A Strategy for Adding Multiple Reporter Groups to Oligonucleotides by Tom Fleming

7/27/2019 A Strategy for Adding Multiple Reporter Groups to Oligonucleotides by Tom Fleming

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UNIVERSITY OF SOUTHAMPTON

FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES

School of Chemistry

MChem 3rd Year Dissertation

A STRATEGY FOR ADDING MULTIPLE REPORTER GROUPS TO

OLIGONUCLEOTIDES

by Thomas Fleming

Supervisors:

Dr. Nittaya GaleProfessor Tom Brown


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Abstract

Advances in DNA functionalisation impact on medical, material and

forensic science, bio, nano and gene technology.1 Oligonucleotides as genetic

probes offer rapid detection of complementary sequences with sufficient

sensitivity to distinguish between sequences containing single base pair

mismatches and normal gene sequences. Fluorescent labelling is a central

feature of probe technologies. Single labelling is easily accomplished, but with

increasingly sophisticated probes and demanding application conditions, such as

sub-femtomolar target sequence concentrations, the demand for a convenient,

effective and flexible, multiple internal labelling strategy is of rising importance.

Current labelling strategies commonly use expensive dye-functionalisedphosphoramidite nucleosides with limited range of dye options. Alternatively,

monomers bearing appropriate functional groups can be incorporated into

oligonucleotides and subsequently reacted with functionalised labels amine-

NHS ester coupling, for example. This approach is constrained by compatibility

with the phosphoramidite method and labelling reaction efficiencies.

A synthetic strategy is reported that offers an efficient route to multiple

labelling or modification of an oligonucleotide. Orthogonal protecting group

strategies enable solid phase conjugation to phosphoramidite derivatives of label

molecules. Five modified nucleoside phosphoramidites have been synthesised

and incorporated into DNA oligonucleotides. Modifications at the 2- and 5-

position of pyrimidine nucleosides with Fmoc, Levulinyl and TBDMS protecting

groups have been investigated. A HyBeacon probe, multiply labelled with FAM

was synthesised and melting studies indicated successful discrimination between

mutant and wild-type target sequences, with biophysical results very close toconventional probe technologies. Advantages of this strategy include

compatibility with the phosphoramidite method, practical convenience, broad

scope of label options and high-yielding label installation.


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Contents

ABSTRACT' I!

ACKNOWLEDGEMENTS' 3!

ABBREVIATIONS'&'ACRONYMS' 4!

INTRODUCTION' 1!

Nucleic'Acid'Structure5' 2!Nucleosides!&!Nucleotides! 2!Watson1Crick!Base!Pairing!Specificity! 4!Monomers!to!Oligos! 5!

Oligonucleotides' 7!Oligonucleotide!Synthesis! 7!The!Phosphoramidite!Monomer! 9!The!Phosphoramidite!Cycle! 0!

Oligonucleotides'in'Genetic'Analysis' 18!

HyBeacon'Probes' 19!

Labelling'Strategies' 21!Previous!Strategies!for!Multiple!Labelling!of!Oligonucleotides! 25!

A!New!Strategy!for!Adding!Multiple!Reporter!Groups!to!Oligonucleotides! 3!

Retrosynthesis'&'Alternative'Approaches' 34!

RESULTS'&'DISCUSSION' 37!

Monomer'Design' 37!

Synthetic'Approach' 41!51O1(4,41Dimethoxytrityl)121O1(21(91fluorenylmethyloxycarbonyl)ethyl)151methyluridine131 O1

(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!)! 4!51O1(4,41Dimethoxytrityl)151(31(91fluorenylmethyloxycarbonyl)1propynyl)121deoxyuridine131 O1

(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!2)! 46!51O1(4,41Dimethoxytrityl)121O1(61(tert1butyldimethylsilyloxy)hexanamido)ethyl)151

methyluridine131O1(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!3)! 49!51O1(4,41Dimethoxytrityl)151(31(61(tert1butyldimethylsilyloxy)1hexanamido)1propynyl)121

deoxyuridine131O1(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!4)! 55!51O1(4,41Dimethoxytrityl)151(31(61(levulinyloxy)1hexanamido)1propynyl)121deoxyuridine131 O1

(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!5)! 56!51O1(4,41Dimethoxytrityl)151(31(61(91fluorenylmethyloxycarbonyl)1hexanamido)1propynyl)121

deoxyuridine131O1(O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!6)! 57!

Oligonucleotide'Synthesis'Strategy' 59!Approach!A! 59!


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Approach!B! 6!Approach!C! 66!HyBeacon!Studies! 67!

CONCLUSION'&'FUTURE'WORK' 71!

Conclusion' 71!

Future'Work' 72!Variant!Labelling! 72!Branching!Oligonucleotides! 73!Cell!Imaging! 74!Combinatorial!Labelling! 74!

EXPERIMENTAL' 77!

General'Methods' 77!General! 77!Spectroscopic! 77!Phosphitylation! 78!The!Phosphoramidite!Method! 78!Oligonucleotide!Purification!&!Characterisation! 79!Melting!Analysis! 79!

Synthesis'of'Modified'Monomers' 80!MONOMER!! 80!MONOMER!2! 85!MONOMER!3! 88!MONOMER!4! 92!MONOMER!5! 94!

REFERENCES' 99!

APPENDICES' 1!

Interim'Report' 1!Introduction! !Results!&!Discussion! 4!Proposals!for!Future!Work! 7!References! 0!

Oligonucleotide'Synthesis'Optimisation' 11!Conditions! !

Mass'Spectrometry'Results' 12!HPLC1MS!&!Deconvoluted!Peaks! 4!


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Acknowledgements

Thank you to Professor Tom Brown for taking account of my interests and

personal ambitions and assigning me a project that suited them so well. To be

given a project with real significance in the progression of the field is an honour

and knowing that my work is of some importance has been a great motivator.

I would very much like to thank Dr. Nittaya Gale for working closely with

me on this project and for making it both enjoyable and the success that it has

been. I have learnt the rewards of persistence in problem solving and I have also

learnt to remain positive when faced with disappointment or mistakes an asset

to a healthy work-attitude that extends beyond the laboratory. I have been

privileged to work alongside someone who has demonstrated and passed on

such a high standard of laboratory skills and practices.

Thank you to Dr. Simon Gerrard for always taking the time to answer my

questions. Thank you to Xiaomei Ren for submitting many spectroscopic samples

on my behalf. Thank you to Dr. Afaf El-Sagheer for on-hand advice, for keeping

an eye on me and for making me go home when its getting late. Working

alongside such hard-working, exceptional people has been inspiring.

For their love, support and interest in all that I do, thank you to my Mum

and Dad.


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Abbreviations & Acronyms

A adenine

angstrom(s)

Ac acetyl

AcOH Acetic acid

Act activating group

aq. aqueous

bp base pair(s)

br broad (spectral)

Bu n-Bu normal (primary) butyl

tBu tert-butylBz benzoyl

C cytosine

C degrees Celsius

cat catalytic

CEP-(Cl) 2-Cyanoethyl N,N-diisopropyl(chloro) -

phosphoramidite

cm centimetre(s)

conc. concentrated

COSY correlation spectroscopy

CPG controlled pore glass

chemical shift in parts per million downfield from

tetramethylsilane

d day(s); doublet (spectral); deci; deoxy

DCA dichloroacetic acid

DCC N,N-dicyclohexylcarbodiimideDCM dichloromethane

DEA diethylamine

DEPT distortionless enhancement bypolarisation transfer

DIC diisopropylcarbodiimide

DIPEA diisopropylamine (Hnigs base)

DMAP 4-(N,N-dimethylamino)pyridine

DMF dimethylformamide

DMSO dimethylsulfoxide


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DMTr 4,4-dimethoxyltrityl

DNA 2-deoxyribonucleic acid

ds double-stranded

E1CB

unimolecular (conjugate base) elimination

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA ethylenediaminetetraacetic acid

eq. equivalent

ESI electrospray ionisation

Et ethyl

Et2O diethyl ether

EtOAc ethyl acetate

EtOH ethanolFAM fluorescein amidite

FCC flash column chromatography

FISH fluorescence in-situhybridisation

Fmoc 9-fluorenylmethoxycarbonyl

FT-ICR Fourier transform ion cyclotron resonance

G guanine

g gram(s)

h hour(s)

HPLC high-performance liquid chromatography

HRMS high-resolution mass spectrometry

Hz hertz

iB iso-butyl

iPrOH iso-propanol

J coupling constant (in NMR spectrometry)

K Kelvin(s) (absolute temperature)

L litre, dm3

Lev levulinyl

LPE light petroleum ether

wavelength

lit. literature value (abbreviation used with full stop)

LRMS low-resolution mass spectrometry

micro

m multiplet (spectral); metre(s); milliM molar (moles per litre); mega


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M+ parent molecular ion

Me methyl

MeCN acetonitrile

MeOH methanol

MHz megahertz

min minute(s)

mM millimolar (millimoles per litre)

mol mole(s); molecular (as in mol wt)

