AMINIMIDE-CONTAINING MOLECULES AND MATERIALS AS MOLECULAR RECOGNITION AGENTS

MOLECULES ET PRODUITS A BASE D'AMINIMIDE, AGENTS DE RECONNAISSANCE MOLECULAIRE

English Abstract

The design and synthesis of novel aminimide-based molecular modules and the use of the modules in the construction of
new molecules and fabricated materials is disclosed. The new molecules and fabricated materials are molecular recognition
agents useful in the design and synthesis of drugs, and have applications in separations and materials science.

Claims

-102-
THE CLAIMS
What is claimed is:
1. A composition having the structure
<IMG>
wherein
a. A and B are the same or different,
and each is a chemical bond; hydrogen; an
electrophilic group; a nucleophilic group; R;
an amino acid derivative; a nucleotide
derivative; a carbohydrate derivative; an
organic structural motif; a reporter element;
an organic moiety containing a polymerizable
group; or a macromolecular component, wherein A
and B are optionally connected to each other or
to other structures and R is as defined below;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;

- 103 -
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
2. The composition of claim 1 wherein n > 2.
3. The composition of claim 1 wherein at least one
of R and R' includes a hydroxyl containing substituent.
4. The composition of claim 1 wherein G includes
at least one of an aromatic ring, a heterocyclic ring, a
carbocyclic moiety, an alkyl group or a substituted
derivative thereof.
5. The composition of claim 1 wherein A and B are
the same.
6. The composition of claim 1 where R and R' are
different so that the composition is chiral.
7. The composition of claim 1 wherein at least one
of A and B is a terminal-structural moiety of formula T-
U, wherein:
a. U is selected from the group
consisting of aliphatic chains having from 2
to 6 carbon atoms, substituted or unsubstituted
aryl, substituted or unsubstituted cycloalkyl,
and substituted or unsubstituted heterocyclic
rings; and
b. T is selected from the group
consisting of -OH, -NH2, -SH, (CH3)3N+-, -
SO3-, -COO-, CH3, H and phenyl.
8. The composition of claim 1 wherein at least one
of A and B is HO-CH2-(CHOH)n-.
9. The composition of claim 1 wherein A and B are
part of the same cyclic moeity.
10. A peptide mimetic having the structure

- 104 -
<IMG>
wherein:
a. A and B are the same or different,
and at least one is an amino acid derivative of
the form (AA)m, wherein AA is a natural or
synthetic amino acid residue and m is an
integer, and A and B are optionally connected
to each other or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
11. A nucleotide mimetic having the structure:

- 105 -
<IMG>
wherein:
a. A and B are the same or different,
and at least one is a nucleotide derivative,
wherein A and B are optionally connected to
each other or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
12. The nucleotide mimetic of claim 11 wherein A is
a nucleotide derivate of the form (NUCL)1, wherein 1 is an
integer, such that (NUCL)1 is a natural or synthetic
nucleotides when l=1, a nucleotide probes when l=2-25 and
an oligonucleotides when l>25 including both deoxyribose
(DNA) and ribose (RNA) variants.

- 106 -
13. A carbohydrate mimetic having the structure:
<IMG>
wherein:
a. A and B are the same or different,
and at least one is a carbohydrate derivative;
wherein A and B are optionally connected to
each other or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? l;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.

- 107 -
14. The carbohydrate mimetic of claim 13 wherein A
and B each is a natural carbohydrate, a synthetic
carbohydrate residue or derivative thereof or a related
organic acid thereof.
15. A pharmaceutical compound having the structure:
<IMG>
wherein:
a. A and B are the same or different,
and at least one is an organic structural
motif; wherein A and B are optionally connected
to each other or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary

- 108 -
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
16. The pharmaceutical compound of claim 15 wherein
the structural motif of the organic compound is obtained
from a pharmaceutical compound or a pharmacophore or
metabolite thereof and has specific binding properties to
ligands.
17. A reporter compound having the structure:
<IMG>
wherein:
a. A and B are the same or different,
and at least one is a reporter element; wherein
A and B are optionally connected to each other
or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for

- 109 -
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
18. The reporter compound of claim 17 wherein the
reporter element is a natural or synthetic dye or a
photographically active residues which possesses reactive
groups which may be synthetically incorporated into the
oxazolone structure or reaction scheme and may be
attached through the groups without adversely interfering
with the reporting functionality of the group.
19. The reporter compound of claim 17 wherein the
reactive group is amino, thio, hydroxy, carboxylic acid,
acid chloride, isocyanate alkyl halide, aryl halide or an
oxirane group.
20. A polymer having the structure:
<IMG>
wherein:
a. A and B are the same or different,
and at least one is an organic moiety
containing a polymerizable group; wherein A and
B are optionally connected to each other or to
other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more

- 110 -
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
21. The polymer of claim 20 wherein the
polymerizable group of the organic moiety is a vinyl
group, oxirane group, carboxylic acid, acid chloride,
ester, amide, lactone or lactam.
22. A substrate having the structure:
<IMG>
wherein:
a. A and B are the same or different,
and at least one is a macromolecular component,

- 111 -
wherein A and B are optionally connected to
each other or to other structures;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds
and R and R' are the same, A and B are different and one
is other than H or R.
23. The substrate of claim 21 wherein the
macromolecular component is a surface or structures which
is attached to the oxazolone module via a reactive group
in a manner where the binding of the attached species to
a ligand-receptor molecule is not adversely affected and
the interactive activity of the attached functionality is
determined or limited by the macromolecule.
24. The substrate of claim 23 wherein the
macromolecule component has a molecular weight of at
least about 1000 Daltons.
25. The substrate of claim 24 wherein the molecular
component is in the form of an ceramic particle, a

- 112 -
nanoparticle, a latex particle, a porous or non-porous
beads, a membrane, a gel, a macroscopic surface or a
functionalized or coated version or composite thereof.
26. A chiral composition of matter having the
structure
<IMG>
wherein
a. A is a chemical bond; hydrogen; an
electrophilic group; a nucleophilic group; R;
an amino acid derivative; a nucleotide
derivative; a carbohydrate derivative; an
organic structural motif; a reporter element;
an organic moiety containing a polymerizable
group; or a macromolecular component, wherein R
is as defined below;
b. Y represents a chemical bond or one
or more atoms of carbon, nitrogen, sulfur,
oxygen or combinations thereof;
c. W is -H? or -H2 X? where X? is an anion;
d. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached; and
e. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen; provided
that if G is a chemical bond, Y includes a

- 113 -
terminal carbon atom for attachment to the
quaternary nitrogen.
27. The composition of claim 26 wherein X is a
halogen or tosyl anion.
28. The composition of claim 26 wherein A is a
terminal-structural moiety of formula T-U, wherein:
a. U is selected from the group
consisting of aliphatic chains having from 2 to
6 carbon atoms, substituted or unsubstituted
aryl, substituted or unsubstitùted cycloalkyl,
and substituted or unsubstituted heterocyclic
rings; and
b. T is selected from the group
consisting of -OH, -NH2, -SH, (CH3)3N+-,
-SO3-, -COO-, CH3, H and phenyl.
29. The composition of claim 27 wherein A is
HO-CH2-(CHOH)n-.
30. The composition of claim 26 where R and R' are
different so that the composition is chiral.
31. The composition of claim 26 wherein Y is a
chemical bond, G is
<IMG>
and A is -COO? or -COOR and W is -H?, where R and R' differ
from each other and are as described above.
32. The composition of claim 26 wherein Y is a
chemical bond, G is
<IMG>
and A is -COO? or -COOR and W is -H2 X?, where R and R'
differ from each other and are as described above.

-114-
33. A process of synthesizing a chiral composition
having the structure
<IMG>
wherein
a. A and B are the same or different, and each is
a chemical bond; hydrogen; an electrophilic group; a
nucleophilic group; R; an amino acid derivative; a
nucleotide derivative; a carbohydrate derivative; an
organic structural motif; a reporter element; an organic
moiety containing a polymerizable group; or a
macromolecular component, wherein A and B are optionally
connected to each other or to other structures and R is
as defined below;
b. X and Y are the same or different and each
represents a chemical bond or one or more atoms of
carbon, nitrogen, sulfur, oxygen or combinations
thereof;
c. R and R' are the same or different and each is
an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or
a substituted or heterocyclic derivative thereof,
wherein R and R' may be different in adjacent units and
have a selected stereochemical arrangement about the
carbon atom to which they are attached;
d. G is a chemical bond or a connecting group
that includes a terminal carbon atom for attachment to
the quaternary nitrogen and G may be different in
adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds and R

-115-
and R' are the same, A and B are different and one is other
than H or R;
wherein the process comprises the steps of:
acylating an asymmetric hydrazinium salt with a molecule
capable of functioning both as an acylating and as an
alkylating agent to form an aminimide;
reacting the aminimide with an asymmetrically
disubstituted hydrazine to form a diastereomeric mixture of
aminimide-hydrazinium salts.
34. The process of claim 33 which further comprises:
resolving the diastereomeric mixture and isolating a
selected diastereomer;
acylating the diastereomer with a second molecule
capable of functioning both as an acylating and as an
alkylating agent to form an aminimide;
capping the resulting aminimide; and
repeating the preceding steps at least once, if
necessary, to form the desired structure.
35. The process of claim 33 wherein the asymmetric
hydrazinium salt is bound to a support surface.
36. A process of synthesizing a chiral composition
having the structure
<IMG>
wherein
a. A and B are the same or different, and each is
a chemical bond; hydrogen; an electrophilic group; a
nucleophilic group; R; an amino acid derivative; a
nucleotide derivative; a carbohydrate derivative; an
organic structural motif; a reporter element; an organic
moiety containing a polymerizable group; or a
macromolecular component, wherein A and B are optionally

-116-
connected to each other or to other structures and R is
as defined below;
b. X and Y are the same or different and each
represents a chemical bond or one or more atoms of
carbon, nitrogen, sulfur, oxygen or combinations
thereof;
c. R and R' are the same or different and each is
an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or
a substituted or heterocyclic derivative thereof,
wherein R and R' may be different in adjacent n units
and have a selected stereochemical arrangement about the
carbon atom to which they are attached;
d. G is a chemical bond or a connecting group
that includes a terminal carbon atom for attachment to
the quaternary nitrogen and G may be different in
adjacent n units; and
e. n ? 1;
provided that, (1) if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and (2) if n is 1, X and Y are chemical bonds and R
and R' are the same, A and B are different and one is other
than H or R;
wherein the process comprises the steps of:
alkylating an asymmetrically distributed acyl hydrazide
with a molecule capable of functioning both as an acylating
and as an alkylating mixture of aminimide isomers; and
reacting the racemic mixture with an asymmetrically
disubstituted hydrazine to form a racemic mixtures of
aminimide-acyl hydrazide isomers.
37. The process of claim 36 which further comprises:
resolving the mixture of aminimide-acyl hydrazide
isomers to isolate a desired isomer;
reacting the isolated isomer with a monofunctional
alkylating agent to produce an aminimide; and
capping the aminimide.
38. The process of claim 36 which further comprises:

-117-
reacting the mixture of aminimide-acyl hydrazide isomers
with a second molecule capable of functioning both as an
acylating and as an alkylating agent to form a racemic
mixture of aminimide isomers;
repeating the preceding steps at least once, if
necessary, to form the desired structure.
39. The process of claim 36 wherein the asymmetrically
disubstituted acyl hydrazide is bound to a support surface.
40. A composition prepared according to the process of
any one of claims 33 to 39.
41. A lipid mimetic composition having the structure:
<IMG>
wherein Q is a chemical bond; an electrophilic group; a
nucleophilic group; R; an amino acid derivative; a nucleotide
derivative; a carbohydrate derivative; an organic structural
motif; a reporter element; an organic moiety containing a
polymerizable group; a macromolecular component; or a
substituent X(T) or X(T)2; wherein X is an alkyl, cycloalkyl,
aryl, aralkyl or alkaryl group or a substituted or
heterocyclic derivative thereof, and T is a linear or
branched chain hydrocarbon having between 12 and 20 carbon
atoms some of which are optionally substituted with oxygen,
nitrogen or sulfur atoms or by an aromatic ring; and provided
that at least two T substituents are present in the structure
of the composition.
42. The composition of claim 41 wherein at least one Q
is attached to the .alpha.-carbon of a naturally occurring amino
acid, or at least one Q is a carbohydrate.
43. A functionalized polymer having the structure:
<IMG>

-118-
wherein
a. X and Y are connecting groups;
b. Rn or R'n (where n = an integer) each represent
hydrogen, alkyl, cycloalkyl, aryl, aralkyl and
alkaryl;
c. (Surface) is a macromolecular component; and
d. n ? 1.
44. A functionalized polymer having the formula:
<IMG>
wherein
a. X and Y are connecting groups;
b. Rn or R'n (where n = an integer) each represent
alkyl, cycloalkyl, aryl, aralkyl and alkaryl;
c. (Surface) is a macromolecular component; and
d. n ? 1.
45. A method of producing an aminimide-functional
support comprising the steps of:
reacting a polymer or oligomer containing pendant
moieties of OH, NH or SH with a compound of the formula:
<IMG>
wherein R1 and R2 each represent alkyl, cycloalkyl,
aryl, aralkyl or alkaryl, and R3 is an amino acid
derivative; a nucleotide derivative; a carbohydrate
derivative; an organic structural motif; a reporter
element; an organic moiety containing a
polymerizable group; or a macromolecular component;
b. coating the reacted polymer or oligomer onto a
support to form a film thereon; and

-119-
c. heating the coated support to crosslink the film.
46. A method of producing an aminimide-functional
support comprising the steps of:
coating a mixture of multifunctional esters and
multifunctional epoxides onto a support to form a film
thereon; and
reacting the coated support with 1,1'-dialkylhydrazine
to crosslink the film.
47. A method of producing an aminimide-functional
support comprising the steps of:
coating a mixture of an aminimide-functional vinyl
monomer, a difunctional vinyl monomer and a vinyl
polymerization initiator onto a support to form a film
thereon; and
heating the coating support to form a crosslinked film.
48. An aminimide-functionalized support prepared
according to the method of one of claims 45, 46 or 47.
49. A three-dimensional crosslinked random copolymer
containing, in copolymerized form:
about 1 to 99 parts of a free-radically polymerizable
monomer containing an aminimide group;
up to 98 parts of a free-radically addition-
polymerizable comonomer; and
about 1 to 50 parts of at least one crosslinking
monomer.
50. The copolymer of claim 49 wherein the comonomer is
water-soluble.
51. The copolymer of claim 50 wherein the comonomer is
water-insoluble.
52. The copolymer of claim 50 wherein the copolymer is
fashioned into a water-insoluble bead, a water-insoluble
membrane or a latex particle.
53. The copolymer of claim 50 wherein the copolymer is
a swollen aqueous gel suitable for use as an electrophoresis
gel.

-120-
54. A three-dimensional crosslinked random copolymer
that is the reaction product of:
about 1 to 99 parts of a condensation-polymerizable
monomer containing a moiety cluster selected from the group
consisting of (1) at least three epoxy groups, (2) at least
three ester groups, (3) at least one epoxy and at least two
ester groups and (4) at least one ester and at least two
epoxy groups;
about 1 to 99 parts of a second condensation-
polymerizable monomer containing a moiety cluster selected
from the group consisting of (1) at least two ester groups,
(2) at least two epoxy groups and (3) at least one ester and
one epoxy group; and
an amount of 1,1-dialkylhydrazine equivalent, on a molar
basis, substantially equal to the total molar content of
epoxy groups.
55. The copolymer of claim 54 wherein the copolymer is
fashioned into a water-insoluble bead, a water-insoluble
membrane or a latex particle.
56. The copolymer of claim 55 wherein the copolymer is
a swollen aqueous gel suitable for use as an electrophoresis
gel.

Description

--WO94/01102 2 1 3 9 ~ 4 9 PCT/US93/06241
- AMINIMIDE-CONTAINING MOLECULES AND
MATERIALS AS MOLECULAR RECOGNITION-AG-ENTS
1. FIELD OF THE INVENTION
The present invention relates to the l ogical
development of biochemical and biopharmaceutical aqents
and of new materials including fabricated materials such
as fibers, beads, films, and gels. Specifically, the
invention relates to the development of molecular modules
based on aminimide and related structures, and to the use
of these modules in the assembly of molecules and
fabricated materials with tailored properties, which are
determined by the contributions of the individual
building modules. The molecular modules of the invention
are preferably chiral, and can be used to synthesize new
compounds and fabricated materials which are able to
recognize biological receptors, enzymes, genetic
materials, and other chiral molecules, and are thus of
great interest in the fields of biopharmaceuticals,
separation and materials science.
2. BACKGROUND OF THE INVENTION
The discovery of new molecules has
traditionally focused in two broad areas, biologically
active molecules, which are used as drugs for the
treatment of life-threatening diseases, and new
materials, which are used in commerciai, especially high-
technological applications. In both areas, the strategy
used to discover new molecules has involved two basic
operations: (i) a more or less random choice of a
molecular candidate, prepared either via chemical
synthesis or isolated from natural sources, and (ii) the
testing of the molecular candidate for the property or
properties of interest. This discovery cycle is repeated
indefinitely until a molecule possessing the desirable
properties is located. In t~e n~ajori~y o~ cases, the

WO94/01102 ~7 ' zi~9 3 49 PCT/US93/0~
-- 2
molecular types chosen for testing have belonged to
rather narrowly defined chemical classes. For example,
the discovery of new peptide hormones has involved work
with peptides; the discovery of new therapeutic steroids
has involved work with the steroid nucleus; the discovery
of new surfaces to be used in the construction of
computer chips or sensors has involved work with
inorganic materials, etc. As a result, the discovery of
new functional molecules, being ad hoc in nature and
relying predominantly on serendipity, has been an
extremely time-consuming, laborious, unpredictable, and
costly enterprise.
A brief account of the strategies and tactics
used in the discovery of new molecules is described
below. The emphasis is on biologically interesting
molecules; however, the technical problems encountered
in the discovery of biologically active molecules as
outlined here are also illustrative of the problems
encountered in the discovery of molecules which can serve
as new materials for high technological applications.
Furthermore, as discussed below, these problems are also
illustrative of the problems encountered in the
development of fabricated materials for high
technological applications.
2.l Druq Desiqn
Modern theories of biological activity state
that biological activities, and therefore physiological
states, are the result of molecular recognition events.
For example, nucleotides can form complementary base
pairs so that complementary single-stranded molecules
hybridize resulting in double- or triple-helical
structures that appear to be involved in regulation of
gene expression. In another example, a biologically
active molecule, referred to as a ligand, binds with
another molecule, usually a macromolecule referred to as

_ 3 _ PCT/U593/~ ~l
ligand-acceptor (e.g. a receptor or an enzyme), and this
binding elicits a chain of molecular events which
ultimately gives rise to a physiological state, e.g.
normal cell growth and differentiation, abnormal cell
growth leading to carcinogenesis, blood-pressure
regulation, nerve-impulse-generation and -propagation,
etc. The binding between ligand and ligand-acceptor is
geometrically characteristic and extraordinarily
specific, involving appropriate three-dimensional
structural arrangements and chemical interactions.
2.1.1 Desiqn and SYnthesis of Nucleotides
Recent interest in gene therapy and
manipulation of gene expression has focused on the design
of synthetic oligonucleotides that can be used to block
or suppress gene expression via an antisense, ribozyme or
triple helix mechanism. To this end, the sequence of the
native target DNA or RNA molecule is characterized and
standard methods are used to synthesize oligonucleotides
representing the complement of the desired target
sequence (see, S. Crooke, The FASEB Journal, Vol. 7, Apr.
1993, p. 533 and references cited therein). Attempts to
design more stable forms of such oligonucleotides for use
in vivo have typically involved the addition of various
functional groups, e.g., halogens, azido, nitro, methyl,
keto, etc. to various positions of the ribose or
deoxyribose subunits (cf., The Organic Chemistry of
Nucleic Acids, Y. Mizuno, Elsevier Science Publishers BV,
Amsterdam, The Netherlands, 1987).
- 30 2.1.2 GlYcoPe~tides
As a result of recent advances in biological
carbohydrate chemistry, carbohydrates increasingly are
being viewed as the components of living systems with the
enormously complex structures required for the encoding
of the massive amounts of information needed to

WO94/~1102 2 1 ~ 9 3 ~ 9 PCT/U593/06~1
orchestrate the processes of life, e.g., cellular
recognition, immunity, embryonic development,
carcinogenesis and cell-death. Thus, whereas two
naturally occurring amino acids can be used by nature to
convey 2 fundamental molecular messages, i.e., via
formation of the two possible dipeptide structures, and
four different nucleotides convey 24 molecular messages,
two different monosaccharide subunits can give rise to 11
unique disaccharides, and four dissimilar monosaccharides
can give rise to up to 35,560 unique tetramers each
capable of functioning as a fundamental discreet
molecular messenger in a given physiological system.
The gangliosides are examples of the
versatility and effect with which organisms can use
saccharide structures. These molecules are glycolipids
(sugar-lipid composites) and as such are able to position
themselves at strategic locations on the cell wall:
their lipid component enables them to anchor in the
hydrophobic interior of the cell wall, positioning their
hydrophilic component in the agueous extracellular
millieu. Thus the gangliosides (like many other
saccharides) have been chosen to act as cellular
sentries: they are involved in both the inactivation of
bacterial toxins and in contact inhibition, the latter
being the complex and poorly understood process by which
normal cells inhibit the growth of adjacent cells, a
property lost in most tumor cells. The structure of
ganglioside GM, a potent inhibitor of the toxin secreted
by the cholera organism, featuring a branched complex
pentameric structure is shown below.

