U.S. patent application number 10/890892 was filed with the patent office on 2005-03-24 for compositions and methods for separating heterocyclic aromatic amine bases, nucleosides, nucleotides, and nucleotide sequences.
This patent application is currently assigned to IBC Advanced Technologies, Inc.. Invention is credited to Bruening, Milton, Bruening, Ronald L., Dearden, David Vernell, Haymore, Barry L., Krakowiak, Krzysztof E..
Application Number | 20050064468 10/890892 |
Document ID | / |
Family ID | 26841798 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050064468 |
Kind Code |
A1 |
Bruening, Ronald L. ; et
al. |
March 24, 2005 |
Compositions and methods for separating heterocyclic aromatic amine
bases, nucleosides, nucleotides, and nucleotide sequences
Abstract
The compositions of the present invention comprise one or more
palladium bound ligands that are covalently bonded to inorganic or
organic solid supports. These palladium bound ligands bonded to
solid supports can be used for single heterocyclic amine base
separation, or can be used to separate nucleotide chain containing
specific sequences from other nucleotides or nucleotide chains. In
one aspect of the invention, each ligand present is individually
complexed to a single Pd(II) ion. If there are from 2 to 4 ligands
present in the composition, then each ligand present must be
separated from the other ligands by at least 3 atoms, preferably
from 3 to 20 carbon atoms or equivalent spacing.
Inventors: |
Bruening, Ronald L.;
(American Fork, UT) ; Krakowiak, Krzysztof E.;
(Provo, UT) ; Dearden, David Vernell; (Mapleton,
UT) ; Haymore, Barry L.; (St. Louis, MO) ;
Bruening, Milton; (Springville, UT) |
Correspondence
Address: |
THORPE NORTH & WESTERN, LLP.
8180 SOUTH 700 EAST, SUITE 200
P.O. BOX 1219
SANDY
UT
84070
US
|
Assignee: |
IBC Advanced Technologies,
Inc.
|
Family ID: |
26841798 |
Appl. No.: |
10/890892 |
Filed: |
July 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10890892 |
Jul 13, 2004 |
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10144245 |
May 10, 2002 |
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6774082 |
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60290577 |
May 11, 2001 |
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Current U.S.
Class: |
435/6.12 ;
536/24.3; 544/225 |
Current CPC
Class: |
C07H 21/00 20130101;
C12N 15/1006 20130101 |
Class at
Publication: |
435/006 ;
536/024.3; 544/225 |
International
Class: |
C12Q 001/68; C07H
021/04; C07F 015/00 |
Claims
1-42. (Canceled)
43. A method for concentrating, removing, or separating a desired
heterocyclic aromatic amine base or a nucleotide sequence
containing said desired heterocyclic aromatic amine base from a
source solution comprising the steps of: (a) contacting a source
solution having a first volume with a composition comprising a
palladium(II)-bound ligand covalently bonded to a solid support,
said source solution containing a desired heterocyclic aromatic
amine base and at least one undesired heterocyclic aromatic amine
base, wherein the palladium(II)-bound ligand is preferentially
selective of the desired heterocyclic aromatic amine base; (b)
removing the source solution from contact with said composition to
which said desired heterocyclic aromatic amine base has been
complexed; (c) contacting said composition having said desired
heterocyclic aromatic amine bases associated therewith with a
smaller volume of an aqueous receiving solution in which said
desired heterocyclic aromatic amine bases are stripped from the
composition; and (d) recovering said desired heterocyclic aromatic
amine bases in concentrated form in said receiving solution.
44. A method as in claim 43 wherein said desired heterocyclic
aromatic amine base is guanine (G), and the ligand portion of the
palladium(II)-bound ligand comprises the sequence
--[B--C.sub.x--B--(C.su- b.x--B).sub.y]wherein C is carbon, each B
is independently nitrogen, sulfur, or phosphene; x is 2 or 3; and y
is 0, 1 or 2.
45. A method as in claim 43 wherein said desired heterocyclic
aromatic amine base is adenine (A), and the ligand portion of the
palladium(II)-bound ligand comprises the sequence
-[D-C--N--C-D]wherein C is carbon, N is nitrogen, and D is a
negative binder.
46. A method as in claim 43, wherein said desired heterocyclic
aromatic amine base is thymine (T), and the ligand portion of the
palladium(II)-bound ligand comprises the sequence 111
47. A method as in claim 43, wherein said desired heterocyclic
aromatic amine base is uracil (U), and the ligand portion of the
palladium(II)-bound ligand comprises the sequence 112
48. A method as in claim 43, wherein said desired heterocyclic
aromatic amine base is cytosine (C), and the ligand portion of the
palladium(II)-bound ligand comprises the sequence 113
49. A method as in claim 43, wherein the aqueous receiving solution
is desired from the group consisting of ammonia, ammonia buffer,
ethylene diamine, and thiocyanate.
50. A method as in claim 49, wherein the aqueous receiving solution
is ammonia or ammonia buffer.
51. A method as in claim 43, wherein the aqueous receiving solution
is strong enough to displace the heterocyclic aromatic amine base
from the composition without altering its configuration and is not
strong enough to displace the Pd(II) from the composition.
52. A method as in claim 43, further comprising a washing step to
remove the aqueous receiving solution from the composition for
further use.
53. A method as in claim 43, wherein the composition is configured
for separating individual heterocyclic aromatic amine bases.
54. A method as in claim 43, wherein the composition is configured
for separating heterocyclic aromatic amine bases that are part of
nucleotide sequences.
55. A method for concentrating, removing, or separating a
nucleotide chain containing a specific nucleotide sequence from a
source solution comprising the steps of: (a) identifying a
nucleotide chain that contains a desired nucleotide sequence that
is desired for concentrating, removing, or separating from a source
solution; (b) providing a composition that will selectively bind
the nucleotide sequence; (c) contacting the source solution having
a first volume with the composition wherein a chemical attraction
between the composition and the nucleotide sequence occurs; (d)
removing the source solution from contact with the composition,
said composition having the nucleotide sequence attracted thereto;
(e) contacting the composition having the nucleotide sequence
attracted thereto with a smaller volume of an aqueous receiving
solution in which said nucleotide chain is stripped from the
composition; and (f) recovering the nucleotide chain in
concentrated form in the receiving solution.
56. A method as in claim 55, wherein the nucleotide sequence
contains from 2 to 4 heterocyclic amine bases.
57. A method as in claim 55, wherein the source solution contains
other non-desired nucleotide sequences are present in the source
solution
58. A method as in claim 55, wherein the composition has from 2 to
4 palladium(II)-bound ligands, said palladium(II)-bound ligands
being tethered together by an organic backbone having from 3 to 20
atoms between each palladium(II)-bound ligand, thereby forming a
palladium(II)-bound ligand backbone grouping, said
palladium(II)-bound ligand backbone grouping covalently bonded to a
solid support, wherein each palladium(II)-bound ligand is
independently selective of a desired heterocyclic aromatic amine
base of the nucleotide sequence.
59. A method as in claim 55, wherein the nucleotide chain is the
same as the nucleotide sequence.
60. A method as in claim 55, wherein the nucleotide chain is longer
than the nucleotide sequence.
61-67. (Canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/290,577 filed on May 11, 2001.
FIELD OF THE INVENTION
[0002] The present invention is drawn to compositions and methods
for separating desired heterocyclic aromatic amine bases,
nucleosides, nucleotides, or sequences of DNA using separation
techniques, preferably non-chromatographic separation
techniques.
BACKGROUND OF THE INVENTION
[0003] Deoxyribonucleic Acid (DNA) is generally comprised of four
different types of nucleotides. These nucleotides are comprised of
three components: (1) heterocyclic aromatic amine base; (2)
2-deoxy-D-ribose; and (3) phosphoric acid. The bases are generally
from two N-heterocyclic categories, namely pyrimidine and purine,
the general structures of which are shown below; respectively.
1
[0004] In DNA, the two pyrimidine-type bases are cytosine (C) and
thymine (T). Additionally with respect to DNA, the two purine-type
bases are adenine (A) and guanine (G).
[0005] Likewise, Ribonucleic Acid (RNA) is similar to DNA in that
they too are comprised of long, unbrancehd chains of nucleotides
joined by phosphodiester bonds between the 3'-hydroxyl of one
pentose and the 5'-hydroxyl of an adjacent pentose. However, there
are three main differences between DNA and RNA including: (1) the
pentose unit in the RNA is D-ribose rather than 2-deoxy-D-ribose;
(2) the pyrimidine base found in RNA is uracil (U) rather than
thymine (T); and (3) RNA is single stranded rather than double
stranded. The three pyrimidine-type heterocyclic aromatic amine
bases found in DNA and RNA are shown below: 2
[0006] The two purine-type heterocyclic aromatic amine bases found
in both DNA and RNA are shown below. 3
[0007] In the case of DNA, a nucleoside is a glycoside in which
nitrogen 9 of a purine or nitrogen 1 of a pyrimidine base is bonded
to 2-deoxy-D-ribose. An example is shown below. 4
[0008] Further, with respect to DNA, a nucleotide is a nucleoside
monophosphate ester in which a molecule of phosphoric acid is
esterified with a free hydroxy group of 2-deoxy-D-ribose. An
example is shown below. 5
[0009] In the prior art, the separation of nucleotides has
primarily been accomplished by utilizing separation devices that
selectively bind to the phosphoric acid portion, or the
2-deoxy-D-ribose portion of the DNA. For example, ligand complexes
containing zinc, copper, and nickel have been used to selectively
bind the phosphoric acid portion of a nucleotide. Though the
selectivity of such compounds has been somewhat effective in
separating nucleotides from other impurities, these systems and
methods do not exhibit high selectivity properties when the desire
is to separate one nucleotide from another, e.g., A from T. With
these systems, if the desired heterocyclic amine is merely in the
form of a nucleoside, there is no phosphoric acid portion present
and the separation cannot occur. Additionally, with such
compositions, lone heterocyclic aromatic amine bases cannot be
separated without being present as an intact nucleotide.
SUMMARY OF THE INVENTION
[0010] The compositions and methods of the present invention
comprise one or more palladium bound ligands that are covalently
bonded to inorganic or organic solid supports. These palladium
bound ligands bonded to solid supports can be used to separate
single heterocyclic amine bases, nucleosides, nucleotides, or
nucleotide chains containing specific sequences from other
non-desired molecular units present. In one aspect of the
invention, each ligand present as part of the composition is
individually complexed to a single Pd(II) ion. If there are from 2
to 4 ligands present on a single composition, then each ligand
present can be bound to a palladium ion and each ligand should be
separated from the other ligands by at least 3 atoms, preferably
from 3 to 20 carbon atoms or an equivalent spacer chain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In the accompanying drawings which illustrate embodiments of
the invention;
[0012] FIG. 1 is a schematic representation of an embodiment of the
present invention wherein nucleotide sequence A-G is selected;
and
[0013] FIG. 2 is a schematic representation of an embodiment of the
present invention wherein nucleotide sequence A-G-T-G is
selected.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Before the present invention is disclosed and described, it
is to be understood that this invention is not limited to the
particular process steps and materials disclosed herein because
such process steps and materials may vary somewhat. It is also to
be understood that the terminology used herein is used for the
purpose of describing particular embodiments only. The terms are
not intended to be limiting because the scope of the present
invention is intended to be limited only by the appended claims and
equivalents thereof.
[0015] It is noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the content clearly dictates otherwise.
[0016] "Lower alkyl" includes straight chain alkyl groups having
from 1 to 10 atoms, and branched chain alkyl groups having from 3
to 10 carbon atoms.
[0017] "Heterocyclic aromatic amine bases" include the five bases
that are present in RNA and DNA, namely: cytosine (C), thymine (T),
uracil (U), adenine (A), and guanine (G).
[0018] "Nucleoside" is a glycoside between a nitrogen of the
heterocyclic aromatic amine base and 2-deoxy-D-ribose. For example,
adenine bonded to 2-deoxy-D-ribose is known as deoxyadenosine.
[0019] "Nucleotide" is a nucleoside monophosphate ester. For
example, the nucleoside deoxyadenosine can be esterified with a
free hydroxy group of the 2-deoxy-D-ribose to become
2'-deoxyadenosine 5'-monophosphate.
[0020] "Organic backbone" or "Backbone" refers to an organic chain
having from about 3 to 20 atoms, including carbon atoms, that are
present to tether multiple ligands to one another. Other
constituents that can form part of or be attached to the backbone,
other than carbon, include oxygen, sulfur, nitrogen, and the like,
as long as such atoms do not substantially interfere with the
palladium-bound ligands being supported by the backbone.
[0021] "Palladium(II)-bound ligand backbone grouping" shall include
from 2 to 4 ligands that are tethered together by an organic
backbone. Between each ligand, from about 3 to 20 atoms can be
present to appropriately space the ligands from one another. The
palladium(II)-bound ligand backbone grouping is typically used when
separating nucleotide sequences rather than individual heterocyclic
aromatic amine bases.
[0022] When referring to a specific purine and pyrimidine ring
found in DNA and/or RNA, e.g., cytosine (C), thymine (T), uracil
(U), adenine (A), and/or guanine (G), its isolated form as well as
its nucleoside and nucleotide forms are included. For example, in
the specification and claims where it is stated that a
palladium(II)-bound ligand is selective of adenine, or where
adenine is bound to a palladium(II)-bound ligand, such a statement
shall include not only adenine itself, but deoxyadenosine and
2'-deoxyadenosine 5'-monophosphate. In other words,
adenine-containing molecules are included as the selectivity
described in the present invention is typically with respect to the
adenine-portion of the molecule, as opposed to the 2-deoxy-D-ribose
and/or the monophosphate portion of the nucleotide and/or
nucleoside.
[0023] With these definitions as a reference, a composition for
selectively binding a desired nucleotide sequence can comprise from
2 to 4 palladium(II)-bound ligands. The palladium(II)-bound ligands
can be tethered together by an organic backbone having from 3 to 20
atoms between each palladium(II)-bound ligand, thereby forming a
palladium(II)-bound ligand backbone grouping. The
palladium(II)-bound ligand backbone grouping can also be covalently
bonded to a solid support, wherein each palladium(Il)-bound ligand
is independently selective of a desired heterocyclic aromatic amine
base of the nucleotide sequence. In an alternative embodiment, an
alternative composition can comprise a palladium(II)-bound ligand
covalently bonded to a solid support, wherein the
palladium(II)-bound ligand is further complexed to a heterocyclic
aromatic amine base.
[0024] In a further detailed aspect, the compositions of the
present invention can comprise one or more ligands that are
covalently bonded to an inorganic or organic solid support through
a spacer and are represented by Formula I as follows:
SS-A-X-B.sub.m[L:Pd(II)].sub.n Formula 1
[0025] wherein SS is an inorganic or organic solid support, A is a
covalent linkage mechanism, X is a spacer grouping, B is an organic
backbone, and each L present is a ligand independently selective of
a desired heterocyclic aromatic amine base that may be present in a
source solution with other undesired heterocyclic aromatic amine
bases. As such, n can be an integer from 1 to 4 such that when n is
1, there is one ligand and one Pd(II) ion present; when n is 2,
there are two ligands and two Pd(II) ions present; and so forth. In
one aspect of the invention, each ligand present is individually
complexed to a single Pd(II) ion. When n is 1, m is 0, e.g., there
is no backbone B, and spacer grouping X is attached directly to the
ligand L. When n is from 2 to 4, m is 1 (denoting a backbone B is
present). Though the spacer grouping is shown attached to the
organic backbone, it can also be attached directly to the ligand,
such as when n is 1, and even sometimes when n is from 2 to 4. For
example, the spacer grouping can be attached to a first ligand, and
a second (or third or fourth) ligand can be tethered to the first
ligand by the organic backbone. If there are from 2 to 4 ligands
present in the composition, then each ligand present should be
separated from the other ligands by at least 3 atoms of the
backbone, preferably from 3 to 20 carbon atoms or equivalent spacer
atoms.
[0026] The SS-A-X-- portion of Formula 1 is well known for use with
ion binding ligands. Therefore, when referring to the SS-A-X--
portion of the formula, reference will be made to a solid support
covalently bonded to a ligand (or palladium(II)-bound ligand), or a
ligand covalently bonded to a solid support.
[0027] With respect to specific embodiments of the SS-A-X-- portion
of Formula 1, certain preferred embodiments will be shown and
described. These embodiments are not meant to be limiting, as other
solid supports and covalent linkage mechanisms may be used with
similar success.
[0028] In Formula 1, the solid support (SS) is an inorganic and/or
organic particulate support material selected from the group
consisting of silica, silica gel, silicates, zirconia, titania,
alumina, nickel oxide, glass beads, phenolic resins, polystyrenes,
agarose, sepharose, and polyacrylates. However, other organic
resins or any other hydrophilic organic and/or inorganic support
materials meeting the above criteria can also be used.
[0029] The use of organic ion binding ligands attached to an
SS-A-X-- solid support by means of a covalent linkage spacer
grouping is illustrated in U.S. Pat. Nos. 4,943,375; 4,952,321;
4,959,153; 4,960,882; 5,039,419; 5,071,819; 5,078,978; 5,084,430;
5,173,470; 5,179,213; 5,182,251; 5,190,661; 5,244,856; 5,273,660;
and 5,393,892. These patents, which disclose various spacers that
can be used in forming an organic ligand attached to a solid
support, are fully incorporated herein by reference.
[0030] When the solid support (SS) is an inorganic material such as
silica, silica gel, silicates, zirconia, titania, alumina, nickel
oxide, or glass beads, the covalent linkage (A) is a silane such
that A-X can be represented by Formula 2 as follows: 6
[0031] where each Z is independently selected from the group
consisting of Cl, Br, I, lower alkyl, lower alkoxy, substituted
lower alkyl or substituted lower alkoxy and S (as used herein,
lower alkyl or lower alkoxy means a group having 1 to 10 carbon
atoms); and X can be, for example, a spacer grouping represented by
Formula 3 as follows:
(CH.sub.2).sub.a[OCH.sub.2CHR.sup.1CH.sub.2].sub.b Formula 3
[0032] wherein R.sup.1 is a member selected from the group
consisting of H, SH, OH, lower alkyl, and aryl; a is an integer
from 2 to about 10; and b is 0 or 1. In Formula 3, the terminal
carbon (or --CH.sub.2-- group most distal to the solid support) can
attach to the ligand (or group of ligands) by any suitable bond. It
is preferred that the terminal carbon on the spacer be covalently
bonded to a nitrogen or another carbon present on the ligand or
ligand backbone. However, it is not the purpose of the invention to
describe the point of attachment between the SS-A-X-- portion of
the composition and the L portion of the composition. Any
functional point of attachment may be implemented.
[0033] When the particulate solid support (SS) is an organic resin
or polymer, such as phenolic resins, polystyrenes, and
polyacrylates, it will generally be a hydrophilic polymer or
polymer derivatized to have a hydrophilic surface and contain polar
functional groups. The ligand (L) (or backbone supporting a group
of ligands) will then generally contain a functional grouping
reactive with an activated polar group on the polymer. The covalent
linkage (A) and the spacer (X) can then be integrated, and may
actually be a single linkage, formed by the reaction between the
activated polar group from the polymer and the functional group
from the ligand and may be represented by Formula 4 below:
--(CH.sub.2).sub.u--(Y).sub.v--(CH.sub.2).sub.z-- Formula 4
[0034] where u is 0 or 1; v and z are independently 0 or an integer
from 1 to 10; and Y is a functional group or aromatic linkage such
as an ether (O), sulfide (S), imine (C.dbd.N), carbonyl (CO), ester
(COO), thioester (CSO), amide (CONH), thioamide (CSNH), amine (NH),
lower alkylamine (NR), sulfoxide (SO), sulfone (SO.sub.2),
sulfonamide (SO.sub.2NH), phenylene (C.sub.6H.sub.4), benzylene
(CH.sub.2--C.sub.6H.sub.4), and the like. At least one of x, y or z
must be 1.
[0035] The SS-A-X-- portion of Formula I can be attached to any
functional location of a palladium(II)-bound ligand backbone
grouping, e.g., the --B.sub.m[L:Pd(II)].sub.n portion of Formula 1.