MS mass spectrometry

MW

mol. wt. molecular weight

m/z mass-to-charge ratio

n normalNHS N-hydroxysuccinimide

NMI N-methylimidazole

nm nanometre(s)

NMR nuclear magnetic resonance

NTP nucleoside triphosphate

OD optical density

ONT oligonucleotide

PAGE polyacrylamide gel electrophoresis

PG protecting group

Ph phenyl

pip piperidine

ppm part(s) per million

Pr propyl

iPr isopropyl

PS polystyrene

py pyridine

q quartet (spectral)

Rep reporter group

Rf

retention factor (in chromatography)

RNA ribonucleic acid

RP-HPLC reverse phase high performance liquid

chromatography

rs residue (gene, protein)rt room temperature


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s singlet (spectral); second(s)

SN1 unimolecular nucleophilic substitution

SN2 bimolecular nucleophilic substitution

SNP singlenucleotide polymorphism

STR short tandem repeats

Su succinimide

t triplet (spectral)

t time; temperature in units of degrees Celsius (C)

T absolute temperature in units of Kelvins (K); thymine

Tac2O tertbutylphenoxyacetyl acetic anhydride

TAMRA carboxytetramethylrhodamine

TBAF tetrabutylammonium fluorideTBDMS tert-butyldimethylsilyl chloride

TCA trichloroacetic acid

TEA triethylamine

TEAA tetraammonium acetate

tert tertiary

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin-layer chromatography

TM

melting temperature

TMS trimethylsilyl; tetramethylsilane

TR

retention time

UV ultraviolet

vis visible

vol volume

v/v volume per unit volume (volume-to-volume ratio)

wt. weight

w/w weight per unit weight (weight-to-weight ratio)


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Introduction

DNA is a biological information storage molecule that contains the genetic

code a sequence made from an alphabet consisting of only four letters (A, G, C

& T), yet holds the fundamental instructions for growth and proliferation of

almost all living systems.3 With entire complex life forms operating on such

fundamentally simple instructions, it is not surprising that small errors in this

code can have serious consequences. Diagnostic techniques that interrogate the

genetic code are invaluable tools for forensic and medical science.4


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Nucleic Acid Structure5

Nucleosides & Nucleotides

DNA is a biological macromolecule (figure 1). In nature, it exists

predominantly in the double stranded DNA double helix form. Each strand of the

helix is a polymer of nucleotides, figure 6, of which there are four possibilities,

dependent on which of the four heterocyclic bases they contain (figure 2).

Figure 1 B-DNA -The Iconic Double Stranded Helix2


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Figure 2 Purines and pyrimidines - the four heterocyclic bases of DNA, nucleoside & nucleotide

structure and atom numbering convention

The 2-deoxyterminology indicates the absence of a hydroxyl group at the

2-position of the ribose sugar, the main structural differentiation between DNA

and ribonucleic acids (RNA), which has a hydroxyl group at the 2-position. The

other distinguishing feature between DNA and RNA is the replacement of the

pyrimidine thymine (T), DNA, with uracil (U), RNA (figure 3).

Figure 3 Distinctions in heterocyclic bases in DNA/RNA

N

NN

N

NH2

NH

NN

N

O

NH2

N

N

NH2

O

NH

N

O

O

O

OH

ROBase

Base =(A) (G) (C) (T)

Purine Pyrimidine

R = H, Nucleoside

R = PO32-, Nucleotide

1'

2'3'

4'

5'

1

2

3

4

5

67

1

2

3

4

5

68

9

A = Adenine

G = Guanine

C = Cytosine

T = Thymine

Heterocyclic

NH

N

O

O

NH

N

O

O

Uracil (RNA) Thymine (DNA)


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Watson-Crick Base Pairing Specificity

The two antiparallel strands of the DNA duplex are said to be

complementary in their pairing. Each base hydrogen bonds specifically to its

complementary, or opposite, partner. The two strands are bound primarily by

this specific hydrogen-bonding pattern and the overall structure is additionally

stabilised by other non-covalent interactions such as base-stacking and entropic

contributions from the internally-positioned hydrophobic bases. It is the specific

hydrogen bonding between bases, however, that confers information storage

ability.

Figure 4 Watson-Crick Base Pair Hydrogen Bonding Scheme

James Watson and Francis Crick elucidated the structure of the DNA double

helix in 1953 with the help of the work of several other prominent researchers.

The base-pairing scheme (figure 4) was named in their honour. Watson-Crick

base pairing specificity is the basis upon which DNA can accurately copy itself.

During semi-conservative DNA replication, the helix is separated and the twoseparated halves of the parent provide all the information required for the

formation of two exact-copy daughter strands (figure 5).


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Figure 5 The semi-conservative nature of DNA replication, a consequence of Watson-Crick base

pairing specificity2

The key concept is that DNA is an accurate and consistent source of

biological information.

Monomers to Oligos

Polymeric nucleoside strands are connected through a 3,5-

phosphodiester bond (figure 6) giving DNA/RNA strands intrinsic directionality.

At physiological pH (7.4), the phosphate groups exist in their deprotonated,

anionic form, hence the name - nucleic acid. The two strands of a double helix

run in opposite directions to each other, termed antiparallel.

Parent Strand

Daughter Strands


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Figure 7 Three nucleic acid duplex conformations, A, B & Z. B is the most common2

Oligo, from Greek few, is the prefix attached to nucleotides to describe

polymeric single nucleic acid strands from anywhere between a dinucleotide

(dimer) to several hundred mers. Oligonucleotides will bind readily to form

stable duplexes with complementary sequences. It is this key aspect of the

oligonucleotide that makes it a perfect tool for investigating specific DNA

sequences, a tool with applications in diverse fields due to the significance of the

information that DNA contains.

Oligonucleotides

Oligonucleotide Synthesis

The chemical synthesis of oligonucleotides follows a solid-phase strategy

conceived in 1959 by Bruce Merrifield. His invention that was originally designed

for the task of peptide synthesis was awarded the 1984 Nobel Prize in Chemistry.

Application of the solid-phase approach to oligonucleotide synthesis retains all

the advantages of the Nobel Prize-winning conception.6

The phosphoramiditemethod has opened new doors in hybrid DNA research, stimulating scientific

A B Z


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progress in biochemistry, molecular biology, pharmacology, drug design &

development and gene technology.

Synthesis on a solid matrix enables synthesis of nucleotide polymer

lengths of up to around 200 - far longer than would otherwise be possible.1b

Complex purifications are replaced by quick, simple and efficient washes that

regularly achieve stepwise yields in excess of 99.5% - a prerequisite for the

viability of longer oligonucleotide syntheses. Large reagent excesses can be

applied, driving reactions rapidly to completion. The nature of the process is

well-suited to computerised automation, enabling highly efficient and

reproducible syntheses.

Table 1 Impact of coupling efficiencies on overall yield

Length

of Oligo

Average Stepwise Yield (Coupling Efficiency)

90% 95% 97% 98.5% 99.5%

10 38.7 63.0 76.0 87.3 95.6

FinalYield(

%)20 13.5 37.7 56.1 75.0 90.9

50 - 8.1 22.5 47.7 78.2

100 - - 4.9 22.4 60.9

150 - - 1.1 10.5 47.4

200 - - - 4.9 36.9

As demonstrated in table 1, high-yielding coupling reactions are necessary

for the successful synthesis of oligonucleotides of reasonable length.


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The Phosphoramidite Monomer

The key aspect of nucleoside building blocks is the phosphoramidite

moiety at the 3-alcohol (figure 8), which undergoes reaction with exposed 5-

alcohol groups under mild acid catalysis. The DMTr group that protects the 5-

alcohol is also of importance as it prevents off-target polymerisation of the

monomers.

Figure 8 Highlighted features of a dT phosphoramidite monomer

HN

O

O N

O

O

O

PO

NN

O

O

Dimethoxytrityl moiety

2-Cyanoethyl N,N-diisopropylphosphoramidite moiety


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The Phosphoramidite Cycle

Oligonucleotide synthesis via the phosphoramidite method begins with a

single protected nucleoside that is covalently attached to a solid support, often

polystyrene or controlled-pore glass (CPG) granules 500-3000 in diameter,

within the confines of a column. Elongation of the nucleotide chain occurs

through the sequential addition of phosphoramidite monomers by a series of

chemical processes that constitute the phosphoramidite cycle (figure 10).

Figure 9 Diagram of solid-phase oligonucleotide column

The functionalised resin column (figure 9) is loaded onto the DNA

synthesiser and subjected to the synthesis cycle.

