chemistry - berry & assoc · 2012-11-09 · into the chemistry of nucleic acids, resulting in a...

Chemistry

Berry & Associates • 2434 Bishop Circle East • Dexter, Michigan • 48130 • 800-357-1145 • www.berryassoc.com

CH

EMISTR

Yfrom

Berry &

Asso

ciates|V

olu

me 1

, Issue 1

|Win

ter 20

06

|A New Dark Quencher | Oligonucleotide Purification | N ew

Phosphoramidites

Founded in 1989 with roots

in the nucleoside field, Berry

& Associates soon moved

into the chemistry of nucleic

acids, resulting in a current

portfolio of more than 130

phosphoramidites and solid

phase-linked monomers for

oligonucleotide synthesis as

well as hundreds of nucleosides,

nucleotides, carbohydrates,

spacers, fluorescent markers,

quenchers, and heterocycles–all

proudly made at our facility just

outside of Ann Arbor, Michigan.

Although our company is small,

the credentials of our highly

trained staff of chemists include

over 400 publications and 80

patents in synthetic organic

and medicinal chemistry. High

quality chemicals, timeliness,

and personalized service are the

hallmarks of Berry & Associates.

In This Issue

A New Dark Quencher

Oligonucleotide PurificationOligonucleotide Purification

New Phosphoramidites:

– Zebularine CEP

– 2'-Deoxypseudoisocytidine CEP

– dF CEP

from Berry & Associates Volume 1, Issue 1 | Winter 2006

While in the excited state, fluorophores hile in the excited state, fluorophores are sensitive to their environment are sensitive to their environment

and may lose excitation energy by several and may lose excitation energy by several processes besides emission of a fluorescence processes besides emission of a fluorescence photon. Such fluorescence quenching can occur by collision or molecular motion (dynamic quenching), excited state reaction with other molecules (photobleaching), contact quenching (the formation of a non-fluorescent ground state complex, also known as static quenching), or energy transfer to another molecule (fluorescence resonance energy transfer, or FRET). Many nucleic acid fluorescence detection techniques use probes that bear both a fluorophore and a quencher, relying on FRET- or contact-mode quenching to diminish fluorescence until a hybridization event occurs. Upon hybridization, the fluoro-phore and quencher are separated in space, resulting in an increase in fluorescence. The efficiency of FRET from the fluorophore to the quencher (i.e., the magnitude of quench-ing) depends on the relative orientation of their transition dipoles, the distance between them, and how well the absorption spec-trum of the quencher overlaps the emission spectrum of the fluorophore. The efficiency of contact quenching depends on the ability of the fluorophore and quencher to form a ground-state complex, and correlates with the mutual affinity of the two species and the distance between them. Quenchers may themselves be fluorescent, emitting a photon at a longer wavelength than the acceptor fluo-rophore. More conveniently, dark quenchers can be used, which are non-fluorescent chro-

mophores that, in FRET mode, can absorb energy from the excited state of the fluoro-phore, preventing emission of a fluorescence photon. The resultant excited state of a dark quencher relaxes to the ground state by radiationless decay (heat). In contact mode, the dark quencher forms a non-fluorescent ground-state complex with the fluorophore, masking its fluorescence until the complex is disrupted by a hybridization event.

The archetypal dark quencher is dabcyl, which is often installed at the 3'-terminus of an oligonucleotide probe and is useful for quenching relatively short wavelength fluoro-phores such as carboxyfluorescein (FAM). Its ability to quench longer wavelength fluoro-phores is limited by its absorption spectrum,

KEY FEATURES

■ Excellent quencher of long-wavelength fluorescence in both FRET and contact modes.

■ λmax ~ 650 nm; useful absorbance between 550 and 750 nm.

■ Ammonia stable; compatible with standard oligonucleotide synthesis chemistry.

■ Internal, 3', or 5' installation.

