U.S. patent application number 13/283220 was filed with the patent office on 2012-02-16 for nucleotide analogs.
This patent application is currently assigned to HELICOS BIOSCIENCES CORPORATION. Invention is credited to Xiaopeng Bai, Jayson Bowers, Philip R. Buzby, J. William Efcavitch, Mirna Jarosz, Edyta Krzymanska-Olejnik, Subramanian Marappan, Judith Mitchell, Atanu Roy, Suhaib Siddiqi.
Application Number | 20120040340 13/283220 |
Document ID | / |
Family ID | 46331863 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040340 |
Kind Code |
A1 |
Efcavitch; J. William ; et
al. |
February 16, 2012 |
NUCLEOTIDE ANALOGS
Abstract
The invention provides for nucleotide analogs and methods of
using the same, e.g., for sequencing nucleic acids.
Inventors: |
Efcavitch; J. William; (San
Carlos, CA) ; Siddiqi; Suhaib; (Wakefield, MA)
; Buzby; Philip R.; (Brockton, MA) ; Mitchell;
Judith; (Brighton, MA) ; Krzymanska-Olejnik;
Edyta; (Brookline, MA) ; Marappan; Subramanian;
(Acton, MA) ; Bai; Xiaopeng; (Watertown, MA)
; Roy; Atanu; (Woburn, MA) ; Jarosz; Mirna;
(Arlington, MA) ; Bowers; Jayson; (Cambridge,
MA) |
Assignee: |
HELICOS BIOSCIENCES
CORPORATION
Cambridge
MA
|
Family ID: |
46331863 |
Appl. No.: |
13/283220 |
Filed: |
October 27, 2011 |
Related U.S. Patent Documents
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12098196 |
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8071755 |
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13283220 |
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11929084 |
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11929084 |
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11803339 |
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11295406 |
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11603945 |
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11286626 |
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11295406 |
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11295155 |
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7476734 |
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11295406 |
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11295155 |
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11295155 |
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11295406 |
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11295155 |
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11496274 |
Jul 31, 2006 |
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11803339 |
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11496262 |
Jul 31, 2006 |
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11496274 |
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11496275 |
Jul 31, 2006 |
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11603945 |
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11496274 |
Jul 31, 2006 |
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11496275 |
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60574389 |
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Current U.S.
Class: |
435/6.1 ;
530/322; 536/26.26 |
Current CPC
Class: |
C07H 21/04 20130101 |
Class at
Publication: |
435/6.1 ;
530/322; 536/26.26 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 19/14 20060101 C07H019/14; C07H 19/10 20060101
C07H019/10; C07K 9/00 20060101 C07K009/00 |
Claims
1. A method for sequencing a nucleic acid, the method comprising
the steps of: exposing a nucleic acid duplex comprising a template
portion and a primer portion to a nucleotide analog comprising an
inhibitor that is charged or capable of becoming charged, and a
polymerase, under conditions that permit template-dependent
incorporation of the analog into the primer; detecting
incorporation of the analog; removing or neutralizing the
inhibitor; and repeating the exposing, detecting, and removing
steps at least once, thereby to determine the sequence of the
template.
2. The method of claim 1, wherein the template portion and/or the
primer portion is directly or indirectly anchored to a support.
3. The method of claim 1, wherein the detecting step comprises
detecting individual analogs.
4. The method of claim 1 further comprising the step of removing
unincorporated analog.
5. The method of claim 1, wherein the inhibitor is selected from
the group consisting of one or more carboxylic acid, one or more
phosphate, one or more amino acid, one or more peptide, one or more
sulfate, one or more caproic acid, and any combination thereof.
6. The method of claim 5, wherein the amino acid is a negatively
charged amino acid.
7. The method of claim 6, wherein the amino acid is selected from
the group consisting of aspartic acid, glutamic acid, histidine,
lysine, and arginine.
8. The method of claim 5, wherein the peptide is from about 2 to
about 10 amino acids in length.
9. The method of claim 1, wherein the inhibitor comprises multiple
charged groups.
10. The method of claim 1, wherein the inhibitor is negatively
charged.
11. The method of claim 1, wherein the inhibitor is positively
charged.
12. The method of claim 1, wherein the inhibitor does not cause
steric inhibition of the polymerase.
13. A nucleotide analog, comprising a nucleoside triphosphate; an
inhibitor comprising (a) one or more multiply charged groups or
groups capable of becoming multiply charged, or (b) two or more
singly charged groups or two or more groups capable of becoming
singly charged; a detectable label; and a linker connecting the
inhibitor and the label to the nucleoside triphosphate.
14. The analog of claim 13, wherein the linker is cleavable.
15. The analog of claim 14, wherein after the linker is cleaved,
the residual analog has the structure of: ##STR00083## wherein
B.sup.1 is selected from the group consisting of purine bases,
pyrimidine bases, and derivatives of purine and pyrimidine bases;
R' is independently selected from the group consisting of --OH,
--O--P(O)(OH).sub.2, --O--C(O)--R.sup.x, --NHR.sup.y, and an
--O-blocking agent, wherein R.sup.x and R.sup.y are alkyl groups;
R'' is independently selected from the group consisting of H and
--OH; R.sup.7 is a phosphodiester or a phosphoryl group; and z is
an integer from about 1 to about 5.
16. The analog of claim 13, wherein the charged groups consist of
between about 2 to about 10 charged groups.
17. The analog of claim 13, wherein the charged groups are selected
from any combination of one or more carboxylic acid, one or more
phosphate, one or more amino acid, one or more peptide, one or more
sulfate, and one or more caproic acid.
18. The analog of claim 13, wherein the label is optically
detectable.
19. The analog of claim 18, wherein the label is a fluorescent
label.
20. The analog of claim 13, wherein the inhibitor is not a steric
inhibitor of a polymerase enzyme.
21. The analog of claim 13, wherein the nucleoside triphosphate is
selected from ATP, GTP, CTP, TTP, UTP, dATP, dGTP, dCTP, dTTP,
dUTP, or an analog of any of the foregoing.
22. A nucleotide analog of the following Formula II: ##STR00084##
wherein NTP is a nucleoside or nucleotide triphosphate or an analog
of either capable of incorporating onto the 3' end of a
polynucleotide strand hybridized to a template presenting the
complement of the NTP; L is a detectable label that facilitates the
identification of the nucleotide analog; Inhibitor comprises (a)
one or more multiply charged groups or groups capable of becoming
multiply charged, or (b) two or more singly charged groups or two
or more groups capable of becoming singly charged; R.sub.1 and
R.sub.2 are independently a bond or a group, wherein at least one
of R.sub.1 and R.sub.2 comprises a cleavable bond, which upon
cleavage results in de-association of NTP from both L and
Inhibitor; R.sub.3 is a bond or group linking R.sub.2 to the
Inhibitor moiety; and R.sub.4 is a bond or group linking R.sub.2 to
a L.
23. The nucleotide analog of claim 22, wherein the Inhibitor does
not comprise a nucleotide or nucleoside or analogs thereof.
24. The nucleotide analog of claim 22, wherein the Inhibitor
comprises a negatively charged group or a group capable of becoming
negatively charged.
25. The nucleotide analog of claim 22, wherein the Inhibitor
comprises a positively charged group or a group capable of becoming
positively charged.
26. The nucleotide analog of claim 22, wherein the Inhibitor
comprises two or more charged groups.
27. The nucleotide analog of claim 22, wherein the Inhibitor
comprises a charged group selected from the group consisting of
--COOH, --PO.sub.4, --SO.sub.4, --SO.sub.3, --SO.sub.2,
--NR.sub.wR.sub.v, where R.sub.w and R.sub.v independently is H, an
alkyl or aryl group.
28. The nucleotide analog of claim 22, wherein the Inhibitor
comprises ##STR00085## wherein R.sub.8 and R.sub.9 independently is
a H or an alkyl group; each of x and y is an integer from 0 to
about 10.
29. The nucleotide analog of claim 28, wherein R.sub.8 and R.sub.9
are H atoms and x=1 and y=2.
30. The nucleotide analog of claim 22, wherein the Inhibitor does
not comprise a --PO.sub.4 group.
31. The nucleotide analog of claim 22, wherein the Inhibitor does
not comprise an aryl group.
32. The nucleotide analog of claim 22, wherein the Inhibitor
comprises an amino acid group or an amino acid analog group.
33. The nucleotide analog of claim 32, wherein the Inhibitor
comprises a peptide of 2 to 20 units of amino acids or analogs.
34. The nucleotide analog of claim 32, wherein the Inhibitor
comprises a group selected from the group consisting of Glu, Asp,
Arg, His, Thr, Trp, Gln, Tyr and Lys.
35. The nucleotide analog of claim 22, wherein R.sub.3 comprises
##STR00086## wherein R.sub.5 is a H or an alkyl group; p is an
integer from 0 to about 10.
36. The nucleotide analog of claim 35, wherein p is 5 or 6.
37. The nucleotide analog of claim 22, wherein R.sub.1 comprises a
C--C triple bond.
38. The nucleotide analog of claim 22, wherein R.sub.1 comprises a
S--S bond.
39. The nucleotide analog of claim 22, wherein R.sub.1 comprises a
C--C triple bond and a S--S bond.
40. The nucleotide analog of claim 22, wherein R.sub.1 comprises
##STR00087## wherein R.sub.6 is a H or an alkyl group; q and r
independently is an integer from about 1 to about 10.
41. The nucleotide analog of claim 40, wherein q is 1 or 2 and r is
1, 2 or 3.
42. The nucleotide analog of claim 22, wherein NTP is selected from
dATP, dGTP, dCTP, dTTP, dUTP, ATP, GTP, CTP, TTP, UTP or an analog
thereof.
43. The nucleotide analog of claim 22, wherein the L is an
optically-detectable moiety.
44. The nucleotide analog of claim 43, wherein the
optically-detectable moiety comprises a fluorophore.
45. The nucleotide analog of claim 44, wherein the fluorophore is
Cy5 or ATTO 647N.
46. The nucleotide analog of claim 22, wherein the group of the
Inhibitor that is charged or capable of becoming charged is from
about 5 to about 60 bonds away from the NTP.
47. The nucleotide analog of claim 36, wherein p is 5 and the
detectable label comprises ATTO 647N.
48. The nucleotide analog of claim 22, wherein R.sub.3 comprises
##STR00088## wherein k is an integer from about 1 to about 5.
49. The nucleotide analog of claim 48, wherein the Inhibitor
comprises a --COOH group.
50. The nucleotide analog of claim 49, wherein the Inhibitor
comprises two or more --COOH groups.
51. The nucleotide analog of claim 22, wherein R.sub.3 comprises
##STR00089## wherein R.sup.1, R.sup.2 are independently H or alkyl
groups, and may together form 3, 4, 5, or 6-member rings, and j is
an integer from about 1 to about 5.
52. The nucleotide analog of claim 22, wherein R.sub.1 comprises
##STR00090## wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
independently H or alkyl groups, and two or more of which may
together form one or more 3, 4, 5, or 6-member rings, and j is an
integer from about 1 to about 3.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part (CIP) of U.S.
application Ser. No. 11/929,084 filed Oct. 30, 2007, which is a
continuation of Ser. No. 11/803,339 filed May 14, 2007, which is a
CIP of Ser. No. 11/603,945 filed Nov. 22, 2006, which is a CIP of
Ser. No. 11/295,406 filed Dec. 5, 2005, which is a CIP of Ser. No.
11/286,626 filed Nov. 22, 2005; Ser. No. 11/803,339 filed May 14,
2007 is a CIP of Ser. No. 11/295,155 filed Dec. 26, 2005, which is
a CIP of Ser. No. 11/295,406 filed Dec. 5, 2005; Ser. No.
11/803,339 filed May 14, 2007 is a CIP of Ser. No. 11/496,262 filed
Jul. 31, 2006, which is a CIP of Ser. No. 11/295,155 filed Dec. 26,
2005, which is a CIP of Ser. No. 11/295,406 filed Dec. 5, 2005;
Ser. No. 11/803,339 filed May 14, 2007 is a CIP of Ser. No.
11/496,274 filed Jul. 31, 2006, which is a CIP of Ser. No.