Solvents & reagent solutionspass through freely

Filters retain growingoligonucleotides onsolid supports

Insoluble solid support(resin) particles anchoroligonucleotides withinthe column


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Table 2 A typical DNA oligonucleotide synthesis cycle for a 0.2 mol scale synthesis

Operation Reagent/Solvent Time/s

Wash Acetonitrile 30

Detritylate 3% TCA/DCM 50

Monitor trityl - -


Flush Argon 10

Couple0.1 M phosphoramidite monomer and 0.5 M tetrazole in

acetonitrile30


Flush Argon 10

CapAcetic anhydride/py/THF 1/1/8 and 17.6% w/v N-methyl

imidazole in acetonitrile30


Flush Argon 10

Oxidise 0.02 M iodine in water/pyridine/THF 2/20/78 45


Flush Argon 10

Detritylation

The resin is functionalised with protected nucleoside monomers. The

DMTr protecting group prevents polymerisation during functionalisation, but

must be removed to allow coupling to the next base (figure 11). The acid-

catalysed detritylation is achieved with a 3% TCA/DCM or 3% DCA/DCM solution.


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Figure 11 Acid-catalysed (TCA or DCA) detritylation mechanism

The trityl cation is a highly-conjugated and stable species, absorbing

strongly in the visible wavelength range. Automatic and real-time UV-Vis

absorption analysis with a radiation source (495 nm

) of the column effluent

quantifies the efficiency of the synthesis, terminating it if yields fall below a

certain threshold to preserve reagents.7

Activation & Coupling

Coupling to the next base (figure 12) requires a mild acid catalyst,

tetrazole (or derivative of), to activate the phosphorous of the incoming

nucleoside phosphoramidite group towards nucleophilic attack. The protonation

of the diisopropylamino group makes it a better leaving group and facilitates

O

O

O

O

O

BH2

O

O

NH

O

OCl

Cl

ClH

O

O

O

O

O

BH2

O

O

NH

O

OCl

Cl

Cl

H

O

O

HO

O

O

NH

BH2

O

O

O

O

etc.

DMT cation (orange)

= Resin (CPG, PS)


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5-alcohol groups to prevent further participation of erroneous oligonucleotides

in further elongation cycles (figure 13).

Figure 13 An electrophilic acylation cocktail prevents deletion-mutation oligonucleotide inclusion

in further elongation cycles

The reaction of acetic anhydride with NMI creates a strong nucleophile

and also an equivalent of acetic acid. Pyridine is therefore included in the

capping cocktail to maintain the alkaline conditions required to avoid premature

detritylation.

Oxidation

The product of the coupling reaction yields a phosphorous (III) linkage

between the bases. This phosphite triester must undergo oxidation to an acid-

stable phosphorous (V) phosphotriester before the next acidic detritylation takesplace (figure 14). Treatment with iodine in water and pyridine affords the

cyanoethyl-protected PV backbone.

O

O

HOB

O

OHN

NN

O O

O

O

NN

O

O

OB

O

OHN

O

HO

ON

NN


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Figure 15 Ester hydrolysis of succinyl linker - cleavage from solid support

Once cleaved from the solid support, the oligonucleotide/conc. aq.

ammonia solution is heated at 55C for 5 h in order to remove the phosphorous

backbone-protecting cyanoethyl groups and any exocyclic nucleobase nitrogen-

protecting groups (figure 16), however, the choice of protecting group varies

depending on the modification within the oligonucleotide e.g. those unstable to

standard conditions.8

Figure 16 Standard protection of exocyclic amines for phosphoramidite oligonucleotide synthesis

The oligonucleotide can then be purified by the method appropriate to the

oligonucleotide and application e.g. gel filtration, HPLC (reverse phase or ion

exchange-HPLC) or PAGE.

O

O

OB

O

OHNNH4

OH

O

OHN

OH

O

OH

OB+/- H

NH3

N

HN

ON

O

OH

HO

O

PhNH

N

N

O

NHN

O

OH

HO

O

N(4)-benzoyl dC (dCBz) N(2)-isobutyryl dG (dGiB)

N

NN

N

HN

O

OH

HO

Ph

O

N(1)-benzoyl dA (dABz)


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Oligonucleotides in Genetic Analysis

Watson-Crick base pairing specificity makes short

oligonucleotides ideal as reliable and sequence-specific probes of

complementary sequences. Labelling of such oligonucleotides is

required, as an oligonucleotide possesses no inherent

detectability. Labelling probes with fluorescent organic molecules

(dyes) confers many useful properties which are exploited in the

various synthetic oligonucleotide applications.1b Many different probe

technologies have been developed, a common theme being the generation of a

fluorescent signal upon hybridisation to complementary sequences.9

Figure 17 Components of a FAM dye phosphoramidite used for labelling ONTs at 5 terminus

The labelling of these probes is achieved through covalent linkage

between the oligonucleotide and the corresponding functionalised derivatives of

the labels. Fluorescent dyes, quenchers and affinity tags with pre-installed

coupling functionality (figure 17) are commercially available and their properties

are well-documented.10 Many applications, such as HyBeacon probes, require,

or can be enhanced by, the inclusion of multiple fluorescent labels.1b

O

O

O

O

ONH

O

O

O

OP

O

N

N

Reactivegroup

Spacer Signalling moiety


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HyBeacon Probes

An excellent application and measure of success of the fluorescent

labelled oligonucleotide is the HyBeacon probe. HyBeacons are linear single-

strand oligonucleotides labelled with fluorescent groups, such as fluorescein.

HyBeacons are the simplest format of oligonucleotides used in genetic analysis,

for example in probing single nucleotide polymorphisms (SNP)s and short

tandem repeats (STRs).4a The principal of the HyBeacon probe is to produce an

increase in fluorescence upon hybridisation to a complementary sequence (figure

18). This fluorescent enhancement upon hybridisation occurs due to the

reduction of distance-dependent fluorescence quenching interactions between

fluorophores and between fluorophores and nucleobases.11

Upon hybridisation,the formation of the ordered and relatively rigid helical duplex reduces the

proximity between fluorophores and quenching structures reliably increasing

fluorescence.12

Figure 18 Signal generation principle of a HyBeacon probe

Correctly-paired Watson-Crick bases contribute to a more

thermodynamically stable duplex than one containing mismatches.13 The

analytical technique exploiting this property is the melt study. The melting

temperature (TM) is the temperature at which the two complementary strands

make the transition between the separate, free strands and the hybridised

duplex. Raising the temperature of a duplex eventually provides enough thermal

energy to overcome the inter-strand attractive interactions and the duplex

denatures; upon cooling the process is reversed. Duplex formation/denaturation

is tracked by changes in fluorescence; the TM

is taken as the point of inflection


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on a fluorescence vs. temperature plot maxima of the first derivative (figure

19).

Figure 19 Melt curve and first derivative of a oligonucleotide duplex, TM= 53C, with correct

Watson-Crick base pairing

Melting experiments quantify duplex stability and can accurately

differentiate between perfect and imperfectly paired gene sequences.

Experiments using HyBeacon probes provide a quick and cheap means of

detecting SNPs, STRs and mutations through fluorescent measurement.

-50

0

50

100

150

200

250

300

350

400

450

7,000

8,000

9,000

10,000

11,000

12,000

35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71

-dFI/

dT

/K-1

Fluoresc

entIntensityat

525

(FI)

/

ArbitraryUnits

Temperature (T) / C

Melt Curve and First Derivative of anOligonucleotide Duplex

Raw Melt Data

First Derivative


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Labelling is most commonly achieved through phosphoramidite ligation,

a), by the inclusion of a modified monomer containing the functional moieties or

linker arms described that react with the incoming phosphoramidite label.15

Phosphoramidite labelling is advantageous in that it is high-yielding and

compatible with the automation of the DNA synthesiser.

Each type of labelling reaction has its strengths and limitations: Methods

b, c, and d) are post-synthetic techniques requiring amino or thiol-modified

monomers and corresponding electrophilic label derivatives. They offer a

strategy for introducing labels that arent stable to oligonucleotide deprotection

conditions. They may suffer from low chemoselectivity due to reactivity of the

electrophilic labels with amino groups of the oligonucleotide bases.

16

Additionally, the corresponding resultant functional group connections are

susceptible to hydrolysis, an outcome only partially avoidable via careful

handling of pH with appropriate buffer system.16 They are not the most efficient

strategies.

Enzymatic incorporation of labelled NTPs, strategy e), is limited to small

scales, but does allow for high-density multiple label incorporation.16 Due to

enzyme-substrate specificity, however the nature of the dye is limited.16 Other

conceivable labelling strategies include the Staudinger ligation and Diels-Alder

type reactions (figure 21).16


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Figure 21 Labelling by h) & i) Diels-Alder [4+2] cycloadditions and j) Staudinger ligation

Compatibility with solid-phase synthesis; retention of dsDNA helix

stability; retention of Watson-Crick pairing specificity and practical synthetic

convenience are challenges in the area of oligonucleotide labelling. Certain in

vivoapplications add the demands of bioorthogonality and toxicity to this list.17

The properties of a particular label must be considered carefully to assess

suitability. The labelling process must be compatible with oligonucleotide

chemistry i.e. easy to attach with mild reagents. Its mode of attachment mustretain the required properties of the label e.g. fluorescence. Interference with

oligonucleotide function, i.e. specific hybridisation and duplex formation, should

be minimal. Additionally, the stability of the labelled oligonucleotide should be

sufficient to withstand storage at -20C.

