in J. Ethnopharm., 51, 29-38
(1996), describes the increased complexities that come with
screening plant extracts using a competitive screening program that
requires a structured approach and the latest scientific
techniques. Shu, in J. Nat. Prod., 61, 1053-1071 (1998), promotes
the value of novel chemicals that have been isolated from plants,
and lists points a screening laboratory must achieve to improve the
success rate when testing plant extracts. The points on the list
are not easily accomplished and include challenges such as making a
screen suitable for natural libraries, removing all interferences,
and accelerating dereplication.
[0005] Preparing plant extracts for screening has always been
recognized as laborious, and published literature suggests that the
method of preparation is more important than previously and
currently understood. Plants have numerous ubiquitous compounds
that may mask an effect or interfere with the mechanism of action
of a biological assay. Ingkaninan, J. Nat. Prod., 62(6), 912-914
(1999), Kato, J. Steroid Biochem., 34(1), 219-227 (1989), Vallette
et al., in Endocrin., 129(3), 1363-1369 (1991), and Kang et al, in
Biochem J., 303, 795-802 (1994), have reported that fatty acids,
phospholipids, and tri-, di-, and monoglycerides cause
noncompetitive or mixed noncompetitive inhibition on some receptors
or modify the structure or confirmation of receptors in cell-based
assays. Numerous plant solvent extracts have high molecular weight
compounds that make up greater than 70% of the mass of the extract
and that never could be approved drugs. Numerous laboratories do
not remove or have not removed these compounds before screening.
This may result in false positives or false negatives during
subsequent biological assays. Tan, et al., in J. Nat. Prod., 54(1),
143-154 (1991), Cardellina II, et al., in J. Nat. Prod., 56(7),
1123-1129 (1993), Claeson, et al., in J. Nat. Prod., 61(1), 77-81
(1998), Lee, et al., in J. Nat. Prod., 61(11), 1407-1409 (1998),
and Patil, et al., in J. Nat. Prod., 60(3), 306-308 (1997),
describe false positives that may be attributed to polyphenols and
tannins. Some laboratories remove these compounds before screening,
while others remove these compounds only after a potential false
positive has occurred, believing that these compounds cannot cause
a false negative. Phillipson, in J. Pharm. Pharmacol. 51:493-503
(1999), has suggested further that partially purified plant
extracts without common metabolites may prove attractive to
screening laboratories.
[0006] A general mantra in preparing plant extracts states, "it is
not what you miss, but what you hit." This approach has led
laboratories to put ease of preparation and number of plant
extracts prepared and screened ahead of a scientifically based
approach for success. Laboratories typically prepare one to three
extracts per plant for screening. These extracts may contain one
hundred to thousands of chemical compounds per extract.
[0007] Because the collision frequency and proper orientation of a
ligand and its receptor play a role in binding, screening plant
extracts with numerous compounds may interfere with the detection
of potential biological effects. In addition, increased
dipole-dipole interactions, hydrogen bonding, or steric effects
that exist in physiological conditions could also contribute to the
disruption of ligand binding. Haberlein, in Planta Medica,
62(3):227-31 (1996), indicate that two different concentrations of
the same ethanolic plant extract cause positive and negative
allosteric regulation of a GABA receptor. In contrast to accepted
principles, interferences may result in false positives as well as
false negatives. Menzies, in Eur. J. Pharm., 350(1), 101-108
(1998), suggests that the bioactivity of a known compound in a
plant extract is not observed in an opioid assay because of an
interfering compound canceling out its activity. Phillipson, in J.
Pharm. Pharmacol. 51:493503 (1999), states that the activity of an
isolated compound is not always directly comparable to the plant
extract from which it was isolated. These suggestions, empirical
data, and hypotheses show that many variables exist in the
screening process. Because the process of scientific investigation
and discovery should reduce the number of variables and operate in
a closed system, the removal of all potential interferences from a
plant extract before entering a biological screen and further
separation of the drug-like chemical compounds to further reduce
interference and enable each chemical compound to be tested at its
detectable screening concentration are essential.
SUMMARY OF THE INVENTION
[0008] In one embodiment, the present invention is directed to a
method of producing a chemical compound library comprising
extracting at least one extract from at least one species of plant;
processing at least one of the extract(s) to remove at least one
type of chemical interference to produce a processed extract;
chromatographically separating the processed extract into a
plurality of chromatographic fractions, each containing an amount
of chemical compounds; determining the amount of chemical compounds
in at least one of the chromatographic fractions; and normalizing
the chromatographic fractions in which the amounts were determined
to produce normalized chromatographic fractions, each such fraction
comprising from about 1 microgram to about 500 micrograms of each
of from one to seven chemical compounds that were present in lower
concentrations in the extract and that each have a log P of from
about -1 to about 5 and a molecular weight less than about 1000
Daltons; thereby to produce a chemical compound library from at
least one species of plant.
[0009] In another embodiment, the present invention is directed to
a chemical compound library comprising a plurality of
chromatographic fractions from at least one biological source. The
fractions are substantially free of compounds that interfere with
biological assays. The majority of the chromatographic fractions
comprising a normalized quantity of from about 1 microgram to about
500 micrograms of seven or fewer chemical compounds. The majority
of said chemical compounds having log P of from about -1 to about 5
and a molecular weight less than about 6,000 Daltons.
[0010] The present invention is further directed to a subset of
such library comprising chemical compounds having similar
predetermined molecular ions.
[0011] The present invention is further directed to a method of
preparing a sublibrary of such library wherein the fractions have
associated data comprising at least one of molecular ion data,
chromatographic elution data, NMR spectra, MS/MS fragmentation
patterns, comprising selecting fractions having at least one set of
similar data.
[0012] The present invention is also directed to a method of
preparing a diverse chemical compound library comprising providing
such library wherein each of the chromatographic fractions have
associated data comprising at least one of molecular ion data for
said chemical compounds, chromatographic elution data for said
fractions, NMR spectra for said fractions and MS/MS fragmentation
patterns for the chemical compounds selecting fractions having at
least one characteristic selected from
[0013] (a) similar molecular ions;
[0014] (b) similar chromatographic elution data;
[0015] (c) similar log Ps; and
[0016] (d) a plurality of biological sources.
[0017] The present invention is further directed to a method of
identifying biologically active chemical compounds comprising
screening such library for biological activity of said chemical
compounds.
[0018] The present invention is also directed to a chemical
compound library comprising: a plurality of arrayed chromatographic
fractions derived from one or more biological sources, the majority
of the fractions comprising seven or fewer individual
nonproteinaceous chemical compounds, and each of the isolates being
free of compounds that interfere with biological testing of said
individual compounds. In this embodiment of the invention, each of
said individual compounds is present in a normalized quantity and
has a log P suitable for biological testing of said individual
compound, the normalized quantity being sufficient to prepare a
sample from said isolate having a concentration of each of the
individual compounds suitable for biological testing.
[0019] The present invention is also directed to a method for
preparing an array of compounds of normalized concentrations from a
natural product comprising a mixture of compounds of various
concentrations. The method comprises normalizing concentrations of
the compounds; and producing an array of samples of the compounds
of normalized concentrations. The invention is also directed to
that array itself.
[0020] The present invention is further directed to a method for
screening a natural product comprising a mixture of compounds of
various concentrations. The method comprises normalizing
concentrations of the compounds; producing an array of samples of
the compounds of normalized concentrations; and screening the
array.
[0021] Further objectives and advantages will become apparent from
a consideration of the description, drawings, and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The invention is better understood by reading the following
detailed description with reference to the accompanying figures, in
which like reference numerals refer to like elements throughout,
and in which:
[0023] FIG. 1 is a schematic representation of liquid
extraction;
[0024] FIG. 2 is a schematic representation of the removal of
interferences;
[0025] FIG. 3 is a picture of an example of a suitable liquid
handling system;
[0026] FIG. 4 is a schematic representation of solid phase
extraction as a first chromatography step;
[0027] FIG. 5 is a picture of a Robotic Manipulator Arm placing an
array of 96 solid phase extraction columns and a vacuum plate over
a microtiter plate in a preferred embodiment of the invention;
[0028] FIG. 6 is a diagram showing the fraction collector system
that may comprise part of the solid phase extraction apparatus or
the purification apparatus, and explaining the spectroscopic
control of fraction collection, including monitoring of multiple
wavelengths and setting of thresholds, and the presence of a diode
array spectrum for each pure sample in the wells of the collection
plate;
[0029] FIG. 7 is a diagram of the contents in wells of a collection
plate containing isolates of the invention, wherein the contents of
three randomly selected wells is described;
[0030] FIG. 8 is a schematic representation of a second
chromatography or purification step;
[0031] FIG. 9 is a schematic representation of fractions of
chemical compounds in a microtiter plate with mass spectrum and
evaporative light scattering chromatogram of an individual fraction
within the collection of chemical compounds;
[0032] FIG. 10 illustrates spectra of molecular ions and
chromatograms of elution conditions of two different isomeric
chemical compounds;
[0033] FIG. 11 illustrates chromatograms of elution conditions and
MS/MS fragmentation patterns of two different isomeric chemical
compounds;
[0034] FIG. 12 is a 1D proton NMR spectrum of two different
isomeric chemical compounds;
[0035] FIG. 13 is a schematic representation of a data table used
to compare molecular ions and chromatographic elution conditions of
the entire collection of chemical compounds;
[0036] FIG. 14 is a schematic presentation of the method for
high-throughput natural product drug discovery;
[0037] FIG. 15 is a group of chromatograms, each yielding 40
natural product library fractions; (A) to (D) showing the channels
1 to 4 in one parallel four-channel preparative HPLC run;
[0038] FIG. 16 is a series of spectra of eight LC-MS chromatograms
of samples (S001208-17 to 24) by a single parallel LC-MS run from
eight natural product library fractions;
[0039] FIG. 17 is a spectrum showing that a bioactive library
fraction containing 4 components was separated into pure compounds
by adopting the preparative HPLC condition as described in Example
3, below; 4A: partial TIC chromatogram of 4 components; 4B-E:
partial LC-ELDS chromatograms of each single compound after
purification;
[0040] FIG. 18 is a schematic representation of the 5 .mu.L
microcoil flow probe NMR configuration with syringe pump and
injection valve for low microgram sample handling;
[0041] FIG. 19 is a graph of the Taxus library that was made using
a high-throughput method of the present invention showing weights
of each well in mg; and
[0042] FIG. 20 is a 1H NMR spectrum of 50 .mu.g paclitaxel in 3
.mu.L CD3OD acquired by 5 .mu.l microcoil flow probe on a 600 MHz
NMR spectrometer.
DETAILED DESCRIPTION OF THE INVENTION
[0043] This application is related to PCT application
PCT/USOO/30195, published as WO 01/33193, and U.S. patent
application Ser. Nos. 60/280,739 and 60/328,788, all of which are
incorporated by reference in their entirety in the present
specification.
[0044] In accordance with the present invention, it has been
discovered that by removing from a mixture of compounds, such as a
biological sample, certain components that tend to be present in
relatively high concentrations but are of little interest and
normalizing the concentration of compounds in the mixture that are
of potential interest, and normalizing the concentrations of those
compounds of potential interest, an array of samples of the
normalized compounds can be produced that is suitable for
high-throughput screening of the array for identification of
activity of compounds of potential interest that are otherwise
missed by such screening techniques either due to masking by the
components of little interest or the low ambient concentrations of
the compounds of potential interest. Remarkably, such techniques
have been discovered to permit the detection of desirable
properties for thousands of compounds that are missed by
conventional screening techniques.
[0045] Accordingly, the present invention allows hundreds of
chemical compounds per biological source to be tested in biological
systems at optimal screening concentrations. Prior to the present
invention, this remarkable advantage was not attainable. Because
extracts from biological sources being tested typically have
constituted approximately 80% by weight of non-drug-like chemical
compounds, conventional techniques have allowed only the major
drug-like chemical compounds from the extract to be tested at
screening concentrations familiar to those skilled in the art of
biological screening for the discovery of new medicines or
agricultural products.
[0046] Thus, the present invention solves problems inherent in the
prior art of preparing compounds from biological sources for
biological screening and, in contrast to the prior art, may be used
to produce from a biological source large numbers of
chromatographic fractions that contain chemical compounds of
potential usefulness, but that are predominately free of
non-drug-like chemical compounds. The fractions not only may
include scientifically useful information, but are organized in a
logical manner. Moreover, by identifying duplicate chemical
compounds in biological collections, thus removing the possibility
of discovering the same chemical compound from a different
biological source, the invention also provides further advantages
that were not previously appreciated. Results obtained using the
inventions also provide for rapid isolation and structural
elucidation of therapeutically useful compounds.
[0047] This invention also achieves a goal previously thought to be
unattainable, that of harnessing plant biodiversity with the
high-throughput approaches of combinatorial chemistry and automated
screening. Combinatorial chemists have typically viewed plant
material as too complex to use as starting material for the
structural variation that is typical in generating combinatorial
libraries. By providing an ordered collection of relatively pure
phytochemicals, the invention permits the study of a vast new array
of phytochemical structures and their evaluation for biological
activity such as in treating diseases or providing useful
agricultural traits. The present invention has aspects in the
mature art of collections of chemical compounds from biological
sources and other aspects in the crowded field of high throughput
screening, none of which aspects had been combined before.
[0048] The separation method of the present invention, therefore,
produces a library that contains a much greater number of purified,
relevant drug-like chemical compounds per plant for biological
screening than currently used by those skilled in the art. The
library allows hundreds of chemical compounds per plant to be
tested in parallel in biological assays at optimal screening
concentrations and is particularly amenable for use in the
high-throughput screening of chemical compounds purified from
plants in biological assays.
[0049] This invention is contrary to the teachings of the prior art
and differs from the prior art in several modifications neither
previously known nor suggested. For example, prior art plant
extraction methods have generally relied on liquid extraction of
plant biomass to provide a single extract or a few (generally less
than four) extracts into different solvents. The general belief is
that a single extract or small number of extracts is adequate for
biological assays because of the sensitivity of bioassays, and, in
addition, it is easy and routine to process plant material in this
fashion. The National Cancer Institute and many commercial
pharmaceutical research laboratories follow this approach. There
has traditionally been no motivation to provide further
fractionation of the extracts before screening, the philosophy
being that if an extract provides a hit, bio-assay guided
fractionation can be performed thereafter. To the extent more than
one extract was desired, the effort has been to accomplish this
goal by selective liquid extraction (e.g. low polarity first, then
increasingly high polarity extraction solvents), in the belief that
this would provide different populations of phytochemicals.
[0050] The prior art has avoided pre-screening chromatography
presumably because it was seen as complicated and unnecessary.
Counter to the accepted view, it has been found that the prior
extraction-based approach to providing fractions of phytochemicals
is flawed because concentrations of most chemical compounds in such
fractions are below optimal screening concentrations and are not
present in normalized to quantities. Moreover, numerous
non-drug-like chemical compounds in such fractions reduce the
effective concentrations of drug-like chemical compounds and
interfere with the activities of the drug-like compounds.
[0051] The library of the present invention, therefore, provides
advantages that were not enjoyed in the prior art. For example, the
library of the present invention can provide a comprehensive
purified collection of the potentially biologically-active
phytochemicals from plants, without significant concentrations of
non-selectively interfering chemical compounds from the plants,
thus resulting in a population of the potential, selective
biologically-active phytochemicals susceptible to testing and
screening by conventional techniques. The separation of the
chemical compounds in the collection surprisingly according to the
present invention reduces problems in high-throughput screening,
such as masking of biological activities of one chemical by another
and allosteric regulation of receptors by multiple components. The
compounds are present in the library of the present invention in
normalized amounts, and have a tight range of molecular weights,
yielding a tight range of concentrations suitable for assays. The
association with data allows for rapid extraction of meaningful
date from the biological assays. The library of the present
invention contains chemical compounds having a higher yield of
phytochemicals of interest as compared to conventional methods,
both in terms of chemical diversity and higher total mass.
