U.S. patent number 5,151,507 [Application Number 07/713,906] was granted by the patent office on 1992-09-29 for alkynylamino-nucleotides.
This patent grant is currently assigned to E. I. Du Pont de Nemours and Company. Invention is credited to Frank W. Hobbs, Jr., George L. Trainor.
United States Patent |
5,151,507 |
Hobbs, Jr. , et al. |
* September 29, 1992 |
Alkynylamino-nucleotides
Abstract
Alkynylamino-nucleotides and labeled alkynylaminonucleotides
useful, for example, as chain terminating substrates for DNA
sequencing are provided along with several key intermediates and
processes for their preparation. For some applications, longer,
hydrophilic linkers are provided.
Inventors: |
Hobbs, Jr.; Frank W.
(Wilmington, DE), Trainor; George L. (Wilmington, DE) |
Assignee: |
E. I. Du Pont de Nemours and
Company (Wilmington, DE)
|
[*] Notice: |
The portion of the term of this patent
subsequent to September 10, 2008 has been disclaimed. |
Family
ID: |
27109068 |
Appl.
No.: |
07/713,906 |
Filed: |
June 12, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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57565 |
Jun 12, 1987 |
5047519 |
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881372 |
Jul 2, 1986 |
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Current U.S.
Class: |
536/26.7;
544/244; 536/27.14; 536/28.52; 536/27.2; 536/28.53; 544/243 |
Current CPC
Class: |
C07D
405/04 (20130101); C07D 405/12 (20130101); C07D
405/14 (20130101); C07D 471/04 (20130101); C07F
9/65586 (20130101); C07F 9/6561 (20130101); C07F
9/6512 (20130101); C07H 19/10 (20130101); C07H
19/20 (20130101); C07H 21/00 (20130101); C12Q
1/6869 (20130101); G01N 27/44721 (20130101); G01N
27/44726 (20130101); C07H 19/06 (20130101) |
Current International
Class: |
C07H
19/00 (20060101); C07F 9/6561 (20060101); C07H
19/10 (20060101); C07D 405/00 (20060101); C07D
405/14 (20060101); C07D 405/12 (20060101); C07D
405/04 (20060101); C07H 19/20 (20060101); C07H
21/00 (20060101); C12Q 1/68 (20060101); C07F
9/00 (20060101); C07H 19/06 (20060101); C07D
471/04 (20060101); C07F 9/6512 (20060101); G01N
27/447 (20060101); C07D 471/00 (20060101); C07F
9/6558 (20060101); C07H 001/00 () |
Field of
Search: |
;536/23,24,26,27,29
;544/243,244 |
References Cited
[Referenced By]
U.S. Patent Documents
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5047519 |
September 1991 |
Hobbs, Jr. et al. |
|
Other References
Bergstrom et al., J. Am. Chem. Soc., vol. 98; p. 1587 (1976). .
Langer et al., Proc. Natl. Acad. Sci., U.S.A., vol. 78; No. 11, pp.
6633-6637. .
Draper, Nucleic Acid Research; vol. 12, No. 2, 989-1022 (1984).
.
Barr et al., J. Chem. Soc.; Perkins Trans I, pp. 1263-1267 (1978).
.
Bergstrom et al., J. Am. Chem. Soc.; vol. 100, p 8106 (1978). .
Vincent et al., Tetrahedron Letters, vol. 22, pp. 945-947 (1981).
.
Robins et al., J. Org. Chem.; vol. 48, 1854-1862 (1983). .
Sonogashira et al., Tetrahedron Letters; No. 50, pp. 4467-4470
(1975). .
Edo et al., Chem. Pharm. Bull.; vol. 26, No. 12, pp. 3843-3850
(1978). .
Seda et al., Chem. Bor., vol. 11; 2925-2930 (1978). .
Schram et al., J. Carbohyd., Nucles., Nucleotis, vol. 2, No. 2, pp.
177-184 (1975). .
Bergstrom et al., J. Org. Chem., vol. 46, No. 7; pp. 1423-1431
(1981). .
Bergstrom et al., Nucleic Acid Research, vol. 8; pp. 6213-6219
(1980). .
Haralambidis et al., Nucleic Acid Research, vol. 15, No. 12, pp.
4857-4867 (1987). .
Gibson et al., Nucleic Acid Research, vol. 15, No. 16, pp.
6455-6467 (1987)..
|
Primary Examiner: Brown; Johnnie R.
Assistant Examiner: Wilson; J. Oliver
Attorney, Agent or Firm: Frank; George A.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No.
057,565, filed Jun. 12, 1987, now U.S. Pat. No. 5,047,519 which, in
turn, is a continuation-in-part of application Ser. No. 07/881,372,
filed Jul. 2, 1986, now abandoned, and is related to U.S. Pat. No.
4,833,332 and application Ser. No. 07/057,566, filed on Jun. 12,
1987, now abandoned, which is also a continuation-in-part of Ser.
No. 07/881,372, filed Jul. 2, 1986.
Claims
We claim:
1. An alkynylamino-nucleotide having the structure:
wherein
R.sub.1 is --CH.sub.2 OCH.sub.2 (CH.sub.2 OCH.sub.2).sub.n CH.sub.2
-- and n=2-5
R.sub.2 and R.sub.3 are, independently, H, C.sub.1 -C.sub.4 alkyl
or a protecting group, and
Nuc is R.sub.4 -Het having the structure ##STR18## Z is H or
NH.sub.2, and R.sub.4 is ##STR19## wherein R.sub.5 is H, PO.sub.3
H.sub.2, P.sub.2 O.sub.6 H.sub.3, P.sub.3 O.sub.9 H.sub.4 or salts
thereof, and
(i) when R.sub.7 =R.sub.8 =H, then R.sub.6 =H, OH, F, N.sub.3 or
NH.sub.2 ; or
(ii) when R.sub.7 =H and R.sub.8 =OH, then R.sub.6 =H or OH; or
(iii) when R.sub.7 =OH and R.sub.8 =H, then R.sub.6 =OH.
2. The alkynylamino-nucleotide of claim 1 wherein n=2.
3. The alkynylamino-nucleotide of claim 1 wherein R.sub.2 is H and
R.sub.3 is H or a protecting group.
4. A labeled alkynylamino-nucleotide having the structure:
wherein
R.sub.1 is --CH.sub.2 OCH.sub.2 (CH.sub.2 OCH.sub.2).sub.n CH.sub.2
-- and n=2-5,
R.sub.2 is H, C.sub.1 -C4 alkyl or a protecting group, and
Nuc is R.sub.4 -Het having the structure ##STR20## Z is H or
NH.sub.2, and R.sub.4 is ##STR21## wherein R.sub.5 is H, PO.sub.3
H.sub.2, P.sub.2 O.sub.6 H.sub.3, P.sub.3 O.sub.9 H.sub.4 or salts
thereof, and
(i) when R.sub.7 =R.sub.8 =H, then R.sub.6 =H, OH, F, N.sub.3 or
NH.sub.2 ; or
(ii) when R.sub.7 =H and R.sub.8 =OH, then R.sub.6 =H or OH; or
(iii) when R.sub.7 =OH and R.sub.8 =H, then R.sub.6 =OH and
R.sub.3 is a reporter group.
5. The labeled alkynylamino-nucleotide of claim 4 wherein R.sub.3
is a fluorescent group.
6. The labeled alkynylamino-nucleotide of claim 4 wherein n=2.
7. An alkynylamino-nucleotide having the structure:
wherein
R.sub.1 is a diradical moiety of 1-20 atoms,
R.sub.2 is H, C.sub.1 -C.sub.4 alkyl or a protecting group,
R.sub.3 contains a biotin moiety, and
Nuc is R.sub.4 -Het having the structure ##STR22## Z is H or
NH.sub.2, and R.sub.4 is ##STR23## wherein R.sub.5 is H, PO.sub.3
H.sub.2, P.sub.2 O.sub.6 H.sub.3, P.sub.3 O.sub.9 H.sub.4 or salts
thereof, and
(i) when R.sub.7 =R.sub.8 =H, then R.sub.6 =H, OH, F, N.sub.3 or
NH.sub.2 ; or
(ii) when R.sub.7 =H and R.sub.8 =OH, then R.sub.6 =H or OH; or
(iii) when R.sub.7 =OH and R.sub.8 =H, then R.sub.6 =OH.
8. The alkynylamino-nucleotide of claim 7 wherein R.sub.1 is
--CH.sub.2 --.
9. An alkynylamino-nucleotide having the structure:
wherein R.sub.1 is --CH.sub.2 OCH.sub.2 (CH.sub.2 OCH.sub.2).sub.n
CH.sub.2 -- and n=2-5
R.sub.2 and R.sub.3 are, independently, H, C.sub.1 -C.sub.4 alkyl
or a protecting group, and
Nuc is R.sub.4 -Het having the structure ##STR24## Z is H or
NH.sub.2, and R.sub.4 is ##STR25## wherein R.sub.5 is H, PO.sub.3
H.sub.2, P.sub.2 O.sub.6 H.sub.3, P.sub.3 O.sub.9 H.sub.4 or salts
thereof, and
(i) when R.sub.7 =R.sub.8 =H, then R.sub.6 =H, OH, F, N.sub.3 or
NH.sub.2 ; or
(ii) when R.sub.7 =H and R.sub.8 =OH, then R.sub.6 =H or OH; or
(iii) when R.sub.7 =OH and R.sub.8 =H, then R.sub.6 =OH, and
R.sub.3 is a reporter group comprising biotin.
Description
FIELD OF THE INVENTION
This invention pertains to alkynylamino-nucleotides and especially
to their use in preparing fluorescently-labeled nucleotides as
chain-terminating substrates for a fluorescence-based DNA
sequencing method.
BACKGROUND OF THE INVENTION
DNA sequencing is one of the cornerstone analytical techniques of
modern molecular biology. The development of reliable methods for
sequencing has led to great advances in the understanding of the
organization of genetic information and has made possible the
manipulation of genetic material (i.e. genetic engineering).
There are currently two general methods for sequencing DNA: the
Maxam-Gilbert chemical degradation method [A. M. Maxam et al.,
Meth. in Enzym., Vol. 65, 499-559 (1980)] and the Sanger dideoxy
chain termination method [F. Sanger, et al., Proc. Nat. Acad. Sci.
USA, Vol 74, 5463-5467 (1977)]. A common feature of these two
techniques is the generation of a set of DNA fragments which are
analyzed by electrophoresis. The techniques differ in the methods
used to prepare these fragments.
With the Maxam-Gilbert technique, DNA fragments are prepared
through base-specific, chemical cleavage of the piece of DNA to be
sequenced. The piece of DNA to be sequenced is first 5'-end-labeled
with .sup.32 P and then divided into four portions. Each portion is
subjected to a different set of chemical treatments designed to
cleave DNA at positions adjacent to a given base (or bases). The
result is that all labeled fragments will have the same 5'-terminus
as the original piece of DNA and will have 3'-termini defined by
the positions of cleavage. This treatment is done under conditions
which generate DNA fragments which are of convenient lengths for
separation by gel electrophoresis.
With Sanger's technique, DNA fragments are produced through partial
enzymatic copying (i.e. synthesis) of the piece of DNA to be
sequenced. In the most common version, the piece of DNA to be
sequenced is inserted, using standard techniques, into a
"sequencing vector", a large, circular, single-stranded piece of
DNA such as the bacteriophage M13. This becomes the template for
the copying process. A short piece of DNA with its sequence
complementary to a region of the template just upstream from the
insert is annealed to the template to serve as a primer for the
synthesis. In the presence of the four natural deoxyribonucleoside
triphosphates (dNTP's), a DNA polymerase will extend the primer
from the 3'-end to produce a complementary copy of the template in
the region of the insert. To produce a complete set of sequencing
fragments, four reactions are run in parallel, each containing the
four dNTP's along with a single dideoxyribonucleoside triphosphate
(ddNTP) terminator, one for each base. (.sup.32 P-Labeled dNTP is
added to afford labeled fragments.) If a dNTP is incorporated by
the polymerase, chain extension can continue. If the corresponding
ddNTP is selected, the chain is terminated. The ratio of ddNTP to
dNTP's is adjusted to generate DNA fragments of appropriate
lengths. Each of the four reaction mixtures will, thus, contain a
distribution of fragments with the same dideoxynucleoside residue
at the 3'-terminus and a primer-defined 5'-terminus.
The terms "terminator", "chain terminator" and "chain terminating
substrate" are used interchangeably throughout to denote a
substrate which can be incorporated onto the 3'-end of a DNA or RNA
chain by an enzyme which replicates nucleic acids in a
template-directed manner but, once incorporated, prevents further
chain extension. In contrast, the natural deoxynucleotide
substrates can be considered to be "chain propagating
substrates".
The term "nucleoside" is used throughout to denote a heterocyclic
base-sugar unit composed of one molecule of pyrimidine or purine
(or derivatives thereof) and one molecule of a ribose sugar (or
derivatives or functional equivalents thereof). The term
"nucleotide" is used throughout to denote either a nucleoside or
its phosphorylated derivative.
In both the Sanger and Maxam-Gilbert methods, base sequence
information which generally cannot be directly determined by
physical methods has been converted into chain-length information
which can be determined. This determination can be accomplished
through electrophoretic separation. Under denaturing conditions
(high temperature, urea present, etc.), short DNA fragments migrate
as if they were stiff rods. If a gel matrix is employed for the
electrophoresis, the DNA fragments will be sorted by size. The
single-base resolution required for sequencing can usually be
obtained for DNA fragments containing up to several hundred
bases.
To determine a full sequence, the four sets of fragments produced
by either Maxam-Gilbert or Sanger methodology are subjected to
electrophoresis in four parallel lanes. This results in the
fragments being spatially resolved along the length of the gel. The
pattern of labeled fragments is typically transferred to
photosensitive film by autoradiography (i.e. an exposure is
produced by sandwiching the gel and the film for a period of time).
The developed film shows a continuum of bands distributed in the
four lanes, often referred to as a sequencing ladder. The ladder is
read by visually scanning the film (starting with the short, faster
moving fragments) and determining the lane in which the next band
occurs for each step on the ladder. Since each lane is associated
with a given base (or combination of bases in the Maxam-Gilbert
case), the linear progression of lane assignments translates
directly into base sequence.
The Sanger and Maxam-Gilbert methods for DNA sequencing are
conceptually elegant and efficacious but they are operationally
difficult and time-consuming. Analysis of these techniques shows
that many of the problems stem from the use of a single
radioisotopic reporter. [A reporter can be defined as a chemical
group which has a physical or chemical characteristic which can be
readily measured or detected by appropriate physical or chemical
detector systems or procedures. Ready detectability can be provided
by such characteristics as color change, luminescence,
fluorescence, or radioactivity; or it may be provided by the
ability of the reporter to serve as a ligand recognition site to
form specific ligand-ligand complexes which contain groups
detectable by conventional (e.g., colorimetric, spectrophotometric,
fluorometric or radioactive) detection procedures. The
ligand-ligand complexes can be in the form of protein-ligand,
enzyme-substrate, antibody-antigen, carbohydrate-lectin,
protein-cofactor, protein-effector, nucleic acid-nucleic acid or
nucleic acid-ligand complexes.]
The use of short-lived radioisotopes such as .sup.32 P at high
specific activity is problematic from both a logistical and a
health-and-safety point of view. The short half-life of .sup.32 P
necessitates the anticipation of reagent requirements several days
in advance and prompt use of the reagent. Once .sup.32 P-labeled
DNA sequencing fragments have been generated, they are prone to
self-destruction and must be immediately subjected to
electrophoretic analysis. The large electrophoresis gels required
to achieve single base separation lead to large volumes of
contaminated buffer leading to waste disposal problems. The
autoradiography required for subsequent visualization of the
labeled DNA fragments in the gel is a slow process (overnight
exposures are common) and adds considerable time to the overall
operation. Finally, there are the possible health risks associated
with use of such potent radioisotopes.
The use of only a single reporter to analyze the position of four
bases lends considerable operational complexity to the overall
process. The chemical/enzymatic steps must be carried out in
separate vessels and electrophoretic analysis must be carried out
in four parallel lanes. Thermally induced distortions in mobility
result in skewed images of labeled DNA fragments (e.g. the smile
effect) which, in turn, lead to difficulties in comparing the four
lanes. These distortions often limit the number of bases that can
be read on a single gel.
The long times required for autoradiographic imaging along with the
necessity of using four parallel lanes force a "snapshot" mode of
visualization. Since simultaneous spatial resolution of a large
number of bands is needed, very large gels must be used. This
results in additional problems: large gels are difficult to handle
and are slow to run, adding more time to the overall process.
Finally, there is a problem of manual interpretation. Conversion of
a sequencing ladder into a base sequence is a time-intensive,
error-prone process requiring the full attention of a highly
skilled scientist. Numerous attempts have been made to automate the
reading and some mechanical aids do exist, but the process of
interpreting a sequence gel is still painstaking and slow.
To address these problems, replacement of .sup.32 P/autoradiography
with an alternative, non-radioisotopic reporter/detection system
has been considered. Such a detection system would have to be
exceptionally sensitive to achieve a sensitivity comparable to
.sup.32 P; each band on a sequencing gel contains on the order of
10.sup.-16 mole of DNA. One method of detection which is capable of
reaching this level of sensitivity is fluorescence. DNA fragments
could be labeled with one or more fluorescent labels (fluorescent
dyes). Excitation with an appropriate light source would result in
a characteristic emission from the label thus identifying the
band.
The use of fluorescent labels, as opposed to radioisotopic labels,
would allow easier tailoring of the detection system to this
particular application. For example, the use of four different
fluorescent labels distinguishable on the basis of some emission
characteristic (e.g. spectral distribution, life-time,
polarization) would allow linking a given label uniquely with the
sequencing fragments associated with a given base. With this
linkage established, the fragments could be combined and resolved
in a single lane and the base assignment could be made directly on
the basis of the chosen emission characteristic.
So far two attempts to develop a fluorescence-based DNA sequencing
system have been described. The first system, developed at the
California Institute of Technology, has been disclosed in L. M.
Smith, West German Pat. Appl. #DE 3446635 Al (1984); L. E. Hood et
al., West German Pat. Appl. #DE 3501306 Al (1985); L. M. Smith et
al., Nucleic Acids Research, Vol. 13, 2399-2412 (1985); and L. M.
Smith et al., Nature, Vol. 321, 674-679 (1986). This system
conceptually addresses the problems described in the previous
section but the specifics of the implementation render Smith's
approach only partially successful. For example, the large
wavelength range of the emission maxima of the
fluorescently-labeled DNA sequencing fragments used in this system
make it difficult to excite all four dyes efficiently with a single
monochromatic source. More importantly, the significant
differential perturbations in electrophoretic mobility arising from
dyes with different net charges make it difficult or impossible to
perform single-lane sequencing with the set of dyes used in this
system. These difficulties are explicitly pointed out by Smith et
al.
In general, the methodology used to prepare the
fluorescence-labeled sequencing fragments creates difficulties. For
Maxam-Gilbert sequencing, 5'-labeled oligonucleotides are
enzymatically ligated to "sticky ended", double-stranded fragments
of DNA produced through restriction cleavage. This limits one to
sequencing fragments produced in this fashion. For Sanger
sequencing, 5'-labeled oligonucleotides are used as primers. Four
special primers are required. To use a new vector system one has to
go through the complex process of synthesizing and purifying four
new dye-labeled primers. The same thing will be true whenever a
special primer is needed.
The use of labeled primers is inferior in other respects as well.
The polymerization reactions must still be carried out in separate
vessels. As in the Maxam-Gilbert and Sanger sequencing systems,
effectively all fragments derived from the labeled primer will be
fluorescently labeled. Thus, the resulting sequencing pattern will
retain most of the common artifacts (e.g. false or shadow bands,
pile-ups) which arise when enzymatic chain extension is interrupted
by processes other than incorporation of a chain terminator.
In a second approach, W. Ansorge et al., J. Biochem. Biophys.
Methods, Vol. 13, 315-323 (1986), have disclosed a
non-radioisotopic DNA sequencing technique in which a single
5'-tetramethylrhodamine fluorescent label is covalently attached to
the 5'-end of a 17-base oligonucleotide primer. This primer is
enzymatically extended in four vessels through the standard
dideoxynucleotide sequencing chemistry to produce a series of
enzymatically copied DNA fragments of varying length. Each of the
four vessels contains a dideoxynucleotide chain terminator
corresponding to one of the four DNA bases which allows terminal
base assignment from conventional electrophoretic separation in
four gel lanes. The 5'-tetramethylrhodamine fluorescent label is
excited by an argon ion laser beam passing through the width of the
entire gel. Although this system has the advantage that a
fluorescent reporter is used in place of a radioactive reporter,
all of the disadvantages associated with conventional sequencing
and with preparing labeled primers still remain.
Until now, no one has created a DNA sequencing system which
combines the advantages of fluorescence detection with terminator
labeling. If appropriate fluorescently-labeled chain terminators
could be devised, labeled sequencing fragments would be produced
only when a labeled chain terminator is enzymatically incorporated
into a sequencing fragment, eliminating many of the artifacts
associated with other labeling methods. If each of the four chain
terminators needed to sequence DNA were covalently attached to a
different distinguishable fluorescent reporter, it should be
possible, in principle, to incorporate all four terminators during
a single primer extension reaction and then to analyze the
resulting sequencing fragments in a single gel lane. If such
fluorescently-labeled chain terminators could be devised, these
compounds would probably also be useful for other types of
enzymatic labeling of nucleic acids. In particular, analogs of
fluorescently-labeled chain terminators could be designed to use
other, non-fluorescent, reporters or to serve as chain-propagating
substrates for enzymes which replicate nucleic acids in a
template-directed manner (e.g., reverse transcriptase, RNA
polymerase or DNA polymerase). Introducing a reporter into DNA in a
manner useful for sequencing is one of the most difficult nucleic
acid labeling problems. Compounds and/or strategies developed for
DNA sequencing are also likely to be applicable to many other
labeling problems.
To be useful as a chain-terminating substrate for
fluorescence-based DNA sequencing, a substrate must contain a
fluorescent label and it must be accepted by an enzyme useful for
sequencing DNA. Suitable substrate candidates are expected to be
derivatives or analogs of the naturally-occurring nucleotides.
Because of the expectation that a fluorescent label and a
nucleotide will not fit into the active site of a replication
enzyme at the same time, a well-designed substrate must have the
fluorescent label separated from the nucleotide by a connecting
group of sufficient length and appropriate geometry to position the
fluorescent label away from the active site of the enzyme. The
nature of the connecting group can vary with both the label and the
enzyme used. For ease of synthesis and adaptability to variations
in label and/or enzyme requirements, however, it is most convenient
to consider the connecting group as consisting of a linker which is
attached to the nucleotide and to the fluorescent label.
In the design of fluorescently-labeled chain terminators for DNA
sequencing, the linker must satisfy several requirements:
1) one must be able to attach the same or a functionally equivalent
linker to all four bases found in DNA;
2) the linker must not prevent the labeled nucleotide from being
utilized effectively as a chain terminating substrate for an enzyme
useful for DNA sequencing;
3) the linker (plus optional spacer and label) must perturb the
electrophoresis of oligonucleotides to which it is attached in a
manner which is independent of the base to which it is
attached;
4) the attachment of the linker to the base and the spacer or label
must be stereoselective and regioselective to produce a single,
well-defined nucleotide substrate; and
5) the linker should preferably contain a primary or secondary
amine for coupling with the label.
Although five different types of amine linkers have been disclosed
for attaching labels to nucleotides and oligonucleotides (see
below), none of these linkers meet all five of the requirements
listed above for use in a chain terminating substrate useful in DNA
sequencing.
Bergstrom et al., J. Am. Chem. Soc., Vol. 98, 1587 (1976), disclose
a method for attaching alkene-amino and acrylate side-chains to
nucleosides by Pd(II)-catalyzed coupling of 5-mercurio-uridines to
olefins. Ruth, PCT/US84/00279, discloses the use of the above
side-chains as linkers for the attachment of reporters to
non-enzymatically synthesized oligonucleotides. Langer et al.,
Proc. Nat. Acad. Sci. USA, Vol. 78, 6633 (1981), disclose the use
of allylamino linkers for the attachment of reporters to
nucleotides. The disadvantages of these linkers include the
difficulty of preparing regioselectively the appropriate mercurial
nucleotide precursors, the difficulty of separating the mixture of
products generated by some of these nucleotide/olefin coupling
reactions, and the potential lability of vinyl substituted
nucleosides. Furthermore, the only reporters which have been
incorporated with this linker are biotin and digoxigenin, Schmitz
et al., Analytical Biochemistry, Vol. 192, 222-231 (1991). These
reporters have the disadvantage that they must be detected via a
complex with avidin, streptavidin, or anti-digoxigenin antibodies,
proteins which bind tightly to them. These proteins, and thus
indirectly biotin and digoxigenin, are detected by attaching
fluorescent or enzymatic reporters to them. For some applications,
such as fluorescent in situ hybridization, direct fluorescent
tagging would provide a superior method for tagging DNA. Klevan et
al., WO 86/02929, disclose a method for attaching linkers to the N4
position of cytidine and the N6 position of adenosine. The
disadvantage of this method is that there is no analogous site in
uridine and guanosine for attaching a linker.
Another potential linker which might satisfy the five requirements
listed above is an alkynylamino linker, in which one end of the
triple bond is attached to the nucleoside and the other end of the
triple bond is attached to a group which contains a primary or
secondary amine. To insure chemical stability, the amine should not
be directly attached to the triple bond. Some methods of attaching
alkyne groups to nucleosides have been disclosed (see below).
Barr et al., J. Chem. Soc., Perkins Trans. I, 1263-1267 (1978),
disclose the syntheses of 5-ethynyluridine,
2'-deoxy-5-ethynyluridine, 5-ethynylcytosine, 5-ethynylcytidine,
2'-deoxy-5-ethynylcytidine and the .alpha.-anomers of the
2'-deoxyribonucleosides. The 2'-deoxyribonucleosides were prepared
by constructing the heterocycles, coupling with a functionalized
2-deoxy sugar, separating the anomeric mixtures, and removing the
protecting groups on the sugars.