For example, when m is 1, B is a 5 carbon chain, and n is 2, the
structure can be
[L.sup.1:Pd(II)]-[(CH.sub.2).sub.5]-[L.sup.2:Pd(II)]. With this
arrangement, the SS-A-X-- portion can be covalently attached to
L.sup.1 directly, L.sup.2directly, or any of the 5 carbons
separating L.sup.1 from L.sup.2. In another example, when m is 1, B
is a 14 carbon chain, and n is 3, one possible structure of the
palladium(II)-bound ligand backbone grouping can be
[L.sup.1:Pd(II)]-[(CH.sub.2).sub.5]-[L.sup.2:Pd(-
II)]-[(CH.sub.2).sub.9]-[L.sup.3:Pd(II)]. With this arrangement
then, the SS-A-X-- portion can be covalently attached to L.sup.1
directly, L.sup.2 directly, L.sup.3 directly, or any of the 14
carbons present on the organic backbone. If other atoms are present
on the organic backbone, then the SS-A-X-- portion of the
composition can be attached to those constituents as well.
[0036] Turning now to the specifics of the ligand (L) portion of
Formula 1, several ligands can be used to complex with specific
heterocyclic aromatic amine bases. When these ligands are complexed
with Pd(II) and tethered to a solid support, nonchromatographic
separations can be carried out that are selective of one
heterocyclic aromatic amine base over another. Additionally, if
from 2 to 4 properly selected ligands are each complexed with
Pd(II), tethered to a solid support, and appropriately spaced, then
specific nucleotide sequences can be separated from other
nucleotide sequences.
[0037] When the desired heterocyclic aromatic amine base to be
selected and/or separated is guanine (G) (including that in its
nucleoside or nucleotide form), then a ligand (L) containing the
sequence of formula 5 below, complexed to a Pd(II) ion can be
used.
--[B--C.sub.x--B--(C.sub.x--B).sub.y] Formula 5
[0038] In the above formula, each B can individually be nitrogen,
sulfur, or phosphorus; x can be 2 or 3; and y can be 0, 1 or 2.
Pendent functional groups or R groups (not shown) can be present on
each of the carbons that include H or straight or branched chained
lower alkyl, aryl, or lower alkyl aryl. An example of a pendent
alkyl group is shown in Formula 5i. Alternatively, one or more
heterocyclic or carbon aromatic ring(s) can be present that
incorporates two adjacent carbons of Formula 5. An example of a
pendent aryl group that utilizes this arrangement is shown in
Formula 5h. This being stated, it is not the purpose of the
invention to describe each and every possible side chain that can
be present on the ligand. All that is required is that the ligand
contain a section as shown in Formula 5. For example, a pyridine
ring, or the like, could be used to provide one or more B groups of
Formula 5, and still be within the scope of Formula 5. A symbol for
a bond and brackets are shown to denote that the ligand will be
attached to a solid support, such as through a covalent linkage
mechanism, a spacer grouping, and/or an organic backbone, depending
on the embodiment.
[0039] Below are included, by way of illustration, examples of
ligands that can be used for the nonchromatographic selection
and/or separation of guanine (G) (including its nucleoside or
nucleotide) from other heterocyclic aromatic amine bases and their
nucleotides and nucleosides. Each of these ligands (5a-5k) are
included within the scope of Formula 5. 789
[0040] It is important to note that the ligands shown above are
not, in and of themselves, the novel feature of the invention. The
invention resides in the compositions and methods of using the
compositions for separating desired heterocyclic aromatic amine
bases, nucleosides, nucleotides, or sequences of DNA or RNA using
non-chromatographic separation techniques. These ligands must be
complexed to Pd(II) and bound to a solid support in order for the
separation to be effectuated.
[0041] An example of a ligand of Formula 5, bound to a solid
support, and complexed to Pd(II) is shown below. 10
[0042] The above structure (using ligand 5d) is particularly
adapted for the separation of guanine (G) from other nucleotides,
nucleosides, or heterocyclic aromatic amine bases because three
coordination sites are used to bind the Pd(II) to each of the
sulfur groups, leaving one additional coordination site on the
Pd(II) ion to bind with the guanine (G). Likewise, any of the
ligands where y is I (as shown in Formula 5) generally provide the
highest selectivity in separations. The point of attachment where
the SS-A-X-- portion attaches to the ligand is shown by way of
example, and does not necessarily provide better separation results
compared to structures where the point of attachment is
different.
[0043] Turning now to the separation of adenine (A) (including that
in its nucleoside or nucleotide form) from other undesired
heterocyclic aromatic amine bases, certain ligands when complexed
to Pd(II) provide for effective separations. These ligands (L)
contain the sequence of Formula 6 below:
[D-C--N--C-D] Formula 6
[0044] In Formula 6, N and C are nitrogen and carbon, respectively;
and each D is independently a negative binder in addition to that
provided by nitrogen. Appropriate negative binders include carboxyl
moieties, phosphonic moieties, and sulfonic moieties. Additionally,
C--N--C can either be covalently bonded as shown, or can combine to
be part of a heterocyclic ring. Again, pendent functional groups or
R groups (not shown) can be present on each of the carbons, or if
applicable, on the aromatic ring. Such groups can include H or
straight or branched chained lower alkyl, aryl or lower alkyl aryl.
Additionally, any other of such R groups can be present, provided
they do not significantly intefere with the binding of adenine (A).
This being said, it is not the purpose of the invention to describe
each and every possible side chain that can be present on the
ligand. All that is required is that the ligand contain a section
as shown in Formula 6. A symbol for a bond and brackets are shown
to denote that the ligand will be attached to a solid support, such
as through a covalent linkage mechanism, a spacer grouping, and/or
an organic backbone, depending on the embodiment.
[0045] Below are included, by way of illustration, examples of
ligands that can be used for the nonchromatographic separation of
adenine (A) (including its nucleoside or nucleotide) from other
heterocyclic aromatic amine bases and their nucleotides and
nucleosides. Each of these ligands (6a-6c) shown are within the
scope of Formula 6. 11
[0046] Of the three examples shown, dipicolinic acid (6b) and
aminodiphosphate (6c) are the most preferred ligands for use with
the present invention. Additionally, though an example having a
sulfonic negative binder is not shown, such structures may also
provide effective separation results.
[0047] Again, it is important to note that the ligands shown above
(or other ligands within the scope of Formula 6) are not of
themselves the invention. The invention is found in the
compositions and methods for separating desired heterocyclic
aromatic amine bases, nucleosides, nucleotides, or sequences of DNA
or RNA using non-chromatographic separation techniques. Thus, these
ligands must be complexed to Pd(II) and bound to a solid support in
order for the separation to be effectuated.
[0048] An example of a ligand of Formula 6, bound to a solid
support, and complexed to Pd(II) is shown below. 12
[0049] With respect to the above structure, or any other ligand
structure within the parameters set forth in Formula 6, the point
of attachment where the SS-A-X-- portion attaches to the ligand is
shown by way of example, and does not necessarily provide better
separation results compared to structures where the point of
attachment is different. Adenine (A) is considered to be more
hydrophobic than some of the other heterocyclic aromatic amine
bases. Thus, the use of an organic solid support such as styrene is
preferred, though inorganic solid supports such as silica are also
functional.
[0050] When the desired heterocyclic aromatic amine base to be
separated is thymine (T) or uracil (U), then a ligand (L)
containing the sequence of formula 7 below, complexed to a Pd(II)
ion can be used. 13
[0051] Though many ligands containing this structure are useful, it
is preferred that a phenyl group be attached to a carbon of one or
more of the heterocyclic aromatic amine groups, such as is shown
below in Formula 8: 14
[0052] With respect to Formulas 7 and 8, pendent functional groups
or R groups (not shown) can be present at any location off of the
aromatic rings, as long as they do not significantly interfere with
the binding of the Pd(II) ion to the nitrogens on the aromatic
ring. Some additional R groups may, in fact, help the selectivity
of the ligand in certain circumstances, as illustrated by the
additional phenyl group included as part of Formula 8 compared to
Formula 7. In both Formula 7 and Formula 8, a symbol for a bond as
well as brackets are shown to denote that the ligand will be
attached to a solid support, such as through a covalent linkage
mechanism, a spacer grouping, and/or an organic backbone, depending
on the embodiment.
[0053] The ligand containing the sequence shown in Formula 7 can be
tethered to the solid support from any functional location. One
such structure is shown below wherein the ligand of Formula 7
(having an extra phenyl group attached thereto as in Formula 8) is
complexed to Pd(II) and is further attached to a solid support.
15
[0054] Again, with respect to the above structure, the point of
attachment where the SS-A-X-- portion attaches to the ligand is
shown by way of example, and does not necessarily provide better
separation results compared to structures where the point of
attachment is different. Attachment to the phenyl group, for
example, may also be desirable.
[0055] The ligand of Formula 7 (and preferably of Formula 8)
functions for the separation of thymine (T) and uracil (U) because
the structure aromatically lines up the nitrogen atoms in a desired
stereo configuration. Thus, the structure is both planer and bulky.
This configuration may also work because the Pd(II) ions are
protected by bulky groups. Guanine (G) is also attracted to the
Pd(II) bound ligand of Formulas 7 and 8, but because guanine (G) is
larger than thymine (T) and uracil (U), guanine (G) has difficulty
binding. Conversely, thymine (T) and uracil (U) are small enough
such that binding can be more easily occur.
[0056] When the desired heterocyclic aromatic amine base to be
separated is cytosine (C) (including its nucleoside or nucleotide),
then an appropriately configured ligand complexed to a Pd(II) ion,
and bound to a solid support can also be used. Formula 9 below,
which is included by way of example, provides a ligand that can be
used for the nonchromatographic selection and/or separation of
cytosine (C) from at least some of the other heterocyclic aromatic
amine bases and their nucleotides and nucleosides: 16
[0057] A symbol for a bond and brackets are shown to denote that
the ligand will be attached to a solid support, such as through a
covalent linkage mechanism, a spacer grouping, and/or an organic
backbone, depending on the embodiment. A specific example of a
ligand of Formula 9, bound to a solid support, and complexed to
Pd(II) is shown below. 17
[0058] The above structure is provided by way of example, and is
particularly adapted for the separation of cytosine (C) from other
nucleotides, nucleosides, or heterocyclic aromatic amines,
particularly uracil, thymine, and adenine. The point of attachment
where the SS-A-X-- portion attaches to the ligand is also shown by
way of example, and does not necessarily provide better separation
results compared to structures where the point of attachment is
different. The unpaired carboxy group can provide some steric
hindrance and provides symmetry to the ligand, and assists in the
selectivity.
[0059] Turning to the loading of the palladium to the ligands
described herein, Pd(II) can be loaded on the ligands described
herein by one of a number of techniques. Before loading Pd(II) on a
specific ligand, a basal capacity (mmol/g) of the ligand should be
ascertained. The basal capacity is typically determined by shaking
the specific ligand with a dilute buffered solution of a base metal
known to be complexed by the ligand, or by shaking with a dilute
solution of Pd(II) at a pH from about 2 to 3. The amount of Pd(II)
needed to load a specific mass of ligand can then be calculated as
described below in Formulas 10 and 11:
mmol Pd(II)=[ligand mass (g)].times.[desired loading
(mmol/g)].times.[excess factor (1.01-3)] Formula 10
volume of solution (ml)=[mM Pd(II)/1000].times.[mmoles Pd(II)
required] Formula 11
[0060] Larger excess factors are needed if the ligand is to be
Pd(II) loaded above its 1:1 stoichiometric basal level.
Additionally, the concentration of Pd(II) can be increased 5-10
fold if the Pd(II) is to be loaded above its basal capacity.
Normally, I mM Pd(II) is used for loadings at or below the basal
capacity.
[0061] Pd(II) can be loaded onto ligand bound solid supports by any
functional method. Two examples are provided herein by way of
example. A first method of loading Pd(II) onto a ligand bound solid
support is described below. First, a Pd(II) solution is made by
diluting a stock solution of Pd(NO.sub.3).sub.2 or PdCl.sub.2 into
0.5M or 0.125M NaHPO.sub.4 at a pH of about 6-7. The pH is NaOH
adjusted and brought to a final volume using H.sub.2O. Next, the
ligand or ligand bound to a solid support is mixed into the Pd(II)
solution and shaken until the composition becomes clear or
colorless. This can take from a few minutes to a few hours.
Alternatively, if color remains, then the mixing should be allowed
to occur until equilibrium is reached, e.g., from about 2 to 15
hours. The Pd(II) loaded ligands (attached to the solid supports)
can then be washed with 3-4 aliquots of 0.5M NaHPO.sub.4, decanted,
blotted, and stored wet for a short period of time, or allowed to
dry for longer period storage.
[0062] A second method for loading a ligand bound solid support
with Pd(I[) can be as follows. With respect to some ligands, the
use of a phosphate buffer is either so kinetically slow, or
otherwise inhibited such that it is not practical to load Pd(II)
using a phosphate solution. In these circumstances, the Pd(II)
solution can be diluted with water and pH adjusted to 2-3 with acid
or base as needed. Above pH 4, Pd(OH).sub.2 often forms a
precipitate. Following the shaking of the ligand bonded to the
solid support in a low pH Pd(II) solution, the composition is
washed using 0.5M phosphate buffer at pH 7 or water until a pH of
about 7 is reached.
[0063] The ability to separate one heterocyclic aromatic amine base
from others using the palladium bound compositions disclosed herein
is advantageous. Typically, with this type of technology,
selectivity is provided in large part due to the choice of metal.
In other words, the metal dominates the selectivity. With the
present invention, no matter what heterocyclic aromatic amine base
is desired for separation, Pd(II) is the metal ion that is used.
Thus, the specified ligand (L) combined with the Pd(II) is what
provides the selectivity, whereas the Pd(II) provides a significant
amount of the attraction.
[0064] Thus far, description has been given with respect to
compositions described in Formula 1 where "n" is 1. However, if the
desire is to separate out nucleotide sequences from other
nucleotide sequences, i.e., when "n" is from 2 to 4, then any of
the Pd(II) bound ligands described above can be bound to another
Pd(II) bound ligand(s) described above (or like ligands) by a
ligand supporting backbone. With each Pd(II) bound ligand
covalently attached to one or more other Pd(II) bound ligands via a
backbone, such a combination is in condition to bind nucleotide
sequences. However, in order to effectuate the separations of the
present invention, the ligand supporting backbone (or one of the
ligands bound to the ligand supporting backbone) is also tethered
to a solid support as described previously. All that is required is
that the ligand supporting backbone be of a functional length such
that desired heterocyclic aromatic amine bases of a nucleotide
sequence can be bound to the Pd(II) bound ligands. Optimally, the
backbone can be from 3 to 20 carbons in length (between any two
adjacent ligands), though this is a preferred range only and other
configurations may also be functional. Additionally, the ligand
supporting backbone does not have to consist solely of carbon
atoms. For example, oxygen and/or sulfur may also be present, as
long as their presence does not substantially interfere with
nucleotide binding at the ligand/Pd(II) site or sites. Examples of
structures having a ligand supporting backbone wherein "n" of
Formula I is other than 1 are shown in FIG. 1 and FIG. 2.
[0065] In FIG. 1, a structure is shown that can be used to bind
adenine-guanine nucleotide sequences. A solid support 10 is shown.
In this embodiment, the solid support 10 is silica gel. Attached to
the solid support 10 is a covalent silane linkage mechanism 12. The
covalent linkage mechanism is used to bridge the gap between
organic chains and the inorganic solid support 10. A spacer
grouping 14 is also shown. The spacer grouping 14 in this example
contains glycidyloxypropyl. The spacer grouping 14 is bound to a
ligand supporting backbone 16. In this embodiment, the ligand
supporting backbone is a 5 carbon straight chain group having the
point of attachment to the spacer grouping at C3. Attached at each
end of the ligand supporting backbone 16, i.e., C1 and C5, are two
Pd(II) bound ligands 18a, 18b. Pd(II) bound ligand 18a is
preferably a Pd(II) bound ligand such as that described in
connection with Formula 6. Pd(II) bound ligand 18b is preferably a
Pd(II) bound ligand such as that described in connection with
Formula 5.
[0066] Shown attached to the structure described above is a desired
nucleotide sequence 28. The desired nucleotide sequence is
comprised of adenine and guanine which collectively comprise the
heterocyclic aromatic amine bases 20. Each base 20 is bound to
2-deoxy-D-ribose 24 by a .beta.-N-glycoside bond 22. Connecting
each 2-deoxy-D-ribose is a monophosphate 26. The 5' end 30 and the
3' end 32 are also shown.
[0067] Turning now to FIG. 2, a structure is shown that is
selective for the nucleotide sequence
adenine-guanine-thymine-guanine. In this figure, a solid support 10
is shown that is organic. Specifically, in this embodiment, the
solid support 10 is polystyrene. Because the solid support 10 is
organic, there is no need for a separate covalent linkage
mechanism. In accordance with Formula 4, the covalent linkage
mechanism (not shown) and a spacer grouping can be integrated into
a single spacer grouping 14. In some cases, the spacer grouping may
actually be a single linkage formed by the reaction between an
activated polar group from the polymer and a functional group from
the ligand (or ligand backbone). In this embodiment, the spacer
grouping 14 is more than a single linkage and is attached to a
ligand supporting backbone 16. The point of attachment is at C4 of
a 15 carbon alkyl chain. Though it has been described that the
spacing between respective ligands is preferably from 3 to 20
carbons in length, to be clear, that is meant to mean carbon atoms
of the chain between each individual ligand. Additionally, carbon
is not the only constituent that can be present in the backbone. In
FIG. 2, there is a 15 carbon chain backbone, however spacing
between each ligand is 3 carbons, 5 carbons, and 3 carbons,
respectfully. Additionally, the four remaining carbons on the 15
carbon chain are attached to a Pd(II) bound ligand 18a-d. Pd(II)
bound ligand 18a is preferably a Pd(II) bound ligand such as that
described in connection with Formula 6. Pd(II) bound ligands 18b,
18d are preferably Pd(II) bound ligands such as those described in
connection with Formula 5. Pd(II) bound ligand 1 8c is preferably a
Pd(II) bound ligand such as that described in connection with
Formula 7 or Formula 8.
[0068] Shown attached to the structure described above is a desired
nucleotide sequence 28. The desired nucleotide sequence is
comprised of adenine, guanine, and thymine which are individually
heterocyclic aromatic amine bases 20 in a nucleotide sequence form.
Each base 20 is bound to 2-deoxy-D-ribose 24 by .beta.-N-glycoside
bond 22. Connecting the each 2-deoxy-D-ribose is a monophosphate
26. The 5' end 30 and the 3' end 32 are also shown. It is noted in
FIG. 2 that the nucleotide sequence contains six nucleotide bases.
As such, two unbound bases 34 are shown. Thus, the present
invention can separate nucleotide sequences that contain more bases
than the Pd(II) bound ligands 18a-d are designed to coordinate or
complex with.
[0069] Although in both FIG. 1 and FIG. 2, the spacer grouping 14
is attached to the ligand supporting backbone 16, this is not
required. This being the case, the spacer grouping 14 can be
attached to one of the Pd(II) bound ligands 18 itself. For example,
if the Pd(II) bound ligand is an ethyleneamine, then the spacer
grouping 14 can be covalently attached directly to a nitrogen. In
this situation, the ligand supporting backbone 16 is used for the
single purpose of separating one Pd(II) bound ligand from other
Pd(II) bound ligands (whereas in FIG. 1 and FIG. 2, the backbone
also acts as the point of attachment).
[0070] The separation methods of the present invention can be
carried out by utilizing the compositions previously described.
Steps that can be carried out in the separation methods of the
present invention can include the following: 1) identifying the
heterocyclic aromatic amine base, nucleoside, nucleotide, or
nucleotide sequence that is desired to be separated from other
undesired amines or substances; 2) selecting corresponding Pd(II)
bound ligands that are selective for the heterocyclic amine base,
nucleoside, nucleotide; or alternatively, selecting a corresponding
appropriately spaced series of Pd(II) bound ligands, i.e.,
preferably from 2 to 4 Pd(II) bound ligands, that are selective for
the nucleotide sequence; 3) tethering the Pd(II) bound ligands (or
series of Pd(II) bound ligands) to solid supports, forming the
selective composition; 4) loading the selective composition in a
separation device with a source solution, thereby loading the
Pd(II) bound ligands with desired heterocyclic aromatic amine base,
nucleoside, nucleotide, or nucleotide sequence; 5) washing the
loaded column with a biologically acceptable and compatible buffer
(if in the form of nucleotides, the buffer cannot hydrolyze the
nucleotide(s)); 6) eluting with a smaller amount of receiving
solution; and 7) washing the receiving solution from the column as
part of a post-elution wash. Though these steps are all mentioned
together, these steps are not all critical. For example, some steps
can be removed, depending on the circumstances.
