Metal free, click and click-click conjugation of ribonucleosides and 2′-OMe oligoribonucleotides on the solid phase

Article abstract of DOI:10.1039/b918463e

A fast and practical metal free conjugation of ribonucleosides and 2′-OMe 4-mer oligoribonucleotides has been accomplished by a nitrile oxide alkyne click cycloaddition reaction on the solid-phase, the methodology is suited to modification at either, or both, the 3′- or the 5′-terminus of the oligoribonucleotide substrate.

Full text of DOI:10.1039/b918463e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Metal free, “click and click–click” conjugation of ribonucleosides and
2¢-OMe oligoribonucleotides on the solid phase†
Ishwar Singh and Frances Heaney*
Received 8th September 2009, Accepted 16th October 2009
First published as an Advance Article on the web 26th November 2009
DOI: 10.1039/b918463e
A fast and practical metal free conjugation of ribonucleosides and 2¢-OMe 4-mer oligoribonucleotides
has been accomplished by a nitrile oxide alkyne click cycloaddition reaction on the solid-phase, the
methodology is suited to modification at either, or both, the 3¢- or the 5¢-terminus of the
oligoribonucleotide substrate.
Chemically modified oligonucleotides are finding increasing appli-
cation as therapeutic agents and as tools for genomic research.1,2
The conjugation to an oligonucleotide of a group capable of
cellular association or surface recognition is hailed as one potential
solution to the problem of inefficient delivery of these therapeutic
molecules.3,4 Whilst a variety of methods describe conjugation
to DNA,5 modification to the chemically more sensitive RNA
provides a greater challenge.1 We wish here to report nitrile
oxide alkyne click cycloaddition chemistry as an effective, fast
and high yielding method for conjugation to ribonucleoside and
2¢-OMe oligoribonucleotide substrates directly on a solid support.
The Cu(I) catalysed azide–alkyne click reaction discovered by
Meldal6 and Sharpless7 has become one of the most ubiqui-
tous chemical ligation methods. It has recently been applied
to monitor RNA transcription8 and has been developed as a
reliable method for DNA modification.9 As a bioconjugation
tool it has numerous advantages over the postsynthetic reaction
of amino- or thioalkyl modified oligonucleotides with active
esters; conjugate yields are often higher and product purification
more facile. The azide–alkyne reaction generally introduces the
ligating group at the nucleobase, along the backbone, or at the
5¢-terminus. Conjugation at the oligonucleotide 3¢-terminus is
less well studied. However, during the preparation of this work
a Cu(I) promoted, microwave assisted, method for 3¢-,5¢-bi-click
DNA conjugation was reported.10 Since copper mediated DNA
functionalisation is sensitive to the availability of a sufficient
amount of a proper copper(I) complexing ligand11 an orthogonal
click ligation avoiding the copper catalyst is highly desirable.
Recently reported solid phase12a,b and solution phase12c nitrile
oxide click modification of DNA by cycloaddition to alkynes
and strained alkenes make important contributions to this field.
Further examples of catalyst free click cycloadditions include the
azide-ring strained cycloalkyne pairing13a and the photoinduced
nitrile imine–alkene cycloadditions.13b The objective of the current
work was to develop a solid phase, catalyst free, click methodology
suited to 2¢-OMe oligoribonucleotide modification at either the 3¢-
or the 5¢-terminus, and suited to formation of 3¢,5¢-bisconjugates.