2 1 3 9 3 1 9 PCI`/US93/06241
_ WO94/01102
X N ~
=~ /r lll
V~ ~
O
~
Ia--O Z
O J~ ~--I
d
2 0 8~I t~ ~
I I I~ ~L
~ Z Z
O ITI
2 5 0--
-
I
I ~ J
O
SUB~ I I I ~JTE SHEET

WO94/01102 21393~9 PCT/US93/0~
The oligosaccharide components of the
glycoproteins (sugar-protein composites) responsible for
the human blood-group antigens (the A, B, and O blood
classes) are shown below.
HOCH2
HO~CHz ~ i pro~ein
~H \~ ~ ¦~\H
H H HNAc
H OH
H~ o
HO~o~H
CH3
13LOOD GROUP O ANTIGEN, TYPE II
HOCH2
HOCHz H ~ ~ pro~ein
HO~ ~ OH H~
2 5 I~H /\ H HNAc
HO ~ 13100dF~opb Y-OH
~LOOD GI~OUI' A AND 1~ AN l'lGENS

WO94/01102 ~1 3 9 3 ~ 9 PCT/US93/06241
Interactions involving complementary proteins
and glycoproteins on red blood cells belonging to
incompatible blood classes cause formation of aggregates,
or clusters and are the cause for failed transfusions of
human blood.
Numerous other biological processes and
macromolecules are controlled by glycosylation (i.e., the
covalent linking with sugars). Thus, deglycosylation of
erythropoetin causes loss of the hormone's biological
activity; deglycosylation of human gonadotropic hormone
increases receptor binding but results in almost complete
loss of biological activity (see Rademacher et al., Ann.
Rev. Biochem 57, 785 (1988); and glycosylation of three
sites in tissue plasminogen activating factor (TPA)
produces a glycopolypeptide which is 30% more active than
the polypeptide that has been glycosylated at two of the
sites.
2.l.3 Design and Synthesis of Mimetics
of Biological Liqands
A currently favored strategy for development of
agents which can be used to treat diseases involves the
discovery of forms of ligands of biological receptors,
enzymes, or related macromolecules, which mimic such
ligands and either boost, i.e., agonize, or suppress,
i.e., antagonize the activity of the ligand. The
discovery of such desirable ligand forms has
traditionally been carried out either by random screening
of molecules (produced through chemical synthesis or
isolated from natural sources), or by using a so-called
"rational" approach involving identification of a lead-
structure, usually the structure of the native ligand,
and optimization of its properties through numerous
cycles of structural redesign and biological testing.
Since most useful drugs have been discovered not through
the ~rational" approach but through the screening of

WO94/01102 ~<~ PCT/US93/06 ~.
randomly chosen compounds, a hybrid approach to drug
discovery has recently emerged which is based on the use
of combinatorial chemistry to construct huge libraries of
randomly-built chemical structures which are screened for
specific biological activities. (S. Brenner and R.A.
Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381)
Most lead-structures which have been used in
~rational" drug design are native polypeptide ligands of
receptors or enzymes. The majority of polypeptide
ligands, especially the small ones, are relatively
unstable in physiological fluids, due to the tendency of
the peptide bond to undergo facile hydrolysis in acidic
media or in the presence of peptidases. Thus, such
ligands are decisively inferior in a pharmacokinetic
sense to nonpeptidic compounds, and are not favored as
drugs. An additional limitation of small peptides as
drugs is their low affinity for ligand acceptors. This
phenomenon is in sharp contrast to the affinity
demonstrated by large, folded polypeptides, e.g.
proteins, for specific acceptors, e.g. receptors or
enzymes, which is in the subnanomolar range. For
peptides to become effective drugs, they must be
transformed into nonpeptidic organic structures, i.e.,
peptide mimetics, which bind tightly, preferably in the
nanomolar range, and can withstand the chemical and
2S biochemical rigors of coexistence with biological fluids.
Despite numerous incremental advances in the
art of peptidomimetic design, no general solution to the
problem of converting a polypeptide-ligand structure to a
peptidomimetic has been defined. At present, ~rational"
peptidomimetic design is done on an ad hoc basis. Using
numerous redesign-synthesis-screening cycles, peptidic
ligands belonging to a certain biochemical class have
been converted by groups of organic chemists and
pharmacologists to specific peptidomimetics; however, in
the majority of cases the results in one biochemical

WO94/01102 213~ 3 ~ 9 PCT/US93/06241
_ 9
area, e . g. peptidase inhibitor design using the enzyme
substrate as a lead cannot be transferred for use in
another area, e . g . tyrosine-kinase inhibitor design using
the kinase substrate as a lead.
In many cases, the peptidomimetics that result
from a peptide structural lead using the ~rational"
approach comprise unnatural ~-amino acids. Many of these
mimetics exhibit several of the troublesome features of
native peptides (which also comprise a-amino acids) and
are, thus, not favored for use as drugs. Recently,
fundamental research on the use of nonpeptidic scaffolds,
such as steroidal or sugar structures, to anchor specific
receptor-binding groups in fixed geometric relationships
have been described (see for example Hirschmann, R. et
al., 1992 J. Am. Chem. Soc., 114:9699-9701; Hirschmann,
R. et al ., 1992 J. Am. Chem. Soc., 114:9217-9218);
however, the success of this approach remains to be seen.
In an attempt to accelerate the identification
of lead-structures, and also the identification of useful
drug candidates through screening of randomly chosen
compounds, researchers have developed automated methods
for the generation of large combinatorial libraries of
peptides and certain types of peptide mimetics, called
~peptoids~, which are screened for a desirable biological
activity. For example, the method of H. M. Geysen, (1984
Proc. Natl. Acad. Sci. USA 81:3998) employs a
modification of Merrifield peptide synthesis wherein the
C-terminal amino acid residues of the peptides to be
synthesized are linked to solid-support particles shaped
as polyethylene pins; these pins are treated individually
or collectively in sequence to introduce additional
amino-acid residues forming the desired peptides. The
~ peptides are then screened for activity without removing
them from the pins. Houghton, (1985, Proc. Natl. Acad.
Sci. USA 82:5131; and U.S. Patent No. 4,631,211) utilizes
individual polyethylene bags ("tea bags") containing

WO94/01102 ~3~13~9 PCT/US93/06~1
-- 10 --
C-terminal amino acids bound to a solid support. ~hese
are mixed and coupled with the requisite amino acids
using solid phase synthesis techniques. The peptides
produced are then recovered and tested individually.
Fodor et al., (1991, Science 251:767) described light-
directed, spatially addressable parallel-peptide
synthesis on a silicon wafer to generate large arrays of
addressable peptides that can be directly tested for
binding to biological targets. These workers have also
déveloped recombinant DNA/genetic engineering methods for
expressing huge peptide libraries on the surface of
phages (Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378).
In another combinatorial approach, V. D.
Huebner and D.V. Santi (U.S. Patent No. S,182,366)
utilized functionalized polystyrene beads divided into
portions each of which was acylated with a desired amino
acid; the bead portions were mixed together and then
split into portions each of which was subjected to
acylation with a second desirable amino acid producing
dipeptides, using the techniques of solid phase peptide
synthesis. By using this synthetic scheme, exponentially
increasing numbers of peptides were produced in uniform
amounts which were then separately screened for a
biological activity of interest.
Zuckerman et al., (1992, Int. J. PePtide
Protein Res. 91:1) also have developed similar methods
for the synthesis of peptide libraries and applied these
methods to the automation of a modular synthetic
chemistry for the production of libraries of N-alkyl
glycine peptide derivatives, called ~peptoids", which are
screened for activity against a variety of biochemical
targets. (See also, Symon et al., 1992, Proc. Natl.
Acad. Sci. USA 89:9367). Encoded combinatorial chemical
syntheses have been described recently (S. Brenner and
R.A. Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381).

- W O 94/01102 i ~ 2 l 3 9i3 ~ 9 PC~r/US93/06241
-- 11 --
In addition to the lead structure, a very
useful source of information for the realization of the
preferred ~rational drug discovery is the structure of
the biological ligand acceptor which, often in
conjunction with molecular modelling calculations, is
used to simulate modes of binding of the ligand with its
acceptor; information on the mode of binding is useful in
optimizing the binding properties of the lead-structure.
However, finding the structure of the ligand acceptor, or
preferably the structure of a complex of the acceptor
with a high affinity ligand, requires the isolation of
the acceptor or complex in the pure, crystalline state,
followed by x-ray crystallographic analysis. The
isolation and purification of biological receptors,
enzymes, and the polypeptide substrates thereof are time-
consuming, laborious, and expensive; success in thisimportant area of biological chemistry depends on the
effective utilization of sophisticated separation
technologies.
Crystallization can be valuable as a separation
technique but in the majority of cases, especially in
cases involving isolation of a biomolecule from a complex
biological mllieu, successful separation is
chromatographic. Chromatographic separations are the
result of reversible differential binding of the
components of a mixture as the mixture moves on an active
natural, synthetic, or semisynthetic surface; tight-
binding components in the moving mixture leave the
surface last en masse resulting in separation.
The development of substrates or supports to be
used in separations has involved either the
polymerizationtcrosslinking of monomeric molecules under
various conditions to produce fabricated materials such
as beads, gels, or films, or the chemical modification of
various commercially available fabricated materials e.g.,
sulfonation of polystyrene beads, to produce the desired

WO94/01102 2~ 9 5 ~ 9 PCT/US93/06 ~l
- 12 -
new materials. In the majority of cases, prior art
support materials have been developed to perform specific
separations or types of separations and are thus of
limited utility. Many of these materials are
incompatible with biological macromolecules, e.g.,
reverse-phase silica frequently used to perform high
pressure liquid chromatography can denature hydrophobic
proteins and other polypeptides. Furthermore, many
supports are used under conditions which are not
compatible with sensitive biomolecules, such as proteins,
enzymes, glycoproteins, etc., which are readily
denaturable and sensitive to extreme pH's. An additional
difficulty with separations carried out using these
supports is that the separation results are often
support-batch dependent, i.e. they are irreproducible.
Recently a variety of coatings and composite-
forming materials have been used to modify commercially
available fabricated materials into articles with
improved properties; however the success of this approach
remains to be seen.
If a chromatographic support is equipped with
molecules which bind specifically with a component of a
complex mixture, that component will be separated from
the mixture and may be released subsequently by changing
the experimental conditions (e.g. buffers, stringency,
etc.) This type of separation is appropriately called
affinity chromatography and remains an extremely
effective and widely used separation technique. It is
certainly much more selective than traditional
chromatographic techniques, e.g chromatography on silica,
alumina, silica or alumina coated with long-chain
hydrocarbons, polysaccharide and other types of beads or
gels which in order to attain their maximum separating
efficiency need to be used under conditions that are
damaging to biomolecules, e.g. conditions involving high

- WO94/01102 21 39 3 4 9 PCT/US93/06~1
- 13 -
pressure, use of organic solvents and other denaturing
agents, etc.
The development of more powerful separation
technologies depends significantly on breakthroughs in
the field of materials science, specifically in the
design and construction of materials that have the power
to recognize specific molecular shapes under experimental
conditions resembling those found in physiological media,
i.e. these experimental conditions must involve an
aqueous medium whose temperature and pH are close to the
physiological levels and which contains none of the
agents known to damage or denature biomolecules. The
construction of these ~intelligent~ materials frequently
involves the introduction of small molecules capable of
specifically recognizing others into existing materials,
e.g. surfacesl films, gels, beads, etc., by a wide
variety of chemical modifications; alternatively
molecules capable of recognition are converted to
monomers and used to create the intelligent" materials
through polymerization reactions.
3. SUMMARY
A new approach to the construction of novel
molecules is described. This approach involves the
development of aminimide-based molecular building blocks,
containing appropriate atoms and functional groups, which
may be chiral and which are used in a modular assembly of
molecules with tailored properties; each module
contributing to the overall properties of the assembled
molecule. The aminimide building blocks of the invention
can be used to synthesize novel molecules designed to
mimic the three-dimensional structure and function of
- native ligands, and/or interact with the binding sites of
a native receptor. This logical approach to molecular
construction is applicable to the synthesis of all types
of molecules, including but not limited to mimetics of

WO94/01102 ^ -~ PCT/US93/06~1
2139349 - 14 -
peptides, proteins, oligonucleotides, carbohydrates,
lipids, polymers and to fabricated materials useful in
materials science. It is analogous to the modular
construction of a mechanical device that performs a
specific operation wherein each module performs a
specific task contributing to the overall operation of
the device.
The invention is based, in part, on the
following insights of the discoverer. (l) All ligands
share a single ùniversal architectural feature: they
consist of a scaffold structure, made e.g. of amide,
carbon-carbon, or phosphodiester bonds which support
several functional groups in a precise and relatively
rigid geometric arrangement. (2) Binding modes between
ligands and receptors share a single universal feature as
well: they all involve attractive interactions between
complementary structural elements, e.g., charqe- and ~-
type interactions, hydrophobic and Van der Waals forces,
hydrogen bonds. (3) A continuum of fabricated materials
exists spanning a dimensional range from lO0 A to l cm in
diameter comprising various materials of varied
construction, geometries, morphologies and functions, all
of which possess the common feature of a functional
surface which is presented to a biologically active
molecule or a mixture of molecules to achieve recognition
2~ between the molecule (or the desired molecule in a
mixture) and the surface. And (4) Aminimide structures,
which have remained relatively unexplored in the design
and synthesis of biologically active compounds and
especially of drugs, would be ideal building blocks for
constructing backbones or scaffolds bearing the
appropriate functional groups, that either mimic desired
ligands and/or interact with appropriate receptor binding
sites; furthermore, aminimide modules may be utilized in
a variety of ways across the continuum of fabricated
materials described above to produce new materials

_- W O 94/01102 2 1 ~ 9 3 4 9 PC~r/US93/06241
- 15 -
capable of specific molecular recognition. These
aminimide building blocks may be chirally pure and can be
used to synthesize molecules that mimic a number of
biologically active molecules, including but not limited
to peptides, proteins, oligonucleotides, polynucleotides,
carbohydrates, lipids, and a variety of polymers and
fabricated materials that are useful as new materials,
including but not limited to solid supports useful in
column chromatography, catalysts, solid phase
immunoassays, drug delivery vehicles, films, and
"intelligent" materials designed for use in selective
separations of various components of complex mixtures.
Working examples describing the use of
aminimide-based modules in the modular assembly of a
variety of molecular structures are given. The molecular
structures include functionalized silica surfaces useful
in the optical resolution of racemic mixtures; peptide
mimetics which inhibit human elastase, protein-kinase,
and the HIV protease; polymers formed via free-radical or
condensation polymerization of aminimide-containing
monomers; and lipid-mimetics useful in the detection,
isolation, and purification of a variety of receptors.
In accordance with the objectives of the
present invention, the aminimide-based molecules of
interest possess the desired stereochemistry and, when
required, are obtained optically pure. In addition to
the synthesis of single molecular entities, the synthesis
of libraries of aminimide-based molecules, using the
techniques described herein or modifications thereof
which are well known in the art to perform combinatorial
chemistry, is also within the scope of the invention.
Furthermore, the aminimide-containing molecules possess
enhanced hydrolytic and enzymatic stabilities, and in the
case of biologically active materials, are transported to
target ligand-acceptor macromolecules in vivo without
causing any serious side-effects.