Multiple labelling is often desirable. Detection of complementary

sequences is often limited by the concentrations at which the targets are

present. Low concentrations yield low intensity fluorescence upon hybridisation.

LOligo NL

Oligo

h)

i)

O

O

O

N

O

O

O

LOligo NL

Oligoj)

O

N

O

O

O

LN3Oligo

OligoO

O

Ph2P

O

O

NH

Ph2P

O

L

O

L Oligo= Label with Spacer, = Oligonucleotide


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Although highly sensitive equipment exists, detection of target sequences

at biological concentrations i.e. picomolar or lower, can prove challenging.18

Enhancing the fluorescent signal of a probe through addition of multiple

fluorescent reported groups helps address this problem. Fluorescence in-situ

hybridisation (FISH) is a technique for imaging living cells and visualising the loci

genes in a chromosome. FISH typically uses 100-1000 bp probes with a labelling

frequency of 1 fluorescent label per 30 nucleotides. For greater specificity in

probe-binding, shorted sequences are desirable, but limitations come with the

low levels of fluorescence this produces. FISH probes are typically synthesised by

enzymatic incorporation of fluorescently-labelled nucleotide triphosphates in a

polymerase process such as nick translation, random priming or the polymerase

chain reaction.

9a

Enzymatic incorporation of fluorescent nucleotides, however,limits the diversity of fluorescent groups that can be attached due to enzyme-

substrate specificity.


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Previous Strategies for Multiple Labelling of Oligonucleotides

Currently, the most common strategy for multiple labelling is via the

inclusion of commercially-available phosphoramidite monomers with pre-

installed dye functionality, such as the monomer shown in figure 22. Prominent

companies offering such compounds include Glen Research8, Link Technologies19

and Invitrogen20.

Figure 22 Components of a general reporting phosphoramidite monomer, such as thosecommercially available from Glen Research8

Such dye phosphoramidite monomers are, however, often expensive and

they are limited by the requirement that the dye and conjugating functional

groups (amide bonds in the above example) must withstand the oligonucleotide

synthesis cycles.

Another current approach is through the inclusion of monomers bearing

the functional groups outlined in figure 22. Amide coupling yields, however,

rarely match those attainable by phosphoramidite coupling. A commercially

available amino modifier is shown in figure 23.

O

O

O

N

HN

O

O

O

NH

HN

O

O

O

P O

NN

Rep

Phosphoramidite nucleobase Linker Reporter group(Rep)


38/140

Figure 23 Fmoc Amino-Modifier C6 dT, 5'-(4,4-Dimethoxytrityl)-5-[N-((9-fluorenylmethoxycarbonyl)-aminohexyl)-3-acrylimido]-2'-deoxyUridine,3'-[(2-cyanoethyl)-( N,N-diisopropyl)]-phosphoramidite, commercially available from suppliers, such as Glen Research8

Incorporation of monomers bearing orthogonal protecting groups into the

oligonucleotide is less well-documented, but offers several advantages over the

previously mentioned approached. Post-synthetic deprotection and exposure of

alcohol groups permits coupling to dye phosphoramidites that do not require the

same levels of resilience to the oligonucleotide phosphoramidite synthesis cycle.

This broadens the scope of potential dyes/labels/reporter groups and may offer

a cheaper and high-yielding multiple labelling strategy.

Previous methods, using modified, protected phosphoramidite monomers,

have achieved successful internal labelling (figure 24).21 These strategies typically

involve interruption of the DNA oligonucleotide solid-phase synthesis at the

point of modification addition. This is then allows manual deprotection of the

modification, and labelling with phosphoramidite-functionalised fluorescent

dyes, such as TAMRA or FAM, before returning to the DNA synthesiser to

complete the sequence.

O

O

O

N

HN

O

O

O

N

H

HN

O

O

O

O

P O

NN


39/140

Figure 24 Internal labelling monomer used in previous work by Dobson et al. 2-Fmoc uridineCEP, 5-O-(4,4-Dimethoxytrityl)-2-O-[2-(9-fluorenylmethyloxycarbonyloxy)]ethyl uridine

The oligonucleotide synthesis strategy for the incorporation and labelling

of such monomers is outlined in figure 25.

O

O

O

N

NH

O

O

O

O

PO

NN

OO O

O


40/140

Figure 25 Previous synthetic approaches to internal labelling21

The implication of this strategy is that, in order to achieve multiple

labelling, the automated DNA synthesis cycle must be interrupted at the point of

3'5'

OPG

3'5'

OH

Couple to reporter phosphoramidite

3'5'

3'5'

Cleavage & exocyclicnucleobase deprotection

3'5'

Extend

Extend

Remove, deprotect

Return to synthesiser forextension until sequence complete

PG = Protecting Group

Modified Monomer

Solid Support (PS/CPG)

Rep = Reporter Molecule

PO

O

O

O

Rep

PO

O

O

O

Rep

PO

O

O

O

Rep

5'


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42/140


43/140


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45/140


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Results & Discussion

Monomer Design

For most applications, the success of a particular oligomer modification is

judged primarily by two factors: signal sensitivity upon hybridisation and

retention of normal hybridisation behaviour. A consequence of the highly

sophisticated and precise nature of the DNA double helix is that random

modifications to its components are more likely to interfere with, rather than

improve duplex stability and Watson-Crick complementarity.

Modification at 2-position does not directly interfere with the

phosphoramidite synthesis method, but steric bulk may reduce coupling

efficiencies.22 Distancing of bulky groups from the 3-CEP group with alkyl

spacers is expected to reduce steric inhibition of the coupling reaction.

Modification at the C-5 position of pyrimidines is a common target since it

is not involved base-pair hydrogen bonding. The modification is projected into

the major groove of the helix, allowing for significant steric tolerance and

minimal inhibition of helix formation (figure 33).

Figure 33 Modification at pyrimidine C-5 projects into major groove

N

NN

NN

NN

O

O

HH

H NN

N

N O

N

NN

N

O

H

HH

H

H

Major

Groove

Minor

Groove

Major

Groove

Minor

Groove

= Modification

A T G C


50/140

The protecting groups chosen must be orthogonal to the conditions used

to remove the DMTr group (acid labile). The fluorenylmethoxycarbonyl (Fmoc)

moiety was used as an alcohol-protecting group, introduced by nucleophilic acyl

substitution and removed by -elimination (figure 34). -elimination is easily

achieved with mild bases, such as morpholine, piperidine, piperazine, for

example, due to the electron-withdrawing fluorene ring system. Decarboxylation

also provides a strong thermodynamic driving force.

Figure 34 Fmoc cleavage by (1,2)-elimination with piperidine

The 2-deoxyuridine version of monomer 1 has been used previously by

Dobson et al. in the successful synthesis of a dual-labelled probe using Fmoc

chemistry.15 This monomer is now synthesised via an improved syntheticstrategy. Use of this monomer to construct a multiply-labelled oligonucleotide

RO O

OH

RO O

O

14 e , 4n+ 2, n= 3Planar Aromatic System

HN

NHH

NH

RO

O

O

NHH

R OH

- CO2

N

- eliminationE1cb


51/140

was attempted, but afforded disappointing yields indicating the need for

optimisation of the synthesis protocol.

The second generation of protecting groups were levulinyl and TBDMS.

The levulinyl protecting group was introduced vianucleophilic acyl substitution

from the corresponding acid anhydride. Hydrazinolytic cleavage was achieved

with hydrazine monohydrate in pyridine/acetic acid (figure 35).

Figure 35 Post-synthetic removal of levulinyl protecting group by hydrazinolysis

The tert-butyldimethylsilyl (TBDMS) protecting group was introduced by

nucleophilic substitution (SN1) and removed with fluoride chemistry, usingTEA.HF as fluoride source (figure 36).

