A Strategy For Adding Multiple Reporter Groups To Oligonucleotides by Tom Fleming

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 Gale Professor Tom Brown
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-functionalised phosphoramidite nucleosides with limited range of dye options. Alternatively, monomers bearing appropriate functional groups can be incorporated into oligonucleotides and subsequently reacted with functionalised labels amineNHS 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 5position 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 to conventional probe technologies. Advantages of this strategy include compatibility with the phosphoramidite method, practical convenience, broad scope of label options and high-yielding label installation.
Contents
ABSTRACT' ACKNOWLEDGEMENTS' ABBREVIATIONS'&'ACRONYMS' INTRODUCTION'
Nucleic'Acid'Structure5' Nucleosides!&!Nucleotides! Watson1Crick!Base!Pairing!Specificity! Monomers!to!Oligos! Oligonucleotides' Oligonucleotide!Synthesis! The!Phosphoramidite!Monomer! The!Phosphoramidite!Cycle! Oligonucleotides'in'Genetic'Analysis' HyBeacon'Probes' Labelling'Strategies' Previous!Strategies!for!Multiple!Labelling!of!Oligonucleotides! A!New!Strategy!for!Adding!Multiple!Reporter!Groups!to!Oligonucleotides! Retrosynthesis'&'Alternative'Approaches' 2! 2! 4! 5! 7! 7! 9! 10! 18! 19! 21! 25! 31! 34!
I! 3! 4! 1!
RESULTS'&'DISCUSSION'
Monomer'Design' 37!
37!
Synthetic'Approach' 41! 51O1(4,41Dimethoxytrityl)121O1(21(91fluorenylmethyloxycarbonyl)ethyl)151methyluridine131O1 (O121cyanoethyl1N,N1diisopropyl)!phosphoramidite!(Monomer!1)! 41! 51O1(4,41Dimethoxytrityl)151(31(91fluorenylmethyloxycarbonyl)1propynyl)121deoxyuridine131O1 (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)121deoxyuridine131O1 (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' Approach!A! 59! 59!
Approach!B! Approach!C! HyBeacon!Studies!
61! 66! 67!
CONCLUSION'&'FUTURE'WORK'
Conclusion' Future'Work' Variant!Labelling! Branching!Oligonucleotides! Cell!Imaging! Combinatorial!Labelling! 71! 72! 72! 73! 74! 74!
71!
EXPERIMENTAL'
General'Methods' General! Spectroscopic! Phosphitylation! The!Phosphoramidite!Method! Oligonucleotide!Purification!&!Characterisation! Melting!Analysis! Synthesis'of'Modified'Monomers' MONOMER!1! MONOMER!2! MONOMER!3! MONOMER!4! MONOMER!5! 77! 77! 77! 78! 78! 79! 79! 80! 80! 85! 88! 92! 94!
77!
REFERENCES' APPENDICES'
Interim'Report' Introduction! Results!&!Discussion! Proposals!for!Future!Work! References! Oligonucleotide'Synthesis'Optimisation' Conditions! Mass'Spectrometry'Results' HPLC1MS!&!Deconvoluted!Peaks! 1! 1! 4! 7! 10! 11! 11! 12! 14!
99! 1!
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.
Abbreviations & Acronyms

A Ac AcOH Act aq. bp br Bu
t
adenine angstrom(s) acetyl Acetic acid activating group aqueous base pair(s) broad (spectral) n-Bu normal (primary) butyl tert-butyl benzoyl cytosine degrees Celsius catalytic 2-Cyanoethyl N,N-diisopropyl(chloro) phosphoramidite centimetre(s) concentrated correlation spectroscopy controlled pore glass chemical shift in parts per million downfield from tetramethylsilane
Bu
Bz C C cat CEP-(Cl) cm conc. COSY CPG
d DCA DCC DCM DEA DEPT DIC DIPEA DMAP DMF DMSO
day(s); doublet (spectral); deci; deoxy dichloroacetic acid N,N-dicyclohexylcarbodiimide dichloromethane diethylamine distortionless enhancement by polarisation transfer diisopropylcarbodiimide diisopropylamine (Hnigs base) 4-(N,N-dimethylamino)pyridine dimethylformamide dimethylsulfoxide
DMTr DNA ds E1CB EDC EDTA eq. ESI Et Et2O EtOAc EtOH FAM FCC FISH Fmoc FT-ICR G g h HPLC HRMS Hz
i
4,4-dimethoxyltrityl 2-deoxyribonucleic acid double-stranded unimolecular (conjugate base) elimination 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ethylenediaminetetraacetic acid equivalent electrospray ionisation ethyl diethyl ether ethyl acetate ethanol fluorescein amidite flash column chromatography fluorescence in-situ hybridisation 9-fluorenylmethoxycarbonyl Fourier transform ion cyclotron resonance guanine gram(s) hour(s) high-performance liquid chromatography high-resolution mass spectrometry hertz iso-butyl iso-propanol coupling constant (in NMR spectrometry) Kelvin(s) (absolute temperature) litre, dm3 levulinyl light petroleum ether wavelength literature value (abbreviation used with full stop) low-resolution mass spectrometry micro multiplet (spectral); metre(s); milli molar (moles per litre); mega
B PrOH
J K L Lev LPE lit. LRMS m M
M+ Me MeCN MeOH MHz min mM mol MS MW m/z n NHS NMI nm NMR NTP OD ONT PAGE PG Ph pip ppm Pr
i
parent molecular ion methyl acetonitrile methanol megahertz minute(s) millimolar (millimoles per litre) mole(s); molecular (as in mol wt) mass spectrometry mol. wt. molecular weight mass-to-charge ratio normal N-hydroxysuccinimide N-methylimidazole nanometre(s) nuclear magnetic resonance nucleoside triphosphate optical density oligonucleotide polyacrylamide gel electrophoresis protecting group phenyl piperidine part(s) per million propyl isopropyl polystyrene pyridine quartet (spectral) reporter group retention factor (in chromatography) ribonucleic acid reverse phase high performance liquid chromatography residue (gene, protein) room temperature
Pr
PS py q Rep Rf RNA RP-HPLC rs rt
s SN1 SN2 SNP STR Su t t T Tac2O TAMRA TBAF TBDMS TCA TEA TEAA tert TFA THF TLC TM TMS TR UV vis vol v/v wt. w/w
singlet (spectral); second(s) unimolecular nucleophilic substitution bimolecular nucleophilic substitution singlenucleotide polymorphism short tandem repeats succinimide triplet (spectral) time; temperature in units of degrees Celsius (C) absolute temperature in units of Kelvins (K); thymine tertbutylphenoxyacetyl acetic anhydride carboxytetramethylrhodamine tetrabutylammonium fluoride tert-butyldimethylsilyl chloride trichloroacetic acid triethylamine tetraammonium acetate tertiary trifluoroacetic acid tetrahydrofuran thin-layer chromatography melting temperature trimethylsilyl; tetramethylsilane retention time ultraviolet visible volume volume per unit volume (volume-to-volume ratio) weight weight per unit weight (weight-to-weight ratio)
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
Nucleic Acid Structure5
Figure 1 B-DNA - The Iconic Double Stranded Helix2
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).
Heterocyclic Base = (A) NH2 N

8 9N 4 3 7 5 6
(G) O N N N NH NH2
5 6
(C) NH2
4
(T) O NH O Pyrimidine N O
N1
2
N3
2
N1
Base RO
4' 3' 5'
Purine
O
1'
OH
2'
A = Adenine G = Guanine
R = H, Nucleoside R = PO32-, Nucleotide
C = Cytosine T = Thymine
Figure 2 Purines and pyrimidines - the four heterocyclic bases of DNA, nucleoside & nucleotide structure and atom numbering convention
The 2-deoxy terminology 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).
O NH N O
O NH N O
Uracil (RNA)
Thymine (DNA)
Figure 3 Distinctions in heterocyclic bases in DNA/RNA
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 two separated halves of the parent provide all the information required for the formation of two exact-copy daughter strands (figure 5).
Daughter Strands
Parent Strand
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.
NH2 N 5' HO O N O O P OO O N N N O NH NH2 N NH2 N N O O O O P OO O O O P OO3' NH N O (C) (G) N (A) 5' HO O
O NH N O NH2 N N O O N O O P OO O (T) N N N O O P OO O O O P OO3'

AGCT 3' ! 5')
(T)
O O P OO O
(C)
O O P OO O
NH NH2 NH2 N N
(G)
(A)
Figure 6 The intrinsic directionality of nucleic acids (AGCT 5' ! 3'
The helical macromolecular structure of double-stranded DNA (dsDNA) is known as a duplex, shown in figure 7. There are many possible duplex conformations that can be formed, but the most common conformation, B-DNA, coils in a right-handed fashion with a periodicity of 34 , each turn consisting of 10-11 base pairs. A-DNA is a shorter and more compact conformation that is only formed under dehydrated conditions, such as those used in crystallographic experiments. Z-DNA is a left-handed helix whose formation is generally unfavourable, although can be promoted by certain conditions.
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 phosphoramidite method has opened new doors in hybrid DNA research, stimulating scientific
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
Average Stepwise Yield (Coupling Efficiency) Length of Oligo 10 20 50 100 150 200 90% 38.7 13.5 95% 63.0 37.7 8.1 97% 76.0 56.1 22.5 4.9 1.1 98.5% 87.3 75.0 47.7 22.4 10.5 4.9 99.5% 95.6 Final Yield (%) 90.9 78.2 60.9 47.4 36.9
As demonstrated in table 1, high-yielding coupling reactions are necessary for the successful synthesis of oligonucleotides of reasonable length.
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 5alcohol groups under mild acid catalysis. The DMTr group that protects the 5alcohol is also of importance as it prevents off-target polymerisation of the monomers.
Dimethoxytrityl moiety O O O O O O O N P N HN O N
2-Cyanoethyl N,N-diisopropylphosphoramidite moiety
Figure 8 Highlighted features of a dT phosphoramidite monomer
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).
Solvents & reagent solutions pass through freely
Insoluble solid support (resin) particles anchor oligonucleotides within the column
Filters retain growing oligonucleotides on solid supports
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.
DMTrO
O O P
B2
DMTrO O O O
B1
N O
DMTrO
O O
B2
HO
Commencement (Detritylation)
B1
O O O
Step 1 Activation & Coupling
N O O
P O O B1
O O
Continue to next cycle Step 3 Oxidation

HO O O N O O O O
O O
Step 2 Capping
O
O O O B1
B2
O O
P O O O B1
Step 4 Detritylation
N
DMTrO
O O
B2
P O O O B1
Cleavage of completed oligonucleotide from support

HO B2
HO O O O P O O
O O
B2
Deprotection
N O P O O O B1
B1
HO
HO
Figure 10 Oligonucleotide synthesis cycle of the phosphoramidite method
The
timing
and
reagents
used
in
each
step
of
typical
DNA
oligonucleotide synthetic cycle are outlined and simplified in table 2.
Table 2 A typical DNA oligonucleotide synthesis cycle for a 0.2 mol scale synthesis
Operation Wash Acetonitrile
Reagent/Solvent
Time/s 30 50 30 10 30 30 10 30 30 10 45 30 10
Detritylate 3% TCA/DCM Monitor trityl Wash Flush Couple Wash Flush Cap Wash Flush Oxidise Wash Flush Acetonitrile Argon 0.1 M phosphoramidite monomer and 0.5 M tetrazole in acetonitrile Acetonitrile Argon Acetic anhydride/py/THF 1/1/8 and 17.6% w/v N-methyl imidazole in acetonitrile Acetonitrile Argon 0.02 M iodine in water/pyridine/THF 2/20/78 Acetonitrile Argon
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 acidcatalysed detritylation is achieved with a 3% TCA/DCM or 3% DCA/DCM solution.
O O H O Cl Cl Cl O = Resin (CPG, PS)
H O O O O O NH O O O NH BH2 O O O O BH2 O Cl O Cl Cl
HO O O O
BH2
etc. O NH O O DMT cation (orange)
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 certain threshold to preserve reagents.7
nm
) of the column effluent
quantifies the efficiency of the synthesis, terminating it if yields fall below a
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
substitution. The 5-alcohol displaces diisopropylamine from the phosphorous centre via an SN2 substitution.
DMTr O O O N O P N
DMTr O O O N O P NH
B +/- H N HO O O O B
DMTr O O O P O O O O O
N N N N
HN HN O
H N
NH N N
Figure 12 Acid-catalysed activation and coupling of the phosphoramidite group with exposed 5alcohol
The incoming nucleoside phosphoramidite monomer is applied in large excess and coupling yields of >99.5% are expected for syntheses using normal phosphoramidite monomers. Unreacted, 5-alcohols present a problem if left untreated, complicating purification and lowering final oligonucleotide purity and quality. The deletion error mutant which would arise in an oligonucleotide where a single base that had escaped a coupling cycle would be difficult to separate from the correct sequence oligonucleotides. This would interfere with hybridisation experiments, producing unreliable results that are entirely
unacceptable, given their applications in both forensic and medical science. A capping process serves to terminate these erroneous sequences and their truncation aids purification.
Capping Treatment with a solution of acetic anhydride with N-methylimidazole (NMI) and pyridine (py) in tetrahydrofuran (THF) acetylates remaining unreacted
5-alcohol groups to prevent further participation of erroneous oligonucleotides in further elongation cycles (figure 13).
O N N O O O N N O O HN HN O N HO O O O O HO O B O O O B
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 acidstable phosphorous (V) phosphotriester before the next acidic detritylation takes place (figure 14). Treatment with iodine in water and pyridine affords the cyanoethyl-protected PV backbone.
O O O P
B H2O
O O O P O I O N
B I
O N
O O O
O O
- pyridinium iodide
PIII
O O O P O O O H
O O O P O O O N
N N
O O
O O
PV
Figure 14 Oxidation of the Phosphorous (III) Backbone
To optimise the yield for long oligonucleotides following oxidative stabilisation of the backbone, a second capping step that displaces water from the column, which can inhibit the next coupling step, is incorporated. The capping cocktail is applied; residual water reacts with acetic anhydride to form two equivalents of acetic acid that are neutralised and eluted with the py/THF solution.
Cleavage from Solid Matrix Oligonucleotides are most commonly ligated to their solid supports via a succinyl linker. Completed oligonucleotides are cleaved from their solid supports in a manner appropriate for their particular linker. The succinyl linker is cleaved by ester hydrolysis using concentrated aqueous ammonia at room temperature for 1 h (figure 14).
O O O O
B O +/- H OH HN O OH O O OH B
HN
NH4 NH3
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 nitrogenprotecting 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
O HN N HO O OH OH N(4)-benzoyl dC (dCBz) N(2)-isobutyryl dG (dGiB) OH N O HO O N N O Ph O N HN NH NH HO O N N N
O Ph N
N(1)-benzoyl dA (dABz)
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.
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
O O O O N P O O N H O O N Reactive group Spacer Signalling moiety O O
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
HyBeacon Probes
An excellent application and measure of success of the fluorescent labelled oligonucleotide is the HyBeacon probe. HyBeacons are linear singlestrand 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
13
more The
thermodynamically stable duplex than one containing mismatches.
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
on a fluorescence vs. temperature plot maxima of the first derivative (figure 19).
Melt Curve and First Derivative of an Oligonucleotide Duplex

