U.S. patent application number 11/483483 was filed with the patent office on 2006-11-09 for synthesis and use of anti-reverse mrna cap analogues.
Invention is credited to Edward Darzynkiewicz, Robert E. Rhoads, Janusz Stepinski.
Application Number | 20060252115 11/483483 |
Document ID | / |
Family ID | 28793968 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060252115 |
Kind Code |
A1 |
Darzynkiewicz; Edward ; et
al. |
November 9, 2006 |
Synthesis and use of anti-reverse mRNA cap analogues
Abstract
The ability to synthesize capped RNA transcripts in vitro has
been of considerable value in a variety of applications. However,
one-third to one-half of the caps have, until now, been
incorporated in the reverse orientation. Such reverse caps impair
the translation of in vitro-synthesized mRNAs. Novel cap analogues,
such as P.sup.1-3'-deoxy-7-methylguanosine-5' P.sup.3-guanosine-5'
triphosphate and P.sup.1-3'-O,7-dimethylguanosine-5'
P.sup.3-guanosine-5' triphosphate, have been designed that are
incapable of being incorporated into RNA in the reverse
orientation. Transcripts produced with SP6 polymerase using
"anti-reverse" cap analogues were of the predicted length. Analysis
of the transcripts indicated that reverse caps were not formed. The
in vitro translational efficiency of transcripts with the novel
"anti-reverse" cap analogues was significantly higher than that of
transcripts formed with conventional caps.
Inventors: |
Darzynkiewicz; Edward;
(Warsaw, PL) ; Rhoads; Robert E.; (Shreveport,
LA) ; Stepinski; Janusz; (Warsaw, PL) |
Correspondence
Address: |
PATENT DEPARTMENT;TAYLOR, PORTER, BROOKS & PHILLIPS, L.L.P
P.O. BOX 2471
BATON ROUGE
LA
70821-2471
US
|
Family ID: |
28793968 |
Appl. No.: |
11/483483 |
Filed: |
July 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10150718 |
May 17, 2002 |
7074596 |
|
|
11483483 |
Jul 10, 2006 |
|
|
|
60367404 |
Mar 25, 2002 |
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Current U.S.
Class: |
435/68.1 ;
435/320.1; 536/23.1; 536/26.1 |
Current CPC
Class: |
C12P 21/02 20130101;
C12P 19/34 20130101 |
Class at
Publication: |
435/068.1 ;
435/320.1; 536/023.1; 536/026.1 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/02 20060101 C07H021/02; C07H 19/04 20060101
C07H019/04 |
Goverment Interests
[0002] The development of this invention was partially funded by
the United States Government under grant number GM20818 awarded by
the National Institutes of Health. The United States Government has
certain rights in this invention. The development of this invention
was partially funded by the Government of Poland under grant number
6 P04A 055 17 awarded by the Polish Committee for Scientific
Research (KBN).
Claims
1. A composition comprising ##STR9## wherein: X is selected from
the group consisting of H, OH, OCH.sub.3, and OCH.sub.2CH.sub.3; Y
is selected from the group consisting of H, OH, OCH.sub.3, and
OCH.sub.2CH.sub.3; n is 1,2,3,4, or5; wherein, if Y is OH and n is
1, then X is neither H nor OH; and B is selected from the group
consisting of ##STR10##
2. A composition as recited in claim 1, wherein X is OH, Y is H,
and n is 1.
3. A composition as recited in claim 1, wherein X is OH, Y is
OCH.sub.3, and n is 1.
4. An RNA molecule whose 5' end incorporates a composition as
recited in claim 1.
5. An RNA molecule whose 5' end incorporates a composition as
recited in claim 2.
6. An RNA molecule whose 5' end incorporates a composition as
recited in claim 3.
7. A method for synthesizing an RNA molecule as recited in claim 4
in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
8. A method for synthesizing an RNA molecule as recited in claim 5
in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
9. A method for synthesizing an RNA molecule as recited in claim 6
in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
10. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim 4
in a cell-free protein synthesis system, wherein the RNA molecule
comprises an open reading frame, under conditions conducive to
translating the open reading frame of the RNA molecule into the
protein or peptide encoded by the open reading frame.
11. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim 5
in a cell-free protein synthesis system, wherein the RNA molecule
comprises an open reading frame, under conditions conducive to
translating the open reading frame of the RNA molecule into the
protein or peptide encoded by the open reading frame.
12. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim 6
in a cell-free protein synthesis system, wherein the RNA molecule
comprises an open reading frame, under conditions conducive to
translating the open reading frame of the RNA molecule into the
protein or peptide encoded by the open reading frame.
13. A composition comprising ##STR11## wherein n is 1, 2, 3, 4, or
5.
14. An RNA molecule whose 5' end incorporates a composition as
recited in claim 13.
15. A method for synthesizing an RNA molecule as recited in claim
14 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
16. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim
14 in a cell-free protein synthesis system, wherein the RNA
molecule comprises an open reading frame, under conditions
conducive to translating the open reading frame of the RNA molecule
into the protein or peptide encoded by the open reading frame.
17. A composition comprising ##STR12## wherein: X is selected from
the group consisting of H, OH, OCH.sub.3, and OCH.sub.2CH.sub.3; Y
is selected from the group consisting of H, OH, OCH.sub.3, and
OCH.sub.2CH.sub.3; n is 1, 2, 3, 4, or 5; and Z is selected from
the group consisting of C.sub.1 to C.sub.4 substituted or
unsubstituted alkyl, C.sub.6 to C.sub.8 substituted or
unsubstituted aryl, or C.sub.1 to C.sub.4 substituted or
unsubstituted alkoxy; and wherein, if Z is CH.sub.3 and Y is OH and
n is 1, then X is neither H nor OH.
18. An RNA molecule whose 5' end incorporates a composition as
recited in claim 17.
19. A method for synthesizing an RNA molecule as recited in claim
18 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions-conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
20. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim
18 in a cell-free protein synthesis system, wherein the RNA
molecule comprises an open reading frame, under conditions
conducive to translating the open reading frame of the RNA molecule
into the protein or peptide encoded by the open reading frame.
21. A composition comprising ##STR13## wherein: X is selected from
the group consisting of H, OH, OCH.sub.3, and OCH.sub.2CH.sub.3; Y
is selected from the group consisting of H, OH, OCH.sub.3, and
OCH.sub.2CH.sub.3; R is selected from the group consisting of H,
CH.sub.3, CH.sub.2C.sub.6H.sub.5, CH.sub.2COC.sub.6H.sub.5,
CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH.dbd.CH.sub.2, C.sub.1 to
C.sub.4 substituted or unsubstituted alkyl, and C.sub.6 to C.sub.8
substituted or unsubstituted aryl; n is 1, 2, 3, 4, or 5; Z is
selected from the group consisting of C.sub.1 to C.sub.4
substituted or unsubstituted alkyl, C.sub.6 to C.sub.8 substituted
or unsubstituted aryl, or C.sub.1 to C.sub.4 substituted or
unsubstituted alkoxy; and B is selected from the group consisting
of guanine, adenine, uridine, and cytosine; and wherein, if Z is
CH.sub.3, and R is H, and Y is OH, and n is 1, and B is guanine;
then X is neither H nor OH.
22. An RNA molecule whose 5' end incorporates a composition as
recited in claim 21.
23. A method for synthesizing an RNA molecule as recited in claim
22 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a
composition as recited, and a polynucleotide template; in the
presence an RNA polymerase; under conditions conducive to
transcription by the RNA polymerase of the polynucleotide template
into an RNA copy; whereby some of the RNA copies will incorporate
the composition as recited to make an RNA molecule as recited.
24. A method for synthesizing a protein or a peptide in vitro, said
method comprising translating an RNA molecule as recited in claim
22 in a cell-free protein synthesis system, wherein the RNA
molecule comprises an open reading frame, under conditions
conducive to translating the open reading frame of the RNA molecule
into the protein or peptide encoded by the open reading frame.
Description
[0001] This application is a divisional of patent application Ser.