Bergstrom et al., J. Am. Chem. Soc., Vol. 100, 8106 (1978),
disclose the palladium-catalyzed coupling of alkenes with 5-mercuri
or 5-iodo derivatives of uracil nucleosides. This method was
reported to fail in analogous reactions of alkynes with uracil
nucleoside derivatives.
Vincent et al., Tetrahedron Letters, Vol. 22, 945-947 (1981),
disclose the synthesis of 5-alkynyl-2'-deoxyuridines by the
reaction of 0-3',5'-bis(trimethylsilyl)deoxyuridine with
alkynylzinc reagents in the presence of palladium or nickel
catalysts [dichloro-bis(triphenylphosphine)palladium(II),
dichloro-bis(benzonitrile)palladium(II) or
dichloro(ethylene-(bis(diphenylphosphine))nickel(II)].
Robins et al., J. Org. Chem., Vol. 48, 1854-1862 (1983), disclose a
method for coupling terminal alkynes, HC.tbd.CR (R=H, alkyl,
phenyl, trialkylsilyl, hydroxyalkyl or protected hydroxyalkyl), to
5-iodo-1-methyluracil and 5-iodouracil nucleosides (protected as
their p-toluyl esters) in the presence of
bis(triphenylphosphine)palladium(II) chloride and copper(I) iodide
in warm triethylamine. When
3',5'-di-O-acetyl-5-iodo-2'-deoxyuridine was reacted with hexyne,
4-(p-toluyloxy)butyne, 4-(tetrahydropyranyloxy) or
4-(trityloxy)butyne, the major products were the cyclized
furano[2,3-d]pyrimidin-2-ones rather than the desired
alkynyluridines.
None of the above references discloses a method for attaching an
alkynylamino linker to nucleosides. The methodology of Bergstrom
fails, and that of Barr is not directly applicable. The catalysts
used by Robins et al. and Vincent et al. have the potential to
promote numerous undesirable side reactions (e.g., cyclization or
intermolecular nucleophilic addition of the amine to an alkyne)
when the alkyne contains an amino group. Coupling reactions have
been reported only with iodonucleosides which contain an
electron-deficient uracil base. Since Pd-catalyzed coupling
reactions generally work best with electron-deficient aryl iodides,
problems may be anticipated in coupling alkynes to any of the other
three bases (which are all more electron-rich than uracil).
There remains a need for alkynylamino nucleotides and for methods
permitting their preparation.
SUMMARY OF THE INVENTION
The compounds of this invention are alkynylamino-nucleotides having
the structure:
wherein R.sub.1 is a substituted or unsubstituted diradical moiety
of 1-20 atoms. R.sub.1 can be straight-chained alkylene, C.sub.1
-C.sub.20, optionally containing within the chain double bonds,
triple bonds, aryl groups or heteroatoms such as N, O or S. The
heteroatoms can be part of such functional groups as ethers,
thioethers, esters, amines or amides. For DNA sequencing, R.sub.1
is preferably straight-chained alkylene, C.sub.1 -C.sub.10 ; most
preferably R.sub.1 is --CH.sub.2 --. For enzymatic incorporation of
multiple reporters (labels) into DNA, R.sub.1 can be a diradical
moiety of an unsubstituted chain of 10-20 carbon atoms optionally
containing oxygen, nitrogen and/or sulfur atoms in place of one or
more carbon atoms. Most preferably, R.sub.1 is --CH.sub.2 OCH.sub.2
(CH.sub.2 OCH.sub.2).sub.n CH.sub.2 --, where n=2-5. Substitutents
on R.sub.1 can include C.sub.1 -C.sub.6 alkyl, aryl, ester, ether,
amine, amide or chloro groups;
R.sub.2 and R.sub.3 are independently H, C.sub.1 -C.sub.4 alkyl, or
a protecting group such as acyl, alkoxycarbonyl or sulfonyl.
Preferably R.sub.2 is H, and R.sub.3 is H or trifluoroacetyl;
Nuc (nucleotide) is R.sub.4 -Het (heterocyclic base): ##STR1## and
wherein R.sub.5 is H, PO.sub.3 H.sub.2, P.sub.2 O.sub.6 H.sub.3,
P.sub.3 O.sub.9 H.sub.4 or salts thereof, and when R.sub.7 =R.sub.8
=H, then R.sub.6 =H, OH, F, N.sub.3 or NH.sub.2 ; or when R.sub.7
=H and R.sub.8 =OH, then R.sub.6 =H or OH; or when R.sub.7 =OH and
R.sub.8 =H, then R.sub.6 =OH.
The labeled alkynylamino-nucleotides of this invention are
structure I where R.sub.3 is a reporter (label), preferably
comprising biotin.
DETAILED DESCRIPTION OF THE INVENTION
The strategy used to incorporate reporters in the DNA sequencing
fragments in a base-specific fashion is a critical feature of any
DNA sequencing system. The use of the alkynylamino-nucleotides of
this invention permit the modification of the Sanger methodology
most advantageously by attaching a reporter (label) to an
alkynylamino-nucleotide chain terminator. Although the reporter can
be chosen from a wide variety of readily detectable groups
(labels), for convenience, the preferred approach is illustrated
below using fluorescent reporters.
This approach offers a number of operational advantages. Most
importantly, terminator labeling firmly links the attached reporter
with the base-specific termination event. Only DNA sequencing
fragments resulting from bona fide termination events will carry a
reporter. This eliminates many of the artifacts observed in
conventional sequencing. This approach also affords complete
flexibility in the choice of sequencing vector since no special
primers are involved. Automation is facilitated by the fact that
the reporters are carried by four low molecular-weight reagents
which can be selectively introduced in a single reaction.
There are no inherent operational disadvantages; the problems with
this approach are encountered in the design stage. In general, the
enzymes used for sequencing DNA are highly substrate selective and
there is no reason a priori to expect to be able to make a
nucleoside triphosphate with a covalently attached reporter that is
an efficient chain-terminating substrate for a sequencing enzyme.
It might be thought that attachment of a reporter to a substrate
would cause sufficiently large changes in the steric and electronic
character of the substrate to make it unacceptable to the enzyme
or, even if accepted, it would not be incorporated in the DNA
chain. It has been found, however, that the small size of the
alkynylamino linker of this invention and the ability to attach the
alkynylamino linker to the 5-position of the pyrimidine nucleotides
and the 7-position of the purine nucleotides provide labeled
chain-terminating substrates that do not interfere excessively with
the degree or fidelity of substrate incorporation.
The alkynylamino-nucleotides of this invention will be illustrated
through the description of fluorescently-labeled alkynylamino
nucleotide chain terminators. To delineate the structural scope and
rationale of fluorescently-labeled alkynylamino-nucleotides of this
invention, it is useful to break the labeled structure (I) into
five components: ##STR2##
(i) a triphosphate moiety, R.sub.5
(ii) a "sugar", R.sub.4
(iii) a heterocyclic base (Het),
(iv) a linker (--C.tbd.CR.sub.1 NR.sub.2 --), and
(v) a fluorescent label, R.sub.3.
(i) Triphosphate Moiety (R.sub.5)
The triphosphate moiety or a close analog (e.g.,
.alpha.-thiotriphosphate) is an obligate functionality for any
enzyme substrate, chain terminating or otherwise. This
functionality provides much of the binding energy for the substrate
and is the actual site of the enzyme-substrate reaction.
##STR3##
(ii) Sugar (R.sub.4)
The "sugar" portion corresponds to the 2'-deoxyribofuranose
structural fragment in the natural enzyme substrates. This portion
of the molecule contributes to enzyme recognition and is essential
for maintaining the proper spatial relationship between the
triphosphate moiety and the heterocyclic base. To be useful for DNA
sequencing, when the "sugar" is a ribofuranose, the
3'-.alpha.-position must not have a hydroxyl group capable of being
subsequently used by the enzyme. The hydroxyl group must either be
absent, replaced by another group or otherwise rendered unusable.
Such sugars will be referred to as chain-terminating sugars. It is
known that a number of modified furanose fragments can fulfill this
requirement, including:
2',3'-dideoxy-.beta.-D-ribofuranosyl [(a), F. Sanger et al., Proc.
Nat. Acad. Sci. USA, Vol. 74, 5463-5467 (1977)],
.beta.-D-arabinofuranosyl, [(b) F. Sanger et al., Proc. Nat. Acad.
Sci. USA, Vol. 74, 5463-5467 (1977)],
3'-deoxy-.beta.-D-ribofuranosyl [(c), Klement et al., Gene Analysis
Technology, Vol. 3, 59-66 (1986)],
3'-amino-2',3'-dideoxy-.beta.-D-ribofuranosyl [(d), Z. G.
Chidgeavadze et al., Nuc. Acids Res., Vol. 12, 1671-1686
(1984)],
2',3'-dideoxy-3'-fluoro-.beta.-D-ribofuranosyl [(e), Z. G.
Chidgeavadze et al., FEBS Lett., Vol. 183, 275-278 (1985)], and
2',3'-dideoxy-2',3'-didehydro-.beta.-D-ribofuranosyl [(f), Atkinson
et al., Biochem., Vol. 8, 4897-4904 (1969)]. ##STR4##
Acyclonucleoside triphosphates, (AcyNTP's), in which the so-called
sugar is an acyclic group [e.g., 2-oxyethoxymethyl, (g)], can also
be used as chain terminators in DNA sequencing by the Sanger
methodology. The use of AcyNTP's as chain-terminating substrates
has been demonstrated by carrying out conventional Sanger
sequencing (.sup.32 P reporter) with the AcyNTP's substituting for
the ddNTP's. The sequencing ladders produced with AcyNTP's were
virtually identical to those produced with ddNTP's, except that a
higher concentration of AcyNTP (approximately 10.times.) was
required to obtain a similar distribution of DNA fragments. The
AcyNTP's are effective with both DNA Polymerase I (Klenow fragment)
and AMV reverse transcriptase. The alkynylamino derivatives of
AcyNTP's are therefore also expected to function as
chain-terminating substrates.
The AcyNTP's have the advantage of being more easily synthesized
than the ddNTP's. While the synthesis of ddNTP's is not a major
problem in conventional sequencing, it is significant when
structurally complex, fluorescently-labeled chain terminators are
being prepared. The use of the 2-oxyethoxymethyl group as a sugar
greatly simplifies reagent synthesis while maintaining acceptable
performance.
Medicinal research has identified other sugar modifications which
can be useful for DNA sequencing. For example,
3'-azido-2',3'-dideoxythymidine, [AZT; Mitsuya et al., Proc. Nat.
Acad. Sci. USA, Vol. 82, 7096-7100 (1985)] and
9-[2'-hydroxy-1'-(hydroxymethyl)ethoxymethyl]guanine [DHPG; Aston
et al., Biochem. Biophys. Res. Comm., Vol. 108, 1716-1721 (1982)]
are two antiviral agents which are presumed to act by being
converted to triphosphates which cause chain termination of DNA
replication. Nucleoside triphosphates with such sugar units can
also be useful for DNA sequencing.
(iii) Heterocyclic Base (Het)
The heterocyclic base functions as the critical recognition element
in nucleic acids, acting as a hydrogen-bonding acceptor and donor
in a particular spatial orientation. The heterocyclic base elements
are essential for incorporation with the high fidelity necessary
for accurate sequencing. This structural part is also the site of
attachment of the linker.
Preferred heterocyclic bases include: uracil (h), cytosine (i),
7-deazaadenine (j), 7-deazaguanine (k), and 7-deazahypoxanthine
(l). The unnatural 7-deazapurines can be employed to attach the
linker without adding a net charge to the base portion and thereby
destabilizing the glycosidic linkage. In addition, other
heterocyclic bases which are functionally equivalent as
hydrogen-bonding donors and acceptors can be used, e.g.,
8-aza-7-deazapurines and 3,7-dideazaadenine can be used in place of
7-deazapurines and 6-azapyrimidines can be used in place of
pyrimidines. (To simplify the nomenclature, the heterocyclic bases
are named and numbered throughout as 7-deazapurines.) ##STR5##
(iv) Linker
The linker is an alkynylamino group in which one end of the triple
bond is attached to an amine through a substituted or unsubstituted
diradical moiety, R.sub.1, of 1-20 atoms; the other end of the
triple bond is covalently attached to the heterocyclic base at the
5-position for pyrimidines or the 7-position (purine numbering) for
the 7-deazapurines. The amine nitrogen of the alkynylamino group is
attached to a reactive functional group (e.g., carbonyl) on the
fluorescent label. The linker must not significantly interfere with
binding to or incorporation by the DNA polymerase. The diradical
moiety can be straight-chained alkylene, C.sub.1 -C.sub.20,
optionally containing within the chain double bonds, triple bonds,
aryl groups or heteroatoms such as N, O or S. The heteroatoms can
be part of such functional groups as ethers, thioethers, esters,
amines or amides. Substituents on the diradical moiety can include
C.sub.1 -C.sub.6 alkyl, aryl, ester, ether, amine, amide or chloro
groups. For DNA sequencing, the diradical moiety preferably is
straight-chained alkylene, C.sub.1 -C.sub.10 ; most preferably the
diradical is --CH.sub.2 --.
For other applications, such as enzymatic incorporation of multiple
reporters (labels) into DNA, substrates with longer linkers are
incorporated more efficiently by many enzymes. Linkers which are
flexible and hydrophilic appear to have advantages over those which
lack these properties. For multiple labeling applications, the
preferred linker is a diradical moiety of an unsubstituted chain of
10-19 carbon atoms optionally containing oxygen, nitrogen, and/or
sulfur atoms in place of one or more carbon atoms. The most
preferred linker is --CH.sub.2 OCH.sub.2 (CH.sub.2 OCH.sub.2).sub.n
CH.sub.2 -- wherein n=2-.
(v) Fluorescent Label (R.sub.3)
The fluorescent label provides detectable, emitted radiation
following excitation by absorption of energy from an appropriate
source, such as an argon ion laser. It is desirable to have unique,
distinguishable fluorescent reporters for each DNA base encountered
in sequencing applications.
A family of reporters useful for fluorescent labeling in a DNA
sequencing method based on labeled chain-terminators can be derived
from the known dye, 9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene [S.
Biggs et al., J. Chem. Soc., Vol. 123, 2934-2943 (1923)]. This
xanthene family has the general structure, 1, ##STR6## where
R.sub.9 and R.sub.10 include H, lower alkyl, lower alkoxy, halo,
and cyano.
A preferred set of dyes suitable for DNA sequencing is structure 1,
a) R.sub.9 =R.sub.10 =H, abs. 487 nm, emis. 505 nm; b) R.sub.9 =H,
R.sub.10 =CH.sub.3, abs. 494 nm, emis. 512 nm; c) R.sub.9
=CH.sub.3, R.sub.10 =H, abs. 501 nm, emis. 519 nm; and d) R.sub.9
=R.sub.10 =CH.sub.3, abs. 508 nm, emis. 526 nm. The instruments
described by L. M. Smith, L. E. Hood et al., L. M. Smith et al. and
W. Ansorge et al. are capable of detecting sequencing fragments
labeled with any one of these dyes at concentrations suitable for
DNA sequencing, but these instruments are not capable or
discriminating among the above set of four dyes. A method for
discriminating the dyes and using this information to determine DNA
sequences is disclosed in application Ser. No. 07/057,566, filed on
Jun. 12, 1987, hereby incorporated by reference.
This application discloses a system for sequencing DNA, comprising
a means for detecting the presence of radiant energy from
closely-related yet distinguishable reporters, which are covalently
attached to compounds which function as chain terminating
nucleotides in a modified Sanger DNA chain elongation method. One
distinguishable fluorescent reporter is attached to each of four
dideoxynucleotide bases represented in Sanger DNA sequencing
reactions, i.e., dideoxynucleotides of adenine, guanine, cytosine,
and thymine. These reporter-labeled chain terminating reagents are
substituted for unlabeled chain terminators in the traditional
Sanger method and are combined in reactions with the corresponding
deoxynucleotides, an appropriate primer, template, and polymerase.
The resulting mixture contains DNA fragments of varying length that
differ from each other by one base which terminate on the 3' end
with uniquely labeled chain terminators corresponding to each of
the four DNA bases. This new labeling method allows elimination of
the customary radioactive label contained in one of the
deoxynucleotides of the traditional Sanger method.
Detection of these reporter labels can be accomplished with two
stationary photomultiplier tubes (PMT's) which receive
closely-spaced fluorescent emissions from laser-stimulated
reporters attached to chain terminators on DNA fragments. These
fragments can be electrophoretically separated in space and/or time
to move along an axis perpendicular to the sensing area of the
PMT's. The fluorescent emissions first pass through a dichroic
filter having both a transmission and reflection characteristic,
placed so as to direct one characteristic (transmission) to one
PMT, and the other characteristic (reflection) to the other PMT. In
this manner, different digital signals are created in each PMT that
can be ratioed to produce a third signal that is unique to a given
fluorescent reporter, even if a series of fluorescent reporters
have closely spaced emissions. This system is capable of detecting
reporters which are all efficiently excited by a single laser line,
such as 488 nm, and which have closely spaced emissions whose
maxima usually are different from each other by only 5 to 7 nm.
Therefore, the sequential base assignments in a DNA strand of
interest can be made on the basis of the unique ratio derived for
each of the four reporter-labeled chain terminators which
correspond to each of the four bases in DNA.
Since these xanthene dyes contain reactive functional groups, the
above application also discloses the design and preparation of
reagents which are useful for attaching these dyes to the amino
group of an alkynylamino linker. N-hydroxysuccinimide esters 2
(where R.sub.9 and R.sub.10 are as defined structure 1) are
preferred examples of such reagents. During the preparation of 2, a
sarcosine group is added to the basic dye structure to minimize
side reactions. In the above application, this optional sarcosine
group is referred to as a "spacer". Since only the preferred
reagents are used herein, the fluorescent part of a labeled
alkynylamino chain terminator is considered to include a sarcosine
spacer. After the N-hydroxysuccinimide leaving group has been
displaced by the amino group of the linker, the fluorescent dye
(part structure 1) is liberated by treatment with concentrated
ammonium hydroxide. N-hydroxysuccinimide esters are acylating
agents which react selectively with highly-nucleophilic amino
groups such as the ones present in the alkynylamino linkers
described above. Control experiments have demonstrated that
N-hydroxysuccinimide esters similar to 2 react much more slowly
with the amino groups of the heterocyclic base than with the linker
amino group. If reaction occurs to a small extent with the
heterocyclic amino groups, it has been discovered that the
resulting amides are hydrolyzed by the ammonium hydroxide treatment
which liberates the dye. It is therefore possible to use esters
such as 2 to attach selectively any reporter such as a fluorescent
dye to the amino group of the linker without modifying the
nucleotide in an unwanted fashion.
Fluorescently-labeled alkynylamino-nucleotide chain-terminators of
this invention function as well in DNA sequencing with AMV reverse
transcriptase as the corresponding substrates containing allylamino
linkers which are disclosed in the above application. However, the
compounds of this invention are easier to synthesize than the
corresponding substrates containing allylamino linkers because the
alkynylamino linkers are more easily attached to a preselected
position on the various bases needed for DNA sequencing. In
addition, the alkynylamino linkers can be attached to the
nucleotides in higher yield. Finally, nucleotides containing an
alkyne in conjugation with a heterocyclic ring are expected to be
more stable than corresponding nucleotides containing an alkene.
Suitable fluorescently-labeled chain terminators derived from
alkynylamino-nucleotides are shown by structure 3, where Nuc,
R.sub.1 -R.sub.4 and R.sub.6 -R.sub.8 are as defined above, R.sub.5
=HO.sub.9 P.sup.-3 and provided that when R.sub.8 is H or OH,
R.sub.6 must not be OH. ##STR7##
Scheme 1 describes methods for preparing the alkynylamino
nucleotides of this invention where the sugar is a
2,3-dideoxyribofuranosyl group. These methods are compatible with
all of the sugars of this invention. When combined with known
methods for modifying the sugars of nucleotides, these methods can
be used to prepare alkynylamino nucleotides in which the
2,3-dideoxyribofuranosyl group is replaced by the other sugars of
the invention. ##STR8##
A variety of routes can be used to prepare the first key
intermediates, the iodonucleosides (4). (In some instances, the
corresponding bromonucleosides can be used in place of the
iodonucleosides.)
5-Iodo-2',3'-dideoxyuridine can be prepared by treating
2',3'-dideoxyuridine [Pfitzer et al., J. Org. Chem., Vol. 29, 1508
(1964)] with ICl [Robins et al., Can. J. Chem., Vol. 60, 554-557
(1982)].
5-Iodo-2',3'-dideoxycytidine can be prepared by converting
2',3'-dideoxycytidine (Rayco Co.) to the corresponding 5-mercurio
nucleoside [Bergstrom et al., J. Carbohydrates, Nucleosides and
Nucleotides, Vol. 4, 257-269 (1977)] and then treating with
iodine.
Although methods for preparing 7-deazaguanosine and
2'-deoxy-7-deazaguanosine are known, it has been shown [Seela et
al., Chem. Ber., Vol. III, 2925-2930 (1978)] that electrophilic
attack on 7-deazaguanines occurs at both the 7- and 8-positions.
However, it has now been found that the desired
7-iodo-7-deazapurines can be obtained by treatment of
6-methoxy-2-thiomethyl-7-deazapurines with N-iodosuccinimide,
followed by replacement of the 2-thiomethyl and 6-methoxy
substituents as shown in Scheme 2 and described in Example 3. The
use of N-iodosuccinimide for regioselective iodination of a
7-deazapurine ring system is unprecedented. ##STR9##
7-Iodo-2',3'-dideoxy-7-deazaadenosine can be prepared by
deoxygenation of tubercidin, followed by mercuration/iodination
(Scheme 3). The deoxygenation reactions were adapted from
procedures disclosed by Moffatt et al. [J. Am. Chem. Soc., Vol. 95,
4016-4030 (1972)] and Robins et al. [Tetrahedron Lett., Vol. 25,
367-340 (1984)] to give an improved synthesis of
2',3'-dideoxy-7-deazaadenosine as shown in Scheme 3 and described
in Example 4.
7-Iodo-7-deazaadenosine can be prepared by regioselective
mercuration/iodination of tubercidin (7-deazaadenosine), as
reported by Bergstrom et al., J. Carbohydrates, Nucleosides and
Nucleotides, Vol. 5, 285-296 (1978) and Bergstrom et al., J. Org.
Chem., Vol. 46, 1424 (1981). ##STR10##
Alternative routes to 7-iodo-2',3'-dideoxy-7-deazaadenosine can be
used which do not use tubercidin, an expensive fermentation
product, as a starting material. These routes are shown in Schemes
4 and 5 and are described in Examples 5 and 6. In one of these
routes, the problem of regioselectively introducing an iodine in
the 7-position was solved by another unprecedented iodination. In
this case, treatment of 6-chloro-7-deazapurine 32 with iodine
monochloride afforded only the 7-iodo regioisomer 33. ##STR11##
Although a method for coupling terminal alkynes with protected
5-iodouracil nucleosides using a Pd(II)/Cu(I) catalyst has been
reported by Robins et al. [Tetrahedron Lett., Vol. 22, 421-424
(1981)], this method does not effect the desired coupling between
alkynylamines (e.g., propargylamine) and the unprotected
5-iodo-pyrimidine or 7-iodo-purine nucleosides. The ability to use
alkynylamines in direct coupling was highly desirable to provide
directly compounds with an amine group for subsequent attachment of
the fluorescent label. Similarly, a method using unprotected
nucleosides was sought to provide a more direct route to the
desired compounds by eliminating an otherwise unnecessary series of
protection/deprotection reactions.
Under the conditions described below, alkynylamines were
successfully coupled to a variety of halonucleosides in excellent
yields using a Pd(0)/Cu(I) catalyst. This coupling reaction was
also successful when the alkynylamine nitrogen was protected by an
acyl group such as acetyl and trifluoroacetyl, alkoxycarbonyl group
such as 9-fluorenylmethyloxycarbonyl group, and a sulfonyl group
such as p-toluenesulfonyl group. Unexpectedly, the number of carbon
atoms between the amino group and the triple bond was found not to
be critical in the procedure described below; 3-amino-1-propyne
(propargylamine), 5-amino-1-pentyne,
N-(2-propynyl)trifluoroacetamide, N-(4-pentynyl)trifluoroacetamide
and N-(11-dodecynyl)trifluoroacetamide were all successfully used
in the coupling reaction.
The broad success of this Pd(0)/Cu(I) catalyzed coupling reaction
is unexpected in view of the art. For example, Bergstrom et al. [J.
Am. Chem. Soc., Vol. 100, 8106 (1978)] noted that alkynes failed to
couple to 5-mercuri or 5-iodo derivatives of uracil nucleosides
using Pd catalysts. Also, Robins et al. [J. Org. Chem., Vol. 48,
1854-1862 (1983)]disclosed that the Pd(II)/Cu(I) catalyzed
reactions of 3',5'-di-O-acetyl-5-iodo-2'-deoxyuridine frequently
produced cyclized products. When the process described below is
used, the coupling succeeds even with alkynylamines (such as
5-amino-1-pentyne) which have the potential to cyclize readily.
Typically, the alkynylamino-nucleosides of this invention can be
prepared by placing the halonucleoside and Cu(I) in a flask,
flushing with Ar to remove air, adding dry dimethylformamide,
followed by addition of the alkynylamine, triethylamine and Pd(0).
The reaction mixture can be stirred for several hours, or until
thin layer chromatography (TLC) indicates consumption of the
halonucleoside. When an unprotected alkynylamine is used, the
alkynylamino-nucleoside can be isolated by concentrating the
reaction mixture and chromatographing on silica gel using an
eluting solvent which contains ammonium hydroxide to neutralize the
hydrohalide generated in the coupling reaction. When a protected
alkynylamine is used, methanol/methylene chloride can be added to
the reaction mixture, followed by the bicarbonate form of a
strongly basic anion exchange resin. The slurry can be then stirred
for about 45 min, filtered, and the resin rinsed with additional
methanol/methylene chloride. The combined filtrates can be
concentrated and promptly purified by flash-chromatography on
silica gel using a methanol-methylene chloride gradient.