12,000 Raw Melt Data 11,000 First Derivative
450 400 350 300 250
Fluorescent Intensity at / Arbitrary Units
525
(FI)
10,000
200 150 100
9,000
8,000
50 0
7,000 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71
-50
Temperature (T) / C
Figure 19 Melt curve and first derivative of a oligonucleotide duplex, TM = 53C, with correct Watson-Crick base pairing
Melting differentiate
experiments between
quantify and
duplex
stability
and
can
accurately sequences.
perfect
imperfectly
paired
gene
Experiments using HyBeacon probes provide a quick and cheap means of detecting SNPs, STRs and mutations through fluorescent measurement.
-dFI/dT /K-1
Labelling Strategies
In general, labelling can be achieved either in solid or solution phase, using the chemistry shown in figure 20.
N a) O O P N Oligo L O O P O O Oligo
HO
b) O L O N O c) L N C S d) L O N O e) O L OH O P O O O N O O OH f) L g) L N3 L N3 Oligo L N O P O O O NH2 HS Oligo L O N O S Oligo O HN N O O OH N N N Oligo N L H2N Oligo L H N S H N Oligo H2N Oligo L O O N H Oligo
Oligo
N N N
Oligo
L = Label with Spacer, Oligo = Oligonucleotide
Figure 20 Common oligonucleotide labelling reactions: a) reaction of phosphoramidite derivative of labelling group; b) amide coupling to NHS-ester or other activated carbonyl (e.g. isothiocyanate, c) of labelling group; d) reaction of a thiol-modified group with an ,-unsaturated ketone; e) reaction of an amino-modified NTP with carboxy-activated label for enzymatic incorporation into DNA during PCR or other DNA extension process; f) & g) labelling conjugation by triazole formation (Huisgen reaction), Click chemistry.14
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
h) O Oligo
O N O L O
O N O Oligo L
i) O Oligo N O j) O Oligo N3 O Ph2P O L Oligo L
O N O Oligo O N H Ph2P O L O L
L = Label with Spacer, Oligo = Oligonucleotide
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 vivo applications 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 must retain 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.
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 enzymesubstrate specificity.
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 preinstalled dye functionality, such as the monomer shown in figure 22. Prominent companies offering such compounds include Glen Research8, Link Technologies19 and Invitrogen20.
O HN O O O O O
O N H
H N O
Rep
P O N
Phosphoramidite nucleobase
Linker
Reporter group (Rep)
Figure 22 Components of a general reporting phosphoramidite monomer, such as those commercially 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 HN O O O O O
O N H
H N O
P O N
Figure 23 Fmoc Amino-Modifier C6 dT, 5'-(4,4-Dimethoxytrityl)-5-[N-((9fluorenylmethoxycarbonyl)-aminohexyl)-3-acrylimido]-2'-deoxyUridine,3'-[(2-cyanoethyl)-(N,Ndiisopropyl)]-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 NH N O
O O O O N P N O O O O
Figure 24 Internal labelling monomer used in previous work by Dobson et al. 2-Fmoc uridine CEP, 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.
OPG
Extend
5' OH
3' Remove, deprotect
5' Rep O P O O O
3' Couple to reporter phosphoramidite
5'
3' Return to synthesiser for extension until sequence complete
Rep O P O Extend O O 5' 3' Cleavage & exocyclic nucleobase deprotection Rep O P O O O 5' 3' Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule
5'
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
each modification insertion and labelling a labour intensive and timeconsuming process, which soon becomes unfeasible as the extent and frequency of internal labelling rises (figure 26).
OPG Extend
5'
OH
5'
3' Couple to reporter phosphoramidite
O P O O O
5'
Rep
3' Return to synthesiser for extension until next modication
PG O
5'
Rep O O P O Extend O
Rep Rep Rep Rep O O O O P O P O P O P O O O O Extend O O O O O

5' 3' Cleavage & exocyclic nucleobase deprotection
Rep Rep O Rep Rep O O O P P O O O P O P O O O O O O O O

5' 3' Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule
Figure 26 Processes required for multiple labelling following previous strategies
Whilst these strategies have proved successful, they are of limited use in the synthesis of multiply-labelled oligonucleotides.
A New Strategy for Adding Multiple Reporter Groups to Oligonucleotides Reported here is strategy that offers the benefits of multiple internal labelling without additional practical workload. The simultaneous deprotection and simultaneous labelling at multiple modified loci within the oligonucleotide reduces the workload significantly (figure 27). Success of the strategy relies on the orthogonality of the protecting group strategy with respect to phosphoramidite synthesis conditions and resilience of the reporter group to oligonucleotide synthesis, deprotection and purification.
Synthesise entire ONT OPG OPG OPG OPG 3' Remove, deprotect
5'
OH 5' 5'
OH
OH
OH 3'
Couple to reporter phosphoramidite Rep O O P O O 5' 5' Rep O O P O O Rep O O P O O Rep O O P O O
3' Cleavage & exocyclic nucleobase deprotection
Rep O O P O O 5' Rep O O P O O
Rep O O P O O Rep O O P O O 3'
Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule
Figure 27 A general strategy for adding multiple reporter groups to oligonucleotide
To achieve the above strategy, a suitable protecting group(s) must be found. The following monomers have been designed, synthesised and their properties explored (figure 28).
O NH DMTr O O
CEP
O HN DMTr O O O N
Fmoc
N O
O Fmoc
CEP
Fmoc =
1
O NH DMTr O O CEP O N O N H O O O DMTr O TBDMS CEP HN O O N O
2
O N H O
5 TBDMS
DMTr =
TBDMS =
4
Si
O
O O HN DMTr O O O CEP O N N H O
Lev =
5 Lev
O
CEP =
N P O
Figure 28 Overview of modified monomers
Once incorporated into the oligonucleotide, the protecting groups may be removed whilst still on the solid supports and coupled to reporter phosphoramidites (figure 29). Purification need only be performed once, saving time, cost and yield compared with post-synthetic labelling strategies that require purification labelling purification.
O N O N P O O H N O O O O O O
Figure 29 6-FAM-phosphoramidite, commercially available from Glen Research and Sigma Aldrich
Choice of label is subject to certain restrictions. The acid or alcohol derivative of the label must be obtainable in order to phosphitylate. Alcohol derivatives may undergo simple phosphitylation whilst acid derivatives can be coupled via their corresponding NHS-esters to an amine linker, protected if necessary, and then phosphitylated (figure 30).
O O O O O OH O O OH O N O O HO O O O HN OH O OH
HO
HO O O
O O HN
OH
O O O O
P O N
Figure 30 Conversion of an acid-derivative of fluorescein to 6-FAM-phosphoramidite
It is also necessary that the chosen label phosphoramidite is stable to standard exocyclic nucleobase deprotection and oligonucleotide cleavage conditions. Nucleophilic functionality in a label must be protected. The major ambitions of this project is the investigation of a strategy that allows simultaneous multiple internal labelling that is compatible with phosphoramidite oligonucleotide synthesis and is practically convenient. The success of the syntheses and properties of the produced labelled oligonucleotide products will be evaluated.
Retrosynthesis & Alternative Approaches

The retrosynthesis for C-5 modified monomers affords several different orders of disconnection. Conceivable strategies are shown in figures 31 & 32.
O OGP N H O NH N O O OH O PGO HN O
O NH N O O O
O PGO OAct
OH
O PGO OAct H2N O NH N O PG O OH HOAct O O OH O
O PG O O PG-X HO OH H2N OH
O NH N O O OH O
O X = Halide H2N X N NH O O OH O PG = Protecting Group (TBDMS, Lev, Fmoc) Act = Activating Group (NHS)
Figure 31 Retrosynthetic analysis of C-5 modified monomer
O OGP N H O NH N O O OH O
O O OGP N H N NH O O OH O
O OGP N H O X N NH O O O OGP HN OH O NH N O O OH O O O O O
O X O OGP OH H2N
O X NH N O O OH OH Cl
X = Halide PG = Protecting Group (TBDMS, Lev, Fmoc)
Figure 32 Retrosynthetic analysis of C-5 modified monomers
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).
A T
Major Groove
Major Groove H H N N O N N H N N H H Minor Groove O N N
H N N N
N H N
O H N O N
Minor Groove
= Modication
Figure 33 Modification at pyrimidine C-5 projects into major groove
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.
HN
14 e , 4n + 2, n = 3 Planar Aromatic System O R O O
O R O O
- elimination E1cb
NHH
O R O O
NH
H NH
- CO2
N R OH
Figure 34 Fmoc cleavage by (1,2)-elimination with piperidine
The 2-deoxy uridine 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 synthetic strategy. Use of this monomer to construct a multiply-labelled oligonucleotide
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 via nucleophilic acyl substitution from the corresponding acid anhydride. Hydrazinolytic cleavage was achieved with hydrazine monohydrate in pyridine/acetic acid (figure 35).
O O O H2N NH2
+/- H
OH
H2N
H N O
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, using TEA.HF as fluoride source (figure 36).
Si F
O F Si O F
Si
HO
Figure 36 TBDMS removal via hypervalent 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
Synthetic Approach
5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9-fluorenylmethyloxycarbonyl)ethyl)-5methyluridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 1)
O NH N HO O OH OH O (i) HO O O OH N
O N N (ii) DMTr O O O OH
(iii)
7
O NH DMTr O O OH O OH N O (iv) DMTr O O OH O O N O NH O (v) Fmoc DMTr O O CEP O O N O
8
NH O
Fmoc
10
Scheme 1 Synthetic route to Fmoc-protected monomer 1 (i) Diphenyl carbonate (1.1 eq.), NaHCO 3 (cat), DMF, 100C, 18 h, 79%; (ii) DMT-Cl (1.4 eq.), py, rt, 24 h, 67%; (iii) Ti(iPrOH)4 (1.4 eq.), ethylene glycol (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%.
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
O NH N HO O OH OH O HO O O N H HO O HO O H O -CO2 O N HO O O OH N O O O O N HO O O O O O
O NH N HO O OH O O Ph O O O NH N O O Ph Ph O O
O NH N HO O OH O
O NH N HO O O
O O O Ph
Figure 37 Proposed mechanism for the formation of the anhydro ring
The loss of carbon dioxide from the 2,3-cyclic carbonate is a powerful driving force in the formation of the anhydro ring (figure 37). This synthesis was performed on a scale 16 times smaller than in the literature, therefore transfer losses etc. diminished the yield more significantly. The reaction proceeded as described in the literature without need for additional reagents or prolonged reaction time. Purification by recrystallisation was straight-forward and effective producing sufficiently-large crystals to allow rapid filtration and withstand quick washing. The purpose of this transformation was the selective protection of the 2-alcohol group. The second transformation requires selective reactivity at the 5-position between the 5-alcohol and a tertiary alkyl halide.
The second transformation, the protection of the 5-alcohol with the trityl group via a 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 through formation 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.
498 70 000 dm3 cm-1 mol-1
1.5
Absorbance (unitless) / Arbitrary Units
410 28 960 dm3 cm-1 mol-1 1.0
0.5
0.0
400
450
500 Absorption, / nm
550
600
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 higher apparent 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 via TLC until satisfactorily clean.
Ti(OiPr)4
2 HO
OH
Ti(OCH2CH2O)2
H N DMTr O O O OH
O HN N DMTr O O O OH O Ti(OCH2CH2O) O O
O HN N +/- H +/- L OH O TiL3 O DMTr O O O
OH
Figure 39 Nucleophilic alkoxide ring opening mechanism
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 the phosphoramidite 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, as no further purification would take place before the compound entered the
oligonucleotide synthesis process. The phosphitylation, a well-documented and reliable, although delicate, reaction, proceeded predictably and purification was achieved rapidly with ease.25
5-O-(4,4-Dimethoxytrityl)-5-(3-(9-fluorenylmethyloxycarbonyl)-propynyl)-2deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 2)
O HN DMTr O O O OH N (i) I DMTr HN O O O OH
OH
N (ii)
11
O O Fmoc
O HN DMTr O N O OH
Fmoc
HN DMTr O O CEP O N
(iii)
12
Scheme 2) Synthetic route to Fmoc-protected monomer 2 (i) Propargyl alcohol (2.0 eq.), CopperI iodide (0.2 eq.), Pd(P(Ph)3)4 (0.1 eq.), TEA, DMF, rt, 23 h, 49%; (ii) Fmoc-Cl (0.95 eq.), DIPEA (2.0 eq.), DCM, rt, 2 h, 82%; (iii) CEP-Cl (1.2 eq.), DIPEA (2.0 eq.), DCM, rt, 2 h, 60%.
The first transformation, a carbon-carbon bond formation, was achieved by Sonogashira cross-coupling. The catalytic cycles of the Sonogashira vinyl/aryl halide terminal alkyne cross-coupling mechanism are shown in figure 40.26
R'
R' X
Pd L2
Reductive Elimination
R' L PdII L
Oxidative Addition
trans-cis Isomerisation
L PdII L
The Palladium Cycle
R'
L PdII X L
R'
Transmetallation
Cu X Cu R
Cu
The Copper Cycle

R3N
R3NH X
H Cu
R X
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).
Cu O Et3N H O DMTr O O OH N N DMTr O O OH O OH N N O
Cu OH
Et3N H
OH O N DMTr O O O OH N
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 18 fractions 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.
5-O-(4,4-Dimethoxytrityl)-2-O-(6-(tert-butyldimethylsilyloxy)hexanamido)ethyl)-5methyluridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 3)
The synthesis of monomer 3 was achieved via intermediate, 14, that can be synthesised by two different approaches. The first was the hydrazinolysis of the phthalimide protecting group to bare the primary amine 14. Second, the bimolecular nucleophilic displacement of the mesylate anion by the azide anion (74%) and subsequent Staudinger reduction can theoretically afford 14.28
O NH DMTr O O OH O N O
(i)
O HO
(ii)
OH
O HO
O O TBDMS
(iii)
O O NH DMTr O O OH O N O
(vi)
O O O
TBDMS
O NH
DMTr
O O OH O
O HN NH2
14
(vii)
O TBDMS
(v)
15
O NH DMTr O O DMTr OH O O O O O O HN
5
O NH N O
N N N
(iv)
13
CEP
O TBDMS
O NH DMTr O O OH O O Ms
Ms =
O S O
Scheme 3) Synthetic route to levulinyl-protected monomer 3 (i) H2NNH2" H2O (5 eq.), MeOH, 3.5 h, 100C, 88%; (ii) TBDMS-Cl (1.2 eq.), imidazole (1.2 eq.), DMF/DCM (4/3, v/v), rt, 2 h, 70%; (iii) N-hydroxysuccinimide (1.1 eq.), EDC-HCl (1.1 eq.), DIPEA (2.2 eq.), DCM, rt, 4 h, 85%; (iv) NaN3 (5.0 eq.), 18-crown-6 (0.01 eq.), DCM, rt; (v) PPh3 (2.0 eq.), H2O (5 eq.), THF, 45C; (vi) DIPEA (1.5 eq.), DCM, rt, 4 h, 78%; (vii) CEP-Cl (1.2 eq.), DIPEA (2.0 eq.), DCM, rt, 4 h, 29%.
The mechanisms of hydrazinolysis and the Staudinger reduction are both particularly interesting and are shown in figures 42 and 43, respectively.
O NH DMTr O O OH O O N NH2 NH2 O O +/- H DMTr O O OH O O N H H2N H N O N NH O +/- H N O DMTr O O
O NH N O
OH O NH2
14
O HN HN O
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
O NH DMTr O O OH O N N N N O DMTr O O
O NH N O
OH O NH2 PPh3 H 2O
14
N N
N N PPh3 N
PPh3 N -N2
PPh3
PPh3
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 is thermodynamically 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.
R1
NH2 HO
O R2 +/- H - H2O R1 N H
O R2
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 coupling does not proceed satisfactorily and carbodiimides can be used to drive the reaction. The mechanism of this process is illustrated in figure 45.
N H
G G O N H
G O
O H O R O O G N H R N H
O R
R R'
NH2
O-acylisourea
O R' +/- H
N H
cyclohexyl (DCC) G = isopropyl (DIC) ethyl (EDC)
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).
G HN O R O
O G G N H
N R G N-acylurea
cyclohexyl (DCC) G = isopropyl (DIC) ethyl (EDC)
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).
N G N H O
G R O R O O R
O O
O R
* G N H
O N H G
H2N R' O R' N H R
+/- H * Acid anhydride of carboxylic acid O R OH cyclohexyl (DCC) G = isopropyl (DIC) ethyl (EDC)
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%.
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 panisaldehyde. If this separation proved particularly difficult for a particular compound then the reaction could be performed with phosphitylating reagent in limiting proportions.
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)
O HO OH
(i)
O TBDMS O OH
(ii)
O O HN DMTr O O O OH N
(iii)
O O N O DMTr O HN O O OH
(iv)
TBDMS NH2
O O N H O
5
TBDMS
16
O O HN DMTr O O O O CEP N N H O
5
TBDMS
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 eq.), DIPEA (2.0 eq.), DCM, rt, 2.5 h, 70%.
The TBDMS group was installed via unimolecular 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.
5-O-(4,4-Dimethoxytrityl)-5-(3-(6-(levulinyloxy)-hexanamido)-propynyl)-2deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite (Monomer 5)
O OH O O HO OH O
(i)
O HN I N
(ii)
O O O O O
DMTr O
O O OH
O HO N H
(iii)
Lev
(iv)
N H
17
18
HN DMTr O O O OH
O O N H O
5
Lev
19
(v)
O O HN DMTr O O O O CEP N N H O
5
Lev
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%.
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.
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)
O HN DMTr O O O OH N
NH2
(i)
O HO
(ii)
OH O O O N H O
O HN
NH2 Fmoc O
HN OH DMTr O O O O TMS N
Fmoc
DMTr O
O O O
N
(iii)
TMS
20
21
O O O HN DMTr O O O OH
22
O O
N H
Fmoc DMTr O
HN O O O CEP N
N H
Fmoc
N
(v)
(iv)
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-(6treated with Fmoc-O-
(hydroxy)-hexanamido)-propynyl)-2-deoxy coupling catalyst (figure 48).
uridine,
succinimide did not proceed even with addition of 4-dimethylaminopyridine, a
O O
O O N O O O N N N N O O O N N
O O O
Figure 48 Mechanism of catalytic action of DMAP in an amide coupling reaction
The reaction mixture was worked-up and dried in vacuo and 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-producing reaction, 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.
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 is repeated until the sequence is complete.
OPG Extend
5'
OH
5'
3' Couple to 6-FAM-phosphoramidite
O P O O O
5'
Rep
3' Return to synthesiser for extension until next modication
PG O
5'
Rep O O P O Extend O
Rep O P O Extend O O
5'
Rep O P O O O
Rep O P O O O
5'
Rep O P O O O
3' Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule, FAM
Figure 49 Labelled-oligonucleotide synthesis approach A
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.
Intens. x104
7536.4
Intens. [mAU]
-MS, 8.5-8.7min, Deconvoluted (MaxEnt)
150
100
50
1
7206.4
0 4 5 6 7 8 9 10 11 Time [min]
5314.1 4511.6 4949.0
5650.0 6317.2
6767.4 9028.2
0 4000 5000 6000 7000 8000 9000 10000 11000 m/z
res3210_GB5_01_14311.d: UV Chromatogram, 290 nm
Figure 50 Left: HPLC trace for monomer 1, sequence: GTGGATA1FATGCG1FTCATGG (1F = monomer 1, labelled with FAM) synthesised with Approach A, correct ONT product (2 FAM additions), Tr: 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.
Synthesise entire ONT OPG OPG 3' Remove, deprotect
5'
OH 5' 5'
OH 3' Couple to 6-FAM-phosphoramidite
Rep O O P O O 5' 5'
Rep O O P O O
Rep O O P O O
Rep O O P O O 3'
5'
Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule, FAM
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
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.
Intens. [mAU] 200
Intens. x104 6958.3 4
150
100
2
50
1
0 0 2 4 6 8 10 12 14 16 Time [min]
6629.2 6325.26421.2 0
5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 m/z
Res3261c_RC1_01_14667.d: UV Chromatogram, 290 nm
Figure 52 Left: HPLC trace for res3261, sequence: GTGGATA2FATGCG2FTCATGG3 (2F = monomer 2, labelled with FAM, 3 = propanol), synthesised with Approach B, peak at TR = 7.4 min: no FAM, 8.1 min: only 1 FAM; Right: Mass spec of peak at TR = 8.1 min
Intens. [mAU] 200
Intens. x104 1.25