[0071] For example, in one embodiment, a method for concentrating,
removing, or separating a desired heterocyclic aromatic amine base
or a nucleotide sequence containing the desired heterocyclic
aromatic amine base from a source solution can comprise several of
the above-mentioned steps. In this method, steps can include (a)
contacting a source solution having a first volume with a
composition comprising a palladium(II)-bound ligand covalently
bonded to a solid support, wherein the source solution contains a
desired heterocyclic aromoatic amine base and an undesired
heterocyclic aromatic amine bases, and wherein the
palladium(II)-bound ligand is preferentially selective of the
desired heterocyclic aromatic amine base; (b) removing the source
solution from contact with said composition to which the desired
heterocyclic aromatic amine base has been complexed; (c) contacting
the composition having the desired heterocyclic aromatic amine
bases associated therewith with a smaller volume of an aqueous
receiving solution in which the desired heterocyclic aromatic amine
bases are stripped from the composition; and (d) recovering the
desired heterocyclic aromatic amine bases in concentrated form in
said receiving solution.
[0072] In an alternative embodiment, a method for concentrating,
removing, or separating a nucleotide chain containing a specific
nucleotide sequence from a source solution can comprise the steps
of (a) identifying a nucleotide chain that contains a desired
nucleotide sequence that is desired for concentrating, removing, or
separating from a source solution; (b) providing a composition that
will selectively bind the nucleotide sequence; (c) contacting the
source solution having a first volume with the composition wherein
a chemical attraction between the composition and the nucleotide
sequence occurs; (d) removing the source solution from contact with
the composition, wherein the composition has the nucleotide
sequence attracted thereto; (e) contacting the composition having
the nucleotide sequence attracted thereto with a smaller volume of
an aqueous receiving solution in which the nucleotide chain is
stripped from the composition; and (f) recovering the nucleotide
chain in concentrated form in the receiving solution.
[0073] If a washing step is followed, a preferable buffer for use
is a phosphate buffer, though other buffers can be used as would be
known to those skilled in the art. Additionally, with respect to
the eluting step, preferably at from 0.1 to 1.0 M of ammonia or
ammonia buffer can be used, though other known receiving solutions
will work, e.g., ethylene diamine and thiocyanate. Ammonia is
particularly useful from a chemical perspective because it is a
stronger binder to Pd(II) than the base it displaces. Additionally,
at pH of 9-10, even oligonucleotides are still stable. Thus, the pH
is high enough to strip the nucleotide from the compositions of the
present invention, but is not high enough to alter the properties
of nucleotide or nucleotide sequences. When selecting an elution
composition for use, the desired properties to achieve include
elution substances that are 1) strong enough to displace the base
without altering its configuration; 2) not strong enough to
displace the Pd(II); and 3) can be washed off.
[0074] With respect to the washing step, if ammonia is used as the
elution agent, then a phosphate buffer works well at removing the
ammonia (when the pH of ammonia gets below about 8.5, it is
protonated and becomes an ammonium ion). If ethylenediamine is used
as the elution agent, then the pH must be lowered even further,
making it a less desirable elution material. Likewise, thiocyanate
requires a pH of about 2 to effectuate a wash. This is not a
problem at this stage because the bases have already been removed
in the receiving solution. However, the column must be returned to
its original more neutral state (pH) to reuse the column for a
further separation. Thus, the use of ammonia for the elution stage
and a phosphate buffer for the washing stage is desirable, as
little or no further preparation steps are required with respect to
the separation device before running another separation.
EXAMPLES
[0075] The following examples illustrate embodiments of the
invention that are presently best known. Thus, these examples
should not be considered as limitations of the present invention,
but are merely in place to teach how to make the best known
compositions of the present invention based upon current
experimental data. As such, a representative number of compositions
and their method of manufacture are disclosed herein.
[0076] In the following examples, several abbreviations will be
used will be understandable to one skilled in the art. Some
abbreviations include: DMF (dimethylformamide), RT (room
temperature), Trit (triphenylmethyl), THF (tetrahydrofuran), Me
(methyl), Et (ethyl), DMAP (4-dimethylaminopyridine), Ts
(tosylate), and BOC (t-butoxycarbonyl).
Example 1
Preparation of Diethylenetriamine Bonded to Silica Gel
[0077] To 0.3 L of toluene was added 13 g of
.gamma.-glycidoxypropyltrimet- hoxysilane and 6.24 g of
diethylenetriamine and the mixture was stirred overnight. To the
mixture was added 60 g of silica gel (35-60 mesh) and the product
was heated at 80.degree. C. for 16 hours. A product was filtered
off, washed with water and methanol, and dried under reduced
pressure. The product produced can have one of the following
structure (a or b): 18
Example 2
Preparation of Polystyrene Bonded to Diethylenetriamine
[0078] About 0.5 g of chloromethylpolystyrene (predried by
coevaporation with 50 ml of benzene) was heated at 120.degree. C.
with 10 g of diethylenetriamine for 24 hours. After the product was
cooled, the beads were collected by filtration, washed with
CH.sub.3OH, and dried under reduced pressure. The product produced
can have one of the following structure (a or b): 19
Example 3
Preparation of Tetramethyldiethylenetriamine Bonded to Silica
Gel
[0079] To a well-stirred solution of 1 g
tetramethyldiethylenetriamine and 0.8 ml of triethylamine in 75 ml
DMF at room temperature was added 1.0 ml of
bromopropyltrimethoxysilane dropwise. The resulting mixture was
heated to 80.degree. C. After 16 hours, 11 g of silica gel was
added. After an additional 4 hours, the reaction product was cooled
to room temperature. The solution was decanted and silica gel was
added to the THF, filtered, and washed with excess THF and
methanol. Next, the product was dried under vacuum for 16 hours at
55.degree. C., producing about 11.5 g of
tetramethyldiethylenetriamine bonded to silica gel. The reaction
formula is shown below: 20
Example 4
Preparation of Tetramethyldiethylenetriamine Bonded to
Polystyrene
[0080] About 318 mg of tetramethyldiethylenetriamine and 0.5 g of
the Merrifield resin was refluxed in 25 ml of dioxane. After 16
hours it was cooled, filtered, and the resin was washed with an
excess of THF and methanol and dried under vacuum for 16 hours at
55.degree. C. About 0.55 g of a tetramethyldiethylenetriamine
bonded to polystyrene was produce. The reaction formula is shown
below: 21
Example 5
Preparation of N,N-dimethyl-N'-tosylethylenediamine
[0081] To 10.5 g of N,N'-dimethylethylenediamine in 250 ml of THF
was added 26 g of tosylchloride (TsCl) in small portions at room
temperature. After 16 hours, solvents were evaporated and the
residue was apportioned between H.sub.2O and CH.sub.2Cl.sub.2. A
CH.sub.2Cl.sub.2 layer was separated and washed with brine, dried
(MgSO.sub.4), filtered, and concentrated under reduced pressure.
About 26.5 g of N,N-dimethyl-N'-tosylethylenediamine (93%) was
produced. The reaction steps are shown below: 22
Example 6
Preparation of
N,N'-ditosyl-2-{3-allyloxy-2-[2-(2-hydroxy-ethylamino)-etho-
xy]-propylamine}-ethanol
[0082] To 9.2 g of N-tosylaminoethanol and 55 g of cesium carbonate
in 300 ml of DMF at room temperature was added dropwise a solution
of 10 g of allyloxy ditosylate glycol in 100 ml of DMF. The
temperature was raised to 70.degree. C. After 16 hours, the product
was cooled to room temperature and filtered. The DMF was evaporated
under reduced pressure and the resulting residue was portioned
between H.sub.2O and CH.sub.2Cl.sub.2. A CH.sub.2Cl.sub.2 layer was
washed with brine, dried (MgSO.sub.4), filtered, and concentrated
under reduced pressure. A crude product
N,N'-ditosyl-2-{3-allyloxy-2-[2-(2-hydroxy-ethylamino)-ethoxy]-pr-
opylamine}-ethanol (85%) was formed as an intermediate. The process
steps are shown below: 23
Example 7
Preparation of
N,N'-ditosyl-2-{3-allyloxy-2-[2-(2-tosyloxy-ethylamino)-eth-
oxy]-propylamine}tosyloxyethane
[0083] To a well-stirred solution of 11.8 g of the
N,N'-ditosyl-2-{3-allyl-
oxy-2-[2-(2-hydroxy-ethylamino)-ethoxy]-propylamine}-ethanol in 200
ml of THF at 0.degree. C. was added 6.7 g powdered KOH followed by
a dropwise addition of a solution of 8.5 g tosychloride in 100 ml
of THF. After 16 hours at room temperature, the product was
filtered and the solvents evaporated off. The residue was
apportioned between H.sub.2O and CH.sub.2Cl.sub.2. A
CH.sub.2Cl.sub.2 layer was washed with brine, dried (MgSO.sub.4),
filtered, and concentrated under reduced pressure. The residue was
purified using silica gel and eluted with ethyl acetate/hexane.
About 14 g (75%) of N,N'-ditosyl-2-{3-allyloxy-2-[2-(2-to-
syloxy-ethylamino)-ethoxy]-propylamine}tosyloxyethane was obtained.
The reaction is shown below: 24
[0084] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 7.8-7.5(m, 8H),
7.4-7.2(m, 8H), 5.9-5.7(m, 1H), 5.3-5.1(m, 2H), 4.3-4.1(t, 4H),
3.9(d, 2H), 3.7-3.0(m, 13H), 2.4(s, 6H) 2.3(s, 6H).
Example 8
Preparation of a Tetratosylate of Oxohexaamine
[0085] To a well-stirred solution of 2.7 g of
N,N-dimethyl-N'-tosylethylen- ediamine and 13.5 g of cesium
carbonate in 150 ml of DMF at room temperature was added a solution
containing 4.5 g of
N,N'-ditosyl-2-{3-allyloxy)-2-[2-(2-tosyloxy-ethylamino)-ethoxy]-propylam-
ine}tosyloxyethane in 50 ml of THF. After 40 hours at 70.degree.
C., the product was cooled, filtered, and concentrated under
reduced pressure. The residue was apportioned between H.sub.2O and
CH.sub.2Cl.sub.2. A CH.sub.2Cl.sub.2 layer was separated, washed
with brine, dried (MgSO.sub.4), filtered, and concentrated under
reduced pressure. The residue was purified on silica gel and eluted
with ethyl acetate/methanol (2/1 v/v). About 4.7 g (85%) of
N,N',N",N'"-tetratosyl-N-(3-allyloxy-2-{2-
-[2-(2-dimethylamino-ethylamino)ethylamino]-ethoxy}-propyl-N'-(2-dimethyla-
mino-ethyl)-ethane-1,2-diamine was obtained, as shown by the
reaction steps below: 25
[0086] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 7.6(d, 8H) 7.3(d,
8H), 5.8(m, 1H), 5.2(m, 2H), 3.9(d, 2H), 3.7-3.1(m, 21H),
2.5-2.3(m, 16H), 2.1(s, 12H).
Example 9
Preparation of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-ethylamino]-ethox-
y}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine
[0087] To a well-stirred solution of 4.7 g of a tetratosylate in 50
ml of methanol was added 5.8 g of sodium hydrogen phosphate, 37 g
of a 6% of sodium amalgam and 20 mg of anthracene. The solution was
refluxed and after 5 days, was cooled, filtered, and concentrated
under reduced pressure. The residue was purified using a silica gel
and eluted with methanol/ammonium hydroxide (5/1). About 1.3 g of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-ethylamino]-ethoxy}-propyl-N'-(2--
dimethylamino-ethyl)ethane-1,2-diamine (70%) was produced as a pale
yellow oil. Specifically, the process steps were as follows: 26
[0088] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 5.9(m, 1H), 5.2(m,
2H), 4.0(d, 2H), 3.9(d, 2H), 3.7-3.4(m, 4H), 2.7-2.6(m, 16H),
2.4(t, 4H), 2.1(s, 12H), 1.6(s, 4H).
Example 10
Preparation of N-(3-allyloxy)-2-{2-[2-(2-dimethylamino
ethylamino]-ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine
Bonded to Silica Gel
[0089] To a well-stirred solution of 100 mg of a
N-(3-allyloxy)-2-{2-[2-(2-
-dimethylamino-ethylamino]-ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-
-1,2-diamine in 25 ml of toluene was added 60 mg of
glycidoxypropyltrimethoxysilane at room temperature. After 16
hours, 0.3 g of silica was added and the product was heated to
85.degree. C. After 24 hours at 85.degree. C., the product was
cooled and the solution decanted. The residue was taken in THF,
filtered, washed with excess THF and methanol, and dried under
reduced pressure for 16 hours at 55.degree. C. About 0.35 g of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-ethylamino]--
ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine ligand
bonded to a silica gel was produced. The process steps are shown
below: 27
Example 11
Preparation of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-ethylamino]-ethox-
y}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine Bonded to
Agarose
[0090] To a well-stirred solution of 1.0 g (wet) activated agarose
in 10 ml of phosphate buffer (pH 10.0) was added 400 mg of a
N-(3-allyloxy-2-{2-[2-(2-dimethylamino-ethylamino]-ethoxy}-propyl-N'-(2-d-
imethylamino-ethyl)ethane-1,2-diamine. The reaction was heated to
45.degree. C. After 16 hours, the product was cooled, filtered,
washed with water and acetone, and dried under reduced pressure for
16 hours at 45.degree. C. About 1.1 g of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-et-
hylamino]-ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine
bonded to agarose was prepared. The preparation is shown
schematically below: 28
Example 12
Preparation of a
N-(3-allyloxy)-2-{2-[2-(2-dimethylamino-ethylamino]-ethox-
y}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine bonded to
chloromethylpolystyrene
[0091] To a stirring solution of 0.28 g Merrifield resin in 25 ml
of THF was added 0.24 g of a
N-(3-allyloxy-2-{2-[2-(2-dimethylamino-ethylamino]--
ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-diamine ligand.
After 16 hours at reflux, the product was cooled, filtered, and
washed with methanol, water, methanol, and THF, and dried under
reduced pressure for 16 hours at 55.degree. C. About 0.32 g of
N-(3-allyloxy-2-{2-[2-(2-dimeth-
ylamino-ethylamino]-ethoxy}-propyl-N'-(2-dimethylamino-ethyl)ethane-1,2-di-
amine bonded to chloromethylpolystyrene was produced. The
preparation is represented schematically below: 29
Example 13
Preparation of 1,3-dithiapropane Bonded to Silica Gel
[0092] To 0.4 L of toluene and 17 g of
3-mercaptopropyltrimethoxysilane was added 4.66 g of sodium
methoxide in 50 ml of methanol. Next, 8.72 g of 1,3-dibromopropane
was dropped into the solution over 10 minutes and was heated to
80.degree. C. (some methanol was distilled off). About 100 g of
silica gel (35-60 mesh) was added and then heated and stirred at
80.degree. C. for over 12 hours. After cooling, the product was
filtered and washed with water and methanol. About 105 g of
1,3-dithiapropane bonded to silica gel was prepared, as shown in
the following structure: 30
Example 14
Preparation of 4,13-dithia-7,10-dioxa-1,16-hexadecanediol
[0093] Into 1 liter of DMF were dissolved 33.35 g of
3-bromopropanol and 20 g of 3,6-dioxa-1,8-octanedithiol, followed
by the addition of 225 g of Cs.sub.2CO.sub.3. The mixture was
stirred at 70.degree. for 3 days and then the DMF was evaporated
under reduced pressure. Next, the residue was dissolved in H.sub.2O
and CH.sub.2Cl.sub.2 and a water layer was extracted twice more
with CH.sub.2Cl.sub.2. Organic layers were combined, dried over
MgSO.sub.4, and evaporated. The oil product was distilled
(b.p.=210.degree./0.45 mm Hg) and 19.53 g of
4,13-dithia-7,10-dioxa-1,16-- hexadecanediol (59.8%) was collected.
The preparation scheme is shown below: 31
[0094] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.65(m, 12H),
2.17(dt, 8H), 2.5(s, 2H), 1.8(m, 4H) C.sup.13NMR, 71.5, 70.5, 61.5,
32.3, 31.7, 29.2.
Example 15
Preparation of 4,13-dithia-7,10-dioxa-1,16-hexanedecanediol
ditosylate
[0095] Into 250 ml of CH.sub.2Cl.sub.2 was dissolved 19.53 g of the
4,13-dithia-7,10-dioxa-1,16-hexadecanediol of Example 14 and cooled
to -5.degree. C. Next, 22.34 g of Et.sub.3N was added to the
solution and 27.9 g of TsCl dissolved in 250 ml of CH.sub.2Cl.sub.2
and added dropwise at 0.degree. C. The solution was stirred for 24
hours and then washed 3 times with 200 ml H.sub.2O. The
CH.sub.2Cl.sub.2 layer was dried over MgSO.sub.4 and evaporated.
The resulting oil was purified on silica using ethylacetate/Hexane
(1/1 v/v). About 25.78 g (65%) of
4,13-dithia-7,10-dioxa-1,16-hexanedecanediol ditosylate was
obtained. The reaction is shown below schematically: 32
[0096] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.75(d, 4H),
7.35(d, 4H), 4.1(t, 4H), 3.55(t, 8H), 2.65(t, 4H), 2.55(t, 4H),
2.4(s, 6H), 1.9(q, 4H).
Example 16
Preparation of 4,13-dithia-7,10-dioxahexadecane-1,16-thiobenzoic
Acid
[0097] To 700 ml of CH.sub.3CN was dissolved 25.78 g of
4,13-dithia-7,10-dioxa-1,16-hexanedecanediol ditosylate and 16.41 g
of potassium thiobenzoate. The product was refluxed for 72 hours.
Acetonitrile was evaporated and the crude oil was dissolved in
CHCl.sub.3. The chloroform layer was washed with H.sub.2O and dried
over MgSO.sub.4 and evaporated. The reacting product yielded 22.62
g of 4,13-dithia-7,10-dioxahexadecane-1,16-thiobenzoic acid (100%).
The reaction described is shown below: 33
[0098] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR: 7.95(d, 4H), 7.52(t, 2H),
7.4(t, 4H), 3.6(dt, 8H), 3.2(t, 4H), 2.2(dt, 8H), 1.9(1,4H).
Example 17
Preparation of 1,5,14,18-tetrathia-8,11-dioxaoctadecane
[0099] To 6.38 g of LiA1H.sub.4 in 125 ml THF under argon at
0.degree. C. was added dropwise 22.62 g of
4,13-dithia-7,10-dioxahexadecane-1,16-thiob- enzoic acid dissolved
in 125 ml of THF. After addition, the product was refluxed for 3
days. Following a cooling step, about 50 ml of H.sub.2O was added
dropwise and then the solution was filtered. The filtrate was mixed
with 600 ml of CHCl.sub.3 and stirred overnight. The product was
then filtered, evaporated, and a crude product was purified on
alumina oxide with CHCl.sub.3/Hexane (2/1 v/v) followed by
CHCl.sub.3. About 1.85 g (13%) of
1,5,14,18-tetrathia-8,11-dioxaoctadecane was collected. The process
steps for obtaining this composition are shown schematically below:
34
[0100] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR: (CDCl.sub.3): 3.7(dt, 8H),
2.75(dt, 12H), 2.0(q, 4H).
Example 18
Preparation of 1,5,14,18-tetrathia-8,11-dioxaoctadecane Bonded to
Silica Gel
[0101] About 132 mg of 1,5,14,18-tetrathia-8,11-dioxaoctadecane was
dissolved in 10 ml of toluene. Then 194 mg of
glycidoxypropyltrimethoxysi- lane and 6.4 mg of 25% NaOCH.sub.3 in
methanol was added. The mixture was heated to 80.degree. C. and
stirred overnight with a mechanical stirrer. The reaction product
was cooled to 50.degree. C. and 2 g of 35-60 mesh silica gel was
added to the product and stirred for an additional 3 days. About
2.2 g of 1,5,14,18-tetrathia-8,11-dioxaoctadecane bonded to silica
gel was filtered and washed with toluene and CH.sub.3OH and dried.