[0052] Also, the library of the present invention solves problems
encountered in prior art methods with solubilities of
phytochemicals, because the solubilities of phytochemicals are
revealed, facilitating the design of the screening system (e.g.
identification and selection of solvents to use) and greatly
accelerates preparation and purification of a compound that
provides a hit. That is, the invention provides a solubility and
fractionation profile for each fraction that can be used both as an
initial indicator of the chemical nature of the isolated
chromatographic fraction, can help as a first step in elucidating
the structure of the compound, and the fractionation data can be
used to refractionate a larger quantity of the material for further
analysis in the event it becomes a lead by virtue of a positive
result in a biological assay. The small scale fractionation and
chromatography producing a small mass chromatographic fraction can
be readily scaled up with more plant material and a larger column
to provide the same chromatographic fraction in larger yield,
thereby shortening the time needed to design a purification
procedure and hasten structure elucidation.
[0053] This invention also satisfies a long felt need for a
biologically-suitable, efficient way to test the potentially
selective biologically-active chemical compounds from a wide
variety of different types of biomass with associated data useful
in further research on the chemical compounds.
[0054] In summary, the present invention provides an array of
chromatographic fractions from biological sources with properties
selected for efficient high throughput screening. The selected
fraction properties include fractions that, for example: [0055] a
contain predominately compounds having low molecular weights in a
range where biological activity is likely to be found. This range
may be, for example, molecular weights less than 6000, 3000, 1,000,
600 or even 300 daltons, as desired; [0056] are free of--or at
least free of significant amounts of--compounds likely to interfere
with biological assays by nonspecific binding, such as compounds
with molecular weights greater than about 3000 or 6000 daltons, for
instance, tannins and tetramers or greater of phenolics, and
compounds having log P's greater than about 5 or 6 or less than
about -1 or -2; [0057] contain a limited number of compounds, for
example, less than about 5 or 7 compounds; [0058] contain a known,
normalized mass quantity of each chemical compound suitable for
preparing a sample that may be used for biological screening (which
quantity may be between 1 microgram and about 100 to 500 micrograms
to be suitable for presently available screening methodologies);
[0059] fall within a predetermined range of molecular weights
(typically about 300 to about 1000 daltons, such as from about 250
to about 600 daltons), so that a normalized mass gives a normalized
molar concentration; and [0060] may have associated data used to
identify specific compounds and/or replicate the isolation to allow
accumulation of additional fractions with the same
characteristics.
[0061] The present invention thus provides a chemical compound
library comprising of a plurality of chromatographic fractions,
wherein said chromatographic fractions contain from about 1
microgram to 100 micrograms of primarily of one to five chemical
compounds; wherein said chemical compounds have log Ps of greater
than -1 and less than 5 and molecular weights less than 3,000
Daltons; wherein said library comprises of said chemical compounds
produced from at least fifty different biological sources; and
wherein said library contains at least 200 (such as from about 200
to about 700) of said different chemical compounds from each
different biological source.
[0062] The library may comprise at least 80, 100, or 150
chromatographic fractions from each different biological source and
each chemical compound within the fractions may have a molecular
weight of less than 10,000 Daltons. The library also may include
associated data sufficient to identify the relative amounts of said
chemical compounds in said chromatographic fractions and the
associated data may be used to increase the diversity of chemical
structures of said chemical compounds, and for analysis to select
the biological sources for the library. In preferred embodiments,
the chromatographic fractions are free of tetramers or greater of
polyphenolics, and the weight of the chemical compounds of each
chromatographic fraction is approximately 100 micrograms to about
500 micrograms of solid material of primarily of one to five
chemical compounds.
[0063] The following definitions apply to the indicated terms as
used in the present specification:
DEFINITIONS
[0064] As used herein, the term "natural product" means a product
obtained from biological sources such as plants and animals. A
natural product may be single compound or a mixture of compounds
derived from biological sources.
[0065] As used herein, the terms "screening" and "biological
screening" means any method used to detect biological activity of a
sample. These terms include in vivo and in vitro testing, including
bioassays.
[0066] As used herein, the terms "chemical interference" and
"interference" generally refer to one or more chemicals or other
material present in natural products that may give rise to an
inaccurate result during screening. An inaccurate result may be a
false positive or a false negative.
[0067] As used herein, the term "hit" is a positive result in
screening given by a compound, fraction or other sample.
[0068] As used herein, the term "high throughput screening" means a
screening method able to test in vitro a relatively large number of
samples in a relatively short period of time to detect samples that
exhibit a biological effect. Generally, high throughput systems are
automated and require little human intervention.
[0069] As used herein, the term "progeny" refers to compounds
derived from a common source (e.g. biological resource). Progeny
may be derived from the original source through one or more
purification steps.
[0070] As used herein, the term "fraction" means any sample that is
part of a larger whole.
[0071] As used herein, the term "isolate" means a chromatographic
fraction containing a mixture of one to five drug-like compounds.
It is a final sample suitable for testing. An isolate may be
derived by one or many purification steps. A set of isolates
arranged in an array of the invention may comprise progeny having a
common ancestor source. All isolates are fractions. Fractions may
be used as final samples and when used as such may be referred to
as isolates.
[0072] As used herein, the terms "automation" and "automated
system" refer to a means or apparatus that functions with a minimum
of human intervention. Usually automation requires computer
control. A system is considered automated even though it requires
some human control, input and/or programming.
[0073] As used herein, the term "chromatography" specifically
includes normal and reverse phase systems, solid phase extraction
and other methods conducted using a column, including high pressure
systems and vacuum systems. The definition of chromatography as
used herein is not otherwise limited, but has the general meaning
that is well understood in the art.
[0074] As used herein, the term "identifying system" is any system
capable of correlating a physical entity and information or data
related to that physical data. An identifying system may have a
physical manifestation, such as a bar code or may be only data
stored electronically. Thus, while an identifying system must track
a physical entity, it need not have a component physically attached
to the entity.
[0075] As used herein, the term "assay concentration" refers to the
amount of a compound in a given volume of sample that has been
prepared for a biological assay.
[0076] As used herein, the term "normalized assay concentrations"
refers to concentrations of compounds that are orders of
magnitude--typically, from tens to thousands of times--larger than
the original concentration of a major component in the extract from
a biological source material when a crude extract of the biological
source materials is prepared for biological testing, but within an
order of magnitude of each of the concentrations of the other major
components in the isolate prepared from the extract.
[0077] As used herein, the term "major component" refers to any of
the most prevalent of the potentially selectively bio-active
compounds in a crude extract, at a concentration typically
sufficient to exhibit a primary biological activity. A typical
plant, for example, may contain about 10-20 major components,
typically 12-15 major components, using conventional screening
assays.
[0078] As used herein, the term "detectable compound" refers to a
compound that is detectable by mass spectrometry or evaporative
light scattering detection.
[0079] As used herein, the term "potentially selective bio-active
compounds" refers to such compounds as described above and
elsewhere herein as having the possibility for usefulness as a drug
or other agent that induces a biological response, and typically
includes certain classes of organic compounds in the catalog of
isolates known to those of skill in the art. The classes are broad,
and may for example include some or all of the following, or
others:
TABLE-US-00001 Acetylene Alkaloid Alkaloid glycoside Benzofuran
Benzophenone Cardenolide Chalcone Courmarin Cyclic peptide
Diketopiperazine Diterpene Flavan Flavone Flavonoid Flavinoid
Alkaloid Furanoquinoline Alkaloid Geranylstilbene Hydroquinone
Indolequinone Isoflavanone Isoflavanoid Isomalabaricane diterpene
Lactone Lignan Macrolide Monoterpene Napthoquinone Phenyl Glycoside
Pyranocoumarin Quassinoid Quinoline Sesquiterpene Sesquiterpene
Quinone Steroid Steroidal Saponin Triterpene
[0080] As used herein, an "array" is a set of fractions or isolates
arranged in a particular format, such as a group of chromatographic
fractions from a single biological source, a group of fractions
from different biological sources, a group of fractions selected
for having similar or diverse molecular ion, partition coefficient
(log P), and so forth. An array is typically a microtiter plate
useful for high throughput screening, or an intermediate format
useful for preparing such a plate. At least a useful proportion of
fractions in an array include chemical compounds in an amount
suitable for high throughput screening. For end-user arrays, all
the fractions contain "normalized" amounts of compounds, as will be
defined below.
[0081] As used herein, the term "suitable for high throughput
screening" means an amount, number and format that takes into
account automated screening methodology and/or economics. For
example, using currently available methodology, a suitable amount
of each compound may comprise at least about one microgram per
assay, and/or an amount which is suitable to prepare samples of
about one micromolar solution. Thus, a well with 20 micrograms of
compound per well may be sufficient to conduct about 20 biological
assays. It is understood that, as technology changes, the amount of
a chemical compound corresponding to a suitable amount may increase
or decrease. The economics involved require having a suitable
diversity of compounds, and large enough quantity of material
available to run a number of biological tests so as to justify the
cost of the isolation and disposable materials on which the
fractions are dispersed and the load factor, for running a
biological assay.
[0082] As used herein, the term "chromatographic fractions"
includes subfractions derived from a first chromatographic
fraction.
[0083] As used herein, the term "small number" in reference to
compounds is a number that allows biological activity from any one
compound in the mixture to be detected without interference from
any other compound in the mixture. A number of compounds that is
small enough to prevent interference due to competition and to
minimize collision frequency is typically less than about 7
compounds or about 5 compounds. The small number provides a
substantial purity, e.g. at least about 80%, or 90%, or 95% of the
mass of the compounds in a given well is in seven or fewer, or even
five or fewer compounds. A person of ordinary skill will recognize
that for all the characteristics of the compound isolates, there is
a range of characteristics. For the number of compounds, generally
there will be fewer than about 5% of isolates that include 6-7
detectable compounds, and fewer than 1% that contain 8 or 9
detectable compounds.
[0084] As used herein, the terms "substantially free of compounds
that interfere with biological testing" and "substantially free of
interferences" in reference to a fraction or isolate mean that the
fraction or isolate is substantially free of compounds that may
non-selectively compete with the compounds of interest for the
active site of potential targets of biological activity.
"Substantially free," as used herein, refers to a non-interfering
amount. In other words, in a fraction that is substantially free of
compounds that interfere with biological testing or substantially
free of interferences, any compound present that may so interfere
by non-selectively compete with the compounds of interest for the
active site of potential targets of biological activity is present
at a concentration too low to cause such interference. Such
compounds or "interferences" that are removed include: (a)
compounds having a high molecular weight, for example a molecular
weight greater than 25,000; 10,000; 6,000; or 3,000 dalton;
depending on the situation and the molecular weights of the
compounds of interest; (b) tetramers or greater of phenolics; (c)
tannins; (d) proteins; and (e) peptides. Such fractions are
"non-proteinaceous," meaning that the chemical compounds do not
comprise proteins or peptides. Proteins may interfere with
biological testing of the individual compounds.
[0085] As used herein, the term "an amount sufficient to prepare a
sample of a concentration suitable for biological testing" means an
amount of compound sufficient to prepare a sample having a
concentration of that compound useful for bioassays; that is, an
amount detectable or responsive in the bioassay to be employed.
Typical amounts required for current known bioassays are about
1-100 micrograms. This amount can be used to prepare samples from
the fraction having a concentration of at least about one
micromolar. As bioassay techniques improve and become more
sensitive, the amount of compound and the concentration may be
reduced.
[0086] As used herein, the term "drug-like compounds" refers to
compounds exhibiting molecular weights less than 1000 daltons and
lipophilicities (log P) less than 5 and approximately greater than
0. These specifications were derived from the analysis of drugs by
researchers at Pfizer which demonstrate good permeability across a
membrane bi-layer. See, for example, Lipinski, et. al.,
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings.
Advanced Drug Delivery Reviews 46 (2001) 3-26. Preferably, the
drug-like compounds have molecular weights less than 500
daltons.
[0087] As used herein, therefore, the term "non-drug-like
compounds" refers to compounds that do not correspond to the
definition of "drug-like compounds." Applicants' analytical data
demonstrate that approximately 80% of a plant extract by weight
consists of non-drug-like compounds. Applicants' purification
procedure was specifically developed to remove most of the
non-drug-like compounds during its process and collect the
drug-like compounds into chromatographic fractions.
[0088] As used herein, the term "a log P suitable for biological
testing" means a log P (partition coefficient) within a range
typical of biologically active compounds: -2 to 6 or -1 to 5. This
is determined by the chromatographic parameters. Compounds such as
fatty acids, log P 6-11, are desirably excluded. As methods change,
this range may increase. It may also be possible that the
biological activity of compounds with log P's outside this range
may be measured and the 15 active compounds chemically modified to
bring the log P within a suitable range.
[0089] As used herein, the term "normalized quantity" means a known
mass that may be used to prepare a sample having a known
concentration. A normalized quantity of a compound is ten to
thousands of times greater than that found in an extract, but is
within a single order of magnitude with respect to the
concentration of other normalized concentrations from that extract.
A "normalized quantity" of a chemical compound or compounds is a
known amount of material removed from the fractions of the original
"mother" plate or intermediate plate as needed so that the known
amount of the compounds on the end-user plate is within a
predetermined concentration range and suitable for high throughput
screening. The predetermined concentration range allows specific
activity to be detected directly from a biological assay of the
fraction. It also ensures that the amount of compound is suitable
to provide a positive result from a biological assay if the
compound is biologically active. A normalized quantity may be
prepared using approximate mass data collected by ELSD which gives
information about the total mass in each fraction. A normalized
quantity may be prepared in a separate plate by taking an aliquot
from the fraction sufficient to transfer the predetermined mass
necessary to give a desirable concentration when diluted in the
array, e.g. between about 1 micromolar and 20 micromolar. The
approximations involved are well within the degree of precision
required by persons of ordinary skill in the art to which the
invention relates. Mass spectral data gives information about the
approximate molecular weight of compounds in the fraction. Coupling
such information with the mass permits compound by-compound
calculations of molar concentration.
[0090] As used herein, forms of the term "normalizing" (e.g.,
"normalization" and "normalized: as well as "normalizing") in
reference to quantities or concentrations of a group of compounds,
such as compounds within a fraction or extract, means increasing
and adjusting the quantities or concentrations of the compounds
such that each of the normalized quantities or concentrations is an
amount sufficient to prepare a sample of a concentration suitable
for biological testing--a concentration generally tens to thousands
of times greater than the concentration in the original
extracts--and such that each of the normalized quantities or
concentrations is within about an order of magnitude of each of the
other normalized quantities or concentrations in the group.
Preferably, the quantities or concentrations are such as to be
suitable for high throughput screening.
[0091] As used herein, the term "organism" refers generally to a
plant but can also be, for example, bacteria, insect, marine
microorganisms, and fungi, etc. Different organisms may be, for
example, different genera species of a plant or different types of
microorganisms, for example a plant and a fungus, or combinations
thereof.
[0092] With these definitions in mind, therefore, according to one
embodiment of the present invention, a sample is extracted from a
biological source such as by liquid extraction, interferences are
removed from the sample, fractions are derived from the resulting
interference-free sample such as by automated distribution and
solid phase extraction, the resulting fractions are purified,
concentrations of compounds of potential interest are normalized,
and the normalized compounds are distributed on a screening plate
suitable for screening by high-throughput screening techniques.
[0093] The screening plate as described is a physical array of
progeny isolates obtained by fractionating a single biological
source, each isolate comprising from one to five compounds of about
250 to about 600 Daltons (typically not protein or nucleic acid)
collected on a physical supporting medium, and associated therewith
a data array including the identity of the biological source, the
location of each isolate, the fractionation conditions by which
each isolate was obtained and preferably physical and/or chemical
information regarding the compound, most preferably including its
elucidated structure. The invention thus provides both a
physical/chemical catalog and a data catalog of the organic
compounds found in the biological source. These organic compounds
may be separated into ultraviolet absorbing and non-ultraviolet
absorbing arrays. The physical array and the data array each have
independent value and usefulness for screening in vitro and "in
silico" (virtual screening using computer modeling), but they are
most useful when combined. In short, screening plate is a physical
array of isolate samples wherein each isolate sample comprises a
small number of components at or near the assay concentration. The
small number of components may be less that 100, is preferably less
than about 15 and is most preferably no more than about five. The
isolate sample may be prepared from one or more extracts that have
been subjected to one or more chromatography steps.