Figure 36 TBDMS removal viahypervalent silyl intermediate

TBDMS and levulinyl (Lev) protecting groups were selected as suitable

protecting groups due to their proven orthogonality with DNA oligonucleotide

synthesis, as demonstrated in a previous use in the construction of cross-linked

DNA.23

O

O

O

H2N NH2

+/- HOH

O

OHN

H2N

OSi

F

Si OFSi

F

HO


52/140


53/140

Synthetic Approach

5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9-fluorenylmethyloxycarbonyl)ethyl)-5-

methyluridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 1)

Precursor 9 was synthesised in accordance with the method described by

Richardson et al, 2009. The first transformation in this sequence, (i), was

achieved with 79% yield. This compares well with the reported literature figure of

90% when the relative scales of synthesis are considered.9e

NH

O

ON

O

OHOH

HO

N

O

O N

O

OH

HO(i)

N

O

O N

O

OH

O(ii)

NH

O

ON

O

OOH

O (iv)

NH

O

ON

O

OOH

O (v)

NH

O

ON

O

OO

O

DMTr

DMTr

OHO

FmocCEP O

Fmoc

DMTr DMTr

(iii)

1109

7 8

Scheme 1 Synthetic route to Fmoc-protected monomer 1 (i) Diphenyl carbonate (1.1 eq.), NaHCO3

(cat), DMF, 100C, 18 h, 79%; (ii) DMT-Cl (1.4 eq.), py, rt, 24 h, 67%; (iii) Ti(i

PrOH)4 (1.4 eq.), ethyleneglycol (5.0 eq.), DMF, rt, 22 h, 62%; (iv) Fmoc-Cl (1.1 eq.), DIPEA (2.0 eq.), DCM, rt, 2.5 h, 40%; (v)CEP-Cl (1.2 eq.), DIPEA (2.0 eq.), DCM, rt, 3 h, 71%.


54/140


55/140

The second transformation, the protection of the 5-alcohol with the trityl

group viaa nucleophilic substitution (SN1) reaction with dimethoxytrityl chloride

is both mechanistically and practically simple. Regioselectivity for the 5-alcohol

in preference to the 3-alcohol was accomplished on grounds of steric hindrance.

The DMTr-Cl is a bulky tertiary alkyl halide that does not react readily with the

secondary alcohols at either 2- or 3-positions. The substitution reaction

proceeds with the concomitant loss of hydrogen chloride from the reagents

necessitating the presence of at least one equivalent of a suitable base due to

the acid-ability of the resulting DMTr protecting group. The reactivity of the

tertiary DMTr carbocation with to the hydroxide ion calls for careful exclusion of

water. The necessarily basic conditions of this reaction mean that the presence

of water would lead to rapid undesired consumption of DMTr-Cl throughformation of DMTr-OH.

The reaction was achieved in 67% yield, lower than that reported in the

literature value- 89%.9e The smaller scale predisposed the reaction to a lower

yield, but other factors also contributed. The reaction proved to be stubborn and

additional aliquots of DMTr-Cl, time and patience were required. Eventually (t =

22 h), the reaction was determined incomplete, but was quenched, worked-up

and purified regardless. Upon repetition of this reaction, more rigorous

exclusion of water would be observed.

A notable feature of syntheses whose reagents or products include the

DMTr protecting group is the distinctive orange colour that indicates presence of

the DMTr cation in solution (figure 38). This can alert the chemist to the

presence of moisture in solvents, for example. Due to the strong molar

extinction coefficient of the DMTr cation however, it is rare that reaction

mixtures remain completely colourless as very low levels of the cation confer

colouration. Also, heat-induced detritylation of compounds offers a rapid and

informative TLC plate visualisation technique.


56/140

Figure 38 UV Absorption spectra of DMT cation in acetonitrile24

Opening of the anhydro ring (figure 39) was achieved in a 62% yield,

slightly lower than the literature value of 79%.9e A possible cause for disparities

in apparent yield success may be due to the contamination of the product

reported in the literature with titanium complex remains accounting for higherapparent yield. Thorough, repeated centrifugation of the reaction mixture

ensured that a highly pure product was obtained, but inevitably at the loss of

some compound to the retentate, although this was washed several times and

checked viaTLC until satisfactorily clean.

Figure 39 Nucleophilic alkoxide ring opening mechanism

Absorption, /nm

400 450 500 550 600

0.0

0.5

1.0

1.5

498 70 000 dm3 cm-1 mol-1

410 28 960 dm3 cm-1 mol-1

Absorbance(unitless)/ArbitraryUnits

Ti(OiPr)4HO

OH

N

O

O N

O

OH

ODMTr

Ti(OCH2CH2O)O

O

H

HN

O

O N

O

OH

ODMTr

TiL3

O

O

+/- H

+/- L

HN

O

O N

O

OH

ODMTr

O

OH

2 Ti(OCH2CH2O)2


57/140

The major source of loss was due to compound-impurity overlap during

column chromatographic purification that necessitated the exclusion of impure

fractions from the pure fraction pool. Total recovery of compound with a second

column could be achieved if it were needed for subsequent syntheses, or if a

valuable amount of reagents had been committed and warranted isolation. A

competitive reaction that may have reduced yield involves the isopropoxide

acting as the ring-opening nucleophile. However, due to greater steric bulk of

the isopropoxide and using an excess of ethylene glycol, this pathway does not

appear to interfere significantly.

Fluorenylmethyloxycarbonyl was chosen to protect the remaining primary

alcohol group as it offers orthogonal compatibility with the conditions of thephosphoramidite method (see experimental, the phosphoramidite method).

Nucleophilic substitution of chloride at the chlorocarbonate group of Fmoc-Cl by

the primary alcohol yields the product with the concomitant overall loss of

hydrogen chloride from the reagents. The basic conditions employed in the

reaction protect the DMT-protected alcohol group from acid-induced exposure

and deprotonation of the alcohol assists in its nucleophilic attack at the Fmoc

carbonyl carbon atom.

The reaction was initially attempted using pyridine functioning as both

solvent and base. Bis-addition of Fmoc resulted, as determined by LRMS.

Experimentation with reaction conditions led to the use of DIPEA as base and

DCM as solvent, addition of Fmoc-Cl as solution and at 0C. This had three

advantages. Firstly, the reaction proceeded more reliably and required fewer, if

any, extra additions of Fmoc-Cl. Secondly, addition of Fmoc-Cl as a solution is

practically cleaner than exposing the reaction to the external environment and

allows for more control over addition rate. Thirdly, DCM can be removed quicker

and more completely than pyridine, which was often detectable by NMR in early

experiments. The combination of lower reaction temperatures and very gradual

addition of Fmoc-Cl solution successfully afforded the desired product, 40%.

Further optimisation would benefit this reaction.

The phosphitylation was performed with great care and afforded the

phosphoramidite monomer 1 in 71% yield. High product purity was required, asno further purification would take place before the compound entered the


58/140


59/140

Figure 40 The Palladium & Copper mediated catalytic cycles of the Sonogashira reaction26

The product was only obtained in 49% yield. Possible side products

include the alkyne-alkyne homocoupled dimer and the furano-pyrimidine

nucleoside (figure 41).

ThePalladiumCycle

TheCopperCycle

OxidativeAddition

trans-cisIsomerisation

Reductive

Elimination

RR' R' X

Transmetallation

RCu

Cu X

RH

Cu X

R3N

R3NH X

PdII

L

L

R' X

PdIIR'

L

L

R

RCu

PdII

R'

L

L R

PdL2


60/140

Figure 41 Proposed furano-pyrimidine side-product formation mechanism

Fmoc protection was successful, achieving a yield of 82%. Originally, this

was attempted using the less-reactive reagent - Fmoc-OSu to achieve the

required primary alcohol selectivity, but the reaction did not proceed so gradual

addition and low temperature, 0C, was employed.27

Phosphitylation to yield monomer 2 was moderately successful as

reflected in the yield of 60%. Improvements in yield could be made through

obtaining better separation of crude components. The product eluted over 18fractions indicating that a lower-polarity eluent system would not be suitable to

achieve better separation of components. Instead a larger volume, wider

diameter column could be tried in order to achieve separation with a slightly

more polar system.

N

O

O N

O

OH

O

OH

DMTr

Et3N H

Cu

N

O

O N

O

OH

O

OH

DMTrEt3N

Cu

H

N

O

O N

O

OH

ODMTr

OH


61/140


62/140

The mechanisms of hydrazinolysis and the Staudinger reduction are both

particularly interesting and are shown in figures 42 and 43, respectively.

Figure 42 Mechanism of primary amine deprotection by hydrazinolytic phthalimide cleavage

The phthalimide-protected amine can be installed via an SN2 reaction

between the corresponding primary alkyl halide and potassium phthalimide.29

The combined subsequent phthalimide deprotection constitutes the Gabriel

synthesis.29 This precursor was synthesised in-house by Dr. Montserrat

Shelbourne, following the approach described by Manoharan et al.30

NH

O

ON

O

OOH

ODMTr

N

NH

O

ON

O

OOH

ODMTr

NH2

O

ONH2

NH2NH

O

ON

O

OOH

ODMTr

NH

O

O

HNH2N

+/- H

+/- H

14

O

HN

HN

O


63/140

Figure 43 Primary amine formation mechanism by Staudinger reduction, affording primary amine,

14 upon aqueous work-up

Nucleophilic attack by triphenylphosphine at the terminal azide nitrogen

followed by ring closure forms a phosphazide. Irreversible loss of nitrogen forms

the iminophosphorane. Hydrolysis occurs upon aqueous work-up and isthermodynamically driven by the formation of the strong phosphorous-oxygen

double bond.

