Sonogashira alkynylation of unprotected 8-brominated adenosines and guanosines: fluorescence properties of compact conjugated acetylenes containing a purine ring

Article abstract of DOI:10.1016/j.tetlet.2006.03.100

A practical Sonogashira alkynylation protocol for the preparation of 8-alkynylated adenosines and guanosines has been developed. Protection of the sugar hydroxyl substituents is not required; protection hinders the purification of these products. A prelim

Full text of DOI:10.1016/j.tetlet.2006.03.100

Tetrahedron Letters 47 (2006) 3529–3533
Sonogashira alkynylation of unprotected 8-brominated
adenosines and guanosines: fluorescence properties
of compact conjugated acetylenes containing a purine ring
Andrew G. Firth,a,b Ian J. S. Fairlamb,a,* Kate Darleyc and Christoph G. Baumannb,*
aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, UK
bDepartment of Biology, Area 10, University of York, Heslington, York YO10 5YW, UK
cReplizyme Ltd, Genesis 2 Building, York Science Park, Heslington, York YO10 5DQ, UK
Received 7 February 2006; revised 5 March 2006; accepted 15 March 2006
Available online 5 April 2006
Abstract—A practical Sonogashira alkynylation protocol for the preparation of 8-alkynylated adenosines and guanosines has been
developed. Protection of the sugar hydroxyl substituents is not required; protection hinders the purification of these products. A
preliminary fluorescent study is reported, which shows that the presence of a substituent on the phenylene ring influences the fluo-
rescent properties considerably, an outcome that could be utilized in biological applications.
ꢀ 2006 Elsevier Ltd. All rights reserved.
C-Modified nucleosides are used in numerous applica-
tions, particularly in pharmaceutical agents and biolog-
ical probes or mimetics.1 Covalently modified
fluorescent nucleosides are valuable probes of DNA
and RNA helix-to-coil transitions, DNA and RNA
chain elongation, protein–nucleic acid complexes, mec-
hano-chemical coupling in motor proteins and cellular
signal transduction pathways.2 This drives the innova-
tive discovery of fluorescent nucleoside analogues, espe-
cially those that are sterically less bulky, but which still
possess desirable fluorescent properties for use in biolog-
ical systems.
the incorporation of a conjugated rigid-rod, attached
directly by C-modification at the 8-position on the pur-
ine (guanosine and adenosine), would represent a useful
way to introduce a mutually compact and effective fluo-
rescent moiety (Fig. 1).
O
N
HN
R
N
H2N
HO
N
O
We recognized that donor–acceptor phenylene–ethynyl-
ene substituted p-conjugated organic derivatives,3 pos-
sessing low-lying charge-transfer excited states, could
facilitate the employment of relatively small fluorescent
nucleoside analogues. Clearly, lengthening, shortening,
or broadening (through ring expansion) the p-conju-
gated electron network would alter the fluorophore
properties, in essence allowing one to tailor-make ana-
logues for specific applications. It was speculated that
OH OH
8-alkynylated-guanosine
NH2
N
N
R
N
N
HO
O
OH OH
8-alkynylated-adenosine
*
Corresponding authors. Tel.: +44 (0)1904434091; fax: +44
(0)1904432516 (I.J.S.F.); tel.: +44 (0)1904328828; fax: +44
(0)1904328825 (C.G.B.); e-mail addresses: ijsf1@york.ac.uk;
cb17@york.ac.uk
R = H, Me, OMe, C(O)Me, CF3, Cl, NO2
Figure 1. 8-Alkynylated purine nucleosides with potential as compact
fluorescent probes.
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.100

3530
A. G. Firth et al. / Tetrahedron Letters 47 (2006) 3529–3533
The low lying charge transfer energy states of the alkyn-
yl/aryl groups in these derivatives could also be tuned
through alteration of the para-substituent to modulate
the fluorescence properties, thus minimizing the degree
of steric changes to the parent phenylene–ethynylene–
purine compound. We thus set about preparing a library
of linear p-conjugated 8-alkynylated adenosines and
guanosines.