[0094] For greater clarity, reference is now made to a non-limiting
illustration of the process of the present invention. According to
the present invention, a plant may be processed to produce an
extract from which a sample is loaded onto a flash column,
producing a small number of fractions--say for the sake of
discussion, five solvent fractions labeled E1-F1 (meaning fraction
1 from extract 1), E1-F2 (fraction 2 from extract 1), E1-F3
(fraction 3 from extract 1), E1-F4 (fraction 4 from extract 1), and
E1-F5 (fraction 5 from extract 1). Fraction E1-F1, which is very
lipophilic, is discarded, and some material is lost due to
irreversible bonding to the very hydrophilic column. Because the
discarded and lost materials do not fall within the lipophilicity
range associated with drugs, they are not considered potential
drugs and in prior art processes are interferences that inhibit
detection of legitimate drug possibilities in the sample. Fraction
E1-F2 is loaded onto a preparative HPLC column and numerous
fractions are collected. To distinguish such fractions from the
mother fraction E1-F2, these will be referred to as daughter
fractions. For the sake of discussion, say forty daughter fractions
are collected from mother fraction E1-F2. The same procedure is
followed with fractions E1-F3, E1-F4 and E1-F5, thereby producing
more daughter fractions. Each of the daughter fractions is then
transferred to a well, one daughter fraction per well, and analyzed
by a combination of HPLC, MS and ELSD. The total mass or weight of
each daughter fraction is determined by ELSD. The amounts in the
wells are then normalized. Normalization may be carried out by
transferring approximately the same mass of material (preferably
each mass should be within about twenty percentage points of each
of the other masses) from each well to a corresponding well on a
screening plate. This may be carried out in a two-step procedure by
diluting each daughter fraction, the greater the material the
greater the degree of dilution, and transferring the diluted
daughter fractions from their wells to corresponding wells on a
screening plate. The volume of each diluted daughter fraction that
is transferred is such as to result in approximately the same mass
of material in each well, that mass being "an amount sufficient to
prepare a sample of a concentration suitable for biological
testing" as that term is defined above. Thus, typically such amount
is about 1 to about 100 micrograms. Generally, about 20 micrograms
may be desirable.
[0095] In greater detail, normalization may be carried out as
follows. After preparative HPLC the HPLC fractions are transferred
to 96 well plates for HPLC/ELSD/MS analysis. The results of the
ELSD estimated weight for each well are processed by software and
sent electronically to a Packard Multiprobe II Liquid Handling
system. The software categorizes mass of the well into one of four
categories. The well of each preparative HPLC fraction will
nominally contain 0, 200, 500 or 1000 .mu.g of a preparative HPLC
fraction. Wells with 0 mass are excluded the final plate map
prepared by the Extractor software. Plates prepared for screening
typically contain 10, 20 or 50 .mu.g of isolates/well.
[0096] Before processing with the liquid handling robot, a new
excel spreadsheet is prepared. This spreadsheet is called a client
plate map and contains data from the Extractor spreadsheet, and
four additional columns. The columns are `fill volume` and one
column each for the amount of volume needed to remove the proper
mass (10, 20 or 50 .mu.g). The fill volumes and `aspirate volumes`
are prepared via Microsoft Excels VBA code. The code uses simple
algebra to calculate the fill volume and aspirate volumes. For
example, if the source well contained 200 .mu.g and the client
required 50 .mu.g (1/4 of the mass) the fill volume would be 300
.mu.l and the aspirate volume would be 75 .mu.l (1/4 of the
volume). Technically if the source well contained 1000 .mu.g and
the client plate required only 10 .mu.g (1% of the mass) then the
fill volume would be 500 .mu.l and the aspirate volume would be 5
.mu.l; however, the liquid handling system is not precise below 10
.mu.l. In the case of 1000 .mu.l, samples and 10 .mu.g requirements
the spreadsheet is programmed to calculate a fill volume of 500
.mu.l and an aspirate volume of 10 .mu.l. This allows for more
precise liquid handling, but creates a situation where some wells
have 2.times. concentration of sample.
[0097] In any event, the amounts should be suitable for high
throughput screening. This screening plate is therefore an array
that is a library of the characteristics and advantages as
discussed above. The library is suitable for high throughput
screening that allows an investigation of the characteristics of
individual compounds or groups of compounds on the plate whose
characteristics are missed by conventional screening techniques due
to the presence of the interferences removed with fraction E1-F1,
above, low concentration of compound or group of compounds, or a
combination of the two.
[0098] In more detail, the steps of a process of an embodiment of
the present invention is as follows: Extraction may be carried out
by grinding dried plant material to a homogenous powder and
sonicating the powder in an organic solvent, such as a mixture of
EtOH:EtOAc (50:50), and shaking the resulting mixture vigorously
for exhaustive extractions.
[0099] Next, flash chromatographic separation may be carried out by
dissolving the organic extract in 5 mL of a solvent such as
MeOH:EtOAc (50:50), adsorbing it onto silica powder and bringing
the dried powder onto a silica column and eluted on the flash
chromatography system using a step gradient of 1) 75% hexanes, 25%
EtOAc, 2) 50% hexanes, 50% EtOAc, 3) 100% EtOAc, 4) 75% EtOAc, 25%
MeOH, 5) 50% EtOAc, 50% MeOH. The flash fraction containing highly
lipophilic material unsuitable for drug possibilities may be
discarded, whereas the remaining fractions may be dried, such as by
rotary evaporation. One or more flash fraction may be screened for
the presence of tannins by LC-MS and passed over a polyamide column
if results are positive. See Anderson, K. J.; Teuber, S. S.;
Gobeille, A.; Cremin, P. A.; Waterhouse, A. L.; Steinberg, F. M. J.
Nutr. 2001, 131, 2837-2842.
[0100] An aqueous extract may be dissolved in water and the
resulting mixture centrifuged. The resulting aqueous layer may be
brought onto a C18 column (pre-rinsed with 1 column volume methanol
and 5 column volumes water). Any insoluble material may again be
dissolved in water using sonication and the suspension may be
centrifuged again. The aqueous layer may also be brought onto the
column. The column then may be rinsed with water and the effluent
discarded. The remaining insoluble material subsequently may be
taken into methanol and the methanol layer brought onto the column.
The column may be eluted with methanol to remove water from the
column and a polyamide cartridge in methanol may be attached to the
bottom of the column. The column then may be eluted with methanol.
The resulting fraction may be dissolved in MeOH:H2O (60:40) and
filtered with a molecular weight cut-off of 3000 amu. The
retentate, typically 1-2 mL, may be discarded. Analytical size
exclusion chromatography has shown that the content of high
molecular weight constituents (>3000 amu) in the filtrate is
reduced significantly from up to 75% to less than 10% of the total
amount of material using ELSD detection.
[0101] Preparative HPLC separation may then be carried out. Flash
fraction material may be dissolved into either MeOH:EtOAc (70:30)
or 100% MeOH and filtered where necessary. The fractions are
separated into several dozen, such as 40, fractions using a device
such as a parallel four-channel preparative HPLC system. A
different gradient may be applied to each flash fraction for
adequate separation; for example, flash fraction 2: 40-80%
acetonitrile in water, flash fraction 3: 30-70% acetonitrile in
water, flash fraction 4: 20-60% acetonitrile in water, flash
fractions 5 and 6: 10-50% acetonitrile in water. The tubes (e.g.,
40 tubes for 40 fractions) containing HPLC fractions may be dried
in an evaporator. The HPLC fractions then may be transferred to
plates such as 96-deep-well plates using a liquid handling system
(e.g., Packard MultiProbe II).
[0102] The mass or weight and the molecular weights of the
materials in the samples may be determined by a parallel
eight-channel LC-ELSD-MS system with chromatographic conditions of
5% acetonitrile in water for the first minute, a linear gradient of
acetonitrile from 5% to 95% in eight minutes, followed by 95%
acetonitrile in water for a minute. Under such chromatographic
conditions, the column is equilibrated at 5% acetonitrile in water
after each analysis. Data processing for determining the
appropriate dilution for each sample for normalization may be
performed automatically with computer software to extract all
graphic information, such as retention times, mass spectra, and
peak integrations, and to convert such information to text to allow
it to be transferred to a database for storage and analysis.
[0103] Based on the knowledge of the amounts of material present,
normalization may be carried out so that the mass of material in
each well is about the same (i.e., within about an order of
magnitude, preferably within about 20 percentage points of each
other), and the mass of each significant compound within each well
is about the same as the other significant compound in that well
(i.e., within about an order of magnitude, preferably within about
20 percentage points of each other). The reference to "significant"
compounds as opposed to all compounds is in recognition that in
addition to the small number of compounds to be studied in the
well, there may be additional compounds present in relatively tiny
amounts and those may be ignored. For example, a well might contain
three or four compounds varying in mass from about, say, 30
micrograms to 50 micrograms, as well as extraneous compounds of
mass on the order of, say, 1 to 2 micrograms.
[0104] Such well may be considered normalized notwithstanding the
presence of low amounts of the extraneous compounds. Although a
logically ordered preparation of arrays of synthetic compounds from
certain scaffolds has been previously described, for example in
U.S. Pat. No. 5,962,736, the present invention provides a
systematic method for providing arrays of plant isolates or
isolated pure compounds that are fundamentally different from all
known prior art. For example, although prior art methods prepare an
ordered array of known target compounds, the invention is
fundamentally different in providing an array of unknown,
non-target compounds for testing. In addition, although the prior
art arrays were generated by isolation or synthesis of single
compounds, arrays of the present invention are generated through
fractionation of complex mixtures of compounds. Although the
identifying system of the invention removes the necessity for
preparing orderly arrays, providing for the ordering of the arrays
of isolates in a logical manner is preferred. These arrays may be
constructed from a wide variety of fractionated plant isolates, but
other fractionated natural product isolates or isolated compounds,
including but not limited to marine organisms, microorganisms, and
insects, are within the scope of the invention.
[0105] In a preferred embodiment, the invention provides a layout
of arrays of isolates in microtiter plates that contain various
unknown compounds for screening in biological systems. The
invention provides that the solubility profile of each isolate is
known. The arrays are preferably ordered in such a fashion as to
expedite collection of the isolates and provide insights into the
solubility of the compounds specific to each array. This method has
great utility in accelerating the discovery of compounds by
providing information about the physical properties of the
chemicals in the isolates before the screening process.
[0106] The preferred arrays are constructed from ordered gradient
elution schemes developed with consideration of the origin of the
solvent fractionated isolate and the solid phase extraction sorbent
that may retain compounds in the mixtures. Each group of arrays
consists of sets of solubility related isolates which may contain
approximately one to fifty compounds per isolate with a common
solubility profile and various structural diversity when a
purification step is included in the method, isolates typically
contain one to three compounds. These arrays may be arranged in
larger groups of arrays consisting of sets of arrays and tested to
provide information regarding all of the isolates in the arrays.
For example, the larger groups of arrays may originate from
different regions of the same plant and be ordered by the plant
from which the isolates originate. A set of such arrays would thus
represent a parent plant and a set of isolates that are the progeny
of that plant.
[0107] For a serial fractionation chromatography system having one
or more independent columns (such as eight as described with the
Gilson liquid handling system), the first fraction is collected
e.g. in column 2, row A, the second fraction is collected in plate
1, column 3, row A, and so on until column 11, row A, then the
fractions would be collected in row B from column 11 back to column
2, and so on. An extract being separated might have from 5 to 150
compounds that can be separately isolated. When the last compound
of a sample is collected, the collector then takes another extract
and begins chromatography on that extract, collecting in the well
after the last fraction of the previous extract. Thus, a plate
might have the extracts from several plants arrayed on it, or a
plant might require several plates to capture all the
compounds.
[0108] The array of fractions or isolates thus comprises a large
number of individual isolates that are related as being, for
example, the progeny of a single plant or originating from a
particular taxonomic division of plants. The large number of
isolates in each array is preferably at least about 10 and most
preferably at least 50. It may be larger than the number of wells
on a plate, in which case the array includes several plates. The
number of individual, unique isolates can be represented by M which
is a function of P, S, E, F and A in which:
[0109] P is the number of plants used to generate the extract
samples;
[0110] S is the number of samples obtained from different parts of
each plant;
[0111] E is the number of solvent extracts taken from each
sample,
[0112] F is the number of fractions obtained from each solvent
extract, the fractions F being obtained by a first chromatography
step; and
[0113] A is the number of subfractions collected from a second
chromatography step of each fraction. The maximum value of M may be
represented as:
M=P.times.S.times.E.times.F.times.A (1)
In equation (1), P is defined as the number of plants used to
generate an array of isolates. For a particular set of isolates, P
is preferably from one to ten and is most preferably one. S is the
number of samples obtained from each plant. For example, if leaves,
stem and root are used, S is three. It is preferred that S be from
one to five. E is defined as the number of extracts taken from each
sample. In a preferred embodiment, E is from one to three. Most
preferably, E is two. The number F is defined as the number of
fractions collected from each extract. In the preferred method of
generating the M isolates that comprise the array, E is the number
of fractions collected by solid phase extraction and is preferably
from three to twelve. A most frequently represents the number of
fractions collected from the further purification system, when
utilized. If the further purification system is not utilized, A is
assigned the value of one. In a preferred embodiment of practicing
the invention that includes the further purification step, A is
from one to twelve. Preferably, E*F*A is greater than 10, more
preferably greater than 15.
[0114] It is contemplated that for each of P, S, E, F, and A, there
can be a different number of products. That is, for each plant
there can be a different number of samples; for each sample, a
different number of extracts; for each extract, a different number
of fractions; and for each fraction, a different number of
subfractions. For example, for a given plant sample, there may be
two solvent extracts (polar and non-polar), one of which produces
three fractions and the other of which produces six fractions, for
at total of M=9 isolates.
[0115] The ordered arrays of the invention are unique in that (a)
they include essentially all of the significant phytochemicals
(those having a potential selective bio-activity) because all have
been extracted by thorough extraction, and none have been passed to
waste due to careful detecting of the chromatography eluent, (b)
each well of the ordered array has at least one detected compound
(none are blank), and (c) the wells do not have more than a few
compounds.
[0116] The array of isolates thus represents a physical catalog of
related compounds. Associated with each isolate is a data array.
Thus, in addition to the novelty and utility of arrays of isolates,
each individual isolate and its associated data is also novel and
valuable. Each individual isolate has associated therewith data
useful for replicating test results and for the isolation of
biologically significant compounds. The data array forms a "virtual
catalog" of properties that mirrors or shadows the physical array
of compounds. Much of the valuable information in the physical
catalog is also contained in the virtual catalog. Thus, the data
label itself that is associated with each isolate has value,
particularly if structural data is associated with such data.
[0117] In another embodiment of the invention, isolate samples may
be packaged on, for example, sample plates wherein each sample on
the sample plate contains a small number of detectable compounds.
Preferably, the small number of detectable compounds is less than
about 100, more preferably is less than about 15 and most
preferably is no more than about five. According to this
embodiment, a biological source material is extracted with at least
one extraction solvent to give one or more extracts. The
interferences are preferably removed from each extract. Each
extract is then subjected to at least one automated chromatography
as previously described. Fraction collection may be conducted by an
automated detection system or by time dependent collection. The
individual fractions are then analyzed. Fractions that do not
contain detectable compounds may be discarded.
[0118] Fractions are then distributed on sample plates such that
each isolate sample contains an amount (that is, "quantity" as
opposed to number) of detectable compounds which, upon preparation
for a biological assay, contain the detectable compounds in an
amount equal to or greater than the normalized assay concentration.
Thus, fractions that contain detectable compounds in excess of the
amount required for preparing a normalized assay concentration may
be subdivided into multiple isolate samples and distributed on more
than one sample plate. Fractions containing an amount less than the
amount required for preparing a normalized assay concentration may
be combined with other fractions having a similar composition.
Using this method, about four to five sets of sample plates may be
prepared in a single sequence.
[0119] Thus, in one embodiment, the present invention is an article
of manufacture that is a library or collection of chemical
compounds with unique and identifiable chromatographic separation
parameters. The library of chemical compounds may be distributed,
for example, on a plate, particularly a microtiter plate, that is
suitable for use in an automated bioassay instrument. In preferred
embodiments, a single source material (from one or more organisms)
is used to produce the library of chemical compounds. The source
material may be divided into greater than 80 fractions with a
minimization of overlapping compounds in the various fractions.