BlackBerry™ Quencher 650A new dark quencher of fluorescenceA new dark quencher of fluorescence

Continued on page 2

which tails off in the range where these fluorophores emit. The advent of high-throughput multiplex assays involving the simultaneous use of fluorophores of different colors has fostered the development of dark quenchers that can quench fluorescence at longer wave-lengths. A desirable characteristic of such quenchers is a long-wavelength absorption maximum. Unfortunately, the chemical stability of such compounds is often diminished. Extended π systems and/or the use of stronger donor-acceptor functional group pairings can lead to sensitivity to oligonucleotide synthesis reagents, e.g., oxidants such as iodine and deblocking agents such as ammonia and AMA.

Berry & Associates now introduces BlackBerry Quencher 650 (BBQ-650), a synthesis-stable dark quencher of long wavelength fluorescence (Figure 1). An 8-alkoxyjulolidine moiety was found to be a power-ful π-electron donor, affording a surprising bathochromic shift when compared to related compounds. The absorp-tion spectrum of a BBQ-650-tagged oligonucleotide

is shown in Figure 2. The broad absorbance centered around 650 nm effectively overlaps the emission max-ima of popular long-wavelength fluorophores such as Cy3, TAMRA, Texas Red, ROX, Cy5, and Cy5.5, allowing efficient quenching.

BlackBerry Quenchers may be installed at the 3' ter-minus, internally, or at the 5' position using the reagents shown in Figure 3. Standard phosphoramidites and syn-thesis protocols may be employed with both reagents except that BBQ-650-dT CEP should be dissolved in 4:1 dichloromethane-acetonitrile and coupled for 15 min. Significantly, nucleobase deprotection may be performed with ammonium hydroxide or AMA without degradation of the quencher, e.g., ammonium hydroxide, 12 h, 55 °C.

To evaluate BlackBerry Quencher 650 in contact quenching mode, molecular beacon probes bearing vari-ous 5'-fluorophores (FAM, Cy3, Texas Red, Cy5, Cy5.5) were synthesized using 3'-BBQ-650 CPG. Signal-to-background ratios upon binding to fully-complemen-tary target were excellent, e.g., >90 with Cy5 and >88 with Cy5.5. Melting temperatures showed that BBQ-650 had a particularly high affinity for Cy dyes; labeled stem hybrids melted 11-14 °C higher than non-labeled stem hybrids. The probes were successful in typing C to T transitions at positions 627 and 630 of the human che-mokine receptor 5 gene,1 and produced excellent results in real-time PCR studies.

BlackBerry Quencher 650 was also found to be an excellent FRET-mode quencher. Pairs of complemen-

BlackBerry™ Quencher 650Continued from front page

Figure 2. Absorption spectrum of a 15-mer bearing a 3' BlackBerry Quencher 650.

Figure 1. BlackBerry Quencher 650 (BBQ-650).

Figure 3. Reagents for the incorporation of BlackBerry Quencher 650 into oligonucleotides.

CHEMISTRY FROM BERRY & ASSOCIATES | www.berryassoc.com 2

The fluorous affinity purification of oligonucleotides is a quick

and simple affinity-based method for the purification of oligonucle-otides that relies on the strong interaction of fluorous-tagged oli-gonucleotides (made with fluorous-tagged phosphoramidites, Figure 4) with the fluorinated adsorbent present in Fluoro-Pak™ columns.1

Fluorous affinity purification is operationally similar to DMT-on purification using a reverse-phase (RP) adsorbent, e.g. RP cartridge purification, except that it involves a much stronger affinity interac-tion and is thus able to afford higher selectivities and recoveries, even with longmers. DMT-on RP cartridge purification is limited to relatively short oligonucleotides, typically ≤30-40-mers, since the relative hydrophobic contribution of the lipophilic DMT group dimin-ishes as the chain length increases, resulting in lower overall yields and a diminished selectivity of the

adsorbent for the desired oligonu-cleotide over failure sequences.