11/496,262 filed Jul. 31, 2006; Ser. No. 11/603,945 filed Nov. 22,
2006 is a CIP of Ser. No. 11/496,275 filed Jul. 31, 2006, which is
a CIP of Ser. No. 11/496,274 filed Jul. 31, 2006; this application
is also a CIP of Ser. No. 11/137,928 filed May 25, 2005 which
claims priority to 60/574,389 filed May 25, 2004, the entire
contents of each of the above applications are expressly
incorporated herein by reference for all purposes.
FIELD OF THE INVENTION
[0002] The invention relates to nucleotide analogs and methods for
sequencing a nucleic acid using the nucleotide analogs.
BACKGROUND
[0003] Sequencing-by-synthesis involves the template-dependent
addition of nucleotides to a template/primer duplex. Traditional
sequencing-by-synthesis is performed using dye-labeled terminators
and gel electrophoresis (so-called "Sanger sequencing"). See, e.g.,
Sanger, F. and Coulson, A. R., 1975, J. Mol. Biol. 94: 441-448;
Sanger, F. et al., 1977, Nature. 265(5596): 687-695; and Sanger, F.
et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 75: 5463-5467.
Recently, single molecule sequencing methods have been proposed
that provide increased resolution, throughput, and speed at reduced
cost. For example, a sequencing-by-synthesis method that results in
sequence determination without consecutive base incorporation, has
been proposed by Braslaysky, et al., Proc. Nat'l Acad. Sci., 100:
3960-3964 (2003). These methods do not rely on the user of
terminator nucleotides as in Sanger sequencing. Instead,
template/primer duplex is anchored directly, or indirectly (e.g.,
via a polymerase enzyme) to a surface and labeled nucleotides are
added in a template-dependent manner.
[0004] A challenge that has arisen in single molecule sequencing
involves the ability to sequence through homopolymer regions (i.e.,
portions of the template that contain consecutive identical
nucleotides). Often the number of bases present in a homopolymer
region is important from the point of view of genetic function.
Many polymerase enzymes used in sequencing-by-synthesis reactions
are highly-processive and tend to add bases continuously in a
homopolymer region. It is often difficult to resolve the number of
nucleotides in a homopolymer due to the difficulty in
distinguishing between the incorporation of one or two labeled
nucleotides and the incorporation of a greater number of
nucleotides.
[0005] A need therefore exists for nucleotide analogs that promote
accurate base-over-base incorporation in sequencing-by-synthesis
reactions.
SUMMARY OF THE INVENTION
[0006] The invention provides nucleotide analogs and methods of
using them to allow sequencing-by-synthesis to occur such that, on
average, a single nucleotide is incorporated into the 3' end of a
primer portion of a template/primer duplex per sequencing cycle.
The invention is based, in part, on the discovery that nucleotide
analogs having an attached inhibitory region with one or more
charged groups provide good incorporation of a single nucleotide
into the duplex without allowing a significant, or any, amount of
second, third, etc. base incorporation.
[0007] The invention generally provides nucleotide analogs and
methods of using nucleotide analogs in sequencing. More
particularly, the invention provides compounds, methods and
compositions useful in introduction of a single base at a time in a
template-dependent sequencing-by-synthesis reaction. The invention
allows template-dependent sequencing-by-synthesis through all
regions of a target nucleic acid, including homopolymer regions,
and provides methods for the determination of the number of
nucleotides present in a homopolymer region.
[0008] The invention provides nucleotide analogs that comprise a
nucleotide (or nucleotide analog), a detectable label, and an
inhibitor group. Upon incorporation of the nucleotide, the
inhibitor prevents subsequent nucleotide incorporation into the
same duplex. However, upon removal of the detectable label and the
inhibitor group, the nucleotide analog does not substantially
hinder subsequent nucleotide (or nucleotide analog)
incorporation.
[0009] In one aspect, A method for sequencing a nucleic acid. The
method includes the steps of: exposing a nucleic acid duplex
comprising a template portion and a primer portion to a nucleotide
analog comprising an inhibitor that is charged or capable of
becoming charged, and a polymerase, under conditions that permit
template-dependent incorporation of the analog into the primer;
detecting incorporation of the analog; removing or neutralizing the
inhibitor; and repeating the exposing, detecting, and removing
steps at least once, thereby to determine the sequence of the
template.
[0010] In another aspect, the invention relates to a nucleotide
analog that includes: a nucleoside triphosphate; an inhibitor
comprising (a) one or more multiply charged groups or groups
capable of becoming multiply charged, or (b) two or more (i.e., a
plurality of) singly charged groups or two or more groups capable
of becoming singly charged; a detectable label; and a linker
connecting the inhibitor and the label to the nucleoside
triphosphate. It should be noted that in some embodiments, one or a
single charged group may be sufficient to provide the desired
inhibitory effect.
[0011] In another aspect, the invention relates to nucleotide
analogs of the formula:
NTP-Tether-Inhibitor. I
NTP is a nucleoside or nucleotide triphosphate or an analog thereof
capable of template-dependent incorporation into the 3' end of a
polynucleotide strand hybridized to a template. Inhibitor comprises
a moiety that is charged or capable of becoming charged and that
inhibits subsequent nucleotide incorporation once the first
nucleotide is incorporated. Tether is a bond or a group linking the
NTP to the Inhibitor group. In a preferred embodiment, the
inhibitor is a non-steric inhibitor.
[0012] In another aspect, the invention relates to nucleotide
analogs of Formula II:
##STR00001##
NTP is a nucleoside or nucleotide triphosphate or an analog of
either capable of template-dependent incorporation into the 3' end
of a polynucleotide strand hybridized to a template presenting the
complement of the NTP. L is a detectable label that facilitates the
identification of the nucleotide analog. Inhibitor comprises (a)
one or more multiply charged groups or groups capable of becoming
multiply charged, or (b) two or more singly charged groups or two
or more groups capable of becoming singly charged. R.sub.1 and
R.sub.2 are independently a bond or a group, wherein at least one
of R.sub.1 and R.sub.2 comprises a cleavable bond, which upon
cleavage results in de-association of NTP from both Label and
Inhibitor. R.sub.3 is a bond or group linking R.sub.2 to the
Inhibitor. R.sub.4 is a bond or group linking R.sub.2 to a
Label.
[0013] In another aspect, the invention relates to a method for
sequencing a nucleic acid. The method includes: (a) anchoring a
nucleic acid duplex, or portion thereof, to a surface, the duplex
comprising a template portion and a primer portion hybridized
thereto; (b) exposing the duplex to nucleotide analog of Formula I
or II (as defined herein) in the presence of a polymerase capable
of catalyzing the addition of the nucleotide analog to the primer
portion in a template-dependent manner; (c) removing unincorporated
nucleotide analog and polymerase; (d) detecting incorporation of
the nucleotide analog into the primer portion; and repeating the
exposing, removing, and detecting steps at least once.
[0014] In another aspect, the invention provides methods and
nucleotide analogs for selectively inhibiting the catalytic
function of a polymerase enzyme. As such, nucleotide analogs
comprise an inhibitory portion, such that the nucleotide analog is
capable of being incorporated into a nucleic acid duplex but then
inhibits subsequent nucleotide incorporation until the inhibitory
portion is removed.
[0015] The inhibitory portion of an analog of the invention
preferably is a charged group. The charged group can take any
appropriate form as long as it carries a charge. Preferably, the
charge group is selected from a phosphate, a carboxylic acid (or
carboxylate), a sulfate, caproic acid (or a caproic acid
derivative), a charged amino acid, --SO.sub.3, --SO.sub.2, and
--NR.sub.wR.sub.v, where R.sub.w and independently is H, an alkyl
or aryl group. The charged group can convey a negative or positive
charge, but negative charged groups are preferred. In another
preferred embodiment, the charge group contains multiple charged
portions. For example, the charge group can be a dipeptide, a
di-phosphate, disulfate, or other multiples of charged moieties.
For example, amino acid inhibitors are preferably selected from
aspartic acid, glutamic acid, arginine, lysine, and histidine.
[0016] The invention provides charged inhibitors of subsequent base
incorporation in a sequencing-by-synthesis reaction. By subsequent
base incorporation it is intended that a first nucleotide (or
analog) is incorporated in a template-dependent manner, but second,
third, etc. base incorporation is inhibited by the inhibitor group.
In a preferred embodiment, inhibition occurs by positioning a
charged group in proximity to the active site of a polymerase
enzyme, thus disabling the ability of the polymerase to make
subsequent incorporations. Without being limited to theory, analogs
of the invention, interfere with magnesium present in the active
site of the polymerase, resulting in a reduced ability of the
active site to catalyze subsequent nucleotide incorporation.
[0017] In a preferred embodiment, an analog of the invention
comprises a nucleoside triphosphate, an inhibitor comprising a
plurality of charged groups, a detectable label, and a linker
connecting the charged groups and the label to the nucleoside
triphosphate. Preferred inhibitors comprise a plurality of charged
groups and may be selected from any charged group capable of
conferring a charge in a local area. Preferably, the inhibitor does
not sterically inhibit a polymerase. Also in a preferred
embodiment, the linker is cleavable. Multiple cleavable groups,
such as enzymatically-cleavable group, such as disulfide bonds and
the like.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The invention provides methods and compositions that
facilitate the addition of a single nucleotide to a template/primer
duplex per reaction cycle (i.e., the addition of nucleotides and
polymerase enzyme under conditions that result in
template-dependent nucleotide incorporation into the primer).
Analogs of the invention comprise a charged inhibitory group that,
upon incorporation of a nucleotide in a template-dependent manner,
prevents subsequent nucleotide incorporation until the inhibitory
group is removed. Thus, an analog of the invention comprises a
nucleotide triphosphate, a linker (or tether), a detectable label,
and a charged inhibitory group, wherein the label and the
inhibitory group are removable.
[0019] In one aspect, the invention generally provides nucleotide
analogs of the following Formula I:
NTP-Tether-Inhibitor I
wherein [0020] NTP is a nucleoside triphosphate or an analog
thereof capable of incorporating onto the 3' end of a
polynucleotide strand hybridized to a template presenting the
complement of the NTP; [0021] Inhibitor comprises a group that is
charged or capable of becoming charged, e.g., under reaction
conditions, and that inhibits a subsequent incorporation of a
nucleotide (or analog thereof), and [0022] Tether is a bond or a
group linking the NTP to the Inhibitor moiety. A group is
considered capable of becoming charged if the group is capable of
becoming electrically non-neutral, e.g., under reaction or buffer
conditions. Examples of such groups include --COOH and
--NR.sub.wR.sub.v, where R.sub.w and R.sub.v independently is H, an
alkyl or aryl group.
[0023] In one embodiment, the inhibitor group can cause inhibition
of subsequent nucleotide incorporation without steric hinderance.
In other words, the inhibition is caused by chemical or charge
interaction with the enzyme and not be a physical blocking of the
enzyme. In another embodiment, the charged inhibitor also provides
steric inhibition of enzyme activity. However, in either case, the
inhibitor group is charged.
[0024] Natural NTPs include nucleoside triphosphates, adenosine
triphosphate (ATP), guanosine triphosphate (GTP), cytidine
triphosphate (CTP), thymidine triphosphate (TTP) and uridine
triphosphate (UTP); and nucleotide triphosphates, deoxyadenosine
triphosphate (dATP), deoxyguanosine triphosphate (dGTP),
deoxycytidine triphosphate (dCTP), deoxythimidine triphosphate
(dTTP) and deoxyuridine triphosphate (dUTP). NTPs useful in this
invention include non-nature nucleosides and nucleotides, and
analogs and derivatives thereof.
[0025] In some embodiments, the inhibitor may include a moiety that
is negatively charged or capable of becoming a negatively charged.
In other embodiments, the inhibitor group is positively charged or
capable of becoming positively charged.
[0026] In some other embodiments, the inhibitor is an amino acid or
an amino acid analog. The Inhibitor may be a peptide of 2 to 20
units of amino acids or analogs, a peptide of 2 to 10 units of
amino acids or analogs, a peptide of 3 to 7 units of amino acids or
analogs, a peptide of 3 to 5 units of amino acids or analogs. In
some embodiments, the Inhibitor includes a group selected from the
group consisting of Glu, Asp, Arg, His, and Lys, and a combination
thereof (e.g., Arg, Arg-Arg, Asp, Asp-Asp, Asp, Glu, Glu-Glu,
Asp-Glu-Asp, Asp-Asp-Glu or AspAspAspAsp). Peptides or groups may
be combinations of the same or different amino acids or
analogs.