We selected the nitrile oxide–alkyne cycloaddition14 as the
conjugating reaction of choice. In the current context the chemical
and biological stability of the resultant aromatic isoxazole nucleus
is particularly important. Among the potential sites for oligonu-
cleotide modification the 2¢-position is attractive as substitution
at this position locks the pentofuranose sugar in the C3¢-endo
conformation which is the structure preferred by the RNAi intra-
cellular machinery.15 Thus, from the outset we were interested in
2¢-O-alkynylnucleoside substrates. 2¢-Oligonucleotide conjugates
are not well known, however, recent reports demonstrate 2¢-O-(4-
pentenyl) adenosines16a and 2¢-O-propargyluridines16b as versatile
building blocks for DNA conjugation; 2¢-O-propargyluridine is
also a successful click partner with azidoboron clusters.17
˚
500 A CPG-Succinyl 2¢-O-propargyl nucleosides, with 5¢-DMT
protection and with standard base protecting groups were com-
mercially available and the proposed nitrile oxide–alkyne click
chemistry was initially tested on uridine. Benzaldehyde oxime was
selected as the nitrile oxide precursor and chloramine-T as the
dipole generating agent.18 These components were premixed in
aqueous EtOH (1 : 1) prior to exposure to an eppendorf tube
containing the supported alkyne 1a. After agitation at room
temperature for 15 min the CPG was thoroughly washed prior to
cleavage from the resin. The HPLC profile of the resulting reaction
mixture indicated the desired click chemistry had failed. We
considered steric demands of the 5¢-DMT protecting group may
have contributed to the lack of reaction. Gratifyingly, following
deprotection, 1b readily participated in reaction, and after 15 min
reaction at room temperature, and following work-up, HPLC
analysis indicated near quantitative conversion to the isoxazole–
nucleoside conjugate 3a, Scheme 1. Thus, the retention time of
the ligated adduct 3a is approximately 6 min longer than that of
the parent propargyl uridine 2a (Fig. 1). The MS data is in full
agreement with the expected product [Found 402.1310; (M + H)+
requires 402.1296].
It is necessary to demonstrate the compatibility of the nitrile
oxide alkyne click reaction with all natural RNA bases and before
focusing on the purine bases we explored ligation of the supported
NBz-2¢-O-propargyl cytosine 4a. Click conjugation of 4a proceeded
very efficiently under conditions identical to those described above
for 1b, Scheme 2. However, to show the future potential of this
methodology for commercial RNA modification on an automated
synthesizer the reaction was attempted directly on a synthesis
column. Following DMT deprotection of the commercial material,
Department of Chemistry, NUI Maynooth, Maynooth, Co. Kildare, Republic
of Ireland
† Electronic supplementary information (ESI) available: HPLC traces, MS
data and 1H and 13C NMR spectra. See DOI: 10.1039/b918463e
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a near quantitative reaction. NBz-2¢-O-Propargyl adenosine 4c
was treated similarly, again optimal conversion was found after
15 min at room temperature. HPLC analysis of the unpurified click
product indicated that the major product is the isoxazole conjugate
5c and there is little evidence for the presence of unreacted
alkyne 2d.
To demonstrate the scope of the reaction in terms of the
acceptable range of nitrile oxide partners we experimented with
naphthalene oxime and pyrene oxime, potentially capable of
delivering ligated nucleosides valuable as molecular beacons.19
Thus, support bound 2¢-O-propargyl uridine 1b was treated, in
turn, with a premixed solution of each oxime and chloramine-T,
Scheme 1. With an increase in the aromatic character and the
steric bulk of naphthonitrile oxide the click reaction required 4 h
to reach completion. Almost full conversion to the naphthalene
ligated uridine 3b was evidenced from the HPLC profile of the
crude reaction product. With a further increase in hydrocarbon
character 1-pyrene oxime was difficult to solubilise in 50% aq.
EtOH, however, in 90% ethanol the click reaction progressed
well. The rate of the reaction was again slow and HPLC analysis
indicated complete reaction required 16 h at room temperature.
MS analysis confirmed the structural integrity of both 3b and 3c.
Reaction with the nitrile oxide derived from 2-
phenoxyacetaldehyde oxime 6 (Fig. 1) represents the first
example of click modification of oligoribonucleotides with an
aliphatic nitrile oxide thus revealing the true latitude of the
approach, Scheme 1. After 16 h agitation of 1b with premixed 6
and chloramine-T in aq. EtOH (3 : 2) conversion to the isoxazole
ligated product was complete as evidenced by HPLC analysis
of the cleaved product; product identity is confirmed by MS
analysis. Given that lengthy synthetic procedures may be required
to prepare desired biomarkers or bioconjugating groups bearing
a masked aromatic nitrile oxide moiety the success of the reaction
with an aliphatic nitrile oxide click partner is a significant advance
over examples reported to-date.12
Scheme 1 Solid supported click conjugation of 2¢-O-propargyluridine.
Fig. 1 Structures of the cleaved and deprotected 2¢-O-propargyl nucleo-
sides 2a–d and the aliphatic oxime 6.
the column was thoroughly washed and dried. The resulting 4a
reacted on passing a pre-mixed solution of benzaldehyde oxime
and chloramine-T between two syringes over a 15 min period.