Thus, most separate fraction includes between about 1 and 5
compounds detectable by mass spectrometry, evaporative light
scattering or other detection means. In addition, the
chromatographic fractions may be treated to remove or minimize
interferences from tannins and polyphenolics. Thus, chemical
compounds prepared according to the invention do not contain
interferences or polymeric compounds, but may include, for example,
non-detectable amounts of other chemical compounds without changing
the basic and novel characteristics of the invention.
[0120] Although all of the primary (mother) fractions collected may
contain material which is detectable using, for example,
evaporative light scattering, TIC mass spectral analysis, or other
mass spectroscopic detection techniques, in preferred embodiments,
the wells or fractions containing detectable compounds are placed
in a secondary well or on a secondary plate for further analysis.
These thus form secondary (daughter) plates of compounds. The
secondary daughter plates may in turn be used as a stockpile or
intermediate to prepare normalized end-user plates. In this way,
the analytical plate that forms the article of the invention may
exclude wells from the original chromatographic separation that do
not include detectable organic compounds (blanks).
[0121] Thus, the invention provides a library of chemical
compounds, typically contained on a microtiter plate, where all of
the chemical compounds fall within a predetermined range of
chromatographic characteristics. These characteristics may include,
for example, solubility, log P, molecular weight, molecular size,
polarity, mass, concentration, etc. Moreover, each of the compounds
within each single chromatographic fraction have common
chromatographic characteristics. For example, typical
chromatographic procedures provide chromatographic fractions
containing chemical compounds with similar log P and solubility.
These chromatographic fractions contain co-elutable compounds under
the particular chromatographic conditions used for the
separation.
[0122] The array may, however, include sets of fractions having
differing characteristics such as a broad range of sizes, molecular
ions, log P's, polarity, etc. Thus, arrays of the invention may
represent a diversity of compounds. This diversity may arise by
selecting fractions with diverse characteristics from a single
source or by constructing the array from a diversity of biological
sources. Diversity increases the likelihood of detecting a
biologically active compound. However, the natural diversity of
mass and concentration of biological compounds is not helpful in
biological screening, and is reduced by normalization as discussed
in more detail elsewhere.
[0123] In a typical embodiment, the chromatographic separation of
the biological source material is conducted in such a way that low
molecular weight organic compounds are selectively placed in the
wells. Low molecular weight compounds are those having molecular
weight of less than 10,000 daltons, more preferably less than about
3,000 daltons, even more preferably less than about 1,000 daltons.
The molecular weights of drug-like compounds are typically less
than about 600, such as about 250 to about 600, daltons, and
average about 400. In addition, the initial chromatography of the
biological source material is conducted in a chromatographic system
which has been calibrated in such a way that particular fractions
elute at different times based on their log P values. Preferably,
compounds are selected having a log P of between about -1 and 5.
This is the desirable range for biological assays.
[0124] In addition to the chromatographic fractions of the
invention, compilation of data describing characteristic
chromatographic and spectral data regarding the contents of
individual wells is contemplated by the invention. For example,
each well will have associated with it data sufficient to reproduce
the chromatographic conditions used to prepare the chromatographic
fraction, including liquid chromatographic data and retention
times. In addition, spectral data may include mass spectral data
sufficient to identify the M+H ion, that is the molecular ion plus
a proton, mass spectral fragmentation data, and NMR spectra. This
data is sufficient to allow for a more rapid structure elucidation
for various components in the fraction. Structural elucidation is
performed manually. Thus, in addition to simply identifying the
source material and chromatographic conditions used to isolate the
various components from the biological source, the data is
generally sufficient to replicate the separation in order to
collect additional material for further characterization and
identification of the specific compound obtained. Data sufficient
for structural elucidation may be compiled during the initial
preparation of chromatographic fractions, e.g. chromatography, or
after biologically active or potentially biologically active
chromatographic fractions are identified.
[0125] In a preferred embodiment, compounds from any one particular
biological source are compared to data obtained from libraries of
compound from different biological sources. The various biological
sources may be different species of the same plants, different
plants within the same family, or any other combination of
biological sources.
[0126] Thus, the invention is environmentally useful in
establishing the ranges of plant diversity thus encouraging the
conservation of plants from as many sources as possible. The
invention permits economic value of biological diversity to be
realized, thus increasing the value of preserves of high biological
diversity such as rainforests, and increasing conservation. The
chromatographic and spectral data may be used to form sub-libraries
of the parent libraries as separate articles of manufacture. These
sub-libraries may be organized so as to include chemical compounds
having similar, i.e. more narrow ranges, of log P. In addition, the
sub-libraries may be arranged by looking at materials with similar
or relatively narrow molecular weight ranges having a common
biological source.
[0127] The libraries obtained by the present invention are
particularly suitable for screening for biological activity. They
have an enriched selectively bio-active population of compounds
suitable for screening because they can be detected without
interference. The libraries are also comprehensive, in that they
contain the full range of compounds in a particular plant or other
organism. Because the invention provides fractions having
sufficient mass for use in the biological assay, for example, about
1 microgram to about 1 milligram of total material or individual
compounds, or, more preferably, about 1 microgram to about 100
micrograms of total material or individual compounds, rapid
throughput screening is readily applied to the libraries. Examples
of biological assays which may be used are those which detect
compounds useful for the treatment of disease or compounds useful
for the control of biological tests. Thus, the library of chemical
compounds are particularly useful when organized in well plates,
for example, a microtiter well plate. Biological screening
apparatus comprising these well plates are also contemplated by the
invention.
[0128] After active chromatographic fractions or chemical compounds
have been identified, it is within the scope of the invention to
further chemically modify those compounds to provide additional
libraries. In particular, the techniques of forming combinatorial
chemical libraries of compounds may be applied. Thus, from a
particular biological source, which may yield several hundred
detectable compounds, biological testing will identify
chromatographic fractions having biologically active compounds. A
particular source may contain one or many of such compounds.
Biologically-active compounds may then be further modified using a
combinatorial chemistry approach to yield even more compounds
suitable for biological screening. Thus the present invention
provides not only for chromatographic fractions from biological
sources, but derivatives of those chromatographic fractions.
[0129] The analysis of the library may be conducted by high
throughput screening. Thus, an application of this invention is the
rapid screening of isolates containing compounds that have been
semi-purified and concentrated. An array of isolates is screened
and the optimum isolates may be chosen for further structure
elucidation and activity confirmation. The invention is extremely
powerful primarily for three reasons, 1) chemicals in the plant
extracts are semi-purified and concentrated when compared to
traditional methods of preparing plant extracts which provides an
increased probability of showing biological activity free from
interferences and numerous other secondary interactions with other
chemicals in the plant extracts, 2) physical property information
about the chemicals in the plant extracts are provided with each
extract which will decrease the time of elucidating the structure;
and 3) the physical array of isolates can be used in existing high
throughput screening systems developed for synthetic combinatorial
chemistry applications, extending these systems to the field of
biodiversity prospecting.
[0130] A method for the screening of foods for nutraceuticals has
been previously described in U.S. Pat. No. 5,955,269. Methods for
the biological screening of libraries of synthetic chemicals
prepared from biologically active scaffolds have been described in
U.S. Pat. No. 5,908,960. It is the object of the present invention
to provide isolates from natural sources in an ordered array of
microtiter plates that is more suitable than known to the prior art
for the high throughput screening of biological assays. Steps of
the preparation of the isolates have been optimized for plants to
increase the success of discovering a chemical that may have a
therapeutic value.
[0131] The data labels of the present invention may be
independently useful screening tools. In addition to bioassays for
screening of compounds, the advent of high speed computers and the
wealth of knowledge regarding structure-activity relationships in
recent years allows for "virtual screening" of chemical compounds.
In virtual screening, a computer analyzes possible structure
activity relationships in drug discovery. Thus, rather than that in
vitro assays, virtual screening provides for "in silico" screening
of drug candidates. The compound itself is not necessary for in
silico screening, only data.
[0132] FIGS. 9 through 13 illustrate examples of the well plates of
the invention, the data which may be associated with those well
plates, and the file structure which may be used in a database for
organizing the data obtained. FIG. 9 is a schematic representation
of fractions of chemical compounds in a microtiter plate with mass
spectrum and evaporative light scattering chromatograms of an
individual fraction within the collection. As shown in FIG. 9, any
particular well, shown here as well designated H3, has associated
with it an evaporative light scattering detector (ELSD)
chromatogram, a total ion current (TIC) chromatogram and a mass
spectrum of the bulk material.
[0133] FIG. 10 illustrates mass spectra of molecular ions and
chromatogram of elution conditions for two different isomeric
chemical compounds. As can be seen in FIG. 10, the liquid
chromatogram is able to distinguish the two isomeric compounds
depicted. The two isomers each have a molecular weight of about
286, and thus, the M+H peak in both chromatograms shows up at
approximately 287. As is well known to persons skilled in the art,
the mass spectrum can be used to determine not only molecular
weight but the molecular formula of compounds. Thus, these
chromatograms are useful for detecting replication of particular
compounds within separate natural library libraries by comparing
chromatographic and/or chemical properties.
[0134] FIG. 11 illustrates chromatograms of elution conditions and
mass spectral fragmentation patterns of two different isomeric
chemical compounds. Because the fragmentation patterns of different
compounds are unique, this particular technique is useful in aiding
in structural elucidation and identification of various compounds.
As is known, and is further illustrated in FIG. 11, different
isomeric compounds have different mass spectral fragmentation
patterns. Thus, further evidence of the unique character of the two
compounds is obtained and chromatographic and spectral
characteristics for the individual compounds are known.
[0135] FIG. 12 illustrates a one-dimensional proton NMR spectra of
the two different isomeric compounds. Again, the NMR spectra differ
and are useful in further elucidating the structure of individual
components derived from the same biological material.
[0136] FIG. 13 is a schematic representation of a data table that
may be used to compare molecular ions and chromatographic elusion
conditions for entire collections of chemical compounds derived
from the same or different biological sources. The information
contained within the data table shows the well from which
particular files originate, the liquid chromatography mass spectral
file name containing data, the number of components within any
particular well, the retention time of individual components, the
M+H data value for each of the individual components, and the
relative abundance of each component within the particular well. As
can be seen, there may be some overlap between individual wells
which contain common compounds. In addition to the data depicted in
FIG. 13, data can also be collected which describes or identifies
overlapping biological sources, i.e. those compounds which appear
in more than one biological source.
[0137] The examples set forth below further illustrate the process
used to produce the invention and the chemical compound library
according to the invention. The non-limiting paragraphs which
follow the examples, describe and outline various aspects of the
present invention.
EXAMPLE 1
[0138] Listed below are procedures that have been used with some
variations as noted in U.S. Ser. No. 60/280,739 and PCT/USOO/30195,
to produce a chemical compound library from greater than two
hundred plants.
[0139] Liquid Extraction Procedure
[0140] E1. Ethanol/Ethyl Acetate (50:50): Weigh the plant material.
Appropriately grind the plant material to a fine powder. Transfer
the ground plant material into a 4 L flask. Transfer 1 L of
ethanol/ethyl acetate (50:50) solution to the 4 L flask. Agitate
for approximately 18 to 22 hours. Pour the solution through a low
ash filter paper and funnel into an appropriate round bottom
evaporator flask. Reduce to approximately 20 ml of liquid at less
than 40.degree. C. Turn off heat. Repeat and combine with first
extract. Reduce to dryness under vacuum. Weigh the dried extract.
Combine with the Ethanol Extract (E1). Mix the extract thoroughly.
Vials are store under nitrogen at -20.degree. C.
[0141] E2. Methanol/Water (70:30): Return any insoluble material in
the low ash filter paper and funnel to the original 4 L flask.
Transfer 1 L of methanol/water (70:30) solution to the 4 L flask.
Agitate for approximately 18 to 22 hours. Pour the solution through
a low ash filter paper and funnel into an appropriate round bottom
evaporator flask. Reduce to approximately 20 ml of liquid at less
than 40.degree. C. Turn off heat. Repeat and combine with first
extract. Reduce to dryness under vacuum. Weigh the dried extract.
Mix the extract thoroughly. Vials are store under nitrogen at
-20.degree. C.
[0142] Flash Chromatography Procedure
[0143] The Flash Chromatographic separations are performed on Flash
Master 11 system, made by Jones Chromatography. The E1 extracts are
separated by silica column and E2 extracts are separated by C-18
column. The gradients for E1 and E2 are as following: [0144] E1:
[0145] 1. E1-F1-hexane/ethyl acetate ( 75/25) fraction [0146] 2.
E1-F2-hexane/ethyl acetate (50150) fraction [0147] 3. E1-F3-ethyl
acetate fraction [0148] 4. E1-E4-ethyl acetate/methanol ( 75/25)
fraction [0149] 5. E1-F5-ethyl acetate/methanol ( 50/50) fraction
[0150] E2: [0151] 1. E2-F1-water fraction [0152] 2. E2-F2-methanol
fraction
[0153] Procedure for the Removal of Tetramers or Greater of
Polyphenolics;
[0154] Method 1
[0155] 1. Column Preparation. (Step 1)
[0156] Place a new IST 2.5 g polyamide column cartridge onto the
"VacMaster Sample Processing. Station" equipped with 20 ml
borosilicate collection tubes. Elute the column with 10 ml
methanol, then elute with 40 ml of water. Leave the column loaded
with additional 20 ml water overnight to swell the resin.
[0157] 2. Extract Detannification. (Step 2)
[0158] 2.1 Sample Preparation.
[0159] Weight 1 g of the organic or aqueous extract into a labeled
vial. Dissolve in 10 mL water (aqueous extract) or methanol
(organic extract). Sonicate if necessary. Spin down any insoluble
material in the Savant SpeedVac.
[0160] 2.2 Sample Loading.
[0161] Outfit the "VacMaster Sample Processing Station" with clean,
empty 20 mL borosilicate collection tubes.
[0162] After spinning the sample in the Savant, bring the liquid
onto the column. Run the column until the liquid reaches the
surface of the frit. Re-dissolve the insoluble material that
settled in the Savant using additional water or methanol (aqueous
extract) or methanol (organic extract). Bring the liquid onto the
column. It may be necessary to sonicate again and if any insoluble
material persists to spin the sample. Repeat this process until all
extract has been brought onto the column.* *Some insoluble material
may be present in the samples resulting from the grinding and
extraction methods. If repeated attempt to dissolve this fail, the
material can brought onto the column using methanol. It will be
filtered out during elution of the sample.
[0163] 2.3 Sample Elution.
[0164] Rinse the column with 40 to 60 mL methanol. Collect into
borosilicate tubes and combine all the eluent into a 1 L round
bottom flask for drying. Rotavap the sample to 20 dryness. It is
now ready for further processing using Flash Chromatography.
##STR00001##
[0165] Procedure to Remove Compounds with Molecular Weights Greater
than 3000 Daltons
[0166] 1. Select the sample for filtration through a Centricon
Filter unit.
[0167] 2. Weigh approximately 100 mg into a scintillation vial.
Dissolve into 50 mL methanol. Transfer to outer unit of the
Centricon Filter. Replace the inner part of the filter and the cap.
Place the filter unit into a centrifuge and spin for 14 hours at
3000 g.
[0168] 3. Poor out the contents of the inner unit into a clean
tarred scintillation vial.
[0169] 4. Dry the fraction overnight in the SpeedVac.
[0170] Parallel Preparative HPLC Procedure
[0171] 1. Sample Preparation. 50 mg or the total amount of sample,
whichever less, is injected.
[0172] 2. Method [0173] 1. 1000 pl of sample is injected. [0174] 2.
30 minute linear gradients are used for elution. The exact gradient
depends on the sample being process (see below). All gradients are
followed by a 5 minute wash with 100% acetonitrile. [0175] a. E1F2:
60-100% water-acetonitrile [0176] b. E1F3: 30-70%
water-acetonitrile [0177] c. E1F4: 5-40% water-acetonitrile [0178]
d. E1F5 5-40% water-acetonitrile [0179] e. E2F2: 5-45%
water-acetonitrile
[0180] 3. Flow rate is 20 ml/minute
[0181] 4. Fractions are collected every 1 minute for the 35 minutes
of the run. [0182] a. 18.times.150 mm glass test tubes are used to
collect the fractions
[0183] 3. Materials and reagents
[0184] 1. Equipment [0185] b. Gilson 215/849 autosampler with 1000
gl injection loops [0186] c. Gilson 204 fraction collectors [0187]
d. Beckman Coulter 126P pumps and 166 detectors [0188] e. Beckman
Coulter
[0189] 2. Reagents [0190] a. Water and Acetonitrile HPLC grade
[0191] b. Columns C-18 Betasil (Keystone), 20.times.lOOmm [0192] c.