7535.4
150
1.00
100
0.75
0.50
50
5394.0
6011.1
0.25
0 0 2 4 6 8 10 12 14 16 Time [min]
9007.6 9420.4
0.00 5000 6000 7000 8000 9000 10000
10528.8
13192.5
11000
12000
13000
14000
m/z
Figure 53 HPLC UV 290 nm chromatrogram for res3263, sequence GTGGATA1FATGCG1FTCATGG3 (1F = monomer 1, labelled with FAM, 3 = propanol), peak at TR = 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
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.
Intens. [mAU] 150
100
50
0 0 2 4 6 8 10 12 14 16 Time [min]
RES3373_RC4_01_16570.d: UV Chromatogram, 290 nm
Figure 54 HPLC UV 290 nm chromatogram for res3373, using polystyrene support (TBDMS deprotection incompatible with CPG, fluoride ion reacts with the Si-O structure of the CPG)
Monomer 5 was incorporated into the following sequence: GTGGATA5FATGCG5FTCATGG
Table 3 Experimentation with ONT synthesis
ONT ID res3371 res3390 res3391
Conditions CPG resin Treated with 20% DEA/MeCN, 25 min to deprotect the acetal capping of the CPG hydroxyl groups CPG resin No treatment with DEA (control experiment) PS resin
Chromatogram A B 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.
A
Intens. [mAU] 40
30
20
10
10
12
14
16
Time [min]
B
Intens. [mAU]
60
40
20
0 0 2 4 6 8 10 12 14 16 Time [min]
C
Intens. [mAU]
30
20
10
10
12
14
16
Time [min]
Figure 55 HPLC UV 290 nm chromatograms, A) res3371, B) res3390 & C) res 3391 sequence: GTGGATA5FATGCG5FTCATGG (5F = monomer 5, labelled with FAM), peak at TR = 7.5 min: unlabelled; TR = 8.2 min: 1 FAM addition; TR = 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.
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 (DMTr OFF) 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.
Synthesise entire ONT (DMTr OFF), cap with hex-5-ynyl phosphoramidite ('X') OPG X 5' Remove, deprotect OPG 3'
OH X 5' 5'
OH 3' Couple to 6-FAM-phosphoramidite
Rep O O P O O X 5' 5'
Rep O O P O O
Rep O O P O O X 5'
Rep O O P O O 3'
Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule, FAM
Figure 56 Labelled-oligonucleotide synthesis approach C
This strategy was used to synthesis the following oligonucleotide (res3415) with monomer 5 on a PS solid support: 5-XGTGGATA5FATGCG5FTCATGG
Intens. [mAU] 1000
Intens. x105
-MS, 8.7-9.0min, Deconvoluted (MaxEnt) 7743.3
1.5
800
600
1.0
400
0.5
200
0 0 2 4 6 8 10 12 14 16 Time [min] RES3415CR1_RB3_01_17066.d: UV Chromatogram, 290 nm
7206.2 0.0
5000 5500 6000 6500 7000
F
7500
8000
F
8500 m/z
Figure 57 Left: HPLC UV 290 nm chromatogram for res3255, sequence: GTGGATA5 ATGCG5 TCATGG (5F = monomer 5, labelled with FAM), peak at TR = 8.6 min: [M-134], unknown, peak at TR = 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 conventionallylabelled 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.
OH O HO O HN O O O O A HO O O HN O O O O B N N H O P O O O N H O O OH O O N O N H H N O O O
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 Probe 1 Probe 2 Probe 3 WT Target MT Target res1137 res3210 res3391 res3375 res3376
Sequence GTGGATATAFGCGFTCATGG GTGGATATA5GCG1TCATGG GTGGATATA5GCG5TCATGG CCATGAACACATATATCCAC CCATGAACGCATATATCCAC
Duplex TM / C Wild Type Mutant 43.0 42.0 45.0 53.0 52.0 54.0 ---
TM / C 10.0 10.0 9.0
The probes successfully detected the SNP and forms a target with very similar physical properties to the probe synthesised with the commercially available FAM-dT-phosphoramidite monomer (figures 59 & 60).
Figure 59 Raw melting curve data of ONT probe-target duplexes
Figure 60 First derivative of melt curve results to show ONT duplex melt peaks
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.
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).
Synthesise Entire ONT OPGA 5' Remove, deprotect PGA OPGB OPGA OPGB 3'
OH 5'
OPGB
OH
OPGB 3'
Couple to reporter phosphoramidite A O(Rep)A 5' Deprotect PGB O(Rep)A 5' OH O(Rep)A OH OPGB O(Rep)A OPGB 3'
3' Couple to reporter phosphoramidite B O(Rep)A O(Rep)B O(Rep)A O(Rep)B 3' Cleavage & exocyclic nucleobase deprotection O(Rep)A O(Rep)B O(Rep)A
5'
O(Rep)B 3'
5'
Modied Monomer Solid Support (PS/CPG) PG = Protecting Group Rep = Reporter Molecule
Figure 61 Capacity for multiple and variant labelling
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.
O HN DMTr O O O O O OPG2 A) N
OPG1 DNA O
O HN O O O DNA B) O O N
Rep1 or ONTs O
CEP
Rep2 or ONTs
Rep1 or ONTs
Rep3 or ONTs
Rep5 or ONTs
C)
Rep2 or ONTs
Rep4 or ONTs
Rep6 or ONTs
Figure 62 A) Orthogonally protected phosphoramidite monomer, B) Reporter groups or branch extensions 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 Ramanactive 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
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 spermine group and fluorescent groups to oligonucleotides may be of use in cell imaging research.
TFA N
TFA N
N TFA
N TFA
P N
O N
O O TFA = triuoroacetyl protecting group F F F
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 costefficient, essential for viability of commercial applications.
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.
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. Reactions were performed under the positive pressure of an inert Argon atmosphere in oven-dry glassware with the inclusion of freshly microwave-activated molecular sieves (3 ). TLC analysis was achieved with Merck Kieselgel 60 F24 0.22 mm, Aluminium-backed silica plates containing fluorescent indicator (60F, 254) visualised with UV-irradiation (254 nm) and staining with p-anisaldehyde, ninhydrin, ceric sulphate, Marys Reagent or potassium permanganate solutions. Column chromatographic purification used Fischer scientific DAVISIL (60 pore size, 3570 m particle size) silica under positive air or Argon (see phosphitylation method).
Spectroscopic C nuclides were interrogated at 75 or 100 MHz and 1H at 300 or 400
13
MHz with the Bruker AC300 and Bruker DPX400, respectively. Chemical shift are reported in ppm relative to the 1H singlet of tetramethylsilane at 0 ppm and Jvalues are reported in Hz, correct to within 0.5 Hz. Individual spectra were internally referenced relative to the known signal of the corresponding residual undeuterated solvent. 1H assignment was assisted by proton-proton correlation spectroscopy (1H-1H COSY) experiment data and 135 (DEPT-135) experiment data. Low-resolution mass spectra were obtained by either positive or negative electrospray ionisation techniques (ESI+/-) on a Fisons VG platform instrument or Waters ZMD quadrupole mass spectrometer. High-resolution mass spectra were
13
C carbon atom hydrogen
substitution multiplicity by distortionless enhancement by polarisation transfer,
obtained by electrospray ionisation on a Bruker APEX III FT-ICR mass spectrometer.
Phosphitylation The phosphitylation reaction is a particularly sensitive process where the exclusion oxygen is essential. Dimethoxytrityl-protected hydroxy groups are acid-labile whilst the fluorenylmethoxycarbonyl-protected hydroxy groups, for example, are base-labile. Hence, the reaction must be handled extremely carefully. All glassware, including test tubes, will be freshly oven-dried and purged with Argon before being sealed. All transfers are made swiftly beneath a blanket of Argon. All solvents, eluents and aqueous washes were thoroughly degassed with Argon. Solvents and eluents are over molecular sieves. Rotary evaporation, column purification and compound desiccation are performed under Argon. Column silica is freshly oven-dried and column eluent contains 0.1% pyridine to neutralise silica acidity.
The Phosphoramidite Method Standard DNA phosphoramidites, FAM, solid supports and additional reagents were purchased from Link Technologies Ltd. unless stated otherwise. All oligonucleotides were synthesised on an Applied Biosystems 394 automated DNA/RNA synthesiser using a standard 0.2 mole phosphoramidite cycles of acid-catalysed detritylation, coupling, capping and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and in all cases were >98.0%. All cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use. The coupling time for normal (A, G, C, T) monomers was 40 s and the coupling time for the modified monomers was extended to 600 s, unless otherwise stated. Cleavage of oligonucleotides from the solid support and deprotection was achieved by exposure to concentrated aqueous ammonia for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 C. The solvent was removed in vacuo to afford the crude oligonucleotide in preparation for further purification.
Oligonucleotide Purification & Characterisation Reversed-phase (RP-) HPLC purification was carried out on a Gilson system using an ABI Aquapore column (C8), 8 mm x 250 mm, pore size 300 . The following protocols were used: run time 30 min, flow rate 4 mL min-1, binary system, gradient: time in min (% buffer B); 0 (0); 3 (0); 5 (10); 21 (40); 21 (60); 25 (100); 27 (0); 30 (0). Elution buffer A (0.1 M TEAA, pH 7.0), buffer B (0.1 M TEAA + 50% acetonitrile, pH 7.0). Elution of oligonucleotides was monitored by UV absorption (290 nm). Following HPLC purification, oligonucleotides were desalted twice using Sephadex G25 columns (NAP-25, followed by NAP-10, GE Healthcare), aliquoted into Eppendorf tubes and stored at 20 C. All oligonucleotides were characterised by Micro-TOF HPLC-MS.
Melting Analysis Duplex melt curve analysis was performed using a CFX96 instrument. Reactions were performed in 20 L reaction volumes in buffer (GoTaq flexi buffer, Promega, UK), comprising 0.1 M of the probe, 0.5 m M of targets, 3.0 mM of MgCl2, reactions were heated to and held at 95C for 30 sec then cooled to and held at 25C for 1 min. Melting curve analysis was then performed by heating reactions from 30C to 98C at 0.1C s-1. The fluorescence melting curve data were converted to the first derivatives giving the melting peaks ((FI)/T) where FI is fluorescent intensity and T is temperature in C).
Synthesis of Modified Monomers

