REDMOND RED™AND YAKIMA YELLOW™DYES, AND ECLIPSE™ …€¦ · maximum for Eclipse Quencher is...

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R E D M O N D R E D ™ A N D Y A K I M A Y E L L O W ™ D Y E S , A N DE C L I P S E ™ N O N - F L U O R E S C E N T Q U E N C H E R

V O L U M E 1 5

N U M B E R 1

M A R C H 2 0 0 2

NEW DYES, QUENCHERS

3'-PT-AMINO-MODIFIER

PYRROLO-dC

NOVEL MONOMERS

CARBOXY-MODIFIER

Glen Research is happy to confirm our agreementwith Epoch Biosciences, Inc., announced on February6, 2002, to distribute several of Epoch’s proprietaryproducts designed for the synthesis of novel DNAprobes. Initially we will provide products based onEpoch’s Redmond Red™ and Yakima Yellow™fluorophores and Eclipse™ non-fluorescentquencher, which will be described in detail in thisarticle. We will also supply PPG, a modifiednucleoside, which is covered in the article on Page8 of this newsletter. It is a pleasure for us to expandour relationship with Epoch by helping to providebroad access for these compounds to researchmarkets worldwide.

As part of the preamble to this article, it is instructiveto quote from the Epoch press release, whichpositions these products perfectly. They note thatthe products covered by this agreement are asfollows:

“all are innovative components for DNA probes, theworkhorses of genetic analysis. Probes hybridize orbind to target DNA and then provide a signal sothat the target can be detected. Many probes alsocarry a quencher that masks the signal until thebinding has occurred. Redmond Red and YakimaYellow are fluorescent dye tags that can beincorporated into synthetic DNA molecules to allowprobe detection on a variety of platforms. The EclipseQuencher is a new non-fluorescent quencher thatallows DNA detection probes to be used for real timePCR applications such as measuring gene expressionor detecting single nucleotide polymorphisms(SNPs). The PPG modified base is the first in a seriesof modified bases that Epoch has developed thatallow DNA probes to be designed for regions ofgenomes that cannot be interrogated by probescontaining normal nucleotide bases.”

Epoch Fluorophores and Quencher

The use of fluorescent tags as an alternative toradiolabels in DNA probes and primers hasblossomed over the years. Fluorescence is safelymeasured with inexpensive instrumentation and itis very straightforward to multiplex assays forexceptionally high throughput. Molecular beaconand fluorescence resonance energy transfer (FRET)probes can be used in assays, which can be carriedout in closed tube formats with less sample handlingat higher throughput. These probes are also suitablefor use in techniques that include amplification ofthe target DNA.

Molecular beacon and FRET probes require efficientquenching until the probe is hybridized to the target.Molecular beacon probes are hairpin structureswherein the fluorescence is quenched by theproximity of the fluorophore to the quenchermolecule. When the probe hybridizes to the target,it becomes linear, quenching is disrupted, and theprobe fluoresces ready for detection. In FRET assays,when the probe is hybridized to the target, it is

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FIGURE 1: STRUCTURES OF EPOCH DYES AND QUENCHER

FIGURE 2: ABSORPTION AND EMISSION SPECTRA OF REDMOND RED AND YAKIMA YELLOW

(Continued from Front Page)

digested by nuclease activity in thepolymerase being used for amplification oftarget copies. The fluorophore, releasedfrom the target and separated from thequencher, is now highly fluorescent andready for detection. Fluorophore/quencherpairs can be chosen based on spectralproperties – the emission of the fluorophoreshould overlap the absorption of thequencher. The quencher may absorb itspartner’s fluorescence and emit thefluorescence at a new wavelength or, in thecase of a non-fluorescent quencher, as heat.

The new Epoch products offer two newfluorescent dyes, available immediately asphosphoramidites and supports, as shownin Figure 1. Their absorbance and emissioncharacteristics are shown in Figure 2.Yakima Yellow has an absorption maximumat 530 nm and emission maximum at 549nm, while Redmond Red’s absorption andemission maxima are at 579 nm and 595nm, respectively.

Over the years, the dabcyl molecule hasproved to be an excellent universal non-fluorescent (dark) quencher for molecularbeacon probes. In addition, dabcyl is a verystable molecule and synthesis of doubly-labelled probes containing a dabcylquencher is quite straightforward. Themechanism of quenching relies on the closeproximity of the fluorophore to the dabcylgroup, generally called static quenching,which is independent of spectral overlapbetween fluorophore and quencher.However in FRET probes, dabcyl’s ability toact as a dark quencher is limited by itsabsorption spectrum to use with dyesemitting at 400 – 550 nm. Other fluorescentquenchers can be used to cover a broadspectrum of dyes, but synthesis of thedoubly-labelled probes is made much moredifficult by the lack of stability of some ofthe potential candidates to the conditionsof oligo deprotection. These probes are bestprepared in a two-step process requiringpost-synthesis conjugation of the seconddye, usually with purification problems.