Highly fluorinated organic compounds are both hydrophobic and lipophobic, preferring instead to associate with other fluorinated substances.2 For example, perfluoro-hexane is insoluble in both water and hexane. Organic molecules that have both an organic domain (e.g. an oligonucleotide) and a perfluoroalkyl domain (e.g., a lin-ear perfluoroalkyl “ponytail”) are known as fluorous molecules, and may be separated from non-fluo-rous molecules by interaction with fluorinated separation media such as Fluoro-Pak columns. Fluorous-fluorous interactions are strong and selective. Fluorous affinity interac-tions are well-documented in the chemistry literature, and are now finding application in more biologi-cal areas, e.g., proteomics3 and now oligonucleotides.1,4

The fluorous affinity purifica-tion of oligonucleotides is similar to

DMT-on RP cartridge purification, and we have developed protocols that are as “plug-and-play” as pos-sible.5 The first step is to install a single nucleotide at the 5'-termi-nus of the oligonucleotide using a fluorous-tagged phosphoramidite (Figure 4). The fluorous tag takes the form of a fluorous DMT group (“FDMT” group), where a fluorous

Fluorous Affinity Purification of OligonucleotidesA higher affinity alternative to RP cartridge purification

tary strands were designed that would bring a 5'-Cy5.5 fluorophore to within 5 or 10 base pairs (20-40 Å) of a 3'-BBQ-650 upon hybridization,2 where quenching efficiencies of ≥98.3 and ≥98.9%, respectively, were observed. Melting temperatures of these hybrids were unchanged from non-labled hybrids, showing that these quenching efficiencies were due to FRET quenching and not contact quenching.

Acknowledgement: We thank Dr. Salvatore A. E. Marras for helpful discussions and for probe studies involving BlackBerry quenchers.

References

(1) Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Methods in Molecular Biology 2003, 212, 111-128.

(2) Marras, S. A. E.; Kramer, F. R.; Tyagi, S. Nucleic Acids Res. 2002, 30, e122.

KEY FEATURES

■ A higher affinity alternative to RP cartridge purification.

■ One-pass loading without ammonia removal.

■ High recoveries (typically 70-100%).

■ High selectivity for removal of failure sequences, even with long oligonucleotides.

■ Excellent for longer oligonucleotides (e.g. 50-100+ mers); recoveries nearly quantitative.

■ Does not require new techniques.

BlackBerry Quencher Products — Ordering Information

Item Catalog No. Size/pack Price (USD)

3'-BBQ-650 CPG BL 2010

200 nmol columns (pkg of 4)* $75.00

1 µmol columns (pkg of 4)* $210.00

100 mg $135.00

1 g $1050.00

BBQ-650-dT CEP BL 1010

50 µmol $150.00

100 µmol $275.00

0.25 g $650.00

5'-BBQ-650 CEP BL 1020 coming soonBL 1020 coming soon

BBQ-650 NHS ester BL 3010

5 mg $95.00

25 mg $375.00

* Add “-E” to part number for Expedite columns, “-A” for ABI-style crimp columns

Continued on page 4

3

Chemistry from Berry & Associates

CHEMISTRY FROM BERRY & ASSOCIATES | www.berryassoc.com

ponytail is attached via an ethylene spacer to a normal DMT group. The FDMT group is designed so that it behaves just like a DMT group: the rate of detritylation is very similar to that observed for the DMT group and the absorbance maximum of FDMT cation (504 nm) is identi-cal to DMT cation. Please note that only one coupling of a 5'-O-FDMT phosphoramidite is required; nor-mal DMT amidites are used for the earlier steps in the synthesis. The synthesis is run in DMT-on mode, leaving the FDMT group in place. FDMT-bearing amidites are entirely soluble in acetonitrile and couple normally.

The FDMT-on oligonucleotide is cleaved from the solid support

as usual and the base protecting groups are removed according to standard methods. If ammonia is used, it is not necessary to evaporate it, since Fluoro-Pak columns use a pH-stable polymeric matrix. HPLC analysis of a crude 75-mer mixture shows that the fluorous-tagged oligonucleotide is strongly retained, even on an RP-HPLC column (Figure 5).