No. 10/150,718, filed May 17, 2002, now U.S. Pat. No. 7,074,596,
issued Jul. 11, 2006; which claimed the benefit of the Mar. 25,
2002 filing date of provisional patent application Ser. No.
60/367,404 under 35 U.S.C. .sctn. 119(e).
[0003] In eukaryotes, the 5' end of most mRNA is blocked, or
"capped." In addition, there are some other forms of RNA that are
also capped. The cap contains a 5'-5' triphosphate linkage between
two nucleotides, and also contains methyl groups. The capping of
RNA promotes its normal function in cells.
[0004] The ability to synthesize capped RNA molecules in vitro is
therefore useful, because it allows workers to prepare RNA
molecules that behave properly as mRNA transcripts in a variety of
in vitro applications. Such applications include both research
applications and commercial production of certain polypeptides in
an in vitro translation system, for example the production of
polypeptides containing an "unnatural" amino acid at a specific
site.
[0005] The method most frequently used to make capped RNAs in vitro
is to transcribe a DNA template with either a bacterial RNA
polymerase or a bacteriophage RNA polymerase in the presence of all
four ribonucleoside triphosphates and a cap dinucleotide such as
m.sup.7G(5')ppp(5')G. The polymerase initiates transcription with a
nucleophilic attack by the 3'-OH of the Guo moiety in m.sup.7GpppG
on the .alpha.-phosphate of the next templated nucleoside
triphosphate, resulting in the initial product m.sup.7GpppGpN. The
alternative, GTP-initiated product pppGpN is suppressed by setting
the ratio of m.sup.7GpppG to GTP between 5 and 10 in the
transcription reaction mixture.
[0006] Synthetic RNAs may be synthesized by cell-free transcription
of DNA templates. See R. Contreras et al., "Simple, efficient in
vitro synthesis of capped RNA useful for direct expression of
cloned eukaryotic genes," Nucl. Acids Res., vol. 10, pp. 6353-6362
(1982); J. Yisraeli et al., "Synthesis of long, capped transcripts
in vitro by SP6 and T7 RNA polymerases, pp. 42-50 in J. Dahlberg et
al. (Eds.), Meth. Enzymol., vol.180., pp. 42-50 (1989); and D.
Melton et al., "Efficient in vitro synthesis of biologically active
RNA and RNA hybridization probes from plasmids containing a
bacteriophage SP6 promoter," Nucl. Acids Res., vol.12, pp.
7035-7056 (1984).
[0007] Capped RNAs thus produced are active in in vitro splicing
reactions. See M. Konarska et al., "Recognition of cap structure in
splicing in vitro of mRNA precursors. Cell, vol. 38, pp. 731-736
(1984); and I. Edery et al., "Cap-dependent RNA splicing in a HeLa
nuclear extract," Proc. Natl. Acad. Sci. USA, vol. 82, pp.
7590-7594 (1985).
[0008] Capped mRNAs are translated more efficiently than are
non-capped mRNAs. See S. Muthukrishnan et al., "5'-Terminal
7-methylguanosine in eukaryotic mRNA is required for translation,"
Nature, vol. 255, pp. 33-37 (1975); L. Chu et al., "Paradoxical
observations on the 5' terminus of ovalbumin messenger ribonucleic
acid," J. Biol. Chem., vol. 253, pp. 5228-5231 (1978); E.
Darzynkiewicz et al., ".beta.-Globin mRNAs capped with m.sup.7G,
m.sub.2.sup.2.7G or m.sub.3.sup.2.2.7G differ in intrinsic
translation efficiency," Nucl. Acids Res., vol 16, pp. 8953-8962
(1988); and E. Darzynkiewicz et al., "Inhibition of eukaryotic
translation by nucleoside 5'-monophosphate analogues of mRNA
5'-cap: Changes in N7 substituent affect analogue activity,"
Biochem., vol. 28, pp. 4771-4778 (1989).
[0009] 5.dbd.-Unmethylated mRNAs have been reported to be
translationally less active than 5'-methylated mRNAs. See G. Both
et al., "Methylation-dependent translation of viral messenger RNAs
in vitro," Proc. Natl. Acad. Sci. USA, vol. 72, pp. 1189-1193
(1975).
[0010] E. Darzynkiewicz et al., "Chemical synthesis and
characterization of 7-methylguanosine cap analogues," Biochem.,
vol. 24, pp.1701-1707 (1985) reported the synthesis of derivatives
of 7-methylguanosine 5'-phosphate that were modified in the ribose
moiety by 2'-O or 3'-O-methylation, or by conversion to the
2'-deoxy or arabinosyl form, and reported that these derivatives
retained cap analogue activity.
[0011] F. Sanger et al., "DNA sequencing with chain-terminating
inhibitors," Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467
(1977) reported a method for determining DNA nucleotide sequences
with 2',3'-dideoxy and arabinonucleoside analogues of normal
deoxynucleoside triphosphates, in which the analogs act as specific
chain-terminating inhibitors of DNA polymerase.
[0012] M. Kadokura et al. 1997, "Efficient synthesis of
.gamma.-methyl-capped guanosine 5'-triphosphate as a 5'-terminal
unique structure of U6 RNA via a new triphosphate bond formation
involving activation of methyl phosphorimidazolidate using
ZnCl.sub.2 as a catalyst in DMF under anhydrous conditions,"
Tetrahedron Lett., vol. 38, pp. 8359-8362 (1997) reported the
synthesis of CH.sub.3pppG from GDP and the imidazolide of methyl
phosphate in DMF, obtaining a yield of 39% in the absence of
ZnCl.sub.2, and a yield of 98% in the presence of ZnCl.sub.2.
[0013] A. Pasquinelli et al., "Reverse 5' caps in RNAs made in
vitro by phage RNA polymerases," RNA, vol. 1, pp. 957-967 (1995)
reported that bacteriophage polymerases also use the 3'-OH of the
7-methylguanosine moiety of m.sup.7GpppG to initiate transcription,
demonstrating that approximately one-third to one-half of RNA
products made with this cap analogue actually contain the "reverse
cap" Gpppm.sup.7GpN. Such reverse-capped RNA molecules behave
abnormally. The same authors reported that when reverse-capped
pre-U1 RNA transcripts were injected into Xenopus laevis nuclei,
they were exported more slowly than natural transcripts. Similarly,
cytoplasmic reverse-capped U1 RNAs in the cytoplasm were not
properly imported into the nucleus. The presence of a cap on mRNA
strongly stimulates translation of an mRNA transcript into protein.
To the knowledge of the present inventors, there have been no
previous reports directly addressing whether, and at what rate,
reverse-capped mRNAs are translated into protein. However, based on
what is known about recognition of the cap structure by eIF4E, one
would expect reverse-capped mRNAs to be translated no more
efficiently than uncapped RNAs.
[0014] Z. Peng et al., "Synthesis and application of a
chain-terminating dinucleotide mRNA cap analog," Org. Lett., vol.
4, pp. 161-164 (2002; published on the Web, December 2001; and
including the supporting information for this article as reprinted
from the journal's web site) reported the synthesis of a
chain-terminating mRNA cap dinucleotide, 3'-O-Me-m.sup.7G(5')pppG,
and its use in the in vitro transcription of homogeneously capped
RNA. Computer modeling was said to indicate that RNA capped with
the compound would be a substrate for cap-dependent
translation.
[0015] Because existing synthetically capped RNAs contain about
one-third to one-half reverse caps, the overall translational
activity of such a RNA preparation is reduced considerably. Other
functional properties of the mRNA may also be affected. There is a
previously unfilled need for a way to prepare capped RNA molecules
in vitro, in which all or essentially all the caps have the proper
orientation.
[0016] We have discovered and synthesized cap analogues that will
not be incorporated into an mRNA molecule in the reverse
orientation. In experiments in which we synthesized and tested two
prototype "anti-reverse" cap analogues (ARCAs), we found that both
were exclusively incorporated into mRNA molecules in the correct
orientation. Furthermore, both behaved like natural RNA caps in
interactions with the translational machinery. The resulting mRNAs
were considerably more active translationally than are traditional
in vitro-prepared RNAs containing a mixture of caps in both the
correct and the reversed orientations.