[0027] In one embodiment, the invention relates to an
oligonucleotide with at least one nucleotide analog of the
invention incorporated therein.
[0028] In some embodiments, the Tether comprises
##STR00002##
wherein L is detectable label that facilitates the identification
of the nucleotide analog after incorporation onto a template;
[0029] R.sub.1 and R.sub.2 are independently a bond or a group,
wherein at least one of R.sub.1 and R.sub.2 comprises a cleavable
bond, which upon cleavage results in de-association of NTP from
both L and Inhibitor; [0030] R.sub.3 is a bond or group linking
R.sub.2 to the Inhibitor moiety; and [0031] R.sub.4 is a bond or
group linking R.sub.2 to a L.
[0032] In another aspect, the present invention is directed to
nucleotide analogs of Formula II:
##STR00003##
wherein [0033] NTP is a nucleoside triphosphate or an analog
thereof capable of incorporating onto the 3' end of a
polynucleotide strand hybridized to a template presenting the
complement of the NTP; [0034] L is a detectable label to facilitate
the identification of the nucleotide analog after incorporation
onto the template; [0035] Inhibitor is a moiety that substantially
inhibits a subsequent incorporation of a nucleotide (or analog
thereof). In some embodiments, the Inhibitor moiety includes a
nucleotide or nucleoside or analogs thereof, in other embodiments,
the inhibitor is not a nucleotide or analog thereof; [0036] R.sub.1
and R.sub.2 are independently a bond or a group, wherein at least
one of R.sub.1 and R.sub.2 comprises a cleavable bond, which upon
cleavage results in de-association of NTP from both Label and
Inhibitor; [0037] R.sub.3 is a bond or group linking R.sub.2 to the
Inhibitor moiety; and [0038] R.sub.4 is a bond or group linking
R.sub.2 to L.
[0039] In some embodiments, NTP is a compound having the following
formula:
##STR00004##
wherein B.sup.1 is selected from the group consisting of purine or
pyrimidine bases, as well as derivatives of purine and pyrimidine
bases; R' is independently selected from the group consisting of
--OH, --O--P(O)(OH).sub.2, --O--C(O)--R.sup.x, --NHR.sup.y, and an
--O-blocking agent, where R.sup.x and R.sup.y are alkyl groups; R''
is independently selected from the group consisting of H and
--OH.
[0040] Non-limiting examples of representative purine and
pyrimidine bases include adenine, cytosine, guanine, thymine,
uracil, or hypoxanthine. Non-limiting examples of derivatives of
purine and pyrimidine bases include naturally-occurring and
synthetic derivatives of a base, including
pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo),
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine,
7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,
imidazo[1,5-a] 1,3,5 triazinones, 9-deazapurines,
imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines,
pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.
[0041] Base B.sup.1 of the invention permits a nucleotide to be
incorporated into a polynucleotide chain by a polymerase and forms
base pairs with a base on an antiparallel nucleic acid strand. The
term base pair encompasses not only the standard AT, AU or GC base
pairs, but also base pairs formed between nucleotides and/or
nucleotide analogs comprising non-standard or modified bases,
wherein the arrangement of hydrogen bond donors and hydrogen bond
acceptors permits hydrogen bonding between a nonstandard base and a
standard base or between two complementary non-standard base
structures. One example of such non-standard base pairing is the
base pairing between the nucleotide analog inosine and adenine,
cytosine or uracil, where two hydrogen bonds are formed.
[0042] The Inhibitor may include a charged moiety (e.g., a
negatively charged moiety, a positively charged moiety, or both) or
a moiety that is capable of becoming charged. The Inhibitor can
include two or more charged groups. The Inhibitor may have a
charged group selected from the group consisting of --COOH,
--PO.sub.4, --SO.sub.4, --SO.sub.3, --SO.sub.2, --NR.sub.wR.sub.v,
where R.sub.w and R.sub.v independently is H, an alkyl or aryl
group. In other embodiments, the Inhibitor moiety does not comprise
a --PO.sub.4 group. In some other embodiments, the Inhibitor moiety
does not comprise an aryl group. In certain other embodiments, the
Inhibitor does not include a nucleotide or nucleoside or analogs
thereof.
[0043] Inhibitor may be a compound having the following
formula:
##STR00005##
wherein R.sub.8 and R.sub.9 independently is a H or an alkyl group;
each of x and y is an integer from 0 to about 5. In some
embodiments, R.sub.8 and R.sub.9 are H atoms and x=1 and y=2.
[0044] R.sub.3 of a nucleotide analog of Formula II may include a
group having the formula of
##STR00006##
wherein R.sub.5 is a H or an alkyl group; p is an integer from 0 to
about 10. In some embodiments, p is 5 or 6.
[0045] In some embodiments, R.sub.3 of a nucleotide analog of
Formula II may include a group having the formula of
##STR00007##
wherein k is an integer from about 1 to about 5. In some
embodiments, k is an integer from about 2 to about 4. In some
embodiments, k is 3.
[0046] In some embodiments, R.sub.3 of a nucleotide analog of
Formula II may include a group having the formula of
##STR00008##
wherein R.sup.1, R.sup.2 are independently H or alkyl groups, and
may together form one or more 3, 4, 5, or 6-member rings, and j is
an integer from about 1 to about 5. In some embodiments, R.sub.3 of
include a group having the formula of
##STR00009##
[0047] In some embodiments, R.sub.1 of a nucleotide analog of
Formula II may include a group having the formula of
##STR00010##
wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are independently H
or alkyl groups, and two or more of which may together form one or
more 3, 4, 5, or 6-member rings, and j is an integer from about 1
to about 3. In some embodiments, R.sub.1 of include a group having
the formula of
##STR00011##
[0048] R.sub.1 of a nucleotide analog of Formula II may include a
C--C triple bond, a S--S bond, or both a C--C triple bond and a
S--S bond.
[0049] In some embodiments, R.sub.1 in the nucleotide analog of
Formula II includes a group having the formula of
##STR00012##
wherein R.sub.6 is a H or an alkyl group; q and r independently is
an integer from about 1 to about 10.
[0050] In some embodiments, q is 1 or 2 and r is 1, 2 or 3.
[0051] In some embodiments of the invention, the location of the
charged moiety within the inhibitor group and/or the distance of
the charged group to the NTP plays an important role in the
effectiveness of inhibiting a subsequent nucleotide incorporation.
In some embodiments, the charged moiety of the inhibitor is from
about 5 to about 60 bonds away from the NTP. In some other
embodiments, the charged moiety of the inhibitor is from about 10
to about 40 bonds away from the NTP. In some other embodiments, the
charged moiety of the inhibitor is from about 10 to about 35 bonds
away from the NTP. In some other embodiments, the charged moiety of
the inhibitor is from about 10 to about 30 bonds away from the NTP.
In some other embodiments, the charged moiety of the inhibitor is
from about 10 to about 20 bonds away from the NTP.
##STR00013##
[0052] For example, the above compound (about 17.times. fold
inhibition) exhibits an inhibiting effect that is much less than
the following compound (about 70.times. fold inhibition).
##STR00014##
[0053] The label (or "L") may be any moiety that can be attached to
or associated with, e.g., directly or via a linker or spacer, an
oligonucleotide and that functions to provide a detectable signal,
and/or to interact with a second label to modify the detectable
signal provided by the first or second label, e.g. fluorescence
resonance energy transfer (FRET). In one embodiment, the label is
an optically-detectable moiety (e.g., a fluorophore). Non-limiting
examples of types of optically-detectable labels include a
fluorescent, chemiluminescence, or electrochemically luminescent
label. Examples of fluorescent labels include, but are not limited
to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid;
acridine and derivatives thereof such as acridine, acridine
isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 15 1); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylaminolnaphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives
of sulforhodamine 101 (Texas Red);
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5;
Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalocyanine;
naphthalocyanine; any of the fluorescent labels available from
Atto-Tec, such as Atto 390, Atto 425, Atto 465, Atto 488, Atto 495,
Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto
610, Atto 611X, Atto 620, Atto 633, Atto 635, Atto 637, Atto 647,
Atto 647N, Atto 655, Atto 680, Atto 700, Atto 725, Atto 740, etc.;
any of the fluorescent labels available from Dyomics such as
DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, Dy-647,
Dy-648, DY-649, Dy-650, Dy-651, DY-652, etc.; any of the
fluorescent labels available from Pierce such as DyLight 405,
DyLight 488, DyLight 549, DyLight 633, DyLight 649, DyLight 680,
DyLight 800, etc.; any of the fluorescent labels available from
AnaSpec such as HiLyte Fluor.TM. 488 dyes, HiLyte Fluor.TM. 555
dyes, HiLyte Fluor.TM. 647 dyes, HiLyte Fluor.TM. 680 dyes, HiLyte
Fluor.TM. 750 dyes, HiLytePlus.TM. 555 dyes, HiLytePlus.TM. 647
dyes, HiLytePlus.TM. 750 dyes, etc.; any of the fluorescent labels
available from Denovo Biolables such as Oyster 500, Oyster 550 P,
Oyster 550 D, Oyster 556, Oyster 645, Oyster 650 P, Oyster 650 D,
Oyster 656, etc.; IRDye.RTM. 680, IRDye.RTM. 700, IRDye.RTM. 700DX,
IRDye.RTM. 800, IRDye.RTM. 800 RS, IRDye.RTM. 800 CW, etc.; any of
the fluorescent labels available from SETA Biomedicals such as Seta
K1-204, Seta K5-3212, Seta K8-1342, Seta K8-1352, Seta K8-1357,
Seta K8-1407, Seta K8-1642, Seta K8-1644, Seta K8-1663, Seta
K8-1664, Seta K8-1669, Seta K8-3002, Seta K4-1082, Seta K8-1669,
Seta K7-545, Seta K7-547, Seta K7-549, Seta K8-1252, Seta K8-1261,
Seta K8-1262, Seta K8-1320, Seta K8-1344, Seta K8-1367, Seta
K8-1377, Seta K8-1382, Seta K8-1446, Seta K8-1667, Seta K8-1752,
Seta K8-1762, Seta K8-1767, Seta K8-1777, Seta K8-1782, etc.; Q
Dots; and dyes having the following structures:
##STR00015## ##STR00016##
wherein each R.sub.x is independently selected from the group
consisting of H, alkyl, and substituted alkyl.
[0054] The above exemplary label moieties include any derivatives
containing the chromophore of any of the labeling moieties
exemplified or described herein, attached to the nucleotide analog
by means of any suitable chemical linking group. For example, the
chromophore can be attached to the nucleotide analog via an alkyl
chain bonded to the nucleotide analog by a functional group such as
an amide, ester, ether, amine, thiol, disulfide, urea, urethane,
carbonate, etc. In one embodiment, the label is a fluorescent label
such as cyanine-3 and cyanine-5.
[0055] Labels other than fluorescent labels are contemplated as
part of the invention, including other optically-detectable labels.
Any appropriate detectable label can be used according to the
invention, and numerous other labels are known to those skilled in
the art.
[0056] The invention also relates to methods for nucleic acid
sequence determination using the nucleotide analogs described
herein. The nucleotide analogs of the invention are particularly
suitable for use in single molecule sequencing techniques. Such
techniques are described for example in U.S. patent application
Ser. Nos. 10/831,214 filed April 2004; Ser. No. 10/852,028 filed
May 24, 2004; Ser. No. 10/866,388 filed Jun. 10, 2005; Ser. No.
10/099,459 filed Mar. 12, 2002; and U.S. Published Application
2003/013880 published Jul. 24, 2003, each of which is herein
incorporated in its entirety for all purposes. In general, methods
for nucleic acid sequence determination include exposing a target
nucleic acid (also referred to herein as template nucleic acid or
template) to a primer that is complementary to at least a portion
of the target nucleic acid, under conditions suitable for
hybridizing the primer to the target nucleic acid, forming a
template/primer duplex.
[0057] The invention also relates to methods for nucleic acid
sequence determination using the nucleotide analogs described
herein. The nucleotide analogs of the invention are particularly
suitable for use in single molecule sequencing techniques. Such
techniques are described for example in U.S. patent application
Ser. Nos. 10/831,214 filed April 2004; Ser. No. 10/852,028 filed
May 24, 2004; Ser. No. 10/866,388 filed Jun. 10, 2005; Ser. No.