This procedure was repeated with a second, portion of reactants.
Following deprotection and cleavage from the resin (NH4OH),
HPLC analysis showed a complete absence of any starting alkyne
2b and near quantitative conversion to the isoxazole conjugate 5a.
Gratifyingly, the purine nucleobases adenosine and guanosine
also submit to the nitrile oxide alkyne click protocol, Scheme 2.
Thus, reaction of NiBu-2¢-O-propargyl guanosine 4b delivered the
click product 5b as evidenced from MS analysis. The presence
of a small peak characteristic of the parent guanosine–alkyne
2c, in the HPLC of the unpurified product is consistent with
To address the need for the click reaction to be compatible
with oligoribonucleotides the 5¢-alkyne functionalized 2¢-OMe
4-mer oligoribonucleotide 8 was targeted, Scheme 3. Thus, CPG-
bound 2¢-OMe-U4 (U4) was purchased from the market and
¯
following 5¢-DMT deprotection the pendant alkyne was intro-
duced by phosphoramidite coupling of 7.12a HPLC analysis on the
cleaved 9 indicated effective formation of the oligoribonucleotide–
alkyne 8. Exposure of 8 to premixed benzaldehyde oxime and
Scheme 2 Solid supported click conjugation of 2¢-O-propargylribonucleosides.
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Scheme 3 Solid supported 5¢-click conjugation of RNA–alkyne 8.
chloramine-T in aq. ethanol (3 : 2) lead to formation of the
isoxazole click product 10 in near quantitative yield as shown
on HPLC analysis of the unpurified reaction mixture. MALDI
TOF MS analysis confirmed the success of the reaction.
important as it demonstrates the versatility of the approach
for the preparation of 2¢-OMe oligoribonucleotide conjugates
functionalized at 5¢- or the 3¢-terminus.
The potential for high density functionalisation to tune the ma-
terial properties of modified oligoribonucleotides is attractive11b
and in this context, we wished to demonstrate the value of the
nitrile oxide alkyne conjugation toolbox for click–click bisfunc-
tionalisation with the substrate 14, Scheme 5. The required
bisalkyne was prepared by coupling of the phosphoramidite 712a
to 1b, HPLC analysis on 15 indicated effective formation of the
support bound bisalkyne 14. Subsequent exposure to the standard
oxime, chloramine-T protocol resulted in near quantitative conver-
sion of 14 to the click–click product as evidenced by HPLC analysis
of the cleaved, but unpurified product 16. LCMS TOF analysis
confirmed the success of the double functionalisation reaction.
With the high level of cycloaddition efficiency retained in the
click–click reaction the potential of the nitrile oxide alkyne pairing
for future application to high density functionalisation is apparent.
Whilst the modification of oligoribonucleotides at the
5¢-terminus by nitrile oxide alkyne click cycloaddition represents
an improvement over known methods, some applications will
desire modification at the 3¢-terminus,1,20 this presents a greater
synthetic challenge. Accordingly, the possibility to click func-
tionalize an RNA at the terminus of choice is an important
marker for the generality of our protocol. To this end, the support
bound dinucleotide 11, prepared by manual solid phase synthesis,
was exposed to the click conjugation protocol, Scheme 4. The
formation, after 4 h at room temperature, of the isoxazole ligated
product in quantitative yield, was judged by comparison of the
HPLC profile the parent alkyne 12 with the cleaved, but unpurified
product 13. LCMS TOF analysis confirmed the structural integrity
of both the alkyne 12 and the conjugate 13. The reaction is
Scheme 4 Solid supported 3¢-click conjugation of RNA–alkyne 11.
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Scheme 5 Solid supported 3¢,5¢-click–click conjugation of RNA 14.
In conclusion, we present a series of model reactions which
illustrate that solid-phase nitrile oxide–alkyne click chemistry
represents a convenient and fast approach towards a variety of
ribonucleoside and short 2¢-OMe oligoribonucleotide conjugates
in an environment free of any metal catalyst. It is compatible
with all four natural nucleobases bearing the standard protecting
groups. It lends itself to the preparation of either 5¢- or 3¢-click-
conjugates or 3¢,5¢-click–click conjugates. The reaction is very
simple and could be applied to more complex conjugates so
unveiling potential for future application in preparation of high
density functionalised RNA conjugates, and in provision of
substrates for genomic research.
chased from Link Technologies, UK. When required the 5¢-DMT
group was removed by treatment with dichloroacetic acid (2.5% in
DCM) for 5 min followed by washed with acetonitrile and drying.