This process is represented schematically below: 35
Example 19
Preparation of 4-allyloxy-1,10-dithia-18-crown-6
[0102] Into a solution containing 1600 ml of CH.sub.3CN and 40.6 g
of Cs.sub.2CO.sub.3 was dropped at 65.degree. C. a mixture of 60 g
of ditosylate and 20.6 g of 1,10-dithia-4,7-dioxadecane dissolved
in 200 ml of CH.sub.3CN. The reaction mixture was refluxed 36
hours. The precipitate was filtered off and the filtrate was
concentrated to a predetermined dryness. The residue was purified
by column chromatography (aluminum oxide, CH.sub.2Cl.sub.2 then
CH.sub.2Cl.sub.2/CH.sub.3COOC.sub.- 2H.sub.57/1 v/v). About 10 g
(24%) of an allyloxydithiacrown was collected and the compound was
used without further purification in the next step. The reaction is
shown below: 36
Example 20
Preparation of 4-hydroxypropyloxymethyl-1,10-dithia-18-crown-6
[0103] To 3.11 g of the 4-allyloxy-1,10-dithia-18-crown-6
(collected in Example 19) in 10 ml of THF, 40 mg of sodium hydride
was added and stirred for 1 hour at room temperature. Next, the THF
was removed under reduced pressure and 30 ml of diglyme and 1.18 g
of trimethylamine N-oxide dihydrate was added. The mixture was
refluxed overnight, the solvent was removed under reduced pressure
and the residue was purified by column chromatography, (aluminum
oxide, acetone/CH.sub.2Cl.sub.2/CH.su- b.3OH 40/1 v/v). About 2.6 g
(83%) of a hydroxypropyldithiacrown was collected. The reaction is
shown generally below: 37
Example 21
Preparation of 1,16-Bis(5'-methoxy-1',10'-dithia-18-crown-6)-10
alkyloxy-4,7,10,13-tetraoxahexadecane
[0104] To 560 mg of dissolved hydroxypropyldithiacrown (collected
in Example 20) in 15 ml dry THF was added 40 mg of sodium hydride
and the mixture was stirred for 1 hour. A solution of 343 mg of
ditosylate in 20 ml THF was added dropwise. After addition, the
reaction mixture was refluxed 3 days and then cooled, filtered, and
evaporated under reduced pressure. The residue was purified by
column chromatography, (aluminum oxide,
acetone/CH.sub.2Cl.sub.25%-10%-15%-20% v/v). An essentially pure
1,16-Bis(5'-methoxy-1',10'-dithia-18-crown-6)-10-alkyloxy-4,7,10,13-tetra-
oxahexadecane (35%) was collected, as shown below: 38
[0105] .sup.1HNMR (CDCl.sub.3): 5.989-5.82(m, 1H), 5.33-5.16(m,
2H), 4.0-3.44(m, 58H), 2.88-2.77(m, 16H), 1.85(t, 4H) HRMS (FAB):
m/z for C.sub.42H.sub.84O.sub.15S.sub.4(M+1).sup.+=953.4461, found:
953.4454.
Example 22
Preparation of
1,16-Bis(5'-methyloxy-1'10'-dithia-18-crown-6)-10-hydroxypr-
opyloxymethyl-4,7,10,13-tetraoxahexadecane
[0106] To 0.58 g of
1,16-Bis(5'-methoxy-1',10'-dithia-18-crown-6)-10-alkyl-
oxy-4,7,10,13-tetraoxahexadecane in 0.5 THF was added 0.21 ml 1 M
BH.sub.3-THF. The mixture was stirred at 0.degree. C. for 1 hour,
and then further stirred for 4 hours at room temperature. After
evaporation of the solvent, 81 mg (CH.sub.3).sub.3N.sup.+--O.sup.-
and 2 ml diglyme was added and refluxed for 2 hours, and then the
solvent was removed under reduced pressure. The residue was then
purified by column chromatography (aluminum oxide, 5%
acetone/CH.sub.2Cl.sub.2), and a
1,16-Bis(5'-methyloxy-1'10'-dithia-18crown-6)-10-hydroxypropyloxymethyl-4-
,7,10,13-tetraoxahexadecane (34%) was collected, as shown below:
39
[0107] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.82-3.43(m, 59H),
2.88-2.77(m, 16H), 1.83(m, 6H) MS(FAB): m/z 993 (M+Na).sup.+.
Example 23
Preparation of
1,16-Bis(5'-methyloxy-1'10'-dithia-18crown-6)-10-hydroxypro-
pyloxymethyl-4,7,10,13-tetraoxahexadecane Bonded to Silica Gel
[0108] A mixture of 364 mg of
1,16-Bis(5'-methyloxy-1'10'-dithia-18crown-6-
)-10-hydroxypropyloxymethyl-4,7,10,13-tetraoxahexadecane, 27 mg NaH
(95%), and 10 ml dry 1,4-dioxane was stirred for 1 hour. Then, 375
mg of epoxy silica gel was added and stirred at 90.degree. C. After
3 days, the product was filtered, washed with THF and MeOH. About
426.5 mg of
1,16-Bis(5'-methyloxy-1'10'-dithia-18crown-6)-10-hydroxypropyloxymethyl-4-
,7,10,13-tetraoxahexadecane bonded to silica gel was obtained. The
reaction is shown schematically below: 40
Example 24
Preparation of Chelidamic Acid Bonded to Polystyrene
[0109] A suspension having 3.4 g of Cs.sub.2CO.sub.3 in 150 ml of
DMF containing 3 g of dimethyl ester of chelidamic acid was stirred
for 1 hour at room temperature. About 2.5 g of Merrifield resin was
added and allowed to react for 48 hours at 75.degree. C. After
cooling, the solid was filtered off, washed with water,
dioxane/water (3/1 v/v), dioxane, and methanol. The resin was dried
overnight under vacuum. This intermediate resin in 200 ml of
1,2-dichloroethane was treated with 30 ml of an aqueous solution of
50% potassium hydroxide containing 2 g of ADOGEN 464. The resulting
mixture was allowed to react for 48 hours at 70.degree. C. under
nitrogen. After cooling down, the solid was filtered off and washed
with water, methanol, water, tetrahydrofuran/water (3/1 v/v),
tetrahydrofuran, acetone, methylene chloride, and water. The washed
product was dried under reduced pressure at 50.degree. C. About 3.0
g of chelidamic acid bonded to polystyrene was prepared, the
reaction is shown schematically below: 41
Example 25
Preparation of iminodi(methylphosphonic Acid) Bonded to Silica
Gel
[0110] About 32 ml of 6 N HCl and 13.5 g of phosphorous acid was
added and stirred to allow the phosphorous acid to dissolve
completely in a 100 ml flask. About 17.7 g of
aminopropyltrimethoxysilane was added dropwise at 25.degree. C.,
refluxed ten minutes, and cooled down to 65.degree. C. Next 25.6 g
of formaldehyde was added dropwise, refluxed 1.5 hours, and cooled
down to 25.degree. C. The solution was transferred into a 1000 ml
flask that was equipped with a mechanical stirrer where 320 ml of
4N HCl was added. Next, 100 g of silica gel was added and the
product was heated to 65.degree.-70.degree. C. and kept overnight
at that temperature. After cooling, the mixture was filtered, and
the solid was washed with H.sub.2O, dried under a hood, and dried
in a vacuum oven. About 110 g of iminodi(methylphosphonic acid)
bonded to silica gel was prepared. 42
Example 26
Preparation of 4-aminomethyl-1,8-octanediamine Bonded to Silica
Gel
[0111] A mixture of 5.2 g of 4-aminomethyl-1,8-octanediamine and
0.7 g of glycidoxypropyltrimethoxysilane in 50 ml toluene was
stirred at room temperature for 5 hours. Next, 2 g (60-100 mesh)
silica gel was added, and stirred at 85.degree. C. for 3 days. The
product was then cooled, filtered, and washed with MeOH. About 2.3
g of 4-aminomethyl-1,8-octanedi- amine bonded to silica gel was
prepared. The reaction is shown below: 43
Example 27
Preparation of Penta(methylphosphonic
Acid)-4-aminomethyl-1,8-octanediamin- e Bonded to Silica Gel
[0112] A mixture of 2 g of 4-aminomethyl-1,8-octanediamine bonded
to silica gel, 1.64 g of phosphorous acid, and 1.08 g of
paraformaldehyde in 6N HCl (10 ml H.sub.2O, 10 ml conc HCl) was
stirred at 65.degree.-70.degree. C. for 3 days. The product was
filtered, washed with H.sub.2O and MeOH, and dried to give 2.3 g of
penta(methylphosphonic acid)-4-aminomethyl-1,8-octanediamine bonded
to silica gel. This reaction is shown below: 44
Example 28
Preparation of 4-hydrophenyl Terpyridine Bonded to Silica Gel
[0113] To a stirred solution of 1.14 g of 4-hydrophenyl terpyridine
in 75 ml DMF was added 350 mg of NaH (60%) and 0.766 g of
3-bromopropyltrimethoxysilane at room temperature. The product was
heated at 70.degree. C. for 16 hours and 3 g of silica gel was
added. Continued heating was applied for an additional 16 hours.
The reaction was then cooled and the solution decanted. The silica
gel was washed in THF, filtered, and washed with excess MeOH and
THF. Then, the product was dried under reduced pressure for 16
hours at 65.degree. C. About 3.5 g of 4-hydrophenyl terpyridine
bonded to silica gel was prepared, the reaction being shown below:
45
Example 29
Preparation of 5-hydroxymethyl-1,4,7,10-tetraoxadecanediol
Ditosylate
[0114] A mixture of 15 g of
5-allyloxymethyl-1,4,7,10-tetraoxadecanediol, 0.86 g of 10% Pd/C,
and 0.3 g of p-toluenesulfonic acid in 100 ml of ethanol was
stirred and heated at 40.degree. C. overnight. The reaction was
monitored by TLC (silica plates, CH.sub.2Cl.sub.2/MeOH 20:1). When
no more starting compound remained, the mixture was filtered off.
The filtrate was then evaporated and the residue was purified on
column chromatography (silica gel, CH.sub.2Cl.sub.2/CH.sub.3OH 40/1
v/v). About 8.18 g (59%) of
5-hydroxymethyl-1,4,7,10-tetraoxadecane-1,10-diol ditosylate was
collected, as shown below: 46
[0115] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.7(d, 4H), 7.3(d,
4H), 4.2-3.4(m, 13H), 2.1(t, 1H).
Example 30
Preparation of
1,10-phenylterpyridine-5-hydroxymethyl-1,4,7,10-tetraoxatet-
radecane
[0116] To 100 ml of DMF was dissolved 2.45 g of the hydroxymethyl
triethylene glycol ditosylate prepared in Example 29, 3.25 g of
terpyridylphenol, and 2.0 g of K.sub.2CO.sub.3. The mixture was
heated at 105.degree. C. for 48 hours and then cooled. After
cooling, DMF was removed under reduced pressure. The residue was
dissolved in 200 ml of CHCl.sub.3 and washed with H.sub.2O. The
organic layer was dried over MgSO.sub.4, filtered, and the
CHCl.sub.3 layer was concentrated. The crude product was purified
using column chromatography (Aluminum oxide, CHCl.sub.3/CH.sub.3OH
60/1 v/v). About 2.50 g of a
1,10-phenylterpyridine-5-hydroxymethyl-1,4,7,10-tetraoxatetradecane
was collected in a 63% yield. The formulation is shown
schematically below: 47
[0117] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 8.8-8.5(m, 12H),
7.7-7.6(m, 8H), 7.3(d, 4H), 7.0(d, H), 4.2-3.0(m, 13H), 2.1 (t,
1H).
Example 31
Preparation of
1,10-phenylterpyridine-5-hydroxymethyl-1,4,7,10-tetraoxatet-
radecane Bonded to Silica Gel
[0118] About 1.20 g of a
1,10-phenylterpyridine-5-hydroxymethyl-1,4,7,10-t-
etraoxatetradecane prepared in accordance with Example 30 was
heated with 0.8 g of an epoxy silica gel, 30 ml of dioxane, and 80
mg of NaH at 85.degree. C. for 48 hours. After cooling, the silica
gel was washed with an excess of CH.sub.3OH and dried under reduced
pressure at 55.degree. C. overnight. About 0.84 g of
1,10-phenylterpyridine-5-hydroxymethyl-1,4,7,1-
0-tetraoxatetradecane bonded to silica gel was collected. The
reaction is shown schematically below: 48
Example 32
Preparation of [4'-(2,2',6',2"-terpyridine)
phenyloxy]ethoxy-ethoxy-ethano- l
[0119] A mixture of 5.09 g of terpyridylphenol, 3.91 g of
(chloroethoxy)ethoxyethanol, and 3.8 g of potassium carbonate was
heated in 130 ml DMF at 120.degree. C. overnight. The insoluble
material was filtered off and washed with DMF. The filtrate was
then concentrated under vacuum and the residue was co-evaporated
with 100 ml of toluene twice. Finally, the residue was dissolved in
toluene, washed with H.sub.2O, dried over MgSO.sub.4, filtered, and
concentrated to give 7.51 g of a thick light brown oil which
solidified upon standing overnight. The solid was then crushed,
stirred in Et.sub.2O, filtered, dried in the air to give 5.81 g
(83%) of a [4'-(2,2',6',2"-terpyridine)
phenyloxy]ethoxy-ethoxy-ethanol composition. The reaction is shown
below: 49
[0120] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 3.65(t, 2H),
3.70-3.80(m, 6H), 3.92(t, 2H), 4.21(t, 2H), 7.06(d, 2H), 7.35(m,
2H), 7.89(m, 4H), 8.66-8.75(m, 6H).
Example 33
Preparation of
tris-{[4'-(2,2',6',2"-tepyridine)phenyloxy]ethoxy-ethoxy-et- hoxy}
pentaerythrityl-bromide
[0121] About 4.25 g of [4'-(2,2',6',2"-terpyridine)
phenyloxy]ethoxy-ethoxy-ethanol prepared in Example 32, 1.14 g of
pentaerythrityl tetrabromide, and 0.97 g of potassium hydroxide was
heated in 35 ml of DMSO at 60.degree. C. for 36 hours. After
cooling, the reaction mixture was poured onto a stirring mixture of
ice/H.sub.2O . A solid was collected by filtration and washed with
water. After the solid was dissolved in CHCl.sub.3, 2N HCl was
added. A bright yellow solid formed and was collected by
filtration. Next, the solid was stirred with 3N NaOH until the
bright yellow color faded to off-white. Then the product was
extracted with CHCl.sub.3 and filtered. To the insoluble material
was added water and then it was extracted with CHCl.sub.3. The
above procedure was repeated from 3 to 4 times, and the combined
chloroform layers were dried over MgSO.sub.4, filtered, and
concentrated to give 2.50 g of a pale-brown thick oily
tris-{[4'-(2,2',6',2"-tepyridin- e)phenyloxy]ethoxy-ethoxy-ethoxy}
pentaerythrityl-bromide. The reaction is shown below: 50
[0122] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 3.45-3.75(m, 32H),
3.85-3.95(m, 6H), 4.16-4.25(m, 6H), 7.0(d, 6H), 7.35(m, 6H),
7.85(m, 12H) 8.6-8.756(m, 18H) MS(FAB) {[M+Na]-2}.sup.+=1539.
Example 34
Preparation of
tris{[4'-(2,2',6',2"-terpyridine)phenyloxy]ethoxy-ethoxy-et-
hoxy}pentaerythrityl-tetraethylene Glycol
[0123] To a preheated solution (40.degree. C.) of 2.47 g of
tris-{[4'-(2,2',6',2"-terpyridine)phenyloxy)ethoxy-ethoxy-ethoxy}-pentaer-
ythrityl bromide prepared as in Example 34 and 40 ml of
tetraethylene glycol was added 0.75 g of sodium (cautiously added
piece by piece). The reaction mixture was heated at 125.degree. C.
overnight. After cooling, the reaction mixture was poured into 300
ml of stirred H.sub.2O. Crude product was extracted by CHCl.sub.3
(150 ml.times.3), dried over MgSO.sub.4, filtered, and concentrated
to give 2.39 g of a brown oil which was redissolved in 6 ml of
CHCl.sub.3 and treated with 2N HCl. A greenish sticky material
formed that was separated from the top of the aqueous phase by
decantation, and washed with a small amount of 2N HCl. To the
residue was added 3N NaOH to pH 11 and the product was extracted
with CH.sub.2Cl, dried over MgSO.sub.4, filtered, and concentrated
to give 1.60 g (61%) of a yellow oily
tris{[4'-(2,2',6',2"-terpyridine)pheny-
loxy]ethoxy-ethoxy-ethoxy}pentaerythrityl-tetraethylene glycol. The
reaction is shown below: 51
[0124] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(CDCl.sub.3): 3.4-3.75(m, 48H,
including 25 singlets at 3.44 & 3.61), 3.86(t, 6H), 4.16(t,
6H), 7.0(m, 6H), 7.31(m, 6H), 7.84(m, 12H), 8.58-8.72(m, 8H)
MS(FAB) [(M+Na)-1].sup.+=1652.
Example 35
Attachment of
tris-{[4'-(2,2',6',2"-terpyridine)phenyloxy]ethoxy-ethoxy-et- hoxy}
Pentaerytrityl Tetraethylene Glycol to Silica Gel
[0125] To a preheated solution of 0.8 g
tris-{[4'-(2,2',6',2"-terpyridine)- phenyloxy]ethoxy-ethoxy-ethoxy}
pentaerytrityl tetraethylene glycol in 30 ml dioxane was added 0.09
g of NaH cautiously. After stirring at room temperature for 10
minutes, 0.82 g of glycidyl-silica gel was added, and the
temperature was brought up to 80.degree. C. After stirring
overnight at 80.degree. C., the reaction was quenched by the
addition of CH.sub.3OH. The silica gel with
tris-{[4'-(2,2',6',2"-terpyridine)phenylo- xy]ethoxy-ethoxy-ethoxy}
pentaerytrityl tetraethylene glycol attached thereto was then
collected by filtration, washed with H.sub.2O, CH.sub.3OH,
CH.sub.2Cl.sub.2, CH.sub.3OH, and H.sub.2O successively, and
finally dried in a vacuum oven at 55.degree. C. overnight. About
1.0 g of
tris-{[4'-(2,2',6',2"-terpyridine)phenyloxy]ethoxy-ethoxy-ethoxy}
pentaerytrityl tetraethylene glycol bonded to silica gel was
prepared. The reaction is shown below: 52
Example 36
Preparation of
3-t-butyldimethylsilyloxymethyl-triethyleneglycolditosylate
[0126] A 4.90 g amount of 3-hydroxymethyl-triethylene glycol
ditosylate and 1.36 g of imidazole were dissolved in 50 ml of DMF.
A solution of 1.51 g of t-butyl dimethyl silylchloride in 20 ml of
DMF, was added dropwise into the system. After stirring 2 hours at
room temperature DMF was removed under reduced pressure. The
residue was purified by a silica gel column eluted with
CHCl.sub.3/CH.sub.3OH (70/1). About 1.77 g of product was obtained.
The reaction is shown schematically below: 53
[0127] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1H NMR (CDCl.sub.3) 7.8(d, 4H), 6.8(d,
4H), 3.4-4.2(m, 13H), 2.5(s, 6H), 0.8(s, 9H), 0.1(s, 6H).
Example 37
Preparation of t-butyldimethyl Silyloxymethyl Triethylene Glycol
Terpyridine Dimer
[0128] Under Argon, 3.16 g of hydroxy methyl terpyridine
triethylene glycol dimer was dissolved in 100 ml of dry DMF. Then,
0.32 g of NaH was added to the system and stirred at room
temperature for half an hour. Next, 1.51 g of
3-t-butyldimethylsilyloxymethyl-triethylene glycol ditosylate was
added to the system in one portion. After stirring at 105.degree.