WO94/01102 ~ PCT/US93/0~1
- 16 -
4. DETAILED DESCRIPTION OF THE INVENTION
To the extent necessary to further understand
any portion of the detailed description, the following
earlier filed U.S. patent applications are expressly
incorporated herein by reference thereto: AMINIMIDE
COMPOSITIONS AND BIOLOGICALLY USEFUL DERIVATIVES THEREOF,
Serial No. 07/906,770, filed June 30, 1992; AMINIMIDE-
BASED SUPPORT MATERIALS AND FUNCTIONALIZED SURFACES,
Serial No. 07/906,769, filed June 30, 1992; DIRECTED PURE
CHIRAL-ISOMER LIGANDS, RECOGNITION AGENTS AND
FUNCTIONALLY USEFUL MATERIALS FROM SUBSTITUTED AMINIMIDES
AND DERIVATIVES CONTAINING AN ASYMMETRIC CENTER, Serial
No. 08/041,559, filed April 2, 1993.
4.l Physical and Chemical Properties of the
Aminimide Functional GrouP
Aminimides are zwitterionic structures
described by the resonance hybrid of the two
energetically comparable Lewis structures shown below.
R2 R2
R1 '~ N- N+ R3 ~ ~ R1 C. N N+ R3
O R4 - R4
The tetrasubstituted nitrogen of the aminimide
group can be asymetric rendering aminimides chiral as
shown by the two enantiomers below.
~ ~ R~ ~R
R4 /\ N+ R4 /\ N+

_- W O 94/01102 ~3 ~9~ PC~r/US93/06241
- 17 -
As a result of the polarity of their
structures, but lack of net charge, simple aminimides are
freely soluble in both water and (especially) organic
solvents.
Dilute aqueous solutions of aminimides are
neutral and of very low conductivity; aminimide conjugate
acids are weakly acidic, pK. _ 4.5. A striking property
of aminimides is their hydrolytic stability, under
acidic, basic, or enzymatic conditions. For example,
boiling trimethyl amine benzamide in 6 N NaOH for 24 hrs
leaves the aminimide unchanged. Upon thermolytic
treatment, at temperatures exceeding 180C, aminimides
decompose to give isocyanates as follows.
lR2 l2
Rl- C= N -I+-R4 ~ Rl- N -C= + I-R4
O~ R R3
4.1.1 Use of the Aminimide Group as a
Mimetic of the Amide Group
The aminimide group mimics several key
structural features of the amide group, such as overall
geometry (e.g. both functional groups contain a planar
carbonyl unit and a tetrahedral atom linked to the
acylated nitrogen) and aspects of charge distribution
(e.g. both functional groups contain a carbonyl with
significant negative charge development on the oxygen).
These structural relationships can be seen below, where
the resonance hybrids of the two groups are drawn.
CR~N'"R ~ ' ~C~N~N,~55S

WO94/01102 ~ 2 i393 ~9 PCT/US93/06~1
- 18 -
~ ~ C~N,C ~ ~ ~N+ ~
Being hydrolytically and enzymatically more
stable than amides and possessing novel solubility
properties due to their zwitterionic structures,
aminimides are valuable building blocks for the
construction of mimetics of biologically active molecules
with superior pharmacological properties. For the
construction of these mimetics, the aminimide backbone is
used as a scaffold for the geometrically precise
attachment of structural units possessing desired
stereochemical and electronic features, such as suitable
chiral atoms, hydrogen-bonding centers, hydrophobic and
charged groups, ~-systems, etc. Furthermore, multiple
aminimide units can be linked in a variety of modes,
using likers of diverse structures, to produce polymers
of a great variety of structures. Specific molecular
forms are chosen for screening and further study using
several criteria. In one instance a certain aminimide
structure is chosen because it is novel and has never
been tested for activity as a biopharmaceutical agent or
as material for device construction. In a preferable
instance an aminimide ligand is chosen because it
incorporates structural features and properties suggested
by a certain biochemical mechanism. In another
preferable case the aminimide structure is the result of
assembly of molecular modules each making a specific

_ WO94/01102 ~- 2 1 ~ 9 3 ~ 9 PCT/US93/06~1
-- 19 --
desirable contribution to the overall properties of the
aminimide-containing molecule.
Summarizing, aminimides are functional groups
with unusual and very desirable physiochemical
properties, which can be used as molecular modules for
the construction of molecular structures that are useful
as biopharmaceutical agents and as new materials for high
technological applications.
4.2 General Synthetic Routes to Aminimides
4.2.1 Aminimides via Alkylation of
N,N-Disubstituted Hydrazones
Alkylation of an acyl hydrazide (hydrazone)
followed by neutralization with a base produces an
aminimide.
O O
R1~ N C (1) R3X , N C
R2~ `N' `R4 (2) neu~ on R2~ `N' `R4
H
This alkylation is carried out in a suitable
solvent such as a hydroxylic solvent e.g. water, ethanol,
isopropanol or a dipolar aprotic solvent e.g., DMF, DMSO,
acetonitrile, usually with heating.
The acyl hydrazide is produced by the reaction
of a 1,1-disubstituted hydrazine with an activated acyl
derivative or an isocyanate, in a suitable organic
solvent, e.g. methylene chloride, toluene, ether, etc. in
the presence of a base such as triethylamine to
neutralize the haloacid generated during the acylation.
2,N -NH2 + 1I R2' N R4

WO94/01102 2 ~3 9 - 20 - PCT/US93/06~1
Activated acyl derivatives include acid chlorides,
chlorocarbonates, chlorothiocarbonates, etc.; the acyl
derivative may also be replaced with a suitable
carboxylic acid and a condensing agent such as
dicyclohexylcarbodiimide (DCC).
The alkylating agent R3X used in the hydrazone
alkylation may be an alkyl halide (X = Cl, Br, I), a
tosylate (X = OTs), or some other suitable reactive
species, such as an epoxide. The conversion of phenyl
isocyanate to an aminimide using the commercially
available l,l-dimethylhydrazine and ethylene oxide as the
hydrazone alkylating agent is given below:
(I)
Cl 12--Cl i2
,~ &1~3 ,~ 1~ 1l 1~ ~C1~3 \o/
~=~N=C=O + NH2--N~ =,~ C1~3 (2) O
~N--C--N---N~-CI32CH2OI~
The desired l,l-disubstituted hydrazines may be
readily prepared in a number of ways well known in the
art; one is the reaction of a secondary amine with NH2Cl
in an inert organic solvent.
Rl\ Rl\
~NH + H2N- Cl ~ N-NH2-HC1
R2 ~2
The above route to aminimides is broadly
applicable and allows the incorporation of a wide variety
of aliphatic, aromatic and heterocyclic groups into
various positions in the aminimide structure.

21~93~9
-- W O 94/01102 PC~r/US93/06241
- 21 -
4.2.2 Aminimides via Acylation of l,l,l-Trialkyl
Hydrazinium Salts
Acylation of a suitable trialkyl hydrazinium
salt by an acyl derivative or isocyanate in the presence
of a strong base in a suitable organic solvent, e.g.
dioxane, ether, acetonitrile, etc. produces good yields
of aminimides.
~I- Nt-NH2 X- + R~- C- oR5 basc R~ N~ R4
The acyl derivatives for the acylation reaction are the
same as those required for the synthesis of the
hydrazones outlined above.
The required hydrazinium salts may be prepared
by routine alkylation of a l,l-disubstituted hydrazines
or by treatment of a tertiary amine with a haloamine (see
78 J. Am. Chem. Soc. 1211 (1956)).
Hydrazinium salts, being chiral at nitrogen,
may be resolved, e . g . by treatment with a chiral acid
followed by separation of the diastereomers (e . g . using
chromatography or fractional crystallization and the
resulting enantiomers used in stereoselective syntheses
Of aminimides~
When one of the alkyl groups in a hydrazinium
salt is an ester group, the ester may be saponified
efficiently using LioH in a mixture of methanol and
water, producing a useful ~-hydrazinium acid after
neutralization of the reaction mixture with an acid.
R2 R3 1. LiO~ R \ ,R
H2N CH2 2 neutralization H2N CH2
X

WO94/01102 2 13 9 3 ~ 9 - 22 - PCT/US93/06~1
Suitably protected hydrazinium carboxylates may be used
in condensation reactions to produce aminimides.
Procedures analogous to those known to be useful in
effecting peptide bond formation are expected to be
useful; e.g. DCC or other carbodiimides may be used as
condensing agents in solvents such as DMF.
Rl~ /NH A NHsl Rl\ N\ ~ N~s
N + ROOC N . N: ~ N
-~, R4~ ~R5 R2~ ~3 a R4~ ~R5
X X'
Alternatively, the hydrazinium carboxylate units may be
coupled with ~-amino-acids or with other nucleophiles,
such as amines, thiols, alcohols, etc., using standard
techniques, to produce molecules of wide utility as
ligand mimetics and new materials for high technological
applications.
The ~-hydrazinium esters may in turn be
produced by the alkylation of a 1,1-disubstituted
hydrazine with a haloester under standard reaction
conditions, such as those given above for the alkylation
of hydrazones.
R2\ R \ ,R
N-NH2 + Cl\ ,CO2Me ~ N\ ,CO2Me
Rl CH2 H2N CH2
Cl
Alternatively, these hydrazinium esters may be produced
by standard alkylation of the appropriate ~-hydrazino
ester.

21393~9
~- WO94/01102 1 ~ PCT/US93/06241
- 23 -
H2N ~C~ 3 R4X H2N H,C~cO R3 X~
The required l,1-disubstituted hydrazine for
the above reaction may be obtained by acid or base
hydrolysis of the corresponding hydrazone (see 108 J. Am.
Chem. Soc. 6394 (1986)); the alkylated hydrazone is
produced from the monosubstituted hydrazone by the method
of Hinman and Flores (24 J. Orq. Chem. 660 (1958)).
15 ~CH2-O--C--NH-NH-C--CO R3 R~X e~3CH2-o--C--N--NH-C--Co2R3
CF3CO21
1~ 1
1 12N 11--C~Co R3
The monosubstituted hydrazones required above may be
obtained by reduction of the Schiff base formed from an
~-keto ester and a suitable hydrazone. This reduction
may also be carried out stereoselectively, if desired,
using DuPHOS-Rhodium catalysis (114 J. Am. Chem. Soc.
- 6266 (1992); 259 Science 479 (1993)), as shown:

WO94/01102 ~ ~ ; PCT/US93/0~
2139~49 - 24 - 1~
Il N O
,C=O + H2N C N C~
R300C ~ R300C ~R2
/[PII (El-DupHos)]
H N~ "O
R2OOC ' -` R2
In a variation of the synthesis given above, ~-halo
aminimides are prepared using an ~-halo acyl halide.
(CH3)3N~N~2 OTs- + ClCH2COCI (CH3)3N~N-COCH2CI
These halo aminimides may be reacted with nucleophiles
containing reactive hydroxyl, thio, or amino groups to
give complex aminimide structures.
4.2.3 Aminimides via the Hydrazine-Epoxide-Ester
Reaction
A very useful and versatile synthesis of
aminimides involves the one-pot reaction of an epoxide,
an asymetrically disubstituted hydrazine, and an ester in
a hydroxylic solvent, usually water or an alcohol, which
is allowed to proceed usually at room temperature over
several hours to several days.

2 1 3 9 3 ~ 9 Pcr/US93/0624l
NO 94/01102
- 2
R2~
R1 - CH--f H2 + N NH2 ~ R4--CooR5
R3 R~
R1-- CH--CH2--N--N--C--R4 -1- RsO~

WO94/01102 ,~ ~ PCT/US93/06~J
2139319 - 26 -
In the equation above, Rl, R2 and R3 are selected from a
set of diverse structural types (e.g. alkyl, cycloalkyl,
aryl, aralkyl, alkaryl or many substituted versions
thereof), and R4 and R5 are alkyl or cycloalkyl.
The rates for the above reaction increase with
increasing electrophilicity of the ester component.
Generally, a mixture of O.l mol of each of the reactants
in 50-lO0 ml of an appropriate solvent is stirred for the
required period at room temperature (the reaction may be
monitored by thin layer chromatography). At the end of
this period the solvent is removed in vacuo to give the
crude product.
If substituent R4 of the ester component in the
above aminimide formation contains a double bond, an
aminimide with a terminal double bond results which may
be epoxidized, e.g. using a peracid under standard
reaction conditions, and the resulting epoxide used as
starting material for a new aminimide formation; thus a
structure containing two aminimide subunits results. If
the aminimide-formation and epoxidation sequence is
repeated n times, a structure containing n aminimide
subunits results; thus for R4 = propene, n repetition of
the sequence results in the structure shown below:
R2 R2n
R1 CH--CH2--N~--N ~C CH2--CH--CH2--N~--N- ' CO--CH2--CH=CH2
OH ~ OH R3 .-n n- I
where the designations R2 and R3 are used to illustrate
the manner in which the hydrazine substituents R2 and R3
can be varied in each polymerization step to produce
oligomers or polymers of diverse structures.

- WO94/01102 21 3 9 3 ~ 9 PCT/US93/06~1
- 27 -
A related aminimide polymerization sequence
utilizes an ester moiety bonded directly to the epoxide
- group.
An additional related polymerization sequence
involves the use of bifunctional epoxides and esters of
the following form
H2C~--HC--(Y)--CH--CH2 and R O C (X~--C- O--R
so as to produce polymers of the following structure
(shown for the case of reaction with dimethyl hydrazine):
~ '
O O IH3 OH OH IH3
( ) N 7 CH2--CH--(Y)--CH--CH2--Nt--N
CH3 CH3
where X and Y are alkyl, cycloalkyl, aryl, aralkyl or
alkaryl linkers.
4.2.4 SYnthesis of Enantiomerically-Pure Aminimides
Enantiomerically-pure aminimides may be
produced by a~ylation of chiral hydrazinium salts as
shown in the example below.
HOCH2CH2 ~ 1- C2HsCO2CH3 HO CH2cH2\
H2N CH3 ~ N-~ CH3
C2~3s

WO94/01102 2~'3g3~9 PCT/US93/06~1
- 28 -
Chirally-pure hydrazinium salts may be obtained
by resolution of the racemates; resolution can be
effected by forming salts with optically pure acids, e.g.
tartaric acid, and separating the resulting diastereomers
by means of chromatography or fractional crystallization
(see, e.g., 103 J. Chem. Soc. 604 (1913)); alternatively
the racemic modification is resolved by subjecting it to
chromatographic separation using a chiral stationary
chromatographic support, or if feasible, by the use of a
suitable enzyme system.
Alternatively, enantiomerically-pure aminimides
may be obtained by resolution of the racemic
modifications using one of the techniques described above
for the resolution of racemic hydrazinium salts (for an
example, see 28 J. org. Chem. 2376 (1963)).
An additional approach to the synthesis of
chiral aminimides involves chiral synthesis; an example
is provided by the reaction of (S)-(-)-propylene oxide
with 1,1-dimethylhydrazine and methyl-(R)-3-hydroxy-
butyrate, all of which are commercially available.
CH3 ~ + ~ N-NHz + H0" ~ o/CH3
H3C ~ I ~ ~lo H
A variety of chiral epoxides, produced by
chiral epoxidations such as those developed by Sharpless
(Asynn. Syn., J.D. Morrison ed., Vol. 5, Ch. 7 + 8, Acad.
Press, New York, N.Y., 1985), and chiral esters, produced

-- W O 94/01102 Z:1~3 9 3 ~ 9 PC~r/US93/06241
- 29 -
by standard procedures, may be used to produce a wide
variety of chiral aminimides.
Chirally-pure aminimide molecular building
blocks are especially preferred since they will be used
to produce a vast array of molecules useful as new
materials for high technological applications and as
molecular recognition agents, including biological ligand
mimetics to be used as drugs, diagnostics, and separation
agents.
4.4 Synthesis of Specific Classes of Aminimides
4.4.1 Synthesis of Chiral Aminimide-Containing
Coniu~ates
The synthetic routes outlined above may be
utilized to produce a wide variety of chiral aminimide
conjugates of the following general structure:
O R
Il ~
A - X - C - N-- N+-Y - B
R2
The substituents A and B shown may be of a
variety of structures and may differ markedly in their
physical or functional properties, or may be the same;
they may also be chiral or symmetric. A and B are
preferably selected from
1) an amino acid derivative of the form
(AA) n~ which would include natural and synthetic
amino acid residues (n=l), peptides (n=2-30),
polypeptides (n=31-70) and proteins (n>70).
2) a nucleotide derivative of the form
(NUCL) n~ which would include natural and
synthetic nucleotides (n=1), nucleotide probes
(n=2-25) and oligonucleotides (n>2S) including
both deoxyribose (DNA) and ribose (RNA)
3S variants.

2139349 ~,T/US93/ 0 6 2 4 1
30~ ~ec'd PCT/P~O 14 OC~ t994
3) a carbohydrate derivative of the form
(CH) n. This would include natural
physiologically active carbohydrates (glucose,
galactose, etc.) including related compounds
such as sialic acids, etc. (n=l), synthetic
carbohydrate residues and derivatives of these
(n=1) and all of the complex oligomeric
permutations of these as found in nature (n>l)
cf. Scientific American, January 1993, p. 82.
4) a naturally occurring or synthetic
organic structural motif. This term includes
any of the well known base structures of
pharmaceutical compounds including
pharmacophores or metabolites thereof. These
structural motifs are generally known to have
specific desirable binding properties to ligand
acceptors of interest and would include
structures other than those recited above in
1), 2) and 3).
5) a reporter element such as a natural
or synthetic dye or a residue capable of
photographic amplification which possesses
J reactive groups which may be synthetically
incorporated into the aminimide structure or
reaction scheme and may be attached through the
groups without adversely interfering with the
reporting functionality of the group.
Preferred reactive groups are amino, thio,
hydroxy, carboxylic acid, acid chloride,
isocyanate alkyl halides, aryl halides and
oxirane groups.
6) an organic moiety containing a
polymerizable group such as a double bond or
other functionalities capable of undergoing
condensation polymerization or
copolymerization. Suitable groups include
A~NGED SH~ET

PCT/US 9;~ / 0 6 2 4 1 --
- 3l - Q~"~ PCt/Pr~? 1 4 OCT t99G
vinyl groups, oxirane groups, carboxylic acids,
acid chlorides, esters, amides, lactones and
lactams.
7) a macromolecular component, such as a
macromolecular surface or structures which may
be attached to the aminimide modules via the
various reactive groups outlined above in a
manner where the binding of the attached
species to a ligand-receptor molecule is not
adversely affected and the interactive activity
of the attached functionality is determined or
limited by the macromolecule. The molecular
weight of these macromolecules may range from
about 1000 Daltons to as high as possible.
They may take the form of nanoparticles (~=100- -
lOOOA), latex particles (dp=lOOOA-5000A), porous
or non-porous beads (dp=0.5~-1000~), membranes,
gels, macroscopic surfaces or functionalized or
coated versions or composites of these.
Under certain circumstances, A and/or B may be a chemical
bond to a suitable organic moiety, a hydrogen atom, an
organic moiety which contains a suitable electrophilic
group, such as an aldehyde, ester, alkyl halide, ketone,
nitrile, epoxide or the like, a suitable nucleophilic
group, such as a hydroxyl, amino, carboxylate, aminde,
carbanion, urea or the like, or one of the R groups
defined below. In addition, A and B may join to form a
ring or structure which connects to the ends of the
repeating unit of the compound defined by the preceding
formula or may be separately connected to other moeities.
A more generalized presentation of the
composition of the invention is defined by the structure
R~ n
A -X I CO -N--N+-GI-~' ~ Y-B
I 'I ...n
, n
AMENDED SHEET

WO94/01102 2 1 3 9 3 ~ 9 PCT/US93/06~1
- 32 -
wherein:
a. at least one of A and B are as
defined above and A and B are optionally
connected to each other or to other compounds;
b. X and Y are the same or different and
each represents a chemical bond or one or more
atoms of carbon, nitrogen, sulfur, oxygen or
combinations thereof;
c. R and R' are the same or different
and each is an alkyl, cycloalkyl, aryl, aralkyl
or alkaryl group or a substituted or
heterocyclic derivative thereof, wherein R and
R' may be different in adjacent n units and
have a selected stereochemical arrangement
about the carbon atom to which they are
attached;
d. G is a chemical bond or a connecting
group that includes a terminal carbon atom for
attachment to the quaternary nitrogen and G may
be different in adjacent n units; and
e. n ~ l.
Preferably, if G is a chemical bond, Y includes a
terminal carbon atom for attachment to the quaternary
nitrogen; and if n is l and X and Y are chemical bonds, R
and R' are the same, A and B are different and one is
other than H or R.
In one embodiment of the invention, at least
one of A and B represent an organic or inorganic
macromolecular surface. Examples of preferred
macromolecular surfaces include ceramics such as silica
and alumina, porous and nonporous beads, polymers such as
a latex in the form of beads, membranes, gels,
macroscopic surfaces or coated versions or composites or
hybrids thereof. This functionalized surface may be
represented as follows:

2139349
- `~CTIus~/ 0 62~ l
Q~. ~ac'd PcT/pTo t 4 OCT 1994
Rl O
11
(SURFACE)- Y- N~ C-X - A
R2
In a further embodiment of the invention, the
above roles of A and B are reversed, so that B is the
S substituent selected from the foregoing list and A
represents a functionalized surface, as shown below:
O R
o (SURFACE)--X--C--N---N~-Y--B
R2
In a third preferred embodiment of the
invention, either A, B, or both contain one or more
double bonds capable of undergoing free-radical
polymerization or copolymerization to produce achiral or
chiral oligomers, polymers, copolymers, etc.
Another embodiment of the invention relates to
a composition having the structure
J A Y G - N- - N W
wherein A, Y, R, Ri and G are as defined above and
25W is -H
or -H2X- where X- is an anion, such as a halogen or tosyl
anion.
Yet another aspect of the invention relates to
a lipid mimetic composition having the structure
O
11
(~, N ~--N C Q
AlJlENDED S~rT

WO94/01102 2 1 3 9 3 4 9 PCT/US93/06~41
- 34 -
wherein Q is a chemical bond; an electrophilic
group; a nucleophilic group; R; an amino acid derivative;
a nucleotide derivative; a carbohydrate derivative; an
organic structural motif; a reporter element; an organic
moiety containing a polymerizable group; a macromolecular
component; or the substituent X(T) or X(T)2; wherein R is
an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or a
substituted or heterocyclic derivative thereof, and T is
a linear or branched hydrocarbon having between 12 and 20
carbon atoms some of which are optionally substituted
with oxygen, nitrogen or sulfur atoms or by an aromatic
ring; and provided that at least two T substituents are
present in the structure of the composition.
In the description that follows, R" where n is
an integer will be used to designate a group from the
definition of R and Rl.
Another aspect of the invention relates to
~unctionalized polymers having the structure:
Rl R2 R2~
(SURFACE)--CH--Cl12 ' 1~--N---C--X--lCI---N--~ Cli2-CIH--Y--Cr--CH2 ' I N 1I X ICI OR
Oll Rl O O R2 Oli Ol~ R2n-l o o
5
or
Rl R2
(SURFAOE~--C ~ -N--N~-CH2-Ci-i-(Y)-CH-Cli2-~N--N--C--(~)--C - OR
O Rl 011 01i R2 o o 11
wherein
a. X and Y are connecting groups;
b. R" or R n (where n = an integer) each
represent alkyl, cycloalkyl, aryl, aralkyl
and alkaryl;

21393~9
- WO94/01102 PCT/US93/06241
- 35 -
c. (STRUCTURE) is a macromolecular component;
and
d. n > 1.
The invention also contemplates various methods
of producing an aminimide-functional support. One method
comprises the steps of reacting a polymer or oligomer
containing pendant moieties of OH, NH or SH with a
compound of the formula:
Cl ~ N~ ~/
wherein Rl and R2 each represent alkyl,
cycloalkyl, aryl, aralkyl or alkaryl, and R3 is
an amino acid derivative; a nucleotide
derivative; a carbohydrate derivative; an
organic structural motif; a reporter element;
an organic moiety containing a polymerizable
group; or a macromolecular component;
coating the reacted polymer or oligomer onto a support to
form a film thereon; and heating the coated support to
crosslink the film.
Another method comprises the steps of coating a
mixture of multifunctional esters and multifunctional
epoxides onto a support to form a film thereon; and
reacting the coated support with 1,1'-dialkylhydrazine to
crosslink the film.
A third method comprises the steps of coating a
mixture of an aminimide-functional vinyl monomer, a
difunctional vinyl monomer and a vinyl polymerization
initiator onto a support to form a film thereon; and
heating the coatinq support to form a crosslinked film.

WO94/01102 213 9 3 ~ 9 PCT/US93/06z41
- 36 -
The aminimide-functionalized support prepared
according to the previous methods are another aspect of
the invention.
The ability to derivatize an aminimide scaffold
in numerous ways using the synthetic techniques outlined
above as well as those given below, offers a vast array
of structures capable of recognizing specific molecular
entities via establishment of specific types of binding
interactions. Thus the aminimide shown below is in
principle capable of establishing the following
interactions: ~-stacking involving the phenyl group;
hydrogen bonds; acid-base interactions involving the
anionic nitrogen; salt bridges involving the quarternary
nitrogen; steric interactions with the bulky isopropyl
substituent; and hydrophobic interactions involving the
hydrocarbon chain.
bonds
~
~ N~ ~ Hydrophobic/
Charge/ CH3~ C,oH23
Acid-Base
.~
Ch~ rge Hydrophobic
As a further example, possible interactions
between a recognition target and a specific supported
3 aminimide are shown below. Experimental procedures for
the synthesis of specific chiral aminimides are given
below.

~ WO94/01102 2 1 3 9 3 ~ 9 PCT/USg3/0624l
- 37 -
Charge/
Acid-B~se
~S~ N~3
H-bonds
4.4.2 Sequential Catenation of Aminimide Subunits
Producinq Sequences of Various Sizes
By choosing aminimide building blocks
possessing functional groups capable of establishing
predictable binding interactions with target molecules,
and using synthetic techniques such as those broadly
described above to effect catenation (linking) of the
building blocks, it is possible to construct sequences of
aminimide subunits mimicking selected native oligomers or
polymers, e.g. peptides and polypeptides, which have
better stability and pharmacokinetic properties than
those of the native sequencers. Specific syntheses of
multisubunit aminimides are outlined below.
4.4.2.1 Catenation of Aminimide Subunits
via Alkylation/AcYlation Cycles
The following steps are involved in this
synthesis:
1. Acylation of a chiral hydrazinium salt,
prepared as described above, with a molecule capable of
functioning botn as an acylating and as an alkylating
agent producing an aminimide; BrCH2COCl and other
bifunctional species, such as bromoalkyl isocyanates, 2-
bromoalkyl oxazolones, etc., may be used as acylating
agents under the reaction conditions given above.
C2H5 ~ N+J~ + Br/~CI ' N,N ",CH
H2N ~C1-!3 1- \~0

~ { j~
WO94/01102 ` PCT/US93/06Z41
- 38 -
21393~9
2. Reaction of the product of the above
reaction with an asymmetrically disubstituted hydrazine
to form a diastereomeric mixture of aminimide hydrazinium
salts under reaction conditions similar to those
described above.
HsC2~N+ ~ H3C~
-N 'CH3 ~ N -NH2 r
l H3C
~0
HC~ - ~ ~ t Br
3. Isolation of the diastereomers produced in
step 2 as described above, e.g. by fractional
crystallization or by chromatography using techniques
familiar to those skilled in the art.
4. Acylation of the desired diastereomer from
step 3 with a bifunctional acyl derivative similar to
those listed in step l above producing a dimeric type
structure-
o HsC2 ~ ~
5. Repetition of steps 2, 3 and 4 therequired number of times to build the desired aminimide
subunit sequence.

--~094/01102 2 1 3 9 3 ~ 9 PCT/US93/06241
- 39 -
6. Capping of the assembled sequence if
desired, for example, by reaction with an acylating
agent, such as acetyl chloride.
The experimental conditions (e.g. reaction-
solvent, temperature and time, and purification
procedures for products) for all of the above reactions
were described above and are also well-known and
practiced in the art. As the molecular weight of the
products increases (e.g. in step 5 above) solubility and
reaction-rate problems may develop if the reactions are
run under the conditions that successfully gave products
of much smaller molecular weight. As is well known from
the art of peptide synthesis, this is probably due to
conformational (folding) effects and to aggregation
phenomena, and procedures found to work in the related
peptide cases are expected to be very useful in the case
of aminimide catenations. For example, reaction solvents
such as DMF, or N-methyl pyrollidone, and chaotropic
(aggregate-breaking) agents, such as urea, are expected
to be helpful in alleviating reactivity problems as the
molecular-weight of the product increases.
25CH3 ~ N`N~ o H5C~ ~ CH
4.4.2.2 Catenation of Aminimide Subunits via
30Acylation/Alkylation CYcles
The following steps are involved in this
synthesis; experimental conditions for running the
reactions are given above.
1. Alkylation of an asymmetrically
disubstituted acyl hydrazide, prepared as outlined above,

WO94/01102 2 1 3 9 3 ~ i 40 - PCT/US93/0~1
with a molecule capable of functioning both as an
alkylating and an acylating agent to form a racemic
mixture of aminimides; as before the use of BrCH2COCl is
shown below, but other bifunctional species, such as
bromoalkyl isocyanates, 2-bromoalkyl oxazolones, etc. may
also be used.
2. Reaction of the racemate from above with
an asymmetrically disubstituted hydrazine to form the
hydrazone:
15R3~ ~N~ ~ R" R ~N,N+R~ N--N
+ ~ ~ N--I~H2 - ~ +
2 01~ R2"- ~ R3~ ,N+ I~N--N/
3. Resolution of the racemic modification
from the previous step as described above.
4. Alkylation of the product from step 3 with
a bifunctional molecule capable of alkylation and
acylation, which may be the same as that used in step 1
or different, to form a mixture of diastereomeric
aminimides.
5. Reaction of the diastereomers from step 4
with a suitable asymmetrically disubstituted hydrazine to
form the diastereomeric hydrazones, as shown:
SUBSTITUTE SHEET

--WO94/01102 2 1 3 9 3 ~ 9 PCT/US93/06~1
- 41 -
o
R3~ ,N;~ , N--N;+" 2~ ` R7
R o
0
R3~ ,N+ ~N~jCH2ll,N--N~
6. Separation of the diastereomers as
described above.
7. Repetition of steps 4, 5 and 6 to build
the desired sequence of aminimide subunits.
8. Capping of the sequence, if desired, using
e.g. methyl bromide to produce a sequence such as shown
below.
N R ~ N `CH3
4.4.2.3 Catenation of Aminimide Subunits Using
Hydrazinolysis of an Ester in the
Presence of an Epoxide
The following steps are involved in this
synthesis; experimental conditions for running the
reactions are given above.
1. Formation of an aminimine from the
reaction of an 1,1-asymmetrically disubstituted hydrazine
with an epoxide; the reaction is illustrated for a chiral

WO94/01102 213 9 3 4 9 42 PCT/US93/06~41
epoxide below (the chiral epoxide may be obtained by e.g.
a Sharples epoxidation):
Rl ~ + 3,N-NH2 H ~ ~ 'R3 H ~ ~N~RNH
OH R2 OH R2
The aminimine is normally not isolated, but used directly
for the following reaction.
~ . The aminimine is reacted with an ester-
epoxide to give an aminimine; for the mixture of
diastereomeric aminimides above and the ester-epoxide
shown below, the following is obtained.
o
N~, 3 ~3 N- ~\~
20H ~ R2 R H ~ 2~R3
CO2Me
+
H ~ ~ ~R3 H~ N~ O
OH R2 OH R2 O
~
3. Separation of the diastereomeric
aminimides as described above.
4. Reaction of the desired diastereomeric
aminimide with an asymmetrically disubstituted hydrazine
to form diastereomeric aminimide-aminimines:
SUBSTIT~JTE SHEET

21393~9
~ WO94/01102 ~ PCT/US93/06~1
- 43 -
R ~ +N-N ~
H /H R24 ~ ~i r~; R4 R
5. Repetition of steps 2, 3 and 4 above using
the appropriate hydrazines and epoxy-esters in each step
to produce the desired aminimide sequence.
6. "Capping" of the final sequence, if
desired, by acylation with a simple ester, such as methyl
acetate, to produce the designed aminimide ligand shown:
Z0 R +N ~ "~
4.4.2.4 Catenation of ~-Hydrazinium Esters
or Carboxylic Acids
The following steps are involved in this
synthesis; experimental conditions for running the
reaction are given above.
1. Treatment of a chirally-pure hydrazinium
- salt (produced as described above) with a strong base,
such as NaOMe in an alcohol solvent, to form the imino
anion:

WO94/01102 2 1 3 9 3 4 9 PCT/US93/06~41
- 44 -
~-N X~ NaOMe , +N
2. Addition of an ~-hydrazinium ester (again
produced as discussed above) to an appropriately blocked
imino-anion-containing mixture from step l to form the
hydrazinium-aminimide, as shown.
Rl~ Nl~ ROOC NHB, \N/ ~\ N~-IB,
R2 ~R3 R4~ ~R5 R2~ 'R3 R4~ ~R5
X- X-
In the equation above, Bl is an appropriate
protecting group such as BOC (t-butoxy carbonyl.
3. Removal of Bl followed by repetition of
steps l and 2 the required number of times to obtain the
desired aminimide sequence, followed by a "capping" step,
using a simple ester as acylating agent.
~ N
Alternatively, the ~-hydrazinium carboxylic
acids may be obtained by treatment of the esters with
LioH in MeOH/H2O at room temperature, as described above,
and coupled with each other using condensation reactions

; 2 Ll 3 9 3 ~ ~
- WO94/01102 ~ PCT/US93/06~1
- 45 -
promoted by DCC or other agents. Protecting groups used
in traditional peptide synthesis are expected to be
useful here as well.
4.4.3 Synthesis of Aminimide-Containing
Peptides and Proteins
Aminimide subunits may be introduced into any
position of a polypeptide via chemical synthesis, using
one of the procedures outlined above, including the
techniques for dealing with problematic reactions of high
molecular weight species. The resulting hybrid molecules
are expected to have improved properties over the native
molecules; for example, the aminimide group may confer
greater hydrolytic and enzymatic stability to the hybrid
molecule over its native counterpart.
As an example of a synthesis of an aminimide-
modified peptide, the modification of a peptide attached
to a Merrifield solid phase synthesis support by
alkylation with aminimide-containing molecule is shown
below.
N Br~N`+N~ (I) Coupiin~
~ H~ R4
If moiety B contains a functional group which
can be used to link additional aminimide and natural or
unnatural amino acid subunits, e.g. via acylation
reactions, complex hybrid structures may be obtained
using the experimental procedures outlined above.
SUBSTITUTE SHEET

W O 94/01102 2 13 9 3 4 9 PC~r/US93/06z41
- 46 -
S ~ H 0 ~N~ ,N~ NII~ + (~--<N~ (2) dcl~rllm n~ 7
~H$ ~ N~ N~
4.4.4 Synthesis and Polymerization of Chiral
Aminimide-Containinq Monomers
The conversion of many of the aminimide
structures described above into monomer building blocks
which can be polymerized to give novel macromolecules,
which are useful in a variety of high technological
applications, is contemplated. The following synthetic
approaches are expected to be very useful in the
production of new materials.
(a) Free-Radical Polymerization
of Vinvl Aminimides
Chiral (as well as achiral) vinylaminimide
monomers of the general structures shown below may be
readily prepared, following the procedures outlined
above, and used in free-radical polymerizations,
according to experimental procedures well-known in the
art, to produce a vast array of novel polymeric
materials.
~ R2~ ~R3 0 R1 R2

21393~9
- WO94/01102 ;. PCT/US93/06241
Additional monomeric structures useful in preferred free
radical polymerizations include those shown below; they
produce polymeric chains capable of being crosslinked
into more rigid structures. The monomers shown below may
be prepared using the synthetic procedures outlined
above, and the polymerization/crosslinking reactions may
be run using standard polymerization techniques. See,
for example, Practical Macromolecular Organic Chemistry,
Braun, Cherdron and Kern, trans. by K. Ivin, 3ed., Vol Z,
Harwood Academic Publishers, New York, N.Y. 1984.
R
~ X ,+N~
The monomers shown above may be polymerized
with other alkenes or dienes, which are either
commercially available or readily prepared using standard
synthetic reactions and techniques, to furnish copolymers
with novel structures and molecular recognition
characteristics.
~
~ N
H3C~ _ C2H~
CH2 CH20H

WO94/01102 2i~93~9 PCT/USg3/062-41
- 48 -
(b) Condensation Polymerizations Producing
Aminimide-Containinq Macromolecules
Sequential condensations of aminimide-forming
molecules may be used to produce a variety of novel
polymers of controlled size. An example involving
dimeric epoxides and esters is given below; processes
involving trimeric and more complex epoxides and esters
are also contemplated; and experimental conditions for
running these polymerizations (including techniques for
resolving experimental difficulties as product molecular
weight increases) have been described above.
CO2Et
Y H ~ OH H3C~
~7 + N--NH2
15O H H O HO~ IH H3C~
CO2Et
HO E ~ II3C ~CH3
2 O~ -- '~Y +N ,-N~ ~ , N+ .~ S5S
H H OH H CH3CH30 HO H
When the polymerization reaction is carried out with
25 molecules immobilized on a support, e.g. silica, a
support capable of specific molecular recognition is
produced; an example of such a support is given below:
H3C~ CH3 HO~ H H3C,~ - 3
` H N ` J~ +N / ~, N
~/ 01~ HO H o H3C CH3 OH H HO H

21393~9
- W O 94/01102 - PC~r/US93/06241
- 49 -
4. 4.5 Lipid Mimetics
Aminimide conjugate structures containing two
long-chain alkyl groups capable of producing bilayer
membrane structures are preferred embodiments of the
present invention. Many uses of these amphiphilic,
surface-active compounds are envisioned. They may be
used to isolate and stabilize biologically-active
molecules from the cell-wall; they are useful in the
construction of affinity chromatography supports for the
isolation and purification of amphiphilic macromolecules,
e.g. receptors, enzymes, etc.; and they may serve as
effective delivery systems for the administration of
drugs.
The structure of one preferred lipid mimetic is
shown below. Substituents R may be chosen from a variety
of structures of various sizes including structures of
ligands of biological receptors or enzymes; a preferred
combination of substituents involves sterically small
groups for R~ and R2 and a group such as A or B described
above for R3; the long-chain alkyl groups are 4-20 carbons
in length; group X is a linker composed of atoms chosen
from the set of C, H, N, O, and S.
R2 chain
~1 /
Rl--N-N-C--(X)
R3 chain
A further desirable variation of the surface-
active structure shown above is as follows:
R2 chain
Rl-C-N-N-(X)
Il I \
o R3 chain

WO94/01102 ~393 49 PCT/US93/062-41
In the above structure, X is a linker group (e.g. CH);
one or more substituents R are chosen from the group of
structures A and B described above and the remaining
substituent(s) in preferably a sterically small group,
e.g. H, or CH3. An additional desirable amphiphilic
structure is shown below; substituent structures are
similar to those listed above.
chain
--C--N-N--R2
~ ~,
chaln
An example of a synthesis of a lipid mimetic is
given below; the required experimental conditions for the
reactions that follow are similar to those described
above for related transformations.
Thus,
/ \
CH~(C~2)l4-CH2-CH-CH2 is combined with (CH3)2NNH2 and CH3COOCH3
p duce CH3(CH2)l4CH2CH(OH1CH2N(CH3)2NCOCH3, which is then
ith CH3(CH2)l4CH2Br under alklaline conditions to
produce: CIH3
CH3-tCH2)1s~CIH~CH2~N -N--CO-CH3
o CH3
CH3-(CH2)14-cH2
4.4.6 Fabrication of Aminimide-Containing
Macromolecular Structures Capable of
Specific Molecular Recognition
In an embodiment of the invention aminimide
molecular building blocks may be utilized to construct
new macromolecular structures capable of recognizing
specific molecules ("intelligent macromolecules"). The
SUB~ I I I UTE SHEET