MONOMER 1 2,2-Anhydro-5-methyluridine
O N HO O O OH N
Under an inert atmosphere (Ar) and magnetic stirring, 5-methyluridine (2.5 g, 9.7 mmol, 1.0 eq.) was dissolved in anhydrous DMF (8 mL) with diphenylcarbonate (2.48 g, 11.6 mmol, 1.1 eq.) and NaHCO3 (82 mg, cat). The reaction was heated (100C) for 5 h and monitored (TLC, 25% MeOH / DCM, all spots UV active and visualised with anisaldehyde stain) until determined complete by absence of limiting starting material. The reaction mixture was precipitated into diethyl ether (40 mL) and triturated to produce a gum then the diethyl ether supernatant decanted. The remaining solid was dissolved with the minimum possible amount of hot MeOH then concentrated in vacuo until the first signs of crystallisation were observed. The concentrated solution was gently cooled (4C) to promote crystallisation and left for 18 h. The solid clear crystals were separated from the orange supernatant by filtration. A total of three recrystallisation crops were harvested from the supernatant by this method. The solids were dried under high vacuum, yielding 1.98 g (7, 7.6 mmol, 79%) of crystalline product (7). C10H12N2O5 MW: 240.21 Rf (20 % iPrOH/DCM) = 0.06 H NMR (400 MHz, DMSO-d6) 7.74 (s, 1H, H6), 6.29 (d, J = 5.7 Hz, 1H, 1), 5.86 (s, 1H, 5OH), 5.17 (d, J = 5.6 Hz, 1H, 2), 4.94 (s, 1H, 3OH), 4.37 (s, 1H, 3), 4.06 (br t, 1H, 4), 3.29 3.22 (m, 1H, 5), 3.16 (m, 1H, 5), 1.79 (s, 3H, CH3).
1
C NMR (101 MHz, DMSO-d6) 171.62 (C), 159.34 (C), 132.21 (CH), 116.64 (C), 90.16 (CH), 89.08 (CH), 88.47 (CH), 74.69 (CH), 60.81 (CH2), 13.51 (CH3).
13
LRMS: [ESI-MS, MeCN] m/z (%): 239.2 ([M-H]-, 100%)
2,2-Anhydro-5-methyl-5-O-(4,4-dimethoxytrityl) uridine
O O N O O O OH O N
Compound 7 (1.41 g, 5.9 mmol, 1.0 eq.) was dissolved in anhydrous pyridine (20 mL) and stirred under inert and anhydrous conditions over molecular sieves (3 ) for 30 min. The gradual addition of DMTr-Cl (2.78 g, 8.2 mmol, 1.4 eq.) over the period of 2 h in 8 portions followed until the reaction was determined complete by TLC (10% MeOH/DCM + TEA) at t = 24 h. Remaining DMTr-Cl was quenched with TEA/MeOH (0.5 / 6.5 mL), stirred for a further 10 min then concentrated in vacuo, redissolved in DCM (20 mL) and washed with sat. NaHCO3, dried over anhydrous Na2SO4, filtered, concentrated in vacuo and purified (FCC, Isolera, SNAP 50 g, 0-10% MeOH/ DCM + 0.05% py). Combined pure fractions yielded 2.16 g of white solid (8, 4.0 mmol, 67%) following concentration in vacuo and placement under high-vacuum. C31H30N2O7 MW: 542.58
Rf (10% MeOH/DCM) = 0.77 H NMR (400 MHz, DMSO-d6) 7.83 (s, 1H, H6), 7.29 7.23 (m, 4H, DMT-ArH), 7.20 7.18 (m, 1H, DMTr-ArH), 7.15 7.12 (m, 4H, DMTr-ArH) 6.84 6.80 (m, 4H, DMT-ArH), 6.32 (d, J = 5.5 Hz, 1H, H1), 5.93 (d, J = 4.3 Hz, 1H, 3OH), 5.18 (d, J = 5.5 Hz, 1H, H2), 4.30 (t, J = 3.5 Hz, 1H, H3), 4.23 (dt, J = 6.8, 3.6 Hz, 1H, H4), 3.73 (d, J = 1.8 Hz, 6H, OCH3), 2.93 (m, 1H, H5), 2.79 (m, 1H, H5), 1.79 (s, 3H, CH3).
1
C NMR (100.6 MHz, DMSO-d6) 171.32 (C), 158.82 (C), 158.04 (C), 144.58 (C), 135.11 (C), 132.06 (CH), 129.40 (CH), 127.74 (CH), 127.42 (CH), 126.61 (CH), 116.86 (C), 113.13 (CH), 89.94 (CH), 88.09 (CH), 86.83 (CH), 85.35 (C), 74.82 (CH), 62.99 (CH2), 54.94 (OCH3), 38.89, 13.45 (CH3).
13
LRMS: [ESI+MS, MeCN] m/z (%): found: 565.2 ([M+Na]+, 100%); calcd.: 542.58
5-O-(4,4-Dimethoxytrityl)-2-O-hydroxyethyl-5-methyluridine
O O N O OH O O OH NH O O
NaHCO3 (167 mg, 1.9 mmol, 0.5 eq.), Ti(iPrOH)4 (1.58 g, 5.5 mmol, 1.4 eq.) and ethylene glycol (1.12 mL, 20 mmol, 5 eq.) were dissolved in anhydrous DMF (6 mL) and stirred under positive Argon pressure for 5 min before the temperature was raised to and maintained at 160C for 3.5 h. Compound 8 (2.15 g, 4.0 mmol, 1.0 eq.), dissolved in anhydrous DMF (5 mL) was then added and the reaction maintained under inert atmosphere and heating for 22 h until complete. The cooled reaction mixture was centrifuged to induce the sedimentation of titanium solids. The sediment was washed sequentially and the supernatant pooled and concentrated in vacuo. Purification was achieved by flash column chromatography (FCC, Isolera, SNAP 50 g, 0-5% MeOH/DCM + 0.1% py). The pure product, 9, was isolated as a pale yellow foam (1.49 g, 1.2 mmol, 62%). C33H36N2O9 MW: 604.65 Rf (5% MeOH/DCM) = 0.21 1H NMR (400 MHz, DMSO-d6) 11.37 (s, 1H, NH3), 7.49 (s, 1H, H6), 7.40 (d, J = 7.8 Hz, 2H, DMTr-ArH), 7.34 7.22 (m, 7H, DMTr-ArH), 6.90 (d, J = 8.3 Hz, 4H, DMTr-ArH), 5.86 (d, J = 4.5 Hz, 1H, H1), 5.10 (d, J = 6.2 Hz, 1H, 3OH), 4.73 (t, J = 5.3 Hz, 1H, CH2CH2OH), 4.30 4.21 (m, 1H, H3), 4.13 4.05 (m, 1H, H2), 4.02 3.94 (m, 1H, H4), 3.74 (s, 6H, OCH3), 3.70 3.49 (m, 4H, CH2CH2), 3.29 3.16 (m, 2H, H5), 1.41 (s, 3H, CH3). C NMR (100.6 MHz, DMSO-d6) 163.65 (C), 158.12 (C), 150.37 (C), 144.57 (C), 135.60 (CH), 135.14 (C), 129.68 (CH), 127.91 (CH), 127.63 (CH), 126.76 (CH), 113.22 (CH), 109.49 (C), 86.58 (CH), 85.86 (C), 82.84 (CH), 80.91 (CH), 68.92 (CH), 63.17 (CH2), 62.73 (CH2), 60.23 (CH2), 55.01 (OCH3), 11.64 (CH3).
13
LRMS: [ESI+MS, MeCN] m/z (%): found: 627.2 ([M+Na]+, 100%); calcd.: 604.65
5-O-(4,4-Dimethoxytrityl)-2-O-[2-(9-fluorenylmethyloxycarbonyloxy)]ethyl-5methyluridine
O O N O O O OH O O O O NH O
10
Compound 9 (1.29 g, 2.1 mmol, 1.0 eq.) was dissolved in anhydrous pyridine (5 mL) and stirred for 15 min under an inert and anhydrous conditions. The reaction was cooled to 0C before a solution of Fmoc-Cl (0.61 g, 2.4 mmol, 1.1 eq.) in anhydrous DCM (2 mL) was added gradually over a period of 10 min. The reaction was brought to and maintained at rt for 2.5 h until complete. The reaction mixture was concentrated in vacuo, redissolved in DCM (2 mL) and purified (FCC, Isolera, SNAP 25 g, 10-100% EtOAc/DCM + 0.1% py, 0-25% MeOH/DCM + 0.1% py) to give the pure product, 10, as a colourless foam. Yield: 0.71 g, 0.9 mmol, 40%. C48H46N2O11 MW: 826.89 Rf (6% MeOH/DCM + TEA) = 0.57 H NMR (400 MHz, DMSO-d6) 11.38 (s, 1H, NH), 7.89 (d, J = 7.6 Hz, 2H, Fmoc Ar-H), 7.63 (d, J = 7.4 Hz, 2H, Fmoc Ar-H), 7.48 (s, 1H, H-6), 7.45 7.19 (m, 15H, DMT Ar-H), 6.89 (d, J = 7.0 Hz, 4H, DMT Ar-H), 5.84 (d, J = 4.1 Hz, 1H, 1), 5.20 (d, J = 6.5 Hz, 1H, 3OH), 4.49 (d, J = 6.4 Hz, 2H, Fmoc-CH2), 4.29 (t, J = 6.5, 6.0 Hz, 1H, Fmoc-CH), 4.25 4.19 (m, 2H, 9, 2OCH2CH2), 4.06 (t, J = 5.6 Hz, 1H, 2), 3.98 (s, 1H, 4), 3.81 (dt, J = 19.7, 10.2 Hz, 2H, 2OCH2), 3.73 (s, 6H, OMe), 3.23 (dd, J = 6.4, 3.6 Hz, 2H, 5), 1.42 (s, 3H, Me).
1
C NMR (101 MHz, DMSO-d6) 163.68 (C2), 158.14 (Ar-C), 154.45 (Fmoc C=O), 150.37 (Ar-C), 144.61 (Ar-C), 143.30 (Fmoc Ar-C), 140.74 (Fmoc Ar-C), 135.55 (Ar-CH), 135.37 (Ar-C), 129.73 (Ar-CH), 127.89 (Ar-CH), 127.68 (Ar-CH), 127.14 (Fmoc Ar-CH), 126.78 (Ar-CH), 124.87 (Fmoc Ar-CH), 120.16 (Fmoc Ar-CH), 113.24 (Ar-CH), 109.50 (Ar-C), 86.85 (CH1), 85.83 (DMT-C), 82.75 (CH), 80.86 (CH), 68.70 (CH2), 68.64 (CH2), 67.63 (CH2), 66.64 (CH2), 63.02 (CH2), 54.88 (OMe), 46.23 (Fmoc CH), 11.72 (CH3).
13
LRMS: [ESI+MS, MeCN] m/z (%): 849.3 ([M+Na]+, 100%) HRMS: [ESI+] m/z: found: 849.3009 (M+Na)+; calcd.: 826.8902
5-O-(4,4-Dimethoxytrityl)-2-O-(2-(9-fluorenylmethyloxycarbonyl)ethyl)-5methyluridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite, monomer 1
O NH O O O O O O O N O
O N O
Compound 10 (600 mg, 0.73 mmol, 1.0 eq.) was dissolved into anhydrous DCM (7 mL) with 3 drops of DIPEA to neutralise inherent DCM acidity under an Argon atmosphere and anhydrous conditions. Stirring over molecular sieves was maintained for 15 min before the simultaneous addition of CEP-Cl (0.87 mmol, 206 mg, 195 L, 1.2 eq.) and DIPEA (253 L, 2.0 eq.). Reaction was deemed complete after 3 h and was immediately concentrated in vacuo then purified (column chromatography, 50% EtOAc/n-Hexane). Pure fractions were concentrated to dryness, placed under high-vacuum for 18 h, redissolved in acetonitrile and filtered to remove oxidised (PV) monomers. The pure solution was concentrated to dryness and placed under high vacuum to dry for 18 h, yielding monomer 1 as a pale yellow foam (529 mg, 0.52 mmol, 71%). C57H63N4O12P MW: 1027.10 Rf (80% EtOAc/n-Hex + py) = 0.43
31
P NMR (121 MHz, MeCN-d3) 151.09, 150.68 (PIII-diastereoisomers)
MONOMER 2 5-O-(4,4-Dimethoxytrityl)-5-(3-hydroxypropynyl)-2-deoxyuridine
O
O
OH
HN O O O OH
O
11
Under inert and anhydrous conditions, TEA (10 mL), copperI iodide (CuII) (0.1 g, 0.5 mmol, 0.2 eq.) and propargyl alcohol (0.54 g, 581 L, 2.0 eq.) and 9 (3.28 g, 5.0 mmol, 1.0 eq.) were dissolved in DMF (15 mL) and stirred at room temperature with molecular sieves (3 ) for 15 minutes prior to the addition of tetrakistriphenylphosphino palladium0 (Pd0(PPh3)4) (0.29 g, 0.3 mmol, 0.1 eq). The reaction was determined complete at t = 23 hours, concentrated to dry in vacuo, redissolved in EtOAc (50 mL) and washed with ethylenediamintetraacetic acid (EDTA) (basified to pH 8 with sodium hydroxide (NaOH), 30 mL x 2) and brine (30 mL). The organic phase was concentrated in vacuo yielding crude product as a yellow foam (3.56 g) that was purified (FCC, Isolera, SNAP 50 g, 80 - 100 % EtOAc /LPE (40 60), 0 30 % MeOH / EtOAc) to give pure product, 11, as a colourless foam (1.43 g, 2.5 mmol, 49%). C33H32N2O8 MW: 584.62 Rf (10% iPrOH/ DCM + TEA) = 0.20 H NMR (400 MHz, DMSO-d6) 11.62 (1H, s, NH), 7.89 (1H, s, H6), 7.41-7.20 (9H, m, DMT ArH), 6.89 (4H, d, J = 8.6 Hz, DMT ArH), 6.11 (1H,t, J = 7 Hz, H1), 5.31 (1H, d, J = 4.1 Hz, 3OH), 5.20 (1H, t, J = 6 Hz, OH), 4.25 (1H, br. t, H3), 4.08 (1H, d, J = 6 Hz, CH2OH), 3.92 (1H, br. m, H4), 3.74 (6H, s, OMe), 3.27-3.23 (1H, m, H5), 3.12-3.09 (1H, m, H5), 2.31-2.17 (2H, m, H2)
1
C NMR (100 MHz, DMSO-d6) 161.5 (C), 158.1 (C), 149.3 (C), 144.8 (CH), 143.0 (C), 135.6 (C), 135.3 (C), 129.7 (CH), 129.6 (CH), 127.9 (CH), 127.6 (CH), 126.6 (CH), 113.2 (CH), 98.5 (C), 92.5 (C), 85.8 (CH), 85.0 (CH), 75.9 (C), 70.4 (CH), 63.7 (CH2), 55.0 (CH3), 49.4 (CH2), 39.7 (CH2).
13
LRMS: [ES-MS, MeCN] m/z (%): 583.2 ([M-H]-, 100 %) HRMS: [ESI+] m/z: found: 607.2062 (M+Na)+; calcd.: 584.6189
5-O-(4,4-Dimethoxytrityl)-5-(prop-3-(9-fluorenylmethyloxycarbonyl)-ynyl)-2deoxyuridine
O O
O
HN O O O OH
O
12
Compound 11 (0.75 g, 1.3 mmol, 1.0 eq.) was dissolved in anhydrous DCM (3 mL), basified with DIPEA (100 L) under inert and anhydrous conditions. The solution was stirred in the presence of molecular sieves for 15 min to permit sequestration of water. The reaction mixture was cooled to 0C before Fmoc-Cl (0.32 g, 1.2 mmol, 0.95 eq.) was dissolved in anhydrous pyridine (1 mL) and added slowly over 10 min. The reaction temperature was raised to rt and stirring continued until consumption of Fmoc-Cl was confirmed at 2 h by TLC. The reaction mixture was concentrated in vacuo and purified (FCC, SNAP 10 g, 50-80 EtOAc/LPE (40 60)). The final product, 12, was isolated as a pale yellow foam by concentration of pure fractions and dried to give 0.85 g of 12 (1.1 mmol, 82%). C48H42N2O10 Mw: 806.85 Rf (6% MeOH/DCM + py) = 0.34 H NMR (400 MHz, DMSO-d6) 11.71 (s, 1H, NH3), 7.99 (s, 1H, H6), 7.89 (m, 2H, Fmoc ArH), 7.64 (d, J = 7.6 Hz, 2H, Fmoc ArH), 7.48 7.13 (m, 13H, Fmoc + DMT ArH), 6.88 6.85 (m, 4H, DMT ArH), 6.09 (t, J = 6.6, 6.1 Hz, 1H, H1), 5.32 (d, J = 4.6 Hz, 1H, 3OH), 4.78 4.64 (m, 2H, CCH2OC), 4.55 (d, J = 6.1 Hz, 2H, Fmoc CH2), 4.29 (m, 2H, H3& Fmoc CH), 3.93 (m, 1H, H4), 3.72 (s, 6H, OMe), 3.26 (dd, J = 10.8, 5.4 Hz, 1H, H5), 3.08 (dd, J = 10.5, 3.1 Hz, 1H, H5), 2.31 2.18 (m, 2H, H2).
1
C NMR (100.6 MHz, DMSO) 161.33 (C), 158.03 (C), 153.80 (C), 149.25 (C), 144.72 (C), 143.20 (C), 140.77 (C), 135.54 (C), 135.22 (C), 129.66 (CH), 129.61 (CH), 127.83 (CH), 127.72 (CH), 127.52 (CH), 127.12 (CH), 126.60 (CH), 124.84 (CH), 120.17 (CH), 113.15 (CH), 97.24 (C), 85.87 (CH) 85.81 (C), 85.13 (CH), 79.40 (C), 70.31 (CH), 69.01 (CH2), 63.63 (CH2), 55.76 (CH2), 54.96 (CH3), 46.19 (CH), 39.73 (CH2).
13
LRMS: [ES+MS, MeCN] m/z (%): 829.2 ([M+Na]+, 100 %) HRMS: [ESI+] m/z: found: 829.2731 (M+Na)+; calcd.: 806.8590
5-O-(4,4-Dimethoxytrityl)-5-(3-(9-fluorenylmethyloxycarbonyl)-propynyl)-2deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite, monomer 2
O O HN O O
O O O P
N O
2
All processes described were performed under an Argon atmosphere. Compound 12 (0.75 g, 0.9 mmol, 1.0 eq.) was dissolved in anhydrous DCM (6 mL) with DIPEA (100 L, 0.6 mmole, 0.6 eq) and stirred with molecular sieves for 15 min before the addition of DIPEA (240 L, 1.4 mmol, 1.5 eq.). The simultaneous addition of CEP-Cl (250 L, 1.1 mmol, 1.2 eq.) and DIPEA (160 L, 1.0 mmol, 1.0 eq.) followed and the reaction was continued until completion after 2h. The reaction was concentrated to dryness, redissolved in anhydrous DCM and rapidly washed (degassed sat. KCl (aq.). The organic phase was dried over anhydrous Na2SO4 and concentrated before purification (column chromatography, 50% EtOAc/n-Hexane (degassed) + 0.1 % py). Pure fractions were concentrated to dryness, redissolved (MeCN, 5 mL) and filtered. Finally, solvent was evaporated to yield monomer 2 as a colourless foam (0.57 g, 0.6 mmol, 60%). C57H59N4O11P MW1007.07 Rf (80% EtOAc/n-Hex + 0.1 % py) = 0.53
31
P NMR (121 MHz, MeCN-d3) 151.09, 150.68 (PIII-diastereoisomers)
MONOMER 3 5-O-(4,4-Dimethoxytrityl)-2-O-(2-azidoethyl)-5-methyluridine
O HN O O O OH O N N N N
13
Sodium azide (0.56 g, 8.6 mmol, 5 eq.) and 18-crown-6 (5 mg, 0.002 mmol, 0.01 eq.) were added to a stirred solution of mesyl-protected (1.16 g, 1.7 mmol, 1.0 eq.) in anhydrous DMF (6 mL). The reaction mixture was heated under an inert Argon atmosphere at 60C for 14 h. The solvent was removed in vacuo and the mixture partitioned between DCM and deionised water. The aqueous layer was extracted twice with DCM and the pooled organic washed with sat. NaCl (aq.) then dried over Na2SO4, filtered and concentrated. C33H37N5O8 MW: 631.68 This compound is yet to be purified and analysed.
5-O-(4,4-Dimethoxytrityl)-2-O-(2-aminoethyl)-5-methyluridine
O HN O O O O
OH O NH2
14
(0.5 g,
5-O-(4,4-Dimethoxytrityl)-2-O-(2-N-phthalimidoethyl)-5-methyluridine
0.7 mmol, 1.0 eq.) was dissolved in anhydrous MeOH (10 mL) under an inert Argon atmosphere. To this stirred solution, hydrazine monohydrate (0.17 g, 3.4 mmol, 5 eq.) was added and the temperature raised to reflux for 3.5 h. The completed reaction mixture was purified (FCC, Isolera, SNAP 25 g, 0-10% MeOH/DCM + 0.1% py), solvent evaporated in vacuo and the compound dried in high vacuum. The pure product, 14, was obtained as a pale yellow foam (0.39 g, 0.6 mmol, 88%). C33H37N3O8 MW: 603.66 Rf (20% MeOH/DCM + TEA) = 0.27 H NMR (300 MHz, DMSO-d6) 8.06 (dd, J = 5.9, 3.3 Hz, 1H, NH2), 7.80 (dd, J = 5.9, 3.3 Hz, 1H, NH2), 7.50 (s, 1H, H6), 7.44 7.37 (m, 2H, DMT Ar-CH), 7.36 7.20 (m, 7H, DMT Ar-CH), 6.97 6.85 (m, 4H, DMT Ar-CH), 5.86 (d, J = 4.7 Hz, 1H, H1), 4.24 (t, J = 5.1 Hz, 1H, H3), 4.05 (t, J = 4.9 Hz, 1H, 4OH), 4.00 (m, 1H, H2), 3.74 (s, 6H, OMe), 3.68 (t, J = 5.0 Hz, 1H, CH2CH2NH2), 3.53 (m, 1H, CH2CH2NH2), 3.32 3.17 (m, 2H, CH2NH2), 2.84 2.64 (m, 2H, H5), 1.41 (s, 3H, CH3).
1
C NMR (100.6 MHz, DMSO) 163.62 (C), 158.13 (C), 150.43 (C), 144.59 (C), 131.91 (CH), 129.69 (CH), 127.90 (CH), 127.64 (CH), 126.79 (CH), 113.24 (CH), 109.54 (C), 86.46 (CH), 85.87 (C), 82.93 (CH), 81.21 (CH), 71.33 (C), 68.92 (CH), 55.02 (OMe), 40.31 (CH2), 11.67 (CH3).
13
LRMS: [ES+MS, MeCN] m/z (%): 626.1 ([M+Na]+, 100 %)
5-O-(4,4-Dimethoxytrityl)- 2-O-(6-(tertbutyldimethylsilyloxy)hexanamido)ethyl)-5-methyluridine
O HN O O O O OH O O HN
5
O Si
15
stirred
solution was
of
5-O-(4,4-dimethoxytrityl)-2-O-(2-aminoethyl)-5added a solution of succinimidyl 6-(O-tert-
methyluridine (0.39 g, 0.6 mmol, 1.0 eq.) in anhydrous DCM (3 mL) under an Argon atmosphere butyldimethylsilyl)hexanoate (0.26 g, 0.8 mmol, 1.2 eq.) in anhydrous DCM (3 mL) and DIPEA (170 L, 0.1 mmol, 1.5 eq.). After 4 h, the completed reaction was diluted with DCM (20 mL) and washed with deionised water, dried over anhydrous Na2SO4, filtered, concentrated in vacuo and purified (column chromatography, 80-100% EtOAc/LPE (40-60)). Pure fractions were combined, concentrated to dryness in vacuo and placed under high vacuum, yielding compound 15 as a pale yellow foam (0.42 g, 0.5 mmol, 78%). C45H61N3O10Si MW: 832.07 Rf (7% MeOH/DCM + TEA) = 0.48 H NMR (400 MHz, DMSO-d6) 11.37 (s, 1H, NH3), 7.82 7.73 (m, 1H, NHC=O), 7.48 (d, J = 1.5 Hz, 1H, H6), 7.44 7.34 (m, 2H, DMT ArH), 7.36 7.19 (m, 7H, DMT ArH), 6.95 6.85 (m, 4H, DMT ArH), 5.83 (d, J = 4.0 Hz, 1H, H1), 5.10 (d, J = 6.7 Hz, 1H, 3OH), 4.26-4.21 (m, 1H, H3), 4.05 3.94 (m, 2H, H2, H4), 3.74 (s, 6H, OMe), 3.68 3.50 (m, 4H, NHCH2CH2O, CH2OSi), 3.34 3.16 (m, 4H, H5, CH2NH), 2.04 (t, J = 7.4 Hz, 2H, NHC=OCH2), 1.54 1.37 (m, 7H, 5-CH3, CH2CH2CH2CH2OTBDMS), 1.25 (m, 2H, CH2CH2CH2OTBDMS), 0.84 (s, 9H, C(CH3)3), 0.00 (s, 6H, Si(CH3)2).
1
C NMR (100.6 MHz, DMSO-d6) 172.13 (C), 163.63 (C), 158.13 (C), 150.35 (C), 144.58 (C), 135.44 (CH), 135.36 (C), 135.15 (C), 129.69 (CH), 127.88 (CH), 127.66 (CH), 126.77 (CH), 113.23 (CH), 109.43 (C), 86.81 (CH), 85.82 (C), 82.68 (CH), 80.90 (CH), 68.89 (CH2), 68.78 (CH), 62.96 (CH2), 62.35 (CH2), 62.31 (CH2), 55.01 (OCH3), 38.38 (CH2), 35.36 (CH2), 32.04 (CH2), 25.78 (CH3), 24.97 (CH2), 17.90 (CH3), 11.72 (CH3), -5.35 (SiCH3).
13
5-O-(4,4-Dimethoxytrityl)-2-O-(6-(tertbutyldimethylsilyloxy)hexanamido)ethyl)-5-methyluridine-3-O-(O-2cyanoethyl-N,N-diisopropyl) phosphoramidite, monomer 3
O HN O O O O P N N O O
O O HN
5
O Si
Compound 15 (0.39 g, 0.5 mmol, 1.0 eq.) was dissolved under inert and anhydrous conditions in anhydrous DCM (5 mL) with DIPEA (30 L) to neutralise the still DCM. To this stirred solution, CEP-Cl (126 L, 0.6 mmol, 1.2 eq.) and DIPEA (130 L, 0.9 mmol, 2.0 eq.) were simultaneously added. After 4 h, the completed reaction was immediately concentrated in vacuo then purified (column chromatography, 50-60% EtOAc/n-Hexane (degassed) + 0.1% py). Pure fractions were concentrated to dryness, placed under high vacuum for 18 h, redissolved in acetonitrile and filtered to remove oxidised (PV) monomers. The pure solution was concentrated to dryness and placed under high vacuum to dry for 18 h, yielding monomer 3 as a white foam. (0.14 g, 0.14 mmol, 29%) C54H78N5O11PSi MW: 1032.28 Rf (EtOAc + py) = 0.60
31
P NMR (400 MHz, MeCN-d3) (PIII-diastereoisomers) 149.3
MONOMER 4 5-O-(4,4-Dimethoxytrityl)-5-(prop-3-(6-(tert-butyldimethylsilyloxy)hexanamido)-ynyl)-2-deoxyuridine
O O O HN O O O OH O N N H O
5
Si
16
A solution of 6-O-TBDMS-succinimidyl ester (0.39 g, 1.2 mmol, 1.2 eq.) in anhydrous DCM (5 mL) was added with DIPEA (250 L, 1.4 mmol, 1.5 eq.) to a stirred solution of DMT-5-PROPARGYLAMINE (0.56 g, 1.0 mmol, 1.0 eq.) in anhydrous DCM (5 mL) under an inert Argon atmosphere. The reaction was complete after 4 h, concentrated in vacuo, diluted with DCM (20 mL), washed with sat. NaHCO3
(aq.)
dried
over
anhydrous
Na2SO4
before
filtration, (50-70%
concentration in vacuo and purification by column chromatography colourless foam. (0.59 g, 0.7 mmol, 63%) C45H57N3O9Si MW: 812.03 Rf (80% EtOAc/LPE + py) = 0.23
EtOAc/LPE (40-60)). Combined pure fractions yielded compound 16 as a
H NMR (300 MHz, DMSO-d6) 11.66 (s, 1H, NH3), 8.15 (t, J = 5.3 Hz, 1H, CCH2NH), 7.90 (s, 1H, H6), 7.45 7.16 (m, 9H, DMT ArH), 6.96 6.83 (m, 4H, DMT ArH), 6.10 (t, J = 6.6 Hz, 1H, H1), 5.32 (d, J = 4.4 Hz, 1H, 3OH), 4.27 (m, 1H, H3), 4.00 3.85 (m, 3H, H4, CH2NH), 3.74 (s, 6H, OMe), 3.54 (t, J = 6.3 Hz, 2H, CH2OTBDMS), 3.26 (dd, J = 10.5, 5.4 Hz, 1H, H5), 3.08 (dd, J = 10.2, 3.1 Hz, 1H, H5), 2.35 2.12 (m, 2H, H2), 2.12 1.95 (m, 2H, NHCOCH2), 1.56 1.35 (m, 4H, CH2CH2CH2CH2OTBDMS), 1.33 1.14 (m, 2H, CH2CH2CH2OTBDMS), 0.85 (s, 9H, tBu), 0.01 (s, 6H, SiCH3).
1
C NMR (100.6 MHz, DMSO-d6) 171.61 (C), 161.48 (C), 158.03 (C), 149.26 (C), 144.74 (C), 143.17 (CH), 135.63 (C), 135.16 (C), 129.71 (CH), 127.84 (CH), 127.48 (CH), 126.59 (CH), 113.21 (CH), 98.32 (C), 89.60 (C), 85.80 (CH), 84.96 (CH), 73.92 (C), 70.45 (C), 63.74 (CH2), 62.33 (CH2), 54.98 (OMe), 39.71 (CH2), 35.03 (CH2), 32.03 (CH2), 28.46 (CH2), 25.79 (CH), 24.95 (CH2), 24.86 (CH2), 17.89 (tBu C), -5.34 (SiMe).
13
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
O O O HN O O O O O P N O N N N H O
5
Si
4
Compound 16 (560 mg, 0.68 mmol, 1.0 eq.) was dissolved in anhydrous DCM (7 mL) and DIPEA (100 L) under an inert Argon atmosphere and anhydrous conditions. The solution was stirred for 15 min to allow sequestration of water. To this solution, CEP-Cl (233 L, 0.8 mmol, 1.2 eq.) was added with DIPEA (240 L, 1.4 mmol, 2.0 eq.). Reaction was determined complete after 2.5 h and was concentrated in vacuo, diluted with anhydrous DCM (20 mL) and washed with degassed, sat. KCl (aq.), dried over anhydrous Na2SO4, filtered and again concentrated in vacuo. The concentrated crude solution was immediately subjected to purification by column chromatography (80% EtOAc/n-Hexane (degassed) + anhydrous py). Pure fractions were combined and the solvent evaporated in vacuo. The concentrate was redissolved in MeCN (5 mL) and filtered. The MeCN was removed in vacuo and the pure compound dried under high vacuum for 18 h. Monomer 4 was obtained as a colourless foam (0.48 g, 0.5 mmol, 70%). C54H74N5O10PSi MW1012.25 Rf (EtOAc + py) = 0.60
31
P NMR (400 MHz, Acetonitrile-d3) 149.3
MONOMER 5 N-Prop-2-ynyl-6-hydroxyhexanamide
O N H OH 17
Propargylamine (0.36 g, 5.7 mmol, 1.5 eq.) was added to 6-hydroxyhexanoic acid (0.5 g, 3.8 mmol, 1.0 eq.) in DMF. To this reaction mixture, EDC-Cl (1.09 g, 5.7 mmol, 1.5 eq.) was added and the reaction stirred under an Argon atmosphere for 18 h. Upon completion, the reaction was concentrated to dryness in vacuo, redissolved in DCM (10 mL) and extracted to aqueous at pH 14 through addition of NaOH (aq., 2 M). The aqueous was neutralised to pH 7 with HCl (aq., 2 M) and the product extracted with butan-1-ol. The solution was concentrated in vacuo to give an orange-coloured oil. This oil was diluted with DCM (10 mL) and MeOH (2 mL) and filtered to remove insoluble salts. The filtrate was concentrated in vacuo and purified (column chromatography, 0-5% MeOH/EtOAc) to give compound 17 as a colourless oil. (0.43 g, 2.6 mmol, 67%) C9H15NO2 MW: 169.22 Rf (15% MeOH/DCM + TEA) = 0.44 H NMR (300 MHz, DMSO-d6) 8.20 (t, J = 5.4 Hz, 1H, NH), 4.33 (t, J = 5.1 Hz, 1H, OH), 3.83 (dd, J = 5.5, 2.6 Hz, 2H, NHCH2), 3.43 3.30 (m, 2H, CH2CO), 3.06 (t, J = 2.5 Hz, 1H, CH), 2.06 (m, 2H, CH2OH), 1.581.32 (m, 2H, CH2CH2CH2CH2OH), 1.32 1.13 (m, 2H, CH2CH2CH2OH).
1
C NMR (75 MHz, DMSO) 171.87 (C), 81.36 (C), 72.74 (CH), 60.60 (CH2), 35.13 (CH2), 32.27 (CH2), 27.69 (CH2), 25.19 (CH2), 25.08 (CH2).
13
LRMS: [ES-MS, MeCN] m/z (%): 187.1 ([M+18]-, 100 %) HRMS: [ESI+] m/z: found: 192.0995 (M+Na)+; calcd.: 169.2218
N-prop-2-ynyl-6-levulinyloxyhexanamide
O N H O O 18 O
Levulinic acid (2.32 g, 2.05 mL, 20.0 mmol, 2.0 eq.) was dissolved in anhydrous Et2O and stirred with molecular sieves under an inert atmosphere. To this solution, DDC (2.05 g, 10.0 mmol, 1.0 eq.) was added with instant observable precipitation of the dicyclohexylcarbamide by-product; the reaction was determined complete after 2 h. The reaction mixture was filtered through Celite and washed with Et2O then concentrated in vacuo to give a colourless oil that was dried under high vacuum to give compound 14 as a clear/white liquid/solid (1.33 g, 12.4 mmol, 62%). The anhydride, prepared in situ (0.6 g, 2.8 mmol, 1.4 eq.) was dissolved in anhydrous DCM (3 mL) and anhydrous DMF (1.5 mL), cooled to 0C. Compound 17, (0.36 g, 2.0 mmol, 1.0 eq.) was dissolved in anhydrous DCM (2 mL) and anhydrous DMF (1.5 mL) and added to the stirred, cooled anhydride solution. The reaction mixture was allowed to warm to rt and proceed for 1.5 h. The completed reaction was concentrated to dryness in vacuo, redissolved in DCM (15 mL) and washed with sat. NaHCO3 (aq., 15 mL), followed by NaOH (aq., 0.1 M, 15 mL). The organic phase was concentrated in vacuo and purified (FCC, Isolera, SNAP 10 g, 2-5% MeOH/DCM), the solvent removed an the compound dried under high vacuum to give compound 18 as a colourless viscous oil (0.37 g, 1.4 mmol, 70%) C14H21NO4 MW: 267.32 Rf (15% MeOH/DCM + TEA) = 0.79 H NMR (400 MHz, CDCl3) 5.85 (s, 1H, NH), 4.06 3.93 (m, 4H, CH2O, CH2NH), 2.69 (t, J = 6.4 Hz, 2H, CH2COO), 2.49 (t, J = 6.4 Hz, 2H, CH2CH2COO), 2.29 2.09 (m, 4H, HC, CH3), 1.70 1.52 (m, 4H, CH2CH2CH2CH2O), 1.44-1.36 (m, 2H, CH2CH2CH2O).
1
C NMR (75.5 MHz, CDCl3) 206.90 (C), 172.78 (C), 172.45 (C), 79.66 (C), 71.46 (CH), 64.42 (CH2), 37.96 (CH2), 36.19 (CH2), 29.89 (CH3), 29.11 (CH2), 28.25 (CH2), 27.97 (CH2), 25.53 (CH2), 25.04 (CH2).
13
LRMS: [ES+MS, MeCN] m/z (%): 290 ([M+Na]+, 100 %)
5-O-(4,4-dimethoxytrityl)-5-(prop-3-(6-(levulinyloxy)-hexanamido)-ynyl)-2deoxyuridine
O O O HN O O O OH O N N H O
5
19
Copper iodide (0.05 g, 0.25 eq., 0.3 mmol), TEA (0.5 mL), 5-O-(4,4dimethoxytrityl)-2deoxy-5-iodoruridine (0.32 g, 1.2 mmol, 1.2 eq.), compound 18 (0.66 g, 1.0 mmol, 1.0 eq.) were dissolved in anhydrous DMF (5 mL) and stirred under an inert Argon atmosphere for 15 min before the addition of Pd0(PPh3)4 (0.12 g, 0.1 mmol, 0.1 eq.). The reaction was complete at 1.5 h then concentrated to dry in vacuo. The crude mixture was redissolved in EtOAc (50 mL) and washed with EDTA (pH 8, NaOH (aq.), 20 mL) then brine (20 mL). The organic phase was concentrated in vacuo and purified (FCC, Isolera, SNAP 10 g, 80-100% EtOAc/LPE (40-60), 0-10% MeOH/EtOAc + 0.1% py) to give compound 19 as a yellow foam after concentration in vacuo and drying under high vacuum. (0.47 g, 0.6 mmol, 59%) C44H49N3O11 MW: 795.87 H NMR (400 MHz, DMSO-d6) 11.65 (s, 1H, NH3), 8.16 (t, J = 5.3 Hz, 1H, NHC=O), 7.90 (s, 1H, H6), 7.46 7.37 (m, 2H, DMT ArH), 7.36 7.18 (m, 7H, DMT ArH), 6.94 6.85 (m, 4H, DMT ArH), 6.10 (t, J = 6.7 Hz, 1H, H1), 5.32 (d, J = 4.5 Hz, 1H, 3OH), 4.29-4.25 (m, 4.0 Hz, 1H, H3), 3.97-3.89 (m, 5H, CH2OCO, H4, CH2NH), 3.74 (s, 6H, OMe), 3.26 (dd, J = 10.5, 5.4 Hz, 1H, H5), 3.08 (dd, J = 10.6, 3.1 Hz, 1H, H5), 2.69 (t, J = 6.5 Hz, 2H, CH2C=OCH3), 2.43 (t, J = 6.5 Hz, 2H, CH2C=OCH3), 2.33 2.13 (m, 2H, H2), 2.12 2.01 (m, 5H, NHCOCH2, CH3), 1.58 1.42 (m, 4H, CH2CH2CH2CH2OH), 1.26 (m, 2H, CH2CH2CH2OH).
1
C NMR (101 MHz, DMSO) 206.70 (C), 172.21 (C), 171.54 (C), 161.50 (C), 158.03 (C), 149.27 (C), 144.75 (C), 143.19 (CH), 135.63 (C), 135.16 (C), 129.70 (CH), 129.57 (CH), 127.85 (CH), 127.48 (CH), 126.59 (CH), 113.21 (CH), 98.29 (C), 89.59 (C), 85.80 (CH), 84.95 (CH), 73.95 (C), 70.44 (CH), 63.73 (2CH2), 54.98 (OMe), 39.78 (CH2), 37.38 (CH2), 34.86 (CH2), 29.49 (CH3), 28.47 (CH2), 27.82 (CH2), 27.58 (CH2), 24.99 (CH2), 24.67 (CH2).