C. for 40 hours, DMF was removed under reduced pressure. The
residue was dissolved in CHCl.sub.3 and washed with H.sub.2O and
brine. The CHCl.sub.3 layer was separated and dried with
MgSO.sub.4. After filtration and concentration, the crude product
was purified by a silica gel column, and eluted with
CHCl.sub.3/MeOH (80/1). About 1.8 g of product was obtained. The
reaction is shown schematically below: 54
[0129] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1H NMR (CDCl.sub.3): 8.6(m, 24H),
7.8(t, 16H), 7.2(t, 8H), 7.0(d, 8H), 3.5-4.2(m, 39H), 0.8(s, 9H),
0.1(s, 6H).
Example 38
Preparation of Hydroxy Methyl Triethylene Glycol Terpyridine
Tetramer
[0130] A 0.65 g amount of t-butyl dimethyl silyloxy methyl
triethylene glycol terpyridine tetramer and 0.80 g of ammonium
fluoride were refluxed in 50 ml of CH.sub.3OH for 20 hours. After
removing CH.sub.3OH, the residue was dissolved in CHCl.sub.3 and
washed with H.sub.2O. The CHCl3 layer was dried with MgSO.sub.4.
After filtration and concentration, 0.6 g of product was obtained.
The reaction is shown schematically below: 55
[0131] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1H NMR (CDCl.sub.3): 8.6(m, 24H),
7.8(t, 16H), 7.2(t, 8H), 70(d, 8H), 3.5-4.2(m, 39H).
Example 39
Preparation of Terpyridine Tetramer Bonded to Silica Gel
[0132] A 0.9 g amount of hydroxymethyl terpyridine tetramer was
dissolved in 20 ml of dioxane. Under Argon, 0.12 g of NaH were
added and stirred for 15 minutes at room temperature. After adding
0.40 g of epoxide modified silica gel, the temperature was raised
to 90.degree. C. for 72 hours. Upon cooling, the mixture was
filtered and the silica was washed with a large amount of
CH.sub.3OH. The product was dried in a vacuum oven at 50.degree. C.
for 8 hours. The reaction is shown schematically below: 56
Example 40
Preparation of Triethylene Glycol Tosylate of Dimethyl Ester of
Chelidamic Acid
[0133] A mixture of 4.22 g of a dimethyl ester of chelidamic acid,
32 g of a triethylene glycol ditosylate, and 4.14 g of
K.sub.2CO.sub.3 in 100 ml CH.sub.3CN was refluxed for 3 days,
cooled, filtered, and evaporated. The residue was purified by
column chromatography (silica gel, 2.5% acetone/CH.sub.2Cl.sub.2).
A triethylene glycol tosylate of dimethyl ester of chelidamic acid
(50%) was collected. The reaction is shown below: 57
[0134] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.83 (d, 2H), 7.79
(s, 2H), 7.34 (d, 2H), 4.33-4.18 (m, 8H), 4.02 (s, 6H), 3.92-3.63
(m, 4H), 2.45 (s, 3H).
Example 41
Preparation of
3,6-dioxa-9,12,15-triaza-9,12,15-triethyldodecane-1-O'-chel- idamic
Acid Dimethyl Ester
[0135] A mixture of 2.0 g of a triethylene glycol tosylate of
dimethyl ester of chelidamic acid of Example 36, 3.0 g of
N,N',N"-triethyl-diethyl- enetriamine and 3.40 g of
Na.sub.2CO.sub.3 in 100 ml CH.sub.3CN was refluxed for 3 days,
cooled, filtered, and evaporated. The residue was purified by
column chromatography (silica gel, CH.sub.2Cl.sub.2-30%
MeOH/CH.sub.2Cl.sub.2). A
3,6-dioxa-9,12,15-triamine-9,12,15-triethyldode-
cane-1-O'-chelidamic acid dimethyl ester (45%) was collected, as
shown schematically below: 58
[0136] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.82(s, 2H),
4.32(m, 2H), 4.02(s, 6H), 3.9(m, 2H), 3.74-3.53(m, 6H),
2.72-2.53(m, 16H), 1.17-0.98(m, 9H), MS(FAB): m/z 535
(M+Na).sup.+.
Example 42
Preparation of
3,6-dioxa-9,12,15-triaza-9,12,15-triethyldodecane-1-O'-chel- idamic
Acid
[0137] About 470 mg of a
3,6-dioxa-9,12,15-triaza-9,12,15-triethyldodecane- -1-O'-chelidamic
acid dimethyl ester was added to a solution of 1514 mg of KOH in 25
ml CH.sub.3OH. The product was stirred at room temperature for 20
hours, the solvent was evaporated, the residue was acidified by
adding 3N HCl, and the solvent was again evaporated. A crude
chelidamic acid derivative was collected, as shown below: 59
[0138] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.78 (s, 2H), 4.86
(t, 1H), 3.84-2.61 (m, 26H), 1.36 (m, 6H), 0.99 (m, 3H), MS(FAB):
m/z 485 (M+1).sup.+.
Example 43
Preparation of
3,6-dioxa-9,12,15-triaza-9,12,15-triethyldodecane-1'-O'-che-
lidamic Acid Bonded to Silica Gel
[0139] About 384 mg of a
3,6-dioxa-9,12,15-triaza-9,12,15-triethyldodecane- -1-O'-chelidamic
acid (0.65 mmol) as prepared in Example 38 was dissolved in 2 ml
H.sub.2O and neutralized to pH 7-8 by adding 6N NaOH. To the above
solution was added 10 ml of a pH 10 buffer solution and 300 mg of
silica gel (60-100 mesh). The mixture was stirred at 45.degree. for
3 days. Next, the silica gel with ligand was filtered off and
washed with H.sub.2O and CH.sub.3OH. About 326 mg of chelidamic
acid triethyleneglycoltriethyl diethylenetriamine bonded to silica
gel was collected. The structure and reaction are shown below:
60
Example 44
Preparation of 9,13-Dithia-3,6,16,19-tetraoxaheneicosane-1,21-diol
Ditosylate
[0140] To 20 ml of methylene chloride and 5.1 g of triethylamine
was added 5.6 g of 9,13-dithia-3,6,16,19-tetraoxaheneicosane
1,21-diol. Then 6.1 g of p-toluenesulfonyl chloride at 0.degree. to
5.degree. C. in 20 ml of methylene chloride was added dropwise. The
mixture was stirred overnight at room temperature and then was
washed with 50 ml of water. The organic layer was dried over
MgSO.sub.4. After filtration, the solvent from the filtrate was
evaporated and the residue was purified using silica gel column
chromatography (CH.sub.2Cl.sub.2/CH.sub.3COCH.sub.3) (50:1 v/v).
About 8 g (78%) of
9,13-dithia-3,6,16,19-tetraoxaheneicosane-1,21-diol ditosylate was
collected. The process step for obtaining this composition is shown
schematically below: 61
[0141] Data verifying the composition was collected using H NMR and
is provided as follows .sup.1HNMR (CDCl.sub.3): 1.85(m, 2H),
2.45(s, 6H), 2.70(m, 8H), 3.7-3.5(m, 16H), 4.15(t, 4H), 7.35(d,
4H), 7.75(d, 4H).
Example 45
Preparation of 1,31-ditrityl
1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxah-
eneicosatriacontane
[0142] About 5.97 g of S-trityl-1,3-dithiapropane and
9,13-dithia-3,6,16,19-tetraoxaheneicosane-1,21-diol ditosylate was
added to 50 ml of DMF with 9.14 g of cesium carbonate. The mixture
was stirred at 60.degree. to 70.degree. C. for 72 hours. This
mixture was then cooled down, evaporated under reduced pressure and
the residue dissolved in 100 ml of CH.sub.2Cl.sub.2/Acetone (20/1
v/v). About 3.3 g of
1,31-ditrityl-1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxaheneicosatriaco-
ntane was collected. The process steps for obtaining this
composition are shown schematically below: 62
[0143] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR: (CDCl.sub.3): 1.65(m, 16H),
1.8(m, 2H), 2.25(t, 4H), 2.50(t, 4H), 2.65(m, 12H), 3.80(m, 16H),
7.74-7.70(m, 30H).
Example 46
Preparation of
1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxaheneicosatriaco-
ntane
[0144] A 2.43 g amount of
1,31-ditrityl-1,5,14,18,27,31-hexathia-8,11,21,2-
4-tetraoxaheneicosatriacontane was added to a mixture of 7 ml of
methylene chloride and 10 ml of triflouracetic acid and 0.75 ml of
triethylsilane. The mixture was stirred at room temperature for 1
hour. Then, 100 ml of methylene chloride was added and the reaction
mixture was washed with 100 ml of water. The organic layer was
dried over MgSO.sub.4. The solution was filtered and the filtrate
solvents were evaporated. The crude product was purified on silica
gel column chromatography with CH.sub.2Cl.sub.2/Acetone (5:1 v/v).
About 360 mg of
1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxaheneicosatriacontane
was collected. The process steps for obtaining this composition are
shown schematically below: 63
[0145] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR: (CDCl.sub.3): 1.47(t, 2H),
1.85(m, 6H), 2.65(m, 20H), 3.65(m, 16H).
Example 47
Preparation of
1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxaheneicosatriaco- ntane
Bonded to Silica Gel
[0146] About 280 mg of
1,5,14,18,27,31-hexathia-8,11,21,24-tetraoxaheneico- satriacontane
was dissolved in 80 ml of dioxane under nitrogen. Then 46 mg of
sodium hydride was added and was stirred for 2 hours. Next, 248 mg
of 3-bromopropyltrimethoxysilane was added and was refluxed
overnight. Then, 600 mg of silica gel (Grace) grade 646 was added
and was stirred at 90.degree. C. for 48 hours. After cooling down,
the silica gel was filtered off and was washed with methanol. The
product was dried at 50.degree. C. under vacuum. The process is
represented schematically below: 64
Example 48
Preparation of
1,26-Ditrityl-1,5,9,18,21,26-hexathia-12,15-dioxa-hexacosan- e
[0147] A mixture of 5.97 g of S-trityl-1,3 propanadithiol and 4.7 g
of 4,13-dithia-7,10-dioxa-1,16-hexanadecanediol tosylate and 6.09 g
of cesium carbonate was stirred at 60.degree. to 70.degree. C. in
50 ml of DMF during 72 hours. The solvent was removed under vacuum
and the residue was dissolved in 100 ml of methylene chloride. The
organic layer was washed with 30 ml of brine and dried over
anhydrous MgSO.sub.4. Next, the solids were filtered off and the
solvent was removed from the solution via evaporation. The residue
was purified on silica gel using methylene chloride. About 3 g
(41%) of 1,26-ditrityl-1,5,9,18,22,26-hexathia-12,15--
dioxa-hexacosane was obtained. The reaction is shown below
schematically: 65
[0148] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1H NMR(CDCl.sub.3): 1.65(m, 4H),
1.80(m, 4H), 2.65-2.25(m, 20H), 3.8(t, 8H), 7.25-7.5(m, 30H).
Example 49
Preparation of 1,5,9,18,22,26-hexathia-12,15-dioxahexacosane
[0149] A 2.14 g amount of 1,26
ditrityl-1,5,9,18,22,26-hexathia-12,15-diox- ahexacosane was
dissolved in a mixture of 9.0 ml of triflouroacetic acid and 9.0 ml
of methylene chloride. About 0.7 ml of triethylenesilane was added
and stirred at room temperature for a half hour. About 100 ml of
methylene chloride was added to the mixture and this was washed
with 50 ml of water. The organic layer was then dried with
magnesium sulfate and filtered. The solvent was evaporated from
filtrate and the residue was purified on silica gel using a mixture
of methylene chloride and acetone (5:1 v/v). About 620 mg (58%) of
1,5,9,18,22,26-hexathia-12,15-dioxahexac- osane was obtained. The
preparation scheme is shown below: 66
[0150] Data verifying the compound was collected using H NMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.4(t, 2H), 1.8(m,
8H), 2.7(m, 20H), 3.6(m, 8H).
Example 50
Preparation of 1,5,9,18,21,26-hexathia-12,15-dioxahexacosane Bonded
to Silica Gel
[0151] About 239 mg of
1,5,9,18,21,26-hexathia-12,15-dioxahexacosane was dissolved in 80
ml of dioxane and to this was added 46 mg of sodium hydride. After
2 hours, 255 mg of bromopropyltrimethoxysilane was added and
refluxed overnight. Then, 600 mg of silica gel was added and
stirred for 8 hours at 90.degree. C. The reaction was cooled down
and was filtered. The solid was washed with methanol and dried in a
vacuum oven at 60.degree. C. This process is represented
schematically below: 67
Example 51
Preparation of
1,17-di(4'"-phenyl-4'(2,2':6'2"-terpyridine))-7,11-dithia-1-
,4,14,17-tetraoxaheptadecane
[0152] A 5.68 g amount of tosylate of diethylene glycol
phenyl-4'(2,2':6'2"-terpyridine) and 0.62 g of 1,3-dithiapropanol-2
was added to 6.52 g of Cs.sub.2CO.sub.3 in 50 ml of DMF. The
mixture was stirred and heated 72 hours at 60.degree. C. DMF was
evaporated under reduced pressure and the residue was dissolved in
300 ml of chloroform. The organic layer was washed with 50 ml of
water and 50 ml of brine and was dried over MgSO.sub.4 and
filtered. From the filtrate the solvent was evaporated and the
residue was passed through a short aluminum oxide column. About
5.68 g of product was collected. The reaction formula is shown
below: 68
[0153] Data verifying the compound was collected using H NMR, and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.8(s, 1H),
2.6-2.8(m, 9H), 3.6(t, 4H), 3.8(t, 4H), 4.2(t, 4H), 7.0(d, 4H),
7.3(t, 4H), 7.8(t, 8H), 8.6-8.8(m, 12H).
Example 52
Preparation of 1,17
di-(4'"-phenyl-4'(2,2':6'2"-terpyridine)-7,11-dithia-1-
,4,14,17-tetraoxaheptadecane Bonded to Silica Gel
[0154] About 2.0 g of the above compound and 0.22 g of sodium
hydride was added to 50 ml of dioxane under nitrogen. After 15
minutes of stirring, 1 g of glycidoxyproyl silica gel was added and
this was heated 72 hours at 90.degree. C. After cooling down, 10 ml
of CH.sub.3OH was added dropwise. Then the modified silica gel was
filtered off and was washed with 200 ml of CH.sub.3OH . The product
was dried 24 hours at 60.degree. C. in a vacuum dryer. 69
Example 53
Preparation of Pyridacyl-2-pyridinium Iodide
[0155] To a well-stirred solution of pyridine (400 ml) at room
temperature was added 2-acetylpyridine (50 g, 0.41 mmole) and
iodine (104 g, 0.41 mmole). The resulting mixture was heated to
90.degree. to 100.degree. C. for 5 hours. The mixture was then
cooled to room temperature and allowed to stir at room temperature
for 16 hours. The mixture was filtered and recrystallized from
methanol (Yield: 80 g, 62%). The preparation scheme is shown below:
70
[0156] Data verifying the composition was collected using .sup.1H
NMR, and is provided as follows: .sup.1H NMR(CDCl.sub.3): 6.5(s,
2H), 7.8(m, 1H) 8.1(m, 2H), 8.20(t, 2H), 8.7(t, 1H), 8.8(d, 1H),
9.0(d, 2H).
Example 54
Preparation of 3,5-Dimethoxy Benzylidene-2-acetylpyridine
[0157] To a well stirred solution of NaOH (12 g, 0.3 mmole) in
H.sub.2O (125 ml) and methanol (250 ml) at 0.degree. C. was added
3,5-dimethoxy benzaldehyde (19 g, 0.11 mmole) followed by a
solution of 2-acetylpyridine (14.4 g, 0.12 mmole) in MeOH. The
reaction was stirred at 0.degree. C. for 1 hour and at room
temperature for 16 hours. The precipitate was filtered and
recrystallized from MeOH (Yield: 18 mg, 57%). The preparation
scheme is shown below: 71
[0158] Data verifying the composition was collected using .sup.1H
NMR and is provided as follows: .sup.1H NMR (CDCl.sub.3): 3.8(s,
6H), 6.5(s, 1H, b) 6.9(s, 1H, b), 7.5(m, 1H), 7.8-7.9(m, 2H),
8.2-8.4(m, 2H), 8.7(d, 1H).
Example 55
Preparation of
2,6-Bis(2'pyridyl)-4-(3,5-dimethoxyphenyl)pyridine
[0159] To a well stirred solution of MeOH (300 ml) and NH.sub.4OAc
(66 g, 855 mmole) was added
3,5-dimethoxybenzylidene-2-acetylpyridine (17.7g, 65.8 mmole) and
pyridacyl-2-pyridinium iodide (20.5 g, 65.6 mmole). The resulting
reaction mixture was refluxed for 6 hours. The reaction mixture was
cooled and filtered. The solid residue was washed with MeOH. The
product was purified on Al.sub.2O.sub.3-neutral, and eluted with
CH.sub.2Cl.sub.2/ethyl acetate (1/1) (Yield: 5.4 g, 22%). The
preparation scheme is shown below: 72
[0160] Data verifying the composition was collected using .sup.1H
NMR, and is provided as follows: .sup.1H NMR (CDCl.sub.3): 3.9(s,
6H), 7.1(s, 1H, b), 7.5(s, 2H, b), 7.8(m, 2H), 7.9(t, 2H),
8.6-8.7(m, 6H).
Example 56
Preparation of 2,6-Bis(2'pyridyl)4-(3,5-dihydroxyphenyl)
Pyridine
[0161] To a well stirred solution of
2,6-Bis(2'pyridyl)-4-(3,5-dimethoxyph- enyl) pyridine (5.4 g, 14.6
mmole) in acetic acid (105 ml) was added dropwise a solution of HBr
(105 ml, 49%) at room temperature. The reaction was heated to
120.degree. C. for 8 hours. The reaction was cooled, made basic
with 20% NaOH and brought to pH 12-14. The clear solution was
extracted with CH.sub.2Cl.sub.2 and the pH then brought to 7-8. The
solid was filtered and washed with MeOH (Yield: 1.6 g, 32%). The
preparation scheme is shown below: 73
[0162] Data verifying the composition was collected using .sup.1H
NMR and is provided as follows: .sup.1H NMR (CDCl.sub.3): 6.3(s,
1H), 6.7(s, 2H), 7.5(t, 2H), 8.0(t, 2H), 8.5-8.7(m, 6H).
Example 57
Preparation of
7,10,13-tri-p-toluenesulfonyl-7,10,13-triazatridecane ol.
[0163] A 30.8 g amount of N,N'N"-Tri-p-tosyldiethylenetriamine was
added to 200 ml DMF and 4.4 g of cesium carbonate. At room
temperature, 5 g of 6-bromohexanol was added dropwise. The mixture
was heated at 80.degree. C. for 40 hours. The solvent was
evaporated under reduced pressure and the residue was dissolved in
200 ml of methylene chloride and was washed twice with 50 ml of
water and with 50 ml of brine. The organic layer was dried over
MgSO.sub.4 and was filtered. The solvent was evaporated and the
residue was purified on silica gel with ethyl acetate/hexane. About
19 g of 7,10,13-tri-p-toluenesulfonyl-7,10,13-triazatridecane was
collected. The process steps for obtaining this composition are
shown schematically below: 74
[0164] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1H NMR (CDCl.sub.3): 1.20-1.40(m,
4H), 1.42-1.60(m, 4H), 1.75(t, 1H), 2.40-2.50(m, 9H), 3.0-3.4(m,
10H), 3.50-3.60(t, 2H), 5.40(t, 1H), 7.30-7.40(m, 6H), 7.60-7.80(m,
6H).
Example 58
Preparation of
7,10,13-tri-p-toluenesulfonyl-7,10,13-triazapentadecaneol
[0165] About 9 g of
7,10,13-tri-p-toluenesulfonyl-7,10,13-triazapentadecan- eol and 5.3
g of cesium carbonate was added to 200 ml of DMF and the mixture
was stirred. After 1 hour 2.5 g of iodoethane in 20 ml of DMF was
added dropwise. The mixture was heated for 16 hours at 60.degree.