The crude deprotection solu-tion is diluted with a salt-contain-ing loading buffer and applied to a Fluoro-Pak column. Binding of the FDMT-tagged oligonucleotide occurs in one pass, leaving most of the failure sequences unbound. Washing with 10% acetonitrile in 0.1 M TEAA removes the rest of the failures. Additional failure washes are unnecessary, but they reveal that the fluorous-tagged oligonucleotide is still retained; no leaching from the column is observed, even with 100-mers. Such selectivity is unprec-edented with DMT-on cartridge purification. On-column detrityla-tion with TFA followed by elution of the purified oligonucleotide is then carried out. As an example, fluorous-tagged mixed-base 75-mers (200 nmol scale) were purified by fluorous affinity purification to provide 9-11 A260 units of the fully deprotected material, free from failure sequences. This represented

a nearly quantitative recovery of the available FDMT-tagged oligonucle-otides present in the crude ammo-nia deblock solutions (estimated by HPLC). A representative RP-HPLC chromatogram of a fluorous-puri-fied 75-mer is shown in Figure 6 (compare to the crude mixture in Figure 5). The method was extended to 100-mers, which proceeded with 76-100% recovery of the available tagged oligonucleotides, again free of failure sequences.

Fluoro-Pak™ and Fluoro-Pak™ II Columns

Our Fluoro-Pak adsorbent has fluorinated organic groups bound to a pH-stable polymeric resin and is ideal for the purification of fluorous-tagged oligonucleotides. Multiple pore and particle sizes have been evaluated in order to provide optimal performance with reasonable back-pressure. Flow may be induced by pressure or vacuum. Fluoro-Pak columns should be used with fluorous-tagged oligo-nucleotides; they are not designed for normal DMT-on purifications. Further, Fluoro-Pak columns were designed specifically for the fluorous purification of oligonucle-otides and have not been validated for other uses, although they may provide a good alternative to tradi-tional silica-based fluorous adsor-

Fluorous Affinity Purification of OligonucleotidesContinued from page 3

Figure 5. HPLC analysis of a crude 75-mer, showing that the fluorous-tagged oligonucleotide is strongly retained. Waters Spherisorb ODS-2 (5 µm, 4.6 x 150 mm, 1 mL/min), mobile phase A = 0.1 M aqueous TEAA; mobile phase B = acetonitrile.

Figure 6. HPLC analysis of a fluorous-purified 75-mer. See Figure 5 for HPLC conditions.

Figure 4. Fluorous phosphoramidites featuring a fluorous dimethoxytrityl (FDMT) group.


bents. Two columns are available: Fluoro-Pak Columns, containing 75 mg of adsorbent, useful for up to 0.2 micro mole purifications, and Fluoro-Pak II Columns, containing 150 mg of adsorbent, useful for 1 micromole purifications.

Fluorous versions of other nucleic acid synthesis reagents are now being developed. The newest addition to our lineup is Fluorous Chemical Phosphorylation Reagent II (F-CPR-II CEP, Figure 7), the fluorous version of CPR-II.6 This reagent allows a combination of fluorous affinity purification with 5'-phosphorylation, which is espe-cially attractive when synthesizing longmers that will be used in ligase reactions. Further, if a 5'-phosphate is tolerated in a particular applica-tion, FCPR-II may be used as a common handle for fluorous purifi-cation, obviating the need to choose a different fluorous phosphorami-dite for each different 5'-terminal nucleotide. F-CPR-II CEP is used in the same way as CPR-II,6 i.e.,

a 6 minute coupling with no cap-ping. The final retro-aldol cleavage to afford the free 5'-phosphorylated oligonucleotide is achieved in the same fashion as for CPR-II.

Detailed information on the fluo-rous purification method may be found in our booklet “User Guide: Fluorous Affinity Purification of Oligonucleotides,” which is included Oligonucleotides,” which is included Oligonucleotides,”with each order and may also be downloaded at www.berryassoc.com.5

References

(1) Pearson, W. H.; Berry, D. A.; Stoy, P.; Jung, K.-Y.; Sercel, A. D. J. Org. Chem. 2005, 70, 7114-7122.

(2) Handbook of Fluorous Chemistry; Gladysz, Handbook of Fluorous Chemistry; Gladysz, Handbook of Fluorous Chemistry;J. A.; Curran, D. P.; Horváth, I. T., Eds.; Wiley-VCH: Weinheim, 2004.

(3) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nature Biotechnol. 2005, 23, 463-468.

