Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE
MODIFICATION OF NUCLEIC ACIDS
Related Applications
This application claims the benefit under 35 U.S.C. ~ 119(e) of United States
provisional application 60/378,184, filed May 6, 2002, the disclosure of which
is
incorporated herein by reference.
Field of the Invention
to This invention relates to methods and compositions for the modification of
nucleic
acids.
Sack~round of the Invention
Following traumatic injury (or during surgery), an organism may require a
blood
15 transfusion to prevent death due to blood loss. In humans and certain
domesticated animals,
blood transfusion has enabled the survival of injured individuals who would
otherwise have
died from blood loss.
Whole blood is composed of many different types of proteins and cells. Blood
proteins include antibodies, complement proteins, and proteins involved in the
blood clotting
2o cascade. In addition, each of the different types of blood cells plays a
unique role in
maintaining the health of the organism. Red blood cells, for instance, are
essential for the
transport of oxygen and carbon dioxide gases to and from the cells of a
multicellular
organism. Another type of blood cell, a platelet, is involved in initiating
blood clotting;
thrombocytopeiiia patients have a platelet deficiency and are prone to
bleeding disorders.
25 It is desirable to identify agents capable of inactivating microorganisms
that are found
in biological compositions. Fox blood transfusions, there is the danger of
transmitting blood-
borne viruses from donor blood to a recipient. The transmission of viral
diseases (e.g.,
hepatitis A, B, and C, acquired immunodeficiency syndrome, and cytomegalovirus
infections) by blood or blood products is a significant problem in medicine.
Screening donor
3o blood for viral markers can help reduce the transmission of viruses to
recipients, but many
screening methods are directed to only a few discrete viruses and are
therefore incomplete or
Iess than 100% sensitive. Bacterial infections also pose a significant risk
for transfusion
recipients. Storage conditions may allow contaminating bacteria to multiply,
causing sepsis
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in the transfusion recipients. Ensuring safe products produced from or
including biological
compositions is rendered difficult because any effective treatment should
inactivate a broad
spectmm of cell-free and cell-associated viruses and bacteria without inducing
toxicity or
carcinogenicity.
Furthermore, the manufacture of maximally safe and effective lcilled vaccines
for
human or veterinary use requires methods that completely and reliably render
live
microorganisms, e.g., viruses, bacteria and parasites, noninfectious.
Summary of the Invention
In general, the invention features methods and compositions for the
modification of
nucleic acids. In particular embodiments, methods and compositions are useful
for
inactivating microorganisms, such as viruses, bacteria, or parasites, in
biological
compositions for in vitro or ih vivos use.
Accordingly, in a first aspect, the invention features a method for modifying
nucleic
acid molecules in a biological composition. This method includes the step of
contacting the
biological composition with an aziridino inactivating compound that has the
formula (I):
N (Cf-12)3-5~ N~R~)2
(I)
2o or a salt thereof, wherein each Rl is, independently, selected from the
group consisting of H,
C2_~ all~enyl, phenyl, and benzyl, wherein the contacting is performed under
conditions and
for a period of time sufficient to modify at least some nucleic acid molecules
in the biological
composition. In particular embodiments, the compound is 1-aziridinepropanamine
or 1-
aziridinebutanamine (compounds 1 and 2, respectively):
CN NHS
(1)
NH2
CN
(2).
In a second, related aspect, the invention features another method for
modifying
3o nucleic acid molecules in a biological composition, this method including
the step of
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contacting the biological composition with an aziridino inactivating compound
having the
formula:
~N WR1)2)(2-5).-N~R1)2
(II)
or a salt thereof, wherein each Rl is, independently, selected from the group
consisting of H,
C1_4 alkyl, C2_4 allcenyl, phenyl, and benzyl, provided that at least one Rl
is phenyl or benzyl,
wherein contacting is performed under conditions and for a period of time
sufficient to
modify at least some of the nucleic acid molecules in the biological
composition.
to Exemplary aziridino inactivating compounds that fall within formula (II)
are 3-
phenyl-1-aziridinepropanamine, N,N-dibenzyl-1-aziridineethanamine, and N-
benzyl-N-ethyl-
1-aziridineethanamine, and 2-benzyl-1-aziridineethanamine (compounds 3, 4, 5,
and 6,
respectively).
,r, NH2
(3)
CN ~/ ~N
I,
i5
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CN~N~
~ c5,
C
(6)
In a third aspect, the invention features still another method for modifying
nucleic
acid molecules in a biological composition, this method including the step of
contacting the
biological composition with an aziridino inactivating compound having the
formula:
\N OH2)~z-s> N~R~) OH2O2-s) N
(
or a salt thereof, wherein Rl is selected from the group consisting of H, Cl_4
allfyl, C2_~
alkenyl, phenyl, and benzyl, wherein contacting is performed under conditions
and for a
period of time suff cient to modify at least some of the nucleic acid
molecules in the
biological composition.
Exemplary compounds that satisfy formula (III) are 1,1'-
[iminobis(dimethylene)]bis
aziridine and 1,1'-[iminobis(trimethylene)]bis aziridine (compounds 8 and 9,
respectively).
CNN--~-N~
I
H (g)
H
C N ~~/~/ N ~~/~/ N ~ (9)
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In a fourth aspect, the invention features still another method for modifying
nucleic
acid molecules in a biological composition, this method including the step of
contacting the
biological composition with an aziridino inactivating compound having the
formula:
R3
~N-R~
R2 (IV)
or a salt thereof, wherein Rl is a Cl_4 alkyl and RZ and R3 is each,
independently, H or a Cl~.
alkyl and wherein contacting is performed under conditions and for a period of
time sufficient
to modify at least some of the nucleic acid molecules in the biological
composition. An
exemplary compound of formula (IV) is:
~N~ (10)
to In a fifth aspect, the invention features still another method for
modifying nucleic acid
molecules in a biological composition, this method including the step of
contacting the
biological composition with one of the following aziridino inactivating
compounds:
NHS
CN
NHz (11)
NH2
CN
is NHS (12)
or a salt thereof, wherein contacting is performed under conditions and for a
period of time
sufficient to modify at least some of the nucleic acid molecules in the
biological composition.
In a sixth aspect, the invention features yet another method for modifying
nucleic acid
molecules in a biological composition, this method including the step of
contacting the
2o biological composition with a aziridino inactivating compound having the
formula:
CN-(CH~)~3_51 NH-(CH~)~3_5j NH2 (V)
or a salt thereof, wherein contacting is performed under conditions and for a
period of time
sufficient to modify at least some of the nucleic acid molecules in the
biological composition.
An exemplary compound of formula (V) is:
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H
N N
NH2 (13)
The aziridino ring of the compounds of the invention can be substituted with a
structure X-CH2-CHZ-N-, wherein X is -Cl, -Br, -F, -I, -O-S(=O)2-CH3,
-O-S(=O)2-CH2-C~HS, or -O-S(=O)2-C~H4-CH3. For example, substituted forms of
compounds of formula (I) may have the following formula:
X-CH2-CHZ-N-(CH2)(3-s>-N(Rl)a (VI)
wherein X is -Cl, -Br, -F, -I, -O-S(=O)2-CH3, -O-S(=O)2-CHZ-C6H5, or -O-S(=O)2-
C6H4-CH3,
to each Rl is, independently, selected from the group consisting of H,
CZ_4 alkenyl, phenyl, and benzyl. When X is Cl, Br, F, or I, these compounds
are often
referred to as nitrogen mustards. Nitrogen mustards are strong electrophiles
and alkylate
nucleophilic groups of nucleic bases either directly or through intermediate
conversion into
the respective aziridines. In one embodiment, these substituted compounds may
be employed
as aziridino inactivating compounds to modify nucleic acids and/or inactivate
microorganisms in biological compositions.
The inactivating compounds of the present invention are protonated (i.e.,
positively
charged) on one or more nitrogens at physiological pH. For example, protonated
compounds
of formula (I) (II), and (III) have the following respective formulas:
CN (CH~)~3_5~ NH(R~)2 X_
(VII)
CN (C(R~)~)~2-s)-NH(R~)2 X
(VIII)
N (CH2)~2_5~ NH(R~) (CH2)(2-s)-N\ I X
~~IIC
wherein each R1 is, independently, selected from the group consisting of H,
CZ_4 alkenyl,
phenyl, and benzyl, and X is a pharmaceutically acceptable counter-ion (e.g.,
sulfate, nitrate,
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halide, tosylate, phosphate, and the like). For compounds within formula
(VIII) or (IX), R~
can also be CI_4 allcyl. Compounds falling within formula (VIII) also have at
least one R~ that
is phenyl or benzyl.