To extend the length of the linker at the 2-amino ethoxy dT, an amide

coupling between the NHS-ester and the amine was used to give compound 15 in

78% yield. The activated acid offers good reactivity; the reaction proceeded

rapidly without need for carbodiimide coupling agents. Amide coupling is a

frequently-employed connection approach throughout the syntheses of these

monomers. A general mechanism is given in figure 44.

NH

O

ON

O

OOH

ODMTr

NH214

NH

O

ON

O

OOH

ODMTr

N

N

N PPh3

NNN

PPh3

N

NN

PPh3

N PPh3 N PPh3-N2

H2O


64/140

Figure 44 General mechanism of amide bond formation

NHS-activated acids offer enhanced reactivity, permitting the use of milder

conditions, by providing a more stable leaving group than the hydroxide anion.

They are of intermediate reactivity - between that of carboxylic acids and acid

chlorides. They are easy to handle yet their enhanced reactivity is still significant

enough to offer a real synthetic advantage. Occasionally the amide couplingdoes not proceed satisfactorily and carbodiimides can be used to drive the

reaction. The mechanism of this process is illustrated in figure 45.

Figure 45 Carbodiimide-promoted amide bond formation

Undesired side-reactions can occur from the O-acylisourea and include a

rearrangement for form a stable N-acylurea and a reaction with an additional

carboxylic acid molecule to form an acid anhydride of the carboxylic acid (figure

46).

R1 NH2

HO

O

R2 +/- H

- H2O

NH

O

R2

R1

NC

NG G

NH

CN

G G

O

O

RH

O

O

R

G NH

N

O

G

O R NH2R'

+/- H

R'NH

O

R NH

O

NH

GG

O-acylisourea

G =

cyclohexyl (DCC)

isopropyl (DIC)ethyl (EDC)


65/140

Figure 46 O-acylisourea rearrangement forming stable N-acylurea

The acid anhydride can go on to react with the amine to form the desired

amide and release a carboxylic acid (figure 47).

Figure 47 Reaction of carboxylic acid with O-acylisourea to form dicyclohexylurea and acid

anhydride - further reacting to give desired amide and an equivalent of corresponding carboxylic

acid

Phosphitylation was successful and without complication. Column

purification however, was problematic. Elution was protracted and caused

overlap with excess phosphitylating reagent. The pure fractions yielded only

29%.

G

HN N

O

G

O

R N-acylurea

NH

O

N

O

R

G

G

G =cyclohexyl (DCC)isopropyl (DIC)ethyl (EDC)

O

O

R

G NH

N

O

G

O R+/- H

R'NH

O

R

N

H

O

N

H

GG

* Acid anhydrideof carboxylic acid

R

O

O

O

R

H2N R'

*

R

O

OH G =cyclohexyl (DCC)isopropyl (DIC)ethyl (EDC)


66/140

A common problem is the inclusion of unreacted phosphitylating reagent.

When elution of this reagent, or if PV species overlap with that of the monomer,

the contaminated fraction(s) cannot be combined. Their presence can be

visualised on TLC plates stained with p-anisaldehyde and are observed as a white

spot. This is easily distinguishable from the monomer that stains black with p-

anisaldehyde. If this separation proved particularly difficult for a particular

compound then the reaction could be performed with phosphitylating reagent in

limiting proportions.


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5-O-(4,4-Dimethoxytrityl)-5-(3-(6-( tert-butyldimethylsilyloxy)-hexanamido)-propynyl)-

2-deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 4)

The TBDMS group was installed viaunimolecular nucleophilic substitution

(SN1) of its chloride derivative. The acid was activated to the NHS ester to

promote amide coupling. The NHS group is a better leaving group than the

hydroxide anion and coupling can be achieved with milder conditions.

Amide coupling via the NHS-activated acid proceeded without

complication. The clean reaction facilitated simple column chromatography and

afforded compound 16 in 63% yield. Phosphitylation was achieved in 70% yield.

HN

O

O N

O

O

O

NH

4CEP

O

O

TBDMS5

DMTr

HN

O

O N

O

OH

O

N

H

O

O

TBDMS5

DMTr

OH

O

HO

HN

O

O N

O

OH

O

NH2

DMTr

OH

O

OTBDMS

O

O

OTBDMS

N

O

O

(i)

(ii)

(iii)

(iv)

16

Scheme 4) Synthetic route to TBDMS-protected monomer 4 (i) TBDMS-Cl (1.2 eq.), DIPEA (2.0 eq.),DCM, rt; (ii) NHS (1.3 eq.), EDC-Cl (1.2 eq.), DIPEA (3.0 eq.) DMF, rt; (iii) DIPEA, (1.5 eq.), DCM, DMF, rt,4 h 63% iv CEP-Cl 1.2 e . DIPEA 2.0 e . DCM rt 2.5 h 70%.


68/140

5-O-(4,4-Dimethoxytrityl)-5-(3-(6-(levulinyloxy)-hexanamido)-propynyl)-2-

deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 5)

Compound 17 was produced by a carbodiimide-assisted amide formation

reaction. Synthesis of compound 18 was achieved in 70% yield through reaction

between the alcohol and the acid anhydride, which was prepared in-situ. This

terminal alkyne product was subsequently coupled to the 5-iodouridine by a

Sonogashira cross-coupling reaction in 59% yield.

HN

O

O N

O

O

O

NH

5CEP

O

O

Lev5

DMTr

HNO

O N

O

OH

O

NH

O

O

Lev5

DMTr

OH

O

HO

NH

O

HO

(i)

HN

O

O N

O

OH

ODMTr

I

NH

O

OLev

(iv)

(iii)

17 18

19

O

O

OH

O

O

O

O

O

(ii)

(v)

Scheme 5 Synthetic route to levulinyl-protected monomer 5 (i) Propargylamine (1.5 eq.), EDC-Cl(1.5 eq.), DMF, rt, 18 h, 67%; (ii) DCC, Et2O, rt; (iii) Levulinic anhydride (prepared in-situ) (1.4 eq.),DMAP (0.5 eq.), DCM, DMF, rt, 1.5 h, 70%; (iv) CopperI Iodide (0.25 eq.), Pd0(PPh3)4 (0.1 eq.), DMF, TEA,rt, 1.5 h, 59%; (v) CEP-Cl (1.2 eq.), DIPEA (2.0 eq.), DCM, rt, 3.5 h, 74%.


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5-O-(4,4-Dimethoxytrityl)-5-(3-(6-(9-fluorenylmethyloxycarbonyl)-hexanamido)-

propynyl)-2-deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite

(Monomer 6)

Scheme 6 (i) TMS-Cl (1.2 eq), DIPEA (2.0 eq.), DCM, rt, 3 h, 64%; (ii) Fmoc-Cl (1.2 eq.), DIPEA (2.0

eq.), DCM, rt; (iii) EDC-Cl (1.3 eq.), DIPEA (3.0 eq.), DCM, rt, 2 h, 56%; (iv) Py.HF (1.1 eq.), py, DCM,

unsuccessful; (v) Not yet attempted

Inclusion of a TMS protection step improved a previous approach.

Previously the Fmoc-protected acid was reacted with compound 20 without the

3-O-TMS protecting group and resulted in bis Fmoc coupling. Deprotection of

the TMS group, however, resulted immediate cleavage of the DMTr group. An

attempt to recover the compound was made since the DMTr group could be

selectively reinstalled at the 5-position, however the recouped yield was

insufficient to warrant further efforts.

Approaching monomer 6 via the 5-O-(4,4-Dimethoxytrityl)-5-(3-(6-

(hydroxy)-hexanamido)-propynyl)-2-deoxy uridine, treated with Fmoc-O-

succinimide did not proceed even with addition of 4-dimethylaminopyridine, acoupling catalyst (figure 48).

HN

O

O N

O

O

O

NH

CEP

O

O

Fmoc5

DMTr

HN

O

O N

O

OH

O

NH

O

O

Fmoc5

DMTr

HN

O

O N

O

O

O

NH

O

O

Fmoc5

DMTr

TMS

HN

O

O N

O

O

O

NH2

DMTr

HN

O

O N

O

OH

O

NH2

DMTr

(i)

(iv) (v)

TMS

O

OHHO

O

OHO

Fmoc

(ii)

(iii)

20 21

22 6


70/140

Figure 48 Mechanism of catalytic action of DMAP in an amide coupling reaction

The reaction mixture was worked-up and dried in vacuoand the reaction

attempted at -42C (MeCN, CO2(s)) with the more reactive Fmoc reagent Fmoc

chloride. The reaction produced four DMTr-containing products as identified by

TLC, but which degraded to starting material before purification was achieved. It

is suspected that the presence of pyridine or remaining DIPEA from the reaction

mixture, which was added to maintain basicity during the HCl-producingreaction, was responsible for the premature Fmoc cleavage. In conclusion,

handing of compounds bearing the Fmoc moiety must be treated carefully with

respect to over-exposure to basic conditions.