10/099,459 filed Mar. 12, 2002; and U.S. Published Application
2003/013880 published Jul. 24, 2003, each of which is herein
incorporated in its entirety for all purposes. In general, methods
for nucleic acid sequence determination include exposing a target
nucleic acid (also referred to herein as template nucleic acid or
template) to a primer that is complementary to at least a portion
of the target nucleic acid, under conditions suitable for
hybridizing the primer to the target nucleic acid, forming a
template/primer duplex.
[0058] In another aspect, the invention relates to a method for
sequencing a nucleic acid. The method includes: (a) anchoring a
nucleic acid duplex to a surface, the duplex comprising a template
portion and a primer portion hybridized thereto; (b) exposing the
duplex to nucleotide analog of Formula I or Formula II in the
presence of a polymerase capable of catalyzing the addition of the
nucleotide analog to the primer portion in a template-dependent
manner; (c) removing unincorporated nucleotide analog and
polymerase; (d) detecting incorporation of the nucleotide analog
into the primer portion; and (e) repeating said exposing, removing,
and detecting steps at least once. The method may further include
cleaving L from the nucleotide analog after the detecting step.
[0059] In another aspect, the invention relates to a method for
inhibiting the catalytic function of a polymerase enzyme in a
sequencing-by-synthesis reaction comprising introducing a
nucleotide attached to an inhibitory group. In one aspect, the
invention comprises attaching one or both members of a
template/primer duplex to a surface, introducing a polymerase and a
nucleotide analog comprising a charged inhibitor under conditions
sufficient for template-dependent incorporation of the nucleotide
and inhibition of subsequent incorporation. Such methods further
comprise removing or neutralizing the inhibitor in order to
facilitate further nucleotide incorporation. Finally, nucleotides
of the invention can be detectably labeled to monitor
incorporation.
[0060] Target nucleic acids include deoxyribonucleic acid (DNA)
and/or ribonucleic acid (RNA). Target nucleic acid molecules can be
obtained from any cellular material obtained from an animal, plant,
bacterium, virus, fungus, or any other cellular organism, or may be
synthetic DNA. Target nucleic acids may be obtained directly from
an organism or from a biological sample obtained from an organism,
e.g., from blood, urine, cerebrospinal fluid, seminal fluid,
saliva, sputum, stool and tissue. Any tissue or body fluid specimen
may be used as a source for nucleic acid for use in the invention.
Nucleic acid molecules may also be isolated from cultured cells,
such as a primary cell culture or a cell line. The cells from which
target nucleic acids are obtained can be infected with a virus or
other intracellular pathogen. Nucleic acid molecules may also
include those of animal (including human), wild type or engineered
prokaryotic or eukaryotic cells, viruses or completely or partially
synthetic RNAs or DNAs. A sample can also be total RNA extracted
from a biological specimen, a cDNA library, or genomic DNA.
[0061] Nucleic acid typically is fragmented to produce suitable
fragments for analysis. In one embodiment, nucleic acid from a
biological sample is fragmented by sonication. Test samples can be
obtained as described in U.S. Patent Application 2002/0190663 A1,
published Oct. 9, 2003, herein incorporated by reference in its
entirety for all purposes. Generally, nucleic acid can be extracted
from a biological sample by a variety of techniques such as those
described by Maniatis, et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Generally,
target nucleic acid molecules can be from about 5 bases to about 20
kb, about 30 kb, or even about 40 kb or more. Nucleic acid
molecules may be single-stranded, double-stranded, or
double-stranded with single-stranded regions (for example, stem-
and loop-structures)
[0062] Single molecule sequencing includes a template nucleic acid
molecule/primer duplex that is immobilized on a surface such that
the duplex and/or the nucleotides (or nucleotide analogs) added to
the immobilized primer are individually optically resolvable. The
primer, template and/or nucleotide analogs are detectably labeled
such that the position of an individual duplex molecule is
individually optically resolvable. Either the primer or the
template is immobilized to a solid support. The primer and template
can be hybridized to each other and optionally covalently
cross-linked prior to or after attachment of either the template or
the primer to the solid support.
[0063] In general, methods for facilitating the incorporation of a
nucleotide analog as an extension of a primer include exposing a
target nucleic acid/primer duplex to one or more nucleotide analogs
disclosed herein and a polymerase under conditions suitable to
extend the primer in a template dependent manner. Generally, the
primer is sufficiently complementary to at least a portion of the
target nucleic acid to hybridize to the target nucleic acid and
allow template-dependent nucleotide polymerization. The primer
extension process can be repeated to identify additional nucleotide
analogs in the template. The sequence of the template is determined
by compiling the detected nucleotides, thereby determining the
complementary sequence of the target nucleic acid molecule.
[0064] Any polymerase and/or polymerizing enzyme may be employed. A
preferred polymerase is Klenow with reduced exonuclease activity.
Nucleic acid polymerases generally useful in the invention include
DNA polymerases, RNA polymerases, reverse transcriptases, and
mutant or altered forms of any of the foregoing. DNA polymerases
and their properties are described in detail in, among other
places, DNA Replication 2nd edition, Komberg and Baker, W. H.
Freeman, New York, N.Y. (1991). Known conventional DNA polymerases
useful in the invention include, but are not limited to, Pyrococcus
furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1,
Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et
al., 1996, Biotechniques, 20: 186-8, Boehringer Mannheim), Thermus
thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991,
Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase
(Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32),
Thermococcus litoralis (Tli) DNA polymerase (also referred to as
Vent.TM. DNA polymerase, Cariello et al., 1991, Polynucleotides
Res, 19: 4 193, New England Biolabs), 9''Nm.TM. DNA polymerase (New
England Biolabs), Stoffel fragment, Thermosequenase.RTM. (Amersham
Pharmacia Biotech UK), Therminator.TM. (New England Biolabs),
Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998
Braz J Med. Res, 3 1: 1239), Thermus aquaticus (Taq) DNA polymerase
(Chien et al., 1976, J. Bacteoriol, 127: 1550), DNA polymerase,
Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997,
Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (from
thermococcus sp. JDF-3, Patent application WO 0132887), Pyrococcus
GB-D (PGB-D) DNA polymerase (also referred as Deep Vent.TM. DNA
polymerase, Juncosa-Ginesta et al., 1994, Biotechniques, 16:820,
New England Biolabs), UITma DNA polymerase (from thermophile
Thermotoga maritima; Diaz and Sabino, 1998 Braz J. Med. Res, 3 1:
1239; PE Applied Biosystems), Tgo DNA polymerase (from thermococcus
gorgonarius, Roche Molecular Biochemicals), E. coli DNA polymerase
I (Lecomte and Doubleday, 1983, Polynucleotides Res. 11: 7505), T7
DNA polymerase (Nordstrom et al., 198 1, J Biol. Chem. 256:3 1 12),
and archaeal DP1I/DP2 DNA polymerase II (Cann et al., 1998, Proc
Natl Acad. Sci. USA 95: 14250-5).
[0065] Other DNA polymerases include, but are not limited to,
ThermoSequenase.RTM., 9.degree.Nm.TM., Therminator.TM., Taq, Tne,
Tma, Pfu, Tfl, Tth, Tli, Stoffel fragment, Vent.TM. and Deep
Vent.TM. DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, and
mutants, variants and derivatives thereof. Reverse transcriptases
useful in the invention include, but are not limited to, reverse
transcriptases from HIV, HTLV-1, HTLV-11, FeLV, FIV, SIV, AMV,
MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8 (1997);
Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al., CRC Crit
Rev Biochem. 3:289-347(1975)).
[0066] Unincorporated nucleotide analog molecules may be removed
prior to or after detecting. Unincorporated nucleotide analog
molecules may be removed by washing.
[0067] A template/primer duplex is treated to remove the label
and/or to cleave the molecular chain attaching the label to the
nucleotide. One may repeat the steps of exposing template/primer
duplex to one or more nucleotide analogs and polymerase, detecting
incorporated nucleotides, and then treating to (1) remove the
label, (2) remove the label and at least a portion of the molecular
chain associating the label to the nucleotide or (3) cleave the
molecular chain thereby identifying additional bases in the
template nucleic acid, The identified bases can be compiled to
determine the sequence of the target nucleic acid. In some
embodiments, at least some portions of the remaining molecular
chain and/or label are not removed, for example, in the last round
of primer extension.
[0068] In some embodiments, a nucleotide analog, after removal of
the label and portions of the molecular chain connecting the label
to the nucleotide can be represented by:
##STR00017##
wherein B.sup.1, R', R'', are as described herein, and z is an
integer from about 1 to about 12. R.sup.7 is a phosphodiester
linkage connecting the nucleotide analog to a sugar of an adjacent
nucleotide in the nucleic acid, or a phosphoryl group. In some
embodiments, z is an integer from about 1 to about 5. In some other
embodiments, z is an integer from about 1 to about 3.
[0069] The invention also provides for a method of removing a label
from a labeled base, comprising(a) exposing a base of Formula I or
Formula II:
##STR00018##
as described herein, to a reducing agent for a time sufficient to
produce an unlabelled base of Formula III:
##STR00019##
where B.sup.1 is a part of the NTP of a nucleotide analog in
Formula I or Formula II, and n is an integer from about 1 to about
12. In some embodiments, the reducing agent is tris(2-carboxyl
ethyl)phosphine. In other embodiments, the base is linked to a
sugar selected from the group consisting of ribose, deoxyribose,
and analogs thereof, where the base and sugar together may be
present in a nucleotide in a nucleic acid.
[0070] One embodiment of a method for sequencing a nucleic acid
template includes exposing a nucleic acid template to a primer
capable of hybridizing to the template, a polymerase capable of
catalyzing nucleotide addition to the primer, and a labeled
nucleotide analog disclosed herein under conditions to permit the
polymerase to add the nucleotide analog to the primer. A method for
sequencing may further include identifying or detecting the
incorporated labeled nucleotide. A cleavable bond may then be
cleaved, removing at least the label from the nucleotide analog.
The exposing, detecting, and removing steps are repeated at least
once. In certain embodiments, the exposing, detecting, and removing
steps are repeated at least three, five, ten or even more times.
The sequence of the template can be determined based upon the order
of incorporation of the labeled nucleotides.
[0071] In another embodiment, a method for sequencing a nucleic
acid template includes exposing a nucleic acid template to a primer
capable of hybridizing to the template and a polymerase capable of
catalyzing nucleotide addition to the primer. The polymerase is,
for example, Klenow with reduced exonuclease activity. The
polymerase adds a labeled nucleotide analog disclosed herein. The
method may include identifying the incorporated labeled nucleotide.
Once the labeled nucleotide is identified, the label and at least a
portion of a molecular chain connecting the label to the nucleotide
analog are removed and the remaining portion of the molecular chain
includes a free hydroxyl group. The exposing, incorporating,
identifying, and removing steps are repeated at least once,
preferably multiple times depending on the application. The
sequence of the template is determined based upon the order of
incorporation of the labeled nucleotides.
[0072] Removal of a label from a labeled nucleotide analog and/or
cleavage of the molecular chain linking a nucleotide analog to a
label may include contacting or exposing the labeled nucleotide
with a reducing agent. Such reducing agents include, for example,
dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP),
tris(3-hydroxy-propyl)phosphine, tris(2-chloropropyl)phosphate
(TCPP), 2-mercaptoethanol, 2-mercaptoethylarnine, cystein and
ethylmaleimide. Such contacting or exposing the reducing agent to a
labeled nucleotide analog may occur at a range of pH values, for
example at a pH of about 5 to about 10, or about 7 to about 9.
[0073] The above-described methods for sequencing a nucleic acid
template can further include a step of capping a molecular chain,
for example, after the label has been removed. After addition of
the nucleotide analog to the primer, any optional 3' phosphate
moiety can be removed enzymatically. In one embodiment, an optional
phosphate can be removed using alkaline phosphatase or T.sub.4
polynucleotide kinase. Suitable enzymes for removing optional
phosphate include, any phosphatase, for example, alkaline
phosphatase such as shrimp alkaline phosphatase, bacterial alkaline
phosphatase, or calf intestinal alkaline phosphatase.