All other chemical agents were purchased from Aldrich Chemical
Company unless otherwise noted.
2-Phenoxyacetaldehye oxime 6
To a round bottomed flask containing 2-phenoxyacetaldehyde21
(1.4 g, 10.2 mM) was added sodium acetate (1.8 g, 21.9 mM) and
hydoxylamine hydrochloride (1.5 g, 21.4 mM) in ethanol : water
(9 : 1, 40 ml) and the mixture was heated at reflux for 12 h. The
product was isolated following extraction with dichloromethane
(3 ¥ 80 ml). The organic layer was dried over anhydrous sodium
sulfate and the solvent was removed under reduced pressure. The
crude product was purified by flash column chromatography over
silica gel using hexane and ethyl acetate (8 : 2) as eluant to give the
title compound as a white sticky solid in 56% yield (910 mg).
dH (300 MHz; CDCl3) 8.69 (brs, 1H), 7.33–7.30 (m, 2H), 7.04–
6.89 (m, 4H), 4.90 (d, J = 3.6 Hz, 2H); dC (75 MHz; CDCl3)
158.0, 157.9, 150.1, 147.6, 129.6, 121.5, 121.4, 114.7, 114.4, 64.7,
61.8; HRMS (ESI) calcd for C8H9NO2 152.0706 [M + H]+; found
152.0704.
Experimental
General experimental
Analytical TLC was performed on precoated (250 mm) silica
gel 60 F254 plates from Merck. All plates were visualized by
UV irradiation, and/or staining with 5% H2SO4 in ethanol
followed by heating. Flash chromatography grade silica gel 60
(230–400 mesh) was obtained from Merck. Mass analysis was
performed on an Ettan MALDI-TOF Pro from Amersham
Biosciences with 2,¢4¢,6¢-trihydroxyacetophenone as matrix or
LCMS TOF from Agilent Technologies. The NMR spectra were
obtained (1H at 300 MHz and 13C at 75 MHz) on a Bruker
instrument at 25 ◦C. Chemical shifts are reported in ppm downfield
from TMS as standard. HPLC was carried out using a Gilson
instrument equipped with a UV detector or a diode array
detector and a Nucleosil C18 column. CPG alkynes were bought
from Chem Genes. 5¢-(4,4¢-Dimethoxytrityl)-uridine-2¢-O-methyl-
3¢-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was pur-
General procedure for phosphitylation reactions: preparation of 8,
11 and 14
To manually couple the alkyne phosphoramidite 712a or 5¢-(4,4¢-
dimethoxytrityl)-uridine-2¢-O-methyl-3¢-[(2-cyanoethyl)-(N,N-
diisopropyl)]-phosphoramidite to the oligoribonucleotide/
nucleoside, 500 mL of phosphoramidite (100 mM in anhydrous
CH3CN) was added to the CPG–RNA or nucleoside (1 mmol)
454 | Org. Biomol. Chem., 2010, 8, 451–456
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along with 500 mL of benzylmercaptotetrazole in CH3CN
(0.3 M). The mixture was allowed to react for 15 min at RT with
mixing between two syringes. This procedure was repeated with
a second portion of each of a new solution of phosphoramidite
and benzylmercaptotetrazole. The CPG was washed with
CH3CN (5 ¥ 2 ml), oxidizer (700 mL, 0.1 M iodine solution in
THF : pyridine : water; 78 : 20 : 2) and CH3CN (2 ¥ 5 mL) and
dried yielding CPG–RNA–alkynes 8, 11 and 14. Cleavage from
the resin proceeded by the method described below furnishing
samples of 9, 12 and 15 respectively.
and cleavage from the resin (NH4OH), HPLC analysis indicated
almost quantitative conversion to the isoxazole–nucleotide conju-
gate 5a.