[0017] Transcription by bacteriophage RNA polymerases in the
presence of m.sup.7GpppG is initiated with a nucleophilic attack by
the 3'-OH of either the m.sup.7Guo moiety or of the Guo moiety on
the electrophilic .alpha.-phosphate of the first templated
nucleoside triphosphate. We eliminated one of these two 3'-OH
groups, so that the nucleophilic attack would cause incorporation
only in the correct orientation. We have made two prototype ARCAs.
In the case of
P.sup.1-3'-deoxy-7-methylguanosine-5'P.sup.3-guanosine-5'triphosphate
(FIG. 1, Compound 9, henceforth abbreviated m.sup.73'dGpppG), we
substituted an --H for the 3'-OH. In the case of
P.sup.1-3'-O,7-dimethylguanosine-5' P.sup.3-guanosine-5'
triphosphate (FIG. 1, compound 10, henceforth abbreviated
m.sub.2.sup.7,O3'GpppG), we instead substituted a --OCH.sub.3 for
the 3'-OH.
[0018] We also developed new coupling strategies to synthesize the
prototype ARCAs. To avoid preparing imidazole derivatives from
7-methylated substrates, the activation of which can be difficult,
we developed a new coupling strategy involving guanosine
5'-phosphorimidazolide and the modified 7-methylated nucleoside
diphosphate. We obtained high yields by conducting the coupling
reaction in the presence of ZnCl.sub.2 instead of Mn.sup.2+, and by
using anhydrous dimethylformamide (DMF) instead of water-as a
solvent. See FIG. 1, depicting schematically the synthesis of
"anti-reverse" cap analogs. (In FIG. 1, "ImGMP" refers to guanosine
5'-imidazolide monophosphate.)
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts the synthesis of "anti-reverse" cap
analogs.
[0020] FIG. 2 depicts an analysis of in vitro-synthesized RNAs by
enzymatic digestion and anion exchange HPLC.
[0021] FIG. 3 depicts the inhibition of translation by ARCAs
compared with m.sup.7GpppG and GpppG.
[0022] FIG. 4 depicts the translational activity of ARCA-capped
mRNAs compared to that of other RNAs.
MATERIALS AND METHODS
[0023] Synthesis of Mono- and Dinucleotides
EXAMPLE 1
[0024] 3'-Deoxyguanosine 5-monophosphate (Compound 3).
3'-Deoxyguanosine (Compound 1, commercial product from Sigma, 50
mg, 0.19 mmol) was stirred overnight with trimethylphosphate (2 mL)
and phosphorus oxychloride (53 .mu.L, 0.57 mmol) at 6.degree. C.
The reaction was quenched by adding 20 mL of water and neutralizing
with 1 M NaHCO.sub.3. DEAE-Sephadex chromatography using a linear
gradient of 0-0.9 M TEAB afforded Compound 3 (yield: 45 mg,
43%).
EXAMPLE 2
[0025] 3'-O-Methylguanosine 5'-monophosphate (Compound 4) was
obtained by a procedure analogous to that for Compound 3, but
instead starting with 59 mg of 3'-O-methylguanosine (Compound 2),
which was prepared by the method of J. Kusmierek et al., "A new
route to 2'(3')-O-alkyl purine nucleosides," Nucl. Acids Res.
Special Publ. No. 4, pp. s73-s77 (1978) (yield: 80 mg, 69%).
EXAMPLE 3
[0026] 3'-Deoxyguanosine 5'-diphosphate (Compound 5). Compound 3
(55 mg, TEA salt, 0.1 mmol), imidazole (34 mg, 0.5 mmol) and
2,2'-dithiodipyridine (Aldrich, 44 mg, 0.2 mmol) were mixed in
anhydrous DMF (1.2 mL) and TEA (14 .mu.L). Triphenylphosphine (52
mg, 0.2 mmol) was added, and the mixture was stirred for 5 h at
room temperature. The mixture was placed in a centrifuge tube, and
sodium perchlorate (49 mg, anhydrous) dissolved in acetone (6 mL)
was added. After cooling for 2 h in a refrigerator, the mixture was
centrifuged and the supernatant was discarded. The precipitate was
ground with a new portion of acetone, cooled and centrifuged again.
The process was repeated once more, and the precipitate was dried
in a vacuum desiccator over P.sub.4O.sub.10. The imidazolide thus
obtained was dissolved in 1.2 mL of DMF, and 200 mg of
triethylammonium phosphate was added. (The latter was prepared from
TEA and phosphoric acid followed by drying over P.sub.4O.sub.10 in
a desiccator to obtain a semicrystalline mass.) Finally, 80 mg of
ZnCl.sub.2 were added, and the reaction mixture was stirred at room
temperature for 6.5 h, poured into a beaker containing a solution
of 250 mg EDTA in 15 mL water, and neutralized with 1 M
NaHCO.sub.3. Chromatographic isolation on a DEAE-Sephadex column
using a linear gradient of 0-1 M TEAB gave Compound 5 (yield: 41
mg, 66%).
EXAMPLE 4
[0027] 3'-O-Methylguanosine 5'-diphosphate (Compound 6) was
obtained by a procedure analogous to that for Compound 5, except
starting from 58 mg of Compound 4 (yield: 32 mg, 49%).
EXAMPLE 5
[0028] 3'-Deoxy-7-methylguanosine 5'-diphosphate (Compound 7).
Compound 5 (34 mg, 0.055 mmol) was mixed with 1 mL of
dimethylsulfoxide, 1 mL of DMF, and 100 .mu.L of methyl iodide at
room temperature. After 3 h the reaction mixture was treated with
30 mL of cold water and extracted three times with 10-mL portions
of diethyl ether. After neutralization with NaHCO.sub.3,
chromatographic separation of the aqueous phase on DEAE-Sephadex,
using a linear gradient of 0 to 0.8 M TEAB, gave Compound 7 (yield:
10 mg, 28 %).
EXAMPLE 6
[0029] 3'-O, 7-Dimethylguanosine 5'-diphosphate (Compound 8) was
obtained by a procedure analogous to that for Compound 7, except
that the starting material was 66 mg of Compound 6 (yield: 64 mg,
95%).
EXAMPLE 7
[0030] P.sup.1-3'-Deoxy-7-methylguanosine-5' P-guanosine-5'
triphosphate (Compound 9). GMP (purchased from Sigma, converted to
the TEA salt, 29 mg, 0.05 mmol), imidazole (17 mg, 0.25 mmol), and
2,2'-dithiodipyridine (22 mg, 0.1 mmol, purchased from Aldrich)
were mixed in anhydrous DMF (1.2 mL) and TEA (7 .mu.L).
Triphenylphosphine (26 mg, 0.1 mmol) was added, and the mixture was
stirred for 5 h at room temperature. The mixture was placed in a
centrifuge tube, and sodium perchlorate (25 mg, anhydrous)
dissolved in acetone (6 mL) was added. The procedure for washing
the precipitate with acetone and drying over P.sub.4O.sub.10 was
the same as for Compound 5. The imidazolide of GMP thus obtained
was dissolved in DMF (1.2 mL), and Compound 7 (10 mg, TEA salt,
0.015 mmol) was added. Next ZnCl.sub.2 (40 mg) was added. The
mixture was stirred at room temperature overnight, poured into a
beaker containing a solution of 125 mg of EDTA in 15 mL of water,
and neutralized with 1 M NaHCO.sub.3. Chromatographic isolation as
for Compound 5 gave Compound 9 (13 mg, 88% based on the amount of
Compound 7 used).
EXAMPLE 8
[0031] P.sup.1-3'-O,
7-Dimethylguanosine-5'P.sup.3-guanosine-5'triphosphate (Compound
10) was prepared from GMP and Compound 8 (34 mg) by a procedure
analogous to that for Compound 9 (yield: 23 mg, 78%).
EXAMPLES 9 & 10
[0032] The final products (Compounds 9 and 10) were converted to
their Na.sup.+ salts by ion exchange on a small column of Dowex 50
W.times.8 (Na.sup.+ form), followed by evaporation of the eluates
to a small volume, precipitation with ethanol, and centrifugation
to give amorphous white powders. Parameters from the .sup.1H NMR
spectra of Compounds 9 and 10 are shown in Tables 1 and 2
below.