The alkynylamino-nucleotides of this invention are preferably
prepared from 5-iodopyrimidine or 7-iodo-7-deazapurine nucleosides,
but the analogous bromonucleosides can also be used. [The
Pd(0)/Cu(I) catalyzed coupling reaction can also be used to
introduce alkynylamine groups at other positions on the aromatic or
heteroaromatic ring, provided only that the appropriate
halonucleotide is available.]
Suitable alkynylamines for the Pd(0)/Cu(I) catalyzed coupling
reaction are terminal alkynes wherein the triple bond is attached
to an amine by a diradical moiety of 1-20 atoms. The diradical
moiety can be straight-chain alkylene, (C.sub.1 -C.sub.20, e.g.,
--C.sub.3 H.sub.6 --), or can contain double bonds (e.g., as in
--CH.dbd.CHCH.sub.2 --), triple bonds (e.g., as in
--C--.tbd.C--CH.sub.2 --) or aryl groups [e.g., (para)--C.sub.6
H.sub.4 --, or para--CH.sub.2 -C.sub.6 H.sub.3 --]. The diradical
can also contain heteroatoms such as N, O, or S in the chain as
part of ether, ester, amine, or amido groups. Suitable substituents
on the diradical moiety can include C.sub.1 -C.sub.6 alkyl, aryl,
ester, ether, amine, amide or chloro groups Preferably, the
diradical is a straight-chain alkylene (C.sub.1 -C.sub.10); most
preferably, the diradical is --CH.sub.2 --. Suitable substituents
on the amine are lower alkyl (C.sub.1 -C.sub.4) and protecting
groups such as trifluoroacetyl. In general, the amine of the
alkynylamine can be primary, secondary or tertiary. For use as a
linker, however, the alkynylamine is preferably a primary amine.
The amine of the alkynylamine is usually protected because amine
protection is required in the next step. The coupled product is
also more readily purified when this amine is introduced in
protected form. A trifluoroacetyl protecting group is preferred
because it is easily removed after the coupling product is
converted to the corresponding 5'-triphosphate. Generally, a
1.5-3.0 fold excess of alkynylamine (relative to iodonucleoside)
can be used to insure complete conversion of the iodonucleoside to
an alkynylamino-nucleoside.
Suitable solvents for the coupling reaction include polar solvents
which dissolve the iodo- or bromonucleoside and do not decompose
the Pd(0)/Cu(I) catalyst system. N,N-Dimethylformamide (DMF),
acetonitrile, THF, dimethylsulfoxide (DMSO),
hexamethylphosphoramide (HMPA), and alcohols can all be used;
solvents which contain small amounts of water are also acceptable.
Preferably, the solvent is DMF. Preferably, the concentration of
the halonucleoside is 0.02-1.0M, most preferably 0.2-0.5M.
Suitable Pd catalysts are Pd(0) complexes, for example,
tetrakis(triarylphosphine)Pd(0). Preferably, the Pd(0) catalyst is
tetrakis(triphenylphosphine)Pd(0). The amount of Pd catalyst used
is generally 1-25 mol % (based on iodonucleoside), preferably 5-15
mol %. The larger amounts of catalyst are used to conduct the
reaction on a very small scale or to decrease the reaction time for
coupling.
The Cu(I) co-catalyst is preferably a cuprous halide or
pseudohalide (such as cuprous cyanide), most preferably CuI.
The mole ratio of Cu(I) co-catalyst to Pd(0) catalyst is more than
1.0 but less than 20. When a protected alkynylamine is used, the
preferred mole ratio of Cu/Pd is 2. When the alkynylamine is
unprotected, the unhindered basic nitrogen atom diminishes the
catalytic activity of the copper. In this case, a Cu/Pd ratio of 5
is preferred. In either case, no reaction is observed at room
temperature when Cu/Pd=1. The reaction rate generally increases as
the Cu/Pd ratio increases. With protected alkynylamines and Cu/Pd
ratios greater than 2, however, this increase is accompanied by
increased side-products, as indicated by TLC.
Triethylamine probably serves as an acid-scavenger in this
reaction; other strongly basic amines can also be used. An excess
of the unprotected alkynylamine can also serve as the
acid-scavenger, but preferably triethylamine is added as well.
Protected alkynylamino nucleosides can be converted to the
corresponding 5'-triphosphates by treatment with phosphorus
oxychloride and then tri-n-butylammonium pyrophosphate [Ruth et
al., Mol. Pharmacol., 415 (1981)]. The resulting crude triphosphate
can be purified at this stage by ion-exchange chromatography by
eluting with a volatile buffer such as triethylammonium
bicarbonate. The desired nucleoside triphosphate is well-separated
from side products, but lyophilization results in some removal of
the protecting group on the linker nitrogen when this protecting
group is a trifluoroacetyl group. Deprotection can be completed by
treatment with 14% aqueous ammonia and the product can again be
purified by ion exchange chromatography. Since the nature of the
linker and its protecting group do not appear to block conversion
to a triphosphate, this methodology can be used to prepare a wide
variety of nucleoside mono-, di-, and triphosphates with protected
or unprotected alkynylamino linkers.
After the preparation of the alkynylamino-nucleotides of this
invention, the stage is set for the production of any desired
reporter-labeled alkynylamino-nucleotide of this invention.
The preferred set of four fluorescently-labeled
alkynylamino-nucleotide chain-terminators (34-37) shown below is
derived from alkynylamino-nucleotides of this invention. This set
of labeled compounds is prepared as described in Example 19 by
coupling N-hydroxysuccinimide esters 2 with alkynylamino nucleoside
triphosphate 6, followed by ammonia deprotection. ##STR12##
These four fluorescently-labeled chain terminators were used in
place of the standard dideoxynucleotide chain terminators when
sequencing DNA using AMV reverse transcriptase according to the
procedure of Zagursky et al., Gene Analysis Techniques, Vol. 2,
89-94 (1985). The resulting fluorescently-labeled sequencing
ladders can be analyzed by an instrument designed to detect
fluorescent molecules as they migrate during gel electrophoresis,
preferably by the instrument described in U.S. Pat. No. 4,833,332
to Robertson et al., issued May 23, 1989. A system of this type
using two filters is described in U.S. Pat. No. 4,833,332, the
contents of which are incorporated herein by reference. As
described in Robertson et al., a pair of modules are positioned
above and below a plane in which the reporter exciting light beam
scans multiple lanes on an electrophoresis gel. Each channel
contains reporter-labeled DNA fragments. Each detection module
comprises a photomultiplier tube having a wide entrance area and a
separate wavelength selective filter positioned between its PMT and
the fluorescent species in the gel. These filters are interference
filters having complementary transmission band characteristic which
simulate the dichroic filter action. The filters permit the PMT' s
to generate signals that vary in amplitude in different senses as a
function of the nature of the species. One filter largely passes
the lower emission wavelengths and rejects the high emission
wavelengths while the other filter does precisely the reverse.
Transmission filters may be used with each interference filter to
reject light from off axis angles greater than a predetermined
angle. The wavelength filters have roughly complementary
transmission vs. wavelength characteristics in the emission region
of the four dyes, with the transition wavelengths occurring near
the center of the species radiant energy spectra.
Detection of the fragments can also be carried out by the methods
described by Smith et al., Hood et al., and Ansorge et al.
Simultaneous analysis of four bases in a single lane using the
information provided by this particular set of four fluorescent
dyes, however, can only be done by means of the signal processing
systems described in the above co-pending patent application.
In order to compare the results obtained by fluorescence detection
with those obtained by standard sequencing techniques, this set of
four labeled chain terminators has also been used to generate
fluorescently-labeled sequencing fragments which also contain a
.sup.32 P reporter. (The .sup.32 P reporter can be enzymatically
incorporated during primer extension by adding labeled dNTP or by
5'-labeling of the primer with a kinase.) The resulting
doubly-labeled sequencing ladders can be analyzed both by
autoradiography and by a fluorescent gel reader. These
fluorescently-labeled sequencing ladders are very similar and
functionally equivalent to ladders produced by the standard
dideoxynucleotide chain terminators except that all bands run
approximately two bases slower. The relative intensity of various
bands in these sequencing ladders is modified by the addition of a
linker and dye, but the modified chain terminator does not appear
to cause any bands to be missed. Under appropriate gel
electrophoresis conditions, the linker and dye on these chain
terminators do not cause any of the bands to migrate anomalously: a
faster-moving band contains fewer bases than a slower-moving band.
The spacing between adjacent bands with different dyes varies
slightly depending on which dyes are next to each other. When using
either fluorescent or radioactive detection under optimal
conditions, these minor variations in relative band intensity and
position do not interfere with the use of these chain terminators
to sequence DNA.
Additionally, the alkynylamino linker can be labeled with biotin.
The resulting biotinylated alkynylamino nucleotides can be used to
generate biotinylated chain termination products suitable for
detection via complexation with labeled avidin or streptavidin.
Preferred biotinylated chain terminators are shown as structures
A-D. ##STR13##
The usefulness of alkynylamino nucleotides for the preparation of
labeled chain terminating substrates for DNA sequencing is not
limited to synthesis of only the set of four compounds shown above
(34-37). Other combinations of sugars, bases and dyes have been
assembled by means of an alkynylamino linker and these compounds
are also useful for sequencing DNA.
In addition to their utility in preparing fluorescently-labeled
chain terminators, the alkynylamino nucleotides of this invention
are generally useful for attaching a variety of reporters to
nucleotides or oligonucleotides. Because the most nucleophilic site
in these molecules is the amino group introduced with the linker, a
reporter containing an activated carboxylic acid (e.g.,
N-hydroxysuccinimide ester), an isocyanate, an isothiocyanate, an
activated aryl halide (e.g., 1-fluoro-2,4-dinitrobenzene), or other
electrophilic functional groups of appropriate reactivity, can be
selectively attached to this nitrogen atom. The resulting labeled
adducts can then be used in other applications to be described
below.
Since the heterocyclic base subunit of nucleotides is used in the
genetic code, the function of many nucleotides is often determined
by the nature of the sugar subunit. Likewise, the utility of
alkynylamino nucleotides depends specifically on what type of sugar
subunit is present. This utility will be diminished if the
alkynylamino linker and/or the reporter interfere with a needed
function of the nucleotide. The alkynylamino linker-containing
nucleotides of this invention have distinct advantages such as: the
small steric bulk of the alkynylamino-linker minimizes perturbation
of the nucleotide; positioning the linker on the 5-position of
pyrimidine nucleotides and the 7-position of 7-deazapurine
nucleotides eventually places the linker and reporter in the major
groove when the nucleotide is incorporated into double-stranded DNA
(this will serve to minimize interference with hybridization and
other processes, which require that a double-stranded conformation
be possible); and alkynylamino-nucleotides with a reporter attached
are excellent substrates for AMV reverse transcriptase. Because
functionally-related enzymes tend to interact with their substrates
in similar ways, it is likely that these nucleotides will also be
substrates for other useful enzymes (such as other reverse
transcriptases, DNA polymerases, and RNA polymerases) which perform
template-directed nucleotide polymerization.
The alkynylamino-nucleotides of this invention offer an attractive
alternative for the chemical (non-enzymatic) synthesis of labeled
2'-deoxyoligonucleotides. Ruth International Application Number:
PCT/US84/00279 discloses a method for incorporating a reporter
group into a defined-sequence single-strand oligonucleotide. The
method includes the preparation of appropriately protected and
activated monomeric nucleotides which possess a linker with a
protected amino group, use of these monomeric nucleotides to
synthesize oligonucleotides chemically, followed by the selective
attachment of a reporter to the linker amino group. The small size
of the alkynylamino linkers and their location on the 5-position of
pyrimidine nucleotides and the 7-position of 7-deazapurine
nucleotides are expected to improve the performance of
oligonucleotides containing them. An appropriately protected and
activated monomer, (38), similar to one described by Ruth, could be
prepared from commercially-available 5-iodo-2'-deoxyuridine by the
Pd(0)/Cu(I) catalyzed attachment of an alkynylamino linker,
followed by the selective dimethoxytritylation of the 5'-alcohol,
and finally conversion to a 3'-phosphoramidite with
chloro(diisopropylamino)methoxyphosphine. This monomer and other
similar alkynylamino-containing monomers are expected to be useful
oligonucleotide synthesis and reporter attachment according to the
methods described by Ruth. (The trifluoroacetyl protecting group on
the linker nitrogen is removed by the basic and/or nucleophilic
reagents normally used for final deprotection during the chemical
synthesis of oligonucleotides.) If the reporter is unaffected by
the reactions used in oligonucleotide synthesis, it could also be
attached to the alkynylamino linker at an earlier stage.
##STR14##
Although chemical synthesis of oligoribonucleotides is currently
not as efficient or useful as synthesis of
2'-deoxyoligonucleotides, an appropriate monomer, [(39),
--O--tetrahydropyranyl (OTHP)] could be prepared and used to make
labeled RNA.
In yet another application, the enzymatic labeling of
double-stranded nucleic acids can be facilitated through the use of
the alkynylamino linkers. Langer et al., Proc. Nat. Acad. Sci. USA,
78, 6633 (1981), disclosed a nick-translation method for labeling
double-stranded DNA with biotin reporters. An allylamino linker was
used to attach biotin to the 5-position of 2'-deoxyuridine
triphosphate and uridine triphosphate. The resulting biotinylated
nucleotides are substrates for DNA and RNA polymerases.
Alternatively, an alkynylamino linker could be used for biotin
attachment or, in general, for the attachment of other reporters
such as fluorescent dyes. Adenosine triphosphate analogs (40) and
(41) with alkynylamino linkers could be prepared more easily than
adenosine analogs with an allylamino linker. Nucleotide
triphosphates analogs of (40) and (41) could be used for
nick-translation labeling of DNA or RNA by the enzymatic procedures
of Langer et al. ##STR15##
In yet another application, alkynylamino nucleosides can be used
for the direct enzymatic incorporation of multiple fluorescent dyes
into nucleic acids. Following incorporation of fluorescence-labeled
alkynylamino nucleotides into nucleic acids, no additional steps
are required prior to detection of the reporter. In contrast, the
above biotinylation methodology of Langer et al. produces nucleic
acids which can be detected by fluorescent means only after they
have undergoine complexation with fluorescence-labeled avidin or
streptavidin. Similarly, nucleic acids tagged with digoxigenin have
to be detected by complexation with labeled anti-digoxigenin
antibodies. Direct labeling with fluorescence-tagged alkynylamino
nucleotides has several advantages over these indirect labeling
methods such as labeling can take place intracellularly, fewer
steps are required, non-specific binding by the reporter protein is
eliminated and signal-to-noise in fluorescence detection is
improved, since a reporter protein (avidin, streptavidin, or
anti-digoxigenin antibody) is not needed, and nucleic acid
hybridization probes can be easily labeled with a variety of
fluorescent dyes. Hybridization probes labeled with biotin or
digoxigenin are conventionally first allowed to hybridize to
homologous target strands and then detected by complexation with a
labeled protein. If the probe is first complexed with the labeled
protein, it will not hybridize efficiently with its homologous
target strand. Indirect labeling methods therefore require a
different ligand and ligand-binding protein for every
distinguishable dye used. Indirect labeling of nucleic acids is
therefore currently limited to using one or two dyes at a time.
Direct labeling nucleic acids with alkynylamino nucleotides does
not suffer from this limitation. Some of the above advantages of
direct labeling (fewer steps, elimination of non-specific binding,
and availability of multiple dyes) are particularly useful for
preparing labeled probes for in situ hybridization. Directly-tagged
hybridization probes have been prepared from genomic, cosmid,
plasmid, and yeast artificial chromosome DNA. After in situ
hybridization, these probes give superior results when analyzed by
fluorescence microscopy.
A preferred family of alkynylamino nucleosides useful for direct
fluorescence-labeled of nucleic acids is shown by structure 42,
wherein Het is a heterocyclic base previously defined. In the case
of multiple-labeling of nucleic acids, the preferred fluorescent
dye can be chosen to fit the requirements of the detection system
being used. Such detection systems include fluorescence microscope,
fluorescence spectrometer, fluorescence microtiter plate reader.
The fluorescent labels which can be attached to the linker nitrogen
include, but are not limited to: xanthenes (e.g., fluoresceins,
eosins, erythrosins, and the previously mentioned succinyl
fluoresceins), rhodamines (e.g., tetramethylrhodamine),
benzimidazoles, ethidiums, propidiums, anthracyclines,
mithramycins, acridines, actinomycins, merocyanines, cyanines,
coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes
(e.g., dansyl, 5-dimethylamino-1-napthalenesulfonyl), salicylic
acids, benz-2-oxa-1,3-diazoles (also known as benzofurans, e.g.,
4-amino-7-nitrobenz-2-oxa-1,3-diazole). For a review of the art in
this field, see A. S. Waggoner, Chapter 1, Applications of
Fluorescence in the Biomedical Sciences, ed. by D. L. Taylor, et
al., Alan R. Liss, New York (1986). A wide variety of these dyes
are commercially available (for example, from Molecular Probes,
Inc., P.O. Box 22010, Eugene, Oreg. 97402) in forms suitable for
selective attachment to the linker nitrogen of alkynylamino
nucleosides. Preferred reagents for attaching fluorescent labels to
alkynylamino nucleotides are fluorescein-5-isothiocyanate and
tetramethylrhodamine-6-isothiocyanate. ##STR16##
Fluorescence-tagged alkynylamino nucleotides of type 42 can be
enzymatically incorporated into nucleic acids by a variety of
techniques familiar to one skilled in the art of nucleic acid
analysis. Some common techniques which have been used successfully
include primer extension, random primer extension, nick
translation, nucleic acid amplification, e.g., as described in
applicants' assignee, E. I. du Pont de Nemours and Company, Inc.,
copending applications Ser. No. 07/329,128, filed Mar. 27, 1989 and
Ser. No. 07/329,142, filed Mar. 27, 1989, and 3'-tailing. In
addition to the above in vitro labeling techniques, in vivo
labeling is also possible. When cells are microinjected with a
fluorescence-labeled alkynylamino nucleotide, newly synthesized DNA
in these cells is fluorescence-labeled. Incorporation of labeled
ribonucleotides during transcription with RNA polymerases can also
occur. Nucleic acid polymerases which have been used to incorporate
labeled alkynylamino nucleotides include AMV reverse transcriptase,
T7 DNA polymerase, E. coli DNA polymerase I (Pol I), the Klenow
fragment of Pol I, Replinase.TM. (the DNA polymerase from Thermus
flavus), Taq DNA polymerase, Vent.TM. DNA polymerase (from
Thermococcus litoralis), and terminal transferase. Dyes which have
been successfully incorporated include fluoresceins, rhodamines,
coumarins, and benzofurazans. Given the likelihood that all nucleic
acid polymerases are evolutionarily derived from the same ancestral
polymerase, it is believed that given alkynylamino nucleotides can
be substrates for any useful nucleic acid polymerase.
In yet another application, Kornher et al. have shown that
alkynylamino nucleotides can be used as "mobility-shifting"
nucleotide analogs in a process for distinguishing nucleic acid
segments on the basis of nucleotide differences. See U.S. Pat. No.
4,879,214. In this assay, multiple alkynylamino nucleotides are
enzymatically incorporated into a nucleic acid and the product is
analyzed by gel electrophoresis. The incorporated mobility shifting
groups (the linker and any group attached to the linker) retard the
mobility of the nucleic acid product. A preferred set of mobility
shifting analogs is shown in structure 43, wherein Het is defined
above. Alkynylamino nucleotides 43 have a larger mobility shifting
group and, therefore, provide better resolution during
electrophoretic analysis. ##STR17##
The following Examples illustrate the invention.
All temperatures are in degrees centigrade. (25.degree. refers to
ambient or room temperature). All parts and percentages not
otherwise indicated are by weight, except for mixtures of liquids
which are by volume. The following abbreviations are employed:
DMF--dimethylformamide; DMSO--dimethylsulfoxide;
NHTFA--trifluoroacetamido-group; TEAB--triethylammonium
bicarbonate; Tris-tris(hydroxymethyl)aminomethane;
SF--succinylfluorescein; NMR--nuclear magnetic resonance spectrum;
IR--infrared spectrum; UV--ultraviolet spectrum or detection;
TLC--thin layer chromatography on silica gel; HPLC--high pressure
liquid chromatography; GC--gas chromatography; mp--melting point;
mp d--melting point with decomposition; bp--boiling point. In
reporting NMR data, chemical shifts are given in ppm and coupling
constants (J) are given in Hertz. All melting points are
uncorrected. Ion exchange resins were washed with appropriate
aqueous and organic solvents prior to use. The identity of all
compounds described herein was established by appropriate
spectroscopic and analytical techniques. Unless otherwise noted,
purification by chromatography on silica gel was performed as
described by Still et al., J. Org. Chem., 43, 2923-2926 (1978).
EXAMPLE 1
Preparation of 5-(3-Amino-1-propynyl)-2',3'-dideoxycytidine
5'-triphosphate (42)
(Compound 42 is an example of structure 6 wherein Het is cytosine
(i) and R.sub.1 is --CH.sub.2 --. It is the immediate precursor to
labeled chain terminator 35.)
A. Preparation of N-propargyltrifluoroacetamide (43)
Propargylamine (24.79 g, 0.450 mole; Aldrich, 99%) was added
dropwise over 1 h to methyl trifluoroacetate (69.19 g, 0.540 mole,
1.2 eq, Aldrich) at 0.degree.. After stirring an additional hour at
0.degree., distillation though a 15 cm Vigreux column afforded
62.12 g (91%) of trifluoroacetamide 43 as a colorless liquid (bp
68.5-69.5.degree. at 11 torr). This material was homogeneous by NMR
and GC and was used interchangeably with
spectroscopically-identical material prepared by acylating
propargylamine with trifluoroacetic acid anhydride.
.sup.1 H-NMR (CDCl.sub.3): 6.85 (broad s, 1H, NHTFA), 4.17 (dd,
J=5.6 and 2.5, 2H, CH.sub.2), 2.35 (t, J=2.5, 1H, CH). IR (neat;
cm.sup.-1): 3300 (N--H), 3095 and 2935 (C--H), 2130 (acetylene),
1720 (C.dbd.O), 1550 (N--H), 1430, 1365, 1160, 1040, 998, 918, 857,
829, 772, and 725.
B. Preparation of 5-Iodo-2',3'-Dideoxycytidine (44)
A solution of 2',3'-dideoxycytidine (2.11 g, 10 mmol, Raylo) and
mercuric acetate (3.35 g, 10.5 mmol, Fisher) in 50 mL of methanol
was refluxed for 19 h. The resulting white suspension was diluted
with methanol (50 mL) and dichloromethane (100 mL). Iodine (3.05 g,
12 mmol) was added and the suspension was stirred at 25.degree.
until a clear purple solution was present. After 4 h, the free base
form of AG3 X4A resin (20 mL, 38 meq, Bio-Rad; a weakly basic
polystyrene resin) was added and hydrogen sulfide was bubbled into
the reaction for 15 min. Complete precipitation of mercury(II) was
verified by TLC. The reaction was filtered though filter aid and
the filter aid was washed with 1:1 methanol-dichloromethane. The
filtrate was evaporated onto silica gel 10 g) and the loaded silica
gel was placed on top of a 150 g silica gel column. Elution with
5%, 10% and 20% methanol in dichloromethane afforded 2.79 g (83%)
of iodide 44 as a colorless crystalline solid. Two
recrystallizations from boiling water afforded, after vacuum-drying
at 50.degree., large, analytically-pure prisms (mp: d
178.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 8.50 (s, 1H, H6), 7.73 (broad s, 1H,
--NH.sub.2 a), 6.53 (broad s, 1H, --NH.sub.2 b), 5.86 (dd, J=6.5
and 2.1, 1H, H1'), 5.19 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.75
(ddd, J=12.1, 5.2, and 2.9, 1H, H5'a), 3.53 (dt, J=12.1 and 3.8,
1H, H5'b), and 2.3-1.7 (m, 4H, H2' and H3'). Calculated for C.sub.9
H.sub.12 N.sub.3 O.sub.3 I: C 32.07%, H 3.59%, N12.46%. Found: C
32.05%, H 3.80%, N 12.46%.
C. A General Procedure for Coupling Aminoalkynes to
Iodonucleosides
Preparation of
5-(3-Trifluoroacetamido-1-propynyl)-2',3'-dideoxycytidine (45)
A 50-mL, thee-necked flask was charged with iodocytidine 44 (770
mg, 2.00 mmol) and cuprous iodide (76.2 mg, 0.400 mmol, 0.20 eq;
Aldrich, Gold Label). After flushing the flask with argon, dry
dimethylformamide (10 mL, Aldrich) was added to produce a 0.2M
solution of iodocytidine which contained suspended cuprous iodide.
N-Propargyltrifluoroacetamide (0.70 mL, 6.00 mmol, 3.0 eq) and
triethylamine (0.56 mL, 4.00 mmol, 2.0 eq, stored over molecular
sieves) were added via syringe.
Tetrakis(triphenylphosphine)palladium(0) (231 mg, 0.20 mmol, 0.10
eq) was weighed into a vial in a dry box and added to the reaction
mixture. The cuprous iodide dissolved, affording a yellow solution
which gradually darkened over several hours. The reaction was
allowed to proceed until TLC indicated that the starting material
was completely consumed. After 4 h, the reaction was diluted with
20 mL of 1:1 methanol-dichloromethane and the bicarbonate form of
AG1X8 resin (Bio-Rad, 2.0 g, ca. 6 eq; a strongly basic, anion
exchange, polystyrene resin) was added. After stirring for about 15
min, evolution of gas ceased. After 30 min, the reaction mixture
was filtered and the resin was washed with 1:1
dichloromethanemethanol. The combined filtrates were rapidly
concentrated with a rotary evaporator. (Removal of
dimethylformamide required about 10 min at 45.degree. and 2 torr.)
The residue was immediately purified by chomatography on 100 g of
silica gel using 10%, 15% and 20% methanol in dichloromethane.