These protonated forms of the compounds, described herein, (also referred to
as
"salts"), and their use in the methods of the invention, are specifically
included as being part
of the invention.
The compounds of the invention described herein also include isomers such as
diastereomers and enantiomers, mixtures of isomers, including racemic
mixtures, solvates,
and polymorphs thereof.
to In one embodiment of the first, second, third, fourth, fifth, or sixth
aspect, the
modification of the nucleic acid molecules or microorganism inactivation is
achieved while
the non-nucleic acid components e.g., cells or biopolymers, such as proteins,
carbohydrates
or lipids, also present in the biological composition, are not substantially
modified in their
functionality. In a particularly preferred embodiment of the first second,
third, fourth, fifth,
15 or sixth aspect, the modification of the nucleic acid molecules is achieved
while preserving
the structure and function of non-nucleic acid components, e.g. cells or
biopolymers, such as
proteins, also present in the biological composition. The extent of this
biopolymer
modification can be determined by means known in the art including, for
example, isoelectric
focusing, polyacrylamide gel electrophoresis, HPLC, and other forms of
chromatography
20 with detection by autoradiography or a suitable method. Modification of
cellular function, as
used herein, would be an effect on the metabolic or mechanical integrity of
the cellular
product. Modif cation of cellular function can be determined by monitoring,
using known
methods, metabolic parameters, including the ability of cells to generate
substrates used for
the maintenance of cell function, such as ATP. Mechanical integrity, as used
herein, relates
25 to the maintenance of the deformability of the cell membrane structure. A
decrease in
mechanical integrity will result in an increased propensity for cell lysis
(e.g., hemolysis for
red blood cells) which is detectable using l~nown methods. Substantial changes
in cellular
function, as used herein, will diminish the therapeutic utility of the
cellular product. For
example in the case of red cells, a substantial change would result in less
than 75% of blood
3o cells remaining in circulation 24 hours after infttsion.
In still another embodiment of the first, second, third, fourth, fifth, or
sixth aspect, the
nucleic acid molecules that are modified are those of a microorganism,
resulting in the
inactivation of microorganism (i.e., the inability of the microorganism to
replicate).
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Microorganisms that are inactivated according to the methods of the invention
include, for
example, viruses, bacteria, or parasites that are cell-contained or cell-free.
Viruses inactivated by the methods and compounds of the invention include DNA
and
RNA viruses or viroids. Viruses inactivated by the methods of the invention
include both
enveloped or non-enveloped viruses. In a preferred embodiment, the viruses to
be inactivated
are infectious vertebrate vintses. In a particularly preferred embodiment the
viruses to be
inactivated are infectious human viruses. Exemplary viruses include:
poxviruses, herpes
viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses,
picornaviruses,
rotaviruses, alphaviruses, rubiviruses, influenza virus e.g., including type A
and B,
flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses,
rhabdoviruses,
lyssaviruses, orthmyxoviruses, bunyaviruses, phlebovimses, nairoviruses,
hepadnaviruses,
hepatitis A virus, hepatitis B virus, hepatitis C virus, arenaviruses,
retroviruses including
human immunodeficiency virus, enteroviruses, rhinoviruses and/or the
filoviruses. TTV is an
additional exemplary virus.
Viruses inactivated by the compositions and methods of the invention can be
included
in killed vaccines.
The methods and compounds of the invention can be used to inactivate
infectious
bacteria including, but not limited to Gram-negative or Gram-positive
bacteria. Exemplary
Gram-positive bacteria inactivated by the compounds and methods of the
invention include,
but are not limited to Pasteurella species, Staplz~lococci species, and
Streptococcus species.
Gram-negative bacteria inactivated by the compounds and methods of the
invention include,
but are not limited to, Esclzez°ichia coli, Pseudoznonas species, and
,Salmonella species.
Specific examples of infectious bacteria inactivated by the compounds and
methods of the
invention include but are not limited to: Helicobacter pylori, Borellia
burgdorferi, Legionella
pzzeuzyzophila, Mycobacteria species (e.g., M. tuberculosis, M. aviuzzz, M.
intracellulare, M.
kazzsaii, M. gordonae), Bacillus cereus, Staphylococcus aureus, Staphylococcus
epiderznidis,
Neisseria gozzorrhoeae, Neissez°ia menizzgitidis, Listeria
nzozzocytogenes, Sty°eptococcus
pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B
Streptococcus),
Streptococcus viridans (viridans group), Streptococczzs faecalis,
Streptococcus bovis,
Streptococcus (anaerobic species), Sty°eptococcus pneu3zaoniae,
pathogenic Canzpylobacter
species, Enterococcus species, Haemoplzilus izzfluezzzae, Bacillus
azztlzracis, Cozynebactez°ium
diphtlzeriae, Cozynebacterium diphtlzeroides, Ezysipelothz°ix
rlzusiopathiae, Clostridium
perfringens, Clostz°idiuzn tetazzi, Enterobacter aerogenes,
Entez°obacter cloacae, Klebsiella
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pneunaoniae, Pasteurella naultocida, Bacter~oides species, Fusobacterium
rrucleatunr,
Streptobacillus rrconiliforrnis, Tr~eporaerna pallidiuna, Ti~eponema
per~tenue, Leptospir~a species,
Riclcettsia species, Actinomyces israelli, Esclzer°ichia coli,
Serr°atia mar-cescens, Serratia
liquefacieras, PYOpionibacter°iurn aches, Asper~gillus ter~r~us,
Salnaonella typhinauriurrr,
Salmonella choler~iasuis, Yersinia eater°ocolitica, Pseudomonas
aeruginosa, and
PsezcdorrZOnas putida. Additional exemplary bacteria are Bar°toraella
spp., Tropherynra
whippelii, Mycoplasrna, e.g. Mycoplasma pneumoniae and Ghlamy~lophila, e.g.
Clalanrydoplzila praeumoniae.
Bacteria inactivated by the compositions and methods of the invention can be
io included in killed vaccines.
Examples of parasites inactivated by the methods and compositions of the
invention
are as follow: blood-borne and/or tissues parasites such as Plasrnodiuna,
Babesia micnoti,
Babesia divergerzs, Leishrnarria tr°opica, Leislrmania, Leislunania
br~azilierrsis, Leishmania
donovarri, Tiypanosoma ganabiense and Tryparaosoma r°laodesien,re
(African sleeping
15 sickness), Trypanoson2a cf uzi (Chagas' disease), and Toxoplasma gondii.
Parasites inactivated by the compositions and methods of the invention can be
included in killed vaccines.
The compositions and methods of the invention reduce the number of infectious
microorganisms in the inactivating compound contacted biological composition
relative to
2o the number of microorganisms in biological composition prior to the
inactivation step. In
some embodiments, the number of infectious microorganisms in the inactivating
compound
contacted biological composition is reduced at least 1 log, preferably at
least 2 logs,
preferably at least 3 logs, or more preferably at least 4 logs relative to the
number of
microorganisms in biological composition prior to the inactivation step. In
preferred
25 embodiments, the number of infectious microorganisms in the inactivating
compound
contacted biological composition is reduced at least 5 logs, more preferably
at least 6 logs,
more preferably at least 7 logs, more preferably at least 8 logs, and more
preferably at least 9
logs relative to the number of microorganisms in the biological composition
prior to the
inactivation step. It is possible that the number of infectious microorganisms
in the
3o inactivating compound contacted biological composition is reduced at least
10, 11, 12, 13, 14,
or even 15 logs.
