Note: Descriptions are shown in the official language in which they were submitted.
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
10
NANOMETER SIZED PARTICLES CONTAINING A REACTIVE MONOLAYER
ADSORBED THEREON AND METHODS OF MAKING THE SAME
Field of the Invention
The invention generally relates to manometer-sized particles which have
been chemically modified and methods of making the same.
Background of the Invention
Technology involving the synthesis of manometer-sized particles
("nanotechnology") has gained widespread attention recently. Broadly speaking,
nanotechnology relates to the art and science of building molecular materials
so
that they are capable of functioning as macro-scale structures and/or
exhibiting
physical and chemical properties which are intermediate between molecular and
bulk materials. Applications involving nanotechnology are potentially far
reaching. Areas of possible interest relate to, for example, catalysis,
molecular
electronics, biotechnology, composite materials, solar energy conversion, and
3o the like. Investigative efforts regarding nanotechnology have focused
largely on
understanding the physical behavior and structure of manometer-sized
materials.
Reiss, H., Proceedings of the Welch Foundation 39~" Conference on Chemical
Research: Nanophase Chemistry (1995) 49-fib discusses thermodynamic
behavior associated with nanophase technology. Berry, R.S., Proceedings of
the Welch Foundation 39t" Conference on Chemical Research: Nanophase
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
2
Chemistry, (1995), 71-81 focuses on the surface behavior of nanometer sized
particles. Quate, C.F., Proceedings of the Welch Foundation 40r" Conference on
Chemical Research: Nanophase Chemistry (1996) 87-95, discusses the use of
a scanning probe lithography in fabricating nanostructures. Schon, G., et al.,
4o Colloid Polym. Sci. 273 (1995) 202-218, discusses uses of nanometer-sized
particles relevant to the microelectronics industry.
Nanometer-sized gold particles can be chemically attached to metal
surfaces, such as electrodes. The purpose of such experiments is to allow the
researcher to add functionality onto the immobilized particles in order to add
45 value to the metal surface. For example, Freeman, R.G., et al., Science 267
(1995) 1629-1632, propose linking gold and silver colloidal particles to a
silanized surface. Doron, A., et al., Langmuir 11 (1995) 1313-1317, proposes
linking gold colloid particles to an indium tin oxide surface, followed by
further
functionalizing the exposed edge of the bound colloid particles with
electroactive
5o functional groups. The aforementioned techniques may be limited to surface
immobilized particles which makes characterization of the end-functionalized
material difficult and uncertain. Furthermore, there is a potential general
constraint in that the size of the particle attached to the surface is
significantly
less able to participate in the functionalization reactions. In essence, the
55 aforementioned work may be useful only as a means to add value to a large
(millimeter or micrometer scale) surface.
Metal sots are small particles which are insoluble, and thus suspended, in
the liquid in which they are dispersed. Numerous recent studies have
investigated methods for adding to the complexity of metal sols. For example,
so Mirkin, C.A., et al., Nature 382 (1996) 607-609, proposes attaching
oligonucleotides to gold sots in order to promote aggregation of said sols
upon
addition of an appropriate complementary oligonucleotide to the sol solution.
U.S. Patent No. 4,859,612, to Cole et al. proposes that antibody coated metal
sots can interact with an appropriately coated solid phase particle as a means
for
65 an immunoassay procedure. U.S. Patent Nos. 5,294,369 and 5,384,073 to
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
3
Shigekawa et al. propose that gold sots can be mixed in an alcohol solution
containing alkanethiol derivatives. Upon isolation of the particles, antigens,
antibodies, and ligands can be further linked to the gold particle via a
reaction in
an aqueous buffer. In all of the aforementioned examples, the proposed
7o methodology may be limited to metal sols, reactions in heterogeneous media,
and a specific subset of functionalized materials. The constraints of the
aforementioned methods potentially make analysis of the monolayer difficult
and
unreliable.
In addition to the above, recent efforts have focused on producing
75 manometer-sized particles having other materials chemically or physically
attached thereto. For example, Brust, M., et al., J. Chem. Soc, Chem. Commun.
(1994) 801-802, proposes manometer-sized gold cores which are stabilized by
chemisorbed layers of dodecanethiolate. The resulting rYiaterial is soluble in
non-polar organic solvents and can be repeatedly isolated and reused. Terrill,
R.
8o H., et al., J. Am. Chem. Soc., 117:50 (1995) 12537-12548, proposes
nanometer-
sized gold cores which are stabilized by chemisorbed layers of octane- or
hexadecanethiolate. These monolayer-protected gold clusters were found to be
highly stable as determined by differential scanning calorimetry techniques.
Hostetler, M.J., et al., Langmuir, 12 (1996) 3604-3612, relates to the
evaluation
85 of the physical structure of alkanethiolates of various chain lengths
adsorbed
onto manometer-sized gold cores. Alkanethiolates with shorter chain lengths
were determined to be relatively disordered while materials with longer chain
lengths were found to be in the trams zig-zag conformation. The previous three
descriptions of the art represent manometer-sized gold cores covered with
9o simple, non-derivatized alkanethiols, a circumstance that severely limits
their
applicability.