Removal of solvent from the appropriate fractions afforded 651 mg
(90%) of alkynylamine nucleoside 45 as a pale yellow crystalline
foam which was homogeneous by TLC and NMR. The product from a
similar preparation was established to be a hemi-hydrate by
elemental analysis.
.sup.1 H-NMR (DMSO-d.sub.6): 9.96 (broad s, 1H, NHTFA), 8.32 (s,
1H, H6), 7.76 (broad s, 1H, NH.sub.2 a), 6.78 (broad s, 1H,
NH.sub.2 b), 5.88 (dd, J=6.5 and 2.5, 1H, H1'), 5.13 (t, J=5.1, 1H,
5'OH), 4.28 (d, J=5.0, 2H, --CH.sub.2 --), 4.04 (m, 1H, H4'), 3.73
(ddd, J=12.0, 5.0 and 3.1, 1H, H5'a), 3.53 (dt, J=12.1 and 4.0, 1H,
H5'b), 2.3-1.7 (m, 4H, H2' and H3'). .sup.19 F-NMR (DMSO-d.sub.6):
-74.0 (s). UV (MeOH): maxima at 238.5 (17,100) and 295.5 (9,300).
Calculated for C.sub.14 H.sub.15 N.sub.4 O.sub.4 F.sub.3 1/2H.sub.2
O: C 45.53, H 4.37, N 15.17. Found: C 45.56, H 4.52, N 15.26.
D. Preparation of Tris(tri-n-butylammonium) pyrophosphate
Tetrasodium pyrophosphate decahydrate (4.46 g, 10 mmol) was
dissolved in the minimum amount of water (about 50 mL) and passed
though a column of AG50W X8 resin (100-200 mesh, 4.times.10 cm bed;
a strongly-acidic, cation exchange, polystyrene resin) poured in
water. The column was eluted with water and the eluent was
collected in an ice-cooled flask until pH of the eluent approached
neutrality. Tri-n-butylamine (Aldrich Gold Label, 7.1 mL, 30 mmol)
was added to the eluent and the two phases were stirred vigorously
until all of the amine dissolved. The resulting solution was
lyophilized. The residue was co-evaporated twice with dry pyridine
and once with dry dimethylformamide. The residue was dissolved in
dry dimethylforamide (10 mL) and the resulting 1.0M solution was
stored (for as long as one month) at 0.degree. under argon until
used.
E. A General Procedure for Converting Protected Alkynylamino
Nucleosides to the Corresponding 5'-Triphosphates and Removing the
Trifluoroacetyl Protecting Group
Preparation of 5-(3-Amino-1-propynyl)-2',3'-dideoxycytidine
5'-triphosphate (42)
Alkynylamino nucleoside 45 (361 mg, 1.00 mmol) was dissolved in
trimethyl phosphate (2.0 mL, Aldrich Gold Label) while stirring
under argon in an oven-dried flask. The solution was cooled to
-10.degree. and phosphorus oxychloride (0.093 mL, 1.00 mmol,
Aldrich Gold Label) was added by syringe. After stirring the
reaction mixture at -10.degree. for 30 min, a second aliquot of
phosphorus oxychloride (0.093 mL, 1.00 mmol) was added and the
solution was allowed to warm slowly to 25.degree. while stirring.
Aliquots from the reaction mixture were quenched with 1N aqueous
hydroxide and analyzed by HPLC. When conversion to the
corresponding nucleotide monophosphate was at a maximum (in this
case 100 min after the second addition of phosphorus oxychloride),
the reaction mixture was added dropwise to a precooled
(-10.degree.) solution of tris(tri-n-butylammonium pyrophosphate
(6.0 mL of the above 1.0M solution in dry dimethylformamide). The
solution was allowed to warm slowly to 25.degree. while stirring
under argon. After 100 min, the reaction solution was added slowly
to a precooled (0.degree.) solution of triethylamine (1.4 mL) in
water (20 mL). The solution was stirred with ice-cooling for 15 min
and then allowed to stand overnight at about 2.degree..
The volatiles were removed by vacuum evaporation at 25.degree. and
0.5 torr. The residue was redissolved in water (75 mL) and applied
to a column of DEAE-SEPHADEX ion exchanger (A-25-120, 2.6.times.65
cm bed) that had been equilibrated with: 1) pH 7.6, 1.0M aqueous
TEAB (300 mL), 2) 1.0M aqueous potassium bicarbonate (300 mL), and
3) pH 7.6, 0.1M aqueous TEAB (300 mL). The column was eluted with a
linear gradient of pH 7.6 aqueous TEAB from 0.1M (1 L) to 1.0M (1
L). The column was driven at 100 mL/h while collecting fractions
every 12 min. The elution was monitored by absorbance at 270 nm (40
AUFS). The desired material eluted as a well-separated, major band
near the end of the gradient (Fractions 73-80). The
product-containing fractions were pooled, concentrated (at below
30.degree.), and co-evaporated twice with absolute ethanol. The
residue was taken up in water (20.4 mL) and lyophilized.
The intermediate product was taken up in water (12.5 mL) and
concentrated ammonium hydroxide (12.5 mL) was added. After stirring
for 3.5 h, the solution was stirred under aspirator vacuum for 2 h
to remove the excess ammonia gas and then lyophilized. The residue
was taken up in pH 7.6 0.1M aqueous TEAB (10 mL) and applied to a
column of DEAE-SEPHADEX ion exchange resin (A-25-120, 1.6.times.55
cm bed) that had been prepared as described above. The column was
eluted while collecting 6 mL fractions with a linear gradient of
TEAB from 0.1M (280 mL) to 1.0M (280 mL). The product eluted as a
single major peak. The fractions estimated to contain 10 pure
product (#39-45) were pooled, concentrated (at below 30.degree.),
co-evaporated with absolute ethanol (2.times.), and taken up in
water (9.8 mL). The solution was assayed by UV absorption and HPLC
and then lyophilized.
A dilute solution of the product showed absorption maxima at 240
and 293.5 nm in pH 8.2 50 mM aqueous Tris buffer. Assuming an
absorption coefficient for the product equal to that of the
starting material (9,300), the yield of product, based on the
absorption at 293.5 nm, was 0.32 mmol (32%). HPLC (Zorbax SAX, 0.2M
pH 6.5 aqueous potassium phosphate, monitoring 270 nm) of the final
product showed essentially a single peak (>99%).
.sup.1 H-NMR (D.sub.2 O): 8.57 (s, 1H, H6), 6.03 (dd, J=6.4 and
1.6, 1H, H1'), 4.42 (m, 2H, H4' and H5'a), 4.18 (ddd, J=12, 5.5 and
3, 1H, H5'b), 4.036 (s, 2H, --CH.sub.2 --), 2.5-1.9 (m, 4H, H2' and
H3'), plus counterion (triethylammonium) peaks. .sup.31 P-NMR
(D.sub.2 O) -9.02 (d, J=20, 1P), -9.74 (d, J=20, 1P), -21.37 (t,
J=20, 1P). UV (pH 8.2 aq Tris): maxima at 240 and 293.5 nm.
EXAMPLE 2
Preparation of 5-(3-Amino-1-propynyl)-2',3'-dideoxyuridine
5'-triphosphate (46)
(Compound 46 is an example of structure 6 wherein Het is uracil (h)
and R.sub.1 is --CH.sub.2 --. It is the immediate precursor to
labeled chain terminator 34.)
A. Preparation of 5-Iodo-2',3'-dideoxyuridine (47)
Dideoxyuridine (2.122 g, 10.0 mmol) was dissolved in 30 mL of warm
methanol and, after cooling to 25.degree., iodine monochloride
(4.06 g, 25 mmol, 2.5 eq, Fisher) in methanol (20 mL) was added
over 5 min. The dark purple reaction mixture was heated in a
50.degree. bath under nitrogen for 20 min and then immediately
cooled in an ice-water bath. After standing without stirring for
165 min, the resulting precipitate was collected by filtvration and
washed with cold methanol (2.times.10 mL). Vacuum-drying overnight
afforded 2.232 g (66%) of iodide 47 as off-white microcrystals.
This material was used without further purification in the next
reaction, but other preparations were purified by chomatography or
recrystallization from boiling methanol (30 mL/g) to give white
needles (mp d 160-164.degree.). NMR indicated that the crude
precipitate was homogeneous, but also that the 5'-hydroxyl proton
was very broad due to exchange catalyzed by trace impurities.
Chomatographed or recrystallized materials afforded spectra in
which this proton was, as usual, a sharp triplet.
.sup.1 H-NMR (DMSO-d.sub.6): 11.60 (broad s, 1H, H3), 8.57 (s, 1H,
H6), 5.90 (dd, J=2.0 and 6.6, 1H, H1'), 5.2 (broad s, 1H, 5'OH),
4.06 (m, 1H, H4'), 3.75 and 3.53 (m, 2H, H5'), 2.26, 2.02 and 1.84
(m, 4H, H2' and H3').
B. Preparation of
5-(3-Trifluoroacetamido-1-propynyl)-2',3'-dideoxyuridine (48)
Iodouridine 47 was coupled for 3 h to N-propargyltrifluoroacetamide
following the general method given in Example 1C. Chomatography
with a 0-5% methanol in dichloromethane gradient afforded material
which was homogeneous by TLC, but which was difficult to dry. After
co-evaporating the chomatographed product several times with
chloroform and vacuum-drying, 536.5 mg of alkynylamino nucleoside
48 was obtained as a white foam. This material was homogeneous by
TLC and was pure by NMR except for a small amount (39 mole %;
corrected yield 66%) of chloroform.
.sup.1 H-NMR (DMSO-d.sub.6): 11.61 (s, 1H, H3), 10.07 (distorted t,
1H, NHTFA), 8.35 (s, 1H, H6), 7.26 (s, 0.39H, CHCl.sub.3), 5.89
(dd, J=6.6 and 3.2, 1H, H1'), 5.15 (t, J=5.2, 1H, 5'OH), 4.22
(broad d, 2H, --CH.sub.2 N--), 4.04 (apparent hept, J=3.5, 1H,
H4'), 3.73 and 3.53 (m, 2H, H5'), 2.26, 2.03 and 1.84 (m, 4H, H2'
and H3'). TLC (95:5 dichloromethane-methanol, two elutions, UV):
Starting iodide 47, R.sub.f =0.37; product 48, 0.28; catalysts,
0.95 and 0.80 plus slight streakiness.
Preparation of 5-(3-Amino-1-propynyl)-2',3'-dideoxyuridine
5'-triphosphate (46)
Alkynylamino nucleoside 48 (0.30 mmol) was converted to the
corresponding triphosphate and its trifluoroacetyl group was
removed following the general procedure given in Example 1E. After
addition of the second aliquot of phosphorus oxychloride,
phosphorylation was allowed to proceed for a total of 210 min.
Assuming an absorption coefficient for the product equal to that of
the starting material (13,000), the yield of triphosphate 46, based
on its UV absorption at 291.5 nm, was 18%.
EXAMPLE 3
Preparation of 7-(3-Amino-1-propynyl)-2',3'-dideoxyguanosine
5'-triphosphate (49)
(Compound 49 is an example of structure 6 wherein Het is
7-deazaguanine (k) and R.sub.1 is --CH.sub.2 --. It is the
immediate precursor of labeled chain terminator 37.)
A. Preparation of
6-Methoxy-2-methylthio-9-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosy
l)-7-deazapurine (9)
6-Methoxy-2-methylthio-7-deazapurine (8, 9.2 g, prepared following
the procedure of F. Seela et al., Chem. Ber., Vol. 111, 2925
(1978)) was azeotropically dried by dissolving in 150 mL of dry
pyridine and evaporating to dryness at 30-35.degree.. This material
was suspended in 450 mL of dry acetonitrile at room temperature
under nitrogen and sodium hydride (2.16 g of a 60% suspension in
oil) was added with stirring. After 45 min,
1-chloro-2-deoxy-3,5-di-O-p-toluoyl-.alpha.-D-ribofuranose (18.6 g,
prepared following the procedure of M. Hoffer, Chem. Ber., Vol. 93,
2777 (1960)) was added in thee equal portions over a 20 min. After
stirring the reaction mixture for an additional 45 min at room
temperature, acetic acid (1 mL) and dichloromethane (300 mL) were
added. The mixture was suction filtered though a pad of filter-aid,
and the filtrate was evaporated to dryness. The residue was
dissolved in benzene and this solution was washed with water
(2.times.) and brine (1.times.). After drying the organic layer
over sodium sulfate and evaporating, the residue was dissolved in
methanol (400 mL) and allowed to crystallize affording 19.24 g
(73.8%) of ribosylated product 9 as colorless crystals (mp
106-107.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.42 (s, 3H, toluoyl CH.sub.3), 2.44 (s,
3H, toluoyl CH.sub.3), 2.64 (s, 3H, SCH.sub.3), 2.70 and 2.89 (m,
2H, H2'), 4.08 (s, 3H, OCH.sub.3), 4.56 (m, 1H, H3'), 4.65 (m, 2H,
H5'), 5.74 (m, 1H, H4'), 6.44 (d, J=4, 1H, H7), 6.77 (dd, J=8 and
6, 1H, H1'), 7.05 (d, J=4, 1H, H8) and 7.25 and 7.95 (m, 8H,
toluoyl H). Recrystallization of a sample of the above material
from methanol containing a small amount of dichloromethane afforded
crystals of mp 109-110.degree..
B. Preparation of
6-Methoxy-2-methylthio-9-(2-deoxy-.beta.-D-ribofuranosyl)-7-deazapurine
(10)
A suspension of ester 9 (19 g) and the hydroxide form of REXYN 201
resin (38 g; a strongly basic, anion exchange, polystyrene resin)
in 600 mL of methanol was refluxed for 1 5 h under nitrogen. The
hot suspension was suction filtered to remove the resin and the
filtrate was evaporated to dryness. The solid residue was dissolved
in ether (450 mL) and, after 10 min, the solution was filtered
though a pad of filter aid to remove a small amount of a colored
impurity. The solution was seeded with crystals of the desired
product obtained from a previous reaction and allowed to stand
overnight at 25.degree.. Crystalline diol 10 was collected by
filtration and the mother liquor was concentrated to afford a
second crop. Each crop was washed thoroughly with ether and dried
to afford a total of 8.43 g (78.0%) of diol 10 as colorless
crystals (mp 129-130.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 2.21 and 2.55 (m, 2H, H2'), 2.56 (s,
3H, SCH.sub.3), 3.53 (m, 2H, H5'), 3.82 (m, 1H, H3'), 4.02 (s, 3H,
OCH.sub.3), 4.36 (m, 1H, H4'), 4.90 (t, J=5.5, 1H, 5'OH), 5.30 (d,
J=5.5, 1H, 3'OH), 6.48 (d, J=4, 1H, H7), 6.55 (dd, J=8 and 6, 1H,
H1'), 7.48 (d, J=4, 1H, H8). Recrystallization of a sample of this
material from dichloromethane containing a small amount of methanol
afforded crystals of mp 130-131.degree..
C. Preparation of
6-Methoxy-2-methylthio-9-(5-O-triphenylmethyl-2-deoxy-.beta.-D-ribofuranos
yl)-7-deazapurine (11)
Diol 10 (7.2 g) was azeotropically dried by dissolving in dry
pyridine and evaporating the solution to dryness at 35.degree.. The
residue was dissolved in dry pyridine (100 mL) and triphenylmethyl
chloride (8.0 g), triethylamine (4.0 mL), and
4-(dimethylamino)pyridine (300 mg) were added. After heating the
reaction mixture at 65.degree. under nitrogen for 30 min, a second
addition of triphenylmethyl chloride (1.0 g) was made and heating
was continued for 16.5 h. After cooling, the reaction mixture was
concentrated and the residue was partitioned between
dichloromethane and water. The aqueous layer was extracted with
dichloromethane and the combined organic layers were washed with
0.3N hydrochloric acid, aqueous sodium bicarbonate, and brine.
After drying over sodium sulfate and concentrating, purification of
the crude product by chomatography on silica gel with 0%, 1%, 1.5%
and 2% methanol in dichloromethane afforded 12.1 g (94.5%) of
monotrityl ether 11 as a colorless glass.
.sup.1 H-NMR (CDCl.sub.3): 2.58 (s, 3H, SCH.sub.3), 2.42 and 2.62
(m, 2H, H2'), 3.37 (m, 2H, H5'), 4.04 (m, 1H, H3'), 4.08 (s, 3H,
OCH.sub.3), 4.60 (m, 1H, H4'), 6.40 (d, J=4, 1H, H7), 6.68
(apparent t, J=7, 1H, H1'), 7.00 (d, J=4, 1H, H8), 7.27 and 7.43
(m, 15H, trityl H). This data was obtained from a different batch
of 11 prepared as described above.
D. Preparation of
6-Methoxy-2-methylthio-9-(5-O-triphenylmethyl-2,3-dideoxy-.beta.-D-ribofur
anosyl)-7-deazapurine (12)
A solution of trityl ether 11 (12.1 g), 4-dimethylaminopyridine
(9.2 g), and phenyl chlorothionocarbonate (7.5 mL, Aldrich) in dry
dichloromethane (220 mL) was stirred at 25.degree. for 2 h under
nitrogen. Since TLC analysis indicated that the reaction was
incomplete, phenyl chlorothionocarbonate (4.0 mL) was added and the
reaction mixture was stirred for an additional 1 h. The solution
was diluted with dichloromethane (280 mL) and was washed
sequentially with 0.5N hydrochloric acid (500 mL), 0.5N sodium
hydroxide (500 mL), and brine. The organic layer was dried over
sodium sulfate and evaporated to dryness.
The resulting crude thionocarbonate was dissolved in dry toluene
(350 mL) and azoisobisbutyronitrile (350 mg) and tri-n-butyltin
hydride (10 mL) were added. The resulting solution was heated at
100-105.degree. for 10 min. After cooling, the solution was diluted
with a little ether and was shaken with 10% aqueous potassium
fluoride (350 mL). The two layers were filtered though a pad of
filter aid (to remove a dark sludge) and separated. The organic
layer was washed with 0.75N potassium hydroxide and brine, dried
over sodium sulfate and concentrated. Chomatography of the
resulting oil on silica gel with 1:1 dichloromethane-ether and then
with dichloromethane afforded 9.93 g (84.5%) of dideoxynucleoside
12 as a colorless solid (mp 122-124.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.10, 2.33, and 2.43 (m, 4H, H2' and
H3'), 2.60 (s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.08 (s, 3H,
OCH.sub.3), 4.29 (m, 1H, H4'), 6.36 (d, J=3.7, 1H, H7), 6.53 (dd,
J=7 and 4, 1H, H1'), 7.09 (d, 1H, J=3.7, H8), 7.25 and 7.45 (m,
15H, trityl H).
E. Preparation of
7-Iodo-6-methoxy-2-methylthio-9-(5O-triphenylmethyl-2,3-dideoxy-.beta.-D-r
ibofuranosyl)-7-deazapurine (13)
N-Iodosuccinimide (10.0 g) was added to a solution of deazapurine
12 (9.9 g) in dry dimethylformamide (550 mL). After stirring in the
dark under nitrogen for 16 h, 10% aqueous sodium bicarbonate (2.5
mL) was added and the reaction mixture was concentrated in vacuo at
50.degree. to a volume of 100 mL. This solution was partitioned
between water and ethyl acetate. The organic layer was washed with
5% aqueous sodium hydrosulfite and brine, dried over sodium
sulfate, and concentrated. Chomatography of the slightly impure
product on silica gel with dichloromethane afforded 11.68 g (95.6%)
of iodide 13 as a colorless glassy solid.
.sup.1 H-NMR (CDCl.sub.3): 2.06, 2.24, and 2.41 (m, 4H, H2' and
H3'), 2.58 (s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.10 (s, 3H,
OCH.sub.3), 4.29 (m, 1H, H4'), 6.47 (dd, J=6 and 4, 1H, H1'), 7.19
(s, 1H, H8), 7.30 and 7.46 (m, 15H, trityl H). This data was
obtained from a different batch of 13 prepared as described
above.
F. Preparation of
7-Iodo-2-methylthio-9-(5-O-triphenylmethyl-2,3-dideoxy-.beta.-D-ribofurano
syl)-7-deazapurin-4-one (14)
Sodium thiocresolate was prepared by adding sodium methoxide (1 eq)
to a solution of thiocresol in methanol and then evaporating to
dryness. A mixture of methyl ether 13 (4.0 g), sodium thiocresolate
(4.0 g), and hexamethylphosphoramide (10 mL) in dry toluene (150
mL) was refluxed under nitrogen for 4.5 h. After cooling, the
mixture was partitioned between ethyl acetate and water. The
organic layer was washed with water and brine, dried over sodium
sulfate, and evaporated to dryness. Chomatography of the resulting
crude product on silica gel with 0% and 2% methanol in
dichloromethane afforded 3.80 g (97.0%) of deazapurinone 14 as a
colorless glassy solid.
.sup.1 H-NMR (CDCl.sub.3): 2.05, 2.25, and 2.42 (m, 4H, H2' and
H3'), 2.60 (s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.28 (m, 1H,
H4'), 6.40 (dd, J=7 and 4, 1H, H1'), 7.05 (s, 1H, H8), 7.30 and
7.46 (m, 15H, trityl H), 10.00 (broad s, 1H, H1).
G. Preparation of
7-Iodo-5'-O-triphenylmethyl-2',3'-dideoxy-7-deazaguanosine
(15).
Meta-chloroperoxybenzoic acid (1.23 g, 85%, Aldrich) was added to a
stirred solution of methylthio ether 14 (3.6 g) in dry
dichloromethane (150 mL) at 0.degree. under nitrogen. After 15
minutes, the cooling bath was removed and stirring was continued at
25.degree. for 40 min. This solution was washed with aqueous sodium
bicarbonate and brine and dried over sodium sulfate. Methanol (two
percent by volume) was added and the resulting solution was passed
though a short plug of silica gel to remove polar impurities. The
resulting crude sulfoxide (3.07 9) was dissolved in dioxane (40 mL)
and placed in a glass-lined bomb. Ammonia (10.0 g) was added and
the mixture was heated at 100.degree. for 2 h in an autoclave. The
resulting solution was evaporated to dryness. The residue was
dissolved in dichloromethane (20 mL) and filtered though a pad of
filter-aid. Methanol (40 mL) was added to the solution and, on
cooling, 1.57 g of colorless product crystallized. The mother
liquor was evaporated and purified by medium pressure liquid
chomatography on silica gel with 5% methanol in dichloromethane to
afford an additional 328 mg of product as colorless crystals. The
total yield of deazaguanosine 15 was 1.90 g (55.4%).
.sup.1 H-NMR (CDCl.sub.3): 2.05, 2.23, and 2.35 (m, 4H, H2' and
H3'), 3.29 (m, 2H, H5'), 4.26 (m, 1H, H4') 5 90 (broad s, 2H,
NH.sub.2), 6.24 (dd, J=7 and 4, 1H, H1'), 6.90 (s, 1H, H8), 7.30
and 7.46 (m, 15H, trityl H) 10.90 (broad s, 1H, H1).
Recrystallization of a sample of this material from
methanol-dichloromethane afforded crystals of mp
201-203.degree..
H. Preparation of 2',3'-Dideoxy-7-iodo-7-deazaguanosine (16)
A solution of trityl ether 15 (1.7 g) in formic acid (12 mL) was
stirred at room temperature for 10 min. The resulting yellow
suspension was then quickly evaporated to dryness in vacuo at
30.degree.. Chomatography of the residue on silica gel with 5%, 7%,
and 10% methanol in dichloromethane afforded 940 mg of a colorless
solid. Trituration of this solid with ether containing a little
dichloromethane yielded 838 mg (81.0%) of nucleoside 16 as
colorless crystals.
.sup.1 H-NMR (DMSO-d.sub.6) 1.95, 2.09, and 2.26 (m, 4H, H2' and
H3'), 3.48 and 3.54 (m, 2H, H5'), 3.98 (m, 1H, H4'), 4.90 (broad t,
J=5, 1H, 5'OH), 6.08 (m, 1H, H1'), 6.32 (broad s, 2H, NH.sub.2),
7.12 (s, 1H, H8), 10.46 (broad s, 1H, H1).
I. Preparation of
7-(3-Trifluoroacetamido-1-propynyl)-2',3'-dideoxy-7-deazaguanosine
(50)
Iodide 16 (376 mg, 1.00 mmol) was coupled for 2.25 h to
N-propargyltrifluoroacetamide by the general method given in
Example 1C. Product and starting material were indistinguishable by
TLC, so the reaction was monitored by reverse phase HPLC (10 cm
ODS, 1 mL/min, gradient from 100% water to 100% methanol over 5
min, then 100% methanol, with UV detection at 280 nm: starting
iodide 16, 5.49 min; product 50, 5.75 min; intermediate, 6.58 min).
The crude product was poorly soluble in dichloromethane, so it was
concentrated from a dichloromethane-methanol solution onto silica
gel (5 g) before being loaded onto the chomatography column.
Elution with 2%, 5%, 7% and 10% methanol in dichloromethane
afforded 300 mg (78%) of alkynylamino nucleoside 50 as a yellow
solid.
.sup.1 H-NMR (DMSO-d.sub.6): 1.96, 2.08, and 2.28 (m, 4H, H2' and
H3'), 3.47 and 3.55 (m, 2H, H5'), 3.99 (m, 1H, H4'), 4.22 (broad s,
2H, --CH.sub.2 --), 4.90 (t, J=5, 1H, 5'OH), 6.09 (dd, J=6 and 4,
1H, H1'), 6.33 (broad s, 2H, NH.sub.2), 7.30 (s, 1H, H8), 10.05
(broad s, 1H, NHTFA), 10.50 (broad s, 1H, H1). .sup.1 H-Decoupled
.sup.13 C-NMR (DMSO-d.sub.6) 155.5 (q, J=36.5, trifluoroacetyl
carbonyl), 157.8, 153.1 and 149.9 (C2, C4 and C6}, 122.6 (C8),
115.9 (q. J=288, CF3), 99.4 and 97.5 (C7 and C5), 84.2 and 77.4
(acetylenic), 83.2 and 81.0 (C1' and C4'), 62.9 (C5'), 29.7
(propargylic), 31.8 and 25.8 (C2' and C3'). This .sup.13 C-NMR data
was obtained from a different batch of 50 prepared as described
above.