EXAMPLE 11
[0033] 7-Methylguanosine 3',5'-diphosphate. Guanosine
3',5'-diphosphate was methylated to make the chromatographic
standard pm.sup.7Gp (FIG. 2) by the same procedure as used for
Compound 7.
EXAMPLE 12
[0034] Column Chromatography
[0035] Both final products (Compounds 9 and 10, FIG. 1) and
intermediate nucleotides (Compounds 3-8) were isolated from
reaction mixtures by column chromatography on DEAE-Sephadex (A-25,
HCO.sub.3.sup.- form) using a linear gradient of triethylammonium
bicarbonate (TEAB), pH 7.5, in water. Fractions were collected, and
products peaks (monitored at 260 nm) were pooled and evaporated to
dryness, with ethanol added repeatedly to remove the TEAB buffer.
The products were obtained as TEA salts.
[0036] The purity of intermediates and products was monitored at
260 nm by analytical HPLC using a Spectra-Physics SP8800 apparatus
on a 25-cm LC-18-T reverse phase column (Supelco). The mobile phase
was a linear gradient of methanol from 0 to 25% in 0.1 M
KH.sub.2PO.sub.4, pH 6, over 15 min with flow rate of 1.3
mL/min.
[0037] Mono- and dinucleotides obtained by enzymatic digestion of
in vitro-synthesized RNAs were analyzed by HPLC using a Waters 625
instrument with a 996 PDA detector on a 4.5.times.250 mm Partisil
10SAX/25 column (Whatman). The program for elution of nucleotides
comprised water for the first 5 min; a linear gradient of 87.5 to
500 mM KH.sub.2PO.sub.4, pH 3.5, over 35 min; a linear gradient of
87.5 to 500 mM of KH.sub.2PO.sub.4 over 30 min; and isocratic
elution at 500 mM KH.sub.2PO.sub.4 for 21 min--all at a flow rate
of 1 mL/min.
EXAMPLE 13
[0038] Spectroscopy
[0039] .sup.1H NMR and .sup.13C NMR spectra were recorded on a
Varian UNITY plus 500 MHz instrument in dimethylsulfoxide-d.sub.6
(for nucleoside intermediates) or D.sub.2O (for mono- and
dinucleotides). Absorption spectra were obtained on a Cary 3E
spectrophotometer.
[0040] .sup.1H NMR spectra for Compounds 9 and 10 were run at
25.degree. C. at 1.4 mg/0.7 mL and 0.4 mg/0.7 mL in D.sub.2O,
respectively. Conformations of the sugar moieties were derived from
the vicinal .sup.1H-.sup.1H coupling constants. Conformations of
the phosphate groups were determined from the .sup.1H-.sup.31P
coupling constants.
EXAMPLES 14 & 15
[0041] In vitro Synthesis of RNA
[0042] Two lengths of RNA, either uncapped or capped with one of
the cap analogues, were synthesized by in vitro transcription. The
DNA template used for both lengths of RNA was pSP-luc+ (Promega),
which contains an SP6 bacteriophage promoter and a sequence
encoding luciferase. To generate the short RNAs (43 bases exclusive
of the cap), the plasmid was digested with Ncol. To generate the
long RNAs (1706 bases, containing the entire luciferase coding
region), the plasmid was digested with EcoRI. A typical 20 .mu.L in
vitro transcription reaction contained 40 mM Tris-HCl, pH 7.9, 6 mM
MgCl.sub.2, 2 mM spermidine, 10 mM DTT, 2 .mu.g BSA, 20 units of
RNasin (Promega), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.1 mM GTP, 1
mM cap analogue (GpppG, m.sup.7GpppG, m.sup.73'dGpppG, or
m.sub.2.sup.7,O3'GpppG), 0.2-1.0 .mu.g DNA, and 20 units of SP6
polymerase (Promega). Reactions to synthesize the short RNAs also
contained 28 .mu.Ci of [.alpha.-.sup.32P]ATP (ICN), and those to
synthesize the long RNAs contained 0.8 .mu.Ci of
[.alpha.-.sup.32P]CTP (ICN). Reaction mixtures were incubated for
60 min at 37.degree. C., extracted with phenol and chloroform, and
the solution was made 2 M in sodium acetate. The nucleic acids were
then precipitated with 3 volumes of ethanol on dry ice for 5 min,
and the mixture was centrifuged at 14,000 rpm for 30 min. The
resulting pellet was dissolved in water, and the solution was made
0.2 M in sodium acetate. The nucleic acid was precipitated with 2.5
volumes of ethanol at 4.degree. C. for 30 min, and the mixture was
centrifuged at 14,000 rpm for 30 min. The pellet was allowed to
air-dry and then dissolved in diethylpyrocarbonate-treated
water.
EXAMPLE 16
[0043] Enzymatic Digestion of RNAs
[0044] The short RNAs were digested with 67 U RNase T2 (Life
Technologies) in 15 .mu.L of 0.14 M sodium acetate, pH 4.6, at
37.degree. C. for 60 min. In some cases, the RNAs were subjected to
a two-step digestion instead. The first digestion was with 10 U TAP
(tobacco acid pyrophosphatase) (Epicentre Technologies) in 5 .mu.L
of 50 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1%
.beta.-mercaptoethanol, and 0.01% Triton X-100 at 37.degree. C. for
60 min. The digestion was continued for 60 min at 37.degree. C.
with 67 U RNase T2 in a final volume of 16 .mu.L of 0.12 M sodium
acetate, pH 4.6. Samples were analyzed without further treatment by
anion exchange HPLC as described above.
EXAMPLE 17
[0045] Cell-Free Translation
[0046] A micrococcal nuclease-treated RRL (rabbit reticulocyte
lysate) system was used for in vitro translation as described in A.
Cai et al., "Quantitative assessment of mRNA cap analogs as
inhibitors of in vitro translation," Biochemistry, vol. 38, pp.
8538-8547 (1999). In some cases, the mRNA used in this system was
natural rabbit globin mRNA, and protein synthesis was measured by
incorporation of [.sup.3H]Leu into a trichloroacetic
acid-precipitable form. In other cases, the mRNA was luciferase
mRNA (the long form), synthesized in vitro as described above, and
protein synthesis was assayed by measuring luciferase activity
using beetle luciferin (Promega) as a substrate, and a Monolite
2010 luminometer to measure light emission.
[0047] The ability of cap analogues to inhibit cell-free
translation in the RRL system programmed with globin mRNA was
measured as described in Cai et al. (1999). Data were fit by least
squares minimization to a theoretical rate equation. The
concentrations of cap analogue solutions were measured by UV
absorption at pH 7.0 using the following parameters for .lamda. and
.epsilon..sub.M, respectively: GpppG, 251 nm, 25.5.times.10.sup.3;
m.sup.7GpppG, m.sup.73'dGpppG, or m.sub.2.sup.7,O3'GpppG, 255 nm,
22.6.times.10.sup.3.
Results
[0048] .sup.13C NMR and UV spectra for intermediates were in good
agreement with the predicted structures (data not shown). The
.sup.1H NMR assignments of protons in both prototype ARCAs
confirmed their chemical structures (Table 1). Two sets of sugar
.sup.1H signals in each spectrum pointed to dinucleotides. The
presence of methyl signals at 4.068 ppm (Compound 10) and 4.027
(Compound 9), together with disappearance of the H(8) resonances
due to exchange for solvent deuterium, testified to the presence of
7-methylguanine. In the case of Compound 10, the additional methyl
group was observed at 3.483 ppm, accompanied by a characteristic
upfield shift of the H3' signal. Lack of the 3'-hydroxyl in
Compound 9 gave the characteristic "deoxy" pattern of H3'/H3'' at
2.086-2.148 ppm, with further scalar couplings to H4' and H2'.