interesting lipophilic 8-alkynylated adenosines 7 were
prepared from 3,7 although the successful conditions
involved use of a large excess of Et3N (30 equiv w.r.t. 3)
and terminal acetylene (6 equiv w.r.t. 3). It has generally
been assumed that protection of the hydroxyl or amino
substituents in guanosines is required for efficient cross-
coupling, which is also required for solubility in com-
mon organic solvents.2e,7
Pd-catalyzed cross-coupling reactions4 have proven
effective for the efficient preparation of substituted
nucleosides.5 Alkynylated nucleosides may be accessed
by Sonogashira cross-coupling, although surprisingly
there are no literature examples detailing the alkynyl-
ation of the unprotected 8-bromoguanosine 1 with termi-
nal acetylenes to give 5 (Fig. 2). There are two studies
detailing the Sonogashira alkynylation of 8-bromoade-
nosine 3 to give 8-alkynylated-adenosines 7. In the first
report,6 no yields or structural characterization for 7
were provided. In the second report, however, some very
On a general note, the Pd-mediated processes appear to
be more facile with adenosine derivatives.8 Various
guanine coordination modes are possible, for example,
N-1/N-7 and O-6 coordination, to Pd (and presumably
Cu), which could be a hindrance in cross-coupling bro-
minated guanosine derivatives such as 1. This could in
part explain the unusual lack of studies detailing the
cross-coupling reactions of 1.
Mindful of this, and the potential inhibitory effects of
the guanine moiety in particular, we initially investi-
gated the development of an efficient Sonogashira alkyn-
ylation protocol for the cross-coupling of 1 with
phenylacetylene 9a to give 5a (Scheme 1, Eq. 1).
O
NH2
N
N
N
N
HN
N
Br
Standard conditions2e for Sonogashira alkynylation of 1
were chosen (10 mol % (Ph3P)2PdCl2, 10 mol % CuI,
1.2 equiv 9a and 3 equiv Et3N in DMF at 110 ꢁC for
18 h). This proved to be a generally ineffective protocol,
affording 5a in yields of ca. 20% with poor purity (be-
lieved to be due to the uptake of Pd and/or Cu), which
led us to optimize the conditions. Rapid Pd agglomera-
tion and precipitation were thought to contribute to the
low yields. An established means to overcome this prob-
lem is to employ lower Pd loadings (lower Pd-concentra-
tion), an observation derived from our previous
experiments involving Sonogashira cross-coupling of
aryl halides with terminal acetylenes.9 It was eventually
established that 1 mol % (Ph3P)2PdCl2 and 2 mol % CuI
proved the most effective catalyst/co-catalyst combina-
tion, affording 5a in 95% yield (after only 2 h).
Br
H2N
N
N
O
R'O
R'O
O
OR' OR'
OR' OR'
3 (R' = H)
1 (R' = H)
4 (R' = Ac)
2 (R' = Ac)
O
NH2
N
N
HN
N
R
R
N
N
H2N
R'O
N
N
R'O
O
O
OR' OR'
OR' OR'
5 (R' = H)
7 (R' = H)
6 (R' = OAc)
8 (R' = OAc)
Under similar conditions, the acetate protected guano-
Figure 2.
sine derivative 2 reacted with 9a to produce 6a in 88%
O
N
Pd(PPh3) Cl2 (Y mol%),
CuI (2X mol%)
O
N
N
N
HN
HN
(see text and Table 1),
Br
Ph
1.2 equiv.
N
H2N
H2N
HO
N
9a
HO
[Eq. 1]
O
O
Et3N (3 equiv.), DMF,
110 oC, 2 h,
5a
1
OH OH
OH OH
[1] = 0.12 M
O
O
N
N
HN
HN
as above using
Br
Pd(PPh3)2Cl2 (1 mol%),
CuI (2 mol%), 18 h
N
N
H2N
AcO
N
H2N
AcO
N
[Eq. 2]
O
O
(88%)
OAc OAc
OAc OAc
2
6a
Scheme 1. Sonogashira alkynylation of unprotected and protected 8-bromoguanosines (1 and 2, respectively) with phenylacetylene 9a.