J. Preparation of
7-(3-Amino-1-propynyl)-2',3'-dideoxy-7-deazaguanosine
5'-triphosphate (49)
Alkynylamino nucleoside 50 (0.90 mmol) was converted to the
corresponding 5'-triphosphate and the trifluoroacetyl protecting
group was subsequently removed following the general procedure
given in Example 1E. After the second addition of phosphorus
oxychloride, the reaction was stirred for an additional 165 min.
Assuming an absorption coefficient for the product equal to that of
the starting material (11,900), the yield of 5'-triphosphate 49,
based on its absorption at 272.5 mn, was 18%.
EXAMPLE 4
Preparation of
7-(3-Amino-1-propynyl)-2',3'-dideoxy-7-deazaadenosine
5'-triphosphate (51)
(Compound 51 is an example of structure 6 wherein Het is
7-deazaadenine (j) and R.sub.1 is --CH.sub.2 --. It is the
immediate precursor to labeled chain terminator 36.)
A. Preparation of
2'-Acetoxy-3'-bromo-5'-(2-acetoxyisobutyryl)adenosine (18)
2-Acetoxyisobutyryl bromide (19.5 mL, 150 mmol, 5 eq, prepared
according to the procedure of Russell et al, J. Am. Chem Soc., 95,
4016-4030 (1973)) was added over 15 min to a suspension of
tubercidin (17, 7-deazaadenosine, 6.66 g, 25.0 mmol, sigma) in dry
acetonitrile (250 mL, Aldrich). The suspended solid dissolved in
about 5 min and the reaction was stirred under nitrogen for 22 h at
25.degree.. The reaction mixture was added to a solution of
dipotassium hydrogen phosphate (43.55 g, 300 mmol, 6 eq) in water
(400 mL). After stirring for 30 min, the solution was extracted
with ethyl acetate (1.times.400 mL and 2.times.200 mL). The
combined organic layers were dried over magnesium sulfate and
evaporated to dryness to afford 14.73 g (118%) of white foam. This
material was greater than 95% one slightly broadened spot by TLC
(with UV detection), but NMR showed that one major and at least one
minor product were present. The NMR spectrum was consistent with
the major product being bromoacetate 18.
.sup.1 H-NMR (DMSO-d.sub.6) for the major component 18: 8.08 (s,
1H, H2), 7.34 (d, J=3.7, 1H, H8), 7.12 (broad s, 2H, NH.sub.2),
6.70 (d, J=3.7, 1H, H7), 6.32 (d, J=3.8, 1H, H1'), 5.61 (dd, J=2.4
and 3.8, 1H, H2'), 4.89 (dd, J=2.4 and 4.5, 1H, H3'), 4.43 (m, 1H,
H4'), 4.35 (dd, J=12 and 4, 1H, H5'a), 4.29 (dd, J=12 and 7, 1H,
H5'b), 2.08 (s, 3H, OAc), 2.00 (s, 3H, OAc), and 1.49 (s, 6H,
2CH.sub.3).
B. Preparation of 2',3'-Dideoxy-2',3'-didehydro-7-deazaadenosine
(19)
Zinc-copper couple was freshly prepared by rapidly (total elapsed
time of about 10 min) washing zinc dust (20 g, Mallinkrodt) with 1N
hydrochloric acid (3.times.50 mL), water (2.times.50 mL), 2% cupric
sulfate (2.times.50 mL), water (4.times.50 mL), ethanol (3.times.50
mL) and ether (2.times.50 mL). During each wash, the zinc dust was
stirred in a fritted funnel until it was suspended and the wash was
removed by suction while minimizing exposure of the zinc to air.
The couple was vacuum-dried for 30 min. The above crude
bromoacetate (14.63 g) was dissolved in dry dimethylformamide (150
mL, Aldrich) and approximately 25 mL of solvent was removed with a
rotary evaporator (45.degree., at 2 torr). Fresh zinc-copper couple
(14.63 g, about 9 eq) was added and the resulting suspension was
stirred under nitrogen at 25.degree.. Depending on the quality of
the zinc-copper couple, this reaction can show an induction period
and/or variable rate, so the reaction was allowed to proceed until
TLC (90:9:1 dichloromethane-methanol-concentrated ammonium
hydroxide: starting material R.sub.f =0.45 and products R.sub.f
=0.39 and 0.36) indicated the starting material had been completely
consumed. In this case, the reaction was complete in less than 15
min. After 100 min, saturated aqueous sodium bicarbonate (75 mL)
was added carefully over 10 min to the reaction mixture. The
reaction mixture was filtered though a filter aid and the filter
aid was washed with methanol (2.times.50 mL). The combined
filtrates were evaporated to dryness and the residue was
partitioned between water (150 mL) and ethyl acetate (150 mL). The
aqueous layer was extracted with ethyl acetate (2.times.100 mL) and
the combined organic extracts were dried over magnesium sulfate,
concentrated, and vacuum dried for 1 h.
The resulting dark orange semisolid was dissolved in methanol (100
mL) and then water (25 mL) and REXYN 201 resin (29 g, 4.3 meq/g, 5
eq, hydroxide form) were added. The reaction mixture was refluxed
for a total of 210 min. Monitoring by TLC (85:13:2
dichlormethane-methanol-concentrated ammonium hydroxide:
intermediate, Rf=0.49; final product 19, 0.24) indicated that the
reaction had rapidly halted at about 70% conversion, so after 165
min, additional resin (29 g) was added. Without cooling, the resin
was removed by filtration and washed with 1:1
dichloromethane-methanol (2.times.75 mL). The combined filtrates
were evaporated to dryness and the resulting purple solid was
recrystallized from boiling isopropanol (150 mL) to afford 3.778 g
of olefin 19 as a off-white needles (mp 205-206.degree.). A second
crop of 0.631 g of product (pale purple needles, mp
202-203.degree.) was obtained by concentrating the mother liquors
to 25 mL. Both crops (total 4.409 g, 76%) were homogeneous by TLC
and pure by NMR except for a trace of isopropanol.
.sup.1 H-NMR (DMSO-d.sub.6): 8.07 (s, 1H, H2), 7.15 (d, J=3.6, 1H,
H8), 7.12 (broad s, 1H, H1'), 7.01 (broad s, 2H, NH.sub.2), 6.57
(d, J=3.6, 1H, H7), 6.43 and 6.02 (broad d, J=6.0, 1H each, H2' and
H3'), 4.95 (t, J=6.5, 1H, 5'OH), 4.79 (m, 1H, H4'), and 3.52 (m,
2H, H5').
C. Preparation of 2',3'-Dideoxy-7-deazaadenosine (20)
A 450-mL Parr bottle was charged with olefin 19 (3.80 g), ethanol
(76 mL), 10% palladium on carbon (380 mg, Aldrich) and 40 psi of
hydrogen. After shaking for 4.67 h at 25.degree., 14.5 psi of
hydrogen had been absorbed and hydrogen uptake had ceased. TLC (two
elutions with 85:13:2 dichloromethane-methanol-concentrated
ammonium hydroxide: starting material 19, 0.45; product 20, 0.48)
showed complete conversion to a single Uv-active new product. The
catalyst was removed by filtration though filter aid and washed
with ethanol. Removal of solvent from the filtrate and vacuum
drying overnight afforded 3.98 g (104%) of dideoxynucleoside 20 as
a white foam. NMR indicated that the product was homogeneous except
for the presence of 8 wt % of ethanol (96% corrected yield).
Similar batches of this material resisted crystallization and
became extremely hygroscopic upon azeotropic drying with anhydrous
solvents. Therefore this material was stored under vacuum for about
1 week and used when NMR indicated that the material contained 5 wt
% of ethanol. The lack of crystallinity and spectral
characteristics observed for this product were in accord with those
reported previously by Robins et al., Can. J. Chem., Vol. 55, 1259
(1977).
.sup.1 H-NMR (DMSO-d.sub.6): 8.04 (s, 1H, H2), 7.33 (d, J=3.6, 1H,
H8), 6.97 (broad s, 2H, NH.sub.2), 6.56 (d, J=3.6, 1H, H7), 6.34
(dd, J=5.2 and 6.4, 1H, H1'), 4.96 (t, J=5.6, 1H, 5'OH), 4.33 (t,
J=5.1, 0.43H, ethanol OH), 4.04 (m, 1H, H4'), 3.4-3.6 (m, 2.86H,
H5' H3'), and 1.06 (t, J=7.0, 1.3H, ethanol CH.sub.3).
D. Preparation of 7-Iodo-2',3'-dideoxy-7-deazaadenosine (21)
A mechanically-stirred solution of 95% pure dideoxynucleoside 20
(2.95 g, 11.96 mmol), anydrous sodium acetate (4.13 g, 50.3 mmol, 4
eq), and mercuric acetate (3.81 g, 11.95 mmol, 1.00 eq, Fisher,
99.9%) in water (190 mL) was heated under nitrogen at 65.degree.
for 2 h. After cooling the resulting white suspension of mercurial
to 25.degree., iodine (4.79 g, 18.9 mmol, 1.6 eq) and ethyl acetate
(190 mL) were added. After 1 h, the suspended mercurial had been
consumed and a clear purple solution remained. After 2 h, sodium
sulfite (6.35 g) was added and the purple color disappeared. After
stirring for 30 min, hydrogen sulfide gas was gently bubbled into
the reaction for 15 min. Mercuric sulfide (a black colloid) and
iodide 21 (a white powder) precipitated from the reaction. Complete
precipitation of mercury(II) was assessed by TLC by monitoring the
disappearence of one of the two major UV-active spots. The reaction
mixture was filtered though filter aid and separated into two
layers. The filter aid was washed with boiling ethyl acetate
(9.times.100 mL) until TLC indicated that no further product was
being extracted. Each ethyl acetate extract was washed with the
aqueous layer. The combined ethyl acetate layers were dried over
magnesium sulfate and evaporated to dryness. The resulting crude
solid turned red upon exposure to air. This material was dissolved
in 3:1 dichloromethane-methanol (100 mL) and the free base form of
AG3 X4A anion exchange resin (5.0 g, BioRad, 2.9 meq/g dry) was
added. Hydrogen sulfide was bubbled into the red solution for 10
min and the red color was discharged. A slight cloudiness was
eliminated by briefly warming and the solution was rapidly filtered
though a 2 cm plug (15 g) of silica gel. The silica gel was eluted
with additional 3:1 dichloromethane-methanol (100 mL). silica gel
(50 g) was added to the filtrate and hydrogen sulfide was bubbled
in for 10 min. The solvent was removed from this mixture with a
rotary evaporator and the silica gel was "dried" by co-evaporating
with chloroform (200 mL). This silica gel was rapidly loaded onto a
silica gel column (500 g) which had been degassed with a stream of
nitrogen. Elution under nitrogen with 5% (6 L) and 10% (4 L)
boiling methanol in dichloromethane afforded 2.92 g (64%) of iodide
21 as a white powder and 456 mg (7.5%) of less polar
7,8-diiodo-2',3'-dideoxy-7-deazaadenosine. Recrystallization of the
major product from boiling ethyl acetate (200 mL) afforded 2.626 g
of white needles (mp 158-160.degree.). Concentration of the mother
liquors to 10 mL afforded a second crop of 0.391 g of light red
needles (mp 156-158.degree.). Both crops were homogeneous according
to NMR and TLC and together represent a 64% overall yield of
iodonucleoside 21 from olefin 19.
.sup.1 H-NMR (DMSO-d.sub.6): 8.09 (s, 1H, H2), 7.67 (s, 1H, H8),
6.65 (broad s, 2H, NH.sub.2), 6.34 (dd, J=4.4 and 6.8, 1H, H1'),
4.95 (t, J=5.5, 1H, 5'OH), 4.04 (apparent hept, J=3.5, 1H, H4'),
3.59 and 3.49 (m, 2H, H5'), 2.30, 2.28 and 2.00 (m, 4H, H2' and
H3').
E. Preparation of
7-(3-Trifluoroacetamido-1-Propynyl)-2',3'-dideoxy-7-deazaadenosine
(52)
Iodide 21 (720.3 mg, 2.00 mmol) was coupled for 90 min with
N-propargyltrifluoroacetamide following the standard procedure
given in Example 1C. Chomatography with 7% methanol in
dichloromethane afforded 705.8 mg (92%) of coupling product 52 as
an off white powder which was homogeneous according to NMR and TLC.
Recrystallization from boiling ethyl acetate (10 mL) afforded 372
mg of white microcrystals (mp 169-171.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 10.1 (distorted t, 1H, NHTFA), 8.10
(s, 1H, H2), 7.78 (s, 1H, H8), 6.0-7.5 (very broad s, 2H,
NH.sub.2), 6.34 (dd, J=4.5 and 7.0, 1H, H1'), 4.98 (t, J=5, 1H,
5'OH), 4.31 (slightly broadened s, 2H, --CH.sub.2 N--), 4.10
(apparent hept, J=3.5, 1H, H4'), 3.60 and 3.40 (m, 2H, H5'), 2.37,
2.18 and 2.00 (m, 4H, H2' and H3'). TLC (90:9:1
dichloromethane-methanol-concentrated ammonium hydroxide; UV):
starting iodide 21, R.sub.f =0.36; product 52, 0.26).
F. Preparation of
7-(3-Amino-1-propynyl)-2',3'-dideoxy-7-deazaadenosine
5'-triphosphate (51)
Alkynylamino nucleoside 52 (1.00 mmol) was converted to the
corresponding 5'-triphosphate and the trifluoroacetyl group was
removed following the general procedure described in Example 1E.
After addition of the second aliquot of phosphorus oxychloride, the
solution was stirred for 120 min. Assuming an absorption
coefficient for the product equal to that of the starting material
(12,700), the yield of triphosphate 51, based on the absorption at
279.5 nm, was 40%.
.sup.1 H-NMR (D.sub.2 O): 7.97 (s, 1H, H2), 7.80 (s, 1H, H8), 6.33
(m, 1H, H1'), 4.44 (m, 1H, H4'), 4.27 (m, 1H, H5'a), 4.14 (m, 1H,
H5'b), 4.11 (broad s, 2H, --CH.sub.2 --), 2.6-2.0 (m, 4H, H2' and
H3'), plus counterion (triethylammonium) peaks. .sup.31 P-NMR
(D.sub.2 O): -8.59 (broad d, J=20, 1P), -9.56 (d, J=20, 1P), and
-21.38 (m, 1P). UV (pH 8.2 aq Tris): maxima at 238 and 279.5
nm.
EXAMPLE 5
A Second Preparation of 7-Iodo-2',3'-dideoxy-7-deazaadenosine
(21)
(Compound 21 is an intermediate prepared and used in Example
4.)
A. Preparation of
6-Chloro-2-methylthio-9-(2-deoxy-.beta.-D-ribofuranosyl)-7-deazapurine
(23)
Methanol (210 mL) and concentrated ammonium hydroxide (210 mL) were
added to a solution of
6-chloro-2-methylthio-9-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl
)-7-deazapurine (22, 26.8 g, prepared as described by Kazamierczuk
et al., J. Am. Chem. Soc., Vol. 106, 6379 (1984)) in
dichloromethane (210 mL). The resulting mixture was stirred at room
temperature for 5 d and then evaporated to dryness. The residue was
dried by co-evaporation with ethanol. The crude product was
dissolved in dichloromethane and colorless crystals precipitated
upon standing. The precipitate was collected and washed thoroughly
with ether to afford 14.5 g (79.1%) of diol 23 (mp d
190-192.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 2.26 and 2.55 (m, 2H, H2'), 2.57 (s,
3H, SCH.sub.3), 3.54 (m, 2H, H5'), 3.84 (m, 1H, H3'), 4.37 (m, 1H,
H4'), 4.95 (m, 1H, OH), 5.34 (m, 1H, OH), 6.57 (m, 1H, H1'), 6.63
(m, 1H. H7), 7.80 (m, 1H, H8). This data was obtained from a
different batch of diol 23 prepared as described above.
B. Preparation of
6-Chloro-2-methylthio-9-(5-O-triphenylmethyl-2-deoxy-.beta.-D-ribofuranosy
l)-7-deazapurine (24)
Diol 23 (14.5 g) was dried by co-evaporation with dry pyridine.
Triphenylmethyl chloride (16 g), 4-(dimethylamino)pyridine (600
mg), and triethylamine (8.0 mL) were added to a solution of the dry
diol in dry pyridine (200 mL). After stirring the reaction mixture
at 65.degree. under nitrogen for 6 h, additional triphenylmethyl
chloride (2.0 g) and triethylamine (1.0 mL) were added and heating
was continued for 17 h. After cooling, methanol (3 mL) was added
and the reaction mixture was evaporated to dryness. The residue was
partitioned between dichloromethane and 0.3N hydrochloric acid. The
organic layer was washed with aqueous sodium bicarbonate and brine,
dried over sodium sulfate, and evaporated to dryness. Chomatography
of the resulting crude product on silica gel with 1% and 1.5%
methanol in dichloromethane afforded 22.7 g (88.6%) of monotrityl
ether 24 as a glassy solid.
.sup.1 H-NMR (CDCl.sub.3): 2.48 and 2.60 (m, 2H, H2'), 2.59 (s, 3H,
SCH.sub.3), 3.40 {m, 2H, H5'), 4.08 (m, 1H, H3'), 4.61 (m, 1H,
H4'), 6.43 (m, 1H. H7), 6.68 (m, 1H, H1'), 7.2-7.5 (m, 16H, trityl
H and H8). This data was obtained from a different batch of 24
prepared as described above.
C. Preparation of
6-Chloro-2-methylthio-9-(5-O-triphenylmethyl-2,3-dideoxy-.beta.-D-ribofura
nosyl)-7-deazapurine (25)
4-(Dimethylamino)pyridine (16.5 g) and phenyl chlorothionocarbonate
(13.5 mL) were added to a solution of trityl ether 24 in dry
dichloromethane (300 mL). After stirring the reaction mixture at
room temperature under nitrogen for 2.25 h, dichloromethane (200
mL) was added. The solution was washed with 0.5N hydrochloric acid
(700 mL), 0.5N sodium hydroxide (700 mL), and brine. The organic
layer was dried over sodium sulfate and evaporated to dryness.
The resulting crude thiocarbonate was dissolved in dry toluene (450
mL) and the solution was heated to a gentle reflux.
Azoisobisbutyronitrile (600 mg) and tri-n-butyltin hydride (17.7
mL) were added. After stirring at reflux under nitrogen for 15 min,
additional tri-n-butyltin hydride (2.0 mL) was added and the
reaction mixture was refluxed for another 15 min. After cooling,
the reaction mixture was diluted with ether (200 mL) and washed
with 10% aqueous potassium fluoride (500 mL), 0.75N potassium
hydroxide (500 mL), and brine After drying over sodium sulfate and
concentrating, chomatography of the resulting crude product on
silica gel with 2:1 dichloromethane-ether and dichloromethane
afforded 10.1 g of dideoxynucleoside 25. The impure fractions were
combined and rechomatographed to afford an additional 3.76 g of
pure product. These products were combined to afford 13.9 g (63.0%)
of 25 as a colorless solid (mp 140-142.5.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.11, 2.36, and 2.46 (m, 4H, H2' and
H3'), 2.60 (s, 3H, SCH.sub.3), 3.33 (apparent d, J=4, 2H, H5'),
4.32 (m, 1H, H4'), 6.39 (d, J=3.7, 1H, H7), 6.52 (dd, J=6.7 and
3.7, 1H, H1'), 7.25 and 7.45 (m, 15H, trityl H), 7.32 (d, 1H,
J=3.7, H8). This data was obtained from a different batch of 25
prepared as described above.
D. Preparation of
6-Chloro-2-methylthio-9-(2',3'-dideoxy-.beta.-D-ribofuranosyl)-7-deazapuri
ne (26)
Trifluoroacetic acid (10 mL) was added to a solution of trityl
ether 25 (7.58 g) in 1:1 methanol-dichloromethane (100 mL) and the
solution was stirred at 25.degree. under nitrogen for 17 h. The
reation mixture was partitioned between dichloromethane (500 mL)
and aqueous sodium bicarbonate, and the aqueous layer was
re-extracted with dichloromethane. The combined organic layers were
dried over sodium sulfate and evaporated to dryness. Chomatography
of the residue on silica gel with 0% and 5% methanol in
dichloromethane afforded 4.07 g (97.1%) of nucleoside 26 as a thick
colorless glass.
.sup.1 H-NMR (CDCl.sub.3): 2.16, 2.26, and 2.50 (m, 4H, H2' and
H3'), 2.63 (s, 3H, SCH.sub.3), 2.76 (broad s, 1H, OH), 3.67 and
3.93 (m, 2H, H5'), 4.27 (m, 1H, H4'), 6.38 (dd, J=6.7 and 5.2 Hz,
1H, H1'), 6.51 (d, J=3.7 Hz, 1H, H7), 7.26 (d, 1H, J=3.7 Hz, H8).
This data was obtained from a different batch of 26 prepared as
described above.
E. Preparation of 2',3'-dideoxy-2-methylthio-7-deazaadenosine
(27)
Ammonia (10 g) was distilled into a solution of chloride 26 (1.83
g) in methanol (50 mL) in a glass-lined bomb. The solution was
heated in an autoclave at 100.degree. for 15 h. After cooling, the
reaction mixture was evaporated to dryness. Purification of the
resulting crude product on silica gel with 0%, 3% and 5% methanol
in dichloromethane afforded 1.27 g (80.4%) of deazaadenosine 27 as
a colorless solid (mp 184-185.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 2.01, 2.21, and 2.39 (m, 4H, H2' and
H3'), 2.45 (s, 3H, SCH.sub.3), 3.50 (m, 2H, H5'), 4.02 (m, 1H,
H4'), 4.83 (t, J=5.5, 1H, 5'OH), 6.32 (dd, J=7 and 4.5, 1H, H1'),
6.50 (d, J=3.7, 1H, H7], 7.07 (broad s, 2H, NH.sub.2), 7.20 (d, 1H,
J=3.7, H8).
F. Preparation of 2',3'-Dideoxy-7-deazaadenosine (20)
A mixture of 600 mg of 27 and excess Raney Nickel (Aldrich,
pre-washed with water and methanol) was refluxed under nitrogen
until TLC indicated the disappearence of the starting material (6
h). The hot solution was filtered though filter-aid and the
collected Raney nickel was washed well with methanol. The combined
filtrates were evaporated to afford 424 g (84.9%) of 20 as a
colorless glassy solid identical to the material prepared in
Example 4C.
G. Preparation of 7-Iodo-2',3'-dideoxy-7-deazaadenosine (21)
Dideoxy-7-deazaadenosine 20 was iodinated following the procedure
given in Example 4D.
EXAMPLE 6
A Third Preparation of 7-Iodo-2',3'-dideoxy-7-deazaadenosine
(21)
(Compound 21 is an intermediate prepared and used in Example
4.)
A. Preparation of
6-Chloro-9-(2-deoxy-.beta.-D-ribofuranosyl)-7-deazapurine (29)
A solution of concentrated ammonium hydroxide (100 mL) in methanol
(175 mL) was added to a solution of
6-chloro-9-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl)-7-deazapuri
ne (28, 10.0 g; prepared as described by Z. Kazimierczuk et al., J.
Amer. Chem. Soc., Vol 106, 6379 (1984)) in dichloromethane (100
mL). After stirring the resulting mixture at 25.degree. for 24 h,
additional concentrated ammonium hydroxide (50 mL) was added. After
stirring for a total of 5 d, the reaction mixture was evaporated to
dryness and the crude product co-evaporated with ethanol. The
residue was dissolved in dichloromethane and the desired product
crystallized. Filtration and drying afforded 4.90 g (92%) of
nucleoside 29 as colorless crystals (mp 155.5-158.5.degree.).
.sup.1 H-NMR (DMSO-d.sub.6): 2.30 and 2.55 (m, 2H, H2'), 3.58 (m,
2H, H5'), 3.85 (m, 1H, H3'), 4.40 (m, 1H, H4'), 4.97 (m, 1H, OH),
5.35 (m, 1H, OH), 6.65 (m, 1H, H1'), 6.75 (d, 1H. H7), 8.00 (m, 1H,
H8), 8.65 (s, 1H, H2). This data was obtained from a different
batch of 29 prepared as described above.
B. Preparation of
6-Chloro-9-(5-O-triphenylmethyl-2-deoxy-.beta.-D-ribofuranosyl)-7-deazapur
ine (30)
Nucleoside 29 (2.5 g) was dried by co-evaporation with dry
pyridine. The residue was redissolved in dry pyridine (40 mL) and
triphenylmethyl chloride (2.5 g), 4-(dimethylamino)pyridine (120
mg), and triethylamine (1.6 mL) were added. The reaction mixture
was stirred at 65.degree. for 4 h under nitrogen. Additional
triphenylmethyl chloride (1.0 g) and triethylamine (0.6 mL) were
added and the reaction was stirred at 75.degree. for 18 h. After
cooling, methanol (2 mL) was added and the reaction mixture was
evaporated to dryness. The residue was partitioned between
dichloromethane and 0.5N hydrochloric acid. The organic layer was
washed with aqueous sodium bicarbonate and brine, dried over sodium
sulfate, and evaporated to dryness. Chromatography on silica gel
with 0%, 1.5% and 3% methanol in dichloromethane afforded 2.26 g
(48%) of trityl ether 30 as a glassy solid.
.sup.1 H-NMR (CDCl.sub.3): 2.46 and 2.65 (m, 2H, H2'), 3.40 (m, 2H,
H5'), 4.10 (m, 1H, H3'), 4.65 (m, 1H, H4'), 6.55 (d, 1H. H7), 6.72
(m, 1H, H1'), 7.2-7.5 (m, 16H, trityl H and H8), and 8.60 (s, 1H,
H2).