TABLE-US-00001 TABLE 1 .sup.1H NMR chemical shifts in ppm versus
internal sodium 3-trimethylsilyl- [2,2,3,3-D.sub.4]-propionate
m.sup.73'dGpppG m.sub.2.sup.7,O3'GpppG (Compound 9) (Compound 10)
m.sup.73'dG G m.sub.2.sup.7,O3'G G H8 --.sup.a 8.016 --.sup.a 7.990
H1' 5.796 5.776 5.864 5.785 H2' 4.587 4.650 4.682 4.687 H3' 2.148
4.473 4.109 4.473 H3'' 2.086 -- -- -- H4' 4.728 4.346 4.428 4.339
H5' 4.460 4.26.sup.b 4.384 4.278 H5'' 4.196 4.26.sup.b 4.219 4.239
CH.sub.3 4.027 -- 4.068 (N7) -- 3.483 (3'O) .sup.adeuterated
.sup.bsignal overlapping
[0049] Table 2 provides NMR information concerning conformational
parameters. These data reflected populations of the N form in the
N.revreaction.S dynamic equilibrium of the sugar ring, populations
of the +sc (gauche-gauche) conformer about C4'-C5', and populations
of the ap (gauche'-gauche') conformer of the phosphate group. The
7-substituted Guo moieties showed the characteristic preference for
the N conformer, up to 100% in the case of m.sub.2.sup.7,O3'Guo, as
opposed to Guo, in which the S conformer dominates. The preference
for +sc was also more pronounced in the 7-substituted guanosines.
The conformation of the Guo moiety of ARCAs was similar to that of
Guo in normal caps, in which about 64% has been reported to be in
the S form (36% N) and about 63% in the +sc form. Thus,
m.sub.2.sup.7,O3'Guo and m.sup.73'dG both displayed conformational
features that were characteristic of m.sup.7Guo rather than of Guo.
TABLE-US-00002 TABLE 2 .sup.1H-.sup.1H and .sup.1H-.sup.31P
coupling constants in Hz (.+-.0.2), and conformer populations
(.+-.5%) in the dynamic equilibria N S of the sugar ring, and about
C4'-C5' (% +sc) and C5'-O5' (% ap) bonds m.sup.73'dGpppG
m.sub.2.sup.7,O3'GpppG (Compound 9) (Compound 10) m.sup.73'dG G
m.sub.2.sup.7,O3'G G J(1', 2') 0.0.sup.a 6.2 4.0 6.3 J(2', 3') 4.5
5.2 5.0 5.1 J(2', 3'') 0.0.sup.a -- -- -- J(3', 3'') 14.2 -- -- --
J(3', 4') 10.4 3.7 5.1 3.6 J(3'', 4') 5.1 -- -- -- J(4', 5')
3.0.sup.b 4.0.sup.b 3.0 4.1 J(4', 5'') 2.7 4.0.sup.b 2.6 4.2 J(5',
5'') 11.6 b 11.5 11.8 J(5', P) 5.0 6.0.sup.b 4.4 5.4 J(5'', P) 5.8
6.0.sup.b 5.9 6.5 J(4', P) 1.0.sup.b 1.0.sup.b 1.0.sup.b 1.0.sup.b
% N 100 37 56 36 % +sc.sup.c 80 55.sup.b 80 54 % ap.sup.d 72
66.sup.b 74 66 .sup.aless than the line width, .about.1 Hz
.sup.bapproximate value .sup.c+synclinal, i.e., O5' in gauche
orientation to O4' and C3' .sup.dantiperiplanar i.e., P5' in trans
orientation to C4'
[0050] EXAMPLE 18
[0051] Synthesis of ARCA-Capped RNA Transcripts
[0052] We tested the prototype ARCAs in an in vitro transcription
system. A template DNA was first generated by digesting the plasmid
pSP-luc+ with EcoRI. The theoretical size of an RNA transcript from
this template should be 1706 bases, which was consistent with the
approximate size of the products that was observed by
electrophoretic migration from reactions carried out in the
presence of [.alpha.-.sup.32P]ATP and either GpppG, m.sup.7GpppG,
m.sup.73'dGpppG, or m.sub.2.sup.7,O3'GpppG. (data not shown).
Samples were run on a 1% agarose gel containing 0.12 M formaldehyde
in 0.4 M 3-(N-morpholino)propanesulfonic acid, pH 7.0, 0.1 M sodium
acetate, and 0.01 M EDTA at 70 mA for 5 h. A Phosphorimage was
obtained with a Molecular Dynamics Storm 860 instrument. Standards
used for comparison were rabbit 28S rRNA, 18S rRNA, and
.beta.-globin mRNA.
[0053] In six separate experiments, the yield of RNA product in the
presence of ARCAs was not significantly different from the yield in
the presence of m.sup.7GpppG.
EXAMPLE 19
[0054] Analysis of Cap Orientation in ARCA-Capped RNA
Transcripts
[0055] The structure of the ARCAs was designed to prevent
incorporation into RNA in the reverse orientation. We verified this
property experimentally by digesting RNAs capped with ARCAs with
RNase T2 and TAP. To obtain a higher proportion of radioactivity in
the cap versus the internal positions, a shorter DNA template was
produced by cleaving pSP-luc+ with Ncol instead of EcoRI. This
digestion was expected to yield an RNA product of 43 bases (plus
the cap). The size of this product was confirmed by polyacrylamide
gel electrophoresis in Tris/borate/EDTA/urea (data not shown).
[0056] RNase T2 digests RNA with no base specificity. Thus, it was
expected to generate primarily 3'-NMPs from this RNA. Those
nucleotide residues that were located 5' to an A residue would
acquire a .sup.32P-labeled 3'-phosphate by nearest-neighbor
transfer. The pyrophosphate bonds in the cap, however, are not
susceptible to RNase T2. Since the first nucleotide residue after
the cap in the synthetic RNA is an A, the .alpha.-phosphate of
[.alpha.-.sup.32P]ATP would be transferred to the cap following
RNase T2 digestion. Thus, for RNAs initiated in the normal
orientation with m.sup.7GpppG, the product was m.sup.7GpppGp*
(where p* denotes radioactive .sup.32p). The RNase T2-digestion
products expected from RNAs initiated with GTP or with the four cap
analogues in either normal or reverse orientations are shown in
Table 3. TABLE-US-00003 TABLE 3 Predicted and observed cap
structures from in vitro-synthesized mRNAs after enzymatic
digestion Possible 5' end labeled Product Cap Possible
transcription digestion products observed dinucleotide
Orientation.sup.1 products RNase T2 RNase T2 + TAP RNase T2 + TAP
None N/A pppGP*Ap(Np).sub.40C pppGp* pGp* 100% GpppG N/A
GpppGp*Ap(NP).sub.40C GpppGp* pGp* 100% m.sup.7GpppG Normal
m.sup.7GpppGp*Ap(Np).sub.40C m.sup.7GpppGp* pGp* 67% Reverse
Gpppm.sup.7Gp*Ap(Np).sub.40C Gpppm.sup.7Gp* pm.sup.7Gp* 33%
m.sup.73'dGpppG Normal m.sup.73'dGpppGp*Ap(Np).sub.40C
m.sup.73'dGpppGp* pGp* 100% Reverse Gpppm.sup.73'dGp*Ap(Np).sub.40C
Gpppm.sup.73'dGp* pm.sup.73'dGp* 0% m.sub.2.sup.7,O3'GpppG Normal
m.sub.2.sup.7,O3'GpppGp*Ap(Np).sub.40C m.sub.2.sup.7,O3'GpppGp*
pGp* 100% Reverse Gpppm.sub.2.sup.7,O3'GP*Ap(Np).sub.40C
Gpppm.sub.2.sup.7,O3'Gp* pm.sup.7,O3'Gp* 0% .sup.1"Normal"
orientation means that the 3'-OH of Guo in the structure
m.sup.7G(5')ppp(5')G (or its analogues) is attached to the first
nucleotide residue in the RNA chain by a 3'-5'phosphodiester
linkage. "Reverse" orientation means that the 3'-OH of m.sup.7Guo
is the point of attachment. .sup.2Radioactive atoms (.sup.32P) are
indicated by *.