[0074] Any suitable detection method may be used to identify an
incorporated nucleotide analog. Thus, exemplary detection methods
include radioactive detection, optical absorbance detection, e.g.,
UV-visible absorbance detection, optical emission detection, e.g.,
fluorescence or chemiluminescence. Single-molecule fluorescence can
be carried out using a conventional microscope equipped with total
internal reflection (TIR) objective. The detectable moiety
associated with the extended primers can be detected on a substrate
by scanning all or portions of each substrate simultaneously or
serially, depending on the scanning method used. For fluorescence
labeling, selected regions on a substrate may be serially scanned
one-by-one or row-by-row using a fluorescence microscope apparatus,
such as described in Fodor (U.S. Pat. No. 5,445,934) and Mathies et
al. (U.S. Pat. No. 5,091,652). Devices capable of sensing
fluorescence from a single molecule include scanning tunneling
microscope (STM) and the atomic force microscope (AFM).
Hybridization patterns may also be scanned using a CCD camera
(e.g., Model TE/CCD512SF, Princeton Instruments, Trenton, N.J.)
with suitable optics (Ploem, CCD (Chase-Completed-Device) in
Fluorescent and Luminescent Probes for Biological Activity Mason,
T. G. Ed., Academic Press, Landon, pp. 1-11 (1993), such as
described in Yershov et al., Proc. Natl. Aca. Sci. 93:4913 (1996),
or may be imaged by TV monitoring. For radioactive signals, a
phosphorimager device can be used (Johnston et al.,
Electrophoresis, 13566, 1990; Drmanac et al., Electrophoresis,
13:566, 1992; 1993). Other commercial suppliers of imaging
instruments include General Scanning Inc., (Watertown, Mass. on the
World Wide Web at genscan.com), Genix Technologies (Waterloo,
Ontario, Canada; on the World Wide Web at confocal.com), and
Applied Precision Inc. Such detection methods are particularly
useful to achieve simultaneous scanning of multiple attached target
nucleic acids.
[0075] The present invention provides for detection of molecules
ranging from a single nucleotide to a single target nucleic acid
molecule. A number of methods are available for this purpose.
Methods for visualizing single molecules within nucleic acids
labeled with an intercalating dye include, for example,
fluorescence microscopy. For example, the fluorescent spectrum and
lifetime of a single molecule excited-state can be measured.
Standard detectors such as a photomultiplier tube or avalanche
photodiode can be used. Full field imaging with a two-stage image
intensified CCD camera also can be used. Additionally, low noise
cooled CCD can also be used to detect single fluorescent
molecules.
[0076] The detection system for the signal may depend upon the
labeling moiety used. For optical signals, a combination of an
optical fiber or charge coupled device (CCD) can be used in the
detection step. In those circumstances where the substrate is
itself transparent to the radiation used, it is possible to have an
incident light beam pass through the substrate with the detector
located opposite the substrate from the target nucleic acid. For
electromagnetic labeling moieties, various forms of spectroscopy
systems can be used. Various physical orientations for the
detection system are available and discussion of design parameters
is provided in the art.
[0077] A number of approaches can be used to detect incorporation
of fluorescently labeled nucleotides into a single nucleic acid
molecule. Optical setups include near-field scanning microscopy,
far-field confocal microscopy, wide-field epi-illumination, but are
not limited to, light scattering, dark field microscopy,
photoconversion, single and/or multiphoton excitation, spectral
wavelength discrimination, fluorophore identification, evanescent
wave illumination, and total internal reflection fluorescence
(TIRF) microscopy. In general, certain methods involve detection of
laser-activated fluorescence using a microscope equipped with a
camera. Suitable photon detection systems include, but are not
limited to, photodiodes and intensified CCD cameras. For example,
an intensified charge couple device (ICCD) camera can be used. The
use of an ICCD camera to image individual fluorescent dye molecules
in a fluid near a surface provides numerous advantages. For
example, with an ICCD optical setup, it is possible to acquire a
sequence of images (movies) of fluorophores.
[0078] Some embodiments of the present invention use TIRF
microscopy for two-dimensional imaging. TIRF microscopy uses
totally internally reflected excitation light and is well known in
the art. See, e g., the World Wide Web at
nikoninstrurnents.jp/eng/page/products/tirf.aspx. In certain
embodiments, detection is carried out using evanescent wave
illumination and total internal reflection fluorescence microscopy.
An evanescent light field can be set up at the surface, for
example, to image fluorescently-labeled nucleic acid molecules.
When a laser beam is totally reflected at the interface between a
liquid and a solid substrate (e.g., a glass), the excitation light
beam penetrates only a short distance into the liquid. The optical
field does not end abruptly at the reflective interface, but its
intensity falls off exponentially with distance. This surface
electromagnetic field, called the "evanescent wave", can
selectively excite fluorescent molecules in the liquid near the
interface. The thin evanescent optical field at the interface
provides low background and facilitates the detection of single
molecules with high signal-to-noise ratio at visible
wavelengths.
[0079] The evanescent field also can image fluorescently-labeled
nucleotides upon their incorporation into the attached target
nucleic acid target molecule/primer complex in the presence of a
polymerase. Total internal reflectance fluorescence microscopy is
then used to visualize the attached target nucleic acid target
molecule/primer complex and/or the incorporated nucleotides with
single molecule resolution.
[0080] Fluorescence resonance energy transfer (FRET) can be used as
a detection scheme. FRET in the context of sequencing is described
generally in Braslavasky, et al., Proc. Nat'l Acad. Sci., 100:
3960-3964 (2003), incorporated by reference herein. In an
embodiment, a donor fluorophore is attached to the primer,
polymerase, or template. Nucleotides added for incorporation into
the primer comprise an acceptor fluorophore that is activated by
the donor when the two are in proximity.
[0081] Measured signals can be analyzed manually or preferably by
appropriate computer methods to tabulate results. Preferably, the
signals of millions of analogs are read in parallel and then
deconvoluted to ascertain a sequence. The substrates and reaction
conditions can include appropriate controls for verifying the
integrity of hybridization and extension conditions, and for
providing standard curves for quantification, if desired. For
example, a control nucleic acid can be added to the sample. The
absence of the expected extension product is an indication that
there is a defect with the sample or assay components requiring
correction.
[0082] As another example, the described nucleotide analogs can be
used to facilitate "four color" sequencing by synthesis if each
base (A, C, G, T) is labeled with a dye emitting and/or absorbing
at a different and resolvable wavelength. The sequencing procedure
can be shortened from four separate addition cycles (i.e., one for
each base) to the following: add A, C, G, T (each differently
labeled) with polymerase and an appropriate reaction buffer, rinse,
image the four resolvable dyes and record which base (if any) was
incorporated, cleave and cap the nucleotides, and repeat. The
described nucleotide analogs facilitate this kind of sequencing
because of their ability to incorporate one and only one base at a
time. Without that ability, if all four bases are added to the
incorporation reaction at once multiple bases would be added to a
given strand and the interactions between the proximate dyes would
hinder the ability to resolve the sequence information
correctly.
[0083] For example, the nucleotide analogs described herein can
facilitate sequencing nucleic acids containing homopolymer
sequences, using sequencing by synthesis methodology (e.g., using
the methods of US 2007/0190546, herein incorporated by reference in
its entirety for all purpose. When the template sequence contains a
homopolymer, using a polymerase, nucleotide analog, and reaction
buffer combination that allows for only a single nucleotide analog
incorporation allows for each base in the homopolymer to be
sequenced sequentially. After one base is incorporated into the
homopolymer and detected, the portion of the analog that inhibits
subsequent base incorporation and that contains the fluorescent
label is removed, making incorporation of the next base in the
homopolymer possible during the next addition cycle of the correct
base.
[0084] Reference to the following figures or schemes illustrating
an exemplary reaction scheme and nucleotide analogs is intended in
no way to limit the scope of this invention but is provided to
illustrate how to prepare and use the compounds of the present
invention.
EXAMPLES
Example 1
Caproic-Glu and Caproic-Glu
##STR00020##
##STR00021## ##STR00022##
[0085]
3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-prop-
ionic acid 2,5-dioxo-pyrrolidin-1-yl ester (2)
##STR00023##
[0087] To a solution of Fmoc-Cys(SStBu)-OH (1, 2.15 g, 5.0 mmole,)
dissolved in anhydrous CH.sub.2Cl.sub.2(30 mL) was added
N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC,
1.146 g, 6 mmole), the reaction mixture was stirred for 10 min. at
room temperature (RT) and then added N-hydroxysuccinimide (NHS)
(0.690 g, 6.0 mmole). To this reaction mixture was added catalytic
amount of N,N'-dimethlyaminopyridine and stirred at RT until
completion of reaction tested with TLC. The solvent was evaporated
and the residue obtained was extracted with ethyl acetate (50
mL.times.2), washed with 1M NaHCO.sub.3 (10 mL), followed by brine
solution (20 mL) and dried over anhydrous Na.sub.2SO.sub.4.
Evaporation of the solvent afforded 2 as a white crystalline solid.
Yield. 2.5 g (95%).
6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propiony-
lamino]-hexanoic acid (3)
##STR00024##
[0089] To a solution of 6-Aminohexanoic acid (0.158. g, 1.2 mmole)
dissolved in 0.1M NaHCO.sub.3 (2.0 mL) was added the NHS ester 2
(0.68 g, 1.3 mmole) in 4 mL of anhydrous THF. The reaction mixture
was stirred at RT for 2 hr. The solvent was completely evaporated
and the dried solid residue obtained was dissolved in
CH.sub.3OH/CH.sub.2Cl.sub.2 mixture and purified by silica gel
column chromatography using 10% CH.sub.3OH/CH.sub.2Cl.sub.2 and
obtained 3 as a white solid on evaporation the solvent. Yield: 0.5
g (77%).
6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propiony-
lamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (4)
##STR00025##
[0091] To a solution of (3, 500 mg, 0.92 mmole,) dissolved in
anhydrous CH.sub.2Cl.sub.2/THF (1:1)(5 mL) was added
N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC,
191 mg, 1.0 mmole), followed by NHS (115 mg, 1.0 mmole). To this
reaction mixture was added catalytic amount of
N,N'-dimethlyaminopyridine and stirred at RT until completion of
reaction tested with TLC. The solvent was evaporated and the
residue obtained was extracted with ethyl acetate (50 mL.times.2),
washed with 1M NaHCO.sub.3 (10 mL), followed by brine solution (10
mL) and dried over anhydrous Na.sub.2SO.sub.4. Evaporation of the
solvent afforded 4 as a white crystalline solid. Yield. 0.52 g
(88%).
2-{6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propi-
onylamino]-hexanoylamino}-pentanedioic acid (5)
##STR00026##
[0093] To a stirred solution of Glutamic acid (20 mg, 0.14 mmole)
in 0.2M NaHCO.sub.3 (0.5 mL) was added
6-[3-tert-Butyldisulfanyl-2-(9H-fluoren-9-ylmethoxycarbonylamino)-propion-
ylamino]-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (4, 96 mg,
0.15 mmole) dissolved in (THF-DMF(1:1), 0.5 mL). The reaction
mixture was stirred at RT for 10 min. and analyzed with LCMS which
showed the product (5) peak with mass m/z: 671.95 [M-H]. The
reaction was stirred at RT for overnight and purified by HPLC using
Phenomenex C18 preparative column, (250.times.21.00 mm, gradient:
2% CH.sub.3CN/50 mM TEAB (triethylammonium bicarbonate), pH 8.4, 10
mL/min flow). Fractions containing the compound 5 were collected
together and evaporated the solvent using rotary evaporator and
dried. Yielded 5 as a white solid: 50 mg.
2-{6-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-mercapto-propionylamino]-h-
exanoylamino}-pentanedioic acid (6)
##STR00027##
[0095] A solution of (5) (10 mg, 0.015 mmole) in H.sub.2O-THF (1:1,
1.0 ml) was treated with tris(2-carboxyethyl)phosphine (TCEP, 0.10
mL, 0.5M in H.sub.2O). The reaction was stirred at RT for 4 h until
complete cleavage of disulphide bond (monitored by LCMS) and
purified by HPLC using Phenomenex C18 preparative column,
(250.times.21.00 mm, gradient: 2% CH.sub.3CN/50 mM TEAB, pH 8.4, 10
mL/min flow). Fractions containing the compound 6 were pooled and
used immediately for the subsequent displacement reaction with
dATP-SPDP (SPDP: N-succinimidyl 3-(2-pyridyl dithio)propionate) and
dCTP-SPDP as described below. LCMS: m/s: 583.95[M-H].