The Eclipse Quencher from Epoch solvesmost of the problems inherent in thesynthesis of molecular beacon and FRETprobes. The Eclipse molecule is highly stableand can be used safely in all common oligodeprotection schemes. The absorption

O

N

OO DMT

O P N

O

CH 2CH 2CNN

O

O O

Cl

Cl

O

O

Cl

ClCH 3

O

OOO

O

CH 2CH 2CN

O

NPO

O

NH

OCH 3

N

N

N

ClO2N

N CH 2CH 2CN

O

NPO

DMTO

NH

O

O

CPG

OO

O

N

O

DMTOO

N

O

O

Cl

Cl

Cl

ClCH 3

O

OO

O

O

ONH CPG

O

O

O

O

N

O DMT

OCH 3

N

N

N

ClO2N

O

O

O

NH CPG

N

DMTO

(1) Epoch Redmond Red Phosphoramidite

(2) Epoch Redmond Red CPG

(3) Epoch Yakima YellowPhosphoramidite

(4) Epoch Yakima Yellow CPG

(5) Epoch Eclipse QuencherPhosphoramidite

(6) Epoch Eclipse Quencher CPG

λmax Abs λmax Em Extinc. at max QYYakima Yellow 530.5 nm 549 nm 84,000 0.96Redmond Red 579 nm 595 nm 52,300 (pH 7.1) 0.84

74,000 (pH 9.1)

Measured in 0.1 M sodium phosphate. As indicated, Redmond Red is pH sensitive. YakimaYellow is not and has the same extinction coefficient at pH 7 as pH 9.

The QYs (quantum yield of fluorescence) were determined in 0.1 M Tris-HCl pH 7.4 relative toreference dyes using the dye-labeled T12 oligo. For Yakima Yellow, the reference dye wascarboxy tetramethylrhodamine in methanol. For Redmond Red, the reference dye wassulforhodamine 101 in EtOH.

Yakima Yellow Absorption Emission

Redmond Red Absorption Emission

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O R D E R I N G I N F O R M A T I O N

Item Catalog No. Pack Price($)

Epoch Redmond Red™ Phosphoramidite 10-5920-95 50 µm 205.0010-5920-90 100 µm 410.0010-5920-02 0.25g 995.00

Epoch Yakima Yellow™ Phosphoramidite 10-5921-95 50 µm 205.0010-5921-90 100 µm 410.0010-5921-02 0.25g 995.00

Epoch Eclipse™ Quencher Phosphoramidite 10-5925-95 50 µm 225.0010-5925-90 100 µm 450.0010-5925-02 0.25g 1125.00

Epoch Redmond Red™ CPG 20-5920-01 0.1g 180.0020-5920-10 1.0g 1500.00

1 µmole columns 20-5920-41 Pack of 4 300.000.2 µmole columns 20-5920-42 Pack of 4 150.0010 µmole column (ABI) 20-5920-13 Pack of 1 750.0015 µmole column (Expedite) 20-5920-14 Pack of 1 1125.00

Epoch Yakima Yellow™ CPG 20-5921-01 0.1g 180.0020-5921-10 1.0g 1500.00


Epoch Eclipse™ Quencher CPG 20-5925-01 0.1g 230.0020-5925-10 1.0g 1925.00


maximum for Eclipse Quencher is at 522 nm,compared to 479 nm for dabcyl. In addition,the structure of the Eclipse Quencher (Figure1) is substantially more electron deficientthan that of dabcyl and this leads to betterquenching over a wider range of dyes,especially those with emission maxima atlonger wavelength (red shifted) such asRedmond Red and Cy5. A simple experiment,using a molecular beacon probe, showedthat quenching of the fluorescence of Cy5was 53% more efficient with EclipseQuencher than with dabcyl. Epochresearchers conducted a comparison1 of thequenching ability of the Eclipse Quencherrelative to dabcyl in an enzymatic digestionassay designed to simulate the efficiency ofquenching of FRET probes. Over the rangeof fluorophores from FAM (520 nm) to Cy5(670 nm), Eclipse Quencher outperformeddabcyl in a measurement of signal tobackground ratio (signal, defined asfluorescence after digestion, divided bybackground, defined as initial fluorescence).The improvement was most marked for thehigher wavelength dyes. In addition, withan absorption range from 390 nm to 625nm (Figure 3), the Eclipse Quencher iscapable of effective performance in a widerange of colored FRET probes.

As is normal these days, the use of theseproducts is restricted and users should heedthe following qualification statement:

"These Products are for research purposes only,and may not be used for commercial, clinical,diagnostic or any other use. The Products aresubject to proprietary rights of Epoch Biosciences,Inc. and are made and sold under license fromEpoch Biosciences, Inc. There is no implied licensefor commercial use with respect to the Productsand a license must be obtained directly fromEpoch Biosciences, Inc. with respect to anyproposed commercial use of the Products.“Commercial use” includes but is not limited tothe sale, lease, license or other transfer of theProducts or any material derived or producedfrom them, the sale, lease, license or other grantof rights to use the Products or any materialderived or produced from them, or the use of theProducts to perform services for a fee for thirdparties (including contract research)."Redmond Red, Yakima Yellow, and Eclipse aretrademarks of Epoch Biosciences, Inc.

Reference:(1) E.A. Lukhtanov, M. Metcalf, and M.W.

Reed, American Biotechnology Laboratory,September, 2001.

Ì

FIGURE 3: ABSORPTION SPECTRUM OF 3'-ECLIPSE-LABELLED dT12 OLIGO

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4

A few months ago, we introduced the new3’-Amino-Modifier supports, shown inFigure 1, (1) and (2). In this alternativeapproach1, the nitrogen destined to becomethe 3’-amino group is included in aphthalimide (PT) group which is attached tothe support through an amide group on thearomatic ring. This simple linkage is verystable to all conditions of oligonucleotidesynthesis and contains no chiral center. Inthe last Glen Report2, we compared yieldsand purity of crude oligonucleotidesproduced with 3’-Amino-Modifier C7 CPG(3) and 3’-PT-Amino-Modifier C6 CPG (2).The results indicated that the yield ofproduct from the 3'-PT-Amino-Modifier C6CPG is about 20% lower if deprotected withammonium hydroxide. However, the purityof amino-modified product is significantlyhigher due to the absence of the acetylcapped product.