[0057] The RNase T2-digestion products of normal and reverse
m.sup.7GpppG-capped RNAs (m.sup.7GpppGp* and Gpppm.sup.7Gp*,
respectively) have identical masses and charges; they would
therefore be expected to elute from an anion exchange column at
nearly the same time. However, TAP digestion of normal and
reverse-capped mRNAs should yield two alternate labeled products,
pGp* and pm.sup.7Gp*, that differ in both charge and mass, because
the m.sup.7 group confers a positive charge on G. The nucleotides
pm.sup.73'dGp* and pm.sub.2.sup.7,O3'Gp* have the same charge as
pm.sup.7Gp*. Thus, although RNase T2 digestion alone would not be
expected to distinguish between normal and reverse orientations,
the combination of RNase T2 and TAP should do so (see Table 3).
[0058] RNA was synthesized from the short DNA template in the
presence of: (1) [.alpha.-.sup.32P]ATP; (2) the other three NTPs
(nonradioactive); and (3) either GpppG, m.sup.7GpppG,
m.sup.73'dGpppG, m.sub.2.sup.7,O3'GpppG, or no cap analogue. The
products were digested with RNase T2 and resolved by anion exchange
HPLC. FIG. 2 depicts an analysis of the in vitro-synthesized RNAs
by enzymatic digestion and anion exchange HPLC. The mRNAs were
generated by transcription of Ncol-digested pSP-luc+ with
[.alpha.-.sup.32P]ATP and either no cap dinucleotide (panels A, B),
GpppG (panels C, D), m.sup.7GpppG (panels E, F), m.sup.73'dGpppG
(panels G, H), or m.sub.2.sup.7,O3'GpppG (panels I, J). Aliquots of
5 to 13 ng of RNA were digested with RNase T2 (left panels), or
with both RNase T2 and TAP (right panels). Nucleotides and caps
were separated on a Partisil 10SAX/25 column developed with a
gradient of potassium phosphate, pH 3.5. Fractions of 1 mL were
collected, and their Cerenkov radiation was determined with a
Beckman LS 6500 scintillation counter. The elution times of the
following standard compounds, detected by UV absorption, are also
shown: 3'-CMP; 3'-UMP; 3'-AMP; 3'-GMP; 5'-GDP; 5'-GTP; 3',5'-GDP
(pGp); 3',5'-m.sup.7GMP (pm.sup.7Gp); and GpppG.
[0059] Uncapped RNA yielded primarily 3'-NMPs (Panel A, 20-30 min)
with a small amount of material that may have been the partially
degraded product ppGp* (Panel A, 76 min). The expected product
pppGp* was not observed. Due to its high negative charge, that
species may not have eluted from the column. Its presence, however,
is likely since RNase T2 plus TAP digestion yielded pGp* (Panel B,
56 min) where none had existed previously (compare Panel A).
[0060] In the case of GpppG-capped RNAs, RNase T2 alone yielded a
structure eluting at 89 min (FIG. 2, Panel C), likely GpppGp* (the
presence of a second phosphate ester reduces the charge relative to
pppGp*). The minor peak at 77 min may have been the partially
degraded product ppGp*. Consistent with these assignments, both
compounds disappeared following TAP digestion, coinciding with the
appearance of a new peak corresponding to pGp* at 56 min (FIG. 2,
Panel D). As expected, no pm.sup.7Gp* (42 min) was formed.
[0061] The major, highly-charged RNase T2-resistant product from
m.sup.7GpppG-capped RNA eluted at 73 min (FIG. 2, Panel E), likely
m7GpppGp*. This compound eluted earlier than the peak at 89 min in
Panel C, tentatively assigned the structure GpppGp*, because of the
additional positive charge. The minor peak at 77 min may be the
reverse cap Gpppm.sup.7Gp*, suggesting that the proximity of the
3'-P to the positive charge of the m.sup.7G ring may influence the
charge on the P. These assignments are strengthened by the fact
that TAP digestion converted these products to two labeled
compounds that eluted earlier, pGp* (56 min) and pm.sup.7Gp* (42
min) (FIG. 2, Panel F). The ratio of pGp* to pm.sup.7Gp* suggest
that they were derived from the 73- and 77-min peaks of Panel E,
respectively.
[0062] With the ARCA m.sup.73'dGpppG, an RNase T2-resistant product
was observed at 78 min, likely m.sup.73'dGpppGp* (FIG. 2, Panel G).
It eluted at nearly the same time as the compound thought to be
m.sup.7GpppGp* (78 min versus 77 min for Panels G and E,
respectively). Note that where there were two peaks in this region
for RNA synthesized with m.sup.7GpppG (Panel E), there was only one
peak for RNA synthesized with the ARCA (Panel G), consistent with
the inability of the ARCA to be incorporated in the reverse
orientation. After digestion with TAP, the peak at 78 min
disappeared and a new one appeared at the elution time of pGp*
(Panel H, 56 min). The fact that no pm.sup.7Gp* appeared at 42 min
with the ARCA (Panel H), while it did with m.sup.7GpppG (Panel F),
is further proof that the ARCA was incorporated in only a single
orientation.
[0063] The products observed upon digestion of RNA synthesized with
the second ARCA, m.sub.2.sup.7,O3'GpppG (FIG. 2, Panels I and J),
eluted almost the same as those that had been obtained with the
m.sup.73'dGpppG-capped RNA--again, consistent with the expectation
that the ARCA should be incorporated in only one orientation.
EXAMPLE 20
[0064] Competitive Inhibition of Protein Synthesis by ARCAs
[0065] One measure of the interaction between a cap analogue and
the translational machinery is competitive inhibition of protein
synthesis. The binding of cap analogues to eIF4E, measured in vitro
with purified components, and the resulting competitive inhibition
of protein synthesis in a cell-free translation system have been
correlated for several different cap analogue structures. We
separately tested GpppG, m.sup.7GpppG, and the two ARCAs for their
ability to compete with natural globin mRNA for recognition by the
translational machinery, and thereby to inhibit translation in an
RRL system. GpppG did not act as an inhibitor, and in fact slightly
stimulated protein synthesis at low concentrations. The two ARCAs,
on the other hand, were equally as inhibitory as m.sup.7GpppG. FIG.
3 depicts the inhibition of translation by ARCAs compared with
m.sup.7GpppG and GpppG. Natural rabbit globin mRNA was translated
at 5 .mu./mL in the RRL system, and globin synthesis was detected
by incorporation of [.sup.3H]Leu into protein. The following cap
analogues were included during translation at the indicated
concentrations: GpppG, circles; m.sup.7GpppG, squares;
m.sup.73'dGpppG, triangles; and m.sub.2.sup.7,O3'GpppG,
diamonds.
[0066] One may compare cap analogues as inhibitors quantitatively,
by fitting a theoretical curve to observed translation data. The
value of the dissociation constant, K.sub.I, for the cap
analogueeIF4E complex was varied to obtain the best least-squares
fit. FIG. 3 depicts such curves for m.sup.7GpppG, m.sup.73'dGpppG,
and m.sub.2.sup.7,O3'GpppG, with corresponding K.sub.I values of
27.8.+-.12.6, 27.8.+-.7.1, and 14.3.+-.1.9 .mu.M, respectively.
Although it appeared in this experiment that the
m.sub.2.sup.7,O3'GpppG compound was more inhibitory, in a replicate
of this experiment the K.sub.i values for the ARCAs did not differ
statistically from those of m.sup.7GpppG.
EXAMPLE 21
[0067] Translation of ARCA-Capped mRNAs
[0068] Because one-third to one-half of m.sup.7GpppG was
incorporated into RNA in the reverse orientation, because the novel
ARCAs were incorporated exclusively in the normal orientation, and
because the ARCAs were recognized to the same extent as
m.sup.7GpppG in the translational inhibition experiment, we
predicted that a homogeneous population of in vitro-synthesized
ARCA-capped mRNAs should be more active translationally than
m.sup.7GpppG-capped mRNAs. We tested this prediction using
luciferase mRNAs that were either uncapped, capped with GpppG,
capped with m.sup.7GpppG, or capped with one of the two ARCAs.