Solvents for sample re-suspension are be HPLC grade
[0193] LC/ELSD/MS Procedure for Analysis of Fractions
[0194] 1. LC-ELSD-MS Experimental methods. [0195] a. HPLC
conditions
[0196] Liquid flow is provided by a Waters 600 binary pump. The
flow is split 8 ways by use of a low dead volume eight way
splitter. The separate streams are directed to the Rheodyne
injection valves of a Gilson 889 Liquid handler which is mounted on
a Gilson 215 multiple injection autosampler. All samples are
presented to this system in 96 well microtiter or deep well plates
and an injection volume of 20 uL employed throughout. HPLC
Separation is achieved on Betabasic C 18 columns (4.6.times.5 0 mm,
5 .mu.M) obtained from Western Analytical. The gradient profile
used for elution is outlined in Table 1.
TABLE-US-00002 TABLE 1 HPLC SOP gradient % A (H.sub.2O + 0.1 % B
(CH.sub.3CN + 0.1% Time/min HCOOH) HCOOH) 0 95 5 1 95 5 8.5 25 75
9.0 5 95 9.6 5 95 10.5 95 5 11.0 95 5
[0197] A flow rate of 9.6 ml/min is used providing a flow of 1.2
ml/min through each column. The eluent from each column is passed
through a four way Valco low dead volume cross which splits the 1.2
ml/min flow into three separate streams of 0 1, 0.4 and 0.7 ml/min.
For each column the 0.1 ml/min stream is presented in a separate
line to one inlet of the 8-way multiplexed electrospray source, the
0.4 n-fl/min to the inlet of the Alltech 500 ELSD detector and the
0.7 ml/min stream is routed to waste. [0198] b. MS analysis
[0199] All MS data is acquired on an LCT orthogonal TOF
spectrometer (Micromass) fitted with an eight-way multiplexed
electrospray interface (MUX). In this analysis each liquid stream
is sampled for 0.1 sec. with mass spectra acquired from 200-1000 Da
into eight simultaneously open data files synchronized with the
spray being sampled. The time taken to move to the adjacent
sampling position is 0.05 sec. This cycle produces a data point for
each spray every 0.1 sec. The LCT and MUX are operated under
MassLynx V3.4. [0200] c. ELSD analysis
[0201] ELSD detection was carried out using Alltech 500 units. For
an inlet flow of 0.4 ml/min, the nebulizer gas flow for each unit
was optimized between 2.95 and 3.25 SLPM. The drift tube
temperature for each unit was similarly optimized between 95-105 T
for each stream.
[0202] 2. Completion of Analysis
[0203] The plate report is placed in the completed plate section of
the LC-ELSD-MS analysis log. The Plate is placed in the "completed
plates" section of the plate refrigerator.
[0204] 3. Data Collection and Organization [0205] a. Raw Data
[0206] Each plate or collection of plates is assigned a project
name in the MassLynx software. The project name is either the name
of the plate or the name of a series of plates combined, e.g.
[0207] Acqudb: LC-MS acquisition methods (time pages)
[0208] Curvedb: quantitation data
[0209] Datadb: Raw data (LC-MS and LC-ELSD data)
[0210] Methdb:
[0211] SampleDB: Sample lists. [0212] b. Post Processing.
[0213] Each plate is further processed by entering the required
OpenLynx method into the sample list. Result of this processing is
to produce a *.rpt file for each sample list or plate. This *.rpt
file is then processed to produce a list of masses and retention
times. These masses are then entered into a second sample list for
each plate and subject to a second OpenLynx method whose final
result is also a *.rpt file from which is extracted the retention
time, significant ion and ELSD data for each peak detected by
LC-ELSD-MS. Data from this *.rpt file is then exported to form part
of the final Excel report for each plate which has the following
form:
TABLE-US-00003 Molecular ion + ELSD File No of significant Peak
Dereplication Compound# Well# name components Rt ions area hits
[0214] Storage of Chromatographic Fractions of Chemical
Compounds
[0215] Chromatographic fractions of chemical compounds consist of
less than one microgram of material to greater than one thousand
micrograms of material. The quantity of material in each fraction
is determined by evaporative light scattering from the LC/ELSD/MS
analysis.
[0216] High-Throughput Screening
[0217] A sufficient quantity of each fraction from the mother plate
that contains chemical compounds is added to an individual well in
an appropriate intermediate microtiter plate. Aliquots of the
fractions are removed as needed from the intermediate microtiter
plate and placed in an end-user biological assay microtiter plate
in a normalized predetermined amount based on mass so as e.g. to
achieve an initial screening concentration of 1 to 20 micromolar.
Hundreds to thousands of fractions are screened in parallel for
biological 5 activity using such plates as is known to a person of
ordinary skill.
EXAMPLE 2
[0218] A single plant sample, the leaves and stems of Baptisia
alba, was fractionated to produce a library according to the
invention. Table 1 contains the weights of the extracts and
fractions of the drug-like and non-drug-like compounds of Baptisia
alba.
TABLE-US-00004 TABLE 1 Approximate Weights of Extracts and
Fractions of the Leaves and Stems of Baptisia alba prior to
Parallel Preparative HPLC. Sample Weight (grams) Extract 1 4.1
Extract 2 7.1 E1-F2 0.045 E1-F3 0.035 E1-F4 0.216 E1-F5 0.104 E2-F2
0.039 Discarded compounds from 1.0 gram of Extract 0.628 1 with log
Ps greater than 6 or less than -1, Tetramers or greater of
Polyphenolics, and molecular weights greater than 3000 Daltons
Discarded compounds from 2.0 grams of Extract 1.966 2 with log Ps
greater than 6 or less than -1, Tetramers or greater of
Polyphenolics, and molecular weights greater than 3000 Daltons
[0219] Because approximately 87% of the extracts of the leaves and
stems of Baptisia alba consisted of discarded non-drug-like
compounds, testing crude extracts or pre-fractionated of the leaves
and stems of Baptisia alba would have resulted in screening
approximately 99% or 80%, respectively, of the drug-like compounds
below optimal screening concentrations. (One of the unexpected
advantages of the inventive method is that the same
physico-chemical characteristics used for fractionation also
eliminate non-drug-like compounds, typically those with log Ps
greater than 6 or less than -1, tetramers or greater of
polyphenolics, and molecular weights greater than 3000 5
Daltons.)
[0220] Table 2 contains the molecular ions, evaporative light
scattering areas, retention times, and calculated weights of the
chemical compounds in the wells of the microtiter plates analyzed
by HPLC/MS/ELSD. Table 2 contains greater than 200 distinct
chemical compounds from the leaves and stems of Baptisia alba.
Because the library was produced according to the invention, all of
these chemical compounds may be tested at optimal screening
concentrations. Information is automatically extracted from the raw
data of chromatograms and placed into a database.
[0221] In Table 2:
[0222] MS RT: Retention time using Mass spectral detector
[0223] M+H ion: Molecular weight of M+H ion
[0224] ELSD PKS: Peaks detected by evaporative light scattering
detector
[0225] ELSD RT: Retention time using evaporative light scattering
detector
[0226] ELSD area: Relative area of peaks from evaporative light
scattering detector
[0227] Weight ELSD: Calculated weights of peaks
[0228] Table 2 shows data obtained from the wells of the
intermediate plate prepared from the mother plate. The filename
identifies the plate number and position. Table 2 presents data for
the library of compounds from one plant, in about 125 wells, on
about 6 different intermediate plates. There are about 500 lines of
data, reflecting about 200 compounds. The MS data and the ELSD data
are obtained for each well, and are somewhat independent of each
other. The ELSD is used as the universal detector for normalization
purposes. For the first well, CP0280 well 2, there is an ELSD
weight of 47 (micrograms), indicating at least one compound is
present. However, in this well the compound apparently did not
ionize in the standard conditions being used, and gave no MS or M+H
ion data. For well 3, there is at least one compound having an ELSD
weight of 41, and MS shows three different compounds with MW in the
range of about 300 to 900 daltons. Well 6 shows one MS peak, but
four ELSD peaks. In well 10, the MS data for three peaks is shown,
but the ELSD weight is shown as zero, meaning the mass is below the
calibrated threshold, and so this well would be considered a
blank.
[0229] To prepare a final plate for high throughput screening, a
normalized mass of material is removed from the intermediate plate.
For example, from CP0280 well 6, 10 micrograms of material out of
the total of about 1900 micrograms is loaded on a well of the final
plate for one particular end user (who may run the material through
several assays). Another plate may be prepared with 20 micrograms
for each well. Wells from the intermediate plates which do not have
enough mass are not loaded on the final assay plates.
TABLE-US-00005 TABLE 2 HPLC/MS/ELSD Data on the Chromatographic
Fractions of the Leaves and Stems of Baptisia alba according to the
Invention. FILENAME MS RT M + H Ion ELSD RT ELSD Area Weight ELSD
CP0280-pos-well 2 0.0 0.00 CP0280-pos-well 2 10.3 2552 47
CP0280-pos-well 3 2.5 286.99 CP0280-pos-well 3 7.7 904.40
CP0280-pos-well 3 8.3 903.39 CP0280-pos-well 3 2.6 8469 41
CP0280-pos-well 4 3.6 285.00 CP0280-pos-well 4 5.5 277.15
CP0280-pos-well 4 8.1 297.20 CP0280-pos-well 4 8.6 903.34
CP0280-pos-well 4 3.3 67530 144 CP0280-pos-well 4 3.6 75988 155
CP0280-pos-well 5 3.7 409.07 CP0280-pos-well 5 8.3 889.37
CP0280-pos-well 5 3.9 6057 43 CP0280-pos-well 5 8.1 43056 134
CP0280-pos-well 5 8.4 28297 106 CP0280-pos-well 5 8.8 46210 139
CP0280-pos-well 6 0.0 0.00 CP0280-pos-well 6 4.4 1056 18
CP0280-pos-well 6 4.5 1210 20 CP0280-pos-well 6 6.9 1500 24
CP0280-pos-well 6 8.5 352791 1824 CP0280-pos-well 7 8.0 901.36
CP0280-pos-well 7 8.8 638395 302 CP0280-pos-well 8 0.0 0.00
CP0280-pos-well 8 9.8 843586 792 CP0280-pos-well 8 10.6 15345 64
CP0280-pos-well 8 10.8 2720 3 CP0280-pos-well 10 7.3 411.25
CP0280-pos-well 10 8.2 887.32 CP0280-pos-well 10 8.4 887.32
CP0280-pos-well 10 0.0 0 CP0280-pos-well 11 7.2 599.19
CP0280-pos-well 11 8.2 887.42 CP0280-pos-well 11 0.0 0
CP0280-pos-well 12 7.3 887.52 CP0280-pos-well 12 8.3 889.32
CP0280-pos-well 12 9.4 1838 17 CP0280-pos-well 13 7.9 381.29
CP0280-pos-well 13 8.4 871.34 CP0280-pos-well 13 6.7 4096 34
CP0280-pos-well 13 9.1 5968 43 CP0280-pos-well 14 0.0 0.00
CP0280-pos-well 14 8.8 8046 110 CP0280-pos-well 16 0.0 0.00
CP0280-pos-well 16 9.8 1916 -3 CP0280-pos-well 16 9.8 1115 -10
CP0280-pos-well 17 7.6 683.35 CP0280-pos-well 17 7.4 414816 277
CP0280-pos-well 18 7.5 427.14 CP0280-pos-well 18 7.6 799.38
CP0280-pos-well 18 0.0 0 CP0280-pos-well 19 7.7 554.36
CP0280-pos-well 19 7.7 263.18 CP0280-pos-well 19 8.5 871.34
CP0280-pos-well 19 10.8 871.29 CP0280-pos-well 19 0.0 0
CP0280-pos-well 20 7.8 566.23 CP0280-pos-well 20 8.1 609.16
CP0280-pos-well 20 8.2 871.34 CP0280-pos-well 20 8.6 887.32
CP0280-pos-well 20 10.5 887.32 CP0280-pos-well 20 0.0 0
CP0280-pos-well 21 7.8 609.12 CP0280-pos-well 21 8.0 887.32
CP0280-pos-well 21 8.5 887.32 CP028b-pos-well 21 7.7 1078 17
CP0280-pos-well 21 7.9 214442 320 CP0280-pos-well 22 8.2 593.15
CP0280-pos-well 22 9.4 11919 155 CP0280-pos-well 23 8.2 871.34
CP0280-pos-well 23 8.6 17910 26 CP0280-pos-well 23 10.2 2121 14
CP0280-pos-well 24 0.0 0.00 CP0280-pos-well 24 10.5 6622 27
CP0280-pos-well 25 8.6 887.32 CP0280-pos-well 25 9.3 283863 216
CP0280-pos-well 26 8.0 887.27 CP0280-pos-well 26 8.2 871.34
CP0280-pos-well 26 8.5 871.34 CP0280-pos-well 26 6.4 1087 39
CP0280-pos-well 26 8.9 1350 41 CP0280-pos-well 26 8.9 4990 54
CP0280-pos-well 26 9.4 1255 40 CP0280-pos-well 26 9.7 14074 71
CP0280-pos-well 26 9.9 17711 76 CP0280-pos-well 27 8.3 871.29
CP0280-pos-well 27 8.8 2867 25 CP0280-pos-well 27 8.9 4912 31
CP0280-pos-well 28 7.7 887.42 CP0280-pos-well 28 8.3 887.27
CP0280-pos-well 28 8.4 887.27 CP0280-pos-well 28 8.9 20461 65
CP0280-pos-well 28 9.0 10197 41 CP0280-pos-well 28 9.1 16919 57
CP0280-pos-well 28 9.3 10653 42 CP0280-pos-well 28 10.2 10535 42
CP0280-pos-well 28 10.4 12477 47 CP0280-pos-well 29 7.9 537.25
CP0280-pos-well 29 8.4 871.34 CP0280-pos-well 29 9.8 567.27
CP0280-pos-well 29 9.8 26171 101 CP0280-pos-well 30 0.0 0.00
CP0280-pos-well 30 9.4 3657 54 CP0280-pos-well 30 9.7 100841 815
CP0280-pos-well 30 10.1 4013 59 CP0280-pos-well 31 0.0 0.00
CP0280-pos-well 31 10.3 2605 15 CP0280-pos-well 32 0.0 0.00
CP0280-pos-well 32 9.8 1651 -5 CP0280-pos-well 32 10.2 61537 177
CP0280-pos-well 32 10.5 20734 82 CP0280-pos-well 33 0.0 0.00
CP0280-pos-well 33 9.4 1144 14 CP0280-pos-well 33 10.6 4051 18
CP0280-pos-well 33 10.6 1445 15 CP0280-pos-well 34 8.1 871.34
CP0280-pos-well 34 8.2 871.34 CP0280-pos-well 34 8.3 871.34
CP0280-pos-well 34 8.4 871.29 CP0280-pos-well 34 10.8 2058 45