13
5-O-(4,4-Dimethoxytrityl)-5-(3-(6-(levulinyloxy)-hexanamido)-propynyl)-2deoxyuridine-3-O-(O-2-cyanoethyl-N,N-diisopropyl) phosphoramidite, monomer 5
O O O HN O O O O O P N O N N N H O
5
5
Compound 19 (0.37 g, 0.5 mmol, 1.0 eq.) was dissolved under an inert Argon atmosphere in anhydrous DCM (5 mL) with DIPEA (20 L) and stirred for 15 min with molecular sieves (3 ) to permit the sequestration of water. The simultaneous addition of CEP-Cl (130 L, 0.6 mmol, 1.2 eq.) and DIPEA (170 L, 0.1 mmol, 2.0 eq.) followed. The reaction was determined complete at 3 h - 30 min after the further addition of CEP-Cl (10 L, 0.6 mmol, 0.1 eq.). Reaction was concentrated in vacuo, diluted with anhydrous DCM (20 mL) and washed with degassed, sat. KCl (aq.), dried over anhydrous Na2SO4, filtered then concentrated in vacuo. The concentrate was immediately purified by column chromatography (EtOAc (degassed) + 0.1% anhydrous py). Pure fractions were combined and the solvent evaporated in vacuo. The concentrated monomer was redissolved in MeCN (5 mL) and filtered. The MeCN was removed in vacuo and the monomer dried under high vacuum for 18 h. The pure monomer, 5, was obtained as a colourless foam. (0.36 g, 0.4 mmol, 74%) C53H66N5O12P MW: 996.09
31
P NMR (121 MHz, Acetonitrile-d3) 149.31, 149.38.
5-O-(4,4-Dimethoxytrityl)-5-(3-(6-(hydroxy)-hexanamido)-propynyl)-2deoxyuridine
O O O HN O O O OH O 13 N N H OH
In DMF (7 mL) 6-hydroxyhexanoic acid (0.33 g, 2.5 mmol, 1.2 eq.) and EDC (490 mg, 2.5 mmol, 1.2 eq.) were combined and stirred under Argon with molecular sieves for 15 min. To this reaction mixture, a solution of 5-propargylamine DMT dU (1.20 g, 2.1 mmol, 1.0 eq.) in DMF (3 mL) was added and the reaction stirred for 18 h. Addition 6-hydroxyhexanoic acid (0.33 g, 2.5 mmol, 1.2 eq.) and EDC (0.49 g, 2.5 mmol, 1.2 eq.) was added and the reaction continued for a further 4 h before evaporation to dryness in vacuo and resolvation with DCM (25 mL). The reaction solution was washed with deionised water, concentrated in vacuo and purified (FCC, Isolera, SNAP 25 g, 0-5% MeOH/EtOAc + 0.1% py). Pure fractions were pooled, concentrated in vacuo, dried under high vacuum to yield product 13 as a pale orange foam (0.86 g, 1.2 mmol, 60%). C39H43N3O9 MW: 697.77. This compound is a useful precursor to C5-modified monomers. Rf (12.5% MeOH/EtOAc + py) = 0.36 H NMR (400 MHz, DMSO-d6) 11.65 (s, 1H, NH3), 8.15 (t, J = 5.4 Hz, 1H, NHCO), 7.90 (s, 1H, H6), 7.45 7.35 (m, 2H, DMT ArH), 7.35 7.20 (m, 7H, DMT Ar-CH), 6.94 6.83 (m, 4H, DMT ArH), 6.10 (t, J = 6.7 Hz, 1H, H1), 5.32 (d, J = 4.5 Hz, 1H, 3OH), 4.30 (m, 1H, H3), 3.98 3.87 (m, 3H, H4, CH2NH), 3.74 (s, 6H, OMe), 3.36 (td, J = 6.5, 5.0 Hz, 2H, CH2OH), 3.26 (dd, J = 10.5, 5.5 Hz, 1H, H5), 3.08 (dd, J = 10.5, 3.0 Hz, 1H, H5), 2.31 2.16 (m, 2H, H2), 2.06 (t, J = 7.5 Hz, 2H, CH2CO), 1.53-1.35 (m, 4H, CH2CH2CH2CH2OH), 1.30 1.20 (m, 2H, CH2CH2CH2OH).
1
C NMR (100.6 MHz, DMSO-d6) 171.67 (C), 161.49 (C), 158.01 (C), 149.27 (C), 144.73 (C), 143.19 (CH), 135.61 (C), 135.16 (C), 129.70 (CH), 127.84 (CH), 127.48 (CH), 126.59 (CH), 113.17 (CH), 98.29 (C), 89.60 (C), 85.77 (CH), 84.94 (CH), 73.92 (C), 70.42 (CH), 63.73 (CH2), 60.55 (CH2), 54.97 (OCH3), 39.90 (CH2), 35.09 (CH2), 32.24 (CH2), 28.45 (CH2), 25.19 (CH2), 24.99 (CH2)
13
References
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inspired Materials. Chemistry A European Journal 2012, 18 (15), 4456-4469; (b) Ranasinghe, R. T.; Brown, T., Fluorescence based strategies for genetic analysis. Chem. Commun. 2005, (44), 5487-5502; (c) French, D. J.; Howard, R. L.; Gale, N.; Brown, T.; McDowell, D. G.; Debenham, P. G., Interrogation of short tandem repeats using fluorescent probes and melting curve analysis: A step towards rapid DNA identity screening. Forensic Science International: Genetics 2008, 2 (4), 333-339. 2. 3. Various, Wikipedia Commons Open Source Images. Goldman, N.; Bertone, P.; Chen, S.; Dessimoz, C.; LeProust, E. M.; Sipos, B.;
Birney, E., Towards practical, high-capacity, low-maintenance information storage in synthesized DNA. Nature 2013, 494 (7435), 77-80. 4. (a) Gale, N.; French, D. J.; Howard, R. L.; McDowell, D. G.; Debenham, P. G.; Brown, T., Rapid typing of STRs in the human genome by HyBeacon[registered sign] melting. Organic & Biomolecular Chemistry 2008, 6 (24), 4553-4559; (b) Ben Gaied, N.; Richardson, J. A.; Singleton, D. G.; Zhao, Z. Y.; French, D.; Brown, T., End-capped HyBeacon probes for the analysis of human genetic polymorphisms related to warfarin metabolism. Organic & Biomolecular Chemistry 2010, 8 (12), 2728-2734. 5. (a) G. Michael Blackburn, M. J. G., Nucleic Acids in Chemistry and Biology. Second ed.; Oxford University Press: New York, 1996; (b) Doonan, S., Nucleic Acids. The Royal Society of Chemistry: Cambridge, 2004; (c) Eckstein, F., Oligonucleotides and Analogues - A Practical Approach. Oxford University Press: New York, 1991. 6. 7. Matteucci, M. D.; Caruthers, M. H., Nucleotide chemistry .4. Synthesis of (a) Kaufman, J.; Le, M.; Ross, G.; Hing, P.; Budiansky, M.; Yu, E.; Campbell, deoxyoligonucleotides on a polymer support. JACS 1981, 103 (11), 3185-3191. E.; Yoshimura, V.; Fitzpatrick, V.; Nadimi, K.; et al., Trityl monitoring of automated DNA synthesizer operation by conductivity: a new method of real-time analysis. BioTechniques 1993, 14 (5), 834-9; (b) Kaufman, J.; Le, M.; Ross, G.; Hing, P.; Budiansky, M.; Yu, E.; Campbell, E.; Yoshimura, V.; Fitzpatrick, V.; Nadimi, K., Trityl monitoring of automated DNA synthesizer operation by conductivity: a new method of real-time analysis. BioTechniques 1993, 14 (5), 834-839.
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Glen Research. http://www.glenresearch.com (accessed 10/04/2013). (a) Levsky, J. M.; Singer, R. H., Fluorescence in situ hybridization: past,
present and future. J. Cell Sci. 2003, 116 (14), 2833-2838; (b) Thelwell, N.; Millington, S.; Solinas, A.; Booth, J.; Brown, T., Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 2000, 28 (19), 37523761; (c) Kranaster, R.; Marx, A., Increased single-nucleotide discrimination in allele-specific polymerase chain reactions through primer probes bearing nucleobase and 2'-deoxyribose modifications. Chemistry-a European Journal 2007, 13 (21), 6115-6122; (d) Darby, R. A. J.; Sollogoub, M.; McKeen, C.; Brown, L.; Risitano, A.; Brown, N.; Barton, C.; Brown, T.; Fox, K. R., High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler. Nucleic Acids Res. 2002, 30 (9), e39; (e) Richardson, J. A.; Gerowska, M.; Shelbourne, M.; French, D.; Brown, T., Six-Colour HyBeacon Probes for Multiplex Genetic Analysis. ChemBioChem 2010, 11 (18), 2530-2533. 10. Gerowska, M.; Hall, L.; Richardson, J.; Shelbourne, M.; Brown, T., Efficient reverse click labeling of azide oligonucleotides with multiple alkynyl Cy-Dyes applied to the synthesis of HyBeacon probes for genetic analysis. Tetrahedron 2012, 68 (3), 857-864. 11. Seidel, C., A M., A. Schulz, and M. Sauer. 1996. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem 100, 5541-5553. 12. Marks, A. H. R.; Bhadra, P. K.; McDowell, D. G.; French, D. J.; Douglas, K. T.; Bichenkova, E. V.; Bryce, R. A., Molecular basis of action of HyBeacon (TM) fluorogenic probes: A spectroscopic and molecular dynamics study. Journal of Biomolecular Structure & Dynamics 2005, 23 (1), 49-62. 13. (a) Hunter, W. N.; Brown, T.; Anand, N. N.; Kennard, O., Structure of an adenine[dot]cytosine base pair in DNA and its implications for mismatch repair. Nature 1986, 320 (6062), 552-555; (b) Brown, T.; Kennard, O., Structural basis of DNA mutagenesis. Curr. Opin. Struct. Biol. 1992, 2 (3), 354-360. 14. (a) Shelbourne, M.; Brown, T.; El-Sagheer, A. H., Fast and efficient DNA crosslinking and multiple orthogonal labelling by copper-free click chemistry. Chem. Commun. 2012, 48 (91), 11184-11186; (b) El-Sagheer, A. H.; Brown, T., Click Chemistry: a Versatile Method for Nucleic Acid Labelling, Cyclisation and Ligation. In Royal Society of Chemistry Biomolecular Sciences Series Fox, K. R.; Brown, T., Eds. Royal Society of Chemistry: 2012.
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Dobson, N.; McDowell, D. G.; French, D. J.; Brown, L. J.; Mellor, J. M.;
Brown, T., Synthesis of HyBeacons and dual-labelled probes containing 2[prime or minute]-fluorescent groups for use in genetic analysis. Chem. Commun. 2003, (11), 1234-1235. 16. 17. Weisbrod, S. H.; Marx, A., Novel strategies for the site-specific covalent Shelbourne, M.; Chen, X.; Brown, T.; El-Sagheer, A. H., Fast copper-free labelling of nucleic acids. Chem. Commun. 2008, 0 (44), 5675-5685. click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction. Chem. Commun. 2011, 47 (22), 6257-6259. 18. Wark, A. W.; Lee, H. J.; Corn, R. M., Multiplexed Detection Methods for Profiling MicroRNA Expression in Biological Samples. Angew. Chem. Int. Ed. 2008, 47 (4), 644-652. 19. 20. 21. Link Technologies Ltd. http://www.linktech.co.uk/ (accessed Invitrogen Life Technologies. (a) Lyttle, M. H.; Walton, T. A.; Dick, D. J.; Carter, T. G.; Beckman, J. H.; 13/04/2013). http://www.invitrogen.com/site/us/en/home.html (accessed 13/04/2013). Cook, R. M., New Reagents and Methods for the Synthesis of Internal and 3Labeled DNA. Bioconj. Chem. 2002, 13 (5), 1146-1154; (b) Dobson, N.; McDowell, D. G.; French, D. J.; Brown, L. J.; Mellor, J. M.; Brown, T., Synthesis of HyBeacons and dual-labelled probes containing 2 '- fluorescent groups for use in genetic analysis. Chem. Commun. 2003, (11), 1234-1235. 22. Yamana, K.; Ohashi, Y.; Nunota, K.; Nakano, H., Synthesis, binding and -position of a ribonucleoside. Tetrahedron fluorescence properties of oligonucleotide derivatives having a dansyl fluorescence label attached to the 2 1997, 53 (12), 4265-4270. 23. Sun, G.; Noronha, A.; Wilds, C., Preparation of N3-thymidinebutyleneN3thymidine interstrand cross-linked DNA via an orthogonal deprotection strategy. Tetrahedron 2012, 68 (38), 7787-7793. 24. Tang, J. X.; He, N. Y.; Tan, M. J.; He, Q. G.; Chen, H., A novel substrate for in situ synthesis of oligonucleotide: plasma-treated polypropylene microporous membrane. Colloids Surf. Physicochem. Eng. Aspects 2004, 242 (13), 53-60. 25. Chen, S.-B.; Li, Y.-M.; Luo, S.-Z.; Zhao, G.; Tan, B.; Zhao, Y.-F., THE STUDY OF PHOSPHORAMIDITE AS O-PHOSPHITYLATION AGENT AND ITS REACTIVITY. Phosphorus, Sulfur, and Silicon and the Related Elements 2000, 164 (1), 277-291.
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Competing Mechanisms for the Copper-Free Sonogashira Cross-Coupling Reaction. Organometallics 2008, 27 (11), 2490-2498. 27. 28. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M., The Use of Esters of NLou, C. G.; Shelbourne, M.; Fox, K. R.; Brown, T., 2 '-Aminoethoxy-2-aminoHydroxysuccinimide in Peptide Synthesis. JACS 1964, 86 (9), 1839-1842. 3-methylpyridine in Triplex-Forming Oligonucleotides: High Affinity, Selectivity and Resistance to Enzymatic Degradation. Chemistry-a European Journal 2011, 17 (52), 14851-14856. 29. 30. Gibson, M. S.; Bradshaw, R. W., The Gabriel Synthesis of Primary Amines. Manoharan, M.; Prakash, T. P.; Barber-Peoc'h, I.; Bhat, B.; Vasquez, G.; Angewandte Chemie International Edition in English 1968, 7 (12), 919-930. Ross, B. S.; Cook, P. D., N-(2-Cyanoethoxycarbonyloxy)succinimide: A New Reagent for Protection of Amino Groups in Oligonucleotides. The Journal of Organic Chemistry 1999, 64 (17), 6468-6472. 31. Gale, N.; Kocalka, P.; Mardle, C.; Brown, T., PNA HyBeacons for analysis of human mutations related to statin-induced myopathy. Artif DNA PNA XNA 2011, 2 (3), 79-89. 32. (a) Cao, Y. C.; Jin, R.; Mirkin, C. A., Nanoparticles with Raman Spectroscopic Fingerprints for DNA and RNA Detection. Science 2002, 297 (5586), 1536-1540; (b) Mahajan, S.; Richardson, J. A.; Brown, T.; Bartlett, P. N., SERS-Melting: A New Method for Discriminating Mutations in DNA Sequences. JACS 2008, 130 (46), 15589-15601. 33. Guy-Caffey, J. K.; Bodepudi, V.; Bishop, J. S.; Jayaraman, K.; Chaudhary, N., Novel Polyaminolipids Enhance the Cellular Uptake of Oligonucleotides. Journal of Biological Chemistry 1995, 270 (52), 31391-31396. 34. Lou, C.; Shelbourne, M.; Fox, K. R.; Brown, T., 2 -Aminoethoxy-2-amino-3-
methylpyridine in Triplex-Forming Oligonucleotides: High Affinity, Selectivity and Resistance to Enzymatic Degradation. Chemistry A European Journal 2011, 17 (52), 14851-14856. 35. Ranasinghe, R. T.; Brown, T., Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis. Chem. Commun. 2011, 47 (13), 3717-3735. 36. Goodchild, J., Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconj. Chem. 1990, 1 (3), 165-187.
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Appendices
Interim Report
A Strategy for Adding Multiple Reporter Groups to Oligonucleotides Interim Report Introduction Fluorescent labelling is a commonly-employed technique in the field of molecular genetic analysis, enabling rapid and bio-compatible visualisation of biomolecules.1b Chemically-synthesised oligonucleotides (ONTs) with fluorescent labels can be used to investigate specific nucleic acid sequences by exploiting Watson-Crick base pair complementarity.4a The fluorescence conferred by these probes can be used to reveal the cellular location of the corresponding biomolecules,9a for example, or to identify mutated genetic sequences by measuring differences in duplex melting temperatures, tracked by changes in fluorescence. Many different fluorescent-based probe technologies exist, each varying subtly in their mode of action and or function.1b Different chemical and physicochemical properties are desirable according to the nature of the probe and its requirements. These are finely tuned through different synthetic approaches, as well as structural modifications. Existing labelling strategies can often only label ONTs once at the 5 terminus since coupling to the reporter phosphoramidite (amidite) essentially caps the 5 hydroxyl group, preventing further extension. Multiple labelling achieved via the N-hydroxysuccinimide (NHS) activated ester of a reporter/dye requires an amino-modified C6 d(A,C,G,T), however most of the primary amines are protected by a trifluoroacetic acid (TFA) protecting group (PG) - chemically incompatible with the capping process (acetic anhydride + methyl imidazole) important for ease of purification in later stages. Multiple and mixed labelling can be achieved via the reporter/dye-modified amidite monomers, although commercial availability of such pre-labelled monomers is not exhaustive. This strategy enables multiple, mixed internal labelling with amidite reporter/dyes without comprising the ONT synthetic process, and broadens the spectrum of possible labelling options. An important advantage
offered by this strategy over many others is its capacity for multiple internal labelling. In this project, two Fmoc (fluorenylmethyloxycarbonyl)- protected alcohol on uridine monomers (Fig.1) will be synthesised for incorporation into a linear ONT strand. ONT synthesis will be achieved via solid-phase (polystyrene (PS) or controlled-pore glass (CPG)) phosphoramidite chemistry. Upon completion of the desired base sequence, deprotection of the base-labile Fmoc may be achieved by treatment with a mild base, such as 20% piperidine (pip) in dimethylformamide (DMF) on solid phase. The exposed (5-propargyl or 2-ethoxy) hydroxyl groups can be subsequently coupled to reporter amidites, such as fluorescein or other fluorescent dyes, at each of the modified bases on the automated deoxyribonucleic acid (DNA) synthesiser before oxidation and cleavage from the solid support. Coupling to the reporter/dye may occur in one cycle at all the exposed hydroxyl groups to give a uniform multiply-labelled ONT, or in sequential cycles - allowing mixed multiple labelling. In this strategy, the reporter and the linear ONT are linked through a phosphate group. The resulting properties will be explored and compared with those arising from conventional strategies, which do not include the phosphate linkage. Current applications of such chemistry include fluorescent in-situ hybridisation probes (FISH)9a and HyBeacon4a technologies.
3'
OPG
OPG
OPG
5'
ODMT
O NH N O
deprotection of base labile PG (Fmoc)
DMTO O O O OFmoc
3'
OH
OH
OH
5'
ODMT
O P N
Addition of Reporter amidite
NC
Reporter O O O- O O
Reporter O O O- O P
Reporter FmocO O O-
O NH
3'
P O
5'
ODMT
DMTO O O
i) Deprotection of acid labile (DMT) at 5' ii) Cleavage and deprotection of exocyclic PG
NC
O P N
Reporter O P O OO O O
Reporter O O OO P O-
Reporter O O O
3'
HO
5'
OH
Fmoc
O
DMT
Figure 1 Strategy and modified monomers 1 and 2, PG = Protecting group
Results & Discussion Synthesis of Monomer 1
Scheme 1 Synthesis of monomer 1: conditions (i) 5-methyl uridine, diphenyl carbonate, sodium hydrogen carbonate (NaHCO3), dimethyl formamide (DMF), 100 C, 5 h, 57 %, (ii) dimethoxytrityl chloride (DMT-Cl), pyridine (py), room temperature (rt), 22 h, 67%, (iii) ethylene glycol, titaniumIV isopropoxide (TiIV(OiPr)4), dimethylformamide (DMF), 150 C, 24 h, X %, (iv) Not yet attempted (v) Not yet attempted
The synthesis of the monomer 1 was performed according to the procedure described previously.34 However, the synthesis is still in progress. 5methyl uridine was first converted to compound 4 (step i) with a five-hour reflux with diphenyl carbonate in the presence of sodium hydrogen carbonate in DMF. The compound was recrystallised from methanol to give the pure product as a crystalline, clear solid in 57 % yield (lit. 90 %). Explanation for the significantly lower yield than the reported literature value was partially because a fourth crop, which has not been included in yield calculations and appears substantial, is still crystallising and has yet to be filtered and combined. The protection of the 5 hydroxyl group with dimethoxytrityl PG (step ii) was straightforward via an SN1 substitution reaction with the DMT-Cl in pyridine to solvate and neutralise the hydrochloric acid by-products minimising potential cleavage of the acid-labile PG. The yield, after purification by flash column chromatography (Isolera, SNAP 50 g) with a 0 10 % gradient of methanol (MeOH) in dichloromethane (DCM), was 67 % (lit. 89 %). The pure product was obtained as a colourless foam. Explanations for the lower than expected yield are thought to be due to the high sensitivity of the DMT-Cl to moisture that, despite addition in excess, was rapidly degraded as observed by TLC and became the limiting reagent. Consequently, the reaction was maintained for longer than in the literature report. Reaction of the anhydro compound (step iii) was much slower than reported in literature. A suspected cause was the quality of the titaniumIV isopropoxide, as it is moisture-sensitive. Concerns of side-reactions led to the decision to cease the reaction, although the limiting reagent the anhydro compound - was still detectable (TLC) in the reaction mixture. Purification was attempted (separation followed by column chromatography (1-5 % MeOH / DCM) containing 0.05% triethylamine (TEA)), but none of the collected fractions proved pure (1H NMR). Work will be undertaken to find a better eluent system that can offer better separation of the crude components. This will be followed by a second flash column chromatographic purification (Isolera, SNAP 50 g, 0 5 % MeOH / DCM + 0.05 % TEA), yielding pure product ready for the next synthetic step. To eliminate the difficulty in separating the product from the starting material, the reaction was attempted for the second time with fresh titaniumIV isopropoxide, which proved successful, and the starting material was consumed.
To avoid contamination from the isopropoxide, which is detectable by 1H NMR in the original product, the titanium compounds were removed by precipitation. Initially, precipitation was attempted with DCM, but after three washes, the desired compound was still significantly present in the solid hence the wash system was adjusted to 5 % MeOH in DCM. This afforded much more effective removal of the product from the precipitated impurities. After three cycles of agitation, centrifuge, decantation then washing the product was no longer significantly detectable in the solid. The collected supernatant was concentrated to dryness in vacuo.
Synthesis of Monomer 2
Scheme 2 (i) 2-hydroxypropyne, TEA, DMF, copperI iodide (CuII), tetrakistriphenylphosphino palladium0 (Pd0(PPh3)4), 23 h, 49 % (ii) Fmoc-Cl, DCM, TEA, 24 h, not yet purified (iii) Not yet attempted The coupling of the propargyl alcohol to the 5-DMT-5-iododeoxyuridine (step (i)) implemented the Sonogashira palladium0, copperI catalysed crosscoupling reaction. The product was isolated (concentrated in vacuo to dryness, dissolved in ethyl acetate (EtOAc), separated and washed (EDTA (pH 8) and brine)
then purified by flash column chromatography (Isolera, SNAP 50 g, 80 100 % EtOAc / petroleum ether, 0 30 % MeOH / EtOAc) in 49 % yield. The addition of the Fmoc group (step ii) was initially attempted using Fmoc-succinimide, a slightly less-reactive reagent than Fmoc-chloride. The reasoning was that this would prevent or minimise unwanted reactivity with the slightly less reactive 3-hydroxy group (2 alcohol) and only the desired reaction with the more reactive propargyl hydroxyl group (1 alcohol) would occur. Experimentally, it was found that the reaction did not proceed at all. The starting material was recovered (reaction mixture separated and washed sequentially with saturated sodium hydrogen carbonate and brine solutions then dried over anhydrous sodium sulphate). The reaction was attempted with a more reactive reagent (Fmoc-chloride) at a low temperature (0 C) in an attempt to minimise unwanted reaction at the 3-hydroxyl group. The crude product was recovered from the reaction mixture by separation (deionised water then brine). The crude product will be purified by column chromatography.
Proposals for Future Work Upon successful completion of both syntheses, the compounds will be characterised (mass spectrometry (LR-MS & HR-MS), NMR (1H &
13
C), UV-vis.
spectra ()) to confirm the high levels of purity needed for ONT synthesis. Providing the monomers prove stable to ONT synthesis, further
experimentation can be performed. A multiply-labelled 50-mer could be characterised for use as a fluorescence in-situ hybridisation probe (FISH), exploiting the high fluorescence of the probe to detect the low and unamplified levels of cytosolic mRNA.35 Additionally, fluorescence may be enhanced through the inclusion of hexaethylene glycol (HEG), which may also be incorporated via the phosphoramidite method. This serves as a spacer, distancing the fluorescent group from the fluorescent-quenching environment created by the nucleosides, thus augmenting fluorescence.36 Broadened scope for the addition of dyes to DNA sequences may be achieved through the inclusion, via the phosphoramidite method, of active
(cyclooctyne) alkynes which may in turn couple to azide-functionalised dyes in a bioorthogonal manner.17 This azide-alkyne coupling, colloquially termed click chemistry, is rapid, high yielding and may be used in this project to cross-link ONTs, as well as to ligate dyes. Copper catalysed click chemistry is a well-known and effective strategy for the ligation of ONTs and the addition of reporter groups. However, multiple labelling with this method is a relatively slow process and the required presence of copper in the reaction can degrade the ONTs.37 Hence copper-free click chemistry via the cyclooctyne has proven to resolve this issue as the reactions can be left to proceed without concerns of degradation. Monomers of varying length linker (indicated as n, figure 2) between the exocyclic base C5 and the hydroxyl group or from 2OH may be synthesized. This factor may influence the quality of ONTs synthesis or affect the physical/biological properties of the ONTs containing reporter groups/dyes in various circumstances, such as in enzymatic or chemical ligation or influencing DNA/RNA polymerase activity.
FmocO
O NH N O DMTO O O O NC N O P N O
O NH N O
DMTO
NC
O P
Figure 2 Investigation of varying monomer-reporter linker lengths Branched ONTs (figure 2) with micro-fabrication applications may be investigated, with their synthesis achieved through orthogonal protecting group chemistry.
OFmoc
5'
Reporter 1 or ONTs
O HN O DMTO O O NC O P N O OPG2 N
OPG1
5'
DNA
O
HN O O O O O N
3'
DNA
A)
B)
5'
Reporter 2 or ONTs Reporter 3 or ONTs
Reporter 1 or ONTs
3'
Reporter 2 or ONTs
5'
Reporter 4/ONTs
C)
Figure 3 Branched application. A) Phosphoramidite with orthogonal protecting groups (PG1 and PG2) at 2, 5 and at C5 exocyclic nucleobase of uridine, B) Extension direction of the modified base: 5 end of ONTs or different reporter groups, C) Dendrimeric ONTs a model of two units of modified base with four different ONTs or reporters.
References 1. 2. Ranasinghe, R. T.; Brown, T., Fluorescence based strategies for genetic Gale, N.; French, D. J.; Howard, R. L.; McDowell, D. G.; Debenham, P. G.;
analysis. Chemical Communications 2005, (44), 5487-5502. Brown, T., Rapid typing of STRs in the human genome by HyBeacon[registered sign] melting. Organic & Biomolecular Chemistry 2008, 6 (24), 4553-4559. 3. 4. Levsky, J. M.; Singer, R. H., Fluorescence in situ hybridization: past, Lou, C.; Shelbourne, M.; Fox, K. R.; Brown, T., 2-Aminoethoxy-2-amino-3present and future. Journal of Cell Science 2003, 116 (14), 2833-2838. methylpyridine in Triplex-Forming Oligonucleotides: High Affinity, Selectivity and Resistance to Enzymatic Degradation. Chemistry A European Journal 2011, 17 (52), 14851-14856. 5. Ranasinghe, R. T.; Brown, T., Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis. Chemical Communications 2011, 47 (13), 3717-3735. 6. Goodchild, J., Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjugate Chemistry 1990, 1 (3), 165-187. 7. Shelbourne, M.; Chen, X.; Brown, T.; El-Sagheer, A. H., Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction. Chemical Communications 2011, 47 (22), 6257-6259. 8. 1267. El-Sagheer, A. H.; Brown, T., Click Nucleic Acid Ligation: Applications in Biology and Nanotechnology. Accounts of Chemical Research 2012, 45 (8), 1258-
Oligonucleotide Synthesis Optimisation