[0069] FIG. 4 depicts the translational activity of the ARCA-capped
mRNAs. Luciferase mRNAs were synthesized in vitro using SP6 RNA
polymerase in the presence of all four NTPs, and either no cap
analogue (circles), GpppG (squares), m.sup.7GpppG (diamonds),
m.sub.2.sup.7,O3'GpppG (inverted triangles) or m.sup.73'dGpppG
(triangles). The RNAs were translated for 60 min in the RRL system,
and luciferase activity was measured in triplicate by luminometry
(RLU, relative light units). Translational efficiency for each mRNA
was estimated from the slopes of the curves of luciferase activity
versus mRNA concentration.
[0070] The fact that all the m.sup.7G-containing mRNAs were
translated more efficiently than the uncapped or GpppG-capped mRNAs
indicated that the RRL system we used was highly dependent on the
presence of a cap on the mRNA. As shown in FIG. 4, we found that
the m.sub.2.sup.7,O3'GpppG-and m.sup.73'dGpppGp-capped mRNAs were
more efficient in translation than m.sup.7GpppG-capped mRNA. In six
experiments employing four separate batches of in vitro-synthesized
mRNAs, the mRNAs produced with the novel ARCAs were consistently
more active than those produced with m.sup.7GpppG.
[0071] Pasquinelli et al. (1995) found that the extent of reverse
capping varied between 28% and 48%, depending on the pH of the in
vitro-transcription reaction. In the experiments whose results are
shown in FIG. 2 and summarized in Table 3, the extent of reverse
capping was approximately 33%. Assuming that the novel ARCAs and
normal caps stimulate translation to the same degree, an assumption
that seems likely based on the inhibition data (FIG. 3), we
predicted that the (homogeneous) preparation of ARCA-capped mRNA
should be more active than the (heterogeneous) preparation of
m.sup.7GpppG-capped mRNAs, a prediction that was consistent with
our experimental observations.
[0072] These results showed that the novel ARCAs behaved very
similarly to normal cap analogues, except that they were not
incorporated into RNAs in the reverse orientation, and that they
can cause substantially higher translational activity. The
modifications at the 3'-O-position of m.sup.7Guo did not appear to
substantially alter conformation (Table 2) or interaction with
translational machinery (FIGS. 3 and 4). The ARCAs have the
advantage of being incorporated into RNA exclusively in the normal
orientation, but have no apparent disadvantages. To our knowledge,
the degree to which m.sup.7G is incorporated in place of G in
internal positions of a synthetic RNA chain by bacteriophage
polymerases has not been rigorously determined. Regardless of the
level of such misincorporation, the ARCAs should be essentially
incapable of donating m.sup.7G either internally or at the 5'-end.
A different type of ARCA, e.g., m.sub.4.sup.2,2,7,O3'GpppG or
m.sub.3.sup.2,2,73'dGpppG, would be useful for in vitro synthesis
of U-type snRNAs with 100% normal cap orientation.
EXAMPLE 22
[0073] An Arabinose-Derived ARCA
[0074] Anti-reverse mRNA cap analogues may also be derived from
arabinose, for example: ##STR1##
[0075] For example, where n=1, this ARCA may be synthesized
starting with 9-.beta.-D-arabinofuranosylguanine, which is
commercially available or which may be prepared by the method of
Ikehara et al., "Studies of nucleosides and nucleotides. XLVIII.
Purine cyclonucleosides. 29. A new method for the synthesis of
9-.beta.-arabinofuranosylguanine (Ara G)," J. Carbohydr.
Nucleosides Nucleotides, vol. 3, pp. 149-159 (1976), in place of
the 3'-deoxyguanosine in Example 1.
[0076] Although the examples described above employed particular
cap analogs, other analogs will also work in practicing the
invention, for example: TABLE-US-00004 ##STR2## 1.
m.sub.2.sup.7,3'OGpppG: X = OH, Y = OCH.sub.3, n = 1; 2.
m.sup.73'dGpppG: X = OH, Y = H, n = 1; 3. m.sub.2.sup.7,2'OGpppG: X
= OCH.sub.3, Y = OH, n = 1; 4. m.sup.72'dGpppG: X = H, Y = OH, n =
1; 5. m.sup.72',3'didGpppG: X = H, Y = H, n = 1; 6.
m.sub.3.sup.7,2'O,3'OGpppG: X = OCH.sub.3, Y = OCH.sub.3, n = 1; 7.
m.sup.7et.sup.3'OGpppG: X = OH, Y = OC.sub.2H.sub.5, n = 1; 8.
m.sup.7et.sup.2'OGpppG: X = OC.sub.2H.sub.5, Y = OH, n = 1; 9.
m.sub.2.sup.7,3'OGppppG: X = OH, Y = OCH.sub.3, n = 2; 10.
m.sup.73'dGppppG: X = OH, Y = H, n = 2; 11.
m.sub.2.sup.7,2'OGppppG: X = OCH.sub.3, Y = OH, n = 2; 12.
m.sup.72'dGppppG: X = H, Y = OH, n = 2; 13. m.sup.72',3'didGppppG:
X = H, Y = H, n = 2; 14. m.sub.3.sup.7,2'O,3'OGppppG: X =
OCH.sub.3, Y = OCH.sub.3, n = 2; 15. m.sup.7et.sup.3'OGppppG: X =
OH, Y = OC.sub.2H.sub.5, n = 2; 16. m.sup.7et.sup.2'OGppppG: X =
OC.sub.2H.sub.5, Y = OH, n = 2.
[0077] Note that both 2' and 3' modifications may be used. These
compounds may, for example, be synthesized in a manner generally
analogous to the syntheses described above. For example, the
synthesis of alternative 3 in the above list (X.dbd.OCH.sub.3,
Y.dbd.OH, n=1) may be conducted in a manner similar to that
described in the above Examples, starting by replacing the
3'-O-methylguanosine with 2'-O-methylguanosine in Example 2, the
synthesis of the latter of which is also described in Kusmierek et
al. (1978).
[0078] Likewise, the synthesis of alternative 4 in the above list
(X.dbd.H, Y.dbd.OH, n=1) may be conducted in a manner similar to
that described in the above Examples, starting with
2'-deoxyguanosine 5'-diphosphate, which is commercially available,
in Example 3 in lieu of 2'-deoxyguanosine 5'-diphosphate.
[0079] The synthesis of alternative 5 may be conducted in a similar
manner, starting with 2',3'-dideoxyguanosine (which is commercially
available) in lieu of 3'-deoxyguanosine in Example 1.
[0080] The synthesis of alternative 6 may be conducted by modifying
the procedure of Kusmierek et al. (1978) by using a large excess of
methylation reagent to prepare the starting material to use as
otherwise described in the Examples, starting with Example 2.
[0081] The synthesis of alternative 7 may be conducted by modifying
the procedure of Kusmierek et al. (1978) by using diazoethane
(instead of diazomethane) as alkylating reagent to prepare the
starting material to use as otherwise described in the Examples,
starting with Example 2.
[0082] The synthesis of alternative 8 may be conducted by modifying
the procedure of Kusmierek et al. (1978) by using diazoethane
(instead of diazomethane) as alkylating reagent to prepare the
starting material to use as otherwise described in the Examples,
starting with Example 2.
[0083] The synthesis of alternatives 9-16 (n=2) may be conducted as
otherwise described in the above Examples, or in the above
syntheses of alternatives 3-8 (n=1), as appropriate, but using GDP
instead of GMP in the steps as otherwise described in Example 7.
Likewise, analogues with highervalues of n may be prepared using
guanosine triphosphate, guanosine tetraphosphate, guanosine
pentaphosphate, etc. in lieu of GMP in the steps otherwise
described in Example 7.
[0084] We expect that 2',3'-dideoxy- and 2',3'-dimethyl cap analogs
will function in the present invention. We also expect that
introducing additional phosphate groups into the phosphate bridge
(creating, e.g., dinucleotide tetraphosphates or even penta-,
hexa-, or heptaphosphates (n=3, 4, or 5 in the above
structure))--will produce compounds that may be more effective than
the triphosphates. Other possible substituents X and Y include
OCH.sub.2CH.sub.3. If Y is OH, then it is preferred that X is
neither H nor OH.