C. After cooling the mixture, the mixture was filtered. From the
filtrate, the solvent was evaporated under reduced pressure. To the
residue 100 ml of CH.sub.2Cl.sub.2 was added. The organic layer was
washed 3 times with 50 ml of water and was dried over MgSO.sub.4.
The inorganic salts were filtered off and the organic solvents were
evaporated away from the filtrate. The residue was purified on
silica gel with CH.sub.2Cl.sub.2/acetone (20:1 v/v). About 8.1 g of
7,10,13-tri-p-toluenesulfonyl-7,10,13-triazapentadecaneol was
collected. The reaction is shown below. 75
[0166] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1H NMR (CDCl.sub.3): 1.2(t, 3H),
1.21-1.40(m, 4H), 1.50-1.60(m, 4H), 2.40-2.50(m, 9H), 3.20-3.40(m,
12H), 3.60(t, 2H), 7.30-7.40(m, 6H), 7.60-7.80(m, 6H).
Example 59
Preparation of 7,10,13-triazapentadecaneol
[0167] About 8 g of
7,10,13-tri-p-toluenesulfonyl-7,10,13-triazapentadecan- eol was
added to 3.9 g of sodium hydrogen phosphate and 66 g of sodium
amalgam in 250 ml of methanol. The mixture was stirred and refluxed
for 40 hours. The mixture was cooled down and was filtered. The
solvent was evaporated from the filtrate and to the residue 50 ml
of CH.sub.2Cl.sub.2 and 50 ml of 2N HCl was added. After shaking,
the acidic layer was separated and neutralized with 20% NaOH. From
the mixture, water was evaporated under reduced pressure and to the
residue was added 100 ml of methylene chloride. Next the inorganic
salts were filtered off. About 2.6 g of crude product was
transferred to the next step without further purification. The
reaction is shown below. 76
Example 60
Preparation of
7,10,13-tri(t-butoxycarbonyl)-7,10,13-triazapentadecanol
[0168] To a well stirred solution of 7,10,13-triazapentadecanol in
CH.sub.2Cl.sub.2(150 ml), was added (BOC).sub.2O (15 g, 69 mmole).
After 16 hours at RT, the solvents were evaporated. The residue was
purified on silica gel using CH.sub.2Cl.sub.2/acetone (20/1 v/v).
The product was obtained in quantitative yield. The reaction is
shown below: 77
[0169] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.10(t, 3H),
1.2-1.6(m, 37H), 3.1-3.4(m, 12H) 3.60(t, 2H).
Example 61
Preparation of 7,10,13-tri
(t-butoxycarbonyl)-7,10,13-triazapentadecanol Tosylate
[0170] A 6.1 g amount of 7,10,13-tri
(t-butoxycarbonyl)-7,10,13-triazapent- adecanol, 100 mg of
dimethylaminopyridine, and 4 ml of triethylamine in 100 ml of
CH.sub.2Cl.sub.2 was stirred at 0.degree. C. Dropwise, 2.64 of
tosyl chloride in 20 ml of CH.sub.2Cl.sub.2 was added at 0.degree.
to 5.degree. C. The reaction was then stirred overnight at room
temperature and then 100 ml of CH.sub.2Cl.sub.2 was added. The
reaction mixture was washed with 30 ml of 2N HCl, 30 ml of water
and 30 ml of brine. The organic layer was dried over MgSO.sub.4 and
was filtered. The solvent was evaporated and the residue from the
filtrate was purified using a silica gel column and eluted with
ethyl acetate/hexane (1:2 v/v). About 7.8 g of
7,10,13-tri(t-butoxycarbonyl)-7,10,13-triazapentadecanol tosylate
was collected. The reaction is shown below: 78
[0171] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.0-1.70(m,
3H), 2.50(s, 3H), 3.00-3.40(m, 12H), 4.0(t, 2H), 7.30-7.40(d, 2H),
7.70-7.80(d, 2H).
Example 62
Preparation of
hexa-t-butoxycarbonyl-(triamine).sub.2-hexane-meta-oxa-phen-
yl-terpyridine
[0172] About 500 mg of 3,5-dihydroxyphenylterpyridine and 2.1 g of
cesium carbonate was stirred 1 hour in 70 ml of DMF at 70.degree.
C. Then 2.25 g of
7,10,13-tri-(t-butoxycarbonyl)-7,10,13-triazapentadecanol tosylate
in 20 ml of DMF (dropwise) was added at 70.degree. C. After 16
hours, the mixture was cooled down and the solvent was evaporated
under reduced pressure. To the residue 50 ml of CH.sub.2Cl.sub.2
and 20 ml of H.sub.2O were added. The organic layer was separated
and was washed with 2% NaOH and 10 ml of brine. The mixture was
dried over MgSO.sub.4 and the inorganic salts were filtered off.
From the filtrate CH.sub.2Cl.sub.2 was evaporated. About 1.85 g
(92%) of crude product was collected and transferred to the next
step without further purification. The reaction is shown below:
79
[0173] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.10(t, 6H),
1.30-1.60(m, 66H), 1.70(m, 4H), 3.10-3.30(m, 24H), 4.0(t, 4H),
6.5(s, 1H), 6.9(s, 2H), 7.30(t, 2H), 7.80(t, 2H), 8.60-8.70(m,
4H).
Example 63
Preparation of
N-ethyl-N'-{2-[3-{3-(3-(2-ethylamino-ethylamino)-ethylamino-
}-propoxy}-5-[2,2',6'2"]terpyridin-4'-yl-phenoxy)-propylamino]ethyl}-ethan-
e-1,2-diamine.
[0174] About 800 mg of the product compound of the previous example
was added to 20 ml of triflouroacetic acid and was stirred at room
temperature 16 hours. Then, triflouroactic acid was evaporated
under reduced pressure. About 1.3 g of crude triflauroacetic salt
of the product was obtained. The compound was used in the next step
without further purification. The reaction is shown below. 80
[0175] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR(CDCl.sub.3): (free amine),
1.10(t, 6H), 1.25-1.80(m, 12H), 2.50-2.80(m, 30H), 4.0(t, 4H),
6.50(s, 1H), 7.0(s, 2H), 7.3(m, 2H), 7.90(t, 2H), 8.20(m, 6H).
Example 64
Preparation of
N-Ethyl-N'-{2-[3-(3-{3-[2-(2-ethylamino-ethylamino)-ethylam-
ino]-propoxy{-5-[2,2';6'2"]terpyridin-4'-yl-phenoxy)-propylamino]-ethyl}-e-
thane-1,2-diamine bonded to silica gel.
[0176] A 0.55 g amount of the title compound was neutralized with
5N sodium hydroxide to pH 10. Then 10 ml of buffer solution (pH 10)
was added and 600 mg of glycidoxypropyl silica gel was added. The
mixture was stirred 40 hours at 40.degree. to 45.degree. C. and was
filtered. The modified silica gel was washed with methanol, THF,
and then methanol and finally was dried under vacuum at 50.degree.
C. The preparation scheme is shown below: 81
Example 65
Preparation of 1,4,7,13,16,19,25,28,31
nona(p-toluenesulfonyl)-1,4,7,13,16-
,19,25,28,31-nonaazaheneicosatriacontane
[0177] About 5 g of 7,10,13 tri
(p-toluenesulfonyl)-7,10,13-triaza-4,16-di- oxanonadecane-1,19-diol
ditosylate, 7.92 g of 1-butoxycarbonyl-1,4,7-tri
(p-toluenesulfonyl)-1,4,7-triazaheptane, and 18 g of
Cs.sub.2CO.sub.3 were mixed with 350 ml of DMF at 70 to 80.degree.
C. for 72 hours. The temperature was slowly raised to 130.degree.
C. and stirring continued for 36 hours. The mixture was cooled and
poured over water with ice, and was extracted with
CH.sub.2Cl.sub.2. The organic phase was dried over MgSO.sub.4,
filtered and concentrated under reduced pressure to give 13.52 g of
crude product, which was purified on silica gel and eluted with
CHCl.sub.3/ethyl acetate (50/1 v/v). About 2.5 g of pure product
was obtained. The preparation scheme is shown below: 82
[0178] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 2.4(m, 27H),
3.2-3.6(m, 40H), 7.2-7.4(m, 18H), 7.6-7.8(m, 18H).
Example 66
Preparation of
1,4,7,13,16,19,25,28,31-nonaazaheneicosatriacontane.
[0179] A 2.13 g amount of
1,4,7,13,16,19,25,28,31-nona(p-toluenesulfonyl)--
1,4,7,13,16,19,25,28,31-nonaazaheneicosatriacontane, 2.13 g of
phenol, and 160 ml of a 33% solution of HBr in acetic acid were
heated at 70 to 80.degree. C. for 2 days. The solution was then
cooled. The acetic acid was removed under vacuum; 100 ml toluene
was added and evaporated to remove the rest of the acetic acid. The
residue was allowed to partition between 100 ml CH.sub.2Cl.sub.2
and 50 ml H.sub.2O. The aqueous layer was washed three times with
CH.sub.2Cl.sub.2 and finally concentrated to .about.5 ml: MeOH was
then added until the mixture became cloudy. 500 mg (36%) of
1,4,7,13,16,19,25,28,31-nonaazaheneicosatriacontane.9HBr
crystallized. The preparation is shown below: 83
[0180] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.38-3.75(m, 32H),
3.82(t, 8H).
Example 67
Preparation of 1,4,7,13,16,19,25,28,31 Nonaazaheneicosatriacontane
Bonded to Silica Gel
[0181] About 1 g (0.85 mmole)
1,4,7,13,16,19,25,28,31-nonaazaheneicosatria- contane.9HBr was
neutralized with 2 N NaOH solution to pH.about.8-9. Then, 10 ml of
buffer solution (pH.about.10) and 350 mg glycidoxypropyltrimetho-
xysilane were added and stirred at 45.degree. C. for 2 days. After
cooling, the mixture was filtered and washed with H.sub.2O and MeOH
to give product. The preparation scheme is shown below: 84
Example 68
Preparation of
1,23-di(t-butoxycarbonyl)-1,4,7,17,20,23-hexa(p-toluenesulf-
onyl)1,4,7,17,20,23-hexaazatricosene
[0182] A 18.73 g amount of
1-butoxycarbonyl-1,4,7-tri(p-toluenesulfonyl)-1- ,4,7-triazaheptane
was mixed with 3.5 g of 1,9-dibromononane and 37.5 g of
Cs.sub.2CO.sub.3 in 1000 ml of DMF at 65 to 70.degree. C. for 22
hours. Stirring was continued at room temperature overnight. The
DMF was evaporated under reduced pressure. CH.sub.2Cl.sub.2 was
added, the solid was filtered and the solvent was evaporated to
give 21.97 g of crude product, which was purified on silica gel and
eluted with CH.sub.2Cl.sub.2/ethyl acetate (100/1 to 70/1 v/v). 9.3
g of pure product was obtained. The preparation scheme is shown
below: 85
[0183] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.2(s, 1OH),
1.4(s, 22H), 2.4(m, 18H), 3.1(t, 4H), 3.4(m, 12H), 4.0(m, 4H),
7.3(m, 12H), 7.7(m, 12H).
Example 69
Preparation of
1,4,7,17,20,23-hexa(p-toluenesulfonyl)1,4,7,17,20,23-hexaaz-
atricosene)
[0184] A 3.68 g amount of
1,23-di(t-butoxycarbonyl)-1,4,7,17,20,23-hexa(p--
tolenesulfonyl)-1,4,7,17,20,23-hexaazatricosene and 50 ml of
trifluouroacetic acid were stirred at room temperature for 2 hours.
The solvent was evaporated under reduced pressure to dryness and
the residue was treated with MeOH. The crystals were filtered,
rinsed with MeOH and dried overnight in a vacuum oven. About 2.85 g
of product was obtained. The preparation scheme is shown below:
86
[0185] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.2(s, 14H),
1.5(s, 4H), 2.4(s, 18H), 3.0-3.3(m, 20H), 7.2-7.8(m, 24H).
Example 70
Preparation of
16-butoxycarbonyl-10,13,16-tri(p-toluenesulfonyl)-10,13,16--
triazahexadecanol
[0186] A mixture of 14 g of
1-butoxycarbonyl-1,4,7-tri-(p-toluenesulfonyl)-
-1,4,7-triazaheptane, 7.04 g of 9-bromo-1-nonanol, and 34.25 g of
Cs.sub.2CO.sub.3 in 700 ml of DMF was stirred at 65.degree. C. for
24 hours. The mixture was cooled and concentrated under reduced
pressure. CH.sub.2Cl.sub.2 was added and the remaining solid was
filtered and discarded. The solvent was evaporated to give 22.17 g
of crude product which was purified on silica gel and eluted with
CH.sub.2Cl.sub.2/ethyl acetate (50/1-20/1 v/v). 7.95 g of pure
product was obtained. The preparation scheme is shown below: 87
[0187] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.2-1.7(m,
23H), 2.4(m, 9H), 3.1 (t, 2H), 3.4(m, 6H), 3.6(m, 2H0, 4.0(m, 2H),
7.45(m, 8H), 7.8(m, 8H).
Example 71
Preparation of
16-butoxycarbonyl-10,13,16-tri(p-toluenesulfonyl)-10,13,16--
triazahexadecaneol Tosylate
[0188] To 5 .47 g of
16-butoxycarbonyl-10,13,16-tri(p-toluenesulfonyl)-10,-
13,16-triazahexadecanol in 200 ml of CH.sub.2Cl.sub.2, 2.95 g of
Et.sub.3N was added and the mixture was cooled to 0.degree. C. To
the well stirred solution was added dropwise at 0.degree. C. 0.22 g
of 4-dimethylaminopyridine and then 1.67 g of p-toluenesulfonyl
chloride in 100 ml of CH.sub.2Cl.sub.2 was added. The reaction was
continued overnight at room temperature. A 0.04 g amount of
4-dimethylaminopyridine and 0.33 g of p-toluenesulfonyl chloride
were added at 7.degree. C. and stirring was continued for 4 hours.
The mixture was extracted with water twice. The organic layers were
dried over MgSO.sub.4, filtered and evaporated under reduced
pressure and eluted with CH.sub.2Cl.sub.2 and
CH.sub.2Cl.sub.2/ethyl acetate (100/1 v/v). About 4.21 g of product
was obtained. The preparation scheme is shown below: 88
[0189] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.3(m, 14H),
1.4(m, 9H), 2.45(m, 12H), 3.05(t, 2H), 3.4(m, 6H), 4.0(m, 4H),
7.35(m, 8H), 7.8(m, 8H).
Example 72
Preparation of
1,55-di(t-butoxycarbonyl)-1,4,7,17,20,23,33,36,39,49,52,55--
dodecane(p-toluenesulfonyl)-1,4,7,17,20,33,36,39,49,52,55-dodecanazapentap-
entacontane
[0190] To a well stirred mixture of 2.67 g of
1,4,7,17,20,23-hexa(p-toluen-
esulfonyl)1,4,7,17,20,23-hexaazatricosene in 150 ml of DMF was
added 5.55 g of Cs.sub.2CO.sub.3. The mixture was stirred at room
temperature for 0.5 hours. 4.71 g of
16-butoxycarbonyl-10,13,16-tri(p-toluenesulfonyl)-10-
,13,16-triazahexadecanol in 75 ml of DMF was added dropwise at room
temperature. After addition was completed, the temperature was
raised to 70.degree. C. and stirring was continued for 24 hours.
The reaction mixture was cooled and DMF was evaporated under
reduced pressure. The residue was washed with CH.sub.2Cl.sub.2, the
solid was filtered and discarded, and the solution was concentrated
under reduced pressure to give 7.93 g of crude product, which was
purified on silica gel and eluted with CH.sub.2Cl.sub.2/ethyl
acetate (50/1 v/v). About 3.1 g of pure product was obtained. The
preparation scheme is shown below. 89
[0191] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.2(s, 30H),
1.35(m, 18H), 1.5(m, 12H), 2.4(m, 36H), 3.0-3.5(m, 40H), 4.0(t,
4H), 7.3(m, 24H), 7.8(m, 24H).
Example 73
Preparation of
1,4,7,17,20,23,33,36,39,49,52,55-dodecaneazapentapentaconta- ne
[0192] About 1.5 g
1,55-di(t-butoxycarbonyl)-1,4,7,17,20,23,33,36,49,52,55-
-dodecane{p-toluene
sulfonyl)-1,4,7,17,20,33,36,39,49,52,55-dodecaneazapen-
tapentacontane, 1.22 g of phenol, and 100 ml of a 33% solution of
HBr in acetic acid were heated at 70 to 80.degree. C. for 2 days.
After cooling the purple precipitate was isolated by filtration and
washed thoroughly with Et.sub.2O to give
1,4,7,20,23,33,36,39,49,52,55-dodecaneazapentopent- acontane.12HBr
(100% yield). The preparation is shown below: 90
[0193] Data verifying the composition was collected using HNMR, and
is provided as follows: .sup.1HNMR(D.sub.2O): 1.65(m, 30H), 1.75(m,
12H), 3.2(t, 12H), 3.5(m, 32H).
Example 74
Preparation of
1,4,7,17,20,23,33,36,39,49,52,55-dodecaneazapentapentaconta- ne
Bonded to Silica Gel
[0194] About 870 mg of
1,4,7,20,23,33,36,39,49,52,55-dodecaneazapentopenta- contane.12HBr
was neutralized with 2N NaOH solution to pH 8.about.9. Then, 10 ml
of buffer solution (pH.about.10) and 350 mg of glycidoxypropyl
modified silica were added and stirred at 45.degree. C. for 2 days.
After cooling, the mixture was filtered and washed with H.sub.2O
and MeOH to give silica gel with bounded amine. The preparation
scheme is shown below: 91
Example 75
Preparation of 9,12,15-triaza-3,6-dioxa-pentadecanol
[0195] A mixture of diethylene triamine (460 ml, 4.3 mole) and
(2-iodoethoxy) ethoxyethanol (7.30 g, 28 mmole) was stirred at room
temperature for 1 hour. The excess of diethylenetriamine was
removed by distillation under vacuum. The residue was then treated
with aqueous Satd NaOH, extracted with CH.sub.2Cl.sub.2, dried over
MgSO.sub.4/Na.sub.2CO.s- ub.3, filtered, and concentrated to give a
yellow oil. This was purificated by column chromatography on silica
with CH.sub.3OH/NH.sub.4OH (10:1), then (5:1) to give 1.43 g (33%)
of the title compound as a pale yellow oil. The reaction is shown
schematically as follows: 92
[0196] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 2.2(6s, 2H),
2.8(m, 10H), 3.7(m, 10H).
Example 76
Preparation of
9,12,15-(tri-t-butoxycarbonyl)-9,12,15-triaza-3,6-dioxapent-
adecanol
[0197] A solution of di-t-butyl-dicarbonate (4.75 g, 22 mmole) in
30 ml dioxane was added to a stirring mixture of the triamine
produced from Example 75 (1.43 g, 6.1 mmole), NaOH(1.76, 44 mmole)
in 60 ml H.sub.2O at 15.degree. C. dropwise, and the reaction
mixture was stirred overnight at room temperature. The reaction
mixture was partitioned in Et.sub.2O/H.sub.2O. The ether layer was
dried over MgSO.sub.4, filtered, and concentrated to give 2.03 g
(62%g) of crude material colorless oil. This product was used for
the next step without further purification. The reaction is shown
schematically as follows: 93
[0198] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.4(m, 10H),
3.6(m, 10H).
Example 77
Preparation of
9,12,15-tri-t-butoxycarbonyl-9,12,15-triaza-3,6-dioxapentad-
ecanol-p-toluenesulfonate
[0199] A mixture of the nitrogen protected alcohol (2.0 g, 3.7
mmole), Et.sub.3N (0.42 g, 4.2 mmole), DMAP(0.10 g, 0.82 mmole) and
Tosyl Chloride (0.78 g, 4.1 mmole) in 80 ml of CH.sub.2Cl.sub.2 was
allowed to stir overnight. The reaction mixture was then diluted
with CH.sub.2Cl.sub.2 (200 ml) and washed with H.sub.2O. The
organic layer was dried over MgSO.sub.4, filtered, and concentrated
to give a pale yellow oil, crude material which was purified by
column chromatography on silica with Hexanol/Ethyl Acetone (1:1),
to give 2.13 g (84%) of the compound as a colorless oil. The
reaction is shown schematically below. 94
[0200] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.4(ms, 27H),
2.4(6s, 3H), 3.4-3.5(m, 16H), 3.7(t, 2H), 4.2(t, 2H), 7.3(d, 2H),
7.6(d, 2H).