Conditions General Conditions CPG 1000 solid support Phosphoramidite coupling time 10 min (nucleosides and FAM) Tetrazole and standard phosphoramidite monomers used unless stated
Deprotection of modified monomer PGs Levulinyl: cocktail of hydrazine"1H2O :AcOH:Pyridine = 0.03:0.5:2.0, 12 min Fmoc: 20% piperidine/DMF,10 min unless stated otherwise TBDMS: neat TEA, 45 min, neat TEA.3HF 2x 30 min
Table 5 Oligonucleotide synthesis optimisation conditions
ONT ID Res 3210 Res3253 Res3255 Res3261 Res3263 Res3273 Res3275 Res 3373
Sequence GTGGATA1FATGCG1FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA1FATGCG1FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA1FATGCG1FTCATGG3 GTGGATA4FATGCG4FTCATGG
Conditions CPG, Strategy A CPG, Strategy B, 10 min coupling, 10 min pip CPG, Strategy B, 20 min coupling, 30 min pip CPG, Strategy B, 10 min coupling, 10 min pip, no cap CPG, Strategy B, 10 min coupling, 10 min pip, no cap CPG, Strategy B, 10 min coupling, 10 min pip, no cap, ETT, dGDMF, dCAc CPG, Strategy B, 10 min coupling, 10 min pip, no cap, ETT, dGDMF, dCAc PS, Strategy B, 10 min coupling, TEA, TEA.3HF 30 min x2 CPG, Strategy B, CPG was treated with 20%DEA for 25 min prior to start the synthesis, no capping, dGDMF, dCAc CPG Strategy B, no capping, dGDMF, dCAc PS, Strategy B, no capping, dGDMF, dCAc PS, Strategy C, no capping, 5terminus capped with hexynyl
Res3371
GTGGATA5FATGCG5FTCATGG
Res 3390 Res3391 Res3415
GTGGATA5FATGCG5FTCATGG GTGGATA5FATGCG5FTCATGG XGTGGATA5FATGCG5FTCATGG3
Mass Spectrometry Results