One of our main concerns about the PT-Amino supports was the efficiency of thecleavage reaction from the support byammonium hydroxide, which led to thelower yield of crude oligonucleotide. Also,we felt it was necessary to compare thecleavage efficiencies of the PT-Amino-Modifier supports under a variety of other,commonly used oligonucleotidedeprotection conditions, since a wide rangeof conditions has had to be developed forthe deprotection of modifiedoligonucleotides. For these supports tobecome universally accepted, they have tobe capable of use with the diverse group ofmodified bases and dyes we offer that hasvarious sensitivities to basic conditions.

A series of experiments was set up toexamine the products from 3'-PT-Amino-Modifier C3 and C6 CPG after deprotectionby a variety of popular techniques. As anexperimental control, the 3’-amino-modified oligonucleotides were compared toa thymidyl 20mer prepared from a standard,ester-linked, thymidine CPG.

Results

The experiments were carried out bysynthesizing a thymidyl 20mer on a 15umole scale on each of the followingsupports: 3'-PT-Amino-Modifier C3 and C6CPG, and T-CPG. The initial dimethoxytritylsolutions were collected so that the exact

COMPARISON OF DEPROTECTION METHODS FOR 3'-PT-AMINO-MODIFIER CPG

TABLE 1: COUPLING EFFICIENCIES OF TEST OLIGONUCLEOTIDES

starting scale could be determined, as wellas the coupling efficiencies, shown in Table1.

After synthesis, the supports were dried andcarefully weighed into 1 µmole scalealiquots in screw cap vials. Aliquots werethen subjected, in duplicate, to thedeprotection conditions shown in Table 2.The product was carefully isolated and theyield determined by absorbance at 260 nm.Table 2 shows the results of thesedeprotection experiments, averaged andnormalized to account for differences inactual amount deprotected and fordifferences in coupling efficiencies. Theresults are given as ratios to the highest

yielding deprotection of the thymidinesupport, which was the potassium carbonatein methanol method.

The products were analyzed by PAGE forpurity. The products all looked exceptionalwith little n-1 evident for any of thesyntheses. No evidence of any partiallydeprotected species, shown in Figure 2, wasobserved by mass spectral analyses.

Discussion

The results confirmed that the PT-Amino-Modifier supports do not fully cleave usingammonium hydroxide, although thedifferences of 10% to 20% seem hardly

Support Coupling Efficiency

Thymidine (1) 98.8%Thymidine (2) 98.7%3'-PT-Amino-Modifier C3 CPG (1) 97.4%3'-PT-Amino-Modifier C3 CPG (2) 97.8%3'-PT-Amino-Modifier C6 CPG (1) 96.9%3'-PT-Amino-Modifier C3 CPG (2) 97.8%

FIGURE 1: STRUCTURES OF 3'-AMINO-MODIFIERS

NNH

O

O

CPG

O

ODMT

NNH

O

O

ODMT

CPG

O

-succinyl-lcaa-CPG

FmocNHODMT

O

(3) 3’-Amino-Modifier C7 CPG

(2) 3’-PT-Amino-Modifier C6 CPG

(1) 3’-PT-Amino-Modifier C3 CPG

5

worth considering given the benefits ofhaving no additional chiral center and theabsence of N-acetylated products.2 Ingeneral, the various ammonium hydroxideconditions all yielded equal amounts ofamino labeled product, which was 80-90%of the yield from the thymidine support,after taking into account the slightly lowercoupling efficiencies.

Since neither of the potential partiallyprotected impurities shown in Figure 2 wasobserved, we can assume that the twophthalimidyl amide bonds cleave well beforethe linkage to the support. The aromaticamide link apparently does not cleave underthese conditions, or some of those sideproducts would be observed in the crudeproduct mix.

We were pleasantly surprised that theUltraFast system using AMA yielded higherresults than with the T-CPG. The fact thatthe longer deprotection gave even betterresults lends credence to the data. The 10-minute deprotection with AMA is anexcellent choice for the PT-Amino-Modifiersupport. It is convenient to prepare and wellsuited for today’s high throughputrequirements.

Unfortunately, but not too surprisingly,UltraMild deprotection conditions withpotassium carbonate in methanol yieldedconsiderably less product and should not beconsidered for use with the PT-Amino-Modifier supports.

In conclusion, we found that with oneexception, potassium carbonate inmethanol, the PT-Amino-Modifier supportscan be deprotected with any of the commondeprotection conditions with good results.

We are happy to acknowledge that this workwas carried out by our colleagues at TriLinkBioTechnologies, Inc. We thank RickHogrefe, Paul Imperial and David Colms forexpanding our knowledge of the PT-Amino-Modifier supports.

References:

(1) C.R. Petrie, M.W. Reed, A.D. Adams, andR.B. Meyer, Jr., Bioconjugate Chemistry,1992, 3, 85-7.

(2) Glen Report, 2001, 14.1, 5.