CP0280-pos-well 36 8.2 871.34 CP0280-pos-well 36 8.5 871.34
CP0280-pos-well 36 9.3 1758 17 CP0280-pos-well 36 9.4 1121 15
CP0280-pos-well 36 9.7 1182 15 CP0280-pos-well 37 8.0 1158.98
CP0280-pos-well 37 8.7 901.36 CP0280-pos-well 37 10.4 901.31
CP0280-pos-well 37 0.0 0 CP0280-pos-well 38 0.0 0.00
CP0280-pos-well 38 8.3 3221 48 CP0280-pos-well 38 8.5 1586 26
CP0280-pos-well 39 7.9 871.34 CP0280-pos-well 39 8.2 901.36
CP0280-pos-well 39 8.7 901.31 CP0280-pos-well 39 10.4 5352 17
CP0280-pos-well 40 0.0 0.00 CP0280-pos-well 40 8.7 3366 8
CP0280-pos-well 40 9.3 15515 65 CP0280-pos-well 40 9.5 2419 1
CP0280-pos-well 40 9.7 7411 31 CP0280-pos-well 40 9.9 2738 3
CP0280-pos-well 40 10.1 2915 5 S390E 1 F3-pos-061501- 0.0 0.00
we116 S390E 1 F3-pos-061501- 5.9 1647 35 we116 S390E1
F3-pos-061501-weII7 0.0 0.00 S39OE1 F3-pos-061501-weII7 6.0 176993
250 S390E 1 F3-pos-061501- 0.0 0.00 we118 S390E 1 F3-pos-061501-
5.7 5585 29 we118 S390E 1 F3-pos-061501- 5.8 18067 98 we118 S390E 1
F3-pos-061501- 8.0 437.28 we1120 S39OE1 F3-pos-061501- 0.0 0 we1120
S390E 1 F3-pos-061501- 8.1 705.59 we1122 S390E1
F3-pos-061501-weII22 8.3 553.48 S390E1 F3-pos-061501-weII22 8.2
3589 71 S390E1 F3-pos-061501-weII22 8.3 1545 33 S390E 1
F3-pos-061501- 0.0 0.00 we1123 S390E 1 F3-pos-061501- 8.4 7938 145
we[123 S390E 1 F3-pos-061501- 8.6 139135 228 we1123 S390E 1
F3-pos-061501- 9.1 13241 149 we1123 S390E 1 F3-pos-061501- 0.0 0.00
we1125 S390E 1 F3-pos-061501- 9.3 3760 23 we1125 S390E 1
F3-pos-061501- 9.0 557.51 we1127 S390E 1 F3-pos-061501- 9.0 2536 32
we1127 CP0084-pos-071401-well 42 9.2 297.27 CP0084-pos-071401-well
42 0.0 0 CP0084-pos-071401-well 44 3.9 611.12
CP0084-pos-071401-well 44 4.0 465.06 CP0084-pos-071401-well 44 4.3
449.08 CP0084-pos-071401-well 44 4.6 609.14 CP0084-pos-071401-well
44 4.7 609.14 CP0084-pos-071401-well 44 4.8 609.14
CP0084-pos-071401-well 44 3.2 31400 87 CP0084-pos-071401-well 44
3.4 27533 79 CP0084-pos-071401-well 44 3.7 97219 182
CP0084-pos-071401-well 44 3.8 258754 331 CP0084-pos-071401-well 44
4.0 381435 415 CP0084-pos-071401-well 44 4.3 255354 328
CP0084-pos-071401-well 44 4.6 360258 401 CP0084-pos-071401-well 45
4.0 449.08 CP0084-pos-071401-well 45 4.6 609.14
CP0084-pos-071401-well 45 6.0 284.06 CP0084-pos-071401-well 45 3.7
39613 128 CP0084-pos-071401-well 45 3.8 84079 194
CP0084-pos-071401-well 45 3.9 22269 92 CP0084-pos-071401-well 45
4.0 89048 200 CP0084-pos-071401-well 45 4.6 12875 67
CP0084-pos-071401-well 46 4.4 465.06 CP0084-pos-071401-well 46 4.7
449.08 CP0084-pos-071401-well 46 5.3 609.14 CP0084-pos-071401-well
46 6.4 505.11 CP0084-pos-071401-well 46 4.5 309388 1684
CP0084-pos-071401-well 46 4.8 214596 1341 CP0084-pos-071401-well 46
5.2 188751 1236 CP0084-pos-071401-well 46 6.6 1372 23
CP0084-pos-071401-well 47 4.6 449.08 CP0084-pos-071401-well 47 5.2
609.14 CP0084-pos-071401-well 47 7.4 315.06 CP0084-pos-071401-well
47 4.3 65914 138 CP0084-pos-071401-well 47 4.5 3500 106
CP0084-pos-071401-well 47 4.8 401700 272 CP0084-pos-071401-well 47
5.3 358997 257 CP0084-pos-071401-well 48 0.0 0.00
CP0084-pos-071401-well 48 3.6 53484 161 CP0084-pos-071401-well 48
3.8 101001 240 CP0084-pos-071401-well 48 4.1 8331 36
CP0084-pos-071401-well 48 4.2 8163 35 CP0084-pos-071401-well 48 4.3
97080 235 CP0084-pos-071401-well 48 4.4 7306 31
CP0084-pos-071401-well 49 4.6 609.14 CP0084-pos-071401-well 49 6.4
301.06 CP0084-pos-071401-well 49 4.2 63588 75
CP0084-pos-071401-well 49 4.3 171295 152 CP0084-pos-071401-well 49
4.6 135488 129 CP0084-pos-071401-well 49 4.7 239959 192
CP0084-pos-071401-well 49 4.8 446792 291 CP0084-pos-071401-well 50
4.3 449.08 CP0084-pos-071401-well 50 4.6 609.14
CP0084-pos-071401-well 50 6.4 301.06 CP0084-pos-071401-well 50 6.9
315.06 CP0084-pos-071401-well 50 9.2 297.27 CP0084-pos-071401-well
50 4.2 2769 47 CP0084-pos-071401-well 50 4.3 182613 174
CP0084-pos-071401-well 50 4.7 347900 229 CP0084-pos-071401-well 50
4.8 438416 253 CP0084-pos-071401-well 50 6.4 2520 47
CP0084-pos-071401-well 51 4.7 639.15 CP0084-pos-071401-well 51 4.8
609.14 CP0084-pos-071401-well 51 5.1 609.14 CP0084-pos-071401-well
51 6.6 301.06 CP0084-pos-071401-well 51 7.2 315.06
CP0084-pos-071401-well 51 4.4 7673 39 CP0084-pos-071401-well 51 4.5
3917 28 CP0084-pos-071401-well 51 4.6 4726 30
CP0084-pos-071401-well 51 4.9 1100704 810 CP0084-pos-071401-well 52
4.5 609.14 CP0084-pos-071401-well 52 4.9 609.14
CP0084-pos-071401-well 52 5.0 609.14 CP0084-pos-071401-well 52 6.3
301.06 CP0084-pos-071401-well 52 6.9 315.06 CP0084-pos-071401-well
52 4.1 15611 54 CP0084-pos-071401-well 52 4.3 18267 60
CP0084-pos-071401-well 52 4.5 1123962 760 CP0084-pos-071401-well 52
5.3 9109 38 CP0084-pos-071401-well 52 6.3 7879 35
CP0084-pos-071401-well 53 4.3 609.14 CP0084-pos-071401-well 53 4.5
609.14 CP0084-pos-071401-well 53 4.7 609.14 CP0084-pos-071401-well
53 5.9 301.06 CP0084-pos-071401-well 53 6.5 315.06
CP0084-pos-071401-well 53 3.7 1107 17 CP0084-pos-071401-well 53 3.9
5572 41 CP0084-pos-071401-well 53 4.1 9365 56
CP0084-pos-071401-well 53 4.4 743714 611 CP0084-pos-071401-well 53
4.7 166860 280 CP0084-pos-071401-well 54 5.2 609.14
CP0084-pos-071401-well 54 5.3 609.14 CP0084-pos-071401-well 54 5.7
463.08 CP0084-pos-071401-well 54 6.8 301.06 CP0084-pos-071401-well
54 5.1 3860 57 CP0084-pos-071401-well 54 5.4 54396 525
CP0084-pos-071401-well 54 5.8 5941 84 CP0084-pos-071401-well 55 5.4
593.14 CP0084-pos-071401-well 55 5.8 463.08 CP0084-pos-071401-well
55 5.5 303221 237 CP0084-pos-071401-well 55 5.9 88108 148
CP0084-pos-071401-well 56 0.0 0.00 CP0084-pos-071401-well 56 4.8
13550 58 CP0084-pos-071401-well 57 5.3 463.08
CP0084-pos-071401-well 57 5.4 7767 22 CP0084-pos-071401-well 58 5.4
427.23 CP0084-pos-071401-well 58 9.2 297.27 CP0084-pos-071401-well
58 0.0 0 CP0084-pos-071401-well 60 8.9 323.31
CP0084-pos-071401-well 60 0.0 0 CP0084-pos-071401-well 66 9.4
297.27 CP0084-pos-071401-well 66 0.0 0 CP0084-pos-071401-well 68
9.2 297.27 CP0084-pos-071401-well 68 0.0 0 CP0084-pos-071401-well
76 8.5 537.27 CP0084-pos-071401-well 76 9.3 297.27
CP0084-pos-071401-well 76 8.5 7987 35 CP0084-pos-071401-well 77 7.9
537.27 CP0084-pos-071401-well 77 8.1 515.28 CP0084-pos-071401-well
77 8.7 539.29 CP0084-pos-071401-well 77 9.0 449.25
CP0084-pos-071401-well 77 8.0 1615 20 CP0084-pos-071401-well 77 8.1
36550 122 CP0084-pos-071401-well 77 8.8 5194 39
CP0084-pos-071401-well 80 8.9 595.34 CP0084-pos-071401-well 80 9.1
507.26 CP0084-pos-071401-well 80 0.0 0 CP0074-pos-071301-well 44
2.3 1316.59 CP0074-pos-071301-well 44 9.0 323.28
CP0074-pos-071301-well 44 2.3 5063 27 CP0074-pos-071301-well 44 2.8
15384 54 CP0074-pos-071301-well 44 3.0 18253 60
CP0074-pos-071301-well 44 3.2 89786 173 CP0074-pos-071301-well 44
3.5 42091 105 CP0074-pos-071301-well 45 6.0 284.06
CP0074-pos-071301-well 45 3.2 1624 20 CP0074-pos-071301-well 46 2.9
219.11 CP0074-pos-071301-well 46 4.0 218.10 CP0074-pos-071301-well
46 54 407.17 CP0074-pos-071301-well 46 6.1 217.08
CP0074-pos-071301-well 46 6.8 284.06 CP0074-pos-071301-well 46 1.1
19258 232 CP0074-pos-071301-well 46 4.5 3175 47
CP0074-pos-071301-well 46 5.4 3414 51 CP0074-pos-071301-well 46 5.6
1261 21 CP0074-pos-071301-well 46 6.9 10297 136
CP0074-pos-071301-well 49 4.3 449.08 CP0074-pos-071301-well 49 6.1
505.11 CP0074-pos-071301-well 49 4.1 25263 40
CP0074-pos-071301-well 49 4.4 63256 75 CP0074-pos-071301-well 49
5.4 8915 23 CP0074-pos-071301-well 49 5.5 5862 20
CP0074-pos-071301-well 49 5.9 14723 29 CP0074-pos-071301-well 49
6.1 29956 45 CP0074-pos-071301-well 50 4.6 449.08
CP0074-pos-071301-well 50 5.2 609.14 CP0074-pos-071301-well 50 5.7
463.08 CP0074-pos-071301-well 50 4.4 1559 42 CP0074-pos-071301-well
50 4.6 22816 82 CP0074-pos-071301-well 50 5.3 3572 50
CP0074-pos-071301-well 50 5.7 41999 100 CP0074-pos-071301-well 51
4.7 449.08 CP0074-pos-071301-well 51 5.4 609.18
CP0074-pos-071301-well 51 5.8 463.08 CP0074-pos-071301-well 51 4.7
6529 36 CP0074-pos-071301-well 51 5.8 23113 76
CP0074-pos-071301-well 52 4.5 625.14 CP0074-pos-071301-well 52 5.4
463.08 CP0074-pos-071301-well 52 6.9 315.06 CP0074-pos-071301-well
52 8.8 297.27 CP0074-pos-071301-well 52 4.5 42312 106
CP0074-pos-071301-well 52 4.7 3209 22 CP0074-pos-071301-well 52 4.9
3995 24 CP0074-pos-071301-well 52 5.4 38681 100
CP0074-pos-071301-well 53 4.4 609.14 CP0074-pos-071301-well 53 4.6
609.14 CP0074-pos-071301-well 53 4.8 609.14 CP0074-pos-071301-well
53 5.1 463.12 CP0074-pos-071301-well 53 6.6 315.06
CP0074-pos-071301-well 53 4.3 22471 93 CP0074-pos-071301-well 53
4.4 56644 156 CP0074-pos-071301-well 53 4.5 183171 294
CP0074-pos-071301-well 53 4.6 239990 339 CP0074-pos-071301-well 53
4.8 34417 118 CP0074-pos-071301-well 54 4.1 625.14
CP0074-pos-071301-well 54 4.6 640.16 CP0074-pos-071301-well 54 4.9
609.14 CP0074-pos-071301-well 54 5.3 609.14 CP0074-pos-071301-well
54 6.7 301.06 CP0074-pos-071301-well 54 4.2 7689 105
CP0074-pos-071301-well 54 4.7 16326 202 CP0074-pos-071301-well 54
5.0 84774 3050 CP0074-pos-071301-well 54 5.8 2281 35
CP0074-pos-071301-well 54 6.5 1490 24 CP0074-pos-071301-well 55 4.7
640.16 CP0074-pos-071301-well 55 5.0 609.14 CP0074-pos-071301-well
55 5.3 609.14 CP0074-pos-071301-well 55 5.8 463.08
CP0074-pos-071301-well 55 7.4 315.06 CP0074-pos-071301-well 55 4.3
38220 124 CP0074-pos-071301-well 55 4.6 35784 123
CP0074-pos-071301-well 55 4.8 117214 162 CP0074-pos-071301-well 55
5.0 1625216 586 CP0074-pos-071301-well 55 5.9 113698 160
CP0074-pos-071301-well 57 5.5 463.12 CP0074-pos-071301-well 57 7.0
315.06 CP0074-pos-071301-well 57 4.8 84974 93
CP0074-pos-071301-well 57 5.0 77894 87 CP0074-pos-071301-well 57
5.1 18199 33 CP0074-pos-071301-well 57 5.3 12286 27
CP0074-pos-071301-well 57 5.5 58311 71 CP0074-pos-071301-well 58
5.4 447.09 CP0074-pos-071301-well 58 5.6 463.12
CP0074-pos-071301-well 58 7.3 315.06 CP0074-pos-071301-well 58 5.0
9741 64 CP0074-pos-071301-well 58 5.1 6228 57
CP0074-pos-071301-well 58 5.3 2204 45 CP0074-pos-071301-well 58 5.4
23691 83 CP0074-pos-071301-well 58 5.7 16324 74
CP0074-pos-071301-well 59 5.8 463.12 CP0074-pos-071301-well 59 7.5
315.06 CP0074-pos-071301-well 59 5.1 3218 26 CP0074-pos-071301-well
59 5.3 1925 22 CP0074-pos-071301-well 59 5.8 3736 28
CP0074-pos-071301-well 59 7.5 1397 20 CP0074-pos-071301-well 60 5.4
463.12 CP0074-pos-071301-well 60 5.5 345.21 CP0074-pos-071301-well
60 6.9 315.06 CP0074-pos-071301-well 60 8.7 323.28
CP0074-pos-071301-well 60 4.7 3037 21 CP0074-pos-071301-well 60 5.4
1639 17 CP0074-pos-071301-well 60 6.9 4304 25
CP0074-pos-071301-well 61 5.5 274.17 CP0074-pos-071301-well 61 6.6
315.06 CP0074-pos-071301-well 61 4.5 2946 28 CP0074-pos-071301-well
61 6.6 1247 18 CP0074-pos-071301-well 62 5.0 609.14
CP0074-pos-071301-well 62 7.2 315.06 CP0074-pos-071301-well 62 5.1
4548 66 CP0074-pos-071301-well 63 6.8 338.07 CP0074-pos-071301-well
63 7.4 315.06 CP0074-pos-071301-well 63 5.2 4206 107
CP0074-pos-071301-well 63 7.6 1478 105 CP0074-pos-071301-well 67
8.1 478.29 CP0074-pos-071301-well 67 0.0 0 CP0074-pos-071301-well
68 6.9 315.06 CP0074-pos-071301-well 68 8.8 297.27
CP0074-pos-071301-well 68 0.0 0 CP0074-pos-071301-well 69 6.6
315.06 CP0074-pos-071301-well 69 0.0 0 CP0074-pos-071301-well 75
8.1 677.30 CP0074-pos-071301-well 75 8.1 1599 21
CP0074-pos-071301-well 76 7.5 677.35 CP0074-pos-071301-well 76 9.0
297.27 CP0074-pos-071301-well 76 7.4 2124 18 CP0074-pos-071301-well
76 7.6 44841 110 CP0074-pos-071301-well 77 7.3 677.35
CP0074-pos-071301-well 77 7.8 517.29 CP0074-pos-071301-well 77 7.3
21262 90 CP0074-pos-071301-well 77 7.8 8918 54
CP0074-pos-071301-well 78 9.0 331.27 CP0074-pos-071301-well 78 0.0
0 CP0490-well 3 1.4 235.63 CP0490-well 3 3.4 325.57 CP0490-well 3
2.6 4130 19 CP0490-well 3 2.7 2367 16 CP0490-well 3 2.9 3532 18
CP0490-well 3 2.9 3528 18 CP0490-well 3 3.0 4137 19 CP0490-well 3
3.4 11487 32 CP0490-well 4 1.4 235.63 CP0490-well 4 2.2 251.61
CP0490-well 4 2.8 251.61 CP0490-well 4 1.9 1436 11 CP0490-well 4
2.8 2858 14 CP0490-well 4 3.0 4833 18 CP0490-well 4 3.3 1222 10
CP0490-well 5 1.4 235.63 CP0490-well 5 2.8 188.47 CP0490-well 5 2.5
1836 15 CP0490-well 5 2.9 18201 55 CP0490-well 5 3.3 4614 24
CP0490-well 6 1.3 235.63 CP0490-well 6 2.8 188.47 CP0490-well 6 2.6
1099 12 CP0490-well 6 2.6 1689 18 CP0490-well 6 2.7 1764 19
CP0490-well 6 2.8 2439 25 CP0490-well 6 2.9 4971 47 CP0490-well 7
1.1 235.60 CP0490-well 7 2.9 38531 27 CP0490-well 7 3.4 11926 15
CP0490-well 7 3.4 8896 13 CP0490-well 8 1.2 235.60 CP0490-well 8
2.6 3726 7 CP0490-well 8 2.9 2184 -1 CP0490-well 8 3.1 6001 16
CP0490-well 8 3.2 2479 1 CP0490-well 8 3.2 2038 -1 CP0490-well 8
3.5 13949 39 CP0490-well 9 1.1 235.63 CP0490-well 9 3.4 3586 11
CP0490-well 10 1.4 235.63 CP0490-well 10 3.5 1466 28 CP0490-well 11
1.5 235.63 CP0490-well 11 4.0 1819 14 CP0490-well 12 1.4 235.63
CP0490-well 12 3.8 7450 23 CP0490-well 12 4.0 43248 72 CP0490-well
12 4.1 34152 61 CP0490-well 14 1.3 235.63 CP0490-well 14 3.9 34804
250
CP0490-well 14 4.1 188525 823 CP0490-well 14 4.3 21972 173
CP0490-well 15 1.2 235.63 CP0490-well 15 4.2 287.44 CP0490-well 15