O NH DNA O N O O DNA O O O Fmoc DNA HO O O Fmoc O O NH DNA O N O O DNA HO O O O PG O N H DNA O O NH N O O DNA DNA O DNA O O O O O H N 4 OH O OO P O O N H O NH N O O O O H N 4 OH O OP O DNA O N O O DNA O O NH O O DNA O O O O O NH N O OP O O 4 HO H N O O O O OH
1F
2F
PG = TBDMS = 4 PG = Lev = 5
4F or 5F
Figure 64 Structural transformations of FAM labelling; legend of final structure abbreviations
Table 6 Oligonucleotide mass spectrometry results. Bold = no FAM label, Italic = ONT with one FAM label; Regular = expected product, 2 FAM labels
ONT ID Res 3210 Res3253 Res3255 Res3261 Res3263 Res3273 Res3275 Res 3373 Res3371 Res 3390 Res3391 Res3415
Sequence GTGGATA1FATGCG1FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA1FATGCG1FTCATGG3 GTGGATA2FATGCG2FTCATGG3 GTGGATA1FATGCG1FTCATGG3 GTGGATA4FATGCG4FTCATGG GTGGATA5FATGCG5FTCATGG GTGGATA5FATGCG5FTCATGG GTGGATA5FATGCG5FTCATGG XGTGGATA5FATGCG5FTCATGG
Calc MS 7535.97 7496.94 7496.94 7496.61 7535.97 7496.94 7535.97 7585.03 7585.03 7585.03 7585.03 7744.18
Found MS 7536.41 6420.2, 6958.3 6420.2, 6958.3 6420.2, 6958.3 6461.2, 6998.3 6421.2, 6958.3 6460.2, 6998.3, 7535.4 Unresolvable results 6509.1,7046.2, 7583.3 6509.1,7046.2, 7583.3 6509.1,7046.2, 7583.3 7743.3
HPLC-MS & Deconvoluted Peaks Res3210