Example 78
Preparation of
10,13,16-triaza-1,4,7-trioxahexadecanyl-4(2,2',6'2"-terpyri- dyl)
Phenyl Ether
[0201] A mixture of N-Boc protected sulfonate (1.25 g, 1.8 mmole)
hydroxy-phenylterpyridine (0.6 g, 1.8 mmole), and Cs.sub.2CO.sub.3
(0.90 g, 2.7 mmole) in DMF (50 ml), was heated to .about.55.degree.
C. overnight. The reaction was cooled, and partitioned in
CH.sub.2-Cl.sub.2/H.sub.2O and separated. The aqueous layer was
extracted with CH.sub.2Cl.sub.2 (300 ml .times.2). The combined
organic layers were dried over MgSO.sub.4, filtered, and
concentrated to give 1.0 g (66%) of crude material as a pale yellow
oil which was used for the next step without purification. The
deprotected reaction was as follows: the above/crude material (0.96
g, 0.75 mmole) was stirred in CF.sub.3COOH(15 ml) at room
temperature overnight. The volatiles were then removed under
reduced pressure. The residue was then taken up in CH.sub.2Cl.sub.2
(200 ml), washed with Satd. Na.sub.2CO.sub.3 dried over MgSO.sub.4,
filtered, and concentrated to give the title compound (0.38 g, 94%)
as a pale yellow oil. The scheme is shown schematically below:
95
[0202] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 2.0(6s, 4H),
2.8(m, 10H), 3.6(m, 6H), 3.9(t, 2H), 4.2(t, 2H), 7.3(m, 2H), 7.8(m,
4H), 8.7(m, 6H).
Example 79
Preparation of Silica Bound triamine-dioxa-phenyl-terpyridine
[0203] Triamine-dioxa-phenyl-terpyridine (0.30 g, 0.6 mmole) was
heated with epoxy-activated silica gel (1.0 g) in 50 ml toluene at
800.degree. C. for 20 hours. The product was collected by
filtration, washed with toluene and CH.sub.3OH, and dried in a
vacuum oven at 55.degree. C. for 20 hours. The reaction is shown
schematically below. 96
Example 80
Preparation of
7,10,13-triaza-1,4-dioxa-tridecanyl-4-(2,2'6',2"-terpyridyl- )
Phenyl Ether.
[0204] A mixture of diethylenetriamine (71.69 g, 0.69 mmole) and
0'-terpyridyl-phenyl-diethylglycol-tosylate (2.0 g, 3.5 mmole) was
stirred at room temperature for 21 hours. The excess triamine was
removed by distillation under reduced pressure. The residue was
partitioned in CHCl.sub.3/H.sub.2O. The organic layer was dried,
filtered and concentrated to leave the title compound as a yellow
oil (2.05 g, quantitative). The reaction is shown schematically
below: 97
[0205] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 2.7(m, 10H),
3.6(t, 2H), 3.8(t, 2H), 3.6(t, 2H), 3.8(t, 2H), 4.2(t, 2H), 7.1(m,
2H), 7.3(m, 2H), 7.8(m, 6H), 8.7(m, 6H).
Example 81
Preparation of Silica Bound triamine-trioxa-phenyl-terpyridine
[0206] A mixture of triamine-trioxa-phenyl-terpyridine (0.35 g,
0.65 mmole) and epoxy-activated silica gel (1.20 g) was heated to
80.degree. C. in 60 ml of toluene for 20 hours. The product was
collected by filtration, washed with toluene and methanol, and then
dried in a vacuum oven for 15 hours. The reaction is shown
according to the scheme below. 98
Example 82
Preparation of
7,10,13,22,25,28-hexaaza-1,4,16,19-tetraoxaoctacosanyl-4'"--
(2,2'6'2"-terpyridyl) Phenylether
[0207] A mixture of hexamine (4.09 g, 12.8 mmole),
terpyridylphenyl-diethy- glycol tosylate (1.81 g, 3.2 mmole) was
refluxed in CH.sub.2Cl.sub.2 for 4 days. An oil thus formed was
separated from CH.sub.2Cl.sub.2 by decanting. The oil was washed
with CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 solution was
concentrated to give a crude oil, which was passed through a short
pad of silica gel, eluted with CH.sub.3OH, and then
CH.sub.3OH/NH.sub.4OH. Two major fractions were collected and
checked. The last fraction (0.48 g) was taken up in
CH.sub.2Cl.sub.2, and washed with H.sub.2O to remove trace amount
of SuperLig.RTM. hexamine. After drying and concentration, 0.43 g
(5%) of the title compound was obtained as a pale yellow oil. The
reaction is shown schematically below. 99
[0208] Data verifying the composition was collected using H NMR,
and is provided as follows: .sup.1HNMR (CDCl.sub.3): 1.6(6s, 7H),
2.7(m, 22H), 3.7(m, 10H), 3.8(t, 2H), 4.2(t, 2H), 7.1(m, 2H),
7.3(m, 2H), 7.8(m, 4H).
Example 83
Preparation of Silica Bound
7,10,13,22,25,26-hexane-1,4,16,19-tetraoxa
Octacosanyl-4"(2,2',6',2"-terpyridyl) Phenylether
[0209] An amount of 7,10,13,22,25,26-hexane-1,4,16,19-tetraoxa
octacosanyl-4"(2,2',6',2"-terpyridyl) phenylether (0.18 g, 0.25
mmole) was heated with epoxy activated silica gel (0.46 g) in 50 ml
of toluene at 85.degree. C. for 19 hours. The product was collected
by filtrate, washed with toluene and methanol and then dried in the
vacuum oven for 20 hours. The reaction is shown schematically
below: 100
Example 84
Preparation of 4-N-malonate-tetraaza-12-crown
[0210] A mixture of cyclene (3.0 g, 17.4 mmole), bromomalonate
(1.38 g, 5.8 mmole), and K.sub.2CO.sub.3 (4.80 g, 34.6 mmole) was
heated to gentle reflux for 24 hours. The insoluble material was
filtered off, and washed with CH.sub.3CN. The compound filtrate was
concentrated to leave an oil which was further purified by column
chromatography on Al.sub.2O.sub.3 to give 2.10 g (40%) of the
impure compound. The reaction is shown schematically below. 101
[0211] The 4-N-malonate-tetraaza-12-crown was identified by .sup.1H
NMR spectroscopy as follows: .sup.1HNMR (CDCl.sub.3): 4.9(bs,
--OH--NH), 4.3(m), 3.0(m).
Example 85
Preparation of N-tetraaza-12-crown-4-N-malonate dilithium-salt
[0212] About 1 g of N-malonate tetraaza-12-crown (1.0 g, 3.0 mmole)
was treated with LiOH (0.44 g, 1.8 mmole) in CH.sub.3OH (60 ml) at
room temperature for 20 hours. The reaction mixture was then
concentrated by evaporation. To the residue, CHCl.sub.3 was added
while shaking. The insoluble matter was then filtered and washed
with CHCl.sub.3. The combined CHCl.sub.3 solution was concentrated
to give a yellow solid (0.91 g, 92% crude yield). The reaction is
shown schematically below: 102
[0213] The N-tetraaza-12-crown-4-N-malonate dilithium-salt was
identified by means of H NMR spectroscopy as follows: .sup.1HNMR
(CD.sub.3OD): 4.9(bs,--OH,--NH), 4.5(m), 3.6(m), 2.6(m).
Example 86
Preparation of tetraaza-12-crown-4-N-malonate, dilithium-salt
Bonded to Silica Gel
[0214] The title compound was attached to silica gel according to
the scheme shown below by employing the title compound (0.40 g, 1.4
mmole) and silica gel(0.40 g) in 50 ml of toluene at 80.degree. C.
for 21 hours. The product was filtered, washed with toluene and
dried under vacuum at 55.degree. C. for 20 hours. The reaction is
shown schematically below: 103
Example 87
Preparation of
9,12,15,25,28,31-hexa(p-toluenesulfonyl)-9,12,15,25,28,31-h-
exaaza-3,6-dioxaheneitriacontanol
[0215] A mixture of
9-bis-(N,N'N"-tri-p-tolysulfonyl-diethylenetriamine)no- nane (3.50
g, 2.8 mmole) and cesium carbonate (1.81 g, 5.60 mmole) in DMF (450
ml) was heated to 95.degree. C. Then a solution of
(2-chloroethoxy)ethoxy ethanol (0.479, 2.8 mmole) in DMF (100 ml)
was added dropwise over 30 mins, and the temperature maintained at
95.degree. C. overnight. The volatiles were evaporated off under
reduced 10 pressure and the residue was evaporated with toluene
twice (250 ml.times.2). The final residue was partitioned in
CH.sub.2Cl.sub.2/H.sub.2O. The organic layer was collected, dried
over MgSO.sub.4, filtered, and concentrated to give a crude
material which was further purified by column chromatography on
silica with Hexane/Ethyl Acetate (10:1); (5:1); (2:1); (1:1);
(1:2); to give the title compound (1.80g. 46%). The reaction is
shown schematically below: 104
[0216] Data verifying the composition was collected using HNMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.75 (m, 12H),
7.35(m, 12H), 3.65(m, 1OH), 3.23(m, 22H), 1.50 and 1.20
(2bs/14H)
Example 88
Preparation of
9,12,15,25,28,31-hexa(p-toluenesulfonyl)-9,12,15,25,28,31-h-
exaaza-3,6-dioxatritriacontanol
[0217] To a stirring mixture of
9,12,15,25,28,31-hexa(p-toluenesulfonyl)-9-
,12,15,25,28,31-hexaaza-3,6-dioxahenetriacontanol (1.80 g, 1.3
mmole) and Cs.sub.2CO.sub.3 (0.70 g, 2.0 mmole) in DMF (50 ml),
ethyliodide (0.30 g, 2.0 mmole) was added at room temperature. Then
the temperature was raised to 65.degree. to 70.degree. C. for 4.5
hours. After being cooled, the reaction mixture was partitioned in
CH.sub.2CI.sub.2/H.sub.2O. The organic layer was dried over
MgSO.sub.4, filtered, and concentrated to give a crude oil. This
was further purified by column chromatography with silica and
Hexane/Ethyl Acetate (1:1), then (1:2), to give 1.41g (77%) of
title compound. The reaction is shown schematically below: 105
[0218] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 7.7(m, 12H),
7.35(m, 12H), 3.6(m, 10 H), 3.3(m, 24H), 2.4(s's, 18H), 1.5 and
1.3(2bs, 14H), 1.2(t, 3H)
Example 89
Preparation of
9,12,15,25,28,31-hexaaza-3,6-dioxatritriacontanol
[0219] A mixture of 9,12,15,25,28,31-hexa(p-toluenesulfonyl)-9,
12,15,25,28,31-hexaaza-3,6-dioxatritriacontanol (1.30 g, 0.92
mmole), Na/Hg (10.85 g), and phosphoric acid disodium salt (1.96 g,
13.8 mmole) in methanol/dioxane (70 ml/40 ml) was heated to gentle
reflux for 24 hours. Additional Na/Hg (2.0 g) was then added. The
reaction mixture was maintained at reflux for another 24 hours,
then cooled. The insoluble material was filtered and washed with
CH.sub.3OH. The filtrate was concentrated to give an oily residue
which was then treated with 2 N HCI (aq) and washed with
CH.sub.2C1.sub.2 (200ml.times.2). The aqueous acidic solution was
then made basic with 10% NaOH (aq), extracted with CH.sub.2CI.sub.2
(250ml.times.3), dried over MgSO.sub.4, filtered and concentrated
to give a pale yellow oil (0.25 g, 55% crude yield) which was used
further without purification. The reaction is shown schematically
below: 106
[0220] Data verifying the composition was collected using HNMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.65(m, 10H),
2.75(m, 44H), 1.5 and 1.3(2bs, 14H), 1. 15(t, 3H)
Example 90
Preparation of
9,12,15,25,28,31-hexa(t-butoxycarbonyl)-9,12,15,25,28,31-he-
xaaza-3,6-dioxatritriacontanol
[0221] A solution of di-t-butyl-dicarbonate (0.75 g, 3.4 mmole) in
1,4-dioxane (30 ml) was added to a mixture of
9,12,15,25,28,31-hexaaza-3,- 6-dioxatriacontanol (0.24 g, 0.49
mmole), and sodium hydroxide (0.27 g, 0.68 mmole) in 30ml of
H.sub.2O at 10-15.degree. C. dropwise. After stirring for 2 hours,
the reaction mixture was diluted with water, extracted with
Et.sub.2O (200 ml.times.2), dried over MgSO.sub.4, filtered and
concentrated by evaporation to give 0.36 (66%) of title compound as
a colorless oil. The reaction is shown schematically below: 107
[0222] Data verifying the composition was collected using H NMR and
is provided as follows: .sup.1HNMR (CDCl.sub.3): 3.65(m, 10 H),
3.35(m, 24H), 1.45(s, 54H), 1.45 and 1.3(bs, 14H), 1.1(t, 3H).
Example 91
Preparation of
9,12,15,25,28,31-hexa-(t-butoxycarbonyl)-9,12,15,25,28,31-h-
exaaza-3,6-dioxa-tritriacontanyl-p-tosyl-sulfonate
[0223] A mixture of
9,12,15,25,28,31-hexaaza(t-butoxycarbonyl)-9,12,15,25,- 28,3
1-hexaaza-3,6-dioxatritriacontanol (0.35 g, 0.32 mmole),
triethylamine (0.036 g, 0.36 mmole), 4-dimethylaminopyridine (0.05
g, 4.1 mmole), and tosyl chloride (0.07 g 0.35 mmole) was stirred
at room temperature in CH.sub.2Cl.sub.2 (50 ml) for 20 hours, and
then stirred at 50.degree. C. for 30 minutes. The reaction mixture
was diluted with CH.sub.2Cl.sub.2, washed with H.sub.2O, dried over
MgSO.sub.4, filtered and concentrated to give an oil. The final
title compound was obtained after purification by column
chromotagraphy on silica gel with Ethyl Acetate/Hexanes (2:1), to
give a colorless oil (0.22 g, 55%). The reaction is shown
schematically below: 108
[0224] The title compound was characterized by H NMR (CDCl.sub.3):
7.6(d, 2H), 7.4(d, 2H), 4.2(t, 2H), 3.7(m, 2H), 3.5(m, 6H), 3.4(m,
24H), 2.4(S, 3H), 1.4(S, 54H), 1.48 and 1.2(m, 14H), 1.1(t,
3H).
Example 92
Preparation of
10,13,16,26,29,32-hexaaza-1,4,7-trioxatetratriacontanyl-4'"-
-(2,2',6',2"-terpyridyl) phenyl ether
[0225] A mixture of
9,12,15,25,28,31-hexa-(t-butoxycarbonyl)-9,12,15,25,28-
,31-hexaaza-3,6-dioxa tritriacontanyl-p-toluenesulfonate (0.21 g,
0.17 mmole), 4'(-4-hydroxyphenyl)-terpyridine(0.06 g, 0.19 mmole),
and Cs.sub.2CO.sub.3 (0.06 gm 0.19 mmole) was heated to 80.degree.
C. in DMF (30 ml) overnight, The reaction mixture was partitioned
in CH.sub.2Cl.sub.2/H.sub.2O. The organic layer was dried over
MgSO.sub.4, filtered and concentrated to give an oily crude product
which was then dissolved in 20 ml of CF.sub.3COOH. The reaction
mixture was stirred overnight. The volatiles were evaporated and
the residue was taken up in CH.sub.2Cl.sub.2, washed with
Na.sub.2CO.sub.3(aq), dried over MgSO.sub.4, filtered and
concentrated to give a 10,13,16,26,29,32-hexaaza-
-1,4,7-trioxatetratriacontanyl-4'"-(2,2',6',2"-terpyridyl) phenyl
ether oil (0.07 g, 52%). The reaction is shown schematically as
follows: 109
[0226] The title compound was characterized by H NMR spectroscopy
in CDCl.sub.3: 8.7(m, 6H), 7.8(m, 4H), 7.2(m, 2H), 7.4(m, 2H),
4.2(t, 2H), 3.8(t, 2H), 3.6(m, 6H), 2.6(m, 24H), 1.4 and 1.2(m,
14H), 1.1 (t, 3H).
Example 93
Preparation of
10,13,16,26,29,32-hexaaza-1,4,7-trioxatetratriacontanyl-4'"-
-(2,2',6',2"-terpyridyl) Phenyl Ether
[0227] A mixture of
triamine-nonane-triamine-para-trioxa-phenyl-terpyridin- e (0.07,
0.09 mmole) and epoxy-activated silica gel (0.20 g) was heated to
90.degree. C. in 50 ml of toluene overnight. The product was
collected by filtration, washed with toluene and CH.sub.3OH, and
dried in a vacuum oven for 15 hours. The reaction is shown
schematically below: 110
[0228] Separation and selectivity examples are now provided that
utilize palladium(II)-bound ligands bonded to solid supports as
described in Examples 1 to 93.
Example 94
Separation of Guanosine Monophosphate
[0229] A 0.0406 g (wet) amount of methylated ethylene triamine
covalently bonded to silica material as described in Example 3
loaded with palladium was placed in a column. A 25.degree. C. 4 ml
source solution consisting of 1.25 mMolar each of adenosine
monophosphate (hereinafter "AMP"), cytidine monophosphate
(hereinafter "CMP"), thymidine monophosphate (hereinafter "TMP"),
guanosine monophosphate (hereinafter "GMP"), and uridine
monomonophosphate (hereinafter "UMP") in 0.01 Molar pH 7 phosphate
buffer (Na salts) was drawn through the column at a flowrate of
0.02 ml/min. The column was then washed with 1 ml of 0.01 Molar
phosphate pH 7 buffer at 0.05 ml/min flowrate followed by 2 ml of
2.5 Molar phosphate pH 5 buffer at a flowrate of 0.05 ml/min. The
column was eluted or stripped using 1 ml of 0.5 Molar NH.sub.3 at a
flowrate of 0.01 ml/min. The collected 1 ml of elution contained
guanine monophosphate at a purity of >99% compared to all of the
other mononucleotides present as measured by HPLC (High Pressure
Liquid Chromatography). The other mononucleotides could not be
detected in the 0.161 mMolar guanine monophosphate collected.
Example 95
Separation of Guanosine Monophosphate
[0230] A 0.0421 g (wet) amount of a palladium loaded nonmethylated
diethylene triamine covalently bonded to silica material of Example
1 was placed in a column. A 25.degree. C. 1.9 ml source solution
consisting of 1.25 mMolar each of AMP, CMP, TMP, GMP, and UMP in
0.01 Molar pH 7.3 phosphate buffer (Na salts) was drawn through the
column at a flowrate of 0.02 ml/min. The column was then washed
with 1 ml of 0.01 molar phosphate pH 7 buffer at 0.05 ml/min
flowrate followed by 2 ml of pH 5 2.5 Molar phosphate buffer at a
flowrate of 0.05 ml/min flowrate. Finally, the column was eluted or
stripped using 1 ml of 1 Molar NH.sub.3 at a flowrate of 0.015
ml/min. The collected 1 ml of elution contained guanosine
monophosphate at a purity of >99% versus all of the other
mononucleotides as measured by HPLC (High Pressure Liquid
Chromotography). All of the other mononucleotides were below
detection in the 0.755 mMolar guanosine monophosphate solution.
Greater concentrations can be achieved by using a larger scale.