A. G. Firth et al. / Tetrahedron Letters 47 (2006) 3529–3533
3531
Table 2. Sonogashira alkynylation of unprotected 8-bromoguanosine
yield (Scheme 1, Eq. 2), which was more cumbersome to
1a
purify than 5a vide infra.10
O
N
HN
The recent reports of Cu(I)-free Sonogashira alkynyl-
ation (formally a Heck alkynylation) led us to assess
the affect of Cu(I) on the yields of 5a.11 Maintaining
the initial Pd(II) concentration constant at 1 mol % it
was found that in the absence of Cu(I) 5a was produced
in 18% yield after 2 h (Table 1, entry 1). Moving to 0.5
and 1 mol % Cu(I) gave 5a in 30% and 38% yields,
respectively (Table 1, entries 2 and 3). On going to high-
er Cu(I) loadings (Table 1, entry 5), the yield of 5a was
reduced (77%). The purity of 5a was also questionable at
the higher Cu(I) loading; the solid is a light green colour,
rather than an off-white, which we interpret as being due
to the presence of trace quantities of guanosine derived
copper complexes (bidentate coordination through N-1
and O-6).
R
N
H2N
HO
N
O
5b-g
OH OH
Entry
1
Terminal acetylene
Product (R=)/yield (%)
OMe
OMe
9b
9c
5b (73)
NEt2
O
NEt2
O
2
3
5c (71)
5d (68)
9d
The purification of 5a is straightforward. On complete
reaction, the mixture is cooled to 40 ꢁC and the DMF
removed in vacuo to leave a dark colored solid, which
is transferred to a sintered glass funnel and washed with
boiling water (this removes Et3NÆHCl and any unreacted
1). The solid is then washed with small quantities of
EtOAc and Et2O to remove any traces of homo-coupled
product (produced through the pre-reduction of
(Ph3P)2PdCl2 with alkynylcuprate). The limited solubil-
ity of compound 5a in boiling water, and the majority of
chlorinated and ethereal solvents, facilitates its purifica-
tion (5a is soluble in DMF, DMSO and DMSO/H2O
mixtures, etc.).
CF3
CF3
NO2
4
5
9e
5e (53)
5f (–)
NO2
9f
Cl
Cl
6
9g
5g (29)
a Reagents and conditions: 1 (1 equiv), (Ph3P)2PdCl2 (1 mol %), CuI
(2 mol %), terminal acetylene (1.2 equiv), Et3N (3 equiv), DMF,
110 ꢁC, 18 h.
Using these optimized conditions,12 a series of Sono-
gashira alkynylation reactions of 1 were performed with
several terminal acetylenes 9b-g (Table 2). Good yields
were recorded for electron-rich phenylacetylenes 9b
and 9c (Table 2, entries 1 and 2). The electron-poor phen-
ylacetylenes 9d and 9e (Table 2) also worked well.
However, for 4-nitrophenylacetylene 9f, no cross-cou-
pling occurred (Table 2, entry 5). In the last example,
4-chlorophenylacetylene 9g faired better, although the
low yield could not be improved upon under the reac-
tion conditions employed.
Both 4-acetophenylacetylene 9d and 4-trifluoromethyl-
phenylacetylene 9e gave cross-coupled products 7d and
7e in 74% and 64% yields, respectively (Table 3, entries
4 and 5). As seen with 1, the cross-coupling of 3 with 4-
nitrophenylacetylene 9f resulted in negligible cross-cou-
pling.13 The reaction of 3 with 4-chlorophenylacetylene
9g again produced the cross-coupled product 7g in poor
yield (Table 3, entry 7).
The difference observed in the reactivity of the terminal
acetylenes suggests that the rate of transmetallation and
reductive elimination in the catalytic cycle are both af-
fected.14 In the latter step, a highly sensitive electronic
relationship clearly exists between the guanine and ade-
nine heterocycles and terminal acetylene on palla-
dium(II). This is not unusual for heterocyclic
compounds, albeit not fully understood.15
With a series of derivatives of 5 prepared, we turned our
attention toward cross-coupling the unprotected 8-
bromoadenosine 3 with the same terminal acetylenes
(Table 3). High yields were recorded for compounds
6a and b (Table 3, entries 1 and 2).
4-Diethylaminophenylacetylene 9c reacted to give 7c in
a satisfactory 52% yield (Table 3, entry 3).
From the results illustrated in Tables 2 and 3, a valuable
library of 8-alkynylated guanosines 5 and adenosines 7
are now available (easily prepared in one step from 1
and 3, respectively), which can be performed on gram
scale.