(4) For related work, see: (a) Beller, C.; Bannwarth, W. Helv. Chim. Acta 2005, 88, 171-179 and (b) Tripathi, S.; Misra, K.; Sanghvi, Y. S. Org. Prep. Proc. Int. 2005, 37, 257-263.37, 257-263.37

(5) User Guide: Fluorous Affinity Purification of Oligonucleotides, 2005, Berry & Associates, Inc., http://www.berryassoc.com/literature/fluorousguide.pdf

(6) (a) Guzaev, A.; Salo, H.; Azhayev, A.; Lönnberg, H. Tetrahedron 1995, 51, 9375-9384. (b) Guzaev, S.; Azhayev, A.; Lonnberg, H., U. S. Patent 5,959,090, 1999. Assignee: Glen Research Corporation.

Figure 7. Fluorous Chemical Phosphorylation Reagent II (FCPR-II).

Fluorous Affinity Purification Products - Ordering Information*


Phosphoramidites

FDMT-N6N6N -Bz-dA CEP FL 1000

100 µmol $47.50

250 µmol $107.50

500 µmol $197.50

FDMT-N4N4N -Bz-dC CEP FL 1100

100 µmol $47.50

250 µmol $107.50

500 µmol $197.50

FDMT-N2N2N -iBu-dG CEP FL 1200

100 µmol $47.50

250 µmol $107.50

500 µmol $197.50

FDMT-T CEP FL 1300

100 µmol $47.50

250 µmol $97.50

500 µmol $177.50

F-CPR-II CEP FL 1360

100 µmol $75.00

0.25 g $250.00

Columns

Fluoro-Pak™ columns FP 7210 Pack of 10 $69.50

FP-7210 A (with Luer adaptor) Pack of 10 $69.50

(75 mg adsorbent for ≤200 nmol syntheses)

Fluoro-Pak™ II columns FP7220 Pack of 10 $95.50

FP 7220-A (with Luer adaptor) Pack of 10 $95.50

(150 mg adsorbent, for ≤1 µmol syntheses)

Loading Buffer LB 7100 200 mL $24.50

* Three items are required: (i) One or more fluorous phosphoramidites (ii) Fluoro-Pak columns (iii) Loading Buffer

5



The C-nucleoside 2'-deoxypseudo-isocytidine (ψ-iso-dC) is an iso-

stere of dC that offers an additional hydrogen-bond donor at N3. Interest in the incorporation of ψ-iso-dC and related compounds into oligo-nucleotides has grown due to their success in improving triplex forma-tion between oligonucleotides and duplex DNA.1-3 The C • GC pyrimi-dine-purine-pyrimidine binding motif requires acidic conditions in order to protonate N3 of cytosine in the Hoogsteen strand (i.e., C+ • GC, Figure 8). Unfortunately, this inter-action is disrupted under the higher pH of physiological conditions. Improvements have been observed using 5-methyl-2'-deoxycytidine in place of dC, although protonation of N3 is still required.4 Kan and co-workers1-3 have examined the use of 2'-deoxypseudoisocytidine and 2'-O-methylpseudoisocytidine as neutral replacements for protonated cytidine (ψ-iso-dC • GC, Figure 8). Indeed, oligonucleotides containing these cytidine replacements form improved triplexes under neutral conditions.

We now offer 2'-Deoxy pseudo-isocytidine CEP (ψ-iso-dC CEP, Figure 9)3,5 for incorporation of ψ-iso-dC into oligonucleotides, allowing researchers to explore the alternate hydrogen-bonding motif afforded by this interesting dC sur-rogate. For those interested in the nucleoside, we offer both pseudoiso-cytidine and 2'-deoxypseudoisocyti-dine (see Ordering Information).

dF CEP

Nucleosides bearing a pyrido [2,3-d]pyrimidine-2,7(8H)-H)-H

dione base, known variously as “dF”, “F”, or “P”, have been prepared and

found to be fluorescent (Figure 10).6,7 The ribofuranosyl version fluoresces at 385 nm. The dF het-erocycle offers interesting alternate base pairing schemes, since evidence suggests that two tautomers may be involved.8,9