Hostetler, M.J., et. al., J. Am. Chem. Soc., 118 (199fi) 4212-4213
proposes manometer-sized gold cores stabilized by mixed monolayers of
unsubstituted and co-substituted (cyano, bromo, vinyl, ferrocenyl)
alkanethiolates.
95 A potential limitation of this and other methods are that they typically
rely on
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/0?823
4
either commercially available or easily-prepared t~substituted alkanethiols.
Another deficiency in these methods is that formation of the rv-substituent
often
does not occur on the protected gold cores, causing the need for extensive
purification steps that can only produce a narrow range of compounds.
Notwithstanding the above, there is a need in the art for manometer-sized
materials which exhibit specific chemical and physical properties. More
particularly, there is a need for such materials which can be tailored for
utilization
in a number of defined end use applications. It would be particularly
desirable if
the manometer-sized particles exhibited flexible chemical behavior as well as
X05 multiplicity with respect to reactivity. It would also be particularly
worthwhile if the
reverse engineering needed to design the synthesis of an envisioned multi-
functionalized manometer-sized particle would be straightforward and
understandable. It would be notably beneficial if the reactions forming the
particles were homogeneous, flexible, easy to learn, and reliable. This would
be
11o especially advantageous in analytical applications involving the
solubilization of
the manometer-sized particles in aqueous or organic solvents.
Summary of the Invention
It is therefore an object of the present invention to provide nanometer-
~~5 sized particles which may be chemically tailored to be desirable in a
variety of
applications.
These and other objects and features are provided by the present
invention. In one aspect, the invention relates to a functionalized nanometer-
sized particle comprising a core which comprises at least one metal or metal
~2o alloy; and a monolayer chemically bonded to the core. The monolayer is
formed
during the formation of the core and can be modified at any time following
formation of the core. Advantageously, the monolayer contains at least one
reactive substituent thereon which is coupled to a functional material such
that
the monolayer becomes chemically modified. The reactive substituent may be
X25 selected from a number of groups such as, for example, SH, OH, NH2, NH,
CA 02329859 2000-10-25
WO 99/61911 PC'TNS99/07823
C02H, S030H, P02(OH)2, BO(OH)2, or mixtures thereof. By virtue of the
manometer-sized particle structure, a number of functional materials may be
employed such as catalysts, biomaterials, and materials which are chemically,
electrochemically, or photochemically active. Thus, the particles may be
readily
~3o used in a number of applications.
In another aspect, the invention relates to a method of making a
functionalized manometer-sized particle. The method comprises providing a
manometer-sized particle comprising: (1) a core which comprises at least one
metal or metal alloy and (2) a monolayer chemically bonded to the core, the
X35 monolayer including at least one reactive substituent. The manometer-sized
particle is then coupled with a functional material such that the monolayer
becomes chemically modifed. Importantly, this method allows for the particle
to
be modified based on a small subset of reactive substituents, all of which can
be
readily synthesized or purchased commercially. Adding greater value to said
manometer-sized particle can then be accomplished using a variety of
functional
materials.
Brief Description of the Drawings
FIG. 1 is a representation of a multi-step synthesis of a tripeptide-
functionalized monolayer-protected manometer-sized gold core.
FIG. 2a is a graph illustrating the electrochemical characterization of a
10H-(phenothiazine-10)propionic acid-functionalized monolayer-protected
manometer-sized gold core. Specifically, the figure illustrates the cyclic
~so voltammetry of 0.8 mM 10H (phenothiazine-10)propionic acid (--) in 2:1
toluene/acetonitrile (v/v) at 100mV/s; and
FIG. 2b is a graph illustrating the thin layer coulometry charge Q vs. cell
length (L) (rz=0.98; slope=11.47 X 10-3 C/cm).
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
6
X55 Detailed Description of the Preferred Embodiments
The present invention now will be described more fully hereinafter with
reference to the accompanying specification, examples, and drawings, in which
preferred embodiments of the invention are shown. This invention may,
however, be embodied in many different forms and should not be construed as
limited to the embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and will fully
convey the scope of the invention to those skilled in the art.
In one aspect, the invention relates to a functionalized nanometer-sized
particle. The functionalized nanometer-sized particle comprises a core
165 comprising at least one metal or metal alloy. For the purposes of the
invention,
the core preferably has a diameter ranging from about 1 nm to about 999 nm,
more preferably from about 1 nm to about 100 nm; even more preferably from
about 1 nm to about 20 nm; and most preferably from about 1 nm to about 7 nm.
A monolayer is chemisorbed or chemically bonded to the core. The monolayer
contains at least one reactive substituent as described further herein.
Advantageously, the reactive substituent(s) on the monolayer is/are coupled to
a
functional material so as to chemically modify the monolayer.