In some embodiments of the of the first, second, third, fourth, fifth, or
sixth aspect,
the biological composition is blood, a red blood cell comprising composition,
a red blood cell
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concentrate, a platelet concentrate, blood plasma, a platelet-rich plasma, a
placental extract, a
cell culture product or culture medium, a product of fermentation, or an
ascites fluid. In
another embodiment, the biological composition is serum, a blood cell protein,
a blood
plasma concentrate, a blood plasma protein fraction, a purified or partially
purified blood
protein or other component, a supernatant or a precipitate from any
fractionation of the
plasma, a purified or partially purif ed blood component (e.g., proteins or
lipids), colostrum,
milk, urine, saliva, a cell lysate, cryoprecipitate, cryosupernatant, or
portion or derivative
thereof, compositions containing proteins induced in blood cells, and
compositions
containing products produced in cell culture by normal or transformed cells
(e.g., via
1o recombinant DNA or monoclonal antibody technology). In preferred
embodiments of the
invention, the biological compositions are derived from vertebrates. In other
preferred
embodiments the biological compositions are derived from mammals. In
additional preferred
embodiments of the invention, the biological compositions are derived from
humans. In
particularly preferred embodiments of the invention, the biological
composition is a purified
15 human enucleated cell composition, particularly a purified red blood cell
composition.
If desired, the method of the first, second, third, fourth, fifth, or sixth
aspect can also
include the step of transfusing the inactivating compound-contacted biological
composition
into a mammal such as a human. At least some of said inactivating compound may
be
removed prior to transfusion, e.g., by washing or solid phase removal. W
addition to (or
20 instead of) removing the inactivating compound, the inactivating compound
can be quenched
with a quenching agent after the contacting step. The quenching agent can be
soluble or
bound to a solid support which may subsequently be removed.
Alternatively, the inactivated microorganisms produced by one of the foregoing
methods may be harvested and used as an immunogen for the purpose of
vaccinating an
25 aiumal (e.g., a human) against that microorganism. Accordingly, the method
of the first,
second, third, fourth, fifth, or sixth aspect can also be considered a method
for making a
vaccine or an imlnunogen for administration to a human or other animal.
Compositions
containing inactivated microorganisms, optionally including one or more
adjuvants, also are
provided. In some embodiments, the compositions are vaccine compositions.
3o In a seventh aspect, the invention features a purified compound having the
formula:
~C~R1 )2)(2-5>-N~R1 )2
(II)
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wherein each RI is, independently, selected from the group consisting of H,
C1_4 alkyl, C2_4
alkenyl, phenyl, and benzyl, provided that at least one Rl is phenyl or
benzyl. Inactivating
compounds that fall within formula (II) include, for example, 3-phenyl-1-
aziridinepropanamine, N,N-dibenzyl-1-aziridineethanamine, and N-benzyl-N-ethyl-
1-
aziridineethanamine, and 2-benzyl-1-aziridineethanamine (compounds 3, 4, 5,
and 6,
respectively), depicted above.
In an eighth aspect, the invention features a purified compound selected from:
CN~N~N
I
H (g)~
NH2
CN
1o NHS (11), and
NH2
CN
NH2 (12).
The compounds and methods of this invention can be used to inactivate
15 microorganisms including viruses, bacteria or parasites, particularly blood-
transmitted
viruses, bacteria or parasites, in cell- or protein-containing compositions in
various contexts,
e.g., in the hospital, laboratory, as part of a kit. Since cell compositions
also include a variety
of proteins, the method of microorganism inactivation described herein is also
applicable to
protein fractions such as blood plasma protein fractions or purified blood
products, including,
2o but not limited to, fractions containing clotting factors (such as factor
VIII and factor IX),
serum albumin or immunoglobulins. The microorganism inactivation may be
accomplished
by treating a protein fraction or purified protein with a compound of the
invention, as
described herein.
In some embodiments, more than one inactivating aziridino compound may be
25 contacted with the biological composition. In some embodiments, the methods
and
compositions of the invention can be combined with still other modes of
inactivating viruses.
For example, the compounds and methods of the present invention can be used in
the
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Solvent/Detergent procedure described by Budowslcy et aL, U.S. Patent No.
6,369,048,
incorporated herein by reference. In another example, certain processes used
in the
preparation of medical products (e.g., chromatography in buffers of low pH, or
storage of red
blood cells in acidic solutions containing calcium chelating agents) may have
incidental viral
inactivating properties for selected, sensitive viruses, usually enveloped
viruses.
In another embodiment, a blood product, decontaminated by the methods and
compositions of the invention, is provided.
By "purified" is meant a preparation or composition that contains, by volume
or
weight, at least 50%, more preferably, at least 70%, more preferably at least
85%, even more
to preferably at least 95%, and most preferably, at least 98% of the indicated
component. For
example, a purified preparation of red blood cells contains at least 50% by
volume red blood
cells, while purified 3-phenyl-1-aziridinepropanamine is at least 50% by
weight 3-phenyl-1-
aziridinepropanamine.
By "nucleic acid" is meant both DNA and RNA, both single and double stranded.
15 By "modifying," "modification," or "modify," when referring to nucleic
acids, means
to substantially eliminate the template activity of DNA or RNA, for example,
by destroying
the ability to replicate, or to transcribe or translate a message. For
example, the inhibition of
translation of an RNA molecule can be determined by measuring the amount of
protein
encoded by a definitive amount of RNA produced in a suitable iTZ vitro or ifZ
vivo translation
2o system.
By "inactivating," "inactivation," or "inactivated," when referring to a
microorganism, means eliminating its ability to propagate. When referring to
bacteria or
parasites, the term means reducing the number of living bacteria or parasites
capable of
replicating or reproducing. When referring to viruses, the term means
diminishing or
25 eliminating the number of infectious viral particles measured as a decrease
in the infectious
titer or number of infectious virus particles per milliliter. Such a decrease
in infectious virus
particles is determined by assays well known to a person of ordinary skill in
the art.
"Microorganism inactivating conditions" refers to the conditions under which
microorganisms incubated with a compound of this invention are inactivated.
Variables
3o include, for example, time of treatment, pH, temperature, salt composition,
ionic strength and
concentration of the inactivating compound so as to inactivate the
microorganisms to the
desired extent.
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By "inactivate at least some of the microorganisms" is meant that the number
of
infectious microorganisms is reduced at least 1 log, preferably at least 2
logs, preferably at
least 3 logs, or more preferably at least 4 logs relative to the number of
microorganisms in
biological composition prior to the inactivation step. In preferred
embodiments, the number
of infectious microorganisms in the inactivating compound contacted biological
composition
is reduced at least 5 logs, more preferably at least 6 logs, more preferably
at least 7 logs,
more preferably at least 8 logs, and still more preferably at least 9 logs
relative to the number
of microorganisms in the biological composition prior to the inactivation
step. It is possible
that the number of infectious microorganisms in the inactivating compound
contacted
biological composition is reduced at least 10, 1 l, 12, 13, 14, or even 1S
logs.
Similarly, by "modify at least some of the nucleic acid molecules" is meant
that at
least 50% of the nucleic acid molecules in the treated biological composition
are modified,
preferably at least 70%, preferably at least 80%, still more preferably at
least 90%, still more
preferably at least 95%, still more preferably at least 99% of the nucleic
acid molecules in
the treated biological composition are modified. Methods to detect nucleic
acid modification
include the detection of covalent chemical adducts using either mass
spectrometry or
radiolabeled aziridino compounds. An alternative method to detect nucleic acid
modification
is to employ molecular science techniques of PCR in which the nucleic acid
molecule is
evaluated for its ability to be replicated by polymerase enzymes. The presence
of nucleic
2o acid damage can result in the loss of ability of the nucleic acid molecule
to be replicated if the
appropriate primers are employed.
By "biological composition" is meant a composition containing cells or
proteins.
Cell-containing compositions include, for example, blood, red blood cell
concentrates,
platelet concentrates, blood plasma, platelet-rich plasma, placental extracts,
cell culture
product or culture medium, products of fermentation, and ascites fluid.
Biological
compositions may also be cell-free. Protein-containing biological compositions
include, for
example, serum, blood cell proteins, blood plasma concentrate, blood plasma
protein
fractions, purified or partially purif ed blood proteins or other components,
a supernatant or a
precipitate from any fractionation of the plasma, purified or partially
purified blood
3o components (e.g., proteins or lipids), colostrum, milk, urine, saliva, a
cell lysate,
cryoprecipitate, cryosupernatant, or portion or derivative thereof,
compositions containing
proteins induced in blood cells, and compositions containing products produced
in cell
culture by normal or transformed cells (e.g., via recombinant DNA or
monoclonal antibody
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technology). In preferred embodiments of the invention, the biological
compositions are
derived from vertebrates. In other preferred embodiments the biological
compositions are
derived from mammals. Tn additional preferred embodiments of the invention,
the biological
compositions are derived from humans. In particularly preferred embodiments of
the
invention, the biological compositions are purified human enucleated cells,
particularly
purified red blood cells.