When incorporated into oligo-nucleotides, dF was found to pair selectively with G and A in double-helical DNA (G > A >> C or T), resulting in higher and lower melt-ing temperatures, respectively, than

the corresponding C-G and T-A pairs.10 It has been proposed that dF forms a Watson-Crick base pair with G much as C does, but can, via a backbone shift, form a wobble base pair with A (Figure 11).10

Alternatively, dF may hydrogen bond with A via an alternate tauto-mer as shown.8,9 In triple helices, dF recognizes A-T base pairs with high selectivity.8,9 It can participate in parallel as well as antiparallel triplex formation, perhaps due to

Featured Phosphoramidites:2'-Deoxypseudoisocytidine CEP (ψ2'-Deoxypseudoisocytidine CEP (ψ2'-Deoxypseudoisocytidine CEP ( -iso-dC)

Figure 8. Comparison of pyrimidine-purine-pyrimidine binding motifs for triplex formation involving cationic 2'-deoxycytidine (C+) and neutral 2'-deoxypseudoisocytidine (ψ-iso-dC) nucleotides.

Figure 9. Pseudoisocytosine-based products.


the availability of both heterocycle tautomers.9 It has been proposed that triplex-forming oligonucleo-tides bearing dF may be useful for measuring triplex formation by quenching of the fluorescence of the heterocycle.9

Oligonucleotides containing dF nucleotides may be prepared using the phosphodiester10 or phosphora-midite solid-phase methods.8,9 We now offer the phosphoramidite dF CEP (Figure 12). Standard synthe-sis protocols and reagents may be employed, except that it has been suggested that dmf-dG CEP and room temperature deprotection with 30% NH4OH should be used to minimize potential degradation of the dF heterocycle.9 The nucleoside dF is also available.

Zebularine CEP

In recognition of the increasing importance of oligoribonucleo-

tides, we are expanding our collec-

tion of reagents for RNA synthesis. Our latest addition is Zebularine CEP (Figure 13).

Zebularine (a.k.a. 2-pyrim-idinone-1-β-D-riboside or 4HC) is similar to cytidine except that it lacks a C4-amino group and thus makes one less hydrogen bond to G. Incorporation of zebularine into RNA strands may be accomplished with Zebularine CEP using standard phosphoramidite chemistry.11,12

Substitution of zebularine for cyti-dine amounts to “atomic mutagen-esis”,12 allowing studies of the role of a particular hydrogen bond in RNA duplexes.11-13 Replacing various cytidine residues in a hammerhead ribozyme with zebularine proved to be an effective probe of structure vs. catalytic activity, supporting the assignment of the proposed catalytically-active magnesium ion binding site.11,13 Atomic muta-genesis at the G1:C72 base pair in class II Escherichia coli alanyl-tRNA Escherichia coli alanyl-tRNA Escherichia coli

2'-Deoxypseudoisocytidine CEP and Related Products - Ordering Information


2'-Deoxypseudoisocytidine CEP BA 0236

(ψ-iso-dC CEP) 100 µmol $425.00

0.25 g $1,125.00

2'-Deoxypseudoisocytidine PYA 11005

(ψ-iso-dC) 10 mg $115.00

100 mg $595.00

Pseudoisocytidine • HCl PYA 11060

(ψ-iso-C) 5 mg $46.50

10 mg $78.00

100 mg $295.00

dF CEP - Ordering Information


dF CEP BA 0238

50 µmol $395.00

0.25 g $975.00

dF (nucleoside only) PYA 11100

10 mg $95.00

100 mg $245.50

Zebularine CEP - Ordering Information


Zebularine CEP BA 0254

100 µmol $217.50

0.25 g $495.00

Continued on back page

Figure 10. Base-pairing schemes offered by dF. A = hydrogen-bond acceptor, D = hydrogen-bond donor.

Figure 11. Pyrido[2,3-d]pyrimidine-2,7(8H)-dione nucleosides are fluorescent and may act as C or A replacements in double- or triple-helical DNA.

Figure 12. dF products.

Figure 13. Zebularine CEP for incorporation of 2-pyrimidinone-1-β-D-riboside (a.k.a., 4HC) into oligonucle-otides.