Table 1. Effect of Cu(I) loading on Sonogashira alkynylation of 1
Entry
CuI loading/mol %
Cu(I):Pd(II) ratio
Yield/%
1
2
3
4
5
0
—
18
30
38
95
77
UV–visible spectra reveal an intense absorption in the
UV region for all the compounds evaluated. The fluores-
cence spectral properties of six compounds are presented
in Table 4, which are consistent with the S0 absorption
and S1 emission wavelengths. As expected, the spectrum
0.5
1.0
2.0
4.0
1:2
1:1
2:1
4:1

3532
A. G. Firth et al. / Tetrahedron Letters 47 (2006) 3529–3533
Table 3. Sonogashira alkynylation of unprotected 8-bromoadenosine
3a
NH2
N
N
R
N
N
HO
O
7a-g
OH OH
Entry
1
Terminal acetylene
Product (R=)/yield (%)
9a
7a (85)
OMe
OMe
2
3
4
5
6
7
9b
9c
7b (80)
NEt2
O
NEt2
O
Figure 3. Normalized fluorescence emission spectra for compounds
5a,c and 5e in DMSO.
7c (52)
fer. The spectroscopic properties detailed in Table 4
show that the S0 absorption wavelength and Stokes shift
can be tuned by altering the electron-donating charac-
teristics of the para-substituent on the phenyl ring. The
larger Stokes shifts observed for 5c and 7e are consistent
with energy transfer to the guanine and adenine rings
being enhanced by both electron-donating and elec-
tron-withdrawing substituents through the aromatic
ring, respectively.
9d
7d (74)
7e (64)
CF3
NO2
Cl
CF3
NO2
Cl
9e
9f
7f (–)
In summary, we have a prepared several novel alkynyl-
ated guanosines and adenosines. Many of these deriva-
tives exhibit interesting fluorescent properties that could
prove useful in biochemical applications. The key to
the success of the Sonogashira cross-coupling was low
9g
7g (25)
a Reagents and conditions: 3 (1 equiv), (Ph3P)2PdCl2 (1 mol %), CuI
(2 mol %), terminal acetylene (1.2 equiv), Et3N (3 equiv), DMF,
110 ꢁC, 18 h.
palladium loading (1 mol %; c 1.18 · 10ꢀ3 mol dmꢀ3
)
and an optimum Pd(II)/Cu(I) ratio (1:2). The effect of
a Cu(I) co-catalyst in these reactions is substantial,
and one which indicates a potential secondary role for
this metal in the catalytic cycle (particularly in reactions
employing 1).16 Further mechanistic studies are under-
way to uncover a dual role for Cu(I) in these reactions.
In due course, the biological applications of the fluores-
cent analogs detailed herein will be reported.
Table 4. Fluorescence spectroscopic properties of select compounds in
DMSO
Compound
kex/nm
kem/nm
Stokes shifta/cmꢀ1
5a
5c
5e
7a
7c
7e
370
398
386
350
408
362
438
502
468
397
506
440
4196
5205
4539
3383
4747
4897
Acknowledgements
a Stokes shift = (1/kex ꢀ 1/kem).
We are grateful to the BBSRC, for Ph.D. studentship
funding (A.G.F.) and Replizyme Ltd, for CASE sup-
port. We thank Dr. C. T. O’Brien and Mr. A. R. Kapdi,
for the preparation of some terminal acetylenes and
ESI-MS experiments, respectively. We also thank Pro-
fessor C. Kleanthous, for the use of a fluorimeter and
Miss K. Lilly, for preliminary studies. I.J.S.F. thanks
the Royal Society, for funding in the form of a generous
equipment Grant and University Research Fellowship.
C.G.B. and I.J.S.F. are both grateful to the University
of York for start-up and pump-priming funds. The
authors are grateful to the referees for their constructive
comments.
of 5c is red-shifted from that of compound 5a (the par-
ent compound), revealing a smaller HOMO–LUMO
gap. Compound 5e, also red-shifted relative to 5a,
exhibits a slightly higher HOMO–LUMO gap than 5c.
Similar trends are seen with the adenosine compounds
7a,c and 7e, although the excitation maximum shift of
7c relative to compound 7a (the parent compound) is
greater (see Fig. 3).