--WO94/01102 21 3-9 3~ 9 PCT/US93/06241
"intelligent macromolecules" may be represented by the
following general formula:
P - C - L - R
where, R is a structure capable of molecular
recognition;
L is a linker;
P is a macromolecular structure serving as
a supporting platform;
C is a polymeric structure serving as a
coating which surrounds P.
Structure R may be a native ligand or a
biological ligand-acceptor or a mimetic thereof, such as
those described above.
Linker L may be a chemical bond or one of the
linker structures listed above, or a sequence of subunits
such as amino acids, aminimide monomers, oxazolone-
derived chains of atoms, etc.
Polymeric coating C may be attached to the
supporting platform either via covalent bonds or "shrink
wrapping," i.e. the bonding that results when a surface
is subjected to coating polymerization well known to
those skilled in the art. This coating element may be
1) a thin crosslinked polymeric film 10 - 50 A
in thickness;
2) a crosslinked polymeric layer having
controlled microporosity and variable thickness, or
3) a controlled microporosity gel. When the
support platform is a microporous particle or a membrane,
as described below, the controlled microporosity gel may
be engineered to completely fill the porous structure of
the support platform. The polymeric coatings may be
constructed in a controlled way by carefully controlling
a variety of reaction parameters such as the nature and

WO94/01102 ~i PCT/US93/06`Z41
21393~9- 52 -
degree of coating crosslinking, polymerization initiator,
solvent, concentration of reactants, and other reaction .
conditions, such as temperature, agitation, etc., in a
manner that is well known to those skilled in the art.
The support platform P may be a pellicular
material having a diameter (dp) from lO0 ~ to lO00 ~, a
latex particle (dp O.l - 0.2 ~), a microporous bead (dp l
- lO00 ~), a porous membrane, a gel, a fiber, or a
continuous macroscopic surface. These may be
commercially available polymeric materials, such as
silica, polystyrene, polyacrylates, polysulfones,
agarose, cellulose, etc. or synthetic aminimide-
containing polymers such as those described below.
Any of the elements P, C, L, or R containing an
aminimide-based structure is derived from a form of the
element containing a precursor to the aminimide-based
structure. The multisubunit recognition agents above are
expected to be very useful in the development of targeted
therapeutics, drug delivery systems, adjuvants,
diagnostics, chiral selectors, separation systems, and
tailored catalysts.
In the present specification the terms
"surface", "substrate", and "structure" refer to either
P, P linked to C or P linked to C and L as defined above.
Thus, another aspect of the invention relates
to a three-dimensional crosslinked random copolymer
containing, in copolymerized form about l to 99 parts of
a free-radically polymerizable monomer containing an
aminimide group; up to 98 parts of a free-radically
addition-polymerizable comonomer; and about l to 50 parts
of at least one crosslinking monomer.
The comonomer used in this copolymer may be
water-soluble or water-insoluble, and the copolymer is
fashioned into a water-insoluble bead, a water-insoluble
membrane or a latex particle, or can be a swollen aqueous
gel suitable for use as an electrophoresis gel.

~~ WO94/01102 2 1 3 9 3 1 9 PcT/us93/06~l
-.53 -
? ~ .
This copolymer is preferably the reaction
product of about 1 to 99 parts of a condensation-
polymerizable monomer containing a moiety cluster
selected from the group consisting of t1) at least three
epoxy groups, (2) at least three ester groups, (3) at
S least one epoxy and at least two ester groups and (4) at
least one ester and at least two epoxy groups; about 1 to
99 parts of a second condensation-polymerizable monomer
containing a moiety cluster selected from the group
consisting of (1) at least two ester groups, (2) at least
two epoxy groups and (3) at least one ester and one epoxy
group; and an amount of 1,1-dialkylhydrazine equivalent,
on a molar basis, substantially equal to the total molar
content of epoxy groups.
4.4.6.1 Amminimide Containinq SuPport Materials
Commercially available or readily obtainable
chromatographic support materials for chromatographic and
other applications, as well as other fabricated materials
may be derivatized with tailored aminimide moieties,
through chemical modification, producing novel materials
capable of recognizing specific molecular structures.
The following general structures are
contemplated.
R
I
A~ C -N--N+-(Y)-(SUREACE)
O R2
and
- 30
~-(X)-N+-N--C-(Y)-(SURFACE)
R2 o

WO94/01102 2 13 9 ~ ~ 9 PCT/US93/06~1
In the structures above, A is selected from the group
consisting of amino acids, oligopeptides, polypeptides
and proteins, nucleotides, oligonucleotides,
polynucleotides, carbohydrates, molecular structures
associated with therapeutic agents, metabolites, dyes,
photographically active chemicals, and organic structures
having desired steric, charge, hydrogen-bonding or
hydrophobicity elements; X and Y are chemical bonds or
groups consisting of atoms selected from the set of C, H,
N, O, S; Rl and R2 are chosen from the group of alkyl,
cycloalkyl, aryl, aralkyl, alkaryl and, preferably,
structures mimicking the side-chains of naturally-
occurring amino acids.
Surfaces and other structures functionalized
with multiple aminimide subunits are also preferred;
general structures are shown below.
Rl..n
A -X ~ C~-N--N+-(Y) ~ (SURFACE)
O Rl n
and
Rl...n
A-X ~ N+-N--C -(Y) ~ (SURFACE)
R'l...n o
, 11
In the above structures R' n and R' I- n are used to
illustr~te the manner in which the hydrazine substituents
Rl an~ R2 can be varied in each polymerization step
described above to produce a functional supported
oligomer or polymer.
The following chemical modifications can be
used to prepare aminimide-functionalized surfaces.

21393~9
- WO94/01102 ~ PCT/US93/06241
- 55 --
4.4.6.1.1 Functionalization of Ester and EPOXY Surfaces
A surface bearing ester groups can be treated
with an epoxide, containing desired group B, and a
- disubstituted hydrazine to form an aminimide surface as
follows:
~2
(SURFACE)--COORi -t N--NH2 ~ \ /
R3 ~ oR2 H
(SURFACE)--C---N~ CH2--f--B
O R3 OH
To carry out the above reaction, the surface is
treated with a solution containing a 10% molar excess of
the epoxide (based on the calculated number of reactive
ester groups of the surface), and a stoichiometric amount
of the hydrazine (with respect to the amount of the
epoxide) in an appropriate solvent, such as an alcohol,
with shaking. The mixture is then allowed to stand at
room temperature for 1 week with occasional shaking. At
the end of this period, the solvent is removed by
decantation, and the surface is thoroughly washed with
fresh solvent and air dried.
This approach allows the functionalization of
readily available supports containing ester groups.
The above reaction sequence can also be
employed with an epoxide-functionalized surface:
H R2
(SURFACE)--C~--~CH2 ~ N--NH2 ~ B COOR
R3
R2
(SURFACE)--f--cl~2-l~+--N---C--B
Ol~ R3 O
To carry out the above reaction, the surface is
treated with a solution containing a 10% molar excess of
the ester (based on the calculated number of reactive
epoxide groups of the support), and a stoichiometric
SUBSTITUTE SHEET

WO /4/01102 21393~9 56 - PCT/US~3/06~1
amount of the hydrazine (with respect to the amount of
the ester used), in an appropriate solvent, such as an
alcohol, with shaking. The mixture is then allowed to
stand at room temperature for 1 week with occasional
shaking. At the end of this period, the solvent is
removed by decantation, and the surface is thoroughly
washed with fresh solvent and air dried.
The foregoing reaction can be modified by
utilizing an ester whose substituent B contains a double
bond. After completion of the reaction shown above, the
double bond of the ester can be epoxidized using one of a
variety of reactions including the asymetric epoxidation
of Sharples (e.g., utilizing a peracid under suitable
reaction conditions well-known in the art), and the
product used as the epoxide in a new repetition of the
aminimide-forming reaction. The overall process can be
repeated to form oligomers and polymers.
For example, using ~,~-butenoic acid methyl
ester as the ester, n repetitions of the above reaction
sequence produces a compound of the form:
~ ~
Rl.... n Rn+l
(SURFACE)--CH--CH2 ` 1`+--N---C--CH2--CH-CH2 ~ 1~+--N---C--CH2--CH-CH~
OH ~ I.. n o OH -~n+l o
,n
where the designations R1~ andRI n are used to illustrate
the manner in which the h~drazine substituents R2 and R3
are varied in each polymerization step, if desired, to
produce an oligomer or polymer.
The foregoing reactions can be carried out
using bifunctional esters of the form ROOC-X-COOR', where
X is a linker and R and R' are alkyl groups as defined
above, and/or bifunctional epoxides of the form shown
below,

-WO94/011022 1 3 9 3 ~ 9 PCT/US93/06241
_ 57 -~ ~
~ r . ~
H2C-CH-Y-CH-CH2
\/ \/
O O
wherein Y is a linker as defined above, to form desirable
polymers. If an ester-functionalized surface is reacted
with bifunctional esters and epoxides, the resulting
surface will have the following general structure.
Rl R2
(SURFAOE)--C ' -N--I`+-CH2-CH-(Y)-CH-CH2-~ N--C--(X)--C_OR
O ~1 OH OH ~2 o n
If an epoxide-functionalized surface is reacted
as above the derivatized surface will have the following
general structure.
Rl R2 R2n-l
(S~FACE)--CH--CH2 ~ t` ~--N--C--X--C---N~ CHl-CH--Y--CH--CH~ --N---C--X--C--OR
OH ~ I O O ~2 OH , n o
4.4.6.l.2 Functionalization of Amine Surfaces
An amine-functionalized surface can be
converted to an ester-bearing surface by reaction with an
acrylic ester as shown in sequence (a) below. This
reaction is followed by reaction with hydrazine and an
epoxide as shown in sequence (b).
o o
(SURFACE)--NH2 ~ H2C=CH--C--O--CH3 -- (SURFACE)--NH-CH2--CH2--C--O--CH3
/--N' 2
~/ R2 X
O Rl CH~cll~2
(SUR~ACE)--NH-CH2--CH2--C---N'~ CH2-CH--B B~ --
~2 OH
SUBSTITUTE SHEET

WO94/01102 PCT/US93/06241
2 ~393 ~g - 58 -
For reaction (a), a 10% molar excess of methyl
acrylate ~based on the number of reactive amino groups
the surface as determined by a titration with acid) is
dissolved in an appropriate solvent, such as an alcohol,
and added to the surface. After addition is complete,
the mixture is shaken at room temperature for 2 days.
The solvent is then removed by decantation and the
surface is washed thoroughly with fresh solvent in
preparation for the next step.
For reaction (b) the stoichiometric amount of a
1:1 mixture of the hydrazine and the epoxide, is combined
in an appropriate solvent, such as an alcohol, and
quickly added to the solvent-wet surface from reaction
(a). The mixture is shaken at room temperature for 3
days. The solvent is then removed by decantation, and
the surface is washed thoroughly with fresh solvent and
dried.
The above reaction sequence can also be
employed with an epoxide-functionalized surface, in which
case substituent B in the structure above represents the
surface and the desired functional group bears the amine
moiety. One way of obtaining such a surface is to react
a silica surface with a silicic ester containing an
epoxide group to produce a so-called "epoxy silica", as
shown below.
- Si-OH + (CH3O)3-Si-CH2-CH2-CH2-O-CH2-CH-CH2
/ \
Si--O~ CH2 CH2 CH2--O--CH2-CH--CH2
("Epoxy Silica")

~ WO94/01102 2 13 9 3 4 9 PCT/US93/06241
- 59 -
4.4.6.1.3 Functionalization of Carboxylic-Acid-Containing
Surfaces
A surface functionalized with a carboxylic acid
group can be reacted with an l,1-dialkylhydrazine and a
coupling agent, such as dicyclohexyl carbodiimide (DCC),
to form a hydrazone-containing surface as shown in step
(a) below. This surface can then be coupled with a
desired group B bearing a substituent capable of
alkylating the hydrazone to give an aminimide structure
(after treatment with base), as shown in step (b):
10ll ~Rl (a) ~R
(SURFACE)- C-OH + H2N -N\ DCC ~ (SURFACE)- C-NH-N
~ /
~ B-CHzX
Rl
(SURFACE)- C--N-~-CH2-B
11 ~2
Substituent B is a surface functionalized with
an alkylating agent capable of reacting with a hydrazone.
To perform the above chemical modification of a
carboxyl-bearing surface, the surface is treated with a
10~ molar excess equimolar amounts of the N,N-
dimethylhydrazine and DCC in a suitable solvent, such as
methylene chloride, and the mixture is shaken for 2 hours
at room temperature. The slurry is then removed by
decantation and the surface is washed thoroughly with
fresh solvent to remove any residual precipitated
dicyclohexyl urea. The surface is then treated with a
stoichiometric amount of the alkylating agent in a
suitable solvent, warmed to 70 C and held at this
temperature for 6 hours. The mixture is then cooled, the

WO94~01102 ~ . PCT/US93/06~1
21393~9 - 60 -
solvent is removed by decantation, and the surface is
washed with fresh solvent and dried.
4.4.6.l.4 Funtionalization of Surfaces
Capable of HYdrazide Alkylation
A surface bearing a group capable of alkylating
acyl hydrazones can be functionalized to contain
aminimide groups as follows:
R
N-NH-C- W -B + (SURFACE)- Z -X
R2/
O R
(SURFACE)- Z-~+-N--C- W -B
~2 O
In the equation above, Z and W are linkers composed of
~S atoms selected from the set of C, N, H, O, S, and X is a
suitable leaving group, such as a halogen or tosylate.
A hydrazone bearing a desired group B is
produced by reacting the appropriate l,l'-
dialkylhydrazine with any of a variety of derivatives
containing B via reactions that are well-known in the
art. These derivatives may be acid halides, azlactones
(oxazolones), isocyanates, chloroformates, or
chlorothioformates.
4.4.6.l.5 Functionalization of Surface Bearing -NH, -SH,
or -OH GrouPs with ChloromethYl Aminimides
Surfaces functionalized with -NH2, -SH, or -OH
groups can be functionalized by treating them with
chloromethyl aminimides in the presence of strong base
using the experimental conditions outlined above:
O Rl
Il I
(SURFACE)- XH + Cl-CH2-C--N-N'-B
l2 R
(SURFACE)- X-C~2-C--N-N~-B
SUBSTITUTE SHEET

--~VO 94/01102 2 1 3 9 3 4 9 PC~r/US93/06241
- 61 -
The required chloromethyl aminimides can be
prepared by known literature procedures (See, e.g., 21 J.
Polymer Sci., Polymer Chem. Ed. 1159 (1983)), or by using
the technigues described above.
4.4.6.1.6 ~unctionalization of Oxazolone-Containing
Surfaces
Oxazolone-containing surfaces can be
functionalized by first reacting them with 1,1'-
dialkylhydrazine as shown in step (a) below followed by
alkylation of the resulting hydrazone with an alkylating
agent B-CH2-X as shown in step (b); reaction conditions
similar to those described above are expected to be
effective in carrying out these modifications.
R2~ NH2 + (SURFACE) Az- (SURFAOE)- C-NH-C-C-NU-N~
C?~/
B-CH~
R3 I
(SURFACE)- C-NH-C-C-N~ CH2B
In the structures above, R3 and R4 are derived from the
five membered azlactone ring denoted by AZ.
Although the previous discussions are
specifically directed to the functionaliziation of
surfaces, these reactions can also be used to construct
aminamide linkages to the other speciesof A and B which
are described in this application.
4.4.7 Preparation of Aminimide-Based
Coatinqs for Support ~aterials
It is possible to produce aminimide-
functionalized composite support materials by coating

WO94/01102 2 1 3 9 3 ~ 9 PCT/US93/06`~1
- 62 -
various soluble aminimide formulations on the surfaces of
existing supports, and subsequently crosslinking the
resulting coatings in place to form mechanically stable
surfaces. The coating may be engineered for a particular
appli~tion (e.g., to take the form of a thin non-porous
fil~--~r~to possess localized microporosity for enhanced
surfa~e-area) by judicious selection of process
conditions, monomer loading levels, the crosslinking
mechanism and the amount of crosslinker.
For example, any of the foregoing reactions can
be carried out with a vinyl aminimide in contact with a
selected surface, which is polymerized according to well-
known techniques (see, e.g., U.S. Patent No. 4,737,560).
The polymerization results in a surface coated with a
polymer containing aminimide side-chains. Other coating
procedures employing aminimide functional groups are
described below in greater detail.
H3C~
Epoxy S~ica + ~N NH2 + (~1~5)2- N-CHZ-CH2CO~Hs
H3C
CH3 O
O ~ CH2CH2CH2-O-CH2-CH-CH2-~+-N--C-CH2- CH2-N(~H5)2
OH CH3
4.4.8 Synthesis of Aminimide-Containing Materials Via
Polymerizations of Aminimide-Based Molecules
In addition to utilizing aminimide chemistry to
chemically modify commercially available or readily
obtainable surfaces, new surfaces and other materials can
be fabricated de novo from aminimide precursors bearing
polymerizable groups by polymerizations and/or
copolymerizations in the presence or absence of
crosslinking agents. Depending upon the properties for
the desired material, various combinations of monomers,
crosslinkers, and ratios thereof may be employed. The

213931l9
- WO94/01102 - (~` PCT/US93/06241
- 63 -
resultant support materials may be latex particles,
porous or non-porous beads, membranes, fibers, gels,
electrophoresis gels, or hybrids thereof. Furthermore,
the monomers and crosslinking agents may or may not all
be aminimides.
Vinyl or condensation polymerizations may be
advantageously employed to prepare the desired aminimide-
containing materials. Vinyl polymerization can include
use of one or more monomers of the form CH2=CH-X that are
copolymerizable with aminimides; suitable examples
include styrene, vinyl acetate, and acrylic monomers. If
desired, compatible non-aminimide crosslinkers, such as
divinyl benzene, may be employed (either singly or in
combination as the other such agents).
Condensation polymerization may be accomplished
using multifunctional epoxides and multifunctional esters
with the appropriate amounts of an l,l'-dialkylhydrazine,
using the reaction conditions described above. Either
the ester component or the epoxide component should be at
least trifunctional to obtain three-dimensionally
crosslinked polymer structures; preferably, both
components are trifunctional.
The nature and conditions of processing, the
ratio of the various monomers and the ratio of
crosslinker to total monomer content can be varied to
produce a variety of product structures (e.g., beads,
fibers, membranes, gels, or hybrids of the foregoing) and
to tailor the mechanical and surface properties of the
final product (e.g., particle size and shape, porosity,
and surface area). Appropriate parameters for a
particular application are readily selected by those
skilled in the art.
4.4.9 Combinatorial Libraries of Peptidomimetics
Derived From Aminimide Modules