A number of metals and metal alloys may be used in the core. Preferably,
the metal or metal alloy is selected from the group consisting of
175 a semiconducting material, a metal oxide material, a Group VIIIA element,
a
Group IB element, a Group IIB element, alloys thereof, and mixtures thereof.
More preferably, the metal or metal alloy is selected from the group
consisting of
a Group VIIIA element, a Group IB element, alloys thereof, and mixtures
thereof.
Examples of elements which may be used include, but are not limited to, gold,
o silver, copper, palladium, platinum, nickel, and alloys thereof. Examples of
semiconducting materials include, but are not limited to, cadmium sulfide,
indium
phosphide, and other Group III-V materials. Example of oxide materials
include,
but are not limited to, titanium oxide (titania), aluminum oxide (alumina),
tin
oxide, and iron oxide. The shape of the core is not restricted to any
particular
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
7
185 geometry, thus, for example, rods, spheres, cuboctahedra, and truncated
octahedra, will all satisfy the conditions stated herein.
For the purposes of the invention, the term "monolayer" may be defined
as a layer preferably having a thickness ranging from about 0.4 nm to about
100
nm, and more preferably 1.0 to 20 nm. Although not wishing to be bound by
any one embodiment, the monolayer is typically formed during the formation of
the core, and the monolayer can be modified at any point following formation
of
the core. The monolayer which is adsorbed or chemically bonded to the core
may comprise a number of materials. Examples of these materials include, but
are not limited to, organic compounds (e.g., alkanethiols, arylthiols,
vinylthiols,
195 and their derivatives); inorganic compounds (e.g.,alkyl borates, alkyl
phosphonates, alkyl silicates), organometallic compounds {e.g.,
ferrocenethiol);
biochemical compounds (e.g., cysteine, albumin, coenzyme A), and mixtures
thereof.
In referring to the molecular structure of the monolayer, substituents which
20o may be present on the monolayer include, for example, branched molecules
(e.g., branched alkyl chains, multiply substituted aryl groups, multiply
substituted
cyclic aliphatic compounds), hyperbranched molecules (e.g., dendritic
molecules); and mixtures thereof. As an example of a non-branched substituent,
the monolayer may comprise an alkanethiol or an alkanethiol derivative.
205 Exemplary alkanethiols include those having between 2 and 23 carbon atoms.
As alluded to herein, the monolayer contains at least one reactive
substituent. For the purposes of the invention, the term "reactive
substituent"
refers to those substituents which are chemically active so that, upon
reaction
with a functional material, part of the reactive substituent remains with the
21o product, serving as a linking or coupling group between the original part
of the
molecule and the new portion of the molecule. Examples of reactive
substituents
include, but are not limited to, SH, OH, NH2, NH, C02H, S030H, P02(OH)2,
BO(OH)2, and mixtures thereof. More preferably, OH, NH, C02H, NH2, and
CA 02329859 2000-10-25
WO 99/61911 PCTNS99/07823
8
mixtures thereof are employed. Examples of compounds which may be present
215 on the reactive substituent are described by the general formula:
R~(EH)x
wherein R is selected from the group consisting of an organic compound, an
inorganic compound, an organometallic compound, a biochemical compound,
and mixtures thereof; E is selected from the group consisting of S, O, NH,
COO,
22o S030H, P02(OH)2, BO(OH)2, C02, NH2, and mixtures thereof; n is an integer
ranging from 1 to 5 (more preferably from 1 to 2); and x is an integer ranging
from 1 to 10 (more preferably from 1 to 3). More preferably, E is selected
from O,
NH, CO2, NH2, and mixtures thereof. The chemically modified monolayer may
also include partially reactive and nonreactive compounds or materials.
225 Compounds or materials being "partially reactive" refer to those
containing
groups which do not participate in coupling but instead are capable of
undergoing substitution reactions, elimination reactions, oxidative and
reductive
reactions, and the like. "Nonreactive" compounds or materials refer to those
which do not undergo the above reactions. Reactive, partially reactive, and
23o nonreactive compounds or materials may be used in any ratio so long as at
least
one reactive compound or material is present on the monolayer.
The monolayer is preferably chemically bonded to the core by various
types of bonds. Examples of these bonds include, but are not (united to, core-
element-sulfur bonds, core-element-oxygen-bonds, core-element-boron, core-
235 element-phosphorus, and core-element-nitrogen-bonds. Combinations of these
types of bonds may also be used. In the most preferred embodiment the
monolayer is chemically bonded to the core by various core-element-sulfur
bonds. In the aforementioned embodiments, core-element refers to any elenent
of which the core is composed.
24o In accordance with the invention, the monolayer is coupled to a functional
material such that the monolayer is chemically modified. The term "coupled"
may be interpreted to mean that the monolayer and the functional material are
linked via formation of a new chemical bond. Examples, include, but are not
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
9
limited to amide, thioester, and ester-forming reactions known in the art.