TABLE 2: DEPROTECTION CONDITIONS AND RESULTS

Method Temp Time PT-C3 PT-C6 T-20 Std

NH4OH RT 24 hrs 0.92 0.92 0.99NH4OH RT 48 hrs 0.80 0.87 1.00NH4OH 55°C 15 hrs 0.83 0.82 0.97NH4OH 65°C 4 hrs 0.72 0.79 0.963/1 NH4OH/EtOH RT 48 hrs 0.87 0.91 0.951/1 MeNH2/NH4OH (AMA) 55°C 10 min. 1.04 1.11 0.901/1 MeNH2/NH4OH (AMA) 55°C 15 hrs 1.13 1.18 0.980.4 M NaOH in 4/1 MeOH/H2O RT 15 hrs 0.97 1.00 0.90K2CO3 in MeOH RT 15 hrs 0.15 0.13 1.00

N

O

O

Oligonucleotide

O

HO

NH

O

O

Oligonucleotide

HOCO 2H

FIGURE 2: STRUCTURES OF POTENTIAL IMPURITIES



3'-PT-Amino-Modifier C3 CPG 20-2954-01 0.1g 95.0020-2954-10 1.0g 675.00


3'-PT-Amino-Modifier C6 CPG 20-2956-01 0.1g 95.0020-2956-10 1.0g 675.00


Potential impurity formed byhydrolysis of the amide linkage to

the support.

Further hydrolysis would yield thispotential di-carboxylic acid impurity.

Neither impurity was observed inoligonucleotide products.

6

Researchers have long been searching forfluorescent nucleosides to probe DNAstructure. Fluorescent nucleoside baseswould also be useful in the analysis of DNA-protein interactions.

Etheno-A (1) and etheno-C (2)1 are tworeadily accessible fluorescent structures butthese molecules are both non-hybridizing.Indeed, etheno-dA is a mutagenic nucleosideformed by the action of vinyl chloride onDNA. Other notable fluorescent baseanalogues are the pteridine nucleosideanalogues actively being investigated byPfleiderer, Hawkins and co-workers.Although very challenging from a syntheticstandpoint, these analogues hold greatpromise because of their substantialfluorescence, as well as their potential tohybridize specifically with natural bases. Themost promising analogue described to date2

is the adenosine analogue (3) but guanosineand other analogues have also beenintensively investigated.3-5 2-Aminopurine(4) is a mildly fluorescent base, which hasfound significant use in probing DNAstructures. It is especially useful in that itis capable of hybridizing with T (Figure 4)in Watson-Crick geometry.6

Two years ago, in our constant search fornucleosides with interesting fluorescenceproperties, we introduced furano-dT (7). Wehad no reason to believe that this moleculewould exhibit any base pairing propertiesand we expected it to act like etheno-dA inthat regard. Still, there is always a need fora novel fluorescent nucleoside to aid in DNAstructural analysis, so we launched it as aninteresting alternative fluorescent base.Before long, it became apparent that therewas more to this product than we initiallyobserved. We were aware that this base wasnot very stable to deprotection conditions,although normal ammonium hydroxidetreatment seemed to cause no problems.However, other techniques using potassiumcarbonate or sodium hydroxide causeddecomposition of the base, thereby losingits fluorescent properties. The nextindication we had that there were problemsinherent with this structure came when weexamined mass spectrometric data onoligonucleotides containing multipleadditions of furano-dT. Consistently, themolecular weights were 1 Dalton less peraddition of furano-dT than expected. When

PYRROLO-C - A NOVEL FLUORESCENT NUCLEOSIDE

we checked melting behavior ofoligonucleotides containing furano-dT, theanswer quickly became clear. Melting curvesgenerated using oligos containing “furano-dT” with complementary strands containingA, C, G or T opposite the “furano-dT” sitesclearly revealed the solution. “Furano-dT”did not hydrogen bond to A, C, or T, butformed a perfect base pair with G. Massspec and melting data were trying to tell usthat furano-dT had been transformed in theammonium hydroxide cleavage anddeprotection to the equivalent aminocompound, which we have chosen to callpyrrolo-dC (6) (Figure 1).

A literature search quickly generated furtherevidence that furano-dT is not stable to

ammonia, which converts it to theequivalent pyrrolo-pyrimidine, pyrrolo-dC.Interestingly, researchers at Epoch7 whosynthesized a pyrrolo-dC analogue wereexamining potential base pairs for selectivelybinding complementary (SBC) oligo-nucleotides. They did not note thefluorescent properties of the pyrrolo-pyrimidine ring system. However, they didobserve that their version of this ring system,different from pyrrolo-dC only in lacking themethyl group on the pyrrolo ring, formed amismatch with G and was quite unstable todeprotection conditions.

In the meantime, one group had alreadyapplied “furano-dT” to their researchendeavors. We were able to alert them to

FIGURE 1: STRUCTURES OF FLUORESCENT BASES

N

N

N

N

N NO

N

NN

N

N

NH2N

N

N

NH 2

N

N O

CH 3

N

NO

OCH 3

N

NO

HN CH 3

FIGURE 2: STRUCTURES OF FURAN0-dT AND PYRROLO-dC

O

O P N(iPr)2O CNEt

DMTON

N

O

HN

O

O P N(iPr)2O CNEt

DMTON

N

O

O

(1) Etheno-A (2) Etheno-C (3) 6MAP (4) 2-Aminopurine

(5) Furano-T (6) Pyrrolo-C

Ammonia

(7) Furano-dT-CEPhosphoramidite

(8) Pyrrolo-dC-CEPhosphoramidite

7

the transformation to pyrrolo-dC inammonia. In their publication8, they madeuse of the fact that the fluorescenceintensity of pyrrolo-dC is sensitive to itsenvironment and can be used to providedirect information on local melting of theDNA. This example makes use of the factthat the fluorescence is quenched in duplexDNA relative to its fluorescence in single-stranded DNA.