WO94/01102 ` 213 ~ 3 ~ ~ PCT/US93/06~1
- 64 -
The synthetic transformations of aminimides
outlined above may be readily carried out on solid
supports in a manner analogous to performing solid phase
peptide synthesis, as described by Merrifield and others
(see for example, Barany, G., Merrifield, R.B., Solid
Phase Peptide Synthesis, in The Peptides Vol. 2, Gross
E., Meienhofer, J. eds., p. 1-284, Acad. Press, New York
1980; Stewart, J.M., Yang, J.D., Solid Phase Peptide
Synthesis, 2nd ed., Pierce Chemical Co., Rockford,
Illinois 1984; Atherton, E., Sheppard, R.C., Solid Phase
Peptide Synthesis, D. Rickwood & B.D. Hames eds., IRL
Press ed. Oxford U. Press, 1989). Since the assembly of
the aminimide-derived structures is modular, i.e., the
result of serial combination of molecular subunits, huge
combinatorial libraries of aminimide-based oligomeric
structures may be readily prepared using suitable solid-
phase chemical synthesis techniques, such as those of
described by Lam (K.S. Lam, et al. Nature 3S4, 82 (1991))
and Zuckermann (R.N. Zuckermann, et al. Proc. Natl. Acad.
Ser. USA, 89, 4505 (1992); J.M. Kerr, et al., J. Am Chem.
Soc. 115, 2529 (1993)). Screening of these libraries of
compounds for interesting biological activities, e.g.,
binding with a receptor or interacting with enzymes, may
be carried out using a variety of approaches well known
in the art. With "solid phase" libraries (i.e.,
libraries in which the ligand-candidates remain attached
to the solid support particles used for their synthesis)
the bead-staining technique of Lam may be used. The
technique involves tagging the ligand-candidate acceptor
(e.g.l an enzyme or cellular receptor of interest) with
an enzyme (e.g., alkaline phosphatase) whose activity can
give rise to color production thus staining library
support particles which contain active ligand-candidates
and leaving support particles containing inactive ligand-
candidates colorless. Stained support particles are
physically removed from the library (e.g., using tiny

WO94/01102 ~ 2 1 3 9 3 4 9 PCT/US93/06241
- 65 -
forceps that are coupled to a micromanipulator with the
aid of a microscope) and used to structurally identify
the biologically active ligand in the library after
removel of the ligand acceptor from the complex by e.g.,
washing with 8M guanidine hydrochloride. With "solution-
phase" libraries, the affinity selection techniquesdescribed by Zuckermann above may be employed.
An especially preferred type of combinatorial
library is the encoded combinatorial library, which
involves the synthesis of a unique chemical code (e.g.,
an oligonucleotide or peptide), that is readily
decipherable (e.g., by sequencing using traditional
analytical methods), in parallel with the synthesis of
the ligand-candidates of the library. The structure of
the code is fully descriptive of the structure of the
ligand and used to structurally characterize biologically
active ligands whose structures are difficult or
impossible to elucidate using traditional analytical
methods. Coding schemes for construction of
combinatorial libraries have been described recently (for
example, see S. Brenner and R.A. Lerner, Proc. Natl.
Acad. Sci. USA 89, 5381 (1992); J.M. Kerr, et al. J. Am.
Chem. Soc. 115, 2529 (1993)). These and other related
schemes are contemplated for use in constructing encoded
combinatorial libraries of oligomers and other complex
structures derived from aminimide units.
The power of combinatorial chemistry in
generating screenable libraries of chemical compounds
e.g.,in connection with drug discovery, has been
described in several publications, including those
- 30 mentioned above. For example, using the "split solid
phase synthesis" approach outlined by Lam et al., the
random incorporation of 20 different aminimide units into
pentameric structures, wherein each of the five subunits
in the pentamer is derived from one of the aminimide
units, produces a library of 205 = 3,200,000

WO94/01102 PCT/US93/06~1
21393~9 - 66 -
peptidomimetic ligand-candidates, each ligand-candidate
is attached to one or more solid-phase synthesis support
particles and each such particle contains a single
ligand-candidate type. This library can be constructed
and screened for biological activity in just a few days.
Such is the power of combinatorial chemistry using
oxazolone modules to construct new molecular candidates.
The following is one of the many methods that
are being contemplated for use in constructing random
combinatorial libraries of aminimides-based compounds;
the random incorporation of three aminimides derived from
~-chloroacetyl chloride and the hydrazines shown below to
produce 27 trimeric structures linked to the support via
a succinoyl linker is given below.
Rl R'l R''l
N-NH2 N-NH2 N -NH2
R2 R'2 R"2
(1) A suitable solid phase synthesis support, e.g., the
chloromethyl resin of Merrifield is treated with 4-
- hydroxyl butyric acid in the presence of CsC03
followed by tosylation with p-toluenesulfonyl
chloride, under conditions known in the art;
1.CsCO3
CH2-Cl + HO-(CH2)3-cO2H
2. TsCl
(~CH2-02C - (CH2)3 - OTs
SUBSTITUTE S~EET

- WO94/01102 21 393~9 PCT/US93/06241
- ~7 -
(2) The resulting resin is split into three equal
portions. Each portion is coupled with one of the
hydrazines shown above to give the hydrazinium resin
which is converted to the aminimide by reaction with
chloroacetyl chloride using the experimental
conditions described above.
Rl
1. RN--NH2
(~CH2-o2c-(cH2)3--OTs ,,
2. ClCH2COCl
Rl O
CH2-O2C-(CH2)3-N+-N- ~ Cl
R2
(3) The aminimide resin portions are mixed thoroughly
and split again into three equal portions. Each
resin portion is coupled with a different hydrazine
followed by a coupling with ~-chloracetyl chloride
producing a resin with two linked aminimide
subunits. The resin portions are then mixed
thoroughly and split into three equal portions.
R'l
1 N -NH2
Rl O R~
CH2-o2c-(cH2)3-N+-N- ~ Cl 2
R2 2.
Rl O R'l O
CH2-O2C-(C~2)3-l -N ~ N+_N ~ Cl
R2 R'2
SUBSTITUTE SHEET

WO94/01102 i 1 3 9 3 ~ 9 68 - PCT/US93/06~1
(4) Each resin portion is coupled with a different
hydrazine followed by reaction with an acid chloride
to produce a resin with three linked aminimide
subunits; Rl
1. N -NH2
S Rl O R'l O
CH2-o2c-(cH2)3-N+-N- ~ N+_N ~ Cl R 2
R2 R'2 2. ~
R Cl
Rl O R'l O R''l O
CH2-O2C-(CH~3-N+-N- ~ N+_N ~ I -N ~ R
R2 R'2 R"2
The resin portions are mixed producing a
library containing Z7 types of beads each bead type
containing a single trimeric aminimide species for
screening using the bead-stain method described above.
Alternatively, the aminimides may be detached from the
support via acidolysis producing a "solution-phase"
library of aminimides containing a butyrylated terminal
nitrogen. (Shown in the structure below in which
R=~H~
\CI~ (CH2)3--N~--N~ ~NJI\~
O ~ f~ 2 R-2
H~
HOJ~(C1~233 ¦ \N/J~ ¦ NJ~ ¦ ~N/JI\Q
R2 R'2 R-2
SUBSTITUTE SHEET

- W O 94/01102 . ~ 2 1 ~ 9 3 4 9 PC~r/US93/06241
4.4.10 Design and Synthesis of
Aminimide-Based GlycopePtide Mimetics
A great variety of saccharide and
polysaccharide structural motifs incorporating aminimide
structures are contemplated including, but not limited
- 5 to, the following.
(1) Replacement of certain glycosidic linkages
by aminimide backbones using reactions well known in the
art of sugar chemistry and reactions described above.
1. TsCI ~ J~
HO~ 2. R~ R, H~A~ X
~ ~ R 2 H2N--N~ ~L PO~H
PO\~H \OP H
H op P ~/H
H op
O Rl
~ O o~N~ I ~
\ OP H / H O P
pO \~/ H r \ Rcrnov~l o~ pro~cc~in~ group P
PO\~/H
O R,
OJ~N~ ¦ ~
Ho\~H HO~OH
H OH
SUBS~3TUTE St~5=ET

WO94/01102 2 1 3 g 3 4~ 9 PCT/US93/06~1
- 70 -
(2) Use of aminimide structures as linkers
holding in place a sugar derivative and a tailored
mimetic, or another sugar.
rCO2CH3
Rl 1. I,k
\ P H ~
H2N--NT~ P( ~\~/H
k-- \ H op
P\~opH 2. Deproleclion
Rl 2 0
N'--N- C
H o O~ R~ H~OH
~ OH H ~\ OH H /
HO\~/ H HO \~/ H
H OH H OH
4.4.11 Design and Synthesis of
Aminimide-Containinq Oligonucleotide Mimetics
The art of nucleotide and oligonucleotide
synthesis has provided a great variety of suitably
25 blocked and activated furanoses and other intermediates
which are expected to be very useful in the construction
of aminimide-based mimetics. (Com~rehensive Orqanic
Chemistry, Sir Derek Barton, Chairman of Editorial Board,
Vol. 5, E. Haslam, Editor, pp. 23-176).
A great variety of nucleotide and
oligonucleotide structural motifs incorporating
aminimide-based structures are contemplated including,
but not limited to, the following.
(1) For the synthesis of oligonucleotides
35 containing peptidic aminimide-based linkers in place of

Z139349
- WO94/01102 ~ , PCT/US93/06~1
the phosphate diester groupings found in native
oligonucleotides, the following approach is one of many
that is expected to be useful.
PIO ~ ROOC
~~O\- ~~
R2--N+-RI OP2
NH2
~~0 11
l/o\ severalsteps -d 1~ ~
R2--N+-RI , R2--IN~-Rl
20N O N~o
25OP2
~O--P=O
o\~\
SUBSi I I I UTE SHEET

WO94/01102 2 13g3 4g PCT/US93/06~1
- 72 -
(2) For the synthesis of structures in which
an aminimide grouping is used to link complex
oligonucleotide-derived units, an approach such as the
following is expected to be very useful.
OTms
N~ + X-(CH2)n - N - N - C - O h coupl~g
TmsO N H
1l 3. R2-X
H3CO--C T
H ~ ~,CH3
O~N Rl >
(CH2)n t_~--NH2
o
H~N ~ CH3 severalsteps
2 0(CH2) ~ N JJ ,¢~ r
O--P~
o
N J~,
1 ,11
(CH2)n~ Nll-- \J
-~2
3 0 ~0--p=O
o\~\
SUBS-i ITU~E SH3~ET

2139349
WO94/01102 1 ~ t.i. ~ !~ PCT/US93/~
- 73 -
5. EXAMPLE: SYNTHESIS OF AN AMPHIPHILIC LIGAND
MIMETIC USEFUL IN ISOLATION AND PURIFICATION
OF RECEPTORS BINDING VINCAMINE
To a solution of l.84 g (0.0l mol) of l,2-epoxy
dodecane (I) in a suitable solvent, such as n-propanol,
- 5 is added with stirring 0.61 g (0.0l mol) of l,l-
dimethylhydrazine. The solution is stirred for l hour at
room temperature, cooled to l0 C in an ice bath, and a
solution of 3.54 g (0.0l mol) of vincamine (II) dissolved
in the minimum amount of the same solvent is added. The
reaction mixture is stirred at 0 C for 2 hours, allowed
to come to room temperature, and stirred at room
temperature for 3 days. At the end of this time the
solvent is removed under high vacuum (0.2 torr) and the
crude product is isolated. The conjugate (II) is useful
l as a stabilization agent for the isolation and
purification of receptor proteins which are
therapeutically acted upon by vincamine and by
structurally related molecules.
o~ . ~zHs
2 0 CH30C~` / Cll3(CI12h-CIl--CH2--N--CH3
~N~N~ + cll3(cll2h c~--~CH2~ OH N
~ J ~_=0
. - OH
~5 C ~ ~CZH5
N
6. EXAMPLE: SYNTHESIS OF AMPHIPHILIC LIGAND
MIMETIC USEFUL IN THE ISOLATION AND
PURIFICATION OF SEROTONIN BINDING RECEPTOR
8.61 g (0.l mol) of methyl acrylate is added
over a 15 minute period to a stirred solution of 17.62 g
(0.l mol) of serotonin in l00 ml of a suitable solvent.
The reaction mixture is allowed to come to room

WO94/01102 2 13 9 3 4 9 74 _ PCT/US93/06~1
temperature and stirred at room temperature for 2 days.
The solvent is then removed by freeze drying to yield the
ester (IV). 6.0l g (O.l mol) of l,l-dimethylhydrazine is
added with stirring to a solution of 18.4 g (O.l mol) of
l,2-epoxydodecane in a suitable solvent, such as
propanol. The mixture is stirred at room temperature for
l hour and a solution of (IV) dissolved in the same
solvent is added. The mixture is then stirred at room
temperature for 3 days. At the end of this time the
solvent is removed in vacuo to yield the serotonin
conjugate (V), which is useful as a ligand for the
discovery, stabilization and isolation of serotonin-
binding membrane receptor proteins.
5~ ~ ~ Ho~3~
HO N--~ o
N~2 //
2 0)3~ OC~
~ T~
N ~ C~1~c~(c~l)~ Cl~,
Cl~
25 7, EXAMPLE: SYNTHESIS OF RHODAMINE-B-CONTAINING LIGAND
MIMETIC USEFUL IN THE ISOLATION AND PURIFICATION OF
CODEINE-BINDING PROTEINS
49.74 g (0.l mol) of the acid chloride of
Rhodamine B (VI), prepared from rhodamine B by the
3 standard techniques for preparing acid chlorides from
carboxylic acids, are dissolved in 500 ml of a suitable
solvent and are added, with stirring, over a l-hour
period to a solution of 6.01 g (0.l mol) of l,l-
dimethylhydrazine in lOO ml of the same solvent. The
temperature is kept at l0 C. After the addition is
complete, the mi~ture is stirred at room temperature for
SUBSTITUTE SHEET

21393~!~
- WO94/01102 ; ` :;! PCT/US93/06~1
, . . , ;.. . .
12 hours, and the solvent is stripped away in vacuo to
yield the Rhodamine B dimethylhydrazine (VII).
5.21 g (0.0l mol) of (VII) is dissolved in lOO
ml of a suitable solvent, such as benzene, and 4.69 g
(0.0l mol) of tosyl codeine (VIII), prepared from codeine
by the standard techniques for the tosylation of an
alcohol, in 50 ml of the same solvent is added over a lS-
minute period with stirring. The mixture is heated to
reflux and held at reflux for l hour. The mixture is
then cooled, the solvent is removed in vacuo, the residue
is redissolved in an appropriate alcohol and adjusted to
pH 8 with 10% methanolic KOH. The precipitated salts are
removed by filtration and the solvent is stripped in
vacuo to yield the conjugate (IX), useful as a probe for
the location, stabilization and isolation of receptor
proteins that bind codeine and structurally similar
analogues.
SUBSTlTUrE SHEET

WO 94/01102 213 9 3 4 9 -7s a - PCI/US93/06~1
~\COCI ~CONHN(CH~)2
El,N ~ ~ ~ N--El
IV Et Yll Et
1 0 CH,
C~,
Vl~ +~OTS ~N'--C N~l
20 C~O O C11,O
LX
E I
SUBSTITUTE SHEET

2 13~3 ~
- WO94/01102 PCT/US93/06~1
- 76 -~
8. EXAMPLE: SYNTHESIS OF DISPERSE=_LUE-3-CONTAINING
LIGAND MIMETIC USEFUL IN THE ISOLATION AND
PURIFICATION OF CODEINE-BINDING PROTEINS
To a solution of 0.285 g (0.00l mol) of norcodeine
(X) dissolved in 50 ml of a suitable solvent (such as
benzene) is added a solution of 0.139 g t0.00l mol) of
4,4'-dimethylvinylazlactone (XI) in l0 ml of the same
solvent and the resulting solution is heated to 70 C and
held at this temperature for l0 hrs. At the end of this
time the temperature is brought to l0 C with cooling and
0.06 g (0.00l mol) of l,l-dimethylhydrazine dissolved in
l0 ml of the same solvent is added dropwise. The
solution is then re-heated to 70 C and held at this
temperature for 2 hours. 0.466 g (0.00l mol) of the
Disperse blue 3 tosylate (XII), prepared by the standard
tosylation techniques from a pure sample of the dye
(obtained from the commercial material by standard
normal-phase silica chromatography), is added and the
mixture is heated at 70 C for 2 more hours. The solvent
is then removed in vacuo, the residue is redissolved in
an appropriate alcohol solvent and titrated to pH 8
(measured with moist pH paper) with 10% (w/v) methanolic
KOH. The precipitated salts are then removed by
filtration, and the filtrate is stripped in vacuo to give
conjugate (XIII), useful as a probe for the location and
isolation of receptor proteins that bind codeine and
similar molecules.
SUBSi I I I UTE SHEET

WO94/01102 PCT/US93/06~1
2139399 -76/l c.~,
- c~
N O o~o/\~\~ N
Cl1,0~C~1 N~
~ (C~ 2 / Nl~
~ ~ N--N;~ ~ ~ors
01~ `111CH,
C~,O~--
9. EXAMPLE: SYNTHESIS OF AN AMPHIPHILIC LIGAND
MIMETIC FOR THE ISOLATION AND PURIFICATION OF
CODEINE-BINDING PROTEINS
29.95 g (0.l mol) of octadecylisocyanate is
added slowly to 6.0l g (0.l mol) of l,l-dimethylhydrazine
in l00 ml of benzene. The mixture is stirred for 18
hours at room temperature and 54.2 g (O.l mol) of tosyl
codeine (VIII), prepared by the standard techniques, is
added portionwise over a l/2-hour period. The mixture is
heated to reflux and stirred at reflux for 2 hours. At
the end of this time the solvent is removed in vacuo, the
residue is dissolved in an appropriate solvent (such as
ethanol), and the pH is titrated to 8 (measured with
moist pH paper) with 10% (w/v) methanolic KOH. The
precipitated salts are then removed by filtration and the
solvent is removed i~ vacuo to give the crude conjugate
(XIV), useful for stabilizing and isolating receptor
proteins that bind to codeine and to similar molecules.
SUB~ I I I ~JTE SHEET

--~094/01102 2 1 3 9 3 4 9 PCT/US93/06241
-77-
C~1,
S C~ N
X1V ~
10. EXAMPLE: SYNTHESIS OF A MIMETIC OF
THE PROTEIN-KINASE BINDING PEPTIDE
a. The dodecamer peptide (BEAD)-Asp-His-Ile-
Ala-Asn-Arg-Arg-Gly-Thr-Arg-Gly-Ser-NH2 is obtained
attached to the solid support as shown using standard
FMOC peptide synthesis techniques, after deprotection of
the terminal FMOC group. This peptide is shaken with a
solution of an equivalent molar amount of ClCH2COCl in a
suitable solvent at 50 C for 6 hours. The solvent is
removed by decantation, leaving a terminal -NH-CO-CH2Cl
group attached to the peptide.
b. A solution of equimolar amounts of l,l-
dimethylhydrazine and dicyclohexylcarbodiimide in a
suitable solvent is treated with an equivalent molar
amount of the heptamer peptide H2N-Thr-Thr-Tyr-Ala-Asp-
Phe-Ile-COOH, prepared and obtained in the free state
using the standard FMOC solid phase peptide synthesis
chemistry (e.g., using instruments and methods marketed
by the Milligen Division of Millipore Corp.). The
mixture is stirred for 4 hours at room temperature. The
precipitated N,N'-dicyclohexyl urea is removed by
centrifuging and decantation, and the solution is added
to the functionalized beads prepared in a. above. The
mixture is then heated to 50 C and shaken overnight at
this temperature. The mixture is cooled, the solvent
removed by decantation, and the peptide released from the
bead to yield the aminimide mimetic H2N-Thr-Thr-Tyr-Ala-
3s Asp-Phe-Ile-CO-N-N(CH3)2-CH2-Ser-Gly-Arg-Thr-Gly-Arg-Asn-
SUBSTITUTE SHEET