245 Examples of functionalities which may be present on the functional
materials
include, but are not limited to, SH, OH, NH2, NH, C02H, S030H, P02(OH)2,
BO(OH)2, and mixtures thereof. Examples of reactive substituents include, but
are not limited to, SH, OH, NH2, NH, C02H, S030H, P02(OH)2, BO(OH)2, and
mixtures thereof. More preferably, OH; NH, C02H, NH2, and mixtures thereof
25o are employed. The functional material may also comprise at least one
compound having the general formula:
Ft~(EH)X
wherein R is selected from the group consisting of an organic compound,
an inorganic compound, an organometallic compound, a biochemical compound,
255 and mixtures thereof; E is selected from the group consisting of S, O, NH,
COO,
S030H, POZ(OH)Z, BO(OH)2, COZ, NHZ, and mixtures thereof; n is an integer
ranging from 1 to 5 (more preferably from 1 to 2); and x is an integer ranging
from 1 to 10 (more preferably from 1 to 3). More preferably, E is selected
from
O, NH, C02, NH2, and mixtures thereof. It should be emphasized that the
2so functional material may be present in the form of a number of structures
which
possesses functionality in the manner described herein. For example, the
functional material may be a catalyst, a biomaterial, a material which is
electrochemically active, or combinations thereof. The functional material may
also be one which has a low-lying excited state which is capable of undergoing
2s5 fluorescence or electron-transfer when excited. The functional material
may also
be selected such that the manometer-sized particle is soluble in a solvent.
The
term "soluble" may be defined to mean the particles being dispersed or
dissolved
in the solvent. Examples of suitable solvents include aqueous or organic
solvents.
27o In another aspect, the invention relates to a method of making a
functionalized manometer-sized particle. The method comprises providing a
manometer-sized particle comprising a core which comprises at least one metal
or metal alloy, and a monolayer adsorbed onto the core. The monolayer
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
includes at least one reactive substituent. The manometer-sized particle is
then
275 coupled with a functional material such that the monolayer is chemically
modified.
A number of functional materials may be used in the above method.
Examples of these materials include, but are not limited to, spin labels
(e.g., 4-
amino-TEMPO), metal ligands (e.g., 4-(aminomethyl)-pyridine), amino acids
280 (e.g., glutamic acid di-f butyl ester), chromophores and fluorophores
(e.g., 1-
aminopyrene and 2-naphthaleneethanol), ionophores (e.g., 2-(aminomethyl)-15-
crown-5), molecules susceptible to functional group conversion {e.g., benzyl
amine), electroactive molecules (e.g., ferrocene methanol, 10H-(phenothiazine-
10) propionic acid, anthraquinone-2-carboxylic acid), sugars (e.g., a-D-
glucose),
285 nucleotides {e.g., uridine), and mixtures thereof.
The coupling step is preferably carried out in the presence of a reagent
which may be, for example, a phophonium reagent, a facilitating reagent, as
well
as mixtures thereof. Preferably, the facilitating reagent may be selected from
a
base, a catalyst, and mixtures thereof. Preferred bases include various
pyridine
290 derivatives. Other examples of components which may be employed during the
coupling step include, but are not limited to, BOP (benzotriazol-1-
yloxytris{dimethylamino)phosphonium hexafluorophosphate); HOBt (1-
hydroxybenzotriazole); NMM (4-methylmorpholine); DMAP (4-
dimethylaminopyridine); and mixtures thereof.
2s5 In another aspect, the invention relates to a method of analyzing a
manometer-sized particle. The method comprises subjecting a manometer-sized
particle as defined herein to an analytical technique such that the
composition of
the functional materials of the monolayer on the particle are determined. A
number of analytical techniques may be employed in this method. Examples of
30o such techniques include, but are not limited to, NMR spectroscopy,
electrochemical techniques, fluorescent emission spectroscopies, and infrared
spectroscopies.
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
11
Examples
305 The examples which follow are set forth to illustrate the invention, and
are
not meant as a limitation thereon. Although the following examples explicitly
employ only monolayer-protected manometer-sized gold cores, other monolayer-
protected manometer-sized cores (comprising at least one metal or metal alloy
which is selected from the group consisting of a semiconducting material, an
3~0 oxide material, a Group VIIIA element, a Group IB element, a Group IIB
element,
alloys thereof, and mixtures thereof) have reactivity and stability that allow
their
use with the reaction conditions set forth below; thus, one skilled in the art
could
reasonably expect that the examples set forth below would be applicable to all
embodiments set forth in the claims of this patent.
3~5 In the examples, 10H-(phenothiazine-10)propionic acid was synthesized
according procedures set forth in Peek, B.M. et al., Int. J. Peptide Protein
Res.
38 (1991) 114. 11-mercapto-undecanoic acid and 11-mercapto-undecanol were
either synthesized according to a procedure described in Bain, C.D. et al., J.
Am.
Chem. Soc. 111 (1989) 321; or purchased from the Aldrich Chemical Company
320 of Milwaukee, Wisconsin (95% and 97% purity, respectively).