In response to the instability of furano-dTto base, we have discontinued thephosphoramidite monomer (7) from ourcatalog (although it is still available to anyresearchers who are continuing researchstarted with it). We now introduce Pyrrolo-dC CE Phosphoramidite (8) and look forwardto this monomer having a long, stableexistence as a Glen Research specialty, whileallowing researchers to probe DNA structureand activity using a fluorescent base thatforms a perfect match to G on thecomplementary strand.

Figure 3 shows the normalized excitationand emission spectra of Pyrrolo-dC in asingle-stranded oligonucleotide. Thefluorescence is, of course, partially quenchedin double-stranded oligonucleotides.

In the meantime, we continue to carry outexperiments designed to investigate theproperties of pyrrolo-dC and we will reporton these in detail in the coming months.

Pyrrolo-dC is a joint development of GlenResearch and Berry & Associates.

References:

(1) S.C. Srivastava, S.K. Raza, and R. Misra,Nucleic Acids Res, 1994, 22, 1296-304.

(2) M.E. Hawkins, W. Pfleiderer, O. Jungmann,and F.M. Balis, Anal Biochem, 2001, 298,231-240.

(3) M.E. Hawkins, W. Pfleiderer, F.M. Balis, D.Porter, and J.R. Knutson, Anal Biochem,1997, 244, 86-95.

(4) S.L. Driscoll, M.E. Hawkins, F.M. Balis, W.Pfleiderer, and W.R. Laws, Biophys J,1997, 73, 3277-86.

(5) R. Charubala, et al., Nucleos Nucleot, 1997,16, 1369-1378.

(6) J.M. Jean and K.B. Hall, Proc Natl Acad SciU S A, 2001, 98, 37-41.

(7) J. Woo, R.B. Meyer, Jr., and H.B. Gamper,Nucleic Acids Res, 1996, 24, 2470-5.

(8) C. Liu and C.T. Martin, J Mol Biol, 2001,308, 465-75.

FIGURE 3: EXCITATION AND EMISSION SPECTRA OF PYRROLO-dC

NNN

N O

N H

H

H N

ON

NHN

NN

N

N H

H

ON

N

O

H

FIGURE 4: BASE PAIRS OF FLUORESCENT BASES



Pyrrolo-dC-CE Phosphoramidite 10-1017-95 50 µm 110.0010-1017-90 100 µm 220.0010-1017-02 0.25g 675.00

The sequence is: 5'-GCC TAA CTT CXG GAG ATG T-3', where X is Pyrrolo-dC.The oligo is single-stranded in water.The spectral properties of pyrrolo-dC are the following and the QY (quantum yield offluorescence) was determined relative to quinine sulfate in 1.0 N H2SO4:

λ maxima: 265 and 347 nm hybidization state QYE260: 4000 L/mol cm single-stranded 0.07E347: 3700 L/mol cm double-stranded 0.02

2-Aminopurine ..... T Base Pair G ..... Pyrrolo-C Base Pair

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8

What would a Glen Report be without aselection of new nucleoside analogues? Ofcourse, GR 15.1 is no different!

7-deaza-8-aza-dG (PPG)

One of the perennial problems of DNAresearch occurs when analyzing DNAsegments that are G-rich. Basically, DNAstructure dictated by Watson-Crick basepairing is disrupted in G-rich segmentsbecause of their ability to create inter andintra strand hydrogen bonding. Thisaggregation causes enzymatic disruption sosequencing and PCR experiments becomehighly problematical. In probe experiments,these segments are not accessible due tothis aggregation of G residues on the probeand/or target. The problem arises from extrahydrogen bonding forming at the N7position of G (1).

Traditionally, 7-deaza-G (2) has been usedto overcome these problems with somesuccess. However, the 7-deaza-G - C basepair is destabilized relative to the G - C basepair by around 1° per insertion. Also, 7-deaza-G is relatively unstable to the iodineoxidation in the regular synthesis cycle and,if several insertions have to be made intoan oligonucleotide, a non-iodine containingoxidizer must be used.

A solution to these problems may be foundin the substitution of 7-deaza-8-aza-G(pyrazolo[3,4-d]pyrimidine) (PPG) (3) for G.This modification has been examined1 and7-substituted analogues were evaluatedseveral years ago by Seela’s group. In 7-deaza-8-aza-G, the N7 and C8 atoms of Gare flipped (Figure 1), allowing the modifiedbase to retain the same electron density asthe guanine ring system. The 7-deaza-8-aza-G - C base pair was found to bestabilized relative to G - C by around 1° perinsertion. This stability enhancement hasled to interest in the use of PPG in diagnosticprobe applications.

The 7-deaza-8-aza-dG-CE Phosphoramiditemonomer (4) is a very stable structure and,therefore, requires no changes from theregular synthesis cycles and deprotectionprocedures. It is made available as part ofour distribution agreement with Epoch