By an "enucleated cell" is meant a cell which, when mature, lacks a nucleus.
Examples of enucleated cells are platelets and red blood cells.
By a "solution that does not quench an inactivating compound" is meant a
solution
to that does not contain a quenching agent (e.g., a thiophosphate or a
thiosulfate). A quenching
agent, when contacted with a compound, renders the contacted inactivating
compound
unreactive. Exemplary solutions that do not react with a compound of the
invention axe
unbuffered saline and water.
By a "quenching agent" is meant a compound that when contacted with a compound
15 of the invention, is capable of rendering the contacted compound
unreactive. Exemplary
quenching agents are thiophosphate or a thiosulfate, or a compound containing
a
thiophosphate or a thiosulfate.
Other features and advantages of the invention will be apparent from the
following
description.
Detailed Description of the Invention
The invention features compositions and methods to modify nucleic acids in
biological compositions by contacting the composition with an inactivating
aziridino
compound. For example, the methods and compositions of the invention can be
used to
modify nucleic acid molecules of microorganisms in a biological composition,
resulting in
microorganism inactivation, by contacting the composition with an inactivating
aziridino
compound of the invention, under microorganism inactivating conditions. An
inactivating
aziridino compounds of the invention may have one of the following four
formulas:
~CH2~(3-5) N~R1~2
(I)
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U~R1 )2~(2-5>-N~R1 )2
(II)
N (CH2)~z.5~ N~R~) tCH2)t2-s)
(III)
CN-(CH~)~3_51 NH-(CH2)~3_5j NH2 (V)
wherein each RI is, independently, selected from the group consisting of H,
C2_4 alkenyl,
phenyl, and benzyl. For compounds within formula (II) or (III), Rl can also be
Cl_4 alkyl.
Compounds falling within formula (II) have at least one Rl that is phenyl or
benzyl.
An aziridino inactivating compound of the invention may also have the
following
structures:
R3
~N-R~
R2 (~)
1o wherein Rl is a Cl_4 alkyl and RZ and R3 is each, independently, H or a
C1_~ alkyl.
Additionally, aziridino inactivating compounds of the invention may have one
of the
following structures:
NHS NH2
CN CN
NH2 (11) and NH2 (12).
1s
Nucleic acid molecules, including nucleic acid molecules in microorganisms,
can be
modified by contacting the composition comprising the nucleic acid or
microorganism
comprising nucleic acid with about 0.00001 to about 0.250 M, preferably about
0.0001 to
about 0.015 M of an inactivating aziridino compound of the invention in a
solution having an
2o ionic strength of about 0.01 M to about 0.5 M, at a pH of about 4.5 to 8.5,
preferably about
6.0 to 8.0, preferably about 6.5 to about 7.5, at a temperature of about
4°C to about 60°C,
preferably 4°C to about 30°C, for a time sufficient to modify
the nucleic acid to the desired
extent. The reaction time range from about 1 minute to about 500 hours and can
be, for
example, about 1 minute, about 1 hour, about six hours, about twelve hours,
about 24 hours,
25 about forty-eight hours or about one hLUZdred and forty-eight hours. The
salts used can be
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any of those normally used in biochemical applications, including sodium,
potassium,
acetate, and so on. The practitioner can adjust the pH of the solution using
many buffers
customarily used in the art to handle biopolymers or cells, such as acetate,
HEPES, MOPS,
and so forth.
The practitioner can also adjust factors such as concentration ofthe
reactants,
temperature, and time of incubation. It should be Dept in mind, however, that
reaction rate,
the ionic strength of the reaction solution, temperature and concentration of
aziridino
inactivating compound are interdependent. In general, increasing the
concentration of the
aziridino inactivating compound, reaction temperature and/or r educing the
ionic strength
to results in a shorter reaction time to achieve the desired extent of nucleic
acid modification or
microorganism inactivation. Accordingly, increasing the reaction temperature
allows the
practitioner to decrease the concentration of aziridino inactivating compound,
reaction time
and/or to increase the ionic strength to achieve the desired degree of nucleic
acid
modification or microorganism inactivation. Similarly, increasing the
concentration of
15 aziridino inactivating compound allows the practitioner to reduce the
reaction temperature,
reaction time and/or increase the ionic strength to achieve the desired degree
of nucleic acid
modification or microorganism inactivation. Similarly, increasing the ionic
strength of the
reaction solution results in a longer reaction time, increased concentration
of aziridino
inactivating compound or increased temperature to achieve the desired degree
of nucleic acid
20 modification or microorgansm inactivation.
Aziridino inactivating compounds of the invention are suitable, for example,
for use
in the treatment of a red blood cell preparation in order to inactivate
microorganisms (e.g.,
viruses, bacteria, and/or parasites) contaminating the red blood cell
preparation, while leaving
the red blood cells suitable for use in a therapeutic setting (e.g., for use
in transfusion).
25 Prior to introduction of the treated red blood cells into the recipient
animal, it may be
desirable to remove the aziridino compound from the cells or reduce the
concentration of the
aziridino compound.
One method to remove aziridino compound from a cell containing biological
composition, e.g., treated red blood cell preparation, is to subject the cells
to repeated
3o washings with an appropriate washing solution as described in co-pending
U.S. patent
application serial number 10/055,143, which is herein incorporated by
reference in its
entirety.
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In addition to, or instead of, washing to remove aziridino inactivating
compounds,
aziridino inactivating compounds can be removed from biologic mixtures using
standard
laboratory methodologies including dialysis, diafiltration, size exclusion
chromatography,
affinity chromatography and also by the use of resins which have an affinity
for aziridino
compounds due to either electrostatic or covalent chemical reactions. The
resins can be
presented to the biologic mixture in the free form or as part of a filter
matrix.
Further, in addition to, or instead of washing to remove aziridino
inactivating
compounds, it may be desirable to employ an agent that quenches the reactivity
of the
aziridino inactivating compounds. Optionally, the quenching agent and quenched
1o inactivating compound may be removed from a biological composition by
bonding the
quenching agent to a nucleophile, e.g., a thiosulfate or thiophosphate group,
to a second
moiety (the separation moiety) which supplies particular properties to the
quenching agent,
such that the quenching agent can be completely and reliably separated, along
with the
quenched inactivating compound, from the biological composition. These
modified
15 quenching compounds can react with and quench electrophiles such as the
inactivating
compounds described herein. Methods of quenching are described in co-pending
application
U.S.S.N. 09/260,375 and U.S. Pat. Ser. No. 6,150,109 which are herein
incorporated by
reference in there entirety.
This method has the added advantage that it is compatible with methods to
remove
2o solvent and detergent from protein-containing preparations that are virally-
inactivated by the
Solvent/Detergent procedure described by Budowsky et al., U.S. Patent No.
6,369,04,
incorporated above by reference in its entirety.
The SolventlDetergent method of virus activation is compatible with the
inactivation
of microorganisms by compounds such as those described herein. Thus, one can
perform two
25 methods of inactivation in sequence or simultaneously. Alternatively, the
inactivation of
microorganisms through the use of the methods of the invention, followed by
quenching and
removal of the quenching agent and inactivating compound using the methods
described
herein, can be performed without the use of the Solvent/Detergent method. It
is also
advantageous that the quenching agent be easily detectable in order to monitor
its removal.
3o This can be fulfilled, for example, with the addition of thymidine, which
is readily detected
by its absorbance of 260 nm light.
The thiophosphate groups used in the invention may be substituted, for
example, with
one substituent (e.g., [separation moiety]-OP(=S)(OH)2, also referred to as a
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tluophosphomonoester), substituted with two substituents (e.g., [separation
moiety]-
OP(=S)(OH)(OAIk), a thiophosphodiester), or substituted with three
substituents (e.g.,
[separation moiety]-OP(=S)(OAllc)2, a phosphothiotriester). The substituent
may be, for
example, a linear, branched, or cyclic saturated or unsaturated hydrocarbon
with one to forty
carbons, a benzyl group, a polycyclic aromatic group, an unsubstituted alkyl
group, or an
alkyl group substituted with hydroxyl, amino, azido, or cyano groups.
Polythiophosphate moieties (i.e., moieties having two or more adjacent
phosphate
groups) can also be used in the invention. For example, guanosine diphosphate
(GDP) or
guanosine triphosphate (GTP), in which one or more of the phosphate groups is
a
to thiophosphate group, may be used in the invention. In the case of guanosine
diphosphate,
one or both phosphate groups may be thiophosphate groups. In the case of
guanosine
triphosphate, one, two, or all three of the phosphate groups may be
thiophosphate groups.