[0085] The "non-methylated" guanosine in the ARCA may be replaced
with another nucleoside, e.g., uridine, adenosine, or cytosine:
##STR3## wherein the moiety B is selected from the group consisting
of ##STR4##
[0086] The synthesis of the ARCAs with these alternative
nucleosides may be conducted as otherwise described in the above
Examples, or in the above syntheses of alternatives 1-16, as
appropriate, but using AMP, ADP, ATP, UMP, UDP, UTP, CMP, CDP, CTP,
etc. in lieu of GMP in the steps otherwise described in Example
7.
[0087] Another alternative is to replace the 7-methyl group with
another substituent, such as C.sub.1 to C.sub.4 substituted or
unsubstituted alkyl, C.sub.6 to C.sub.8 substituted or
unsubstituted aryl, or C.sub.1 to C.sub.4 substituted or
unsubstituted alkoxy, as illustrated in the examples below:
TABLE-US-00005 ##STR5## [a] et.sup.7m.sup.3'OGpppG: X = OH, Y =
OCH.sub.3, Z = C.sub.2H.sub.5 n = 1; [b] et.sup.73'dGpppG: X = OH,
Y = H, Z = C.sub.2H.sub.5 n = 1; [c] bn.sup.7m.sup.3'OGpppG: X =
OH, Y = OCH.sub.3, Z = CH.sub.2C.sub.6H.sub.5 n = 1; [d]
bn.sup.73'dGpppG: X = OH, Y = H, Z = CH.sub.2C.sub.6H.sub.5 n = 1;
[e] et.sup.7m.sup.3'OGpppG: X = OH, Y = OCH.sub.3, Z =
C.sub.2H.sub.5 n = 2; [f] et.sup.73'dGpppG: X = OH, Y = H, Z =
C.sub.2H.sub.5 n = 2; [g] bn.sup.7m.sup.3'OGpppG: X = OH, Y =
OCH.sub.3, Z = CH.sub.2C.sub.6H.sub.5 n = 2; [h] bn.sup.73'dGpppG:
X = OH, Y = H, Z = CH.sub.2C.sub.6H.sub.5 n = 2; [i]
pFbn.sup.7m.sup.3'OGpppG: X = OH, Y = OCH.sub.3, Z =
CH.sub.2C.sub.6H.sub.4pF n = 1; [j] pFbn.sup.73'dGpppG: X = OH, Y =
H, Z = CH.sub.2C.sub.6H.sub.4pF n = 1; [k]
pClbn.sup.7m.sup.3'OGpppG: X = OH, Y = OCH.sub.3, Z =
CH.sub.2C.sub.6H.sub.4pCl n = 1; [l] pClbn.sup.73'dGpppG: X = OH, Y
= H, Z = CH.sub.2C.sub.6H.sub.4pCl n = 1; [m]
pFbn.sup.7m.sup.3'OGpppG: X = OH, Y = OCH.sub.3, Z =
CH.sub.2C.sub.6H.sub.4pF n = 2; [n] pFbn.sup.73'dGpppG: X = OH, Y =
H, Z = CH.sub.2C.sub.6H.sub.4pF n = 2; [o]
pClbn.sup.7m.sup.3'OGpppG: X = OH, Y = OCH.sub.3, Z =
CH.sub.2C.sub.6H.sub.4pCl n = 2; [p] pClbn.sup.73'dGpppG: X = OH, Y
= H, Z = CH.sub.2C.sub.6H.sub.4pCl n = 2;
[0088] The synthesis of alternatives [a] and [b] above may be
carried out, for example, as otherwise described in the Examples
above, starting with Example 5 or 6, and replacing the 100 .mu.L of
methyl iodide with 100 .mu.L of ethyl iodide.
[0089] The synthesis of alternatives [c] and [d] above may be
carried out, for example, as otherwise described in the Examples
above, starting with Example 5 or 6, and replacing the 100 .mu.L of
methyl iodide with 100 .mu.L of benzyl bromide. See generally M.
Jankowska et al., "Synthesis and properties of new NH.sub.2 and N7
substituted GMP and GTP 5'-mRNA cap analogues," Collect. Czech.
Chem. Commun., vol. 58, pp. S138-S141 (1993).
[0090] The synthesis of alternatives [e] through [h] above may be
carried out, for example, as otherwise described in the Examples
above, starting with Examples 5 through 8, and replacing the 100
.mu.L of methyl iodide with 100 .mu.L of ethyl iodide or benzyl
bromide, as appropriate, and replacing GMP with GDP.
[0091] The synthesis of alternatives [i] through [p] above may be
carried out, for example, as otherwise described in the Examples
above, starting with Examples 5 through 8, and replacing the 100
.mu.L of methyl iodide with 100 .mu.L of p-chlorobenzyl chloride or
p-fluorobenzyl chloride, as appropriate, and replacing GMP with GDP
when appropriate. See Jankowska et al. (1993).
[0092] Another possible modification is a methyl or other
substitution at the N.sup.2 position: ##STR6##
[0093] where, R may, for example, be H, CH.sub.3,
CH.sub.2C.sub.6H.sub.5, CH.sub.2COC.sub.6H.sub.5,
CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH.dbd.CH.sub.2, or another
substituent, such as C.sub.1 to C.sub.4 substituted or
unsubstituted alkyl, or C.sub.6 to C.sub.8 substituted or
unsubstituted aryl. Such modifications may, for example, be made at
the beginning of the synthetic route, in the initial synthesis of
the nucleoside, prior to carrying out the other steps of the
synthesis. For example, N.sup.2,3'-O-dimethylguanosine
5'-monophosphate may be obtained by a procedure analogous to that
for Compound 3, but instead starting with
N.sup.2,3'-O-dimethylguanosine, which may be prepared by
introduction at the beginning of methyl groups into the N.sup.2
position of guanosine by the method of J. Boryski et al.,
Nucleosides Nucleotides, vol. 4, pp. 595 ff (1985); or Sekine et
al., "A convenient method for the synthesis of
N.sup.2,N.sup.2-dimethylguanosine by reductive C--S bond cleavage
with tributyltin hydride," J. Org. Chem., vol. 56, pp. 1224-1227
(1991). See also J. Boryski, "Application of the
1,N-2-isopropenoguanosine system for synthesis of novel
N-2-substituted derivatives of guanosine and acyclovir," Coll.
Czech. Chem. Commun., vol. 55 (special issue), pp. 85-88
(1990).
[0094] Still more generally, compounds in accordance with the
present invention will include structures such as the following:
##STR7## wherein the substituents R, X, Y, and Z are as previously
described, and the moiety B is selected from the group consisting
of ##STR8##
[0095] Miscellaneous
[0096] The complete disclosures of all references cited in this
specification are hereby incorporated by reference. Also
incorporated by reference are the complete disclosures of the
following publications of the inventors' own work, which are not
prior art to the present application: J. Stepinski et al.,
"Synthesis and properties of mRNAs containing the novel
`anti-reverse` cap analogs 7-methyl(3'-O-methyl)GpppG and
7-methyl(3'-deoxy)GpppG," RNA, vol. 7, pp. 1486-1495 (2001); E.
Darzynkiewicz et al., "New `anti-reverse` 5'-mRNA dinucleotide cap
analogues (ARCA)," Abstract POTH-035, 27th Meeting of the
Federation of European Biochemical Societies (Lisbon, Portugal,
Jun. 30-Jul. 5, 2001); J. Stepinski et al., "Synthesis and
properties of `anti-reverse` cap analogues," Abstract, 6th Meeting
of the RNA Society (Banff, Canada, May 29-Jun. 3, 2001); and J.
Stepinski et al., "Preparation and properties of mRNAs capped with
the novel `anti-reverse` dinucleotide cap analogues," Abstract
P-13, 4th West Coast Meeting on mRNA Stability and Translation
(Seattle, Wash., Oct. 14-16, 2001). In the event of an otherwise
irreconcilable conflict, however, the present specification shall
control.
* * * * *