Example 96
Separation of Guanylyl (3'-5') Guanosine
[0231] A 0.0045 g (wet) amount of a bis-diethylene triamine
covalently bonded to silica material as described in Example 10
loaded with palladium was placed in a column. A 25.degree. C. 1.05
ml source solution consisting of 0.18 mMolar each of adenylyl
(3'-5')adenosine (hereinafter "AA"), cytidylyl (3'-5') cytidine
(hereinafter "CC"), thymidylyl (3'-5') thymidine (hereinafter
"TT"), guanylyl (3'-5') guanosine (hereinafter "GG"), and uridylyl
(3'-5') uridine (hereinafter "UU") in 0.01 Molar pH 7 phosphate
buffer (Na salts) was drawn through the column at a flowrate of
0.05 ml/min. The column was then washed with 0.6 ml of 0.01 Molar
phosphate buffer at 0.05 ml/min flowrate followed by 1.1 ml of pH 5
2.5 Molar phosphate buffer at a flowrate of 0.05 ml/min flowrate.
The column was eluted or stripped using 0.6 ml of Molar NH.sub.3 at
a flowrate of 0.05 ml/min. The collected 0.6 ml of elution
contained GG at a priority of >99% versus all of the other
dinucleotides as measured by HPLC (High Pressure Liquid
Chromatography). The other dinucleotides were not detected in the
0.058 mMolar guanyl (3'-5') guanosine eluent sample.
Example 97
Separation of Adenosine Monophosphate
[0232] A 0.1 ml amount of amino diphosphonic acid covalently bonded
to silica material of Example 25 (and loaded with palladium) was
placed in a column. A 25.degree. C. 6 ml source solution consisting
of 2 mMolar each of AMP, CMP, TMP, GMP, and UMP in 0.01 Molar pH
7.3 phosphate buffer (Na salts) was drawn through the column at a
flowrate of 0.04 ml/min. The column was then washed with 1 ml of
0.01 Molar phosphate pH 7 buffer at 0.04 ml/min flowrate followed
by 2 ml of pH 7 0.5 Molar phosphate buffer (Na salts) at a flowrate
of 0.04 ml/min. Finally the column was eluted or stripped using 1
ml of 0.1 Molar NH.sub.3 at a flowrate of 0.04 ml/min. The
collected 1 ml of elution contained adenosine monophosphate at a
purity of about 96% versus all of the other mononucleotides as
measured by HPLC (High Pressure Liquid Chromatography). The
adenosine monophosphate was collected at 0.497 mMolar in this small
scale example.
Example 98
Separation of Thymidine Monophosphate
[0233] About 0.04 g (wet) of a palladium loaded phenyl terpyridine
covalently bonded to silica material of Example 28 was placed in a
column. A 25.degree. C. 2.5 ml source solution consisting of 1.00
mMolar each of AMP, CMP, TMP, and GMP in 0.01 Molar pH 7.3
phosphate buffer (Na salts) was drawn through the column at a
flowrate of 0.02 ml/min. The column was then washed with 0.55 ml of
0.01 Molar phosphate pH 7 buffer at 0.02 ml/min flowrate followed
by 2 ml of pH 4 2.5 Molar phosphate buffer at a flowrate of 0.02
ml/min flowrate to strip the TMP. The collected 2 ml of elution
contained TMP at a purity of >90% versus all of the other
mononucleotides as measured by HPLC (High Pressure Liquid
Chromatography). Greater concentrations can be achieved by using a
larger scale.
Example 99
Selectivity for TTT over TT and T
[0234] A 0.0106 g (wet) amount of a tertiary phenyl terpyridine
covalently bonded to silica material of Example 35 (loaded with
palladium) was soaked in 1.5 ml of a solution for 24 hours. The
solution contained 0.1 mM each of TMP, TT, and thymidylyl (3'-5')
thymidylyl (3'-5') thymidine (hereinafter "TTT") in 0.01 molar pH7
phosphate buffer (Na salts). The starting and ending concentration
of each of the nucleotides was measured using HPLC (High Pressure
Liquid Chromatography). The results showed the TTT was reduced to
0.0106 mMolar, the TT was reduced to 0.0408 mMolar, and the TMP was
reduced to 0.072 mMolar. Selectivity amongst the three nucleotides
is calculated from the equilibrium data by comparing or calculating
the ratio of the mMolarity removed divided by the final equilibrium
concentration (in mMolar) for each nucleotide. This formula is
shown below: 1 Selectivity = [ Starting conc . nucleotide X - Final
conc . nucleotide X ] Final conc . nucleotide X [ Starting conc .
nucleotide Y - Final conc . nucleotide Y ] Final conc . nucleotide
Y
[0235] Hence, the selectivity of TTT over T (TMP) is 22 and the
selectivity of TTT over TT is 12. This high selectivity shows the
ability to selectively bind the TIT for use in making separations
or analytical detection.
Example 100
Selectivity of TTT over AAA and CCC
[0236] A 0.0119 g (wet) amount of palladium loaded tertiary
phenylterpyridine covalently bonded to silica material of Example
35 was soaked in 1.5 ml of a solution for 24 hours. The solution
contained 0.086 mM each of adenylyl (3'-5') adenylyl (3'-5')
adenosine (hereinafter "AAA"), cytidylyl (3'-5') cytidylyl (3'-5')
cythdine (hereinafter "CCC"), and TTT in 0.01 molar pH 7 phosphate
buffer (Na salts). The starting and ending concentration of each of
the nucleotides was measured using HPLC (High Pressure Liquid
Chromatography). The results showed the TTT was reduced to 0.033
mMolar, the CCC was reduced to 0.0675 mMolar, and the AAA was
reduced to 0.0734 mMolar. Selectivity amongst the three nucleotides
is calculated from the equilibrium data by comparing or calculating
the ratio of the mMolarity removed divided by the final equilibrium
concentration (in mMolar) for each nucleotide. This formula is
shown below: 2 Selectivity = [ Starting conc . nucleotide X - Final
conc . nucleotide X ] Final conc . nucleotide X [ Starting conc .
nucleotide Y - Final conc . nucleotide Y ] Final conc . nucleotide
Y
[0237] Using this formula, the selectivity of TTT over CCC was
determined to be 6.0 and the selectivity of TTT over AAA was
determined to be 9.4. This high selectivity shows the ability to
selectively bind the TTT for use in making separations or
analytical detection.
Example 101
Separation of GG over AA, CC, TT, and UU
[0238] In this example, a 0.31 ml (wet) column of a palladium
loaded Bis propyldithia covalently bonded to silica material of
Example 18 was prepared. A 25.degree. C. 1.75 ml source solution
consisting of 0.2 mMolar each of AA, CC, GG, TT, and UU in 0.01
Molar pH 7.3 phosphate buffer (Na salts) was drawn through the
column at a flowrate of 0.015 ml/min. The column was then washed
with 0.45 ml of 0.01 Molar phosphate pH 7 buffer at 0.015 ml/min
flowrate followed by 1.75ml of pH 5 2.5 Molar phosphate buffer at a
flowrate of 0.015 ml/min flowrate. Finally, the column was eluted
or stripped using 1 ml of 1 Molar NH.sub.3 at a flowrate of 0.015
ml/min. The collected 1 ml of elution contained GG at a purity of
>99% versus all of the other nucleotides as measured by HPLC
(High Pressure Liquid Chromatography). All of the other nucleotides
were below detection in the 19 mMolar guanosine monophosphate
solution. Greater concentrations can be achieved by using a larger
scale.
Example 102
Separation of GG over AA, CC, TT, and UU
[0239] A 0.02 g (wet) amount of a palladium loaded Bis
1,10-dithia-18-crown-6 covalently bonded to silica material of
Example 23 was placed in a column. A 25.degree. C. 5 ml source
solution consisting of 50 mMolar each of AA, CC, GG, TT, and UU in
0.01 Molar pH 7.3 phosphate buffer (Na salts) was drawn through a
column at a flowrate of 0.01 ml/min. The column was then washed
with 1 ml of 0.01 molar phosphate pH 7 buffer at 0.01 ml/min
flowrate. Finally, the column was eluted or stripped using 1 ml of
0.4 Molar NH.sub.3 at a flowrate of 0.01 ml/min. The collected 1 ml
of elution contained GG at a purity of >99% versus all of the
other mononucleotides as measured by HPLC (High Pressure Liquid
Chromatography). All of the other mononucleotides were below
detection in the 0.239 mMolar guanosine monophosphate solution.
Greater concentrations can be achieved by using a larger scale.
Example 103
Separation of TT over AA, CC, and GG
[0240] A 0.13ml (wet) amount of a palladium loaded Bis phenyl
terpyridine covalently bonded to silica material of Example 31 was
placed in a column. A 25.degree. C. 2 ml source solution consisting
of 0.25 mMolar each of AA, CC, GG, and TT was drawn through the
column at a flowrate of 0.012 ml/min. The column was then washed
with 0.45 ml of 0.01 molar phosphate pH 7 buffer at 0.012 ml/min
flowrate followed by 0.95 ml of 0.1 M NH.sub.3 wash at a flowrate
of 0.012 ml/min. Finally, the column was eluted or stripped using
0.95 ml of pH 4 0.5 M Phosphate Buffer at a flowrate of 0.012
ml/min. The collected 0.95 ml of elution contained TT at a purity
of >99% versus all of the other mononucleotides as measured by
HPLC (High Pressure Liquid Chromatography). All of the other
mononucleotides were below detection in the 61 mMolar TT solution.
Greater concentrations can be achieved by using a larger scale.
Example 104
Separation of TTT over GGG and AAA
[0241] A 0.411 g (wet) amount of a palladium loaded Tri Phenyl
Terpyridine covalently bonded to silica material of Example 35 was
placed in a column. A 25.degree. C. 2 ml source solution consisting
of 0.1 mMolar each of AAA, GGG (Guanyl (3'-5') guanyl
(3.varies.-5') guanidine) hereafter referred to as GGG, and TTT in
0.01 Molar pH 7.3 phosphate buffer (Na salts) was drawn through the
column at a flowrate of 0.013 ml/min. The column was then washed
with 0.55 ml of 0.01 molar phosphate pH 7 buffer at 0.013 ml/min
flowrate followed by 1.05 ml of 0.1 Molar NH.sub.3 at a flowrate of
0.013 ml/min. Finally, the column was eluted or stripped using 2.18
ml of 0.5 Molar pH 4 Phosphate Buffer at 0.013 ml/min. The
collected 2.18 ml of elution contained TTT at a purity of >99%
versus all of the other mononucleotides as measured by HPLC (High
Pressure Liquid Chromatography). All of the other mononucleotides
were below detection in the 6 mMolar TTT solution. Greater
concentrations can be achieved by using a larger scale.
Example 105
Selectivity of GMP over AMP, CMP, and TMP
[0242] The propyl dithia on silica material of Example 13
(preloaded with palladium) was soaked in a solution for 24 hours
containing AMP, GMP, CMP, and TMP in 0.01 Molar pH 7 phosphate
buffer (Na salts). The starting and ending concentration of each of
the nucleotides was measured using HPLC and the selectivities
calculated as done for Example 99 and 100. A selectivity of 6.3 for
GMP over AMP, 12.6 for GMP over CMP, and 32 for GMP over TMP was
measured. These selectivities show the ability to selectively bind
the GMP for use in making separations or analytical detection.
Example 106
Selectivity of GG over GT
[0243] The Bis triamine bonded to silica material of Example 10
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of GG and Guanyl (3'-5') Thymidine
(hereafter referred to as GT) in 0.01 Molar pH 7 phosphate buffer
(Na salts). The starting and ending concentration of each of the
nucleotides was measured using HPLC and the selectivities
calculated as done for Examples 99 and 100. A selectivity of 2.0
was measured. This selectivity shows the ability to selectively
bind the GG for use in making separations or analytical detection
even when the very similar GT is present.
Example 107
Selectivity for GG over AA, CC, TT, and UU
[0244] The Bis dithia-18-crown-6 on silica material of Example 23
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of GG, AA, CC, TT, and UU in 0.01
Molar pH 7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 5 for GG over AA, 6 for GG over CC, 5 over GG over
TT, and 4 for GG over UU was measured. These selectivities show the
ability to selectively bind the GG for use in making separations or
analytical detection.
Example 108
Selectivity for GG over AA, CC, and TT
[0245] The Bis dipropylene trithia on silica material of Example 50
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of GG, AA, CC, and IT, in 0.01
Molar pH 7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 7 for GG over AA, 9 for GG over CC, and 2.3 for GG
over TT was measured. These selectivities show the ability to
selectively bind the GG for use in making separations or analytical
detection.
Example 109
Selectivity of AA over GG, TT and UU
[0246] The Bis amino diphosphonic acid on silica material of
Example 27 (preloaded with palladium) was soaked in a solution for
24 hours containing equal concentrations of AA, GG, TT and UU in
0.01 Molar pH 7 phosphate buffer (Na Salts). The starting and
ending concentration of each of the nucleotides was measured using
HPLC and the selectivities calculated as done for Examples 99 and
100. A selectivity of 16 for AA over CC, 8 for AA over GG, and
>100 for AA over TT or UU was measured. These selectivities show
the ability to selectively bind the AA for use in making
separations or analytical detection.
Example 110
Selectivity of TT over GG, and GT
[0247] The Bis phenyl terpyridine on silica material of Example 31
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of TT, GG, and GT in 0.01 Molar pH
7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 4.4 for TT over GG and 2.4 for TT over GT was
measured. These selectivities show the ability to selectively bind
the TT for use in making separation or analytical detection even
when the very similar GT is present.
Example 111
Selectivity of GGG over AAA, CCC, and TTT
[0248] The Tri triamine on silica material of Example 67 (preloaded
with palladium) was soaked in a solution for 24 hours containing
equal concentrations of GGG, AAA, CCC, and TTT in 0.01 Molar pH 7
phosphate buffer (Na Salts). The starting and ending concentration
of each of the nucleotides was measured using HPLC and the
selectivities calculated as done for Examples 99 and 100. A
selectivity of 103 for GGG over AAA, 8 for GGG over CCC, and 3 for
GGG over TTT was measured. These selectivities show the ability to
selectively bind the GGG for use in making separations or
analytical detection.
Example 112
Selectivity of GGG over AAA, CCC, and TTT
[0249] The Tri propylene dithia on silica material of Example 47
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of GGG, AAA, CCC, and TTT in 0.01
Molar pH 7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 33 for GGG over TTT, 13 for GGG over AAA, and 8 for
GGG over CCC was measured. These selectivities show the ability to
selectively bind the GGG for use in making separations or
analytical detection.
Example 113
Selectivity of GGGG over TGGT, TTTT, GGTT, TTGT, and TTTG
[0250] The Tetra triamine on silica material of Example 74
(preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentrations of
Guanyl(3'-5')-Guanyl(3'-5')-Guanyl(3'-5')-Guanidine (hereinafter
"GGGG"), Thymidyl(3'-5')-Guanyl(3'-5')-Guanyl(3'-5')-Thymidi- ne
(hereinafter "TGGT"),
Thymidyl(3'-5')-Thymidyl(3'-5')-Thymidyl(3'-5')-T- hymidine
(hereinafter "TTTT"), Guanyl(3'-5')-Guanyl(3'-5')-Thymidyl(3'-5')-
-Thymidine (hereinafter "GGTT"),
Thymidyl(3'-5')-Thymidyl(3'-5')-Guanyl(3'- -5')-Thymidine
(hereinafter "TTGT"), and Thymidyl(3'-5')-Thymidyl(3'-5')-T-
hymidyl(3'-5')-Guanine (hereinafter "TTTG") in 0.01 Molar pH 7
phosphate buffer (Na Salts). The starting and ending concentration
of each of the nucleotides was measured using HPLC and the
selectivities calculated as done for Examples 99 and 100. A
selectivity of 1.8 for GGGG over GGGT, 2 for GGGG over TGGT, 2.2
for GGGG over TTTT, 1.9 for GGGG over GGTT, 2.3 for GGGG over TTGT,
and 2.7 for GGGG over TPTG was measured. These selectivities show
the ability to selectively bind the GGGG for use in making
separations or analytical detection even in the presence of very
similar nucleotides.
Example 114
Selectivity of TTTT over GGGG, CCCC, and AAAA
[0251] The Tetra phenyl terpyridine on silica material of Example
39 (preloaded with palladium) was soaked in a solution for 24 hours
containing equal concentration of GGGG, TTTT, CCCC, and AAAA in
0.01 Molar pH 7 phosphate buffer (Na Salts). The starting and
ending concentration of each of the nucleotides was measured using
HPLC and the selectivities calculated as done for Examples 99 and
100. A selectivity of 55 for TTTT over GGGG, 5 for TTTT over CCCC,
and >100 for TTTT over AAAA was measured. These selectivities
show the ability to selectively bind the TTTT for use in making
separations or analytical detection.
Example 115
Selectivity of GT over TT, GG, CC, and AA
[0252] The Triamine-phenyl terpyridine on silica with TrioxaBridge
material of Example 79 (preloaded with palladium) was soaked in a
solution for 24 hours containing GT, TT, GG, CC, and AA in 0.01
Molar pH 7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 1.2 was measured for GT over TT, 1.4 for GT over GG,
4 for GT over CC, and 6 for GT over AA was measured. These
selectivities show the ability to selectively bind the GT for use
in making separations or analytical detection with similar or
completely different nucleotides present.
Example 116
Selectivity of GTG over GTG, TTT, GGG, TGT, CCC, and AAA
[0253] The Triamine-phenyl terpyridine-triamine on silica material
with meta-oxa-hexane bridges of Example 64 (preloaded with
palladium) was soaked in a solution for 24 hours containing GTG,
TTT, GGG, TGT, CCC, and AAA in 0.01 Molar pH 7 phosphate buffer (Na
Salts). The starting and ending concentration of each of the
nucleotides was measured using HPLC and the selectivities
calculated as done for Examples 99 and 100. A selectivity of 3 for
GTG over TTT, 1.5 for GTG over GGG, 3 for GTG over TGT, 8 for GTG
over CCC, and 22 for GTG over AAA was measured. These selectivities
show the ability to selectively bind the GTG for use in making
separations or analytical detection with similar or very different
nucleotides present.
Example 117
Selectivity of GGT over GTG, TGT, TTT, GGG, AGA, and CCC
[0254] The Triamine-triamine-phenyl terpyridine on silica material
with a nonane and diethylene trioxa bridge of Example 93 (preloaded
with palladium) was soaked in a solution for 24 hours containing
GGT, GTG, TGT, TTT, GGG, AGA, and CCC in 0.01 Molar pH 7 phosphate
buffer (Na Salts). The starting and ending concentration of each of
the nucleotides was measured using HPLC and the selectivities
calculated as done for Examples 99 and 100. A selectivity of 1.2
for GGT over GTG, 1.3 for GGT over TGT, 3.6 for GGT over TTT, 1.8
for GGT over GGG, 5.1 for GGT over AGA, and 5.4 for GGT over CCC
was measured. These selectivities show the ability to selectively
bind the GGT for use in making separations or analytical detection
with similar or very different nucleotides present.
Example 118
Selectivity of GGT over TGT, TTT, GGG, CCC, and AAA
[0255] The Trimine-triamine-phenyl terpyridine on silica material
with ethylene dioxa bridges of Example 83 (preloaded with
palladium) was soaked in a solution for 24 hours containing GGT,
TGT, TTT, GGG, CCC, and AAA in 0.01 Molar pH 7 phosphate buffer (Na
Salts). The starting and ending concentration of each of the
nucleotides was measured using HPLC and the selectivities
calculated as done for Examples 99 and 100. A selectivity of 1.3
for GGT over TGT, 1.3 for GGT over TTT, 1.3 for GGT over GGG, 3.2
for GGT over CCC, and 8.9 for GGT over AAA was measured. These
selectivities show the ability to selectively bind the GGT for use
in making separations or analytical detection with similar or very
different nucleotides present.
Example 119
Selectivity of CMP over AMP, GMP, and TMP
[0256] In this example, the tetra-aza-12-Crown-4 malonate on silica
material of Example 86 (preloaded with palladium) was soaked in a
solution for 24 hours containing AMP, GMP, CMP, and TMP in 0.01
Molar pH 7 phosphate buffer (Na Salts). The starting and ending
concentration of each of the nucleotides was measured using HPLC
and the selectivities calculated as done for Examples 99 and 100. A
selectivity of 1.6 for CMP over AMP, 1 for CMP over GMP, and 5 for
CMP over TMP was measured. These selectivities show the ability to
selectively bind the CMP for use in making separations or
analytical detection, particularly over AMP and TMP.
[0257] While the invention has been described with reference to
certain preferred embodiments, those skilled in the art will
appreciate that various modifications, changes, omissions, and
substitutions can be made without departing from the spirit of the
invention. It is therefore intended that the invention be limited
only by the scope of the appended claims.
* * * * *