Intens. [mAU]
150
100
50
0 4 5 6 7 8 9 10 11 Time [min]
res3210_GB5_01_14311.d: UV Chromatogram, 290 nm

Intens. x104
6513.2 -MS, 7.6-7.6min, Deconvoluted (MaxEnt)
1.0
0.8
0.6
0.4
0.2
5212.9 5429.3 5819.1 7815.6 6985.2 8468.7 9206.4 9766.9 10476.9 11729.5
4349.3
0.0 4000
Intens. x105 1.0
5000
6000
7000
8000
9000
10000
11000
m/z
0.8
0.6
0.4
0.2
6351.3 4178.7 4726.2 4963.1 5853.1 6961.3
0.0 4000 4500 5000 5500 6000 6500 7000 7500 m/z
Intens. x104
7536.4
1
7206.4 4949.0 5314.1 5650.0 6317.2 6767.4 9028.2
4511.6
0 4000 5000 6000 7000 8000 9000 10000 11000 m/z
Figure 65 Peak at Tr = 7.5 and 7.6, 6513.2 & 5693.1, impurity unrelated to base deletion or missing labels; peak at Tr = 8.6 min, 7536.4, 2 FAM labels, expected product
Res3253
Intens. [mAU] 150
100
50
0 1 2 3 4 5 6 7 8 Time [min]
res3253cr_BB5_01_14508.d: UV Chromatogram, 290 nm

Intens. x104 2.0
1.5
1.0
0.5
6116.2 5130.0 5538.0 5787.1 6837.4
7278.8
7684.1
0.0 5000 Intens. x104 1.50 5500 6000 6500
7000
7500
m/z
1.25
1.00
0.75
0.50
6567.3 6421.2 5746.1 6076.1 6824.2
0.25
5031.2
5433.0 5218.0
0.00 5000 5500 6000 6500 7000 7500 m/z
Figure 66 Peak at Tr = 7.4 min, 6420.2, No FAM; peak at Tr = 8.1 min, 6958.3, 1 FAM
Res3255
Intens. [mAU] 400
300
200
100
0 1 2 3 4 5 6 7 8 Time [min] RES3255 CR_RE3_01_14528.d: UV Chromatogram, 290 nm

Intens. x104
6420.2 -MS, 7.3-7.5min, Deconvoluted (MaxEnt)
6107.1 5129.0
6272.2
0 5000 Intens. x104 6 5500 6000 6500 7000 7500
7705.5
m/z
1
6567.3 5218.7 5434.0 6421.2
0 5000 5500 6000 6500 7000 7500 m/z
Figure 67 Peak at Tr = 7.4 min, 6420.2, No FAM; peak at Tr = 8.1min, 6958.3, 1 FAM
Res3261
Intens. [mAU] 200
150
100
50
0 0 2 4 6 8 10 12 14 16 Time [min]

Intens. x104 6420.2 4 -MS, 7.4-7.7min, Deconvoluted (MaxEnt)
1 6116.2 6272.2 0
6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 m/z
6945.3
7384.0
7715.0
Intens. x104 1.50 6958.3
1.25
1.00
0.75 6421.2 0.50 6567.3
7244.0
0.25 6075.2 0.00

6000 6200 6400
6654.3 6238.2 6317.2

6600
6791.3
7059.5 7660.1
6800
7000
7200
7400
7600
7800
m/z
Figure 68 Peak at Tr = 7.6 min, 6420.2, No FAM, peak at Tr = 8.1 min, 6958.3, 1 FAM
Res3263
Intens. [mAU] 200
150
100
50
0 0 2 4 6 8 10 12 14 16 Time [min]
Intens. x104
6461.2
1.5
1.0
0.5
7091.3
7747.5
9695.0
14550.9
0.0 5000
Intens. x104
6998.3
6000
7000
8000
9000
10000
11000
12000
13000
14000
m/z
2
5473.0
9711.8
0 5000
Intens. x104 1.25
7535.4
6000
7000
8000
9000
10000
11000
12000
13000
14000
m/z
1.00
0.75
0.50
5394.0 6011.1
0.25
9007.6 9420.4
0.00 5000 6000 7000 8000 9000 10000
10528.8
13192.5
11000
12000
13000
14000
m/z
Figure 69 Peak at Tr = 7.6 min, 6461.2, No FAM; peak at Tr = 8.2 min, 6998.3, 1 FAM; peak at Tr = 8.6 min, 7535.4, expected product
Res3273
Intens. [mAU] 300
250
200
150
100
50
0 0 2 4 6 8 10 12 14 16 Time [min] Res3273CR 2ND_RA2_01_14753.d: UV Chromatogram, 290 nm

Intens. x104 6958.3 4 -MS, 7.9-8.1min, Deconvoluted (MaxEnt)
1 6629.2 6325.26421.2 0
5500 5750 6000 6250 6500 6750 7000 7250 7500 7750 8000 m/z
Intens. x104 6421.2
2 6092.1 1 5787.0
5459.0 0
5000 5500
6000
6500
7000
7500
8000
8500
9000
9500
10000 m/z
Figure 70 Peak at Tr = 7.4 min, 6461.2, No FAM; peak at Tr = 8.0 min, 6958.3, 1 FAM
res3275
Intens. [mAU]
300
200
100
0 0 2 4 6 8 10 12 14 16 Time [min]
Res3275CR_RA4_01_14744.d: UV Chromatogram, 290 nm
Intens. 6000
5000
4000
3000
2000 6066.0 1000 5166.4 5653.0 5907.8 6481.3
7063.0
7747.4
8479.6 9094.6 9316.5 9705.69890.2
0
5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 m/z
Intens. x104 6460.2 4
6131.1 5842.1
6000 6500 7000 7500 8000 8500 9000 9500 10000 m/z
5514.1 0
5000 5500
Figure 71 Peak at Tr = 7.5 min, 6460.2, No FAM; peak at Tr = 8.1 min, 6998.3, 1 FAM; peak at Tr = 8.5 min, 7535.4, expected product
res3371
Intens. [mAU] 40
30
20
10
0
Intens. 8000
10
12
14
16
Time [min]

6509.1
6000
4000
2000
5625.0
0 5500
Intens. x104
5931.0
6000
6248.06374.0
6500
6767.9
7000 7500 8000
8205.2
8500 m/z
1.25
1.00
0.75
0.50
0.25
6164.1
0.00 5500 6000
6467.1
6500
6796.1 6913.2
7000 7500 8000 m/z
Intens.
8000
7583.3
6000
4000
2000
6700.2
0 6250 6500 6750 7000 7250
7335.3
7500 7750 8000 8250 m/z
Figure 72 Peak at Tr = 7.4 min, 6509.1, No FAM; peak at Tr = 8.2 min, 7046.2, 1 FAM; peak at Tr = 8.3 min, 7583.3, 2 FAM labels, expected product
res3390
Intens. [mAU]
60
40
20
0 0
Intens. 8000
10
12
14
16
Time [min]

6509.1
6000
4000
2000
5625.0
0 5500
Intens. x104
5931.0
6000
6248.06374.0
6500
6767.9
7000 7500 8000
8205.2
8500 m/z
1.25
1.00
0.75
0.50
0.25
6164.1
0.00 5500 6000
6467.1
6500
6796.1 6913.2
7000 7500 8000 m/z
Intens.
8000
7583.3
6000
4000
2000
6700.2
0 6250 6500 6750 7000 7250
7335.3
7500 7750 8000 8250 m/z
Figure 73 Peak at Tr = 7.4 min, 6509.1, No FAM; peak at Tr = 8.2 min, 7046.2, 1 FAM; peak at Tr = 8.3 min, 7583.3, 2 FAM labels, expected product
res3391
Intens. [mAU]
30
20
10
10
12
14
16
Time [min]
Intens.
8000
6000
4000
2000
5687.5 5254.6
0 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 m/z
5500.7
6700.2
7005.3
7334.3
9500.5
Figure 74 Peak at Tr = 8.7 min, 7584.3, 2 FAM labels, expected product

Intens. [mAU] 250
200
150
100
50
0 0 2 4 6 8 10 12 14 16 Time [min] RES3391A_RA1_01_16992.d: UV Chromatogram, 290 nm
Intens. x104 7583.3 2.5
2.0
1.5
1.0
0.5 5689.8 0.0

5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 m/z
6700.1
7004.2
7333.2
Figure 75 res3391 post purification high purity desired dual-labelled ONT product obtained
res3415
Intens. [mAU] 1000
800
600
400
200
0 0
Intens. x105
10
12
14
16
Time [min]
RES3415CR1_RB3_01_17066.d: UV Chromatogram, 290 nm

1.5
1.0
0.5
7206.2 0.0
5000 5500 6000 6500 7000 7500 8000 8500 m/z
Figure 76 Peak at Tr = 8.8 min, 7743.3, 2 FAM labels, expected product

A Strategy For Adding Multiple Reporter Groups To Oligonucleotides by Tom Fleming

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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

Supervisors: Dr. Nittaya Gale Professor Tom Brown

Approach!B! Approach!C! HyBeacon!Studies!

61! 66! 67!

Abbreviations & Acronyms

Bz C C cat CEP-(Cl) cm conc. COSY CPG

J K L Lev LPE lit. LRMS m M

PS py q Rep Rf RNA RP-HPLC rs rt

Nucleic Acid Structure5

Figure 1 B-DNA - The Iconic Double Stranded Helix2

Heterocyclic Base = (A) NH2 N

R = H, Nucleoside R = PO32-, Nucleotide

Figure 3 Distinctions in heterocyclic bases in DNA/RNA

Figure 4 Watson-Crick Base Pair Hydrogen Bonding Scheme

Monomers to Oligos Polymeric nucleoside strands are connected through a 3,5-

NH2 N 5' HO O N O O P OO O N N N O NH NH2 N NH2 N N O O O O P OO O O O P OO3' NH N O (C) (G) N (A) 5' HO O

O NH N O NH2 N N O O N O O P OO O (T) N N N O O P OO O O O P OO3'

Figure 6 The intrinsic directionality of nucleic acids (AGCT 5' ! 3'

Table 1 Impact of coupling efficiencies on overall yield

2-Cyanoethyl N,N-diisopropylphosphoramidite moiety

Figure 8 Highlighted features of a dT phosphoramidite monomer

Solvents & reagent solutions pass through freely

Filters retain growing oligonucleotides on solid supports

Figure 9 Diagram of solid-phase oligonucleotide column

Step 1 Activation & Coupling

Continue to next cycle Step 3 Oxidation

Cleavage of completed oligonucleotide from support

Figure 10 Oligonucleotide synthesis cycle of the phosphoramidite method

oligonucleotide synthetic cycle are outlined and simplified in table 2.

Operation Wash Acetonitrile

O O H O Cl Cl Cl O = Resin (CPG, PS)

etc. O NH O O DMT cation (orange)

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

) of the column effluent

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

Figure 14 Oxidation of the Phosphorous (III) Backbone

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

O HN N HO O OH OH N(4)-benzoyl dC (dCBz) N(2)-isobutyryl dG (dGiB) OH N O HO O N N O Ph O N HN NH NH HO O N N N

Figure 16 Standard protection of exocyclic amines for phosphoramidite oligonucleotide synthesis

Oligonucleotides in Genetic Analysis

O O O O N P O O N H O O N Reactive group Spacer Signalling moiety O O

Figure 18 Signal generation principle of a HyBeacon probe

thermodynamically stable duplex than one containing mismatches.

Melt Curve and First Derivative of an Oligonucleotide Duplex

450 400 350 300 250

Fluorescent Intensity at / Arbitrary Units

200 150 100

L = Label with Spacer, Oligo = Oligonucleotide

i) O Oligo N O j) O Oligo N3 O Ph2P O L Oligo L

L = Label with Spacer, Oligo = Oligonucleotide

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

Reporter group (Rep)

3' Remove, deprotect

3' Couple to reporter phosphoramidite

3' Return to synthesiser for extension until sequence complete

Figure 25 Previous synthetic approaches to internal labelling21

3' Remove, deprotect

3' Couple to reporter phosphoramidite

3' Return to synthesiser for extension until next modication

3' Return to synthesiser for extension until sequence complete

Rep Rep Rep Rep O O O O P O P O P O P O O O O Extend O O O O O

Rep Rep O Rep Rep O O O P P O O O P O P O O O O O O O O