Compound 7
##STR00028##
[0097] The fractions containing compound 6 in 60% CH.sub.3CN/50 mM
TEAB buffer (4.0 mg, in 4 mL) collected from HPLC were mixed with
dATP-SPDP (3.6 .mu.mole, ref. previous patent) in 4 ml of 30%
CH.sub.3N/50 mM TEAB buffer, pH 8.4 in a round bottom flask and
stirred for 2 h. The reaction solution was concentrated under
reduced pressure, diluted with water and purified with HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 1.5%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and evaporated and
dried. Yielded 7 (3.0 mg) as a white solid. LCMS: m/z: 2121.80
[M-2H], 606.05 [M/2-2H].
Compound 8
##STR00029##
[0099] The compound 7 (2.0 mg) obtained was dissolved in anhydrous
DMF (0.6 mL) added 60 .mu.l of piperidine. The reaction mixture was
then stirred at RT for an hour. The complete cleavage of FMOC group
was monitored by LCMS and the reaction mixture was purified by HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 1.5%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and evaporated and
obtained 8 (1.0 .mu.mole) as a colorless solid. LCMS: 990.95
[M/2-2H].
Compound 9
##STR00030##
[0101] To a solution of 8 (0.5 .mu.mole) in 0.5 mL of 50 mM
K.sub.2HPO.sub.4 was added Cy5-NHS (1 mg, 1.2 .mu.mole) dissolved
in 20 .mu.L of anhydrous DMF and stirred at RT until the complete
disappearance of starting material 8 which was monitored by LCMS.
Then the blue color reaction mixture was purified HPLC (Phenomenex
C18 column, 250.times.21.0 mm, gradient: 1.5% CH.sub.3CN/50 mM TEAB
buffer, 10 mL/min flow rate). Fractions containing the desired were
pooled together and lyophilized. Yielded 9a (0.36 .mu.mole) as a
blue solid. LCMS: 814.40 [M/2-2H].
[0102] Similarly a solution of 8 (0.5 .mu.mole) in 0.5 mL of 50 mM
K.sub.2HPO.sub.4 was added Atto 647N--NHS (2 mg, 2.5 .mu.mole)
dissolved in 40 .mu.l of anhydrous DMF and stirred at RT until the
complete disappearance of starting material 8 which was monitored
by LCMS. Then the blue color reaction mixture was purified HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 2.0%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and lyophilized.
Yielded 9b (0.3 .mu.mole) as a blue solid. LCMS: 1595.2 [M-2H],
797.0 [M/2-2H].
Compound 10
##STR00031##
[0104] The fractions containing compound 6 in 60% CH.sub.3CN/50 mM
TEAB buffer (3.0 mg, in 3 mL) collected from HPLC were mixed with
dCTP-SPDP (3.0 .mu.mole, ref. previous patent) in 3 ml of 30%
CH.sub.3N/50 mM TEAB buffer, pH 8.4 in a round bottom flask and
stirred for 2 hr. The reaction solution was concentrated under
reduced pressure, diluted with water and purified with HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 1.5%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and evaporated and
dried. Yielded 10 (3.0 mg) as a white solid. LCMS: m/z: 1189.85
[M-2H], 594.8 [M/2-2H].
Compound 11
##STR00032##
[0106] The compound 10 (2.0 mg) obtained was dissolved in anhydrous
DMF (0.6 mL) added 60 .mu.l of piperidine. The reaction mixture was
then stirred at RT for an hour. The complete cleavage of FMOC group
was monitored by LCMS and the reaction mixture was purified by HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 1.5%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and evaporated and
obtained 11 (1.2 .mu.mole) as a colorless solid. LCMS: 967.90
[M/2-2H].
Compound 12
##STR00033##
[0108] To a solution of 11 (0.6 .mu.mole) in 0.5 mL of 50 mM
K.sub.2HPO.sub.4 was added Cy5-NHS (1.5 mg, 1.6 .mu.mole) dissolved
in 30 .mu.L of anhydrous DMF and stirred at RT until the complete
disappearance of starting material 11 which was monitored by LCMS.
Then the blue color reaction mixture was purified HPLC (Phenomenex
C18 column, 250.times.21.0 mm, gradient: 1.5% CH.sub.3CN/50 mM TEAB
buffer, 10 mL/min flow rate). Fractions containing the desired were
pooled together and lyophilized. Yielded 12a (0.5 .mu.mole) as a
blue solid. LCMS: 814.40 [M/2-2H].
[0109] Similarly a solution of 11 (0.4 .mu.mole) in 0.5 mL of 50 mM
K.sub.2HPO.sub.4 was added Atto 647N--NHS (2 mg, 2.5 .mu.mole)
dissolved in 40.mu.L of anhydrous DMF and stirred at RT until the
complete disappearance of starting material 11 which was monitored
by LCMS. Then the blue color reaction mixture was purified HPLC
(Phenomenex C18 column, 250.times.21.0 mm, gradient: 2.0%
CH.sub.3CN/50 mM TEAB buffer, 10 mL/min flow rate). Fractions
containing the desired were pooled together and lyophilized.
Yielded 12b (0.35 .mu.mole) as a blue solid. LCMS: 1595.2 [M-2H],
797.0 [M/2-2H].
Example 2
Caproic-Asp-Asp
##STR00034## ##STR00035##
[0110] C* cap-Asp-Asp:
##STR00036## ##STR00037##
##STR00038## ##STR00039##
[0111] .alpha.-N-Fmoc-S-tert-butylthio-L-cysteine (1 g, 2.32 mmol)
was dissolved in anhydrous acetonitrile and solution of
dicyclohexylcarbodiimide (DCC) (573 mg, 2.78 mmol in CH.sub.3CN)
was added followed by solution of NHS (345 mg, 3.01 mmol in
CH.sub.3CN). After 1 hr. dicyclohexylurea was spun down and active
ester used without purification in coupling with
.epsilon.-amino-hexanoic acid (304 mg, 2.32 mmol) dissolved in 50%
aq. DMF. N,N'-Diisopropylethylamine (DIPEA) was added to correct pH
to 8.0. Upon completion reaction mixture was acidified to pH 3 and
partitioned between water and dichloromethane (DCM). Organic layer
was dried over anhydrous Na.sub.2SO.sub.4 and evaporated to give
1.33 g of crude material. Purification using flash chromatography
in DCM/methanol gave 745 mg of pure material (MW=544.75).
##STR00040##
[0112] .alpha.-N-Fmoc-S-tert-butylthio-L-cyst-caproic acid (3, 77
mg, 141 .mu.mols, CH.sub.3CN) was converted to NHS active ester
using DCC (35 mg, 169 .mu.mols, CH.sub.3CN) and NHS (21 mg, 183
.mu.mols, ACN). After 1 hr. precipitate of dicyclohexylurea was
removed by centrifugation and ester used without further
purification in coupling with H-Asp-Asp-OH peptide (12 mg, 48
nmols) dissolved in 0.5M K.sub.2HPO.sub.4, pH of reaction mixture
corrected to 7.5 with DIPEA. Progress of reaction was monitored by
TLC (disappearance of ester) and by LC-MS (formation of product).
Upon completion product was isolated by direct injection on
preparative HPLC (C18 column, 3% CH.sub.3CN gradient in 50 mM TEAB,
pH 8.6). Isolated product was lyophilized to give white powder
(MW=774.9)
##STR00041##
[0113] To free the thiol
.alpha.-N-Fmoc-S-tert-butylthio-L-cyst-caproic-Asp-Asp-OH (15) was
treated with 100 mM DTT in 0.1M K.sub.2HPO.sub.4 during 1 hr. at
RT. Reaction was monitored by LC-MS and upon completion injected
directly on preparative HPLC (C18 column). Purification using 2%
CH.sub.3CN gradient in 50 mM TEAB, pH 8.6 yielded product
(MW=686.7) which was used immediately without evaporation in
displacement reaction with SPDP modified nucleotide
triphosphates.
##STR00042##
[0114] dATP-AP3 and dCTP-AP3 were prepared by a modified procedure
of Hobbs and Cocuzza: a) Pyrophosphate and tributylamine were added
to the reaction mixture rather than vice versa; b) After
pyrophosphate addition the reaction was quenched with 50 mM TEAB
within 15 min.; c) DEAE-Sephadex chromatography was replaced by
preparative HPLC.
##STR00043##
[0115] SPDP modification of dATP-AP3 and dCTP-AP3 was accomplished
using standard protocol: 2 .mu.mols of dNTP-AP3 were dissolved in
250 .mu.l of 0.1N NaHCO.sub.3 and 1.2 equivalent (eqv.) of freshly
prepared 50 mM stock of SPDP in anhydrous DMF was added. Progress
of modification was monitored using LC-MS. Product was isolated
using preparative HPLC (C18 column) with 1% CH.sub.3CN gradient in
50 mM TEAB, pH 8.6 gradient and used in displacement reaction with
thiol without evaporation of HPLC solvents (MW=717.01 for
dCTP-AP3-SPDP, MW=740.03 for dATP-AP3-SPDP).
##STR00044##
[0116] Small aliquots of isolated thiol were added to freshly
isolated dNTP-AP3-SPDP to obtain displacement product. Progress of
reaction was monitored by LC-MS after every addition of thiol.
Reaction was completed when all dNTP-AP3-SPDP was consumed at which
point reaction mixture was concentrated and purified on preparative
HPLC (C18 column) using 1% gradient of CH.sub.3CN in 50 mM TEAB, pH
8.6. Isolated product was lyophilized to give white powder
(MW=1293.06 for cytidine-analog and MW=1316.09 for
adenosine-analog).
##STR00045##
[0117] Removal of Fmoc-protecting group was accomplished using 20%
piperidine in CH.sub.3CN (20 min., RT). Subsequently solvents were
removed and crude reaction mixture purified on preparative HPLC
(C18 column) using 2% CH.sub.3CN gradient. Product was dried down
and OD measured in water at 290 nm for cytidine analog (800 nmols,
MW=1070.8) and 280 nm for adenosine analog (640 nmols,
MW=1093.8).
##STR00046## ##STR00047##
[0118] Dye modified final products were prepared using following
standard conditions: peptide modified dNTPs were re-dissolved in 20
mM K.sub.2HPO.sub.4 and dye-NHS dissolved in anhydrous DMF (5 mg in
100 .mu.l) was added using initially 1.2 eqv. up to 4 eqv. to reach
complete consumption of starting material. Progress of modification
was monitored using LC-MS. Product was isolated using preparative
HPLC (C18 column) with 1% CH.sub.3CN gradient and 50 mM TEAB, pH
8.6. Desired fractions were combined, organic solvent removed under
reduced pressure and products subjected to CH.sub.3OH
repurification on C18 HPLC column (1% CH.sub.3OH gradient). Final
fractions were quantitated at 650 nm using .epsilon..sub.650=250000
M.sup.-1cm.sup.-1 for Cy5 dye and 150000 M.sup.-1cm.sup.-1 for Atto
647N dye.
Example 3
Caproic-Arg-Arg-Arg
##STR00048## ##STR00049##
[0119] Compound 32
[0120] Compound 31 (100 mg, 0.18 mmol) was dissolved in 0.8 ml DMF
and added 0.2 mL piperidine and then kept at RT for 30 min. DMF was
removed and the residue was purified with flash column using
CH.sub.2Cl.sub.2: CH.sub.3OH (2:1). The purified amine (35 mg) was
dissolved in 1 mL DMF and used directly for the next step without
characterization. 3.5 mg of the purified amine in 0.1 mL DMF (10.8
.mu.mol) was added 60 .mu.L DMF and 40 .mu.L DIPEA and then Cy5
Mono NHS Ester (6.63 .mu.mol) in 100 .mu.L anhydrous DMF was added
into the solution. After 30 minutes, the reaction mixture was
purified with HPLC (Waters Delta 600 pump and 2487 Dual .lamda.
Absorbance Detector, Phenomenex C18 preparative column,
250.times.21.00 mm 10 micron, gradient: 100% A for 5 min, then 1%
B/min, buffer A 0.05 M TEAB, buffer B CH.sub.3CN, 10 mL/min flow).