WO94/01102 2 1 3 9 3 4 9 -78- PCT/US93/06~1
Ala-Ile-His-Asp-COOH. This mimetic has the aminimide in
place of alanine in the naturally occurring protein-
kinase binding peptide, UK (5-24), and is useful as a
synthetic binding peptide with enhanced proteolytic
stability.
s
11.1. SYNTHESIS OF A MIMETIC OF A
HUMAN ELASTASE INHIBITOR
This example teaches the synthesis of a
competitive inhibitor for human elastase based on the
structure of known N-trifluoroacetyl dipeptide analide
inhibitors (see 162 J. Mol. Biol. 645 (1982) and
references cited therein).
o I 1~
F3CJ~N' ~ + C~ N ' ~,N~ (1~ ncu~raJiz~lion
H J~ 1~3C ~30 1~ (2) purirlcalion
F]CJ~N ~=~N +~N
To 3.7 g (0.01 mol) of the aminimide N-(p-
isopropylanalido)-methyl)-S-N-methyl-N-
benzylchloromethylacetamide in 50 ml ethanol was added1.86 g (0.01 mol) of 1-methyl-1-isobutyl-2-N-
trifluoroacetyl hydrazone (prepared from the reaction of
trifluoroacetic anhydride with 1-methyl-1-
isobutylhydrazine [from methylisobutylamine and
chloramine~ using standard acylation methods) in 50 ml
SUE~ IJTE SH~=ET

21393~9
- WO94/01102 PCT/US93/06241
. i ~ . i,
-79- ~
ethanol. The mixture was heated to reflux and stirred at
reflux for 4 hours. The mixture was then cooled to room
temperature and titrated with 10% (w/v) KOH in methanol
to the phenolphthalein endpoint. The mixture was then
filtered and the solvent removed in vacuo on a rotary
evaporator. The residue was taken up in benzene and
filtered. Removal of the benzene on the rotary
evaporator yielded 5.l g (95%) of crude mixed
diastereomeric aminimides. The desired (S)-(S) isomer
was obtained by normal-phase chromatographic purification
over silica. This product is useful as a competitive
inhibitor for human elastase, characterized by HPLC on
Crownpack~ CR(+) chiral stationary phase (Daicell
Chemical Indu~tries Ltd.) using pH 2 aqueous mobile
phase. NMR (DMSO-d6): Chemical shifts, peak integrations
and D2O exchange experiments diagnostic for structure.
ll.2. Synthesis of the Chiral
Chloroaminimide Startinq Material
~\ N 1~ ~ + Cl /\~ neu~ u~n
Cl/--~ N.~ `~5J
~,~
SUB~ JTE SHEET

WO94/01102 2 1 ~ 9 3 4 9 PCT/US93/06~1
-80-
A mixture of 4.2 g (0.01 mol) of the
hydrazinium iodide enantiomer prepared as outlined below,
1.0 g (0.0106 mol) chloroacetic acid and 1.24 g (0.011
mol) chloracetyl chloride, contained in a micro reaction
flask equipped with a drying tube, was heated in an oil
bath at 105C for 1 hour. The (homogeneous) reaction
mixture was then cooled to room temperature and extracted
with 4 x 20 ml of ethyl ether to remove chloracetyl
chloride and chloracetic acid, with vigorous stirring
each time. The residual semisolid was dissolved in the
minimum amount of methanol and titrated with 10% KOH in
methanol to the phenolphthalein end point. The
precipitated salts were filtered and the filtrate
evaporated to dryness on a rotary evaporator at 40OC.
The residue was taken up in benzene and filtered. The
solvent was removed on a rotary evaporator to yield 3.37
g (90%) of the (S)-aminimide enantiomer, characterized by
CDC~ NMR spectrum and D20 exchange experiments and
directly used in the next step in the sequence (see
above).
11.3. Synthesis of the Chiral
Aminimide Startinq Material
5
li
~Cl /~ tartarate Kl
H2N--N O ~ r~solution
H.N ~N
SUBSTITUTE SHEET

21393~9
--WO94/01102 :t J;~ PCT/US93/06241
- 81 -
13.6 g (O.l mol) of l-methyl-l-benzyl-hydrazine
(prepared from methyl benzyl amine and chloramine using
standard methods [J. Chem. Ed. 485 (1959)]) in 125 ml of
toluene was cooled to 5C in an ice bath. To this was
gradually added, with vigorous stirring over a one-hour
period, a solution of 21.17 g (O.l mol) of p-
isopropylphenyl chloromethyl analide (prepared from
chloracetyl chloride and p-isopropylphenyl amine)
dissolved in lO0 ml of toluene. Throughout the addition,
the temperature was maintained at 5C. The reaction
mixture was then allowed to warm to room temperature, and
was stirred overnight. The precipitated solid
hydrazinium salt was filtered, washed with cold toluene
and dried in a vacuum oven at 60C/30" to yield 34.3 g
(98%) of racemic product. This racemate was slurried at
room temperature overnight in lO0 ml ethanol and a slight
molar excess of moist silver oxide was added and the
mixture was stirred at room temperature overnight. The
mixture was then filtered into an ethanolic solution
containing an equivalent of D-tartaric acid in the
minimum amount of solvent. The alcoholic filtrate was
concentrated to approximately 20% of its volume and
diethylether was added until turbidity was observed. The
turbid solution was then cooled at 0C overnight and the
crystals were collected by filtration. The solid
substance was purified by recrystallization from
ethanol/ether to yield the desired pure diastereomeric
salt, which was subsequently converted to the iodide form
by precipitation from a water-ethanol solution of the
tartrate (made alkaline by the addition of sodium
carbonate) on treatment with an equivalent of solid
potassium iodide, characterized by HPLC on Crownpack~
CR(+) chiral stationary phase (Daicell Chemical
Industries Ltd.) using pH 2 aqueous mobile phase. NMR
(DMSO-d6): chemical shifts, peak integrations & D2O
exchange experiments diagnostic for structure.

WO94/01102 2 13 9 3 ~ 9 PCT/US93/06~41
- 82 -
14. EXAMPLE: SYNTHESIS OF A PEPTIDE
MIMETIC INHIBITING HUMAN ELASTASE
o
l~3C~ N ~ NH
o~J I33C~ H
~N' ;-~ ~ /(
o
H3C~ N ~ N~-l
0~ ~/o
~ C1~3 0 1~3C 3
To 4.36 g (0.0l mol) of the chloromethylaminimide above
in 50 ml ethanol was added a solution of l.86 g (0.0l
mol) of l-methyl-l-isobutyl-2-N-trifluoroacetyl hydrazone
(prepared from the reaction of trifluoroacetic anhydride
with l-methyl-l-isobutylhydrazine [from methyl isobutyl
amine and chloramine] using standard acylation
conditions) in 50 ml of ethanol. The mixture was heated
to reflux, stirred at reflux for 4 hours, then cooled to
room temperature and titrated with 10% (w/v) KOH in
methanol to the phenolphthalein endpoint. The mixture
was filtered and the solvent was removed in vacuo on a
rotary evaporator. The residue was taken up in benzene
and again filtered. Removal of the benzene on the rotary
evaporator yielded 5.7 9 (95%) of the mixed (R)-(S) and
(S)-(S) aminimide diastereomers. The desired (S)-(S)
isomer was obtained pure by normal-phase chromatographic
purification over silica. This product is useful as a
competitive inhibitor for human elastase, characterized

`--WO94/01102 2 1 3 9 3 4 9 PCT/US93/06~1
- 83 -
by HPLC on Crownpack~ CR(+) chiral stationary phase
(Daicell Chemical Industries Ltd.) using pH 2 aqueous
mobile phase. NMR (DMSO-d6): chemical shifts, peak
integrations & D2O exchange experiments diagnostic for
structure.
SYnthesis of the Chiral Chloroaminimide
1~3C~ ~ JJ` Nl I
~ 1- + Cl~
1l2N C1~3 o
15ll3C`N ~ NH
o~ ~1
0~ ~N,~ ~
A mixture of 4.87 g (0.0l mol) of the
hydrazinium iodide enantiomer prepared as described in
5.2.3, l.0 g (0.0106 mol) chloroacetic acid and l.24 g
(0.0ll mol) chloroacetyl chloride, contained in a micro
reaction flask equipped with a drying tube, was heated at
105C for l hour with an oil bath. The (homogeneous)
reaction mixture was then cooled to room temperature and
extracted with 4 x 20 ml of ethyl ether to remove
chloracetyl chloride and chloracetic acid. The residual
semisolid mass was dissolved in the minimum amount of
methanol and titrated with 10% KOH in methanol to the
phenolphthalein end point. The precipitated salts were
filtered and the filtrate was evaporated to dryness on a
rotary evaporator at 40C. The residue was then taken up
in benzene and filtered. The solvent was removed on a
rotary evaporator to give 3.88 g (89%) of the (S)-

WO94/01102 2 1 3 9 3 4 g PCT/US93/06~41
- 84 -
aminimide enantiomer, characterized by CDC~ NMR spectrum
and D2O exchange experiments and used directly in the next
step in the synthesis (see above).
Synthesis of the Chiral Aminimide
o
"N~CI resolution
N--Nl 12
o
N Nl I
Kl o ~ 1-
N+ ~ N ~
18.4 g (0.1 mol) of 1-(5'[3'-methyl uracil]
methyl)-1-methylhydrazine (prepared by the alkylation of
2-methylphenylhydrazone with 5-chloromethyl-3-methyl
uracil in ethanol, as described in 24 J. Orq. Chem. 660
(1959) and references cited therein, followed by removal
of the benzoyl group by acid hydrolysis) in 100 ml
toluene was cooled to 5C in an ice bath and a solution
of 21.1 g (0.1 mol) p-isopropylphenyl-chloromethylanalide
(prepared from chloracetyl chloride and p-
isopropylanaline), in 100 ml of toluene, was added
thereto with vigorous stirring over a 1-hour period,
maintaining a temperature of 5C. The reaction mixture
was then allowed to warm to room temperature and was
stirred overnight. The solution was cooled to 0C and
the precipitated hydrazinium chloride salt was filtered,
washed with cold toluene and dried in a vacuum oven at
40C/30" to yield 4.77 g (98%) of crude racemic product.
This racemate was slurried in 100 ml ethanol, a slight
molar excess of moist silver oxide was added, and the

- WO94/01102 2 1 3 9 3 ~ 9 PCT/US93/06~1
- 85 -
mixture was stirred at room temperature overnight. This
racemate was resolved via its tartrate salts and isolated
as the iodide using the method of Singh, above,
characterized by HPLC on Crownpack~ CR(+) chiral
stationary phase (Daicell Chemical Industries Ltd.) using
pH 2 aqueous mobile phase. NMR (DMSO-d6): chemical
shifts, peak integrations & D2O exchange experiments
diagnostic for structure.
SYnthesis of 3-methyl-5-chloromethYluracil
A. 74.08 g (1 mol) of N-methyl urea and 216.2
g (1 mol) diethylethoxymethylenemalonate were heated
together at 122C for 24 hours, followed by 170C for 12
hours, to give the 3-methyluracil-5-carboxylic acid ethyl
ester in 35% yield (following recrystallization from
ethyl acetate)-
B. 30 g 3-methyluracil-5-carboxylic acid
ethyl ester was saponified with 10% NaOH to yield the
free acid in 92% yield (after standard work-up and
recrystallization from ethyl acetate).
C. 20 g of 3-methyluracil-5-carboxylic acid
was decarboxylated at 260C to give a quantitative yield
of 3-methyluracil.
D. 3-methyluracil-5-carboxylic acid was
treated with HCl and CH2 using standard chloromethylation
conditions to give 3-methyl-5-chloromethyl uracil in 52%
yield (following standard work-up and recrystallization
from ethyl acetate). NMR (DMSO-d6): chemical
shifts, peak integrations & D2O exchange experiments
diagnostic for structure.

WO94/01102 PCT/US93/06~1
213~3~9- 86 -
13. EXAMPLE: SYNTHESIS OF A PEPTIDE
MIMETIC INHIBITING THE HIV PROTEASE
This example teaches the synthesis of a
competitive inhibitor for the HIV protease with enhanced
stability, based on the insertion of a chiral aminimide
residue into the scissile bond position of the substrate
Ac-L-Ser(Bzl)-L-Leu-L-Phe-L-Pro-L-Ile-L-Val-OMe (see,
e.g., 33 J. Med. Chem. 1285 (l990) and references cited
therein).
o
Ac-Ser(Bzl)-Leu-As~ heJ~N ~ J + Br/~ Val-lle-OC113
O ~ O
~c-Ser-Leu-Phc ~ N ~ ,V~1-11e-OC~3
0.735 gms (l mmol) of Ac-Ser(Bzl)-Leu-Asn-Phe-
CO-NH-NC5H~o is dissolved in the minimum amount of DMF, and
0.344 g of BrCH2CONH-Val-Ile-OMe, prepared by treatment of
H2N-Val-Ile-OMe with (BrCH2CO) 2 according to the method of
Kent (256 Science 221 (1992), is added thereto. The
mixture is heated to 60C and stirred at this temperature
overnight. At this point the DMF is removed under high
vacuum, and the desired (S) isomer is obtained from the
enantiomeric mixture after neutralization by standard
normal-phase silica chromatography to yield the protected
peptide. The side chain blocking groups are subsequently
removed using standard peptide deprotection techniques to
yield the product Ac-Ser-Leu-Asn-Phe-CON~N+(C5Hlo)-CH2-CO-
NH-Val-Ile-OMe, useful as a enhanced stability
competitive inhibitor for the HIV protease.

21393~9
-WO94/01102 PCT/US93/06241
- 87 -
Synthesis of the Tetra~ePtide Hydrazone
Ac-Scr(Bzl)-Leu-Asn-Phe-OI~ + H2N--N~
1/~
Ac-Ser(Bzl)-Leu-Asn-Phe--N
0.653 g (1 mmol) of AcSer(Bzl)-Leu-Asn-Phe-OH,
prepared via standard peptide synthesis techniques (see
33 J. Med. Chem. 1285 (1990) and references cited
therein), is coupled with 0.10 g (1 mmol) of 1-
aminopiperidine using standard peptide-coupling methods
15 and chemistries (see 33 J. Orq. Chem. 851 (1968)) to give
a 97% yield of the hydrazide, isolated by removal of the
reaction solvent in vacuo.
14. EXAMPLE: PREPARATION OF CHIRAL MONOMER
USEFUL IN POLYMERIZATION YIELDING
CROSSLINKED POLYMER-CHAINS
~ ~ Me + ~\I--~o
H5C2~ NH2 Mc
~` N J~N~f
3.18 g (0.01 mol) of (S)-1-methyl-1-ethyl-1-p-
vinyl- benzylhydrazinium iodide, prepared from p-

WO94/01102 i 13 9 3 4 ~ -88- PCT/US93/06~1
vinylbenzyl chloride and 1-methyl-1-ethylhydrazine using
standard alkylation conditions, and isolated as the (S)-
enantiomer by the method of Singh (103 J. Chem. Soc. 604
(1913)), were added to 75 ml of anhydrous t-butanol. The
mixture was stirred under nitrogen and 1.12 g (0.01 mol)
of potassium t-butoxide was added. The mixture was
stirred for 24 hours at room temperature and the reaction
mixture was diluted with 75 ml of anhydrous THF, cooled
in an ice bath and 1.39 g (0.01 mol) of 2-vinyl-4,4-
dimethylazlactone in 50 ml of THF were then added over a
15-min. period. When addition was complete, the mixture
was allowed to warm to room temperature and stirred at
room temperature for 6 hours. The solvent was stripped
under aspirator vacuum on a rotary evaporator to yield
3.0 g (92%) of crude monomer. The product was
recrystallized from ethyl acetone at -30C to yield pure
crystalline momomer, useful for fabricating crosslinked
chiral gels, beads, membranes and composites for chiral
separations, particularly for operation at high pH. NMR
(CDCl3) chemical shifts, presence of vinyl groups in 6 ppm
region, vinyl splitting patterns, peak integrations and
D2O experiments diagnostic for-structure. FTIR absence of
azlactone CO band in 1820 cm~~ region.
SUBSTITUTE SHEET

- W O 94/01102 21 3 9 3 ~ 9 PC~r/US93/06241
-88/1-
15. EXAMPLE: FUNCTIONALIZATION OF SILICA WITH AN
OXAZOLONE FOLLOWED BY CONVERSION TO A CHIRAL
AMINIMIDE USEFUL IN THE RESOLUTION OF RACEMIC
CARBOXYLIC ACIDS
~5~`0 I~N, ~ 3 KO--
~ ~ ~ N- CH3
SUBSTITUTE SHEET

WO94/01102 ~ ~ PCT/US93/06~1
2139~49~ -
- gq-
2.81 g (0.01 mol) of (S)-1-methyl-1-ethyl-1-
phenyl- hydrazinium iodide, prepared by the method of
Singh (103 J. Chem Soc. 604 (1913)), was added to lOO ml
anhydrous t-butanol. The mixture was stirred under
nitrogen and 1.12 g (0.01 mol) potassium t-butoxide was
added. The mixture was stirred for 24 hours at room
temperature, after which the reaction mixture was diluted
with 100 ml anhydrous THF. To this mixture was added 5.0
g silica functionalized with the Michael-addition product
of (S)-4-ethyl-4-benzyl-2-vinyl- 5-oxazolone to
mercaptopropyl-functional silica. This mixture was
stirred at room temperature for 8 hours. The
functionalized silica was collected by filtration and
successively reslurried and refiltered using lOO-ml
portions of toluene (twice), methanol (four times) and
water (twice). The resulting wet cake was dried in a
vacuum oven at 60C under 30" vacuum to constant weight,
yielding 4.98 g of chiral-aminimide-functionalized
silica, useful for the separation of racemic mixtures of
carboxylic acids, such as ibuprofen, ketoprofen and the
like.
16. EXAMPLE: FUNCTIONALIZATION OF SILICA WITH A CHIRAL
AMINIMIDE FOR USE IN THE SEPARATION OF MANDELATES
NO2
Cl~3~
+ N--NH2 + Cl~300C~N~J~ N02
o
NO2
NO2
o
S~IBSTITUTE SHEET

- W O 94/01102 2 1 3 9 3 4 9 PC~r/US93/06241
-- 90 --
10.0 g epoxy silica (15~ Exsil C-200 silica)
was slurried in 75 ml methanol and shaken to uniformly
wet the surface. To this slurry was added 6.01 g (0.01
mol) l,l-dimethylhydrazine, and the mixture was allowed
to stand at room temperature with periodic shaking for 45
min. 32.5 g (0.1 mol) of (S)-3,5-dinitrobenzoylvaline
methyl ester was added and the mixture was allowed to
stand at room temperature with periodic shaking for three
days. The functionalized silica was then collected by
filtration, re-slurried in 100 ml methanol and re-
filtered a total of five times, then dried in a vacuum
oven at 60C/30" overnight to give 9.68 g of the product.
This functionalized silica was slurry packed from
methanol into a 0.46 x 15 cm stainless steel column and
used to separate mixtures of mandelic acid derivatives
under standard conditions.
Preparation of E~oxY Silica
50 g of 5 ~ C-200 Exsil silica (SA 250 ~2/g) was
added to 650 ml toluene in a two-liter three-necked
round-bottomed flask equipped with a Teflon paddle
stirrer, a thermometer and a vertical condenser set up
with a Dean-Stark trap through a claisen adaptor. The
slurry was stirred, heated to a bath temperature of 140C
and the water was azeotropically removed by distillation
and collection in the Dean-Stark trap. The loss in
toluene volume was measured and compensated for by the
addition of incremental dry toluene. 200 g of
glycidoxypropyl trimethoxysilane was added carefully
through a funnel and the mixture was stirred and refluxed
overnight with the bath temperature set at 140C. The
reaction mixture was then cooled to about 40C. The
resulting functionalized silica was collected on a
Buechner filter, washed twice with 50 ml toluene, sucked
dry, reslurried in 500 ml toluene, refiltered, reslurried

WO94/01102 2 1 3 9 3 ~ 9 91 - PCT/US93/06~1
in S00 ml methanol and refiltered a total of four times.
The resulting methanol wet cake was dried overnight in a
vacuum oven set for 30" at 60OC to yield 48.5 g of epoxy
silica.
Svnthesis of N-3 5-DinitrobenzoYl-(S)-Valine MethYl Ester
COCI 02N
02N J~No2 ~;~CO2M~ CO2Me
13.12 g (0.1 mol) of (S)-valine methyl ester
was added with stirring to a solution of 8 g (0.2 mol)
sodium hydroxide in 50 ml of water, cooled to about 10C,
and the mixture stirred at this temperature until
complete solubilization was achieved. 23.1 g (0.1 mol)
of 3,5-dinitrobenzoylchloride was then added dropwise
with stirring, keeping the temperature at 10-15C with
external cooling. After the addition was complete,
stirring was continued for 30 min. To this solution was
added over a 10-min. period 10.3 ml (1.25 mol) of
concentrated hydrochloric acid, again keeping the
temperature at 15C. After this addition was complete,
the reaction mixture was stirred for an additional 30
min. and cooled to 0C. The solid product was collected
by filtration, washed well with ice water and pressed
firmly with a rubber dam. The resulting wet cake was
recrystallized from ethanol/water and dried in a vacuum
oven under 30" vacuum at 60C to yield 28.5 g (90%) of N-
3,5-dinitrobenzoyl-(S)-valine methyl ester. NMR (CDCl3):
chemical shifts, splitting patterns, integrations and D2O
exchange experiments diagnostic for structure.