C. Preparation of
6-Chloro-9-(5-O-triphenylmethyl-2-deoxy-3-thionocarbophenoxy-.beta.-D-ribo
furanosyl)-7-deazapurine (30a)
4-(Dimethylamino)pyridine (1.35 g) and phenyl chlorothionocarbonate
(1.20 mL) were added to a solution of trityl ether 30 in dry
dichloromethane (30 mL). After stirring the reaction mixture under
nitrogen for 2 h at 25.degree., additional dichloromethane (20 mL)
was added and the solution was washed with 0.5N hydrochloric acid,
0.5N sodium hydroxide, and brine. The organic layer was dried over
sodium sulfate and evaporated to dryness. Trituration of the
residue with dichloromethane-ether afforded 1.53 g (76%) of
thiocarbonate 30a as colorless crystals (mp
186.5-188.5.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.85 and 3.00 (m, 2H, H2'), 3.55 (m, 2H,
H5'), 4.50 (m, 1H, H4'), 6.00 (m, 1H, H3'), 6.60 (d, 1H. H7), 6.85
(m, 1H, H1'), 7.1-7.5 (m, 20H, trityl and phenyl H), 7.50 (d, 1H,
H8), and 8.60 (s, 1H, H2).
D. Preparation of
6-Chloro-9-(5-O-triphenylmethyl-2,3-dideoxy-.beta.-D-ribofuranosyl)-7-deaz
apurine (31)
A solution of thiocarbonate 30a (1.2 g), azoisobisbutyronitrile (50
mg), and tri-n-butyltinhydride (0.60 mL) in dry toluene (50 mL) was
heated at 110.degree. under nitrogen for 15 min. After cooling, the
reaction mixture was diluted with 50 mL of ether and washed with
10% aqueous potassium fluoride (50 mL) and brine. The organic layer
was dried over sodium sulfate and evaporated to dryness.
Chromatography of the resulting crude product on silica gel with 0%
and 1.5% methanol in dichloromethane afforded 0.84 g (92%) of
dideoxynucleoside 31 as a colorless solid (mp 60-63.5.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.11, 2.36, and 2.50 (m, 4H, H2' and
H3'), 3.37 (m, 2H, H5'), 4.35 (m, 1H, H4'), 6.50 (d, J=3.7, 1H,
H7), 6.58 (dd, 1H, H1'), 7.25 and 7.45 (m, 15H, trityl H), 7.55 (d,
1H, J=3.7, H8), and 8.60 (s, 1H, H2).
E. Preparation of
6-Chloro-9-(2,3-dideoxy-.beta.-D-ribofuranosyl)-7-deazapurine
(31a)
Trifluoroacetic acid (1.5 mL) was added to a solution of trityl
ether 31 (700 mg) in 1:1 methanoldichloromethane (20 mL). After
stirring under nitrogen at 25.degree. for 17 h, sodium bicarbonate
(1.5 g) was added and the mixture was stirred for 30 min. The
reaction mixture was filtered and evaporated to dryness.
Chromatography of the resulting crude product on silica gel with 0%
and 2% methanol in dichloromethane afforded 300 mg (84%) of alcohol
31a as a colorless glass.
.sup.1 H-NMR (CDCl.sub.3): 2.20, 2.40, and 2.65 (m, 4H, H2' and
H3'), 3.65 and 4.00 (m, 2H, H5'), 3.95 (broad s, 1H, OH), 4.35 (m,
1H, H4'), 6.28 (dd, 1H, H1'), 6.62 (d, J=4, 1H, H7), 7.40 (d, 1H,
J=4,H8), and 8.65 (s, 1H, H2).
F. Preparation of
6-Chloro-9-(5-acetoxy-2,3-dideoxy-.beta.-D-ribofuranosyl)-7-deazapurine
(32)
Acetic anhydride (2.0 mmol) was added to a solution of alcohol 31a
(284 mg) in dry pyridine (10 mL). After stirring the solution for
1.25 h at 25.degree., methanol (10 mL) was added. After stirring an
additional 30 min, the reaction mixture was evaporated to dryness.
The residue was dissolved in dichloromethane and this solution was
washed with 1N hydrochloric acid (2.times.) and brine (1.times.).
The organic layer was dried over sodium sulfate and evaporated to
dryness to afford 295 mg (89%) of crude acetate 32 as a colorless
glass.
.sup.1 H-NMR (CDCl.sub.3): 2.07 (s, 3H, acetyl), 2.20, 2.45, and
2.55 (m, 4H, H2' and H3'), 4.25 and 4.35 (m, 2H, H5'), 4.40 (m, 1H,
H4'), 6.55 (dd, 1H, H1'), 6.65 (d, 1H, H7), 7.50 (d, 1H, H8), and
8.60 (s, 1H, H2).
G. Preparation of
6-Chloro-7-iodo-9-(5-O-acetyl-2,3-dideoxy-.beta.-D-ribofuranosyl)-7-deazap
urine (33)
A solution of iodine monochloride (340 mg) in dichloromethane
(about 1 mL) was added to a solution of acetate 32 (200 mg) in dry
dichloromethane (20 mL). After stirring at 25.degree. for 3 h, the
reaction mixture was partitioned between dichloromethane and
aqueous sodium hydrosulfite. The organic layer was washed with
aqueous sodium bicarbonate and brine, dried over sodium sulfate and
evaporated to dryness. The residue was triturated with
dichloromethane-ether to afford 135 mg (47%) of colorless crystals
(mp 132.5-134.degree.).
.sup.1 H-NMR (CDCl.sub.3): 2.10, 2.40, and 2.55 (m, 4H, H2' and
H3'), 2.17 (s, 3H, COCH.sub.3), 4.27 and 4.37 (m, 2H, H5'), 4.40
(m, 1H, H4'), 6.55 (dd, 1H, H1'), 7.72 (s, 1H, H8), and 8.60 (s,
1H, H2).
H. Preparation of 7-Iodo-2',3'-dideoxy-7-deazaadenosione (21)
Ammonia (4 g) was added to a solution of 125 mg of chloride 33 in
methanol (20 mL) in a glass-lined bomb. The bomb was heated in an
autoclave at 100.degree. for 3 h. After cooling, the reaction
mixture was evaporated to dryness. The residue was dissolved in hot
ethyl acetate and the hot solution was filtered though a pad of
filter aid. After evaporating the filtrate to dryness, the residue
was triturated with ether to afford 85 mg of slightly impure
product 21 as colorless crystals. Further purification of this
material by preparative TLC on a silica gel with 5% methanol in
dichloromethane afforded 67 mg (63%) iodide 21 as a colorless
solid. This material was identical to that prepared in Example
4D.
EXAMPLE 7
Preparation of 7-Iodo-2',3'-dideoxy-7-deazainosine (53)
(Compound 53 is an example of structure 4 wherein Het is
7-deazahypoxanthine (1).)
Water (18 mL) was added dropwise to a suspension of deazaadenosine
21 (720.3 mg, 2.00 mmol) in glacial acetic acid (2.0 mL) under
argon to produce a clear solution. Solid sodium nitrite (1.38 g,
20.0 mmol, 10 eq) was added though a stream of argon in small
batches over 10 min. The resulting cloudy reaction mixture was
mechanically stirred under argon and a gummy precipitate gradually
formed. After 18 h, the reaction was filtered and the precipitate
was washed thoroughly with ethyl acetate (100 mL) and water (about
10 mL). The combined filtrates were partitioned and the aqueous
layer was extracted with ethyl acetate (2.times.50 mL). The
combined organic layers were dried over magnesium sulfate and
evaporated to dryness. According to TLC, both the precipitate and
the ethyl acetate extracts consisted of product 53 and contaminated
with less than 5% of unreacted 21. Both batches of product were
dissolved in 1:1 methanol-dichloromethane, combined, and evaporated
onto silica gel (7 g). The silica gel was co-evaporated with
chloroform (50 mL) and placed on a silica gel column (50 g).
Elution with 8% methanol in dichloromethane afforded 558.3 mg (78%)
of deazainosine 53 as a pale yellow solid. Two crops of white
needles were obtained by recrystallizing this material from boiling
isopropanol. These needles exhibited a melting point with
decomposition that varied between 200.degree. and 210.degree.. The
chromatographed and recrystallized products were homogeneous by TLC
and NMR except for the presence of isopropanol (5 mole %).
.sup.1 H-NMR (DMSO-d.sub.6): 12.04 (broad s, 1H, H1), 7.93 (s, 1H,
H2), 7.56 (s, 1H, H8), 6.29 (dd, J=4.0 and 6.8, 1H, H1'), 4.94 (t,
J=5.0, 1H, 5'OH), 4.04 (apparent hept, J=3.5, 1H, H4'), 2.36, 2.18
and 1.99 (m, 4H, H2' and H3').
EXAMPLE 8
Preparation of 5-(3-Amino-1-propynyl)-2'-deoxycytidine
5'-triphosphate (54)
(Compound 54 is an example of an alkynylamino nucleotide (I)
wherein R.sub.1 is --CH.sub.2 --, Het is cytosine (i), R.sub.2,
R.sub.3, R.sub.7, and R.sub.8 are H, R.sub.6 is OH, and R.sub.5 is
P.sub.3 O.sub.9 H.sup.3-.)
A. Preparation of 5-Iodo-2'-deoxycytidine (55)
A solution of 2'-deoxycytidine monohydrate (1.226 9, 5.00 mmol,
Aldrich) and mercuric acetate (1.753 g, 5.5 mmol, 1.1 eq, Fisher)
in methanol (20 mL) was refluxed for 14.5 h. The resulting white
suspension was diluted with methanol (30 mL) and dichloromethane
(50 mL) and then iodine (1.522 g, 6.00 mmol, 1.2 eq) was added.
After stirring for 60 min, the resulting purple solution had
decolorized and unreacted mercurial was still visible as a white
suspension. After 100 min and 240 min, further additions of iodine
(0.381 g, 1.5 mmol, 0.3 eq and 0.122 g, 0.50 mmol, 0.1 eq;
respectively) were made. After a total of 5 h, the reaction was
crystal clear and purple. AG3 X4A resin in the free base form (5.17
g, 2.9 meq/g, 3 eq, Bio-Rad) was added and then hydrogen sulfide
was bubbled into the reaction mixture for 5 min. Complete
precipitation of mercury(Il) was verified by TLC. The reaction was
filtered though filter aid and the filter aid was washed with 1:1
methanol-dichloromethane. Silica gel (5 g) was added to the
combined filtrates and the reaction mixture was evaporated to
dryness. The silica gel was co-evaporated with chloroform (50 mL)
and placed on a silica gel column (50 g). Elution with 15%, 20% and
30% methanol in dichloromethane afforded 1.378 g (78%) of
iodocytidine 55 as a white powder. Recrystallization from boiling
methanol (35 mL) afforded, after vacuum-drying overnight. 0.953 g
of white needles (mp 179-180.degree.). Concentration of the mother
liquors to 10 mL afforded a second crop of 0.140 g of pale yellow
needles (mp 172-174.degree.). With the exception of a trace of
methanol, both crops (total yield, 62%) were homogeneous according
to TLC and NMR.
.sup.1 H-NMR (DMSO-d.sub.6): 8.28 (s, 1H, H6), 7.8 and 6.6 (broad
s, 2H, NH.sub.2), 6.08 (t, J=6.3, 1H, H1'), 5.20 (d, J=4, 1H,
3'OH), 4.90 (t, J=5, 1H, 5'OH), 4.20 (m, 1H, H4'), 3.77 (distorted
q, 1H, H3'), 3.60 and 3.54 (m, 1H, H5'), 2.12 and 1.98 (m, 1 H,
H2'). TLC (75:20:5 dichloromethane-methanol-concentrated ammonium
hydroxide; UV): starting material, R.sub.f =0.15; product 55, 0.33;
mercury(II), 0.54.
B. Preparation of
5-(3-Trifluoroacetamido-1-propynyl)-2'-deoxycytidine (56)
Iodide 55 (353.1 mg, 1.00 mmol) was coupled for 4 h to
N-propargyltrifluoroacetamide following the general procedure given
in Example 1C. Chomatography of the crude product with a 0-20%
methanol in dichloromethane gradient afforded 3.84 g (102%) of
white powder after vacuum drying overnight. This material was
homogeneous by TLC, but tenaciously retained solvent.
Recrystallization of this powder from boiling isopropanol (10 mL)
and cooling to -20.degree. afforded 299.6 mg (74%) alkynylamino
nucleoside 56 as white needles (mp 168-170.degree.). NMR showed
that the recrystallized product was homogeneous and that the
crystals contained 0.5 molecules of isopropanol per molecule of
product 56.
.sup.1 H-NMR (DMSO-d.sub.6): 9.96 (broad s, 1H, NHTFA), 8.15 (s,
1H, H6), 7.83 and 6.86 (broad s, 2H, NH.sub.2), 6.10 (t, J=6.5, 1H,
H1'), 5.21 (d, J=4.5, 1H, 3'OH), 5.06 (t, J=5, 1H, 5'OH), 4.35 (d,
J=4, 0.5H, isopropanol OH), 4.28 (broad s, 2H, --CH.sub.2 N--),
4.20 (apparent hex, J=3.5, 1H, H4'), 3.79 (m, 1.5H, H3' and
isopropanol CH), 3.56 (m, 2H, H5'), 2.13 and 1.97 (m, 1H, H2'), and
1.04 (d, J=6, 3H, isopropanol CH.sub.3). TLC (85:13:2
dichloromethane-methanol-concentrated ammonium hydroxide, two
elutions; UV): starting iodide 55, R.sub.f =0.31; product 56,
0.27.
C. Preparation of 5-(3-Amino-1-propynyl)-2'-deoxycytidine
5'-triphosphate (54)
Alkynylamino nucleoside 56 (0.275 mmol) was converted to the
corresponding 5'-triphosphate and its trifluoroacetyl group was
removed following the general procedure given in Example 1E. After
addition of the second aliquot of phosphorus oxychloride,
phosphorylation was allowed to proceed for 3.5 h Assuming an
absorption coefficient for the product equal to that of the
starting material (8,780), the yield of triphosphate 54, based on
its UV absorption at 293 nm, was 17%.
EXAMPLE 9
Preparation of 5-(3-Trifluoroacetamido-1-propynyl)-2'-deoxyuridine
(57)
(Compound 57 is an example of an alkynylamino nucleotide (I)
wherein R.sub.1 is --CH.sub.2 --, R.sub.2 is COCF.sub.3, Het is
uracil (h), R.sub.3, R.sub.5, R.sub.7 and R.sub.8 are H, and
R.sub.6 is OH.)
5-Iodo-2'-deoxyuridine (7.08 g, 20.0 mmol, Aldrich) was coupled for
4 h to N-trifluoroacetylpropargylamine following the general
procedure given in Example 1C except that the reaction was run 2.5
times more concentrated than usual. Chromatography of the crude
product on silica gel (500 g) with 10-20% methanol in
dichloromethane afforded, 3.50 g (46%) of alkynylamino nucleoside
57 as a tan solid. According to NMR and TLC, this material was
>95% pure except for the presence of methanol (about 50 mole %)
that was not removed by vacuum-drying.
.sup.1 H-NMR (DMSO-d.sub.6): 11.63 (s, 1H, H3), 10.06 (distorted t,
1H, NHTFA), 8.19 (s, 1H, H6), 6.10 (apparent t, 1H, H1'), 5.23 (d,
J=4, 1H, 3'OH), 5.07 (t, J=5, 1H, 5'OH), 4.23 (m, 3H, --CH.sub.2 --
and H4'), 3.8 (apparent q, J=4, 1H, H3'), 3.58 (m, 2H, H5'), and
2.12 (m, 2H, H2').
EXAMPLE 10
Preparation of
5-(5-Trifluoroacetamido-1-pentynyl)-2',3'-dideoxyuridine (58)
(Compound 58 is an example of structure 5 wherein Het is uracil (h)
and R.sub.1 is --(CH.sub.2).sub.3 --.)
A. Preparation of 5-Trifluoroacetamido-1-pentyne (59)
Sodium hydride (60% dispersion in oil, Alfa) was rendered oil-free
by thoroughly and rapidly washing with pentane and then
vacuum-drying. Oil-free sodium hydride (4.40 g, 0.110 mole, 1.1 eq)
was added in about 20 portions over 25 min to a solution of
5-chloropentyne (10.6 mL, 0.100 mole, 1.0 eq), trifluoroacetamide
(14.13 g, 0.125 mole, 1.25 eq), and sodium iodide (14.99 g, 0.100
mole, 1.0 eq) in dry dimethylformamide (250 mL, Aldrich). The
reaction mixture was stirred at 25.degree. for 4.5 h and at
60.degree. for 21 h. After cooling, the reaction mixture was added
to a solution of potassium dihydrogen phosphate (43.5 g, 0.250
mole, 2.0 eq) in water (500 mL). This solution was extracted with
pentane (2.times.500 mL) and ether (3.times.500 mL). The combined
organic layers were washed with water (1.times.100 mL), dried over
magnesium sulfate, and concentrated with a rotary evaporator.
Fractional distillation twice through a 20 cm Vigreux column
afforded 8.09 g (45%) of 5-trifluoroacetamido-1-pentyne (58) as a
colorless, mobile liquid (bp 68-69.degree. at 13 torr.)
.sup.1 H-NMR (CDCl.sub.3): 6.77 (broad s, 1H, NHTFA), 3.53 (q,
J=6.7 and 2.7, 2H, --CH.sub.2 NHTFA), 2.31 (td, J=6.7 and 2.7, 2H,
HCCCH.sub.2 --), 2.04 (t, J=2.7, 1H, HCCCH.sub.2 --), and 1.83
(quintet, J=6.7, 2H, --CH2CH2CH2--).
C. B. Preparation of
5-(5-Trifluoroacetamido-1-pentynyl)-2',3'-dideoxyuridine (58)
5-Trifluroacetamido-1-pentyne (59) was coupled for 4 h to
5-iodo-2',3'-dideoxyuridine (47, prepared as described in Example
2A) according to the general procedure described in Example 1C.
Chromatography on silica gel (100 g) with a 0-5% methanol in
dichloromethane gradient afforded 647.7 mg of alkynylamino
nucleoside 58 as a light tan foam. This material was homogeneous by
TLC and NMR except for the presence of about 16 mole % of
dimethylformamide. Correcting for the presence of
dimethylformamide, the yield of desired product was 80%.
.sup.1 H-NMR (DMSO-d.sub.6): 11.52 (s, 1H, H3), 9.47 (distorted t,
1H, NHTFA), 5.90 (q, 1H, H1'), 5.12 (t, 1H, 5'OH), 4.04 (m, 1H,
H4'), 3.71 and 3.52 (m, 2H, 5'H), 3.30 (m, 2H, --CH.sub.2 CH.sub.2
CH.sub.2 NHTFA), 2.40 (t, 2H, --CH.sub.2 CH.sub.2 CH.sub.2 NHTFA),
2.23, 2.01 and 1.85 (m, 4H, H2' and H3'), and 1.73 (quintet, 2H,
--CH2CH2CH2NHTFA).
EXAMPLE 11
Preparation of
5-(12-Trifluoroacetamido-1-dodecynyl)-2',3'-dideoxyuridine (60)
(Compound 60 is an example of structure 5 wherein Het is uracil (h)
and R.sub.1 is --(CH.sub.2).sub.10 --.)
A. Preparation of 11-Dodecyn-1-ol (61)
1-Bromo-10-tetrahydropyranyloxydecane (64.26 g, 0.200 mole,
Lancaster, "97+%") was added dropwise over 140 min to a precooled
suspension of lithium acetylide ethylenediamine complex (23.94 9,
0.260 mole, 1.3 eq, Aldrich, 90%) in dry dimethylsulfoxide (100 mL)
so that the internal temperature remained at 5-10.degree.. After
the addition was complete, the cooling bath was removed and the
reaction mixture was stirred for 4.5 h. Water (20 mL) was added
dropwise to the reaction mixture. After stirring for 10 min, the
reaction mixture was poured into water (300 mL). This solution was
extracted sequentially with pentane (2.times.300 mL) and ether
(2.times.300 mL). Each organic layer was washed individually with
water (about 20 mL) and the aqueous washes were combined with the
main aqueous layer for re-extraction. The combined organic layers
were dried over magnesium sulfate and evaporated to dryness to
afford 51.38 g (96%) of crude 12-(tetrahydropyranyloxy)-1-dodecyne
as an oil.
A strongly acidic ion exchange resin (AG-50W-X8, 50 g, 5.1 meq/g,
Bio-Rad) was added to a solution of the above crude product (49.96
g) in a mixture of chloroform (260 mL) and methanol (260 mL). The
suspension was heated at reflux for 4.5 h and then cooled. The
reaction mixture was filtered and the filtrate was concentrated.
Chromatography of the residue on silica gel (500 g) with 10%, 20%
and 30% ethyl acetate in hexanes afforded 31 g of an oil which was
>95% one spot by TLC with detection by phosphomolybdic acid.
Distillation of this material through a 20 cm Vigreux column
afforded, after a 0.78 g forerun, 17.91 g of 11-dodecyn-1-ol (61)
as a thick, colorless oil (bp 104-108 at 1.4 torr) which solidified
to a white solid on standing. This material was 98% one peak by
GC.
.sup.1 H-NMR (CDCl.sub.3) of the chromatographed product before
distillation: 3.64 (t, 2H, --CH.sub.2 OH), 3.37 and 3.33 (m, about
0.2H, impurity) 2.17 (td 2H, HCCCH.sub.2 --) 1.92 (t, 1H,
HCCCH.sub.2 --), and 1.2-1.6 (m, 17H, (CH.sub.2).sub.8 and OH). IR
(thin film of melt): 3392 (O--H), 3311, 2930 and 2854 (C--H), 2160
(acetylene), 1466, 1432, 1394, 1371, 1352, 1328, 1303, 1103, and
1001.
B. Preparation of 12-Iodo-1-dodecyne (62)
Iodine (43.16 g, 170 mmol, 2.0 eq) was added to a suspension of
distilled alcohol 61 (15.50 g, 85 mmol), imidazole (17.36 g, 255
mmol, 3.0 eq), and triphenylphosphine (66.90 g, 255 mmol, 3.0 eq)
in dry toluene (425 mL, stored over molecular sieves). The reaction
mixture was heated at reflux with vigorous stirring for 25 min,
generating a yellow solution with a oily black precipitate. After
colling to 25.degree., saturated aqueous sodium bicarbonate (200
mL) and iodine (23.73 g, 93.5 mmol, 1.1 eq) were added and the
reaction was stirred vigorously for 1 h. Saturated aqueous Sodium
sulfite (40 mL) was added, quenching the purple color. The reaction
mixture was allowed to separate into two layers and the organic
layer was washed with brine. The organic layer was dried over
magnesium sulfate and concentrated. The residue was dissolved in
dichloromethane (50 mL) and ether (200 mL) was added. After
standing for 30 min, the resulting precipitate (triphenylphosphine
oxide) was removed by filtration and washed with ether (100 mL). On
further standing, the combined mother liquor and ether wash
deposited a second crop of crystals which were removed as before.
The combined mother liquors and ether washes were concentrated and
dissolved in warm toluene (200 mL). This solution was placed on a
silica gel column (500 g) and eluted with toluene (3 L) to afford
13.55 g (55%) of iodide 62 as a pale Yellow mobile liquid. This
material was 96% one peak by GC.
.sup.1 H-NMR (CDCl.sub.3): 3.20 (t, 2H, --CH.sub.2 I), 2.17 (td,
2H, HCCCH.sub.2 --), 1.94 (t, 1H, HCCCH.sub.2 --), 1.82, 1.51 and
1.20-1.42 (m, 16H, (CH.sub.2).sub.8).
C. Preparation of 12-Trifluoroacetamido-1-dodecyne (63)
Sodium hydride (60% dispersion in oil, Alfa) was rendered oil-free
by rapidly and thoroughly washing with pentane and vacuum-drying.
Trifluoroacetamide (22.61 g, 200 mmol, 5 eq) was added in about 10
portions over 50 min to a suspension of oil-free sodium hydride
(3.84 g, 160 mmol, 4 eq) in dry dimethylformamide (90 mL, Aldrich).
When it was discovered early in this addition that the reaction
mixture was getting warm, an ice-water bath was added and the rest
of the addition was performed at an internal temperature of about
10.degree.. The ice-water bath was removed and the reaction mixture
was stirred until hydrogen evolution ceased. After stirring an
addition 15 min, a solution of iodide 63 (11.69 g, 40.0 mmol) in
dry dimethylformamide (10 mL) was added dropwise over 10 min to the
reaction mixture. After stirring for 4 h at 25.degree., the
reaction mixture was rapidly poured into a stirred mixture of
saturated aqueous ammonium chloride (200 mL), water (200 mL) and
pentane (200 mL). The reaction vessel was rinsed with a mixture of
water (50 mL), saturated aqueous ammonium chloride (50 mL) and
pentane (200 mL). The combined solutions were allowed to separate
into two layers and the aqueous layer was extracted with pentane
(2.times.200 mL). The combined organic layers were dried over
magnesium sulfate and evaporated to dryness to yield 10.42 g (94%)
of trifluoroacetamide 63 as an oil which solidified to a waxy solid
on standing. Recrystallization of this material from boiling
hexanes (100 mL) with slow cooling to -20.degree. afforded 8.145 g
(73%) of trifluoroacetamide 63 as pale yellow needles (mp
46-47.degree.).
.sup.1 H-NMR (CDCl.sub.3): 6.27 (broad s, 1H, NHTFA), 3.34
(apparent q, 2H, --CH.sub.2 NHTFA), 2.18 (td, 2H, HCCCH.sub.2 --),
1.94 (t, 1H, HCCCH.sub.2 --), 1.20-1.65 (m, 16H, (CH.sub.2).sub.8).
IR (thin film of melt): 3312, 3298, 2932 and 2857 (C--H and N--H),
2117 (acetylene), 1706 (C.dbd.O), 1675 , 1563, 1460, 1448, 1208,
1182, 1166, 722, and 634.
D. Preparation of
5-(12-Trifluoroacetamido-1-dodecynyl)-2',3'-dideoxyuridine (60)
Protected alkynylamine 63 was coupled for 24 h to
5-iodo-2',3'-dideoxyuridine (47, 676.2 mg, 2.00 mmol, prepared as
described in Example 2A) following the general procedure described
in Example 1C. Chromatography on silica gel (100 g) eluting with a
0-5% methanol in dichloromethane gradient afforded a dark red foam.