H2N

O

NN

HN

H2N

O

NN

N

HN

N

N

N

N

NH2

N

N

NN

NH2

N

H2N

O

NN

HN

32

1

6 7

8

94

5

O

O P N(iPr)2O CNEt

DMTO

N

NN

N

N

NMe 2

HN

N

NN

O

N

DMTO

CNEtO

N(iPr)2PO

O

Me2N

O

O P N(iPr)2O CNEt

DMTO

N

N

N

N

F

O

NO2

O

O P N(iPr)2O CNEt

DMTO

N

O

N

N

N

HNOMeMe2N

HN

N

N

N

O

H2NO

N

NH

HN

O

NHN

O

HN

ONH2

NH2

O

N

N

N

N

NN

NMe 2

NMe 2

DMTO

O P N(iPr)2O CNEt

O

O P N(iPr)2O CNEt

OMe

O

N

N

N

HN

DMTO

iPr-PhOAcHN

OMe

DMTO

CNEtO

N(iPr)2PO

O

NHAc

O N

N

OMe

DMTO

CNEtO

N(iPr)2PO

O

NHAcOPh

N

N

N

N

FIGURE 1: STRUCTURES OF THE MODIFIED BASES DESCRIBED

(1) G (3) 7-deaza-8-aza-G(2) 7-deaza-G

(4) 7-deaza-8-aza-dG

(5) A (6) 7-deaza-8-aza-A

(7) 7-deaza-8-aza-dA

(8) 2-F-dI

(9) 8-oxo-G (11) Z(10) Iz

(12) 8-OMe-dG (13) 8-Amino-dA

(14) 2'-OMe-Pac-A (16) 2'-OMe-iPr-Pac-G(15) 2'-OMe-Ac-C(Continued on Page 9)

M O R E A N D S T I L L M O R E N O V E L M I N O R B A S E S

9

Biosciences, Inc., discussed in detail on theFront Page of this issue.

7-Deaza-8-aza-dA

Similarly, the Adenine (5) analogue is 7-deaza-8-aza-A (6). Again, this molecule hasbeen studied in depth over the years bySeela’s group as the unsubstitutednucleoside2, but more recently withsubstituents on the 7-position. The meltingbehavior of 7-deaza-8-aza-A is similar tothe G analogue in that the Tm of the 7-deaza-8-aza-A – T base pair is generallyraised relative to the A - T base pair. Thedifferent electron density of thepyrazolo[3,4-d]pyrimidine ring systemprobably allows for better base stacking ina duplex.3

Again, the 7-deaza-8-aza-dA-CE Phosphor-amidite monomer (7) is a stable structureand there is no need to change conditionsduring its use in oligonucleotide synthesisand deprotection.

2-F-dI

It has been a long time but we are at lastadding to our list of Convertible Nucleosides,2-F-dI-CE Phosphoramidite (8). 2-Fluoro-2’-deoxyInosine (2-F-dI) can be convertedto 2-substituted dG derivatives by reactionwith a primary amine, which displaces thefluorine atom.4,5 The timing of theconversion step is a little tricky becausesmall alkyl primary amines are capable ofdoing the conversion while also cleaving anddeprotecting the oligonucleotide. Forexample, reaction with ethylamine wouldconvert 2-F-dI to N2-ethyl-dG but wouldsimultaneously cleave and deprotect theoligonucleotide. Although that may beinteresting in its own right, we have chosento focus on larger primary amines in ourdevelopment work. For example, treatmentof the oligonucleotide (while still fullyprotected on the synthesis column) withdansyl cadaverine converts the nucleosideto an N2-dansyl-dG derivative, as shown inFigure 2. Further conventional deprotectionof the oligonucleotide leads to the finalproduct. The product oligonucleotide nowhas a fluorescent tag which, whenhybridized to the target strand, will projectinto the minor groove of the double-stranded duplex. In a further example, we

used cystamine to convert the 2-F-dI to aproduct containing a thiol group at the N2position (Figure 2). Once this converted oligois hybridized to the target, the thiol isavailable for cross-linking to, for example,a protein binding to the minor groove. Thethiol can also form a disulfide crosslink witha similarly modified G on the complementarystrand.6

As with all convertible nucleosides, wecaution that these reactions are not trivialand should be undertaken by researcherswith a good background in chemistry andaccess to appropriate analytical techniques.

8-OMe-dG

Oxidative damage to G residues in biologicalsystems leads to the formation of 8-oxo-G(9), the predominant product of G damage.2-Aminoimidazolone (Iz, 10) and itshydrolysis product imidazolone (Z, 11) arealso major oxidation products of G. Accessto these two potential lesions is not possibleduring oligonucleotide synthesis becausethey are so base-labile. A suitable precursor,8-methoxy-dG (8-OMe-dG, 12), to dIz hasnow been described.7 We have, therefore,added the convertible nucleoside monomer8-OMe-dG CE Phosphoramidite (12) to ourseries of products offered for researchingDNA damage and repair.

Oligonucleotide synthesis and deprotectionin the presence of 8-OMe-dG isstraightforward. The conversion of 8-OMe-dG to dIz takes place by irradiation of theoligonucleotide (1 mM) in 50 mM sodiumcacodylate buffer, pH 7, in the presence ofriboflavin (50 µM) for 2 minutes on atransilluminator (366 nm), under aerobicconditions at 4°C. Surprisingly for aphotochemical reaction, the conversion isvirtually quantitative. But the moleculargymnastics occurring are completely mind-blowing and we would invite reactionenthusiasts to review the proposedmechanism.

8-Amino-dA

8-Amino-2’-deoxyAdenosine has beensubstituted for 2’-deoxyAdenosine inoligonucleotides used in the studies of triplehelix formation. It has been shown8 thatoligonucleotides containing this modifiedbase form stable triple helices at neutral pH,whereas regular triple helical structures arenormally observed under acidic conditions.We are happy to make available 8-Amino-dA-CE Phosphoramidite (13), which can beused for oligonucleotide synthesis andsubsequent deprotection without need formodification of normal procedures.