The linear p-conjugated 8-alkynylated purine nucleo-
sides were designed to allow through-bond energy trans-

A. G. Firth et al. / Tetrahedron Letters 47 (2006) 3529–3533
3533
10. Compound 6a and related derivatives need to be isolated
by solvent extraction and purified by laborious column
chromatography.
References and notes
1. (a) De Clercq, E. Biochem. Pharmacol. 1984, 33, 2159; (b)
Minakawa, N.; Kojima, N.; Sasaki, T.; Matsuda, A.
Nucleosides Nucleotides 1996, 15, 251; (c) De Clercq, E.;
Descamps, J.; De Somer, P.; Barr, P. J.; Jones, A. S.;
Walker, R. T. Proc. Natl. Acad. Sci. U.S.A. 1979, 76,
2947; (d) Manfredini, S.; Baraldi, P. G.; Bazzanini, R.;
Marangoni, M.; Simoni, D.; Balzarini, J.; De Clercq, E. J.
Med. Chem. 1995, 38, 199; (e) Hocek, M.; Holy, A.;
11. (a) Li, J. H.; Zhang, X. D.; Xie, Y. X. Eur. J. Org. Chem.
2005, 4256; (b) Anderson, K. W.; Buchwald, S. L. Angew.
Chem., Int. Ed. 2005, 44, 6173; (c) Lemhadri, M.; Doucet,
H.; Santelli, M. Tetrahedron 2005, 61, 9839; (d) Batchu, V.
R.; Subramanian, V.; Parasuraman, K.; Swamy, N. K.;
Kumar, S.; Pal, M. Tetrahedron 2005, 61, 9869; (e)
Shirakawa, E.; Kitabata, T.; Otsuka, H.; Tsuchimoto, T.
Tetrahedron 2005, 61, 9878; (f) Li, J. H.; Zhang, X. D.;
Xie, Y. X. Synthesis 2005, 804; (g) Mery, D.; Heuze, K.;
Astruc, D. Chem. Commun. 2003, 1934; Bohm, V. P. W.;
Herrmann, W. A. Eur. J. Org. Chem. 2000, 3679.
´
´
Votruba, I.; Dvorakova, H. J. Med. Chem. 2000, 43,
1817.
2. (a) Thoresen, L. H.; Jiao, G.-S.; Haaland, W. C.; Metzker,
M. L.; Burgess, K. Chem. Eur. J. 2003, 9, 4603; (b)
Caretta, A.; Cavaggioni, A.; Sorbi, R. T. Eur. J. Biochem.
1985, 153, 49; (c) Langer, P. R.; Waldrop, A. A.; Ward, D.
C. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6633; (d)
Shimkus, M.; Levy, J.; Herman, T. Proc. Natl. Acad. Sci.
U.S.A. 1985, 82, 2593; (e) Crisp, G. T.; Gore, J. Tetra-
hedron 1997, 53, 1523.
12. General procedure:
A solution of 8-bromoguanosine
(520 mg, 1.5 mmol, 1 equiv) in dry DMF (12 mL) was
added to the substituted phenylacetylene (1.8 mmol,
1.2 equiv) and dry triethylamine (0.63 mL, 4.5 mmol,
3 equiv) in a vacuum dried Schlenk tube. PdCl2(PPh3)2
(11 mg, 0.015 mmol, 1 mol %) and CuI (6 mg, 0.030 mmol,
2 mol %) were added and the reaction mixture was left to
stir at 110 ꢁC for 18 h, after which time it was allowed to
cool to 40 ꢁC and the DMF removed in vacuo to leave a
brown solid. The solid was transferred to a sintered glass
filter and was washed with boiling water (5 · 200 mL),
EtOAc (5 · 25 mL) and diethyl ether (2 · 25 mL) yielding
the product as a light cream solid (0.53 g, 91%). Repre-
sentative data for 8-(20-phenylethynyl)guanosine: Mp 234–
236 ꢁC (decomp.); mmax (DMSO solution)/cmꢀ1 1695
(CO), 3126 and 3328 (NH), 3421 (OH); dH (400 MHz;
DMSO-d6) 3.65 (1H, m), 3.88 (1H, m), 3.84 (1H, s), 4.19
(1H, s), 4.99 (2H, m), 5.15 (1H, br s), 5.50 (1H, d, J 6.4),
5.89 (1H, d, J 6.4), 6.62 (2H, s), 7.51 (3H, m), 7.64 (2H,
m), 10.90 (1H, s); dC (128 MHz; DMSO-d6) 61.9, 70.4,
70.8, 79.4, 85.6, 88.2, 92.6, 117.5, 120.4, 128.9, 129.3,
129.9, 131.5, 151.1, 153.9, 156.0; m/z (FAB) MH+ 384
(2.5%), 369, 354, 277, 185 (100%); HRMS (MH+):
384.1299 (calcd for C18H18N5O5 384.1307).