N

N

OO

O

N

O

O

N

N

O

O

N

N

O

O

O

OO

O

N N


71/140

Oligonucleotide Synthesis Strategy

Modified monomers 1-5 were introduced into a HyBeacon probe by three

different approaches. Following deprotection of the modifications, the exposed

groups were labelled with 6-FAM phosphoramidite. The probe sequence was

programmed to differentiate between wild-type and mutant gene for an SNP

implicated in statin-induced myopathy, rs4149056.31

Approach A

In the first approach (figure 49), the modified monomer is incorporated

into the sequence at the desired positions as follows. The sequence is

synthesised until after the coupling of the modified monomer. Keeping the

terminal 5- DMTr group on, the synthesis is paused and the oligonucleotide

column is treated with an appropriate cocktail to remove protecting group (Lev,

Fmoc or TBDMS) of the hydroxyl. The column returned to the synthesiser and the

synthesis cycle resumed at the point of coupling to the next nucleoside in the

sequence. The label phosphoramidite is coupled to the modified monomer and

then the sequence continued until, at the next modification, the process isrepeated until the sequence is complete.


72/140

Figure 49 Labelled-oligonucleotide synthesis approach A

3'5'

OPG

Remove, deprotect

3'5'

OH

Couple to 6-FAM-phosphoramidite

3'5'

Return to synthesiser for

extension until next modification

3'5'

Cleavage & exocyclic nucleobase deprotection

3'5'

Extend

Extend

Return to synthesiser forextension until sequence complete

3'5'


Modified Monomer


Extend

Rep = Reporter Molecule, FAM

PO O

O

ORep

PG

O

PO OO

ORep

PO O

O

O

Rep

PO O

O

O

Rep

PO O

O

ORep

PO O

O

ORep


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This approach was tested with monomer 1 with the following sequence, where

superscript XF represents FAM labelling:

5-GTGGATA1FATGCG1FTCATGG

The crude HPLC-MS and the MS are shown in figure 50. The major peak

corresponds to the correct oligonucleotide product and indicates a yield of

around 80%. This approach afforded a high-purity probe (figure 50), but the

complexity of the procedure may deter many from its use.

The labelled oligonucleotide product was isolated and tested as a

HyBeacon probe in a melting analysis.

Figure 50 Left:HPLC trace for monomer 1, sequence: GTGGATA1FATGCG1FTCATGG (1F = monomer1, labelled with FAM) synthesised with Approach A, correct ONT product (2 FAM additions), T

r: 8.6

min; Right: MS showing the correct expected product calc. MS = 7535.97 Da, found MS =7536.41Da)

Approach B

Approach B (figure 51) offers a less-complicated labelling protocol for the

installation of multiple labels. The entire oligonucleotide is synthesised with the

protected, modified bases in their desired location within the sequence, the

terminal 5-DMTr is left on. The oligonucleotide column is removed from the

synthesiser, treated with DEA (as above), the modification protecting groups

removed and the column returned to the synthesiser at the point of next

coupling. Finally, the phosphoramidite label is applied and the synthesis is

complete, ready for standard deprotection and cleavage.

4 5 6 7 8 9 10 11 Time [min]

0

50

100

150

Intens.

[mAU]

res3210_GB5_01_14311.d: UV Chromatogram, 290nm

4511.6 4949.05314.1

5650.0

6317.2

6767.4

7206.4

7536.4

9028.2

-MS, 8.5-8.7min, Deconvoluted (MaxEnt)

0

1

2

3

4x10

Intens.

4000 5000 6000 7000 8000 9000 10000 11000 m/z


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Figure 51 Labelled-oligonucleotide synthesis approach B

Several oligonucleotides were synthesised following this approach in order

to optimise the conditions. For monomers 1 and 2, the results were

unsatisfactory (figures 52 & 53) 0-15% product was found. Attempts at

optimisation were made, changing capping conditions from acetic anhydride to

Tac2O (milder capping reagent) and no capping. Further optimisation attempts

5'

Remove, deprotect


3'5'

Synthesise entire ONT


Modified Monomer



3'5'

OPGOPG


3'

OHOH

5' 3'

O

5'

5'

P

OO

O

Rep

O

P

OO

O

Rep

O

POO

O

Rep

O

POO

O

Rep


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included doubling the coupling times (10 to 20 mins), concentration of the

applied phosphoramidite monomer (0.1 to 0.2 M) and using fast-deprotecting

nucleosides (dGDMF, dCAc). An example HPLC trace is shown in figure 52, full

details are given in HPLC section of the appendix.

Figure 52 Left: HPLC trace for res3261, sequence: GTGGATA2FATGCG2FTCATGG3 (2F = monomer 2,labelled with FAM, 3 = propanol), synthesised with Approach B, peak at T

R= 7.4 min: no FAM, 8.1

min: only 1 FAM; Right: Mass spec of peak at TR

= 8.1 min

Figure 53 HPLC UV 290 nm

chromatrogram for res3263, sequence GTGGATA1FATGCG1FTCATGG3 (1F= monomer 1, labelled with FAM, 3 = propanol), peak at T

R= 7.4 min no FAM, 8.1 min: only 1 FAM

addition and TR

= 8.6 min: expected product with 2 FAM label. Right: MS of peak at TR= 8.6 min.

Monomer 4 was incorporated into the following sequence (res3373):

5-GTGGATA4FATGCG4FTCATGG

Deprotection of monomer 3 was attempted with TEA (20% in MeCN, 45

min) then TEA.3HF (2 x (0.3 mL, 30 min)). The mass spectrometry results did not

produce any clear peaks (figure 54). Purification was attempted with RP-HPLC,but no separation was achieved. This could be due to the TEA.3HF, a strong

0 2 4 6 8 10 12 14 16 Time [min]

0

50

100

150

200

Intens.

[mAU]

Res3261c_RC1_01_14667.d:UV Chromatogram,290 nm

6325.26421.2

6629.2

6958.3

-MS,7.9-8.1min,Deconvoluted(MaxEnt)

0

1

2

3

4

4x10

Intens.

5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 m/z

0 2 4 6 8 10 12 14 16 Time [min]

0

50

100

150

200

Intens.

[mAU]

Res3263c_RC3_01_14669.d: UV Chromatogram, 290 nm

5394.06011.1

7535.4

9007.6 9420.4 10528.8 13192.5

-MS, 8.6-8.7min, Deconvoluted(MaxEnt)

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Intens.

5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 m/z


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base, prematurely deprotecting the exocyclic nucleobase amine groups causing

FAM to add to free amines of the nucleobases, resulting in numerous products

and preventing successful purification. Optimisation is required.


chromatogram for res3373, using polystyrene support (TBDMSdeprotection incompatible with CPG, fluoride ion reacts with the Si-O structure of the CPG)

Monomer 5 was incorporated into the following sequence:

GTGGATA5F

ATGCG5F

TCATGG

Table 3 Experimentation with ONT synthesis

ONT ID Conditions Chromatogram

res3371

CPG resin

Treated with 20% DEA/MeCN, 25 min to deprotect

the acetal capping of the CPG hydroxyl groups

A

res3390

CPG resin

No treatment with DEA (control experiment) Bres3391 PS resin C

All oligonucleotides in table 3 were treated with DEA (10% DEA/MeCN, 10

min) to deprotect the phosphate backbone prior to levulinyl deprotection.

Levulinyl deprotection conditions were hydrazine monohydrate:acetic

acid:pyridine, 0.031:0.5:2.0 v/v/v, rt, 12 min.

0 2 4 6 8 10 12 14 16 Time [min]

0

50

100

150

Intens.

[mAU]

RES3373_RC4_01_16570.d: UV Chromatogram, 290 nm


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A

B

C


chromatograms, A) res3371, B) res3390 & C) res 3391 sequence:GTGGATA5FATGCG5FTCATGG (5F = monomer 5, labelled with FAM), peak at T

R= 7.5 min:

unlabelled; TR= 8.2 min: 1 FAM addition; T

R= 8.9 min: 2 FAM additions (correct product); MS traces

are shown in appendix (HPLC and Deconvoluted MS)

Treatment with DEA prior to synthesis produces a higher amount of

product than when untreated (figure 55). It is suspected that coupling to FAM

phosphoramidite was reduced by migrating acetyl groups from the CPG-hydroxy

protection. These were removed with the DEA and were therefore not present to

complicate capping.

0 2 4 6 8 10 12 14 16 Time [min]

0

10

20

30

40

Intens.

[mAU]


0 2 4 6 8 10 12 14 16 Time [min]

0

20

40

60

Intens.