7



In This Issue

A New Dark Quencher

Oligonucleotide Purification

New Phosphoramidites:

– Zebularine CEP

– 2'-Deoxypseudoisocytidine CEP

– dF CEP

2434 Bishop Circle East, Dexter, MI 48130 USA

Phone 734-426-3787 • Toll Free 800-357-1145 • Fax 734-426-9077

www.berryassoc.com | [email protected] | [email protected]

“BlackBerry” and “Fluoro-Pak” are trademarks of Berry & Associates, Inc.

“Texas Red” is a registered trademark of Molecular Probes. “Cy,” “Cy5,” and “Cy5.5” are trademarks of Amersham Biosciences Ltd. “FAM,” “ROX,” and “TAMRA” are trademarks of Applied Biosystems, Inc.

Products for fluorous affinity purification of oligonucleotides: Patents applied for, Berry & Associates, Inc. Further, the use of these products is licensed under U.S. Patents 6,673,539, 6,156,896, 5,859,247, and 5,777,121 and one or more pending patents owned or controlled by Fluorous Technologies, Inc.

BlackBerry Quencher technology: Patents applied for, Berry & Associates, Inc.

(1) Ono, A.; Ts’o, P. O. P.; Kan, L. J. Org. Chem. 1992, 57, 3225-3230.57, 3225-3230.57

(2) Ono, A.; Ts’o, P. O. P.; Kan, L. J. Am. Chem. Soc. 1991, 113, 4032-4033.

(3) Chin, T.-M.; Lin, S.-B.; Lee, S.-Y.; Chang, M.-L.; Cheng, A. Y.-Y.; Chang, F.-C.; Pasternack, L.; Huang, D.-H.; Kan, L.-S. Biochemistry 2000, 39, 12457-12464.

(4) Singleton, D. F.; Dervan, P. B. Biochemistry1992, 31, 10995-11003.

(5) For an alternate phosphoramidite for ψ-iso-dC incorporation, see: Mayer, A.; Leumann, C. J. Nucleosides, Nucleotides & Nucleic Acids 2003, 22, 1919-1925.

(6) Bergstrom, D. E.; Inoue, H.; Reddy, P. A. J. Org. Chem. 1982, 47, 2174-2178.47, 2174-2178.47

(7) Bergstrom, D.; Lin, X.; Wang, G.; Rotstein, D.; Beal, P.; Norrix, K.; Ruth, J. Synlett 1992, 179-188.

(8) Staubli, A. B.; Dervan, P. B. Nucleic Acids Res. 1994, 22, 2637-2642.

(9) Durland, R. H.; Rao, T. S.; Jayaraman, K.; Revankar, G. R. Bioconjugate Chem. 1995, 6, 6, 6278-282.

(10) Inoue, H.; Imura, A.; Ohtsuka, E. Nucleic Acids Res. 1985, 13, 7119-7128.

(11) Adams, C. J.; Murray, J. B.; Arnold, J. R. P.; Stockley, P. G. Tetrahedron Lett 1994, 35, 1597-1600.

(12) Beuning, P. J.; Gulotta, M.; Musier-Forsyth, K. J. Am. Chem. Soc. 1997, 119, 8397-8402.

(13) Murray, J. B.; Adams, C. J.; Arnold, J. R. P.; Stockley, P. G. Biochem. J. 1995, 311, 487-494.

synthetase using various G and C analogs (zebularine, 7-deaza-dG, inosine, 2-aminopurine) allowed dissection of the roles of various hydrogen bonds in the major and minor grooves, revealing the nature of the high degree of specificity afforded by major groove interactions at the end of the helix.12 Finally, it should be noted that zebularine is fluorescent. Excitation of a zebularine-containing ribozyme at 298 nm caused emission at 367 nm. Upon annealing with a complementary strand, a small increase in fluorescence intensity (10%) was observed with no change in emission wavelength.11

Featured PhosphoramiditesContinued from page 7

2434 Bishop Circle EastDexter, MI 48130 USA

Featured Phosphoramidites: References

Berry & Associates, Inc.

chemistry - berry & assoc · 2012-11-09 · into the chemistry of nucleic acids, resulting in a...

Documents