Procedure for naphthalene-1-nitrile oxide click reaction on
CPG-alkyne 1b and 11
To solid supported alkynes 1b or 11 and (0.5 mM) in an
eppendorf tube was added a premixed solution (10 min) of
1-naphthylaldehyde oxime22 (45 mg) and chloramine-T mono-
hydrate (59 mg) in ethanol (500 mL) and water (500 mL). The
mixture was agitated at room temperature for 4 h. Following
settling the supernatant liquid was removed by syringe and the
CPG washed firstly with CH3CN (5 ¥ 300 mL) and then H2O (5 ¥
300 mL). Cleavage from the CPG and HPLC analysis followed by
the procedures described above.
General cleavage–deprotection procedure
For analytical purposes a portion of the RNA or nucleoside
was deprotected and cleaved from the CPG by incubating the
◦
supported material in 40% aqueous CH3NH2 (500 mL) at 65 C
for 30 min or in the case of NBz-cytidine with conc. aqueous NH3,
ethanol 3 : 1 for 24 h at 25 ◦C. The CH3NH2/NH3 was evaporated
using a concentrator. The CPG was washed with H2O (5 ¥ 200 mL
aliquots), all solutions and washings were combined to afford an
aqueous solution of the RNA/nucleoside alkynes or cycloaddition
products.
Procedure for pyrene-1-nitrile oxide click reaction on 1b
To solid supported alkyne 1b (0.2 mM) in an eppendorf tube was
added a solution of 1-phenoxyacetaldehyde oxime23 (16 mg) in
ethanol (900 mL) followed by water (100 mL) and chloramine-T
monohydrate (15 mg). The combined mixture was agitated at
room temperature for 16 h. Workup, cleavage and HPLC analysis
followed by the procedures described above.
General method for HPLC analysis
The ribonucleosides and RNA conjugates were analyzed by
reverse-phase HPLC with an analytical column (Nucleosil) under
the following conditions; 200 mL injection loop. For RNA alkynes
and RNA conjugates buffer A: 0.1 M TEAAc, pH 6.5, 5% (v/v)
MeCN. Buffer B: 0.1 M TEAAc, pH 6.5, 65% (v/v) MeCN).
Gradient; 0–5 min, 5% B; 5–20 min, 95% B; 20–28 min, 95% B, flow
rate: 1.0 mL min-1. For ribonucleoside alkynes and click conjugates
of ribonucleosides the eluant was water and acetonitrile (0–5 min,
5% B; 5–15 min, 95% B, 15–25 min, 100% B, flow rate: 1.0 mL min-1
and detection at 260 nm or absorbance was monitored in the range
220–500 nm with a diode array detector and recorded at 260 nm.
Procedure for phenoxyacetaldehyde-1-nitrile oxide click reaction
on 1b
To solid supported alkyne 1b (0.2 mM) in an eppendorf tube was
added a solution of 1-phenoxyacetaldehydeoxime 6 (15 mg) in
ethanol (400 mL) followed by water (600 mL) and chloramine-T
monohydrate (23 mg). The combined mixture was agitated at
room temperature for 16 h. Workup, cleavage and HPLC analysis
followed by the procedures described above.
Acknowledgements
General procedure for benzonitrile oxide click reactions on CPG–
nucleoside–alkynes/CPG–RNA–alkynes 1a,b, 4a–4c, 8 and 14
We thank C. Batchelor, NUI Maynooth for assistance with
MALDI-TOF mass spectral measurements. Financial support
from the Science Foundation of Ireland (Programme code
05/PICA/B838) is gratefully acknowledged
To solid supported alkynes 1a,b, 4a–4c, 8 and 14 (0.5 mM) in an
eppendorf tube was added a premixed solution of benzaldehyde
oxime (40 mg) and chloramine-T monohydrate (75 mg) which
had been premixed in ethanol (500 mL) and water (500 mL). The
combined mixture was agitated at room temperature for 15 min.
Following settling the supernatant liquid was removed by syringe
and the CPG washed firstly with CH3CN (5 ¥ 300 mL) and then
H2O (5 ¥ 300 mL). Deprotection–cleavage and HPLC analysis
followed by the procedures described above.
References
1 J. H. Jeong, H. Mok, Y.-K. Oh and T. G. Park, Bioconjugate Chem.,
2009, 20, 5–14.
2 D. R. Corey, J. Clin. Invest., 2007, 117, 3615–3622.
3 R. Juliano, M. R. Alam, V. Dixit and H. Kang, Nucleic Acids Res.,
2008, 36, 4158–4171.