The red impurity was removed by chromatography on a reverse phase
column (100 g, octadecylsilane on 40 micrometer silica gel, Baker)
with 40% water in methanol. The appropriate fractions were
combined, concentrated, and co-evaporated twice with absolute
ethanol to afford 731 mg of alkynylamino nucleoside 60 as a clear
oil. This material was homogeneous by TLC and NMR except for the
presence of residual ethanol (25 mole %, corrected yield 73%).
.sup.1 H-NMR (DMSO-d.sub.6): 11.49 (broad s, 1H, H3), 9.38
(distorted t, 1H, NHTFA), 8.15 (s, 1H, H6), 5.90 (dd, 1H, H1'),
5.12 (distorted t, 1H, 5'OH), 4.35 (t, 0.25H, CH.sub.3 CH.sub.2
OH), 4.03 (m, 1H, H4'), 3.72 and 3.52 (m, 2H, H5'), 3.43 (m, 0.5H,
CH.sub.3 CH.sub.2 OH), 3.16 (quintet, 2H, --CH.sub.2 NHTFA), 2.34
(t, 2H, propargylic H), 2.16, 2.01, and 1.86 (m, 4H, H2' and H3'),
1.65-1.15 (m, 16H, (CH.sub.2).sub.8), and 1.06 (t, 0.75H, CH.sub.3
CH.sub.2 OH).
EXAMPLE 12
Preparation of 5-(5-Amino-1-pentynyl)-2',3'-dideoxyuridine (64)
(Compound 64 is an example of an alkynylamino nucleotide (I)
wherein Het is uracil (h), R.sub.1 is (CH.sub.2).sub.3, and
R.sub.2, R.sub.3, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are H.)
A. Preparation of 5-Amino-1-pentyne (65)
Ammonia (340 g, 20 mole ) was distilled into a bomb which contained
5-chloropentyne (20.51 g, 0.200 mole) and sodium iodode (7.49 g,
0.050 mole, 0.25 eq). The bomb was sealed and heated in an
autoclave at 100.degree. for 12 h. The ammonia was allowed to
evaporate and the residue was stirred with a two phase mixture
consisting of sodium hydroxide (40 g, 1.0 mole, 5 eq), water (100
mL), and ether (100 mL). The resulting mixture was filtered and
allowed to separate into two layers. The organic layer was dried
over magnesium sulfate and distilled through a 20 cm Vigreux
column. Four fractions (12.46 g, bp 95-127.degree., atmospheric
pressure) were found by GC to contain significant amounts of
product. These fractions were combined and carefully distilled
through a spinning band column to afford 6.55 g (39%) of
5-amino-1-pentyne (65) as a colorless, mobile liquid (bp
125.5-126.degree. ). This material was >99% one peak by GC.
.sup.1 H-NMR (CDCl.sub.3): 2.81 (t, J=7.5, 2H, --CH.sub.2
NH.sub.2), 2.27 (td, J=7.5 and 2.5, 2H, HCCCH.sub.2 --), 1.96 (t,
J=2.5, 1H, HCCCH.sub.2 --), 1.66 (quintet, J=7.5, 2H,
--CH2CH2CH2--), and 1.07 (broad s, 2H, NH.sub.2).
B. A General Procedure of Coupling Unprotected Alkynylamines to
Iodonucleosides
Preparation of 5-(5-Amino-1-pentyne)-2',3'-dideoxyuridine (64)
A dry, 35-mL, round-bottomed flask was charged with
5-iodo-2',3'-dideoxyuridine (47, 676.2 mg, 2.00 mmol, prepared as
described in Example 2A) and then flushed with argon. Dry
dimethylformamide (10 mL, Aldrich), dry triethylamine (0.56 mL, 4.0
mmol, 2.0 eq, stored over sieves), 5-amino-1-pentyne (0.59 mL, 6.03
mmol, 3.0 eq), and tetrakis(triphenylphosphine)palladium(0) (231
mg, 0.200 mmol, 0.1 eq, weighed into a vial in a dry box) were
added. The resulting suspension was stirred for 45 min, but the
palladium catalyst remained at least partly undissolved. Cuprous
iodide (190.4 mg, 1.00 mmol, 0.5 eq, Aldrich Gold Label) was added.
After stirring for 15 min, a homogenous blue solution had formed
and after about 150 min the solution became cloudy. After 200 min,
TLC showed that all of starting iodide 47 had been consumed. After
4 h, the reaction mixture was concentrated with a rotary evaporator
for about 10 min at 45.degree. and 2 torr. The residue was
immediately absorbed onto a silica gel column (100 g) and eluted
with a mixture of dichloromethane, methanol and concentrated
ammonium hydroxide (400 mL each of 90:9:1, 85:13:2, 75:20:5,
65:30:5 and 50:45:5). The fractions containing the major polar
product according to TLC were combined, co-evaporated twice with
ethanol, and vacuum-dried overnight to afford 395.9 g (67%) of
alkynylamino nucleoside 64 as a yellow solid. This material was
homogeneous by TLC and NMR except for the presence of ethanol (33
mole %) which was not removed by vacuum-drying. The yield of 64,
corrected for the presence of ethanol, was 64%.
.sup.1 H-NMR (DMSO-d.sub.6): 8.33 (s, 1H, H6), 5.90 (dd, J=6.6 and
3.0, 1H, H1'), 4.05 (m, 1H, H4'), 3.73 (dd, J=12.1 and 2.8, 1H,
H5'a), 3.53 (dd, J=12.1 and 3.1, 1H, H5'b), 2.80 (broad s, 2H,
--CH.sub.2), 2.45 (t, J=7.0, 2H, propargylic H), 2.28, 2.02 and
1.86 (m, 4H, H2' and H3'), and 1.70 (quintet, J=7.0 Hz, 2H,
--CH.sub.2 CH.sub.2 CH.sub.2 --). This NMR data was obtained
f.COPYRGT.rm a different batch of 64 prepared in a manner similar
to that described above. The signals for the exchangeable hydrogens
(H3, 5'OH, and --NH.sub.2) in NMR samples of both materials were
combined into a single broad (> 2 ppm wide) signal which was
barely resolved from the baseline.
EXAMPLE 13
Preparation of 5-(3-Amino-1-propynyl)-2',3'-dideoxyuridine (66)
(Compound 66 is an example of an alkynylamino nucleotide (I)
wherein Het is uracil (h), R.sub.1 is CH.sub.2, and R.sub.2,
R.sub.3, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are H.)
5-Iodo-2',3'-dideoxyuridine (47, 2.00 mmol) was coupled for 3 h to
propargylamine (6.00 mmol, Aldrich) according to the procedure
described in Example 12B except that propargylamine was used in
place of 5-amino-1-pentyne. Chromatography as described above
returned 794.5 mg of impure alkynylamino nucleoside 66 as a Yellow
solid which afforded a single spot when analyzed by TLC. NMR and
the mass balance of the reaction indicated that this material was
contaminated by ethanol and possibly inorganic impurities.
.sup.1 H-NMR (DMSO-d.sub.6): 11.70 (broad s, 1H, H3), 8.40 (s, 1H,
H6), 8.25 (broad s, 2H, NH.sub.2), 5.89 (dd, J=6.6 and 3.0, 1H,
H1'), 5.13 (t, J=5.0, 1H, 5'OH), 4.07 (m, 1H, H4'), 3.96 (s, 2H,
--CH.sub.2 NH.sub.2), 3.71 and 3.56 (m, 2H, H5'), 2.30, 2.04 and
1.85 (m, 4H, H2' and H3') and signals for ethanol and an unknown
impurity. The above NMR data was taken from different preparation
of 66 performed as above except that 0.2 eq of cuprous iodide was
used and the reaction did not go to completion.
EXAMPLE 14
Preparation of
1-(2-Hydroxyethoxymethyl)-5-(3-amino-1-propynyl)cytosine
triphosphate (67)
(Compound 67 is an example of an alkynylamino nucleotide (I)
wherein R.sub.1 is --CH.sub.2 --, R.sub.2 and R.sub.3 are H, Het is
cytosine (i), R.sub.4 is (g), and R.sub.5 is P.sub.3 O.sub.9
H.sup.3-.)
A. Preparation of 1-(2-Hydroxyethoxymethyl)-5-iodocytosine (68)
A mixture of 1-(2-hydroxyethoxymethyl)cytosine (1.85 g, 10.0 mmol)
and mercuric acetate (3.35 g, 10.5 mmol) was refluxed in methanol
(50 mL) and dichloromethane (100 mL). Iodine (3.05 g, 12.0 mmol)
was added and the reaction mixture was stirred for 1 h. The free
base form of AG3-X4 resin (38 meq) was added and the solution
bubbled with hydrogen sulfide for 15 min. The solids were removed
by filtration and the filtrate stripped down onto silica gel (10
g). The silica was loaded onto a silica gel column (4.times.25 cm)
and eluted 5%, 10% and 20% methanol in dichloromethane. Evaporation
followed by vacuum-drying afforded a colorless solid (1.73 g,
56%).
Recrystallization from 95% ethanol afforded analytically pure
material (mp 172.degree.). Calculated for C.sub.7 H.sub.10 N.sub.3
O.sub.3 I: C 27.03%, H 3.24%, N 13.51%. Found: C 27.08%, H3.41%,
N13.51%. UV (methanol): maximum at 292.5 (5,300). .sup.1 H-NMR
(DMSO-d.sub.6): 3.481 (m, 4H), 4.659 (t, J=5, 1H), 5.070 (s, 2H,
6.665 (broad s, 1H), 7.869 (broad s, 1H), and 8.107 (s, 1H).
B. Preparation of
1-(2-Hydroxyethoxymethyl)-5-(3-trifluoroacetamido-1-propynyl)cytosine
(69)
Iodide 68 (311 mg, 1.00 mmol) was coupled to
N-propargyltrifluoroacetamide (43) according to the general
procedure described in Example 1C. Flash chromatography on silica
gel (3.times.20 cm) with 5%, 10% and 20% methanol in dichlormethane
afforded alkynylamino nucleotide 69 as a pale yellow foam (77.4 mg,
23%).
.sup.1 H-NMR(DMSO-d.sub.6): 3.472 (broad s, 4.276 (d, J=5.0, 2H),
4.653 (broad t, J=4.5, 1H), 5.091 (s, 2H), 6.925 (broad s, 1H),
8.037 (s, 1H), and 9.964 (broad s, 1H).
C. Preparation of
1-(2Hydroxyethoxymethyl)-5-(3-amino-1-propynyl)cytosine (67)
The hydroxyl group of the sugar part of alkynylamino nucleoside 69
(0.167 mmol) was converted to a triphosphate and the
trifluoroacetyl group was removed following the general procedure
given in Example 1E. After addition of the second aliquot of
phosphorus oxychloride, phosphorylation was allowed to proceed for
for 75 min. Assuming an absorption coefficient for the product
equal to that of the starting material (7,790), the yield of
triphosphate 67, based on its UV absorption at 291 nm, was 21%.
EXAMPLE 15
Preparation of N-Hydroxysuccinimide Ester 2a
(A preferred reagent for attaching a 505 nm fluorescent dye to an
alkynylamino-nucleotide wherein R.sub.9 and R.sub.10 are H).
A. Preparation of 9-(Carboxyethylidene)-3,6-dihydroxy-9H-xanthene
(SF-505)
Resorcinol (33.0 g, 0.300 mol) and succinic anhydride (30.0 g,
0.300 mol) were placed in a round bottomed flask and purged with
nitrogen. Methane-sulfonic acid (150 mL) was added and the solution
was stirred at 65.degree. C. for 2 hours under an atmosphere of
nitrogen. The reaction mixture was added dropwise to rapidly
stirred, ice-cooled water (1 L) with simultaneous addition of 50%
aqueous sodium hydroxide to maintain pH 2.5 +/0.5. The product
which appeared as a granular precipitate was collected by
filtration and rinsed with water (3.times.100 mL) then acetone
(3.times.100 mL). The product was air-dried then vacuum-dried
(vacuum oven) at 110.degree. C. for 18 hours to afford a dark red
powder (37.7 g, 88%).
An analytical sample was prepared by dissolving 1.0 g of product in
25 mL of hot 0.3N HCl. The precipitate which formed on cooling was
removed by filtration and discarded. Dilute aqueous sodium
hydroxide was added to raise the pH to 1.25. The resulting
precipitate was collected by filtration, rinsed with water,
air-dried, then vacuum-dried over P.sub.2 O.sub.5 at 140.degree. C.
for 36 hours. Anal: Calc. [C(16)H(12)O(5)] C 67.60, H 4.26. Found:
C 67 37, H 4.34, 0.52% water (K-F). NMR (DMSO-d.sub.6): (mostly
spirolactone form) w 2.690 (t, J=8.6 hz, 2H); 3.070 (t, J=8.6 hz,
2H), 6.530 (d, J=1.8 hz, 2H); 6.676 (dd, J=8.7, 1.8 hz, 2H), 7.432
(d, J=8.7, 1.8 hz, 2H), 7.432 (d, J=8.7 hz, 2H), and 9.964 (s, 2H).
Vis. abs. (pH 8.2; 50 mM aq Tris/HCl): max 486 nm (72,600).
B. Preparation of
9-(2-Carboxyethyl)-3,6-diacetoxy-9-ethoxy-9H-xanthene
(Ac2EtSF-505)
SF-505 (29.3 g, 103 mmol) was added to ice-cold acetic anhydride
(500 mL) followed by pyridine (100 mL). The mixture was stirred in
ice for 20 minutes then added over 20 minutes to rapidly stirred,
ice-cold water (7 L). After stirring for an additional 30 minutes,
the intermediate product was filtered and resuspended in water (4
L) and stirred for another 30 minutes. The solid was collected by
filtration, dissolved in absolute ethanol (1 L), and refluxed for
45 minutes. The solution was concentrated on a rotary evaporator to
200 mL which resulted in crystallization. The product was collected
by filtration, air-dried, then vacuum-dried to afford pale-orange
microcrystals (21.9 g, 51%).
Recrystallization from methylene chloride/cyclohexane gave
colorless microcrystals. M.p.: 142-143.degree. C. Anal: Calc.
[C(22)H(22)O(8)] C 6.63.76, H 5.35. Found: C 63.58, H 5.39. NMR
(DMSO-d.sub.6): w 1.035 (t, J=6.9 hz, 3H), 1.667 (m, 2H), 2.232 (m,
2H), 2.294 (s, 6H), 2.888 (q, J=6.9 hz, 2H), 7.0-7.1 (m, 4H), and
7.575 (d, J=9.1 hz, 2H).
C. Preparation of
9-(2-(N-Succinimidyloxy-carbonyl))-ethyl)-3,6-diacetoxy-9-ethoxy-9H-xanthe
ne (Ac2EtSF-505-NHS)
Ac2EtSF-505 (10.4 g, 25.1 mmol) was mixed with methylene chloride
(300 mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (9.70 g, 50.6 mmol) and N-hydroxysuccinimide (4.32 g,
37.5 mmol) were added. The mixture was stirred for one hour and
then washed with water (5.times.50 mL). The combined aqueous layers
were back extracted with methylene chloride (50 mL) and the pooled
organic layers were dried over sodium sulfate and stripped down.
Trituration with ethanol (75 mL) followed by filtration and
air-drying afforded the crude product as a light yellow solid (c.
10 g). This material was dissolved in methylene chloride (50 mL)
and cyclohexane (50 mL) was added. One teaspoon of charcoal was
added, the mixture was filtered, and the product was brought down
with an additional portion of cyclohexane (100 mL). Collection by
filtration, air-drying, and vacuum-drying afforded colorless
crystals (6.94 g, 54%).
A second crystallization from ethanol afforded an analytical
sample. M.p.: 162-3.degree. C. Anal: Calc. [C(26)H(25)N(1)O(10)] C
61.05, H, 4.93, N 2.74. Found C 60.78, H 5.01, N 2.65. NMR
(DMSO-d.sub.6): w 1.056 (t, J=7.0 hz, 3H), 2.4-2.1 (m, 4H), 2.293
(s, 6H), 2.757 (s, 4H), 2.922 (q, J=7.0 hz, 2H), 7.069 (m, 4H), and
7.617 (p d, J=9.1 hz, 2H).
D. Preparation of
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy
-9-ethoxy-9H-xanthene (Ac2EtSF-505-Sar-OBn)
To a solution of sarcosine benzyl ester* (1.13 g, 6.31 mmol) in
methylene chloride (50 mL) was added Ac2EtSF-505-NHS (2.58 g, 5.05
mmol) and 5% aq sodium bicarbonate solution (30 mL). The two-phase
mixture was stirred rapidly for 20 hours. The layers were separated
and the organic layer washed with 3.times.15 mL water, dried over
sodium sulfate, and concentrated to 25 mL. The solution was diluted
to 150 mL with cyclohexane, charcoal-treated, and reduced to 75 mL
under a stream of nitrogen resulting in the precipitation of the
product. The supernatant was decanted away and the residue
coevaporated with methylene chloride to afford a colorless foam
(1.70 g, 58%).
Extensive vacuum-drying afforded an analytical sample Anal: Calc
[C(32)H(33)N(1)O(9)] C 66.77, H 5.78, N 2.43. Found: C 66.66, H
5.89, N 2.25. NMR (DMSO-d.sub.6): (Shows 5:2 mixture of amide bond
rotamers.) w (major and minor) 1.040 and 1.018 (t, J=6.7 hz, 3H),
1.789 and 1.670 (m, 2H), 2.211 (m, 2H), 2.290 and 2.276 (s, 6H),
2.713 and 2.695 (s, 3H), 2.893 (q, J=6.7 hz, 2H), 3.963 (s, 2H),
5.075 and 5.039 (s, 2H), 7.044 (m, 4H), 7.324 (m, 5H), and 7.573
and 7.516 (p d, J=9.2 hz, 2H).
E. Preparation of
9-(2-(N-Methyl-N-(N'-succinimidyl-oxycarbonylmethyl)carboxamido)ethyl)-3,6
-diacetoxy-9-ethoxy-9H-xanthene (Ac2EtSF-505 -Sar-NHS. Structure
2a)
To a solution of Ac2EtSF-505-Sar-OBn (1.55 g, 2.69 mmol) in
absolute ethanol (60 mL) was added 10% palladium on carbon (0.15
g). The mixture was stirred under balloon pressure of hydrogen for
30 minutes. The catalyst was removed by filtration and the ethanol
stripped off to afford a syrupy residue.
This residue was dissolved in methylene chloride (85 mL) and
N-hydroxysuccinimide (0.495 g, 4.30 mmol) and
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (1.12
g, 5.84 mmol) were added (4.times.25 mL). The solution was
concentrated to 25 mL, diluted to 175 mL with cyclohexane, charcoal
treated, and reduced in volume to 75 mL under a stream of nitrogen.
The solid product was collected by filtration, air-dried, and
vacuum-dried to afford a colorless powder (0.97 g, 62%).
Coevaporation with methylene chloride followed by extensive
vacuum-drying at 40.degree. C. removed traces of cyclohexane and
afforded an analytical sample as an amorphous solid. Anal: Calc.
[C(29)H(30)N(2)O(11)] C 59.79, H 5.19, N 4.81. Found: C 59.37, H
4.62, N 4.62, 0.93% water (K-F). NMR (DMSO-d.sub.6): (Shows a 4:1
mixture of amide bond rotamers.) w (major and minor) 1.034 (t,
J=6.9 hz, 3H), 1.827 and 1.935 (m, 2H), 2.223 (m, 2H), 2.289 (s,
6H), 2.758 (s, 4H), 2.779 and 2.824 (s, 3H), 2.888 (q, J=6.8 hz,
2H), 4.333 and 4.473 (s, 2H), 7.043 (m, 4H), and 7.587 (per d,
J=9.1 hz, 2H).
EXAMPLE 16
Preparation of N-Hydroxysuccinimide Ester 2b
(A preferred reagent for attaching a 512 nm fluorescent dye to an
alkynylamino-nucleotide wherein R.sub.9 is H and R.sub.10 is
CH.sub.3)
A. Preparation of 4-Methylresorcinol
2,4-Dihydroxybenzaldehyde (33.97 gm, 0.246 mol) (recrystallized
from toluene) was dissolved in spectroscopic grade 2-propanol (3 L)
in a round bottom flask fitted with a gas inlet and a bubbler
outlet. 10% Palladium on carbon (1.35 gm) was added followed by
phosphoric acid (3 mL) and the mixture was sparged with nitrogen.
The nitrogen flow was switched to hydrogen and the mixture was
rapidly stirred with ice cooling. After 3 hours hydrogen uptake was
complete and the catalyst was removed by filtration. The filtrate
was stripped down to 200 mL and 200 mL of ethyl acetate was added.
The solution was washed with 4.times.200 mL of water and the
combined water extracts back-extracted with ethyl acetate. These
organic extracts were water washed and the combined organic layers
dried over sodium sulfate and stripped down to afford the product
as a colorless crystalline solid (29.95 gm, 98%). M.p.: 106.degree.
C. (Lit. 106-107.degree. C. [J. C. Bell, W. Bridge, and A.
Robertson, J. Chem. Soc., 1542-45 (1937)]). NMR (DMSO-d.sub.6): w
1.961 (s, Me), 6.076 (dd, H-6, J[5,6]=8 hz, J[2,6]=2 hz), 6.231 (d,
H-2), 6.760 (d, H-5) 8.867 (s, OH), and 9.008 (s, OH).
B. Preparation of
9-Carboxyethylidene-3,6-dihydroxy-2,7-dimethyl-9H-xanthene
(SF-512)
4-Methylresorcinol (25.8 g, 0.208 mol) and succinic anhydride (20.8
g, 0.208 g) were placed in a round bottom flask and the flask was
purged with nitrogen. Methanesulfonic acid (150 mL) was added and
the solution heated under nitrogen to 65.degree. C. for 2 hours.
The solution was added dropwise to 1 L of rapidly stirred,
ice-cooled water with the simultaneous addition of 50% aq sodium
hydroxide to maintain the pH at 2.25 +/-0.25. The product was
collected by centrifugation and washed with water (3.times.) and
acetone (2.times.). The solid was air-dried, then vacuum-dried at
110.degree. C. to afford a brick-red powder (24.1 g, 74%).
Purification was effected by allowing ethyl acetate to slowly
diffuse into a solution of the product in dimethyl sulfoxide. The
precipitate was collected by filtration, air-dried, then
vacuum-dried. NMR (DMSO-d.sub.6): (Shows pure delta form along with
one mole each of water and dimethyl sulfoxide.) w 2.124 (s, 6H),
3.421 (d, J=7.2 hz, 2H), 5.769 (t, J=7.2 hz, 1H); 6.512 (s, 1H),
6.573 (s, 1H); 7.295 (s, 2H), 9.681 (s, 1H), 9.825 (s, 1H), and
12.346 (bs, 1H). Vis. abs. (pH 8.2 aq Tris): max 493.5 nm.
C. Preparation of
9-Carboxyethyl-3,6-diacetoxy-2,7-dimethyl-9-ethoxy-9H-xanthene
(Ac2EtSF-512)
A sample of SF-512 (20.0 g, 64.0 mmol) was added to acetic
anhydride (350 mL) followed by pyridine (80 mL). This was stirred
for 1 hour and then filtered to remove traces of unreacted dye. The
filtrate was poured into 3.5 L of rapidly stirred water. The solid
intermediate was collected by filtration, resuspended in 2 L cold
water, stirred for 15 minutes, then recollected and air-dried to
afford the spirolactone intermediate (20.8 g). This was dissolved
in absolute ethanol (600 mL) and refluxed for 45 minutes. The
solution was charcoal-treated and concentrated to 300 mL. The
product was collected by filtration, rinsed with cold ethanol
(2.times.50 mL), air-dried, and then vacuum-dried to afford
colorless microcrystals (14.9 g, 53%). M.p.: 143.degree. C. Anal:
Calc. [C(24)H(26)O(8)] C 65.15, H 5.92. Found: C 65.31, H 5.97. NMR
(DMSO-d.sub.6): w 1.027 (t, J=6.9 hz, 3H), 1.628 (m, 2H), 2.136 (s,
6H), 2.207 (m, 2H), 2.303 (s, 6H), 2.884 (q, 6.9 hz, 2H), 6.939 (s,
2H), and 7.417 (s, 2H).
D. Preparation of
9-(2-(N-Succinimidyloxycarbonyl)-ethyl)-3,6-diacetoxy-2,7-dimethyl-9-ethox
y-9H-xanthene (Ac2EtSF-512-NHS)
To a solution of Ac2EtSF-512 (9.42 g, 21.3 mmol) in methylene
chloride (175 mL) was added N-hydroxysuccinimide (3.62 g, 31.5
mmol) followed immediately by
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.05
g, 42.0 mmol). The solution was stirred at room temperature for 2
hours. The mixture was washed with water (4.times.100 mL) and the
aqueous washings back-extracted with methylene chloride (2.times.50
mL). The combined organic layers were dried over sodium sulfate and
stripped down to an oil. Absolute ethanol was added and
crystallization was induced by scratching. The product was
collected by filtration, air-dried, then vacuum-dried to afford
pale-orange microcrystals (9.80 g, 85%).
An analytical sample was prepared by dissolving 1 g in methylene
chloride (10 mL) and adding cyclohexane (40 mL). Charcoal treatment
followed by cooling and scratching induced crystallization
affording a colorless crystalline solid. M.p.: 159.degree. C. Anal:
Calc. [C(28)H(29)N(1)O(10)] C 62.33, H 5.42, N 2.60. Found: C
62.06, H 5.71, N 2.39. NMR (DMSO-d.sub.6): w 1.053 (t, J=6.9 hz,
3H), 2.149 (s, 6H), 2.304 (s, 6H), 2.1-2.4 (m, 4H), 2.747 (s, 4H),
2.920 (q, J=6.9 hz, 2H), 6.975 (s, 2H), and 7.464 (s, 2H).