[mAU]


0 2 4 6 8 10 12 14 16 Time [min]

0

10

20

30

Intens.[mAU]



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Approach C

A further improvement in terms of practical simplicity was developed in

approach C (figure 56). Interruption of the synthesis at the point of labelling was

avoided. The terminal DMTr group at the end of synthesis was removed (DMTrOFF) and the 5-alcohol capped with hex-5-ynyl phosphoramidite (X). This

means that no human intervention is required during the synthesis and a

standard phosphoramidite cycle program can be used without further

manipulation.

Figure 56 Labelled-oligonucleotide synthesis approach C

5'

Remove, deprotect


3'5'

Synthesise entire ONT (DMTr OFF), cap with hex-5-ynyl phosphoramidite ('X')


Modified Monomer



3'5'

OPGOPG


3'

OHOH

5' 3'

O

5'

5'

P

OO

O

Rep

O

P

OO

O

Rep

OP

OO

O

Rep

OP

OO

O

Rep

X

X

X

X


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This strategy was used to synthesis the following oligonucleotide

(res3415) with monomer 5 on a PS solid support:

5-XGTGGATA5FATGCG5FTCATGG

Figure 57 Left: HPLC UV 290 nm

chromatogram for res3255, sequence: GTGGATA5FATGCG5FTCATGG(5F = monomer 5, labelled with FAM), peak at T

R= 8.6 min: [M-134], unknown, peak at T

R= 8.8 min:

2 FAM additions (correct product); Right: MS

This approach yielded the correct product (figure 57) in a high-yield and

was accomplished with a practically-uncomplicated protocol. This represents a

successful new strategy for adding multiple reported groups to oligonucleotides.

The dual-labelled oligonucleotide was tested as a HyBeacon probe.

HyBeacon Studies

A melting study provided a direct comparison between a conventionally-

labelled HyBeacon and one synthesised by the new strategy (figure 58). A

HyBeacon was synthesised using fluorescein dT, purchased from Glen

Research. The HyBeacon probes were identical apart from the mode of

fluorescein attachment, permitting an assessment of the effect of the phosphate

linker on fluorescence and duplex stability.

0 2 4 6 8 10 12 14 16 Time [min]

0

200

400

600

800

1000

Intens.

[mAU]

RES3415CR1_RB3_01_17066.d:UV Chromatogram,290nm

7206.2

7743.3

-MS,8.7-9.0min,Deconvoluted (MaxEnt)

0.0

0.5

1.0

1.5

5x10

Intens.

5000 5500 6000 6500 7000 7500 8000 8500 m/z


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Figure 58 Structural comparison of FAM dT monomers: A commercially-available, = F in res1137

and B - as produced by the reported strategy (5F,used in res3391)

The probe sequence was programmed as the complement for an SNP at

rs4149056, implicated in Statin-induced myopathy, forming a G:C base pair in

the matched sequences (WT) and G:A in the mismatched. The melting

temperatures of the probe-target duplexes were investigated. The results are

displayed in table 3.

Table 4 Melting temperature results of HyBeacon(TM) probe comparison study

Oligo.ID SequenceDuplex T / C TM / CWild Type Mutant

Probe 1res1137 GTGGATATAFGCGFTCATGG 43.0 53.0 10.0

Probe 2res3210 GTGGATATA5GCG1TCATGG 42.0 52.0 10.0

Probe 3res3391 GTGGATATA5GCG5TCATGG 45.0 54.0 9.0

WTTarget

res3375 CCATGAACACATATATCCAC --

MTTarget

res3376 CCATGAACGCATATATCCAC --

O

O

O

N

HN

O

O

O

NH

HN

O

O

O

HO

O

OH

O

O

O

N

HN

O

O

NH

O

OPO

O O

NH

O

O

O

HO

O

OH

A

B


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Conclusion & Future Work

Conclusion

This project set out to investigate a new strategy for adding multiple

reporter groups to oligonucleotides through incorporation of protected modified

monomers. Five monomers were synthesised and incorporated into

oligonucleotides. The most successful were tested as HyBeacon probes. The

probes successfully differentiated between the two alleles of a gene implicated in

statin-induced myopathy. The properties of the probe-target duplexes with

probes synthesised by the reported strategy and a probe synthesised using a

FAM-dT-phosphoramidite, purchased from Glen Research8, were compared. The

probes were found to have high similarity, indicating that the structural

differences in the base-label connection do not significantly interfere with either

duplex stability or fluorescent emission. This was a successful demonstration of

the strategy. It is clear that an effective multiple labelling strategy has been

developed.


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Future Work

Variant Labelling

Theoretically, through the use of two or more modified monomers in

which the label-linking functional groups (-OH or NH2) are mutually orthogonal

in their protection, multiple and variant labels could be easily installed (figure

61).

Figure 61 Capacity for multiple and variant labelling

5'

Remove, deprotect PGA


3'5'

O(Rep)AO(Rep)A O(Rep)B O(Rep)B

3'


Modified Monomer


Rep = Reporter Molecule

3'5'

OPGAOPGB OPGBOPGA

Couple to reporter phosphoramidite A

3'

OHOPGB OPGBOH

5'

O(Rep)AO(Rep)A O(Rep)B O(Rep)B

5'3'

O(Rep)AOPGB OPGBO(Rep)A

Deprotect PGB

5' 3'

O(Rep)AOH OHO(Rep)A

Couple to reporter phosphoramidite B

Synthesise Entire ONT


85/140

Variant label installation could prove useful in applications that desire

dendrimeric structures, asymmetric branching, or installation of multi-coloured

probes for wider instrument detection compatibility.

Branching Oligonucleotides

Installing multiple reporter groups could enhance HyBeacon probe

resolution. Ultra-high fluorescence outputs could be achieved through the use of

branching side chains containing multiple fluorescent groups (figure 62).

Similarly, fluorescence in-situ hybridisation (FISH) target detection capability

could be improved through higher fluorophore installation levels.

Figure 62 A) Orthogonally protected phosphoramidite monomer, B) Reporter groups or branchextensions C) Branched oligonucleotide

DNA and RNA sequences can be detected by surface-enhanced Raman

spectroscopy (SERS).32 The probe sequence is usually connected through a

thioether at the 3-terminus to a gold nanoparticle and labelled with a Raman-

active dye. The anchored strand has many degrees of freedom and the proximity

of the Raman dye to the gold surface, required for Raman enhancement, cannot

HN

O

O N

O

O

O

CEP

DMTr

O

OPG2

OPG1HN

O

O N

O

O

O

DNA

DNA

O

O

O

Rep1 or ONTs

Rep2 or ONTs

Rep1 or ONTs

Rep2 or ONTs

Rep3 or ONTs

Rep4 or ONTs

Rep5 or ONTs

Rep6 or ONTs

A) B)

C)


86/140

be guaranteed. Branching oligonucleotides could provide multiple anchor points,

maintaining dye-surface proximity and optimum Raman signal enhancement.

Cell Imaging

For imaging sequences in living cells, the probe must enter the cell. This

can be achieved manually by microinjection or with by addition of a delivery

vehicle, such as lipofectamine, but both techniques can damage the cell and alter

its normal functioning. Natural cellular uptake mechanisms can be encouraged to

accept molecules bearing polyamines (figure 63), which exist in their protonated

cationic form at physiological pH.

33

A route for simple ligation of the sperminegroup and fluorescent groups to oligonucleotides may be of use in cell imaging

research.

Figure 63 Spermine phosphoramidite, commercially available from Glen Research

Combinatorial Labelling

The reported strategy for adding multiple reporter groups to

oligonucleotides is particularly well suited to large-scale synthesis. This is due to

the simple labelling protocol and single purification step. It is time and cost-

efficient, essential for viability of commercial applications.

N N N NO

TFA TFA

TFA TFA

O P O

NN

O

O

TFA = trifluoroacetyl protecting group F

FF

O


87/140

Simultaneous, detection of many specific pathogens, bacterial, viral,

fungal, parasitic, could be achieved with a cocktail of FISH probes, each with

specific pathogen targets. The synthesis of the probe cocktail could be easily

accomplished with a combinatorial process. Complete probe sequences can be

combined on their solid supports then deprotected and labelled in one batch.

The probe cocktail could be tailored to its application. A hand-wash gel,

loaded with the correct probe sequences could simultaneously detect the

predominant bacterial pathogens responsible for hospital infections or provide a

rapid detection system for biological warfare agents for use in high-risk facilities.


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Experimental

General Methods

General

Standard reagents were procured from Aldrich, Avocado or Fluka. Solvents

were distilled as follows: DCM, TEA, DIPEA, py from CaH2

and THF from sodium

with benzophenone as indication of anaerobic, anhydrous quality. Reaction

Documents

A Strategy for Adding Multiple Reporter Groups to Oligonucleotides by Tom Fleming