4.0 1229 9 CP0490-well 15 4.2 238118 100 CP0490-well 15 4.4 359411
135 CP0490-well 16 1.4 235.63 CP0490-well 16 4.3 287.44 CP0490-well
16 4.5 38689 87 CP0490-well 17 1.2 235.63 CP0490-well 17 4.8 6709
14 CP0490-well 18 1.4 235.63 CP0490-well 18 4.8 609.41 CP0490-well
18 5.7 549.39 CP0490-well 18 4.8 236312 129 CP0490-well 18 5.0 1654
28 CP0490-well 18 5.7 13676 47 CP0490-well 19 1.4 235.63
CP0490-well 19 4.7 609.41 CP0490-well 19 7.0 315.49 CP0490-well 19
4.7 271230 243 CP0490-well 19 5.0 4392 20 CP0490-well 19 5.7 3995
19 CP0490-well 19 7.0 6414 23 CP0490-well 20 1.4 235.63 CP0490-well
20 4.7 609.41 CP0490-well 20 4.8 151322 160 CP0490-well 20 5.0 1980
12 CP0490-well 20 5.1 4917 18 CP0490-well 21 0.0 0.00 CP0490-well
21 4.8 1968 15 CP0490-well 21 5.1 14686 48 CP0490-well 21 5.2 1910
15 CP0490-well 22 5.4 463.43 CP0490-well 22 5.0 1305 14 CP0490-well
22 5.1 2619 27 CP0490-well 22 5.4 22542 176 CP0490-well 22 7.2 1089
12 CP0490-well 24 5.7 311.66 CP0490-well 24 5.8 2782 3 CP0490-well
25 5.6 311.66 CP0490-well 25 6.0 2203 10 CP0490-well 28 0.0 0.00
CP0490-well 28 4.8 1029 10 CP0490-well 28 6.5 21523 45 CP0490-well
29 6.4 301.48 CP0490-well 29 6.4 3457 21 CP0490-well 34 1.4 235.65
CP0490-well 34 7.4 285.49 CP0490-well 34 7.8 518.60 CP0490-well 34
7.5 1278 27 CP0490-well 36 1.4 235.65 CP0490-well 36 0.0 0
CP0490-well 37 1.4 235.65
EXAMPLE 3
[0230] This example describes a high-throughput method for the
production, analysis, and characterization of large libraries of
small-molecules from natural resources, which accelerates the
natural product drug discovery process for pharmaceutical and
biotech industries. The library production process integrates
automated flash chromatography, solid phase extraction, filtration,
and parallel four-channel preparative high-performance liquid
chromatography (HPLC) to produce the libraries in 96- or 384-well
plates. The libraries consist of purified fractions with
approximately one to five compounds per well. The libraries are
analyzed prior to biological screening by a parallel eight channel
LC-ELSD-MS system that determines the molecular weight and the
number and quantity of compounds in a fraction. After biological
screening of the libraries, active compounds are rapidly purified
and activities are confirmed. The structures of active compounds
discovered in the libraries are elucidated using a combination of
NMR data, acquired on about 50 micrograms of material using a
Bruker Avance 600 MHz NMR spectrometer equipped with a novel
capillary (5 .mu.L) microcoil flow probe and MS data from the LC-MS
database. Using these high-throughput methods, a natural product
library containing 36,000 fractions from diverse plant collections
were produced and analyzed, and screened in various drug discovery
programs. As a demonstration, a small-molecule library was made
from the stem bark of Taxus brevifolia. Biological screening in the
NCI in vitro panel of 3 cancer cell lines demonstrates that the
library enables the discovery of highly active anticancer
compounds, whose activities are not detected in the flash fractions
from which the library originates.
[0231] According to this example, high-throughput technologies may
be applied to generate large libraries of purified fractions of
small-molecule natural products rapidly for HTS. The production
process begins with the fractionation of polar and non-polar plant
extracts using automated flash chromatography. The resulting flash
fractions are subjected to solid-phase extraction to remove tannins
and to molecular weight cut-off filters to remove high molecular
weight components in flash fractions from polar extracts. Flash
fractions are further separated using a parallel four-channel
reversed-phase preparative HPLC system resulting in fractions,
which contain a mixture of about 5 compounds/well. The fractions
are analyzed by a parallel eight-channel analytical LC-ELSD-MS
system. The fractions containing detectable compounds are
collectively called "the library", from which more focused
libraries are drawn for biological screening. After biological
screening, the individual compounds of the active fractions are
rapidly purified and the activities of the compounds are confirmed.
Using a Bruker Avance 600 MHz NMR spectrometer equipped with a new
capillary (5 .mu.l) microcoil flow probe with an active volume of
only 1.5 .mu.L, 5-10 .mu.g of a pure compound is sufficient for 1H
and COSY experiments to characterize and dereplicate known
structures. To characterize novel structures, approximately 50
.mu.g pure compound is needed to acquire additional experiments
such as a gHMQC and a gHMBC.
[0232] The high-throughput method described herein was used in the
production process of large libraries of small-molecule natural
products and to illustrate the process a library was prepared from
an extract of Taxus brevifolia, the pacific yew tree containing
paclitaxel (Taxol) and many of its derivatives. The resulting Taxus
library was analyzed by parallel eight-channel LC-ELSD-MS. Library
fractions were screened by the National Cancer Institute (NCI) in 3
cancer cell lines. Screening results indicate that the presentation
of natural products to biological screening in the form of
libraries produced as described here, enables the discovery of
highly active, minor metabolites whose activities would otherwise
go undetected. Several flash fractions obtained during the
production of the Taxus library did not show activity in the
assays, whereas several library fractions resulting from these
flash fractions did. The active compounds,
7-(.beta.-xylosyl)-taxol, 7-(.beta.-xylosyl)-taxol C, and
7-(.beta.-xylosyl)-10-deacetyltaxol C, were quickly dereplicated
using their molecular weights determined during LC-ELSD-MS analysis
of the library and the structures were characterized using 1H and
COSY experiments. These compounds were previously reported from the
stem bark of Taxus baccata and possess potent activity against B16
melanoma.
[0233] The instrumentation used in the method of this example was
as follows. Flash chromatography separations were performed on 50
gram Si and C18 flash columns (International Sorbent Technology
Ltd., Mid Glamorgan, UK) using a Flash Master II automated
chromatographic system (Jones Chromatography Inc., Lakewood,
Colo.). The removal of tannins was performed using a 500 mg or a
2.5 g polyamide-filled cartridge (Jones Chromatography Inc.).
Preparative HPLC separations were performed on Betasil C18 columns
(20.times.100 mm, 5 .mu.m, Keystone Scientific Inc., Bellefonte,
Pa.). A parallel four-channel preparative HPLC system was assembled
and consisted of 4 Beckman System Gold 126 gradient HPLC pumps
(Beckman Coulter Inc., Fullerton, Calif.) with system controllers
and four-way solvent delivery modules, 4 Beckman System Gold 166
single wavelength UV detectors with preparative flow cells, a
Gilson 215/849 multiple probe autosampler (Gilson Inc., Middleton,
Wis.), and 4 Gilson 204 fraction collectors. The system was
controlled by Beckman 32 Karat chromatography software. A Mega 1200
evaporator (Genevac Technologies, Suffolk, UK) was used to remove
solvents from the preparative HPLC fractions. The preparative HPLC
fractions were transferred from tubes to 96-deep-well plates by a
Packard MultiProbe II liquid handling system (Packard BioScience
Company, Meriden, Conn.). Focused libraries for screening were
prepared in either 96- or 384-well plates using the same liquid
handling system. A Genevac HT-12 evaporator was used to remove
solvents from the 96- and 384-well plates. A parallel eight-channel
LC-ELSD-MS system was assembled and consisted of a LCT
time-of-flight mass spectrometer with an eight-way MUX electrospray
interface (Micromass Ltd, Manchester, UK), a Waters 600E
Multisolvent Delivery System (Wasters Corporation, Milford, Mass.)
to pump solvents through an eight-way manifold which splits the
flow to 8 HPLC columns (4.6.times.50 mm, 3 .mu.m, Keystone Betasil
C-18), a Gilson 215/889 multiple probe autosampler, and 8 Alltech
500 ELSD detectors (Alltech Associates Inc., Deerfield, Ill.). The
system was controlled by MicroMass MassLynx software. Data analysis
was performed using the OpenLynx Software followed by Extractor, a
customized software package developed for Sequoia Sciences by Koch
Associates, La Jolla, Calif. The isolation of individual compounds
was performed using semi-preparative Keystone Betasil C18 or C8
columns (8.times.250 mm I.D., 5 .mu.m) on a single channel Beckman
HPLC system consisting of a Beckman 168 diode array UV detector,
Sedex ELSD detector (Richard Scientific Inc., Novato, Calif.), and
Gilson 204 fraction collector (a splitter is used to split the flow
in 10:90 to ELSD and fraction collector). Size exclusion
chromatographic analyses were conducted on a single channel
analytical Beckman HPLC system using Macrosphere GPC column
(4.6.times.250 mm, 7 .mu.m, Alltech Associates, Inc.) and Sedex
ELSD detector. NMR data for the structure elucidation of compounds
were acquired utilizing a Bruker Avance 600 MHz NMR system (Bruker,
Rheinstetten, Germany) and a 5 .mu.L capillary microcoil NMR flow
probe with 1.5 .mu.L active volume (Magnetic Resonance
Microsensors, Savoy, Ill.), a Harvard 22 syringe pump (Harvard
Apparatus Inc., Holliston, Mass.) and a Valco 6 port injection
valve (Valco Instruments Co. Inc., Houston, Tex.) and 3 .mu.L loop
as the sample loading device.
[0234] Plant samples consisted of whole plant material or separated
plant parts such as roots, stems, leafs, flowers and fruits, or
various combinations of parts. Plant samples from Gabon were dried
immediately after collection above a gas-powered plant drier. Plant
samples from the USA were shipped frozen. Frozen plants samples
were lyophilized upon arrival. Low purity Taxol (extract of the
stem bark of Taxus brevifolia adsorbed onto silica) was purchased
from Hauser Chemical Research Inc. (Boulder, Colo.).
[0235] Dried plant material (150 g) was ground to a homogenous
powder. The powder was sonicated for 30 minutes in an organic
solvent mixture of EtOH:EtOAc (50:50) followed by vigorous shaking
for exhaustive extractions (two times, 4 and 8 hours each). After
filtration and removing the organic solvents by rotary evaporation
the organic extract was obtained. The remaining residue was
exhaustively extracted using an aqueous solvent mixture of H2O:MeOH
(30:70) (two times, 4 and 8 hours each). The aqueous extract was
obtained after removing the solvents by rotary evaporation. The low
purity taxol powder was exhaustively extracted with EtOH:EtOAc
(50:50). After filtration the taxol preparation was dried by rotary
evaporation. This taxol extract (TX001) was treated as an organic
extract.
[0236] Organic extract material (1 g) was dissolved in 5 mL
MeOH:EtOAc (50:50) and adsorbed onto 5 g of silica powder. The
dried powder was brought onto a 50 g silica column and eluted on
the flash chromatography system using a step gradient of 1) 75%
hexanes, 25% EtOAc, 2) 50% hexanes, 50% EtOAc, 3) 100% EtOAc, 4)
75% EtOAc, 25% MeOH, 5) 50% EtOAc, 50% MeOH. The Flash Master II
was modified to collect large fractions of 250 mL of solvent per
gradient step. The system was set up to perform automated
separations of 10 samples per loading. Flash fraction 1 was
discarded, whereas fractions 2 to 5 were dried by rotary
evaporation. Flash fractions 4 and 5 were screened for the presence
of tannins by LC-MS and passed over a 2.5 g polyamide column if
results were positive. Flash fractions produced from the Taxus
organic extract were named TX002 to TX005. Aqueous extract (2 g)
was dissolved into 10 mL of water and the resulting suspension was
centrifuged. The aqueous layer was brought onto a 50 g C18 column
(pre-rinsed with 1 column volume methanol and 5 column volumes
water). Any insoluble material was again dissolved into 10 mL water
using sonication. The suspension was centrifuged again. The aqueous
layer was also brought onto the column. The column was then rinsed
with 5 column volumes water and the effluent was discarded. The
remaining insoluble material was subsequently taken into 10 mL
methanol. The methanol layer was brought onto the column. The
column was eluted with one column volume methanol to remove water
from the column and a 500 mg polyamide cartridge in methanol was
attached to the bottom of the column. The column was eluted with 5
column volumes methanol. The resulting fraction (flash fraction 6,
100 mg) was dissolved in MeOH:H2O (60:40, 15 mL) and filtered at
3000 g for 8 hours using Centricon filter units with a molecular
weight cut-off of 3000 amu. The retentate, typically 1-2 mL was
discarded. Analytical size exclusion chromatography showed that the
content of high molecular weight constituents (>3000 amu) in the
filtrate was reduced significantly from up to 75% to less than 10%
of the total amount of material using ELSD detection.