E. Preparation of
9-(2-(N-methyl-N-(benzyloxycar-bonylmethyl)carboxamido)ethyl)-3,6-diacetox
y-2,7-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-512-Sar-OBn)
To a solution of sarcosine benzyl ester (0.72 g, 4.02 mmol) in
methylene chloride (25 mL) was added Ac2EtSF-512-NHS (1.73 g, 3.21
mmol) and 5% aq sodium bicarbonate solution (20 mL). The two-phase
mixture was stirred rapidly for 20 hours. The layers were separated
and the organic layer washed with 3.times.15 mL water, dried over
sodium sulfate, and concentrated to 10 mL. The solution was diluted
to 60 mL with cyclohexane, charcoal-treated, and reduced to 25 mL
under a stream of nitrogen resulting in the precipitation of the
product. The supernatant was decanted and the colorless solid
vacuum-dried (1.44 g, 74%).
Recrystallization from methylene chloride/cyclohexane with charcoal
treatment afforded an analytical sample. M.p.: 150-2.degree. C.
Anal: Calc. [C(34)H(37)N(1)O(9)] C 67.65 H 6.18 N 2.32. Found: C
67.42 H 6.08 N 2.33. NMR (DMSO-d.sub.6): (Shows 5:2 mixture of
amide bond rotamers.) w (major and minor) 1.049 and 1.008 (t, J=6.8
hz, 3H), 1.747 and 1.66 (m, 2H), 2.144 and 2.115 (s, 6H), 2.18 (m,
2H), 2.314 and 2.303 (s, 6H), 2.694 (s, 3H), 2.907 and 2.884 (q,
J=6.8 hz, 2H), 3.961 (s, 2H), 5.075 and 5.016 (s, 2H), 6.960 and
6.917 (s, 2H), 7.430 and 7.396 (s, 2H), and 7.30 (m, 5 H).
F. Preparation of
9-(2-(N-Methyl-N-(N'-succinimidyl-oxycarbonylmethyl)carboxamido)ethyl)-3,6
-diacet-oxy-9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene
(Ac2EtSF-512-Sar-NHS, Structure 2b)
To a suspension of Ac2EtSF-512-Sar-OBn (0.45 g, 0.745 mol) in
absolute ethanol (20 mL) was added 10% palladium on carbon (0.05
g). The mixture was stirred under balloon pressure of hydrogen for
30 minutes. The catalyst was removed by filtration and the ethanol
stripped off to afford a syrupy residue.
This residue was dissolved in methylene chloride (25 mL) and
N-hydroxysuccinimide (0.129 g, 1.12 mmol) and
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (0.292
g, 1.52 mmol) were added. The mixture was stirred for 30 minutes
and then washed with water (3.times.15 mL). The solution was dried
over sodium sulfate, concentrated to 10 mL, diluted to 40 mL with
cyclohexane, charcoal treated, and reduced in volume to 20 mL under
a stream of nitrogen. The supernatant was decanted and the residue
subjected to a second precipitation from methylene chloride to
afford a colorless powder (0.27 g, 59%). Anal: Calc.
[C(31)H(34)N(2)O(11)] C 60.98, H 5.61, N 4.59. Found: C 60.28, H
5.71, N 4.40, 1.08% water (K-F). NMR (DMSO-d.sub.6): (Shows a 5:1
mixture of rotamers about the amide bond.) w (major and minor)
1.043 (t, J=7.0 hz, 3H), 1.793 and 1.933 (m, 2H), 2.145 and 2.133
(s, 6H), 2.198 (m, 2H), 2.314 (s, 6H), 2.740 (s, 4H), 2.778 and
2.821 (s, 3H), 2.900 (q, J=7.0 hz, 2H), 4.334 and 4.469 (s, 2H),
6.960 and 6.925 (s, 2H0, and 7.441 (s, 2H).
EXAMPLE 17
Preparation of N-Hydroxysuccinimide Ester 2c
(A preferred reagent for attaching a 519 nm fluorescent dye to an
alkynylamino-nucleotide wherein R.sub.9 is CH.sub.3 and R.sub.10 is
H)
A. Preparation of
9-(2-Carboxyethylidene)-3,6-dihydroxy-4.5-dimethyl-9H-xanthene
(SF-519)
2-Methylresorcinol (37.2 g, 0.300 mol) and succinic anhydride (30.0
g, 0.300 mol) were placed in a round bottomed flask and purged with
nitrogen. Methanesulfonic acid (150 mL) was added and the solution
was stirred at 65.degree. C. for 4 hours under an atmosphere of
nitrogen. The reaction mixture was added dropwise to rapidly
stirred, ice-cooled water (1 L) with simultaneous addition of 50%
aqueous sodium hydroxide to maintain pH 6.0 +/-0.5. The finely
divided solid was collected by centrifugation and rinsed with water
(4.times.250 mL), each time resuspending, spinning down, and
discarding the supernatant. The crude product was suspended in
water (1 L) and sufficient aqueous sodium hydroxide (50%) was added
to raise the pH to 10.2. The solution was filtered and the filtrate
brought to pH 1.2 with concentrated HCl. The product was collected
by centrifugation and rinsed with water (3.times.350 mL) and
acetone (3.times.250 mL) as described above. The resulting solid
was azeotroped with toluene, collected by filtration, and
vacuum-dried at 110.degree. C. to afford a brick-red powder (24.6
g, 53%). Anal: Calc. [C(18)H(16)O(5)] C 69.22 H 5.16. Found: C
68.95 H 5.30, 0.80% water (K-F). NMR (DMSO-d.sub.6) (mostly delta
form): w 2.164 (s, 3H), 2.177 (s, 3H), 3.376 (d, J=7.1 hz, 2H),
5.749 (t, J=7.2 hz, 1H), 6.642 (d, J=8.8 hz, 1H), 6.672 (d, J=8.8
hz, 1H), 7.216 (d, J=8.5 hz, 1H), 7.227 (d, J=8.5 hz, 1H), 9.602
(bs, 1H), and 9.758 (bs, 1H). Vis. abs. (pH 8.2; 50 mM aq Tris/HCl)
max 500 nm (69,800).
B. Preparation of
9-(2-Carboxyethyl)-3,6-diacetoxy-4,5-dimethyl-9-ethoxy-9H-xanthene
(Ac2EtSF-519)
SF-519 (I5.0 g, 48.0 mmol) was added to acetic anhydride (250 mL)
and the solid was pulverized. (Sonication is useful to disperse the
highly insoluble SF-519.) The suspension was ice-cooled, pyridine
(50 mL) was added, and the mixture stirred for 20 minutes. The
solution was filtered and added in a slow but steady stream to
rapidly stirred ice-cold water (4 L). After stirring for an
additional 20 minutes, the intermediate product was filtered,
resuspended in water (3 L), and stirred for another 25 minutes. The
solid was collected by filtration and air-dried. The dried
intermediate was dissolved in absolute ethanol (600 mL) and
refluxed for 1 hour. The solution was concentrated on a rotary
evaporator to 200 mL which resulted in crystallization. The product
was collected by filtration, air-dried, then vacuum-dried to afford
colorless microcrystals (12.13 g, 57%).
An analytical sample was prepared by precipitation from methylene
chloride solution with cyclohexane. NMR (DMSO-d.sub.6): w 1.033 (t,
J=6.9 hz, 3H), 1.674 (m, 2H), 2.189 (s, 6H), 2.19 (m, 2H), 2.348
(s, 6H), 2.878 (q, J=6.9 hz, 2H), 7.006 (d, J=8.6 hz, 2H), and
7.399 (d, J=8.6 hz, 2H).
C. Preparation of
9-(2-(N-Succinimidyloxycarbonyl)ethyl-3,6-diacetoxy-4,5-dimethyl-9-ethoxy-
9H-xanthene (Ac2EtSF-519-NHS)
Ac2EtSF-519 (7.80 g, 17.6 mmol) was mixed with methylene chloride
(175 mL) and N-hydroxysuccinimide (2.75 g, 23.9 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.00
g, 36.5 mmol) were added. The mixture was stirred for 90 minutes
and then washed with water (5.times.100 mL). The combined aqueous
layers were back extracted with methylene chloride (2.times.50 mL)
and the pooled organic layers were dried over sodium sulfate and
stripped down. Trituration with ethanol (100 mL) followed by
filtration and air-drying afforded the product as a light yellow
solid (7.45 g, 78%).
Two recrystallizations from cyclohexane/methylene chloride with
charcoal treatment afforded an analytical sample. M.p.:
164-5.degree. C. Anal: Calc. [C(28)H(29)N(1)O(10)] C 62.33, H 5.42,
N 2.60. Found: C 62.17, H 5.47, N 2.48. NMR (DMSO-d.sub.6: w 1.051
(t, J=7 0 hz, 3H), 2.4-2.1 (m, 4H), 2.191 (s, 6H), 2.337 (s, 6H),
2.715 (s, 4H), 2.912 (q, J=7.0 hz, 2H), 7.015 (d, J=8.6 hz, 2H),
and 7.429 (d, J=8.6 hz, 2H).
D. Preparation of
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy
-4,5-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-519-Sar-OBn)
To a solution of sarcosine benzyl ester (0.557 g, 3.11 mmol) in
methylene chloride (19 mL) was added Ac2EtSF-519-NHS (1.30 g, 2.41
mmol) and 5% aqueous sodium bicarbonate solution (15 mL). The
two-phase mixture was stirred rapidly for 18 hours. The layers were
separated and the organic layer washed with 3.times.10 mL water,
dried over sodium sulfate, and concentrated to 10 mL. The solution
was diluted to 40 mL with cyclohexane, charcoal-treated, and
reduced to 20 mL under a stream of nitrogen resulting in the
precipitation of the product as a sticky solid. The supernatant was
decanted away and the residue coevaporated with methylene chloride
to afford a colorless foam (0.97 g, 67%).
Extensive vacuum drying afforded an analytical sample. Anal: Calc.
[C(34)H(37)N(1)O(9)] C 67.65 H 6.18 N 2.32. Found: C 67.43 H 6.37 N
2.32. NMR (DMSO-d.sub.6) (Shows 5:2 mixture of amide bond
rotamers.): w (major and minor) 1.044 and 1.020 (t, J=7.0 hz, 3H),
1.824 and 1.714 (m, 2H), 2.17 (m, 2H), 2.195 and 2.169 (s, 6H),
2.346 and 2.337 (s, 6H), 2.720 and 2.691 (s, 3H), 2.889 (q, J=7.0
hz, 2H), 3.959 and 3.988 (s, 2H), 5.073 and 5.048 (s, 2H), 7.000
and 6.954 (d, J=8.6 hz, 2H), and 7.45-7.25 (m, 7H).
E. Preparation of
9-(2-(N-Methyl-N-(N'-succinimidyl-oxycarbonylmethyl)carboxamido)ethyl)-3,6
-diacet-oxy-4,5-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-519-Sar-NHS,
Structure 2c)
To a solution of Ac2EtSF-519-Sar-OBn (1.35 g, 2.24 mmol) in
absolute ethanol (50 mL) was added 10% palladium on carbon (0.13
g). The mixture was stirred under balloon pressure of hydrogen for
20 minutes. The catalyst was removed by filtration and the ethanol
stripped off to afford a syrupy residue.
This residue was dissolved in methylene chloride (50 mL) and
N-hydroxysuccinimide (0.39 g, 3.39 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.57
g, 8.19 mmol) were added. The mixture was stirred for 75 minutes
and then washed with water (4.times.15 mL). The solution was dried
over sodium sulfate, concentrated to 25 mL, diluted to 125 mL with
cyclohexane, charcoal treated, and reduced in volume to 50 mL under
a stream of nitrogen. The supernatant was decanted and the
remaining oil taken up in methylene chloride (5 mL) and added
dropwise to rapidly stirred cyclohexane (75 mL) to afford a
colorless powder (0.587 g, 43%).
To provide an analytical sample a portion of the product was taken
up in methylene chloride, dried over molecular sieves, evaporated
under a stream of nitrogen, and finally dried in a drying pistol at
48.degree. C. over phosphorus pentoxide for 20 hours. Anal: Calc.
[C(31)H(34)N(2)O(11)]; C 60.98, H 5.61, N 4.59. Found: C 60.15, H
5.71, N 4.51, water (K-F) 1.51%. NMR (DMSO-d.sub.6) (Shows a 4:1
mixture of amide bond rotamers.): w (major and minor) 1.039 (t,
J=6.9 hz, 3H), 1.841 and 1.945 (m, 2H), 2.19 (m, 2H), 2.194 (s,
6H), 2.345 (s, 6H), 2.767 and 2.744 (s, 4H), 2.778 and 2.825 (s,
3H), 2.888 (q, J=6.9 hz, 2H), 4.328 and 4.461 (s, 2H), 7.000 (d,
J=8.6 hz, 2H), and 7.410 (d, J=8.6 hz, 2H).
EXAMPLE 18
Preparation of N-Hydroxysuccinimide Ester 2d
(A preferred reagent for attaching a 526 nm dye to an
alkynylamino-nucleotide wherein R.sub.9 and R.sub.10 are
CH.sub.3)
A. Preparation of 2,4-Dihydroxy-3-methylbenzaldehyde
Phosphorus oxychloride (80 mL, 0.86 mol) was added to a stirred
mixture of N-methylformanilide (102 mL, 0.82 mol) in ether (250
mL). The mixture was stirred for 1 hour at room temperature and
then cooled in ice. 2-Methyl resorcinol (Aldrich, 100 g, 0.81 mol)
was added and the mixture was allowed to warm to room temperature
while stirring overnight. The precipitated intermediate product was
collected by filtration and rinsed with ether (3x). The
intermediate was hydrolyzed by dissolving in a mixture of acetone
(250 mL) and water (250 mL) and stirring for 30 minutes. Water (2
L) was added, the mixture was brought to a boil, and then allowed
to cool and deposit crystalline product. This was recrystallized a
second time from water (4 L) to afford pure product (70 g, 57%).
M.p. 150.degree. C. (Lit. 152-3.degree. C. [W. Baker et al., J.
Chem. Soc., 2834-5 (1949).]. NMR (DMSO-d.sub.6): w 1.973 (s, 3H),
6.551 (d, J=8.5 hz, 1H), 7.428 (d, J-8.5 hz, 1H), 9.703 (s, 1H),
10.745 (s, 1H), and 11.592 (s, 1H).
B. Preparation of 2,4-dimethylresorcinol
A solution of 2,4-dihydroxy-3-methylbenzaldehyde (30.0 g, 197 mmol)
with isopropanol (3 L) was ice-cooled in a 5 L 3-neck flask fitted
with a magnetic stirrer. Phosphoric acid (4 mL) and 10% palladium
on carbon were added and the solution was sparged with nitrogen,
then hydrogen. When uptake was judged to be complete (c. 1.5 hour)
the solution was again sparged with nitrogen and then filtered
through Celite.RTM.. The solvent was stripped off, the residue
taken up in ethyl acetate, and the resulting solution washed with
water (4.times.100 mL). The water washes were back-extracted with
ethyl acetate and the combined organic layers dried over sodium
sulfate and stripped down. Sublimation (95.degree., 0.05 torr)
afforded a colorless solid (19.6 g, 72%). M.p. 107-8.degree. C.
(Lit. 108-109.degree. C. [W. Baker et al., J. Chem. Soc.,
2834-5(1949).]). NMR (DMSO-d.sub.6): w 1.969 (s, 3H), 2.037 (s,
3H), 6.220 (d, J=8.1 hz, 1H), 6.637 (d, J=8.1 hz, 1H), 7.929 (s,
1H), and 8.785 (s, 1H).
C. Preparation of
9-(2-Carboxyethylidene)-3,6-dihydroxy-2,4,5,7-tetramethyl-9H-xanthene
(SF-526)
2,4-Dimethylresorcinol (28.4 g, 0.205 mol) and succinic anhydride
(20.0 g, 0.200 mol) were placed in a round bottomed flask and
purged with nitrogen. Methanesulfonic acid (231 mL) was added and
the solution was stirred at 70.degree. C. for 20 hours under an
atmosphere of nitrogen. The reaction mixture was added dropwise to
a rapidly stirred mixture of aqueous sodium hydroxide (95 g in 150
mL water) and ice (3 L). Sufficient methanesulfonic acid was added
to bring the final pH from 4.7 to 1.5. The resulting solid was
collected by centrifugation and washed by suspending, spinning
down, and decanting from water (5.times.1.2 L). The final
suspension was collected by filtration, air-dried, then oven-dried
at 110.degree. C. for 6 hours to afford a brick-red solid (30.6 g,
44%).
A second precipitation from alkaline solution, followed by
centrifugation and water washes afforded an analytical sample.
Anal: Calc. [C(16)H(12)O(5)] C 70.57, H 5.92. Found: C 70.39, H
6.00, 0.21% water (K-F). NMR (DMSO-d.sub.6) (mostly spirolactone
form): w 2.172 (s, 12H), 2.508 (m, 2H), 3.342 (m, 2H), and 7.604
(s, 2H). Vis. abs. (pH 8.2; 50 mM aq Tris/HCl): 509 nm
(71,300).
D. Preparation of
9-(2-Carboxyethyl)-3,6-diacetoxy-9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene
(Ac2EtSF-526)
SF-526 (25.2 g, 74 mmol) was added to ice-cold acetic anhydride
(450 mL) followed by pyridine (100 mL) and the mixture was stirred
with ice-cooling for 150 minutes. The reaction mixture was filtered
then added in a slow, steady stream to rapidly stirred, ice-cold
water (7 L). After stirring for an additional 30 minutes, the
intermediate product was filtered, washed with water, resuspended
in water (4 L) and stirred for another 30 minutes. The solid was
collected by filtration and air-dried to afford the spirolactone
intermediate (28.9 g). A portion of this intermediate (18.6 g) was
dissolved in absolute ethanol (1 L), and refluxed for 90 minutes.
The solution was concentrated on a rotary evaporator to 300 mL
which resulted in crystallization. The product was collected by
filtration, rinsed with ethanol, air-dried, then vacuum-dried to
afford colorless microcrystals (11.6 g, 52% based on amount of
intermediate used).
Recrystallization from methylene chloride/cyclohexane with charcoal
treatment gave colorless microcrystals. M.p.: 154-155.degree. C.
Two evaporations from methylene chloride removed traces of
cyclohexane for analysis. Anal: Calc. [C(20)H(20)O(5)] C 70.57, H
5.92. Found: C 70.39, H 6.00, 0.21% water (K-F). NMR (DMSO-d.sub.6)
(mostly spirolactone form): w 2.172 (s, 12H), 2.508 (m, 2H), 3.342
(m, 2H), and 7.604 (s, 2H). Vis. abs. (pH 8.2; 50 mM aq Tris/HCl):
509 nm (71,300).
E. Preparation of
9-(2-(N-Succinimidyloxycarbonyl)ethyl)-3,6-diacetoxy-9-ethoxy-2,4,5,7-tetr
amethyl-9H-xanthene (Ac2EtSF-526-NHS)
Ac2EtSF-526 (4.70 g, 9.99 mmol) was mixed with methylene chloride
(75 mL) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (3.10 g, 16.2 mmol) and N-hydroxysuccinimide (1.50 g,
13.0 mmol) were added. The mixture was stirred for 90 minutes and
then washed with water (4.times.50 mL). The combined aqueous layers
were back extracted with methylene chloride (50 mL) and the pooled
organic layers were dried over sodium sulfate and stripped down.
Trituration with ethanol (75 mL) followed by filtration and
air-drying afforded the crude product as a light yellow solid (c.
4.7 g). This material was dissolved in methylene chloride (50 mL)
and cyclohexane (50 mL) was added. One teaspoon of charcoal was
added, the mixture was filtered, and the product was brought down
with an additional portion of cyclohexane (25 mL). Collection by
filtration, air-drying, and vacuum-drying afforded colorless
crystals (3.14 g, 55%).
A second precipitation from methylene chloride with cyclohexane
afforded an analytical sample. Anal: Calc. [C(30)H(33)N(1)O(10)]; C
63.48, H 5.86, N 2.47. Found: C 63.08, H 6.00, N 2.37. NMR
(DMSO-d.sub.6): w 1.058 (t, J=6.9 hz, 3H), 2.136 (s, 6H), 2.155 (s,
6H), 2.228 (m, 4H), 2.371 (s, 6H), 2.748 (s, 4H), 2.918 (q, J=6.9
hz, 2H), and 7.300 (s, 2H).
F. Preparation of
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy
-9-ethoxy-9H-xanthene (Ac2EtSF-505-Sar-OBn)
To a solution of sarcosine benzyl ester (0.72 g, 4.02 mmol) in
methylene chloride (40 mL) was added Ac2EtSF-526-NHS (1.82 g, 3.21
mmol) and 5% aq sodium bicarbonate solution (30 mL). The two-phase
mixture was stirred rapidly for 20 hours. The layers were separated
and the organic layer washed with 4.times.15 mL water, dried over
sodium sulfate, and concentrated to 15 mL. The solution was diluted
to 100 mL with cyclohexane, charcoal-treated, and reduced to 50 mL
under a stream of nitrogen resulting in the precipitation of the
product. Filtration followed by air-drying afforded a colorless
solid (0.96 g, 47%).
Coevaporation with methylene chloride followed by extensive vacuum
drying afforded an analytical sample. Anal: Calc. for
[C(36)H(41)N(1)O(9)] C 68.45, H 6.54, N 2 22. Found: C 68.29, H
6.70, N 2.07. NMR (DMSO-d.sub.6) (Shows 5:2 mixture of amide bond
rotamers.): w (major and minor) 1.049 and 1.027 (t, J=6.8 hz, 3H),
1.783 and 1.700 (m, 2H), 2.129 and 2.099 (s, 6H), 2.159 and 2.129
(s, 6H), 2.14 (m, 2H), 2.379 and 2.371 (s, 6H), 2.699 and 2.690 (s,
3H), 2.873 (q, J=6.8 hz, 2H), 3.958 and 3.976 (s, 2H), 5.075 and
5.019 (s, 2H), 7.266 and 7.233 (s, 2H), and 7.25-7.40 (m, 5H).
G. Preparation of
9-(2-(N-Methyl-N-(N'-succinimidyloxycarbonylmethyl)carboxamido)ethyl)-3,6-
diacetoxy-9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene
(Ac2EtSF-526-Sar-NHS. Structure 2d)
To a solution of Ac2EtSF-526-Sar-OBn (0.96 g, 1.52 mmol) in
absolute ethanol (40 mL) was added 10% palladium on carbon (0.10
g). The mixture was stirred under balloon pressure of hydrogen for
30 minutes. The catalyst was removed by filtration and the ethanol
stripped off to afford a syrupy residue.
This residue was dissolved in methylene chloride (40 mL) and
N-hydroxysuccinimide (0.26 g, 2.26 mmol) and
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (0.59
g, 3.08 mmol) were added. The mixture was stirred for 30 minutes
and then washed with water (4.times.15 mL). The solution was dried
over sodium sulfate, concentrated to 15 mL, diluted to 100 mL with
cyclohexane, charcoal treated, and reduced in volume to 50 mL under
a stream of nitrogen. The product was collected by filtration, air
dried, and vacuum dried to afford colorless microcrystals (0.573 g,
59%).
Coevaporation with methylene chloride followed by extensive vacuum
drying at 40.degree. C. removed traces of cyclohexane and afforded
an analytical sample as an amorphous solid. NMR (DMSO-d.sub.6): w
1.043 (t, J=6.7 hz, 3H), 1.82 (m, 2H), 2.130 (s, 6H), 2.157 (s,
6H), 2.15 (m, 2H), 2.378 (s, 6H), 2.748 (s, 4H), 2.778 (s, 3H),
2.891 (q, J=6.7 hz, 2H), 4.327 (s, 2H), and 7.275 (s, 2H).
EXAMPLE 19
A General Method for Coupling Alkynylamino-nucleotides with
N-Hydroxysuccinimide Esters 2
Preparation of Fluorescently-labeled Chain Terminating
Alkynylamino-nucleotides 34-37
Alkynylamino-nucleotide triphosphate 49 (10 micromole, from Example
3J) was taken up in water (0.050 mL) and diluted with
dimethylformamide (0.100 mL). A solution of N-hydroxysuccinimide
ester 2a (12.3 mg, 21 micromole, 2.1 eq, from Example 15E) in
dimethylformamide (0.100 mL) was added and the mixture was stirred
at 50.degree. for 4 hours. Concentrated ammonium hydroxide (0.25
mL) was added, the reaction vessel was tightly stoppered, and
heating at 50.degree. was continued for 25 minutes. The resulting
red solution was diluted to 10 mL with water and applied to a
column of DEAE-Sephadex A-25-120 (1.times.19 cm bed) that had been
equilibrated with 1.0M pH 7.6 aqueous TEAB (50 mL) and then 0.2M pH
7.6 aqueous TEAB (50 mL). The column was eluted with a linear
gradient of pH 7.6 aqueous TEAB from 0.4M (150 mL) to 0.7M (150
mL). The column was driven at 100 mL/h collecting fractions every 3
minutes. The eluent was monitored by absorbance at 498 nm (40
AUFS). Two lesser by-product bands eluted first followed by the
stronger product band with baseline resolution. The fractions
estimated to contain pure product were pooled, stripped down
(T<30.degree.), co-evaporated three times with absolute ethanol,
and taken up in water (0.74 mL). The solution was assayed by
visible absorption (pH 8.2 50 mM aqueous Tris buffer) and
lyophilized. A dilute solution of the product displayed an
absorption maximum at 487.5 nm. Assuming an absorption coefficient
for the product equal to that of the free dye (72,600), the yield
of labeled alkynylamino-nucleotide 37 was 4.2 micromole (42%).
The above procedure produced fluorescently-labeled chain terminator
37 wherein Het is a 7-deazaguanine (k). Labeled chain terminators
34 (Het is uracil (h)), 35, (cytosine (i)), and 36
(7-deazaadenosine (j)) were prepared following similar procedures
by coupling alkynylamino-nucleotide triphosphates 46, 42 and 51
with N-hydroxysuccinimides 2d, 2c, and 2b, respectively. Other
fluorescently-labeled nucleotide triphosphates were also prepared
by the same methods.
* * * * *