- WO94/01102 ? 1~ 9 349 PCT/US93/06~1
- 92 -
17. EXAMPLE: PREPARATION OF AMINIMIDE-CONTAINING
ION-EXCHANGE SILICA MATRIX
This example describes preparation of an
aminimide-functionalized ion-exchange silica matrix using
epoxy silica as the support to be modified. The reaction
sequence is:
Epoxy Silica + (CH3)2NNH2 + Et2NCHzCH2COOEt --->
-Si-o-S~CH2CH2CH2oCH2CH(oH)CH2N(CH3)2NCocH2cH2NEt2
25 g of epoxy silica (15~ Exsil AWP 300 silica,
with surface area of 100 m2/g) was slurried in 100 ml
methanol until completely wetted by the solvent. 10.2 g
of 1,1-dimethylhydrazine were then added with swirling
lS and the mixture allowed to stand at room temperature for
3 hours. 24.7 g of Et2NCH2CH2COOEt were then added and the
mixture kept at room temperature with periodic shaking
for 2 days.
The diethylaminoethyl (DEAE) functionalized
silica was collected by filtration, re-slurried in 100 ml
methanol and re-filtered a total of five times. The
packing was dried in a vacuum oven at 60C/30" overnight.
A 1.0 ml bed of this material was then packed in a 15 mM
NaAc buffer at pH 7.7. The column was then equilibrated
with 15 mM NaAc buffer at pH 5.6, and a solution of 1
mg/ml ovalbumin in this buffer run through the bed at a
flow rate of 1.6 ml/min. A total of 59.2 ml of protein
solution was run.
The column was then washed with 41.7 ml of 15
mM NaAc buffer at pH 5.58 and at a flow rate of 3.9
ml/min. The bound protein was eluted using 23.4 ml of
0.5M NaCl at a flow rate of 3.9 ml/min. The eluent (15.2
ml) was then collected and the transmission of an aliquot
measured at 280 m~ with a spectrophotometer. The
ovalbumin concentration was determined from a calibration

WO94/01102 2 i 3 g ~ ~ ~ PCT/US93/06~1
- 93 -
curve. The total amount of ovalbumin collected was 63.7
mg.
18. EXAMPLE: PREPARATION OF AMINIMIDE-CONTAINING
SIZE-EXCLUSION SILICA MATRIX
This example describes preparation of an
aminimide-functionalized size-exclusion silica matrix
using the epoxy silica support described above.
l0.0 g of epoxy silica (15~ Exsil C-200 silica,
with surface area of 250 m2/g) was slurried in 75 ml of
methanol and shaken to uniformly wet the surface. To
this slurry was added 10.2 g of l,l-dimethylhydrazine.
The mixture was allowed to stand at room temperature with
periodic shaking for 45 min.
15 g of ethyl acetate were then added and the
mixture allowed to stand at room temperature with
periodic shaking for 3 days. The functionalized silica
was then collected by filtration, re-slurried in l00 ml
methanol, re-filtered a total of five times and dried in
a vacuum oven at 60C/30" overnight. The functionalized
silica was slurry packed from methanol into a l0mm
interior-diameter jacketed glass column with adjustable
pistons to provide an 8 cm-long packed bed. This packing
was used to separate mixtures of polyethylene glycol
polymers of varying molecular weight with good resolution
using a mobile phase.
In a second experiment, the bulk packing was
found to selectively adsorb polyethylene-glycol
functionalized hemoglobin from serum samples taken from
test animals that had been treated with this derivative
as a blood substitute. Filtration of the serum, after
treatment with the bulk packing, gave a serum free from
the functionalized hemoglobin, thus allowing blood
screening or testing by means of standard methods.
l9. EXAMPLE: COATING OF A SILICA MATRIX WITH
HYDROXYPROPYL CELLULOSE FUNCTIONALIZED WITH

- WO94/01102 2a393~9 PCT/US93/06~1
- 94 -
AN AMINIMIDE
Hydroxypropylcellulose is mono-functionalized
by reaction, under strong alkaline conditions (preferably
provided by a strong base, such as potassium t-butoxide)
with ClCH2CON-N+(CH3)3. The result is replacement of
approximately one hydroxyl group in each saccharide unit
with the aminimide as follows:
OCH2CONN(CH3)3
~ [SACCHARID~ UNlT]n
OH OH
The resulting aminimide derivative is coated
onto a surface (e.g., silica). Upon heating to 140 C,
the N(CH3)3 group leaves, resulting in formation of an
isocyanate moiety:
OCH2NCO
[SACCHARIDE UNIT~n
OH OH
The isocyanate groups then react with unreacted
hydroxyl groups on the saccharide units to produce a
cross-linked coating.
Alternatively, the cellulose can be coated onto
the surface and immobilized using standard techniques
(e~g.~ reaction with bisoxiranes), and then mono, di- or
tri-substituted with desired aminimide derivatives as
described above.
The foregoing reaction sequence can also be
employed with polymers or oligomers bearing NH or SH
groups instead of hydroxyl groups and can also be
utilized to fabricate structures such as crosslinked
cellulose membranes.

WO94/01102 ~21 3 9 3 4 9 PCT/US93/062~1
- 95 -
20. EXAMPLE: COATING OF A SILICA MATRIX VIA
POLYMERIZATION OF AN AMINIMIDE ON THE MATRIX
This example illustrates an alternative
immobilization technique, namely, polymerizing aminimide
precursors containing vinyl groups and which have been
coated onto a surface. The chemistry resembles the
approach described above, except polymerization forms a
sturdy shell around an existing support rather than
creating a solid block of material.
This sequence makes use of the reaction
described above. An epoxide,
H Cl
C 3
(CH3)3-~+-CH2-CH\-/H2
CH3
is combined with methyl methacrylate and
dimethylhydrazine as set forth in 2.a above to form
CH=C( CH3) -CO-NN ( CH3)2-CH2-CH ( OH ) -CH2-N+ ( CH3)3Cl-. 3 . 11 g of
this material and 0.598 g n-methylol acrylamide were
dissolved in 75 ml of methanol, and 3 . 54 ml of water were
then added. To this solution were added 15 g of epoxy
silica (15~ Exsil AWP 300 silica, with surface area of
100 m2/g)
The mixture was stirred in a rotary at room
temperature for 15 min and then stripped using a bath
temperature of 44 C to a volatiles content of 15% as
measured by weight loss (from 25-200 C with a sun gun).
The coated silica was slurried in 100 ml of isooctane
containing 86 mg of VAZ0-64 dissolved in 1.5 ml toluene
which had been de-aerated with nitrogen. The slurry was
thoroughly de-aerated wtih nitrogen and then stirred at
70 C for two hours.
The coated silica was collected by filtration
35 and washed three times in 100 ml methanol and air dried.

- ~094/01102 `~-i' 2 13 9~3 4 9 PCT/US93/06~1
--g6--
The silica was heated at 120 C for 2 hours to cure the
coating. 13.l g of coated silica were obtained. A l ml
bed of this material was packed in an adjustable glass
column and successfully used to separate BSA from
lactoglobulin.
S
2l. EXAMPLE: PREPARATION OF SILICA SUPPORT CONTAINING
CROSSLINKED AMINIMIDE POLYMER CHAINS
In this example, an epoxy-functionalized
surface is reacted with disubstituted hydrazine, a
bisepoxide and a triester to form a crosslinked network
of aminimide chains attached covalently to the surface as
follows:
O O
Il 11
(SURFACE)--CH-~CH2 + H5C2--O--C--CH--C--O--C2Hs + H2C--CH-CH2 CH-CH2
o"C ~o~C2H5
N--NH2
2 0 ~/ R2
Rl Rl
N +--N---C--CH--C--N---N +-CH2-CH-CH2-CH--CH2
2 5 ~ R2 o ¢O O 72 OH OH , n
N-
Rl--I +--R2
CH2
3 0 CH-OH
(SURFACE)
SUB~ I I I ~JTE SHEET

WO94/01102 2 i 3 9 3 4 g PCT/US93/06~1
The reaction can be carried out in water at
room temperature without special conditions.
22. EXAMPLE: PREPARATION OF CROSS-LINKED
POROUS AMINIMIDE ION-EXCHANGE BEADS
This example describes preparation of three-
dimensional cross-linked porous copolymeric aminimide
ion-exchange beads. It involves reaction of three
monomers:
~o Monomer A: CH2=CH-CON-N+(CH3)3
Monomer B: CH2=C(CH3)-CON-N+(CH3)2-CH2-CH(OH)-CH2-
N+(CH3)3Cl-
Crosslinker: CH2=CH-CO-NH-C(CH3)2-CON-N+(CH3)2-CH2-Ph-
CH=CH2
where Ph is phenyl.
Pre~aration of Monomer A: This monomer was preparedaccording to the method described in 21 J. Polymer Sci.,
PolYmer Chem. Ed. 1159 (1983).
Pre~aration of Monomer B: 30.3 g (0.2 mol) of
glycidyl-trimethylammonium chloride was dissolved in 100
ml of methanol and filtered free of insolubles. 22 g
(0.22 mol) of methyl methacrylate was added thereto,
followed by 12 g (0.2 mol) of l,l-dimethylhydrazine. The
solution grew warm and turned slightly pink. It was
allowed to stand for 6 days at room temperature, and was
then treated with charcoal, filtered, and concentrated on
a rotary evaporator at 55 C and 10mm to produce a thick
lavendar-colored, viscous material. This material was
triturated with diethylether and hot benzene and
dissolved in the minimum amount of methanol. The mixture
was then treated with charcoal, filtered, heated to
boiling and brought to the cloud point with ethyl
acetate. The resulting solution was allowed to stand at
0 C for a week. The white crystals that formed were
collected by filtration, washed with cold ethyl acetate

WO94/01102 ~ f 2 1 3 9 3 1 9 PCT/US93/06~1
- 98 -
and dried in a vacuum oven at room temperature to yield
7.3 g of monomer B.
Preparation of Monomer C: 18 g (0.3 mol) of l,l-
dimethylhydrazine was dissolved in 50 ml CH2Cl2 and cooled
in an ice bath with stirring. 4l.7 g (0.3 mol) of
vinylazlactone in 50 ml CH2Cl2 were added slowly to keep
the temperature below 5 C. The clear solution was
stirred and allowed to come to room temperature over l
hour (resulting in formation of a white solid) and was
stirred at room temperature for an additional l.5 hours.
The white solid was collected by filtration, re-slurried
in lO0 ml CH2Cl2 and re-filtered. It was then dried in a
vacuum oven at room temperature overnight to yield a
total of 26.81 g of the intermediate
CH2=CH-CO-NH-C(CH3)2-CO-NH-N-(CH3)2. lO.0 g (0.05 mol) of
this intermediate and 7.66 g (0.05 mol) of vinyl benzyl
chloride were dissolved in a mixture of 50 ml ethanol and
50 ml CH3CN. The solution was refluxed for 4 hours under
a nitrogen stream. It was then cooled to room
temperature and concentrated on a rotary evaporator at 55
C to produce a thick yellow oil. The oil was triturated
three times with diethylether to yield 17.08 g of an off-
white solid. This solid was dissolved in lO0 ml of hot
methanol and filtered through a celite pad to remove a
small amount of gelatinous material, and the clear
filtrate was stripped to yield lO.0 g of Monomer C as a
white solid.
PolYmerization: l ml of the emulsifier Span 80 and
175 ml of mineral oil were introduced into a 500 ml
round-bottomed flask equipped with stirrer and a heating
bath. The mixture was mechanically stirred at 70 RPM and
brought to a temperature of 55 C. 40.5 g of monomer A,
7.2 g of monomer B and 5.7 g of the cross-linker were
dissolved in lO0 ml of demineralized water and heated to
55 C. To this solution were added 150 mg of ammonium
persulfate, and the mixture was then poured into the

WO94/01102 2 1 3 9 3 49 PCT/US93/06~41
_ 99 _
stirred mineral oil. The agitation was adjusted to
produce a stable emulsion with an average droplet
diameter of approximately 75 ~ (as determined with an
optical microscope).
After 15 min, 0.15 ml of TMED was added and
stirring was continued for an additional 45 min. The
reaction mixture was cooled and allowed to stand
overnight. The supernatant mineral oil phase was removed
by aspiration and the beads were collected by
decantation. The beads were washed three times with a
0.05% solution of Triton X-100 in demineralized water to
remove any remaining mineral oil and then washed with
water and allowed to settle. The water was removed by
decantation.
This procedure was repeated a total of five
times. The beads obtained at the conclusion of the
foregoinq steps had a mean diameter of approximately 75
and an ion-exchange capacity of 175 ~eq/ml.
23. EXAMPLE: PREPARATION OF AN AMINIMIDE-BASED
ELECTROPHORETIC GEL
This example describes preparation of an
aminimide electrophoresis gel. As a control, the
standard Sigma protein electrophoresis mix (available
from Sigma Chemical Co., St. Louis, MO) was run on an
acrylamide/methylene bisacrylamide linear gradient gel
prepared using a gradient maker with 5% and 12.5% monomer
solutions, as shown below. The gel was overlayed with
isobutanol and allowed to polymerize overnight.
5% Monomer 12.5%
Monomer
Lower Tris 5.0 ml 5.0 ml
H2O 11.7 ml 4.7 ml
30% Acrylamide 3.3 ml 8.3 ml

213~349
-~VO 94/01102 ` ~ ~ - PC~r/US93/06241
-- 100 --
Glycerol --- 2.0 ml
Ammonium Persulfate 30 ~1 30 ~1
TMED 15 ~1 15 ~1
Lower Tris 1.5M: 6.06 g Tris base, 8 ml 10~ SDS,
volume adjusted to 90 ml with double-distilled water.
The pH was adjusted to 6.0 with concentrated HCl, and the
final volume adjusted to 100 ml with DD water.
AcrYlamide 30~ w/v: 29.2 g acrylamide, 0.8 g of
methylene bisacrylamide and 100 ml DD water.
SDS 10% w/v: 10 g of SDS dissolved in DD water and
adjusted to a volume of 100 ml.
Ammonium Persulfate 10%: 0.1 g ammonium persulfate
was dissolved in 0.9 ml DD water. The solution was used
within 4 hours of preparation.
TMED: used directly as obtained from Sigma Chemical
Co., St. Louis, MO, under the tradename TMEDA.
A second gel was prepared by replacing the
acrylamide with an equal weight of the aminimide monomer
CH2=CH-CO-N-N(CH3)3 and the protein standard was run in the
same way as the first.
Separation of proteins with the aminimide gel
were equivalent to the acrylamide gel, but the aminimide
gel produced Rf (i.e., the ratio of distance traversed by
a particular protein to the distance traversed by the
solvent front) levels approximately 20% higher than those
of the acrylamide gel.
24. EXAMPLE: PREPARATION OF AMINIMIDE-BASED
LATEX PARTICLES
This example describes preparation of latex
particles containing an aminimide comonomer.
591.1 ml of distilled water was charged to a
three-necked round-bottomed flask. A nitrogen dip tube
was placed below the liquid level and the nitrogen flow
rate set to 2 cm3/min. The solution was mechanically

WO94/01102 2 1 3 9 3 ~ 9 PCT/US93/062~
-- 101 --
agitated with a Teflon paddle at 250 RPM and heated to 80
C over a half-hour period. In a separate flask were
dissolved 12l.6 g of butyl acrylate, 54.6 g of ethyl
acrylate, 13.0 g of acrylic acid, 9.97 g of methyl
methacrylate, 59.7 g of the aminimide monomer CH2=CH-C0-N-
N(CH3)2-CH2-CH2-OH and 0.92 g of Aerosol TR-70 so as to
obtain solution without exceeding a temperature of 25 C.
When completely dissolved, l.53 g of additional TR-70
were added and the mixture was then stirred until
solution was achieved.
20.7 ml of distilled water was purged with
nitrogen for lO min and l.59 g of K2S208 is dissolved in
it. This persulfate solution was added to the heated
water in the reaction flask after it stabilized at 80 C.
The nitrogen dip tube was raised and a nitrogen blanket
was maintained. The monomer mix was pumped in at a
steady, calibrated rate such that the constant addition
took exactly 4 hours. When addition was complete, the
latex was post-heated at 80 C for l hour, cooled to 25
C and titrated to pH 5.0 by dropwise addition of
triethylamine tapproximately 20 cm3) over 20 min with
agitation. The latex was then filtered through cheese
cloth and stored. Average particle size was measured at
about 0.14~.
It should be apparent to those skilled in the
art that other compositions and processes for preparing
the compositions not specifically disclosed in the
instant specification are, nevertheless, contemplated
thereby. Such other compositions and processes are
considered to be within the scope and spirit of the
3~ present invention. Hence, the invention should not be
limited by the description of the specific embodiments
dislosed herein but only by the following claims.