Fractions containing the desired compound 32 were pooled and
quantified; (3.0 .mu.mol, 45%, .epsilon..sub.649=250000); ESI-MS
(negative ion mode): m/z=959.20 (M-H).
Compound 33
[0121] The NHS ester of the acid 32 was prepared by dissolving the
acid 32 (3.0 .mu.mol) in DMF (500.0 .mu.L) and
N,N,N',N'-Tetramethyl-O--(N-succinimidyl)uronium
hexafluorophosphate (SbTMU) (4.3 mg, 12 .mu.mol) in 100 .mu.L DMF
was added to the acid solution followed by the addition of DIPEA
(80 .mu.L). After stirring at RT for 1 hr., the reaction mixture
was used immediately for peptide coupling without any purification.
The peptide Arg-Arg-Arg-OH (14.5 mg, 30 .mu.mol) was dissolved in
160 .mu.L 0.5M phosphate buffer, and added to the freshly prepared
NHS ester of the acid 32. The reaction mixture was stirred for 30
minutes and then the crude reaction mixture was purified with HPLC
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.21.00 mm 10 micron,
gradient: 100% A for 5 min, then 1% B/min, buffer A 0.05 M TEAB,
buffer B MeCN, 10 mL/min flow). Fractions containing the desired
compound 33 were pooled and quantified; (0.6 .mu.mol, 20%,
.epsilon..sub.649=250000); ESI-MS (negative ion mode): m/z=713.45
[(M-2H)/2].
Compound 34
[0122] A solution of compound 33 (0.6 .mu.mol) in 3 ml H.sub.2O was
treated with TCEP (300 .mu.L, 1M solution) in an aluminum foil
covered flask. After 30 minutes, the reaction mixture was purified
with HPLC (Waters Delta 600 pump and 2487 Dual .lamda. Absorbance
Detector, Phenomenex C18 preparative column, 250.times.21.00 mm 10
micron, gradient: 100% A for 5 min, then 1% B/min, buffer A 0.05 M
TEAB, buffer B CH.sub.3CN, 10 mL/min flow). Fractions containing
the desired thiol, analyzed with ESI-MS (negative ion mode):
m/z=669.90 [(M-2H)/2], were pooled and immediately added dATP-SPDP
(1 .mu.mol in 1 mL H.sub.2O). After 15 minutes, LCMS analysis
indicated that the completion of the reaction and the reaction
mixture was then partially concentrated under reduced pressure to
remove CH.sub.3CN, then purified with HPLC (Waters Delta 600 pump
and 2487 Dual .lamda. Absorbance Detector, Phenomenex C18
preparative column, 250.times.10.00 mm 10 micron, gradient: 100% A
for 5 min, then 1% B/min, buffer A 0.05M TEAB, buffer B CH.sub.3CN,
5 mL/min flow). Fractions containing the desired compound were
pooled and concentrated and then purified again with HPLC using
CH.sub.3OH and TEAB buffer. The fractions containing the desired
compound 34 were pooled and lyophilized to yield compound 34 as a
bright blue solid (0.37 mol, 62%, .epsilon..sub.649=250000). ESI-MS
(negative ion mode): m/z=983.75 [(M-2H)/2].
Example 4
Cap-Asp-Asp-Asp-Asp
##STR00050## ##STR00051##
[0123] Compound 45
[0124] Cy5 Mono NHS Ester (100.0 .mu.L, 6.63 .mu.mol) in anhydrous
DMF was added to a solution of amine 44 (13.26 .mu.mol, 2 equiv) in
DMF (100 .mu.L) and DIPEA (20.0 .mu.L) in an aluminum foil covered
flask. After 30 minutes, the disappearance of the starting amine
was determined by LCMS or HPLC. The reaction was HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.21.00 mm 10 micron,
gradient: 100% A for 5 min, then 1% B/min, buffer A 0.05 M TEAB,
buffer B CH.sub.3CN, 10 mL/min flow). Fractions containing the
desired product were pooled and quantified; (4.0 mol, 60.3%,
.epsilon..sub.649=250000); ESI-MS (negative ion mode): m/z=959.20
(M-H).
##STR00052##
Compound 46
[0125] The NHS ester of the acid 45 was prepared by dissolving the
acid 45 (4.0 .mu.mol, 1 eqv.) in DMF (700.0 .mu.L) and the SbTMU
5.93 mg, 16.5 .mu.mol, in 200 .mu.L DMF, 4.0 eqv.) was added, to
the acid solution followed by the addition of DIPEA (103.0 .mu.L).
After stirring at RT for 1 hour, the reaction mixture was used
immediately for peptide coupling without any purification. The
peptide (Asp-Asp-Asp-Asp) was dissolved in DMF:H.sub.2O (400.0
.mu.L, 1:1), basified using DIPEA (50.0 .mu.L). To this peptide
solution was added freshly prepared NHS ester of the acid 45. The
reaction mixture was stirred for 30 minutes and it was then
analyzed by LCMS. The crude reaction mixture was HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.21.00 mm 10 micron,
gradient: 100% A for 5 min., then 1% B/min, buffer A 0.05 M TEAB,
buffer B CH.sub.3CN, 10 mL/min flow). Fractions containing the
desired were pooled and quantified; (3.0 .mu.mol, 75.0%,
.epsilon..sub.649=250000); ESI-MS (negative ion mode): m/z=709.20
(1/2M-H).
##STR00053##
Compound 47
[0126] A solution of compound 46 (1.0 .mu.mol) in H.sub.2O was
treated with TCEP (40.0 .mu.L, 19.92 .mu.mol, 0.5 M in H.sub.2O,
19.92 equiv) in an aluminum foil covered flask. After 30 minutes,
the reaction mixture was analyzed by LCMS and was then HPLC
purified (Waters Delta 600 pump and 2487 Dual .lamda. Absorbance
Detector, Phenomenex C18 preparative column, 250.times.21.00 mm 10
micron, gradient: 100% A for 5 min., then 1% B/min, buffer A 0.05 M
TEAB, buffer B CH.sub.3CN, 10 mL/min flow). Fractions containing
the desired were pooled and used immediately for the subsequent
displacement reaction without removing the solvent. ESI-MS
(negative ion mode): m/z=665.45 (1/2M-H).
##STR00054##
Compound 48a
[0127] HPLC fractions containing the thiol 7 (0.34 .mu.mol, 1 eqv.)
were mixed with HPLC fractions containing dCTP-SPDP (0.41 .mu.mol,
1.25 eqv.) in an aluminum foil covered flask. After 15 min. LCMS
analysis indicated that the completion of the reaction and it was
then partially concentrated under reduced pressure to remove
CH.sub.3CN, then HPLC purified (Waters Delta 600 pump and 2487 Dual
.lamda. Absorbance Detector, Phenomenex C18 preparative column,
250.times.10.00 mm 10 micron, gradient: 100% A for 5 min, then 1%
B/min, buffer A 0.05 M TEAB, buffer B CH.sub.3CN, 5 mL/min flow).
Fractions containing the desired were pooled and lyophilized to
yield compound 1 as a bright blue solid (0.17 .mu.mol, 50%,
.epsilon..sub.649=250000). The desired product was HPLC purified a
second time under the same conditions, using CH.sub.3OH instead of
CH.sub.3CN for buffer B. Fractions containing the desired were
pooled and stored at -80.degree. C. without removing the solvent.
ESI-MS (negative ion mode): m/z=968.35 (1/2M-H).
##STR00055##
Compound 49a
[0128] HPLC fractions containing thiol 47 (0.5 .mu.mol, 1 eqv.)
were mixed with HPLC fractions containing dATP-SPDP (0.6 .mu.mol,
1.2 eqv.) in an aluminum foil covered flask. After 15 min. LCMS
analysis indicated that the completion of the reaction and it was
then partially concentrated under reduced pressure to remove
CH.sub.3CN, then HPLC purified (Waters Delta 600 pump and 2487 Dual
.lamda. Absorbance Detector, Phenomenex C18 preparative column,
250.times.10.00 mm 10 micron, gradient: 100% A for 5 min., then 1%
B/min, buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min flow).
Fractions containing the desired were pooled and lyophilized to
yield compound 49a as a bright blue solid (0.35 .mu.mol, 70%,
.epsilon..sub.649=250000). The desired was HPLC purified a second
time under the same conditions, using CH.sub.3OH instead of
CH.sub.3CN for buffer B. Fractions containing the desired were
pooled and stored at -80.degree. C. without removing the solvent.
ESI-MS (negative ion mode): m/z=980.10 (1/2M-H).
Example 5
Caproic-Asp
##STR00056## ##STR00057## ##STR00058##
[0129] NHS Ester
[0130] Fmoc-Cys(StBu)-OH (2.0 g, 4.63 mmol, 1 eqv.) was dissolved
in CH.sub.3CN (10 mL). DCC (1.2 g, 5.81 mmol, 1.26 eqv.) was added,
followed by NHS (0.70 g, 6.08 mmol, 1.31 eqv.) and the reaction was
stirred at RT for 1 hr. White precipitate (DCU) began forming
within five min. The reaction mixture was transferred to Eppendorf
tubes and centrifuged to remove the white precipitate. The
supernatant was then used in subsequent reactions without further
purification.
##STR00059##
Acid
[0131] 6-Aminohexanoic acid (0.60 g, 4.57 mmol, 1 eqv.) was
dissolved in 1:1 H.sub.2O:DMF (6 mL total). DIPEA (0.016 mL) was
added to keep the pH about 8. NHS ester (4.63 mmol in 10 mL
CH.sub.3CN, 1.01 eqv.) was added to the reaction mixture in 1 mL
aliquots over about 10 min. DIPEA (0.02 mL) was added after each
aliquot to keep the reaction basic. After the first aliquot of NHS
ester was added, the reaction became cloudy, and addition of extra
H.sub.2O (0.2 mL) was needed to clear up the solution. The reaction
was stirred at RT for two hours, then quenched with 20 mL 10% HCl
(aq.). The aqueous phase was extracted with CH.sub.2Cl.sub.2
(2.times.50 mL). The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure to yield a brown
oil. Purification by flash column chromatography (100%
CH.sub.2Cl.sub.2 to 5% CH.sub.3OH/CH.sub.2Cl.sub.2) afforded the
desired acid as a white foam (2.14 g, 86%).
##STR00060##
NHS Ester
[0132] The starting acid (0.99 g, 1.82 mmol, 1 eqv.) was dissolved
in CH.sub.3CN (10 mL). DCC (0.46 g, 2.23 mmol, 1.23 eqv.) was
added, followed by NHS (0.28 g, 2.43 mmol, 1.34 eqv.) and the
reaction was stirred at RT for an hour. White precipitate (DCU)
began forming within 5 min. The reaction mixture was transferred to
Eppendorf tubes and centrifuged to remove the white precipitate.
The supernatant was then used in subsequent reactions without
further purification.
##STR00061##
Dimethyl Ester
[0133] L-Aspartic acid dimethyl ester hydrochloride (0.2 g, 1.01
mmol, 2 eqv.) was dissolved in CH.sub.3CN (1 mL) and DIPEA (0.32
mL, 1.84 mmol, 4 eqv.). A solution of NHS ester (0.48 mmol, 1 eqv.)
in CH.sub.3CN (2 mL) was added, and the reaction was stirred at RT
for 12 hr. The reaction was diluted with EtOAc (25 mL), then washed
with brine (1.times.30 mL) and sat. NH.sub.4Cl (aq.) (1.times.30
mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered,
and concentrated under reduced pressure. Purification by flash
column chromatography (100% CH.sub.2Cl.sub.2 to 2%
CH.sub.3OH/CH.sub.2Cl.sub.2) afforded the desired ester as a white
foam (0.12 g, 36%).
##STR00062##
Diacid
[0134] 1M LiOH(aq) (0.18 mL, .about.6 equiv) was added to a
solution of dimethyl ester (0.02 g, 0.029 mmol, 1 eqv.) in THF
(0.30 mL). The reaction was stirred at RT until the starting
dimethyl ester was consumed based on LCMS analysis (about 15 min).