(Continued from Page 8)

(Continued on Page 10)

FIGURE 2: CONVERSION OF 2-F-dI TO 2-AMINO DERIVATIVES

O

NR

N

N

NH

N

H

O

NR

N

N

N

N

NO2

H

O

F

N

N

N

N

NO2

N

NH

N

N

NH(CH 2)5NHS

O

O

O

N(CH 3)2

N

N

N

N

NH(CH 2)5NHS

O

O

N(CH 3)2

NO2

O

N

NH

N

N

NH(CH 2)2SH

O

N

NH

N

N

NH(CH 2)2SS(CH 2)2NH2

O

N

N

N

N

NH(CH 2)2SS(CH 2)2NH2

O

NO2

H2NR

1M DBU in acetonitrile 1M DBU in acetonitrile Cleavage/DeprotectionDTT Reduction

0.5M dansyl cadaverine in DMSO

0.5M cystaminein DMSO

1M DBU in acetonitrile

2-F-dI

10

Most conjugation schemes start with theamino or thiol group on the oligonucleotideand require that the molecule to beconjugated is first converted to either anactive ester for amine reactions, such as asuccinimide, or a thiol reactive maleimide.In either case, it is very costly to conduct ascreen of a wide number of differentconjugates. Also, it is not always possibleto find a convenient starting material inorder to prepare an activated ester. In somecases, the molecule is not even amenableto activation because of other propertiesthat interfere with the chemistry. Anotheroption is to react the oligonucleotide witheither a heterobifunctional or homo-bifunctional linker. This broadens theapplicability by allowing conjugation withother types of molecules but adds steps tothe process and is often difficult toreproduce.

An alternative approach is to place the activeester on the oligonucleotide and conjugatewith a molecule containing a primary amine.A wider range of unusual amines is readilyavailable and generally more affordable. Ifthe amine component is stable to the mildbase used for deprotection, then it can beused with carboxy-modified oligo-nucleotides.

Glen Research is pleased to add 5’-Carboxy-Modifier-CE Phosphoramidite (Figure 2,Back Page) to our range of products for the

A N O V E L R O U T E T O



7-Deaza-8-aza-dG-CE Phosphoramidite 10-1073-95 50 µm 197.50(PPG) 10-1073-90 100 µm 395.00

10-1073-02 0.25g 1095.00

7-Deaza-8-aza-dA-CE Phosphoramidite 10-1083-95 50 µm 177.5010-1083-90 100 µm 355.0010-1083-02 0.25g 975.00

2-F-dI-CE Phosphoramidite 10-1082-95 50 µm 180.0010-1082-90 100 µm 360.0010-1082-02 0.25g 975.00

8-OMe-dG-CE Phosphoramidite 10-1075-95 50 µm 177.5010-1075-90 100 µm 355.0010-1075-02 0.25g 975.00

8-Amino-dA-CE Phosphoramidite 10-1086-95 50 µm 177.5010-1086-90 100 µm 355.0010-1086-02 0.25g 975.00

2'-OMe-Pac-A-CE Phosphoramidite 10-3601-90 100 µm 25.0010-3601-02 0.25g 62.5010-3601-05 0.5g 125.0010-3601-10 1.0g 250.00

2'-OMe-Ac-C-CE Phosphoramidite 10-3115-90 100 µm 20.0010-3115-02 0.25g 50.0010-3115-05 0.5g 100.0010-3115-10 1.0g 200.00

2'-OMe-iPr-Pac-G-CE Phosphoramidite 10-3621-90 100 µm 25.0010-3621-02 0.25g 62.5010-3621-05 0.5g 125.0010-3621-10 1.0g 250.00

Cap Mix ATHF/Pyridine/Pac2O 40-4210-52 200mL 120.00

(Applied Biosystems) 40-4210-57 450mL 260.00

THF/Pac2O 40-4212-52 200mL 115.00(Expedite) 40-4212-57 450mL 250.00

UltraMild 2’-OMe-RNA

The use of UltraMild monomers inoligonucleotide synthesis has allowed verysensitive dyes like TAMRA, HEX and Cy5 tobe used virtually routinely. The DNA andRNA monomers are currently available and,by popular demand, we are adding the setof 2’-OMe-RNA monomers. In our renderingof this chemistry, we use as protectinggroups phenoxyacetyl (Pac) for A, acetyl (Ac)for C, and isopropyl-phenoxyacetyl (iPr-Pac)for G. The structures of the UltraMild 2’-OMe-RNA monomers are shown in Figure1, (14) – (16).

It has become clear9 that acetic anhydridein the conventional capping mix can causetransamidation in situations where an amineprotecting group is quite labile. This leadsto acetyl protection on the amino group thatmay be slow to be removed. Consequently,if many dG residues are included in theoligonucleotide, we recommend the use ofphenoxyacetic anhydride (Pac2O) in Cap A.This modification removes the possibility ofexchange of the iPr-Pac protecting groupon the dG with acetate from the aceticanhydride capping mix.

References are listed at the foot of column3 on this page.


References (from column 2 above):

(1) F. Seela and H. Driller, Helvetica ChimicaActa, 1988, 71, 1191-1198.

(2) F. Seela and K. Kaiser, Helvetica ChimicaActa, 1988, 71, 1813-1823.

(3) F. Seela and G. Becher, Nucleic Acids Res.,2001, 29, 2069-2078.

(4) L.V. Nechev, I. Kozekov, C.M. Harris, andT.M. Harris, Chem Res Toxicol, 2001, 14,1506-1512.

(5) A.R. Diaz, R. Eritja, and R.G. Garcia,Nucleos Nucleot, 1997, 16, 2035-2051.

(6) D.A. Erlanson, J.N.M. Glover, and G.L.Verdine, J. Amer. Chem. Soc., 1997, 119,6927-6928.