4 H. Lonnberg, Bioconjugate Chem., 2009, 20, 1065–1094.
5 Y. Singh, N. Spinelli and E. Defrancq, Curr. Org. Chem., 2008, 12,
263–290.
6 C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67,
3057–3064.
7 V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless, Angew.
Chem., Int. Ed., 2002, 41, 2596–2599.
8 C. Y. Jao and A. Salic, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
15779–15784.
9 F. Amblard, J. H. Cho and R. F. Schinazi, Chem. Rev., 2009, 109,
4207–4220.
Click on the column-preparation of isoxazole conjugate 5a
The click conjugation on NBz-2¢-O-propargyl cytosine 4a was
performed directly on the column subsequent to the 5¢-DMT
deprotection of the commercial material. The resulting 4a was
reacted for 15 min at room temperature by passing an aqueous
ethanolic, pre-mixed solution of benzaldehyde oxime (40 mg) and
chloramine-T monohydrate (75 mg) between two syringes. This
procedure was repeated with a second, new, portion of pre-mixed
oxime and chloramine-T monohydrate. Following deprotection
10 G. Pourceau, A. Meyer, J.-J. Vasseur and F. Morvan, J. Org. Chem.,
2009, 74, 1218–1222.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 451–456 | 455
©

View Article Online
11 (a) P. M. E. Gramlich, S. Warncke, J. Gierlich and T. Carell, Angew.
Chem., Int. Ed., 2008, 47, 3442–3444; (b) P. M. E. Gramlich, C. T.
Wirges, J. Gierlich and T. Carell, Org. Lett., 2008, 10, 249–251.
12 (a) I. Singh, J. S. Vyle and F. Heaney, Chem. Commun., 2009, 3276–
3278; (b) K. Gutsmiedl, C. T. Wirges, V. Ehmke and T. Carell, Org.
Lett., 2009, 11, 2405–2408; (c) V. Algay, I. Singh and F. Heaney, Org.
Biomol. Chem., 2010, DOI: 10.1039/b917450h.
Lachmann, X. Li, O. S. Wolfbeis and H.-A. Wagenknecht, Bioconjugate
Chem., 2009, 20, 558–564.
17 B. A. Wojtczak, A. Andrysiak, B. Gruener and Z. J. Lesnikowski,
Chem.–Eur. J., 2008, 14, 10675–10682.
18 A. Hassner and K. M. L. Rai, Synthesis, 1989, 57–59.
19 P. Conlon, C. J. Yang, Y. Wu, Y. Chen, K. Martinez, Y. Kim, N. Stevens,
A. A. Marti, S. Jockusch, N. J. Turro and W. Tan, J. Am. Chem. Soc.,
2008, 130, 336–342.
20 Y. Ueno, K. Kawada, A. Shibata, K. Yoshikawa, Y. Wataya and Y.
Kitade, Nucleic Acids Symp. Ser., 2008, 52, 503–504.
21 J. Brussee, W. T. Loos, C. G. Kruse and A. Van der Gen, Tetrahedron,
1990, 46, 979–986.
13 (a) J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,
I. A. Miller, A. Lo, J. A. Codelli and C. R. Bertozzit, Proc. Natl. Acad.
Sci. U. S. A., 2007, 104, 16793–16797; (b) W. Song, Y. Wang, J. Qu and
Q. Lin, J. Am. Chem. Soc., 2008, 130, 9654–9655.
14 R. Huisgen, Angew. Chem., 1963, 75, 604–637.
15 M. Manoharan, Biochim. Biophys. Acta, 1999, 1489, 117–130.
16 (a) A. M. Jawalekar, N. Meeuwenoord, J. G. O. Cremers, H. S.
Overkleeft, G. A. van der Marel, F. Rutjes and F. L. van Delft,
J. Org. Chem., 2008, 73, 287–290; (b) S. Berndl, N. Herzig, P. Kele, D.
22 H. E. Master, S. I. Khan and A. K. Poojari, Bioorg. Med. Chem., 2005,
13, 4891–4899.
23 H. Lee, E. Luna, M. Hinz, J. J. Stezowski, A. S. Kiselyo and R. G.
Harvey, J. Org. Chem., 1995, 60, 5604–5613.
456 | Org. Biomol. Chem., 2010, 8, 451–456
This journal is
The Royal Society of Chemistry 2010
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