[0237] Flash fraction material (50 mg) was dissolved into either
1000 .mu.L of MeOH:EtOAc (70:30) (for flash fractions 2 and 3 of
organic extracts) or 100% MeOH (for flash fractions 4 and 5 of the
organic extracts and flash fraction 6 of the aqueous fraction) and
filtered where necessary. The fractions were separated into 40
fractions (20 mL/min, 1 min per collection per tube) using the
parallel four-channel preparative HPLC system. A different 35 min
gradient was applied to each flash fraction for adequate
separation: flash fraction 2: 40-80% acetonitrile in water, flash
fraction 3: 30-70% acetonitrile in water, flash fraction 4: 20-60%
acetonitrile in water, flash fractions 5 and 6: 10-50% acetonitrile
in water. The 40 tubes containing HPLC fractions were dried in the
Mega 1200 Evaporator. The HPLC fractions were transferred to
96-deep-well plates using the liquid handling system (Packard
MultiProbe II). A Taxus library was made from Taxus flash fractions
(TX002 to TX005) consisting of a total of 160 samples named TX002-1
to 40 to TX005-1 to 40, respectively.
[0238] All samples were analyzed by a parallel eight channel
LC-ELSD-MS system with chromatographic conditions of 5%
acetonitrile in water for the first 1.0 minutes, a linear gradient
of acetonitrile from 5% to 95% in 8.0 minutes, followed by 95%
acetonitrile in water for 1.0 minutes. After each analysis the
column was equilibrated at 5% acetonitrile in water for 2.5
minutes. Data processing was performed automatically starting with
OpenLynx, followed by a customized software package, Extractor, to
automatically extract all graphic information, such as retention
times, mass spectra, and peak integrations, and convert it to text
to allow it to be transferred to a database for storage and
analysis.
[0239] In vitro cytotoxicity tests were conducted at the NCI using
an in vitro 3-cell-line panel consisting of MCF7 breast cancer,
NCI-H460 lung cancer, and SF-268 CNS cancer. Each cell line was
inoculated and incubated in microtiter plates. After 24 hours test
samples were added to a final assay concentration of 2 .mu.g/mL and
the culture was incubated for 48 hours. Results for each test
sample were reported as the percentage of growth of the treated
cells when compared to untreated controls. Compounds which reduced
the growth of any one of the cell lines by 32% or less against
standard were considered to be active.
[0240] The constituents of active library fractions were purified
using a single channel HPLC system. The gradient applied to the
separations was based on the elution profile observed during the
preparative HPLC separation that created the fraction and was
optimized for base line separation of the compounds. The
purification required approximately 100 .mu.g per separation and
the yield per compound was typically in the range of 5 to 50 .mu.g
per compound. Pure compounds were dissolved into 3 .mu.L CD3OD and
loaded onto the microcoil NMR flow probe using a syringe pump
equipped with a sample injection valve and capillary tubing. A
sample of 5 to 50 .mu.g was used to run a 1D 1H spectrum (64 scans,
8 or 16 increments) and a gCOSY spectrum (256 scans, 16
increments). The probe was operated at a temperature of 293K. Pulse
widths were 5.5 .mu.s at a power of 23 dB for the 1H spectra. This
information together with the molecular ions from the LC-ELSD-MS
analysis was used to verify the structure of previously reported
molecules. Novel structures for which 50 .mu.g could be obtained
were identified using additional experiments such as gHMQC and
gHMBC and high-resolution mass spectra were generated by TOF mass
spectrometry for the determination of molecular formula.
[0241] Most drug discovery programs today are capable of screening
large numbers of compounds against multiple targets using
micro-gram quantities of material. To meet the demand for large
numbers of structurally diverse compound libraries, a
high-throughput method to accelerate the natural product drug
discovery process was developed. FIG. 14 is a schematic
presentation that depicts the strategy by which the natural product
libraries were produced and screened, active hits were purified,
and biologically active compounds were characterized for
pharmaceutical discovery programs. Reversed-phase, preparative HPLC
is routine for the purification of pharmaceutical compounds, but
the design and application of the automated, parallel four-channel
preparative system has increased the efficiency of this technique
four-fold. A four-channel preparative HPLC system was customized in
the laboratory. It operates 4 gradient pumping systems
independently but simultaneously, permitting 10 parallel
separations of 4 samples per run. Since the systems are delivering
the effluents independently to each preparative column the
separations for each channel have the same efficiency. Because of
the complexity of natural product extracts, the separation of
naturally occurring drug-like compounds with acceptable resolution
and yield requires several steps of cleanup and pre-separation
procedures before loading samples onto a preparative column. The
purification of plant extracts using normal-phase or reversed-phase
flash chromatography, solid phase extraction using polyamide, and
filtration through molecular weight cut-off filters, removes highly
lipophilic and hydrophilic compounds, pigments, large molecular
weight tannins, polysaccharides, and other non-drug-like molecules.
The optimized gradient chromatography separates 50 mg of flash
fractions and collected at 1 minute per tube with a total of 40
tubes per collection. Based on one sample per hour per channel, a
parallel four-channel system can purify 32 samples in a working
day, which generates 1280 fractions (32.times.40). The preparative
HPLC fractions containing quantifiable compounds make up the
library. These fractions consist of approximately 1-5 compounds per
well and primarily from 0.1 to 1 mg of material. FIG. 15 is a
typical example of chromatograms obtained from one run of a
parallel four-channel preparative HPLC purification of library
fractions. The production process was validated by processing a
single sample repeatedly and comparing the resulting LC-ELSD-MS
data of the preparative HPLC fractions. The LC-ELSD-MS information
for plant samples processed repeatedly proved to be very
reproducible and accurate. So far a library with 36,000 fractions
containing 1-5 compounds per well has been produced.
[0242] To enable the analysis of large numbers of natural products,
a method was developed using a parallel eight-channel LC-ELSD-MS
system. Parallel LC-MS technology has recently been introduced to
combinatorial chemistry and originated from U.S. Pat. No.
6,066,848, May 23, 2000. The methods employ a multiple sampling
mass spectrometry interface now referred to as MUX technology. In
addition to a parallel LC-MS interface, the system incorporates
ELSD as a quantitative tool to determine the quantity of compounds
in each fraction. ELSD is considered a universal detector with
relatively good sensitivity and accuracy. After fractions were
produced by parallel preparative HPLC, the fractions were
transferred to 96-deep-well plates (rows 2-11). Prior to analysis
of a plate, row 1 containing a mixture of 3 standards was injected
to ensure acceptable system performance. The system operated at 10
minutes per run with 8 samples per run, one plate per 2.5 hours, 8
plates per day, and 40 plates per week. After large numbers of
samples were analyzed, the retention time of the standard compounds
shifted slightly. The standard compounds always served as points of
reference and retention times were normalized to the standards.
Data processing by the workstation of the parallel eight-channel
LC-ELSD-MS was quite a challenge because each file contained large
amounts of information. Software (Extractor) was developed to
process these data to get information such as the molecular weights
of the compounds, number of compounds per well, retention time and
the integration of each peak in the chromatogram. At the same time
the automated data processing software compares the standards with
the samples to make corrections for retention time shifts from
different channels of the chromatogram. All data in text format are
exported to a database for characterization and dereplication of
the compounds. FIG. 16 shows a series of typical parallel
eight-channel LC-MS data from the natural product library. The mass
spectra of relevant peaks for sample S001208 fraction 17 are as
follows: Rt 3.31, m/z 491.4, Rt 3.41, m/z 404.5, Rt 3.51, m/z
238.5, Rt 3.75, m/z 597.2, Rt 3.83, m/z 475.5; S001208 fraction 18:
Rt 4.12, m/z 371.2, Rt 4.22, m/z 497.5; S001208 fraction 19: Rt
4.65, m/z 509.4, Rt 4.79, m/z 509.4; S001208 fraction 20: Rt 5.22,
m/z 509.4, Rt 5.30, m/z 799.5; S001208 fraction 21: Rt 5.45, m/z
922.2, Rt 5.53, m/z 1207.3, Rt 5.65, m/z 936.3, Rt 5.79, m/z 813.2;
S001208 fraction 22: Rt 6.38, m/z 904.5, Rt 6.50, m/z 537.4, Rt
6.62, m/z 537.2; S001208 fraction 23: Rt 6.80, m/z 1052.2, Rt 6.90,
m/z 918.4; S001208 fraction 24: Rt 7.25, m/z 1066.3, Rt 7.43, m/z
1066.2, Rt 7.65, m/z 1062.4.
[0243] Sample tracking and archiving of data are important when
producing large natural product libraries. The taxonomic identity
of each plant sample was determined after collection by plant
taxonomists at the Missouri Botanical Garden. If the various plant
parts were separated, a single species of plant yielded multiple
samples. Plants samples were numbered and assigned a unique barcode
after collection. The plant samples were weighed and the weights
were recorded electronically into a relational database together
with their taxonomic information, collection location (GPS
coordinates), and any other ecological information. As a plant or a
sample was further fractionated, the resulting extracts or
fractions were assigned new barcodes at every step of the process.
The use of barcodes and computerized balances ensured electronic
data entry into the database. The database was used at every step
of the process in the selection of samples for purification and to
review information on previously purified samples.
[0244] By this method, 36,000 samples of a natural product library
were made. The library has been screened in various drug discovery
programs against different biological targets. The hit rates have
been 0.5% or lower depending upon the biological assay. Active
compounds were isolated in quantities of 5 to 50 .mu.g from
approximately 100 .mu.g of a preparative HPLC fraction. Since
elution conditions, quantities, and molecular ions of the compounds
in the library are known, the isolation of single compounds with
purities greater than 85% has become a process amenable to
standardization. After a single compound is obtained the biological
activities of the compound are confirmed. The structures of active
compounds were elucidated within a fraction of the time and with a
fraction of the amount needed when compared to conventional natural
products chemistry strategies. The standardization of the
high-throughput library production and compound isolation process
as described in this paper significantly reduced the time needed to
identify active materials. In addition, the combination of the
library production process and the high sensitivities of the latest
NMR and LC-MS technologies, greatly reduced the amount of material
needed for structure elucidation and dereplication, and consistent
with the small amounts of material currently needed for HTS assays.
FIG. 17 is an example of the purification of a bioactive library
fraction containing 4 peaks into a total of 4 single components
using a shallow-gradient, semi-preparative HPLC separation. The
sample loading onto the column was about 100 .mu.g and the recovery
of each peak was approximately 5 to 50 .mu.g. The possibility to
work with such small quantities is an important step forward in
natural product drug discovery.
[0245] High-resolution NMR is a routine tool used by chemists to
elucidate the structures of compounds. Since conventional NMR
employs either 5 or 3 mm tubes, most laboratories need low
milligram quantities of sample to acquire all homo- and
hetero-nuclear correlations for structure determination. If
compounds are mass limited, as in the case of natural products drug
discovery, obtaining low milligram quantities of sample requires
multiple steps of separations and weeks or even months of time.
With the advanced NMR technologies, in particular the microcoil
flow probe (capillary-based microliter-volume flow probe) developed
by Dean Olson and Jonathan Sweedler and now commercialized by MRM
(Magnetic Resonance Microsystems, also called .mu.FlowProbe.TM.),
it dramatically improves the acquisition of NMR spectra on samples
in trace quantities. The new 5 .mu.L microcoil flow probe reduces
the active volume inside the coil to 1.5 .mu.L, when compared to
the conventional microliter-volume flow probes (40-200 .mu.L)
currently on the market, and the placement of the coil directly
around the capillary results in further enhancement of mass
sensitivity (signal to noise per mass unit). This characterization
laboratory has implemented a system that permits one to work
routinely with mass limited samples at the low microgram levels. As
shown in FIG. 18, a syringe pump operating at 5 .mu.L per minute
pushed the sample to the microcoil probe in 2.5 minutes and parked
the sample inside the probe. After the acquisition is completed the
sample was collected in recovery vials. Sample loading was
typically done in 3 .mu.L with 5 to 50 .mu.g quantities. 1D 1H NMR
spectra and high-resolution mass spectra can be generated on
isolated compounds of 5 to 50 .mu.g in quantity providing the
necessary data for characterization and dereplication. Working at
the low microgram level, instead of the low milligram level,
appears to be a tremendous advancement in natural products
chemistry. These techniques should open doors enabling chemists to
readily discover bioactive components among the minor constituents
of natural resources.
[0246] Flash fractions (TX002 to TX005) were produced by processing
1 gram of organic extract (TX001) from Taxus brevifolia. As
previously described, above, a Taxus library was produced from
these flash fractions utilizing a parallel four-channel preparative
HPLC system. A total of 160 preparative HPLC 14 fractions were
collected. Analysis of the fractions by parallel LC-ELSD-MS showed
that the fractions primarily contained 1-5 components. The
quantities of the fractions in the library were from 100 .mu.g to
1.7 mg as determined by ELSD (see FIG. 19). A total of 147
compounds from this library were detected by (+) ESI mass
spectrometry. Among those samples, paclitaxel was identified in
sample TX003-17 (flash fraction TX003, fraction 17) at the purity
greater than 95%. All samples, including the original organic
extract (TX001), flash fractions (TX002-005), and the Taxus library
consisting of 160 preparative HPLC fractions were delivered to the
NCI to be screened in vitro in the 3-cell-line anticancer panel.
The results are presented in Table 3 and show that the organic
extract and flash fractions TX003 and TX004 exhibited anticancer
activities. Sample TX0003-17, which proved to be pure paclitaxel,
exhibited activity in all three cell lines. Samples TX005-28 and
30-32 also exhibited activity on the MCF7 breast cancer cell line,
in which the original TX005 did not show the activities among those
cell lines (see Table 3). The NMR data needed to identify the known
taxanes (1D 1H and COSY) were acquired using samples containing
5-50 .mu.g of pure compound in 3 .mu.L of CD3OD in the microcoil
flow probe. The samples were parked in the coil after two
calibration runs using strychnine as a standard. A 1H NMR spectrum
of paclitaxel is shown in FIG. 20 (50 .mu.g of sample in 3 .mu.L of
CD3OD). 1D 1H and 2D (COSY) NMR spectra as well as mass spectra
allowed characterization of the major peaks from TX005-28, 30, and
31 to be 7-(.beta.-xylosyl)-taxol, 7-(.beta.-xylosyl)-taxol C, and
7-(.beta.-xylosyl)-10-deacetyltaxol C, respectively.
[0247] On average 60% of the analyzed fractions contained
detectable compounds with one to five compounds per fraction. A
total of 36,000 fractions were made and these fractions were
collectively called "the library" from which smaller, more focused
libraries are drawn for screening. Screening results indicate that
hit rates are 0.5% or less and the library facilitates the
discovery of minor metabolites whose activity may go undetected
upon the screening of crude extracts or even flash fractions. Since
libraries are drawn using equal amounts from each fraction, the
concentration of minor metabolites in a screening assay is
comparable to that of major metabolites. Active compounds are
rapidly purified from the fractions and the bioactivities of pure
compounds are then confirmed. Characterization and structure
determination of hit compounds has been done using the LC-ELSD-MS
data and 1H and COSY NMR experiments on as little as 5 .mu.g using
a Bruker Avance 600 MHz NMR spectrometer equipped with a new 5
.mu.L-microcoil flow probe. The structures of novel compounds can
be elucidated using as little as 50 .mu.g with reasonable
experiment times. Any naturally occurring active compounds can be
quickly identified using the LC-ELSD-MS data and purified for
structure activity relationship (SAR) studies during the screening
process.
TABLE-US-00006 TABLE 3 Results of One Dose Primary Anticancer
Assaysa Breast H-460 SF-268 CNS Samples MCF-7 cancer Lung cancer
cancer TX001-1 (2 .mu.g/mL) 31 91 75 TX001-2 (5 .mu.g/mL) 23 27 55
TX001-3 (10 .mu.g/mL) 17 25 43 TX002 (2 .mu.g/mL) 94 117 110 TX003
(2 .mu.g/mL) 19 23 29 TX004 (2 .mu.g/mL) 23 61 60 TX005 (2
.mu.g/mL) 85 107 105 TX003-17 (2 .mu.g/mL) 21 30 29 TX005-28 (2
.mu.g/mL) 21 71 49 TX005-30 (2 .mu.g/mL) 30 93 55 TX005-31 (2
.mu.g/mL) 14 43 38 TX005-32 (2 .mu.g/mL) 15 45 52 Paclitaxel (2
.mu.g/mL)b 23 13 30 Cephalomanine (2 .mu.g/mL)c 15 19 30 aThe
numbers presented in the table showed the percentage of the reduced
growth of the cell lines. Samples have less than 32% are active. b,
cThese are the standard compounds for positive control.
* * * * *