(7) H. Ikeda and I. Saito, J. Amer. Chem. Soc.,1999, 121, 10836-10837.

(8) R.G. Garcia, E. Ferrer, M.J. Macias, R.Eritja, and M. Orozco, Nucleic Acids Res.,1999, 27, 1991-1998.

(9) Q. Zhu, M.O. Delaney, and M.M.Greenberg, Bioorg Medicinal Chem Letter,2001, 11, 1105-1107.

11

preparation of modified oligonucleotides.This unique linker is designed to be addedat the terminus of an oligonucleotidesynthesis. It contains an activatedcarboxylic acid N-hydroxysuccinimide (NHS)ester suitable for immediate conjugationwith molecules containing a primary amine,resulting in a stable amide linkage. Afterthe support has been removed from the DNAsynthesis column, the molecule with theamino group is dissolved in an organicsolvent and reacted with the NHS ester.Excess amino compound can be flushed fromthe column and recovered, if necessary. Theoligonucleotide is then deprotected by amethod appropriate for the reactivity of themodified oligonucleotide.

This procedure offers a novel way ofpreparing conjugates with much moreflexibility and was originally designed toallow higher throughput screening of a largenumber of conjugates. The process using5’-Carboxy-Modifier is shown in Figure 1.The modifier is coupled to the terminus,usually at the 5’ end of an oligonucleotide.The linker is now ready to be coupled to anyprimary amine. This greatly increases thenumber of conjugates that may be readilyprepared. Now, researchers can quicklyscreen a large number of interestingconjugates that were chemically difficult, ifnot impossible, before, and do it in acontrolled, single step fashion that enhancesthe probability of success.

The fact that the conjugation chemistry isaccomplished while the oligonucleotide isstill support-bound adds several advantages.Often, successful conjugations are limitedto those that are amenable to therequirement that some water (20-50%) isneeded to dissolve the oligonucleotide in theconjugation mixture. Solid phaseconjugations allow the use of completelyorganic systems, even dichloromethane, forconjugation. This allows the readyconjugation of very lipophilic compounds tooligonucleotides, which can then act aspurification handles for reverse phasepurification of the conjugates.

Another advantage of this system is that thereagent used in excess, the amine, is notaffected by the reaction and thereforerecoverable. In fact, the reagent can be usedagain without further purification for the

]9[O

Base

HO

OP

OO

-

OBase

O

OP

O

O-

O

O

NHR

]n

]9[O

Base

O

OP

OOCNEt

OBase

O

O OP

O

OCNEt

O

O

NHR

[]n

support

[

]9[O

Base

O

OP

OOCNEt

OBase

O

O OP

OO

OCNEt

O

OO

O

N

[]n

support

O

Base

O

OP

OOCNEt

OBase

O

OHO

[]n

support

FIGURE 1: ILLUSTRATION OF THE USE OF 5'-CARBOXY-MODIFIER C10

Couple 5'-Carboxy-Modifier to the 5'-Terminus.

React with Excess of Primary Amine Molecule.Collect Excess Primary Amine for Recovery, if Desired.

Deprotect Oligonucleotide. Purify, if Necessary.

next conjugation reaction. Besides theobvious cost savings, this will also improveoverall efficiency in that much largerexcesses are now feasible even in large-scaleconjugations. By driving the reaction furtherto completion, downstream operations suchas purifications are reduced or eveneliminated.

5’-Carboxy-Modifier C10 is offered for saleunder license from TriLink BioTechnologies,Inc. It is intended for research anddevelopment purposes only, and may not beused for commercial, clinical, diagnostic orany other use. It is covered under US PatentNo. 6,320,041.

Sample Applications

1. Conjugation of an Amino Compound

a. Couple 5’-Carboxy-Modifier C10 to theoligonucleotide using any conventionalcycle. Do NOT allow the synthesizer toproceed to normal cleavage withammonium hydroxide.

Remember that conjugation must occurbefore deprotection using moststandard conditions, especially thoseusing ammonium hydroxide ormethylamine. Ammonium hydroxidewill lead to the amide and methylaminewill form the methylamide.

A C T I VA T E D C A R B O X Y L A T E M O D I F I E D O L I G O N U C L E O T I D E S

(Continued on Back Page)

Product Conjugate

FIGURE 2: STRUCTURE 5'-CARBOXY-MODIFIER C10

N

O

O

O

O

O P N(iPr)2O CNEt



5'-Carboxy-Modifier C10 10-1935-90 100 µm 50.0010-1935-02 0.25g 200.00

5’-Carboxy-Modifier C10


b. Wash the support with acetonitrile andair dry it. Add the support to a vialcontaining 10 equivalents of the aminedissolved in 2 mL of dichloromethaneor other appropriate solvent with 10%triethylamine. Leave the reaction atroom temperature for 4 hours withcontinuous agitation. Decant theamine solution from the support andwash with further dichloromethane. Airdry the support.

Proceed to regular deprotection,keeping in mind the stability of themodified oligonucleotide to thedeprotection conditions.

2. Preparation of Oligonucleotides with 5’Carboxylic Acid Linkers

a. Couple the 5’-Carboxy-Modifier-CEPhosphoramidite to the oligonucleotideusing any conventional cycle. Do NOTallow the synthesizer to proceed tonormal cleavage with ammoniumhydroxide.

b. Deprotect the oligonucleotide overnightat room temperature using 0.4 Mmethanolic sodium hydroxide(Methanol/Water 4:1)

c. Isolate the oligonucleotide by firstpassing the reaction through thedesalting process of your choice, andthen purify as usual. The product mayalso be used crude after desalting.

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