GDP or GTP may be attached to the separation moiety, for example, at the 2' or
the 3'
hydroxyl group or to the heterocyclic base.
The quenching systems of the invention can be used as follows. An inactivating
compound, such as 1-aziridinepropanamine, is added to a biological
composition, under
microorganism inactivating conditions as described herein. At the end of the
time necessary
for microorganism inactivation, the biological composition is contacted with
quenching
agent, e.g., a compound containing one or more thiosulfate or thiophosphate
moieties
2o attached to a separation moiety. The biological composition and the
quenching agent are
allowed to remain in contact for the desired time. An excess of thiosulfate or
thiophosphate
groups per equivalent of inactivating compound is generally used.
The thiosulfate or thiophosphate moieties react with the highly reactive
moieties of
the inactivating compounds or their haloderivative salts, and become
covalently linked to
these compounds. When the coupled thiosulfate or thiophosphate moieties are
removed from
the biological composition, therefore, the quenched inactivating compounds are
removed as
well. The end result is a biological composition that is substantially free of
infectious
microorganisms (e.g., viruses), quenched inactivating compounds, and quenching
agent.
Killed vaccines can be made by contacting a virus or other microorganism with
an
inactivating compound under microorganism-inactivating conditions. The
microorganism-
inactivating conditions may be selected from the methods described herein. In
one example,
virus at a titer of about 10~ to 108 units per mL is incubated with
inactivating agent at about
pH 6.5 to about pH 7.5, in a solution having an ionic strength of less than
about 0.5 M at
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about 4°C to about 40°C. The time of treatment (i.e., the end
point of inactivation) depends
on the structure and composition of the particular virus, temperature of
incubation, ionic
strength, and the number of protonizable or positively charged groups in the
inactivating
agents. However, kinetic studies indicate that depending on pH and the virus
to be
inactivated, incubation time could be as little as a few seconds, and also can
be about 1 hour,
5 hours, 50 hours, 100 hours 300 hours or 500 hours. The killed virus can be
used directly in
vaccine formulations, or lyophilized in individual or multiple dose containers
for subsequent
mixture with the pharmaceutically acceptable carrier. Methods of preparing
vaccines are well
known in the art.
l0 The vaccines of this invention are useful in the prevention of animal or
human
disease. Vaccines capable of conferring the desired degree of immunity will,
of course,
contain an amount of inactivated microorganism effective to evoke an immune
response. In
the preparation of killed vaccines, the sample of microorganism is incubated
with the
aziridino inactivating agents of this invention in amounts and under such
conditions to
inactivate the microorganism wlule retaining immunogenicity.
The vaccine can be administered in or with an adjuvant, i.e., a substance that
potentiates an immune response when used in conjunction with an antigen. The
vaccine can
be given in an immunization dose. An immunization dose is an amount of an
antigen or
immunogen needed to produce or enhance an immune response. The amount will
vary with
the animal and irnmunogen or antigen or adjuvant but will generally be less
than about 1000
~.g per dose. The immunization dose is easily determined by methods well known
to those
skilled in the art, such as by conducting statistically valid host animal
immunization and
challenge studies.
The particular dosage of the vaccine to be administered to a subject will
depend on a
variety of considerations including the nature of the rnicroorganisrn, the
schedule of
administration, the age and physical characteristics of the subject, and so
forth. Proper
dosages may be established using clinical approaches familiar to the medicinal
arts.
Examples
Example 1: Synthesis of 1-aziridinepropanamine
1-Aziridinepropanamine was prepared as follows.
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CN
CNH
~N~CN
Scheme 1
As is shown in Scheme 1, aziridine (19 mL; 0.37 moles) was added dropwise to
20 mL (0.3
moles) acrylonitrile over 1.5 hr. The addition was exothermic. The temperature
was
maintained below 40°C during the addition. The reaction mixture was
stirred at room
temperature overnight, then distilled in a vigreux distillation apparatus
under reduced
pressure. This process resulted in 27.1 g of a clear colorless liquid, 1-
aziridinepropanenitrile
(b.p. 56-57°C/3mm), 99.5% by GC. Thin layer chromatography (TLC; 10%
MeOH: CHC13)
to shows a small amount of more polar impurities. NMR is consistent with the
structure. The
yield was 92%.
HZ ; Raney Ni ; NH3
CN~CN ~ N~NH2
Scheme 2
Referring to Scheme 2, 1-aziridinepropanenitrile (27.1g; 0.2 moles) was
dissolved in 60 mL
methanol; to this was added 4.8 g Raney 2800 nicl~el. The reaction was run in
a Parr 4562
Mini reactor under 1500 psi HZ at 50°C for ~15 hr. The reaction mixture
was cooled,
filtered through celite, then washed with methanol. The filtrate was distilled
in a vigreux
distillation apparatus under reduced pressure. The following fractions were
collected: (#1)
100 mL clear colorless liquid (b.p. 21-40°C/183mm); (#2) 4.2 g clear
colorless liquid (b.p.
32°C/3mm); (#3) 4.6 g clear colorless liquid (b.p. 32°C/3mm);
(#4) 9.0 g clear colorless
liquid (b.p. 32-36°Cl3mm). The pot residue consisted of 5.6 g of a
cloudy green liquid.
Fractions 2-4 were combined and redistilled in a vigreux distillation
apparatus under
reduced pressure, resulting in the following fractions: (#1) 0.2 g clear
colorless liquid (b.p.
22°C/108mm 99.8% methanol by GC); (#2) 0.1 g clear colorless liquid
(b.p. 22-75°C/107mm
96.6% by GC); (#3) 1.2 g clear colorless liquid (b.p. 22-37°C/6 mm
97.5% by GC); (#4) 14.7
g clear colorless liquid (b.p. 29-37°C/6 mm 97.1% by GC). The pot
residue was 0.5 g of a
clear yellow liquid. NMR analysis of fraction #4 is consistent with the
structure of
1-aziridinepropanamine (52% yield).
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Example 2: Synthesis of 1-aziridinebutanamine
1-Aziridinebutanamine was prepared as follows.
Br ~ CN; I~ZCO3
CN~~CN
Scheme 3
Refernng to Scheme 3, 4.84 g (0.035 moles) potassium carbonate (milled,
anhydrous) was
slurried in 14.5 mL (0.28 moles) aziridine. To this mixture was quickly added
4.72 g (0.032
moles) 4-bromobutyronitrile. After ~30 seconds, the reaction mixture
exothermed. The
reaction mixture was chilled in an ice bath; during which time the reaction
refluxed on its
own for an additional 5 min. The reaction mixture was then stirred at room
temperature
to overnight, then distilled in a vigreux distillation apparatus under reduced
pressure. 3.1 g
crude distillate (b.p. 63-73°C/6 mm) was chromatographed on 35.4g
silica gel (70-200 mesh)
eluting with CHC13 then 10% MeOH:CHC13. The appropriate fractions were
concentrated on
a rotary evaporator under reduced pressure (6 mm). The residue was then
lcugelrohr distilled
wider reduced pressure, resulting in 1.3g 1-aziridinebutanenitrile, a clear
colorless liquid (b.p.
25-80°C/6mm; 37% yield). TLC (9/1) (7:1:1:1M40H) (7:1:1:1=nBuOH: HBO:
EtOH:
NH40H) shows one spot. Purity is 99.1 % by GC. NMR is consistent with the
structure.
LiAlH4
CN~~CN ~ N z
NH
Scheme 4
Referring to Scheme 4, 1.10 g (0.027 moles) lithium aluminmn hydride was
weighed
2o into the flask under argon. Ether (54 mL) was added and the mixture chilled
in an ice bath.
3.0 g (0.027 moles) 1-aziridinebutanenitrile was dissolved in 7 mL ether and
added dropwise
over 15 min. The addition was exothermic, with HZ given off. This mixture was
stirred in an
ice bath. After 10 min, added were 1 mL deionized water, 1 mL 20% NaOH, and 2
mL
deionized water. The reaction mixture was placed in the refrigerator for 2 hr,
then filtered.
The solid was washed with 100 mL ether, and the filtrate distilled first under
argon, then
under reduced pressure. The atmospheric pressure distillation was done in a
vigreux
distillation apparatus. The reduced pressure distillation was done in a
kugelrohr apparatus.