3. For leading references, see: (a) Collings, J. C.; Parsons, A.
`
C.; Porres, L.; Beeby, A.; Batsanov, A. S.; Howard, J. A.
K.; Lydon, D. P.; Low, P. J.; Fairlamb, I. J. S.; Marder, T.
B. Chem. Commun. 2005, 2666; (b) Nguyen, P.; Yuan, Z.;
Agocs, L.; Lesley, G.; Marder, T. B. Inorg. Chim. Acta
1994, 220, 289; (c) Nguyen, P.; Todd, S.; Van den
Biggelaar, D.; Taylor, N. J.; Marder, T. B.; Whittmann,
F.; Friend, R. H. Synlett 1994, 299; (d) Biswas, M.;
Nguyen, P.; Marder, T. B.; Khundkar, L. R. J. Phys.
Chem. A. 1997, 101, 1689; (e) Nguyen, P.; Lesley, G.;
Marder, T. B.; Ledoux, I.; Zyss, J. Chem. Mater. 1997, 9,
406; (f) Fasina, T. M.; Collings, J. C.; Lydon, D. P.;
Albesa-Jove, D.; Batsanov, A. S.; Howard, J. A. K.;
Nguyen, P.; Bruce, M.; Scott, A. J.; Clegg, W.; Watt, S.
W.; Viney, C.; Marder, T. B. J. Mater. Chem. 2004, 14,
2395.
4. Fairlamb, I. J. S. Tetrahedron 2005, 61, 9661.
5. (a) Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Chem. Rev.
2003, 103, 1875; (b) Hocek, M. Eur. J. Org. Chem. 2003,
245; (c) Lakshman, M. K. Curr. Org. Synth. 2005, 2, 83.
6. Volpini, R.; Costanzi, S.; Lambertucci, C.; Vittori, S.;
Klotz, K.-N.; Lorenzen, A.; Cristalli, G. Bioorg. Med.
Chem. Lett. 2001, 11, 1931, It is worthy of note that an
unusual rearrangement was seen on cross-coupling 1-
phenylprop-2-yn-1-ol with 3 to give an enone, rather than
the expected alkynol.
13. The molecular ion for the cross-coupled product was
1
detected by FAB-MS, but shown to be impure by H and
13C NMR spectroscopy.
14. It is acknowledged that electron-poor terminal acetylenes
may favour ligation to Pd(0), thus removing active catalyst
from the catalytic cycle. For more insightful detail, see:
Amatore, C.; Bensalem, S.; Ghalem, S.; Jutand, A.;
Medjour, Y. Eur. J. Org. Chem. 2004, 366.
15. Fairlamb, I. J. S.; Ba¨uerlein, P. S.; Marrison, L. R.;
Dickinson, J. M. Chem. Commun. 2003, 632.
7. Flasche, W.; Cismas, C.; Herrmann, A.; Liebscher, J.
Synthesis 2004, 2335.
8. Western, E. C.; Shaughnessy, K. H. J. Org. Chem. 2005,
70, 6378.
16. Preliminary 31P NMR spectroscopic experiments indicate
that the excess Cu(I) reduces the amount of Pd(0) species
bound to 1 or 5, thereby releasing active catalyst into the
catalytic cycle. Complete details of these experiments will
be reported in a full paper.
´
9. Fairlamb, I. J. S.; Kapdi, A. R.; Lee, A. F.; Sanchez, G.;
´
´
´
´
Lopez, G.; Serrano, J. L.; Garcıa, L.; Perez, J.; Perez, E.
Dalton Trans. 2004, 3970.