The crude reaction was then HPLC purified (Waters Delta 600 pump
and 2487 Dual .lamda. Absorbance Detector, Phenomenex C18
preparative column, 250.times.21.2 mm 10 micron, gradient: 90% A
for 3 min., then 5% B/min., buffer A 0.05M TEAB, buffer B
CH.sub.3CN, 10 mL/min. flow). Fractions containing the desired were
pooled and concentrated to yield the desired diacid, which was used
for subsequent reactions without quantifying.
##STR00063##
Thiol
[0135] Diacid (.about.29 .mu.mol, 1 eqv.) was treated with TCEP
(1.7 mL, 0.85 mmol, 0.5M in H.sub.2O, 29 eqv.). The reaction was
stirred at RT until the starting material was consumed based on
LCMS analysis (about 30 min.). The crude reaction was then HPLC
purified (Waters Delta 600 pump and 2487 Dual .lamda. Absorbance
Detector, Phenomenex C18 preparative column, 250.times.21.2 mm 10
micron, gradient: 100% A for 3 min, then 5% B/min., buffer A 0.05M
TEAB, buffer B CH.sub.3CN, 10 mL/min. flow). Fractions containing
the desired were pooled and used for subsequent reactions without
concentrating or quantifying.
##STR00064##
Disulfide
[0136] HPLC fractions containing the thiol (about 10 .mu.mol, 2
eqv.) were mixed with HPLC fractions containing SPDP-dATP (5
.mu.mol, 1 equiv). After the SPDP-dATP was consumed based on LCMS
analysis (about 10 min), the reaction was partially concentrated
under reduced pressure to remove CH.sub.3CN and then HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.15.0 mm 10 micron,
gradient: 100% A for 3 min, then 1% B/min., buffer A 0.05M TEAB,
buffer B CH.sub.3CN, 10 mL/min. flow). Fractions containing the
desired were pooled and lyophilized, then used for subsequent
reactions without quantifying.
##STR00065##
Amine
[0137] The starting carbamate (.about.5 .mu.mol, 1 eqv.) was
treated with 20% piperidine in 1:1 DMF: CH.sub.3CN (2 mL), and
stirred at RT until the starting material was consumed based on
LCMS analysis (.about.15 min). After removing the solvent under
reduced pressure, the reaction was HPLC purified (Waters Delta 600
pump and 2487 Dual .lamda. Absorbance Detector, Phenomenex C18
preparative column, 250.times.21.2 mm 10 micron, gradient: 100% A
for 3 min, then 1% B/min., buffer A 0.05M TEAB, buffer B
CH.sub.3OH, 10 mL/min. flow). Fractions containing the desired were
pooled and lyophilized to yield the product as a white foam (1
.mu.mol, .epsilon..sub.280=12700).
##STR00066##
A* Caproic-Asp
[0138] Atto647N--NHS ester (0.030 mL, 1.8 .mu.mol, 0.06M in
anhydrous DMF, 3.6 eqv.) was added to a solution of amine (0.5
.mu.mol, 1 eqv.) in H.sub.2O (0.25 mL) in 10 .mu.L aliquots. The
reaction was monitored by LCMS to determine how much dye was needed
to consume the starting amine. After disappearance of amine, the
crude reaction was HPLC purified (Waters Delta 600 pump and 2487
Dual .lamda. Absorbance Detector, Phenomenex C18 preparative
column, 250.times.10.0 mm 10 micron, gradient: 100% A for 3 min,
then 2% B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min.
flow). Fractions containing the desired were pooled and
concentrated, then HPLC purified a second time under the same
conditions, using CH.sub.3OH instead of CH.sub.3CN for buffer B.
Fractions containing the desired were pooled and stored at
-80.degree. C. without removing the solvent (0.086 .mu.mol, 17%,
.epsilon..sub.645=150000).
##STR00067##
Disulfide
[0139] HPLC fractions containing the thiol (.about.10 .mu.mol, 6
eqv.) were mixed with HPLC fractions containing SPDP-dGTP (1.5
.mu.mol, 1 eqv.). After the SPDP-dGTP was consumed based on LCMS
analysis (about 10 min), the reaction was partially concentrated
under reduced pressure to remove CH.sub.3CN and then HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.10.0 mm 10 micron,
gradient: 100% A for 3 min., then 1% B/min, buffer A 0.05M TEAB,
buffer B CH.sub.3CN, 5 mL/min. flow). Fractions containing the
desired were pooled and lyophilized, then used for subsequent
reactions without quantifying.
##STR00068##
Amine
[0140] The starting carbamate (.about.1.5 .mu.mol, 1 eqv.) was
treated with 20% piperidine in DMF (0.5 mL), and stirred at RT
until the starting material was consumed based on LCMS analysis
(about 15 min). After removing the solvent under reduced pressure,
the reaction was HPLC purified (Waters Delta 600 pump and 2487 Dual
.lamda. Absorbance Detector, Phenomenex C18 preparative column,
250.times.10.0 mm 10 micron, gradient: 100% A for 3 min, then 1%
B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min. flow).
Fractions containing the desired were pooled and lyophilized to
yield the product as a white foam (0.26 .mu.mol, 17%,
.epsilon..sub.272=11900).
##STR00069##
G* Caproic-Asp
[0141] Atto647N--NHS ester (0.011 mL, 0.66 .mu.mol, 0.06 M in
anhydrous DMF, 2.5 eqv.) was added to a solution of amine (0.26
.mu.mol, 1 equiv) in H.sub.2O (0.50 mL) in small aliquots. The
reaction was monitored by LCMS to determine how much dye was needed
to consume the starting amine. After disappearance of amine, the
crude reaction was HPLC purified (Waters Delta 600 pump and 2487
Dual .lamda.. Absorbance Detector, Phenomenex C18 preparative
column, 250.times.10.0 mm 10 micron, gradient: 100% A for 3 min,
then 2% B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min.
flow). Fractions containing the desired were pooled and
concentrated, then HPLC purified a second time under the same
conditions, using CH.sub.3OH instead of CH.sub.3CN for buffer B.
Fractions containing the desired were pooled and stored at
-80.degree. C. without removing the solvent (0.076 .mu.mol, 29%,
.epsilon..sub.645=150000).
##STR00070##
Disulfide
[0142] HPLC fractions containing the thiol (about 5 .mu.mol, 5
eqv.) were mixed with SPDP-dCTP (1 .mu.mol, 1 eqv.) in H.sub.2O
(0.20 mL). After the SPDP-dCTP was consumed based on LCMS analysis
(about 10 min.), the reaction was partially concentrated under
reduced pressure to remove CH.sub.3CN and then HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.21.2 mm 10 micron,
gradient: 100% A for 3 min, then 3% B/min., buffer A 0.05M TEAB,
buffer B CH.sub.3CN, 5 mL/min. flow). Fractions containing the
desired were pooled and lyophilized, then used for subsequent
reactions without quantifying.
##STR00071##
Amine
[0143] The starting carbamate (about 1 .mu.mol, 1 eqv.) was treated
with 20% piperidine in CH.sub.3CN (0.5 mL), and stirred at RT until
the starting material was consumed based on LCMS analysis
(.about.15 min). After removing the solvent under reduced pressure,
the reaction was HPLC purified (Waters Delta 600 pump and 2487 Dual
.lamda. Absorbance Detector, Phenomenex C18 preparative column,
250.times.10.0 mm 10 micron, gradient: 100% A for 3 min., then 1%
B/min, buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min. flow).
Fractions containing the desired were pooled and lyophilized to
yield the product as a white foam (0.15 mol, 15%,
.epsilon..sub.294=9300).
##STR00072##
C* Caproic-Asp
[0144] Atto647N--NHS ester (0.012 mL, 0.72 .mu.mol, 0.06M in
anhydrous DMF, 3.6 eqv.) was added to a solution of amine (0.15
.mu.mol, 1 eqv.) in H.sub.2O (0.20 mL) in 5 .mu.L aliquots. The
reaction was monitored by LCMS to determine how much dye was needed
to consume the starting amine. After disappearance of amine, the
crude reaction was HPLC purified (Waters Delta 600 pump and 2487
Dual .lamda. Absorbance Detector, Phenomenex C18 preparative
column, 250.times.10.0 mm 10 micron, gradient: 100% A for 3 min.,
then 2% B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min.
flow). Fractions containing the desired were pooled and
concentrated, then HPLC purified a second time under the same
conditions, using CH.sub.3OH instead of CH.sub.3CN for buffer B.
Fractions containing the desired were pooled and stored at
-80.degree. C. without removing the solvent (0.030 .mu.mol, 20%,
.epsilon..sub.645=150000).
##STR00073##
Disulfide
[0145] HPLC fractions containing the thiol (.about.5 .mu.mol, 2.5
equiv) were mixed with SPDP-dUTP (2 .mu.mol, 1 eqv.) in H.sub.2O
(0.13 mL). After the SPDP-dUTP was consumed based on LCMS analysis
(.about.10 min), the reaction was partially concentrated under
reduced pressure to remove CH.sub.3CN and then HPLC purified
(Waters Delta 600 pump and 2487 Dual .lamda. Absorbance Detector,
Phenomenex C18 preparative column, 250.times.10.0 mm 10 micron,
gradient: 100% A for 3 min., then 1% B/min., buffer A 0.05M TEAB,
buffer B CH.sub.3CN, 5 mL/min. flow). Fractions containing the
desired were pooled and lyophilized, then used for subsequent
reactions without quantifying.
##STR00074##
Amine
[0146] The starting carbamate (.about.1 .mu.mol, 1 equiv) was
treated with 20% piperidine in DMF (2 mL), and stirred at RT until
the starting material was consumed based on LCMS analysis (about 15
min). After removing the solvent under reduced pressure, the
reaction was HPLC purified (Waters Delta 600 pump and 2487 Dual
.lamda. Absorbance Detector, Phenomenex C18 preparative column,
250.times.10.0 mm 10 micron, gradient: 100% A for 3 min, then 1%
B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min. flow).
Fractions containing the desired were pooled and lyophilized to
yield the product as a white foam (0.19 .mu.mol, 19%,
.epsilon..sub.289=13000).
##STR00075##
T* Caproic-Asp
[0147] Atto647N--NHS ester (0.010 mL, 0.68 .mu.mol, 0.06M in
anhydrous DMF, 3.6 eqv.) was added to a solution of amine (0.19
.mu.mol, 1 eqv.) in H.sub.2O (0.40 mL) in small aliquots. 1M
K.sub.2HPO.sub.4 (0.40 mL) was also added to accelerate the
reaction after there was little product formed within an hour. The
reaction was monitored by LCMS to determine how much dye was needed
to consume the starting amine. After disappearance of amine, the
crude reaction was HPLC purified (Waters Delta 600 pump and 2487
Dual .lamda. Absorbance Detector, Phenomenex C18 preparative
column, 250.times.10.0 mm 10 micron, gradient: 100% A for 3 min.,
then 2% B/min., buffer A 0.05M TEAB, buffer B CH.sub.3CN, 5 mL/min.
flow). Fractions containing the desired were pooled and
concentrated, then HPLC purified a second time under the same
conditions, using CH.sub.3OH instead of CH.sub.3CN for buffer B.
Fractions containing the desired were pooled and stored at -80
.degree. C. without removing the solvent (0.059 .mu.mol, 31%,
.epsilon..sub.645=150000).
Example 6
Caproic-Asp-Asp-Asp-Asp (Alternative Routes)
##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080##
[0148] Example 7
G* Pro-Pro-Lys-Pro-Asp
##STR00081## ##STR00082##
[0150] The schemes above and variations thereof may be utilized for
syntheses of derivatives and analogs of the exemplary nucleotide
analogs shown above, for example, those having additional amino
groups at the Inhibitor end and/or compounds of different linking
groups.
[0151] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification.
Contemplated equivalents of the nucleotide analogs disclosed here
include compounds which otherwise correspond thereto, and which
have the same general properties thereof, wherein one or more
simple variations of substituents or components are made which do
not adversely affect the characteristics of the nucleotide analogs
of interest. In general, the components of the nucleotide analogs
disclosed herein may be prepared by the methods illustrated in the
general reaction schema as described herein or by modifications
thereof, using readily available starting materials, reagents, and
conventional synthesis procedures.
EQUIVALENTS
[0152] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
INCORPORATION BY REFERENCE
[0153] The entire disclosure of each of the publications and patent
documents referred to herein is incorporated by reference in its
entirety for all purposes to the same extent as if each individual
publication or patent document were so individually denoted.
* * * * *