1.6 g clear colorless liquid (b.p. 25-90°C/6 mm) was obtained. TLC
(9/1) (7:1:1:1/NH40H)
(7:1:1:1=nBuOH: H20: EtOH: NH40H) shows a trace of more and less polar
impurities.
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98.6% by GC. NMR is consistent with the structure of 1-aziridinebutanamine,
with 50%
yield and impurities in the 1-3% range.
Example 3: Synthesis of N-Benzyl-N-ethyl-1-aziridineethanamine
N-Benzy-N-ethyl-1-aziridineethanamine was prepared as follows.
\ ~Br
H
HO~N~ HO~N~
CH3CN ; KZC03
Scheme 5
Referring to Scheme 5, potassium carbonate (31.0 g; 0.22 moles),
l0 2-(ethylamino)ethanol (17.83 g; 0.2 moles), and 100 mL acetonitrile were
slurried together.
Benzyl bromide (26 mL; 0.22 moles) was dissolved in 50 mL acetonitrile and
added to the
slurry dropwise over 1 hr. The addition was exothermic. This mixture was
stirred at room
temperature for 4 hr, then filtered. The filtrate was concentrated on a rotary
evaporator under
reduced pressure (6 mm). The reaction mixture was distilled through a vigreux
distillation
15 apparatus under high vacuum, resulting in the following fractions: (#1)
1.7g clear colorless
liquid (b.p. 20-75°C/0.16 mm), 97% by GC; (#2) 22.4 g clear colorless
liquid (b.p. 75-
80°C/0.15 mm), 99% by GC; (#3) 4.9 g clear colorless liquid (b.p. 74-
79°C/0.15 mm), 98%
by GC; pot residue 8.0 g yellow waxy oil. Fractions 2 and 3 were combined to
give 27.8 g
clear colorless liquid, N-benzyl-2-(ethylamino)ethanol. TLC (20% MeOH: EtOAc)
shows
20 one spot. NMR is consistent with the structure. 77% yield.
\ NEt3; CHZCL~; MsCI O \
I I
HO~N~ ~S~~O~N~
O
Scheme 6
Referring to Scheme 6, N-benzyl-2-(ethylamino)ethanol (5.38 g; 0.03 moles) was
dissolved
and 8.4 mL (0.06 moles) triethylamine in 100 mL dichloromethane. The reaction
mixture
25 was chilled in an ice bath for 0.5 hr. Methanesulfonyl chloride (4.7 mL;
0.06 moles) was
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dissolved in 11 mL dichloromethane and added dropwise to the reaction mixture
over ~10
min. The reaction mixture was allowed to warm to room temperature over 4 hr,
then poured
on a column of 101.4 g silica gel (60-200 mesh) and eluted with hexanes,
followed by 10%
EtOAc:hexanes. The appropriate fractions were concentrated on the rotary
evaporator under
reduced pressure (6 mm). The residue was then kugelrohr distilled under high
vacuum to
yield 3.1 g clear colorless liquid, 1-methsnesulfonyloxy-3-aza-3-benzylpentane
(b.p. 25-
90°C/0.03 mm). TLC (10% EtOAc:hexanes) shows one spot. 40% yield.
O KZCO3; CH3CN; HN
/S\w0~/Nw/ ~ CN~/Nw/
O
Scheme 7
to Referring to Scheme 7, aziridine (1.3 mL; 0.025 moles), potassium carbonate
(3.49 g; 0.025
moles), and 91 mL acetonitrile were mixed together and chilled in an ice bath
for 0.5 hr.
1-Methanesulfonyl-3-aza-3-benzylpentane (3.1 g; 0.012 moles) was dissolved in
13 mL
acetonitrile and added dropwise over 15 min, stirred at room temperature
overnight, and
refluxed for 3 hr. The reaction mixture was filtered and the filtrate
concentrated on a rotary
15 evaporator under reduced pressure (6 mm). The residue was chromatographed
on 60.3 g
silica gel (60-200 mesh) that had been saturated with triethyl amine, and
eluted serially with
EtOAc and 40% MeOH:EtOAc. The appropriate fractions were concentrated, and the
residue
was kugelrohr distilled under high vacuum to yield 1.0 g clear colorless
liquid N-benzyl-N-
ethyl-1-aziridineethanamine (b.p. 25-88°C/0.05 mm). TLC (20%
MeOH:EtOAc) shows a
20 trace of a less polar impurity, 99.4% by GC. NMR is consistent with the
structure. 40%
yield.
Example 4: Synthesis of N,N-Dibenzyl-1-aziridineethanamine
N,N-Dibenzyl-1-aziridineethanamine was prepared as follows.
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Br
I~ZC03; CH3CN; \
N
HO ~NHZ ' HO ~
/
Scheme 8
Referring now to Scheme 8, potassium carbonate (60.06 g; 0.43 moles),
monoethanol amine
(12.22 g; 0.2 moles), and 150 mL acetonitrile were mixed together. The mixture
was chilled
in an ice bath for 0.5 hr. Benzyl bromide (50 mL; 0.42 moles) was dissolved in
50 mL
acetonitrile and added dropwise over 20 min. The reaction mixture was then
stirred at room
temperature for 4 hr, then filtered. The filtrate was concentrated on a rotary
evaporator under
reduced pressure (6 mm). The residue was distilled through a vigreux
distillation apparatus
under high vacuum, resulting in the following fractions: (#1) 6.8 g clear
colorless liquid (b.p.
l0 22-27°C/0.1 mm), 97% benzyl bromide by GC; (#2) 1.4 g clear yellow
liquid (b.p. 22-
123°C/0.06 mm), 94% by GC; (#3) 10.6 g yellow oil (b.p. 123-
133°C/0.06 mm), 98% by GC;
(#4) 29.9 g yellow oil (b.p. 123-133°C/0.06 mm), 98% by GC; pot residue
2.5 g orange oil.
Fractions #3 and #4 were combined, representing 40.5 g N,N-dibenzylethanol
amine. TLC
(20% EtOAc:hexanes) shows a trace of a less polar impurity. NMR is consistent
with the
15 structure. Oil slowly crystallized (m.p. 41-44°C; 83% yield).
NEt3; CHZCIz; MsCl
HON ~ O
/S~~O~N
/ O
\ ~ /
Scheme 10
Referring to Scheme 10, N,N-dibenzylethanolamine (9.65 g; 0.04 moles) and
triethylamine
(6.1 mL; 0.044 moles) were dissolved in 90 mL dichloromethane. To this mixture
was
2o quickly added methanesulfonyl chloride (3.4 mL; 0.044 moles) dissolved in
10 mL
dichloromethane. The addition was exothermic. The reaction was stirred at room
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temperature for 4 hr. The reaction mixture was then chromatographed on 122.4 g
silica gel
(60-200 mesh), eluting with hexanes then 20% EtOAc:hexanes. The appropriate
fractions
were concentrated on a rotary evaporator to yield 4.8 g clear colorless liquid
(2-[bis(phenylmethyl) amino]ethanol methane sulfonate). TLC (10%
EtOAc:hexanes)
showed a small amount of more polar impurities. 37% yield.
/ I /
O v a
S~ N I~ZC03; CH3CN; HN
/ \O O~/ ~ CN~/N
Scheme 11
Refernng now to Scheme 11, aziridine (2.3 mL; 0.044 moles) and potassium
carbonate (6.23
g; 0.045 moles) were mixed together in 90 mL acetonitrile and chilled in ice
bath for 25 min.
l0 2-[bis(phenylmethyl)amino]ethanol methane sulfonate (4.8 g; 0.015 moles)
were dissolved in
mL acetonitrile and added dropwise over 5 min. The resulting mixture was
refluxed for 4
hr, then cooled and filtered. The filtrate was concentrated on a rotary
evaporator under
reduced pressure (6 mm), and the residue chromatographed on 99.5 g silica gel
(60-200
mesh) saturated with triethylamine, then eluted in series with hexanes, 40%
EtOAc:hexanes,
and EtOAc. The appropriate fractions were concentrated, and this residue
kugelrohr distilled
under high vacuum to yield 1.6g clear colorless liquid (N,N-dibenzyl-1-
aziridineethanamine;
b.p. 25-165°C/0.11 mm). TLC (EtOAc) shows a small amount of more and
less polar
impurities, 94% by GC. NMR is consistent with the structure. 40% yield.