Tetrahydrofuran
(less than 16 ppm water content) was used for all coupling reactions. The
synthesis of manometer-sized gold cores which are stabilized by chemisorbed
layers of dodecanethiolate was accomplished in accordance with a procedure
described Hostetler, M.J., et al., Langmuir 14 (1998) 17-30 and addition of w-
Z-
325 alkanethiol (with Z being COOH or OH) onto the manometer-sized gold cores
was accomplished as described in Hostetler, et al., J. Am. Chem. Soc. 118
(1996} 4212-4213. All other reagents were used as received. Nomenclature for
the manometer-sized gold cores protected by a complex monolayer may be
defined as CX:CYZ (a:b) wherein X specifies the length of the alkanethiol
chain,
33o Y specifies the chain length of the place exchanged w-Z-alkanethiol (with
Z being
COOH or OH) and (a:b) specifies the mole ratio of X and Y chains in the
monolayer on the manometer-sized gold core as determined from the
methyI/CH2R'H NMR spectral ratio in solutions of manometer-sized gold cores
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
12
protected by complex monolayers that have been treated with 12 in order to
335 remove the monolayer from the nanometer-sized gold core as taught in
Templeton, A.C., et al., J. Am. Chem. Soc., 120 (1998) 1906-1911. Percent
conversion data was determined using the aforementioned methodology.
With respect to the spectroscopy data,'H NMR spectra (in Ceps, CD2CI2,
or CDC13) were obtained using a Bruker AMX 200 MHz spectrometer sold by
34o Bruker Instruments, Inc. of Billerica, Massachusetts. A line broadening
factor of
1 Hz was used to improve the S/N of the NMR resonances belonging to the
monolayer on the nanometer-sized gold core. Infrared absorbence spectra of
clusters as thin films were acquired using a Biorad 6000 FTIR spectrometer
sold
by Bio-Rad Laboratories, Inc. of Cambridge, Massachusetts. Reported IR bands
345 are those that are unique to each functionalized monolayer-protected
nanometer-sized gold core (an infrared spectra of a nanometer-sized gold core
which is stabilized by chemisorbed layers of dodecanethiolate was used for
background subtraction).
The examples set forth below employ the following abbreviations for the
35o sake of brevity: THF (tetrahydrofuran); DMF (dimethylformamide); MPC
(monolayer-protected nanometer-sized gold core); BOP (benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate); HOBt (1-
hydroxybenzotriazole); NMM (4-methylmorpholine); DMAP (4-
dimethylaminopyridine); 4-amino-TEMPO (4-amino-2,2,6,6-
355 tetramethylpiperidinyloxy, free radical); IR (infrared); NMR (nuclear
magnetic
resonance); EPR (electron paramagnetic resonance); ml (milliliter); mg
(milligram); ppm (parts per million); cm'' (wavenumbers); d+ (symmetric
methylene stretching vibration); d' (antisymmetric methylene stretching
vibration);
br (broad. All reactions were performed within the temperature range 15 to 35
°
360 C, although one skilled in the art would reasonably assume that the
reactions
would also be functional in the temperature range -80 to 100 °C.
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/078Z3
13
ExamJ~les 1-6
Coupling of Amines to C12:C11COOH (a:b)
3s5 Various coupling reactions were performed according to a procedure set
forth in McCafferty et al., Tetrahedron 51 (1995) 1993. In these examples, ca.
100 mg of acid MPC (C12:C11COOH MPC (4:1)) was treated with five
equivalents {relative to moles of MPC acid groups) of BOP (60 mg), HOBt (18
mg), NMM (14 uL), and DMAP (16 mg) in low water THF {concentration of 2 mg
37o cluster/mL). Following a brief activation period (10 minutes), five
equivalents of a
specified amine was added to the reaction mixture and the solution was stirred
at
room temperature for 15 hours. The solvent was then removed under vacuum
and the reacted MPC was collected on a frit where unreacted materials were
removed by washing with 500 mL of acetonitrile followed by
sonication/decanting
375 with 50 mL acetonitrile (3X).
Example 1
4-amino-TEMPO (spin label)
The above procedure was carried out using 4-amino-TEMPO as the
38o amine. The following data were obtained: IR: 2851 (d+), 2922 (d-), 1641,
1537
crri'. The EPR spectrum is shown in Figure 2. The percent conversion to
number coupled ratio was determined to be 95/13.
Example 2
385 4-(aminomethyl)-pyridine (metal ligand)
The above procedure was carried out using 4-(aminomethyl)-pyridine as
the amine. The following data were obtained; NMR (in CDZC12): 8(ppm) = 0.89
(br, 15.6 H), 1.28 (br, H), 1.74 (br, H), 2.21 (br, 3.1 H), 4.32 (br, 1.9 H),
7.12 (br,
2.3 H), 8.43 (br, 2 H). IR: 2850 (d+), 2920 (d-), 1653, 1602, 1539 cm''. The
3so percent conversion to number coupled ratio was determined to be 95/9.5.
CA 02329859 2000-10-25
WO 99/61911 PCTNS99/07823
14
Example 3
glutamic acid di-t-butyl ester (amino acid)
The above procedure was carried out using glutamic acid di-t butyl ester
395 as the amine. The following data were obtained: NMR (in CgDB): 8 (ppm) =
1.02
(br, 27 H), 1.4 (br, 218 H), 2.3 (br, 28 H), 4.8 (br, 2 H). IR: 2850 (d~),
2920 (d-),
1734, 1680, 1650, 1536, 1392, 1367, 1155 cm'. The percent conversion to
number coupled ratio was determined to be 9014.
400 Example 4
1-aminopyrene (chromophore; fluorophore)
The above procedure was carried out using 1-aminopyrene as the amine.
The following data were obtained: NMR (in CD2CI2): 8 (ppm) = 0.85 (br, 3 H),
1.3
(br, 15 H), 1.85 (br, 1.9 H), 3.35 (br, 0.33 H), 3.55 (br, 0.32 H), 7.15 (br,
0.02 H),
405 7.45 (br, 0.04 H), 7.7 (br, 0.06 H), 8.0 (br, 0.18 H). IR: 2851 (d+), 2921
(d-), 1735,
1700, 1655, 1601, 1558, 1517 cm-'. The percent conversion to number coupled
ratio was determined to be 95/7.
Example 5
41o 2-(aminomethyl)-15-crown-5 (ionophore)
The above procedure was carried out using 2-(aminomethyl)-15-crown-5
as the amine. The following data were obtained: NMR (in C6Dg): 8 (ppm) = 0.75
(br, 0.8 H), 1.0 (br, 3 H), 1.45 (br, 18 H), 2.25 (br, 2 H), 3.6 (br, 3.8 H).
IR: 2949
(d+), 2922 (d'), 1734, 1650, 1543, 1122 cm'. The percent conversion to number
415 coupled ratio was determined to be 80/8.
Example 6
benzyl amine (group conversion)
42o The above procedure was carried out using benzyl amine as the amine.
The following data were obtained: NMR (in CD2C12): 8 (ppm) = 0.90 (br, 31 H),
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
1.3 (br, 141 H), 2.2 (br, 4 H), 4.4 (br, 3 H), 7.3 (br, 5 H). IR: 2850 (d+),
2920 (d'),
1734, 1646, 1547 cm'). The percent conversion to number coupled ratio was
determined to be 95/7.
425
Examples 7-10
Coupling of alcohols to C12:C11COOH (a:b)
In reactions set forth in these examples, ca. 100 mg of the acid MPC
(C12: C11COOH (4:1)) was treated with five equivalents (relative to the moles
of
43o MPC acid groups) of BOP (60 mg), HOBt (18 mg), NMM (14 p.and DMAP (16
mg) in low water THF (conc. of 2 mg cluster/mL). Following a brief activation
period (10 minutes), five equivalents of an alcohol described in these
examples
was added to the reaction mixture and the solution was stirred at room
temperature for 15 hours. The solvent was removed under vacuum and the
435 reacted MPC was collected on a frit where unreacted materials were removed
by
washing with 500 mL of acetonitrile followed by sonication/decanting with 50
mL
of acetonitrile (3X).
Example 7
440 2-naphthalene-ethanol (chromophore)
The above procedure was carried out using 2-naphthalene-ethanol as the
alcohol. The following data were obtained: NMR (in CD2C12): 8 (ppm) = 0.92
(br,
10 H), 1.3 (br, 69 H), 2.25 (br, 2 H), 3.4 (br, 1.1 H), 3.6 (br, 2 H}, 5.9
(br, 0.10 H),
6.5 (br, 0.11 H), 7.15 (br, 0:05 H), 7.4 (br, 0.17 H), 7.8 (br, 0.18 H) IR:
2851 (d'),
445 2922 (d-), 1739, 1653, 1118, 1070 crri'. The percent conversion to number
coupled ratio was determined to be 85/11.5.
Example 8
Ferrocene-methanol (electroactive)
45o The above procedure was carried out using ferrocene-methanol as the
alcohol. The following data were obtained: NMR (in CsDs): b (ppm) = 1.0 (br,
27
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07$23
16
H), 1.5 (br, 226 H), 3.9 (br, 5 H), 4.2 (br, 1.3 H), 4.8 (br, 0.9 H) IR: 2851
(d+},
2922 {d-), 1727, 1680, 1594, 1268, 124418: 2851 (d'), 2922 (d'), 1737, 1710,
1653, 1616 cm''. The percent conversion to number coupled ratio was
455 determined to be 50/5.
Example 9
a-D-glucose (sugar)
The same procedure was employed except that a D-glucose was used
46o and a solvent 5:1 THFIDMF was employed to aid glucose solubility. The
following data were obtained: NMR (in CD2C12): 8 (ppm) = 0.9 (br, 3 H), 1.3
(br,
17 H), 2.9 (br, 0.34 H) IR: 2850 (d+), 2920 (d'), 1815, 1733, 1647, 1612, 840,
781, 769, 740 crri'. The percent conversion to number coupled ratio was
determined to be 35/5.
465
Example 10
Uridine (nucleotide)
The same procedure was employed except uridine was used and a
solvent 5:1 THF/DMF was employed to aid uridine solubility. The following data
47o were obtained: NMR (in CDZC12): 8 (ppm) = 0.9 (br, 3 H), 1.3 (br, 17 H),
2.9 (br,
0.85 H) IR: 2851 (d+), 2921 (d-), 1817, 1733, 1700, 1633, 1616, 1491, 1411,
843,
782, 764, 742 cm~'. The percent conversion to number coupled ratio was
determined to be 60/8.
475 Exameles 11-12
Coupling of carboxylic acids to C12:C110H (a:b)
In these examples, five equivalents (relative to moles of MPC alcohol) of a
carboxylic acid was treated with five equivalents of BOP (60 mg), HOBt (18
mg),
NMM (14~zL), and DMAP (16 mg) in low water THF (conc. of 2 mg cluster/mL).
48o Following a brief activation period (10 minutes), ca. 100 mg of the
alcohol
(C12:C110H (4:1)) was added and the solution was stirred at room temperature
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
17
for 15 hours. The solvent was removed under vacuum and the reacted MPC
was collected on a frit where unreacted materials were removed by washing with
500 mL of acetonitrile followed by sonication/decanting with 50 mL
acetonitrile
485 (3X).
Example 11
10H-(phenothiazine-10)propionic acid (electroactive)
The above procedure was carried out using 10H-(phenothiazine-
4so 10)propionic acid as the acid. The following data were obtained: NMR (in
CD2C12): s (ppm) - 0.9 (br, 17 H), 1.3 (br, 150 H), 2.75 (br, 0.2 H), 4.0 (br,
2 H),
4.1 (br, 2 H), 6.8 (br, 5.9 H), 7.1 (br, 5.9 H) IR: 2851 ,(d'), 2921 (d'),
1817, 1733,
1700, 1633, 1616, 1491, 1411, 843, 782, 764, 742 crri'. The electrochemistry
of
this MPC derivative is shown in Figure 3a. The percent conversion to number
495 coupled ratio was determined to be 65/7.4.
Example 12
Anthraquinone-2-carboxylic acid (electroactive; chromophore)
50o The above procedure was carried out using anthraquinone-2-carboxylic
acid as the acid. The following data were obtained: NMR (in CD2Clz): 8 (ppm) _
0.88 (br, 17 H), 1.3 (br, 119 H), 1.8 (br, 14 H), 3.55 (br, 1.8 H), 4.3 (br, 2
H), 7.8
(br, 1.8), 8.3 br, 3.4 H), 8.8 (br, 0.93 H) IR: 2851 (d~), 2922 (d-), 1727,
1680,
1594, 1268, 1244 cm-'. The percent conversion to number coupled ratio was
505 determined to be 75/7.5.
Example 13
Coupling of BOC-phenylalanine
Five molar equivalents of BOC-phenylalanine (168 mg) were activated (10
5~o minutes) by treatment with five equivalents of BOP (280 mg), HOBt (87 mg),
NMM (128 pL), and DMAP (77 mg) in THF (conc. of 2 mg cluster/mL). 200 mg
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
18
of 3:1 C12:C1110H was added to the above and the solution was stirred at room
temperature for 15 hours. The solvent was removed under vacuum and the
reacted MPC was collected on a frit where unreacted materials were removed by
5~5 washing with 500 mL of acetonitrile followed by sonicationldecanting with
50 mL
acetonitrile (3X). The following data were obtained: NMR {in CgDe): 8 (ppm) _
0.88 (br, 12.8 H), 1.35 (br, 48 H), 3.05 (br, 1.75 H), 3.55 (br, 2.2 H), 4.1
(br, 1.7
H), 4.5 (br, 0.3 H), 7.2 {d, 5 H).
520
Example 14
Deprotection of BOC-Phe-terminated MPC.
To a 30 ml solution of 1.0 g of BOC-Phe-terminated MPC (synthesized in
Example 14) in CH2C12 was added 7.5 ml of trifluoroacetic acid. The reaction
525 was stirred at room temperature for 2 hours after which time the reaction
mixture
was diluted with 100 ml of distilled H20. The organic phase was separated, and
the aqueous phase was further washed with 100 ml of CH2C12. The combined
organic phases were washed with 2 x 100 ml of 10% NaHC03 and then removed
in vacuo. The precipitate was then washed with copious quantities of
acetonitrile
53o and was air dried. The following data were collected: NMR (in CD2C12): 8
(ppm)
= 0.88 {br, 26 H), 1.35 (br, 192 H), 7.2 (d, 5 H).
Example 15
Coupling of BOC-alanine
535 BOC-alanine was coupled to the C-terminus of phenylalanine as
described in Examples 2-8 (fve molar excess of reagents). The following data
were obtained; NMR (in CsDs): b (ppm) = 0.85 (br, 13.6 H), 1.35 (br, 115 H),
3.05
(br, 2.6 H), 4.1 {br, 2.5 H), 4.7 (br, 0.6 H), 7.2 (d, 5 H).
CA 02329859 2000-10-25
WO 99/61911 PCT/US99/07823
19
Examale 16
Deprotection of BOC-Ala-Phe-terminated MPC
BOC-alanine was deprotected as described in Example 15. The following
data were obtained: NMR (in CD2C12): 8 (ppm) = 0.85 (br, 16.8 H), 1.35 (br,
156
H), 3.05 (br, 3.4 H), 4.1 (br, 1.9 H), 4.7 (br, 0.7 H), 7.2 (d, 5 H) 7.65 (br,
0.12 H).
545
Examale 17
Coupling of BOC-isoleucine
BOC-isoleucine was coupled to the C-terminus of alanine as described in
Examples 2-8 above (five molar excess of reagents). The following data were
550 obtained: NMR (in CsDs): b (ppm) = 0.85 (br, 18 H), 1.35 (br, 147 H), 3.05
(br,
2.6 H), 4.1 (br, 2.4 H), 4.7 (br, 0.02 H), 7.2 (d, 5 H). IR: 3063, 3029, 2848
(d'),
2917 (d'), 1738, 1718, 1687, 1644, 1513, 1497, 1164 cm''.
Examale 18
555 Deprotection of BOC-Ile-Ala-Phe-terminated MPC
BOC-isoleucine was deprotected as described in Example 15. The
following data were obtained: NMR (in CD2C12): 8 (ppm) = 0.85 (br, 16.8 H),
1.35
(br, 156 H), 3.05 (br, 3.4 H), 4.1 (br, 1.9 H), 4.7 (br, 0.7 H), 7.2 (d, 5 H)
7.65 (br,
0.12 H).
560
Examale 19
Analysis of W-Carboxylic Acid-Alkanethiolate
Functionalized MPCs using 12 Decomposition
5s5 Approximately 50 mg of the c~-carboxylic acid-alkanethiolate
functionalized MPC was dissolved in dichloromethane and stirred with
approximately 3 mg of iodine for one hour. Following disulfide formation,
which
was monitored by a change in solution color from dark brown to clear violet,
the
insoluble brown residue (actual identity of insoluble materials not
identified) was
57o removed and the sample rotovapped to dryness. The NMR of the 12-
CA 02329859 2000-10-25
WO 99/61911 ~ PCT/US99/07823
decomposed C12:C11COOH (4:1) (in CDC13) was: S (ppm) = 0.85 (t, 5.5 H),
1.25 (m, 35.4 H), 1.66 (m, 5.3 H), 2.35 (t,1 H), 2.65 (t,4 H).
Example 20
575 Electrochemical Measurement: Cyclic Voltammetry
Cyclic voltammetry (seen in Figure 2) was performed with a BAS 1008
electrochemical analyzer sold by Bioanalytical Systems, Inc., located in West
Lafayette, Indiana. A platinum 3 mm diameter working electrode was polished
with 0.5 p.m diamond (Buehler) paste followed by rinsing with water, ethanol,
and
5so acetone prior to each experiment. A Pt coil counter electrode and
saturated
calomel reference electrode resided in the same cell compartment as the
working electrode. Solutions (2:1 toluene/acetonitrile as disclosed in Ingram
et
al., J.Am.Chem.Soc. 1996, 51, 1093} of the phenothiazine-MPC (0.8 mM in
phenothiazine) and of phenothiazine monomer (1 mM) were degassed and then
585 bathed throughout with solvent-saturated N2.
Example 21
Electrochemical Measurement: Thin-layer coulometry
Thin-layer coulometry (as seen in Figure 2b) was performed using a BAS
590 1008 electrochemical analyzer. A 4.3 mm diameter Pt working electrode was
polished with 0.5 pm of diamond (Buehler) paste followed by rinsing with
water,
ethanol, and acetone and toluene prior to each experiment. A Pt wire counter
electrode and Ag wire quasi-reference electrode (AgQRE) resided in a locally
designed thin-layer cell as defined in Reilley, C.N., Pure Appl. Chem. 18
(1968)
595 137. A Mitutoyo digital micrometer (1-2", 0.00005" resolution) sold by
Mitutoyo
Corporation of Japan was fitted to the cell and used to define the thin-layer
cell
thickness, L. Charge-time measurements were performed for cell thicknesses of
2-30 pm, and Q values of zero-time intercepts extrapolated from the longer
time
plateaus (20-32 s) of each charge-time plot. The product of the number of
60o phenothiazines per cluster, 0, and of electrons per phenothiazine, n, is
obtained
CA 02329859 2000-10-25
WO 99/61911 PCTNS99/07823
21
from the slope of a Q versus L plot as shown in Figure 3b (Q/L = nFAC = 11.47
X
10-3 C/cm, C = 1.07x10'' mole/cm3).
In the drawings, specification, and examples there have been disclosed
typical preferred embodiments of the invention and, although specific terms
are
soy employed, they are used in a generic and descriptive sense only and not
for the
purposes of limitation, the scope of the invention being set forth in the
following
claims.