2o Example 5: Synthesis of 3-phenyl-1-aziridinepropanamine
3-phenyl-1-aziridinepropanamine was prepared as follows.
N
CN NEt3; DBU; HN~ ~ CN
Scheme 12
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Referring to Scheme 12, a mixture of 10.3 g (0.079 moles) cinnamonitrile, 24.1
g (0.56
moles) aziridine, 5.5 mL (0.39 moles) triethylalnine, and 3.0 g (0.02 moles)
DBU was
refluxed for 76 hr. The reaction mixture was concentrated on a rotary
evaporator. The
residue was chromatographed on silica gel eluting with hexanes, then 50%
EtOAc:hexanes.
The appropriate fractions were concentrated to yield 13.4 g 3-phenyl-1-
aziridinepropanenitrile as an oil. 98% yield.
N
N
CN Raney Ni; Hz; EtOH/NH3
\ ~ ~ \ ~ z
Scheme 13
l0 Referring now to Scheme 13, 3-phenyl-1-aziridinepropanenitrile (3.0 g; 0.17
moles) was
dissolved in 100 mL, 10% ammonia in ethanol. Raney nickel (0.35 g) was then
added, and
the mixture then reduced under 50 psi HZ in a Parr shaker. After 26 hr, the
reaction mixture
was filtered through celite and the filtrate concentrated on the rotary
evaporator. The residue
was chromatographed on silica gel eluting in series with 1:1 MeOH: CH2C12,
MeOH, and 2:1
15 MeOH: triethylamine, and the appropriate fractions were then concentrated
to yield 1.98 g
3-phenyl-1-aziridinepropanamine. NMR is consistent with the structure. 64%
yield.
Example 6: Synthesis of N-[3-(1-aziridinyl)propyl]-1,4-butanediamine
N-[3-(1-aziridinyl)propyl]-1,4-butanediamine was prepared as follows.
~CHO HzN H
~ Ether ~ ~N~CHO MeOH ; NaBH~ ~N~N~~z
Scheme 14
Referring to Scheme 14, 8.2 mL (0.16 moles) aziridine was dissolved in 55 mL
ether
and chilled in an ice bath for 20 min. Ten milliliters (0.15 moles) acrolein
was dissolved in
35 mL ether and added dropwise over 25 min. The reaction mixture was stirred
in an ice bath
for an additional 15 min, then allowed to warm to room temperature and stir
for 2 hr. The
reaction mixture was concentrated on the rotary evaporator under reduced
pressure (6mm) to
give 18.1 g clear colorless oil. TLC shows more and less polar impurities.
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The crude aldehyde was split into four aliquots and reacted in the following
manner.
1,4-diaminobutane (1.4 g; 16.2 mmoles) was dissolved in 40 mL methanol. Cmde
aldehyde
(3.2g; 32rnmoles) was dissolved in 30 mL methanol and added dropwise over 15
min. The
reaction mixture was stirred at room temperature for l5min, and chilled in an
ice bath. To
this chilled mixture was added 2.6g (68 mmoles) sodium borohydride portionwise
over 15
min. The reaction mixture was removed from the ice bath and stirred at room
temperature for
4 hr. To the reaction mixture was then added 6N methanolic HCl dropwise until
H2 was no
longer given off. The reaction mixture was concentrated on the rotary
evaporator under
reduced pressure (6 mm). The residue was dissolved in 40 mL diH20, then made
basic (~pH
l0 12) with 40%NaOH. The solution was extracted with 3 x 120 mL ether, the
combined
organics were dried over Na2SO4 and filtered, and the filtrate concentrated.
The residue was
kugelrohr distilled under high vacuum. The distillate from each of the four
batches was
combined, then distilled through a vigreux distillation apparatus under high
vacuum to yield
1.0 g clear colorless liquid, N-[3-(1-aziridinyl)propyl]-1,4-butanediamine,
b.p. 58-
61 °C/0.06mm (95% by GC). TLC shows less polar impurities. NMR is
consistent with the
structure. 2% yield.
Example 7: Synthesis of 1-ethylaziridine
1-ethylaziridine was prepared as follows.
O
N HZSOø ' H20 S N NaOH ; H20 N
HO~ ~ HO~ \\~O~
O
Scheme 15
Refernng to Scheme 15, a cold solution of 23mL (0.41 moles) concentrated
sulfuric
acid in 80 mL water was added in portions to 40 g ( 0.45 moles) 2-
(ethylamino)ethanol in
80mL water. The water was then distilled under atmospheric pressure until the
temperature
of the reaction mixture reached 115°C. The reaction mixture was then
subjected to distillation
under reduced pressure (~l5mm) while heating. The bath temperature was raised
to 175°C,
and the vacuum applied until the reaction mixture solidified, ~ lhr. A
solution of 56.0 g
(0.85 moles) potassium hydroxide in 60 mL water was added to dissolve the
mixture. The
reaction mixture was heated to a bath temperature of 130°C to distill
off a mixture of water
and desired product. The fraction boiling at 94-97°C was collected and
treated with 50 g
KOH and chilled in an ice bath. The two layers were separated, and the organic
layer dried
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over KOH, filtered, and distilled at atmospheric pressure two times to give
13.8 g clear
colorless liquid, 1-ethylaziridine, by 47°C. NMR is consistent with the
structure. 43% yield.
Example 8: Synthesis of 1,1'-[iminobis(trimethylene)]bis aziridine
1,1'-[iminobis(trimethylene)]bis aziridine was prepared as follows.
CNH RCN CND Rh/C ; MeOH ; HZ CN~N~N
CN
Scheme 16
Referring to Scheme 16, 19 mL (0.37 moles) aziridine was added dropwise to 20
mL
(0.3 moles) acrylonitrile over 1.5 hr. The addition was exothermic. The
temperature was
l0 maintained below 40°C during the addition. The reaction mixture was
stirred at room
temperature overnight, then distilled in a vigreux distillation apparatus
under reduced
pressure to yield 27.1 g clear colorless liquid, 1-aziridinepropanenitrile
(b.p. 56-57°C/3 mm;
99.5% by GC). TLC (10% MeOH: CHC13) shows a small amount of more polar
impurities.
NMR is consistent with the structure. 92% yield.
15 1-aziridinepropanenitrile (19.23 g; 0.2 moles), 1.63g 5% rhodium on carbon,
and 250
mL methanol were mixed in a Parr 4562 Mini-reactor. The mixture was stirred
and heated
under 1000 psi HZ at 30°C for 45 hr. The mixture was filtered and the
filtrate concentrated on
the rotary evaporator under reduced pressure (6 mm). The residue was distilled
in a vigreux
distillation apparatus under reduced pressure twice to yield 4.3 g clear
colorless liquid, 1,1'-
20 [iminobis(trimethylene)]bis aziridine (b.p: 20-76°C/0.02 mm). NMR is
consistent with the
structure. 96.4% by GC. TLC [9/1 7:1:1:l/NH40H (7:1:1:1=
nBuOH:EtOH:H20:NH40H)]
shows a small amount of more polar impurities. 23% yield.
Example 9: Inactivation of Esclzericl2ia coli by 1-aziridinepropanamine
25 The ability of 1-aziridinepropanamine to inactivate E. coli is demonstrated
in the
following assay. E. coli stationary phase culture prepared by overnight
growing in LB media
at 37°C is spiked to a concentration of 10~ to 10' cfu/ml in low ionic
strength medium (LISM:
4.3% Dextrose (239 mM), 12.5 mM Na-phosphate pH 7.1-7.2, osmolality: 285-290
mOsm).
The E. coli culture is divided into four 10 mL aliquots in 50 mL culture
flasks. A
30 1-aziridinepropanamine 20x stock solution is prepared immediately before
use by addition of
a specified quantity of the compound, prepared as described above, to a 0.32 M
NaHZPO4
solution. The freshly prepared 1-aziridinepropanamine 20x stock solution (pH
7.5 to 7.6) is
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kept on ice until it is added to two flasks, to a final concentration of 12
mM. One control
flask, having no 1-aziridinepropanamine, and a flask having 12 mM 1-
aziridinepropanamine
are incubated at 37°C for ten hours. Another control flask, having no
1-aziridinepropanamine, and a flask having 12 mM 1-aziridinepropanamine are
incubated at
22°C for ten hours. One milliliter aliquots are taken sterilely from
the control and treated
flasks every two hours, serially diluted with phosphate buffered saline (PBS)
and are plated
on LB agar plates. The plates are incubated for 48 hrs at 37°C and
bacterial colonies are
counted. The limit of detection is 10° cfu/mL.
The presence 12 mM 1-aziridinepropanamine in low ionic strength medium at
37°C
to resulted in the complete loss of E. coli cells after two hours wlule the
control had a E. coli
cell concentration of about 10~ cfulml. A similar result was obtained when the
cells were
cultured at 22°C, differing in that the time required to inactivate all
of the E. coli cells
increased from two hours to eight hours.
Example 10: Inactivation of Yersinia e~zterocolitica by 1-aziridinepropanamine
and 1-
aziridinebutanamine
Each of 1-aziridinepropanamine and 1-aziridinebutamamine inactivated Yersizzia
(Y.)
efztez-ocolitica 0:20 cells izz vitYO. This was demonstrated by the following
method.
Yez:sizzia etzte>~ocolitica stationary phase culture, prepared by overnight
growing in LB
2o media at 37°C is spiked to a final concentration of 106 to 10~
cfu/ml in low ionic strength
medium (LISM: 4.3% Dextrose (239 mM), 12.5 mM Na-phosphate pH 7.1-7.2,
osmolality:
285-290 mOsm). The Yezsizzia ezzterocolitica stationary phase culture is
divided into three 10
mL aliquots in 50 mL culture flasks. 1-aziridinepropanamine, prepared as
described above
and freshly diluted in 0.32 M NaH2P04, is added as a 20x stock (pH 7.5 to 7.6)
to a final
concentration of 12 mM to one of the flasks. 1-aziridinebutanamine, prepared
as described
above and freshly diluted in 0.38 M NaH2P04, is added to a final concentration
of 12 mM to
another flasks. A control flask, having no 1-aziridinepropanamine or 1-
aziridinebutanamine,
a tube having 12 mM 1-aziridinepropanamine and a flask having 12 mM 1-
aziridinebutanamine are incubated at 22°C for six hours. 1 mL aliquots
are taken sterilely
3o from the control and treated flaslcs every two hours and are plated on LB
agar plates. The
plates are incubated for 2 days at 37°C and bacterial colonies are
counted. The limit of
detection is 10° cfu/mL.
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The presence 12 mM 1-aziridinepropanamine in low ionic strength medium at
22°C
resulted in the complete inactivation of Y. errteYOCOlitica cells after two
hours while the
positive control had a concentration of about 10' cfu/ml at the two hour time
point.
1-aziridinebutamamine reduced the number of Y. eTZterocoliticcz cfu by about 7
logs, after six
hours compared to the positive control which had a concentration of about 109
cfu/ml at the
6 hour time point.
Example 11: Inactivation of porcine parvo virus by 1-aziridinepropanamine.
The ability of 1-aziridinepropanamine to inactivate porcine parvo virus
("PPV") is
to demonstrated in the following assay.
1-aziridinepropanamine is prepared as described above. A 20x stoclc solution
is
prepared by diluting 1-aziridinepropanamine into 0.32 M NaHZP04 (final pH of
20x stock
7.5-7.6), and kept on ice, immediately prior to use.
PPV is prepared according to conventional procedures including purification
and
15 determination of infectivity and stability. Aliquots of the viral stock are
added to tubes of
thawed fresh frozen plasma ("FP") to result in a concentration of 106 - 10~
TCIDSO/ml.
Aliquots of the stock 1-aziridinepropanamine solution are added, to a final
concentration of 6
mM, to the tubes of PPV in FP , except that no 1-aziridinepropanamine is added
to the
control tubes.
2o The tubes are incubated at 22°C and aliquots are taken sterilely
from each reaction
tube and control at 1 hour, 3 hours, and 6 hours. The aliquots are serially
diluted in a
microtiter plate for a total of 8 microtiter wells. 25 ~1 of each dilution is
incubated for 6 days
with PT-1 cells (porcine testicle cells) in microtiter plates. The wells are
checked for a
cytopathic effect. The limit of detection is > 5 TCmso/mL.
25 The presence of 6 mM 1-aziridinepropanamine in FP, incubated at 22°C
for 1 hour, 3
hours and 6 hours, resulted in less than a 2 log, more than a 2 log, and about
a 3 log PPV
reduction, respectively, compared to the positive control at the same time
point.
Example 12: Inactivation of porcine parvo virus by 1,1'-
[iminobis(trimethylene)]bis
3o aziridine.
The ability of 1,l'-[iminobis(trimethylene)]bis aziridine to inactivate PPV is
demonstrated in the following assay.
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1,1'-[iminobis(trimethylene)]bis aziridine is prepared as described above and
a 20x
stoclc solution prepared immediately prior to us by diluting 1,1'-
[iminobis(trimethylene)]bis
aziuidine into 0.5 M NaH2P04 (final pH of 20x stock 7.4), and kept on ice. PPV
is prepared
according to conventional procedures including purification and determination
of infectivity
and stability. Aliquots of the viral stock are added to tubes of thawed fresh
frozen plasma
("FP") and red blood cell concentrate ("RBCC") to result in a concentration of
10~
TCIDSO/mL. Aliquots of the 1,1'-[iminobis(trimethylene)]bis aziridine 20x
stock solution are
added, to a final concentration of 12 mM, to the tubes of PPV in FP and RBCC
except that
no 1,1'-[iminobis(trimethylene)]bis aziridine is added to the control tubes.
l0 The tubes are incubated at 22°C and aliquots are taken sterilely
from each reaction
tube and control at 1 hour, 3 hours, and 6 hours. The aliquots are serially
diluted in a
microtiter plate for a total of 8 microtiter wells. Twenty-five microliters of
each dilution is
incubated for 6 days with PT-1 cells (described above) in microtiter plates.
The wells are
checked for a cytopathic effect. The limit of detection is > 5 TCIDSO/ml.
The presence 12 mM 1,l'-[iminobis(trimethylene)]bis aziridine in FP, incubated
at
22°C for 1 hour, 3 hours and 6 hours, resulted in less than a log, more
than a log, and a 2 log
PPV reduction, respectively, compared to the positive controls. The presence
12 mM
1,1'-[iminobis(trimethylene)]bis aziridine in RBCC, incubated at 22°C
for 1 hour, 3 hours
and 6 hours, resulted in more than a log, more than a 3 log, and more than a 3
log PPV
2o reduction, respectively, compared to the positive controls.
Example 13 : Inactivation of encephalomyocarditis virus by 1-ethylaziridine
The ability 1-ethylaziridine to inactivate encephalomyocarditis virus ("EMCV")
is
demonstrated in the following assay.
1-ethylaziridine is prepared as described above. A 20x stock solution is
prepared by
diluting 1-ethylaziridine in 0.18 M NaHZP04 (final pH of 20x stock 7.1-7.3),
and kept on ice,
immediately prior to use.
EMCV is prepared according to conventional procedures including purification
and
determination of infectivity and stability. An aliquot of the viral stock is
added to a tube of
12.5 mM sodium phosphate, pH 7.2, to arrive at a concentration of 10~ - 10~
TCIDSO/mL. An
aliquot of the stock 1-ethylaziridine is added, to a final concentration of 12
mM, to the tube
of EMCV and sodium phosphate. No 1-ethylaziridine is added to the control
tube.
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The tubes are incubated at 37°C and aliquots are taken sterilely from
the reaction tube
and control 4 hours. The aliquots are serially diluted in a microtiter plate
for a total of 8
microtiter wells. Twenty-five microliters of each dilution is incubated for 6
days with
African green monkey kidney cells in microtiter plates. The wells are checked
for a
cytopathic effect. The limit of detection is > 5 TCmso/mL.
The presence 12 mM 1-ethylaziridine in 12.5 mM sodium phosphate, incubated at
37°C for 4 hours resulted in a greater than 5 log EMCV reduction
compared to the positive
control.
to Other Embodiments
All publications, patent applications, and patents mentioned in this
specification are
herein incorporated by reference.
While the invention has been described in connection with specific
embodiments, it
will be understood that it is capable of further modifications. Therefore,
this application is
15 intended to cover any variations, uses, or adaptations of the invention
that follow, in general,
the principles of the invention, including departures from the present
disclosure that come
within known or customary practice within the art.
What is claimed is:
