Biotin-, fluorescein- and 'clickable' conjugates of phospha-oseltamivir as probes for the influenza virus which utilize selective binding to the neuraminidase

Article abstract of DOI:10.1039/c1ob05384a

The synthesis of conjugates of phospha-oseltamivir to the well established reporter groups fluorescein and biotin and an approach to multimeric inhibitors is described. We report powerful inhibition of the influenza neuraminidase by these probes and quant

Full text of DOI:10.1039/c1ob05384a

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 5625
www.rsc.org/obc
COMMUNICATION
Biotin-, fluorescein- and ‘clickable’ conjugates of phospha-oseltamivir as
probes for the influenza virus which utilize selective binding to the
neuraminidase†
Mathew Stanley,a Stephen R. Martin,b Max Birge,a Benoit Carbaina and Hansjo¨rg Streicher*a
Received 11th March 2011, Accepted 9th June 2011
DOI: 10.1039/c1ob05384a
The synthesis of conjugates of phospha-oseltamivir to the
well established reporter groups fluorescein and biotin and
an approach to multimeric inhibitors is described. We report
powerful inhibition of the influenza neuraminidase by these
probes and quantify fluorescence quenching during binding
of the fluorescein conjugate through titration with the neu-
raminidase. Thus, we show that they could be useful tools
to efficiently inhibit, detect and quantify the virus and the
neuraminidase in biological systems.
negative charge of the latter is indispensable and consequently
present in all three inhibitors to allow for ionic bonding between
the carboxylate and an arginine triad in the active site of the NA
which is an essential feature for strong binding of sialic acids and
sialylmimetics.9,10,11,12
We have argued that these three powerful inhibitors are quite
simply also the strongest binding low molecular weight ligands
for the virus.13,14 For instance, they bind NA much stronger than
anything that has been discovered to bind to the sialic acid-
recognising influenza virus hemagglutinin (HA), at least as long
only monovalent ligands are considered. Consequently, they could
serve as efficient, selective anchors in tools designed to detect,
quantify or otherwise investigate the virus or the neuraminidase. In
particular oseltamivir has a pharmacophore with reduced analogy
to sialic acids that should display very little cross-reactivity with
other sialic acid binding proteins in complex systems.15
Virtually each year, the influenza virus succeeds in keeping the
WHO and national health authorities on high alert because of the
fear of a possible pandemic, coupled with high mortality rates in a
worst case scenario.1 This is particularly true for influenza type A
viruses, manyofwhichareavianviruses thatcaninfecthumans and
are thus the most likely source for new, highly pathogenic strains.
While in the past decades, most winter epidemics in the UK were
caused by the influenza A (H3N2) virus, it has for the time being
apparently been replaced in this role by the H1N1 (‘swine flu’,
‘Mexican flu’) virus, the source of the 2008 pandemic.2 In addition,
the less contagious (for humans), but highly pathogenic H5N1
(‘bird flu’) virus remains a threat and there is little doubt that all
are here to stay as potential sources for dangerous recombinations.
The development of anti-influenza drugs based on inhibition of
the viral neuraminidase (NA) has added new options to combat
influenza.3,4,5 Oseltamivir carboxylate 1,6 available in the form of its
The problem to chemically link oseltamivir without impairing
the pharmacophore has been solved by us through replacement
of the carboxylate by phosphonate monoesters, which retain
the negative charge crucial for binding under physiological
conditions.13,14,16
In this contribution we have chosen an w-ethynylbutyl- and an
w-azidohexyl-derivative (3a and 3b) as they also allow immobil-
isation/conjugation through Huisgen-1,3-dipolar cycloaddition
(‘click-chemistry’) (Fig. 1).17 We have demonstrated the latter
by ‘clicking’ 3b with 1.4-diethynylbenzene to afford a dimeric
inhibitor (→3c) as example for a class of compounds with known
improved bioavailability.18 As reporter groups to be conjugated
with 3b, we have chosen biotin (→3d) and fluorescein (→3e) for
the following reasons: Biotin is universally used to immobilise
or conjugate bioactive compounds, normally via its high-affinity
interaction with avidin or streptavidin, for instance in ELISA-type
assays or in Biacore systems. To give an example, treatment of read-
ily available streptavidin-coated microtiter plates with a solution
of 3d (or an analog with optimised spacer length) should result
in a phospha-oseltamivir-coated plate with a strong affinity for
influenza NA. Fluorescein is one of a family of fluorophores widely
ethyl ester prodrug TamifluTM, zanamivir7 (marketed as RelenzaTM
)
and peramivir8 are mimetics of the sialic acid substrates of
influenza NA and inhibit the enzyme in the subnanomolar range.3,4
So far, carbocyclic oseltamivir 1 has been the most frequently used
in the field due to its advantages in formulation and bioavailability.
The main features of the inhibitor beside the cyclohexene scaffold
are the pentyloxy group mimicking the glycerol sidechain of sialic
acid, the acetamide, the amino group providing important ionic
bonds with the enzyme and the carboxylate group (Fig. 1). The
aChemistry Division, School of Life Sciences, University of Sussex, Brighton,
BN1 9QG, UK. E-mail: h.streicher@sussex.ac.uk; Fax: +44-1273-876687
R
used in biochemistry and virology (e.g. the Alexafluorꢀ group).
This will aid direct fluorimetric detection and quantification of
influenza viruses or influenza NA independent of antibodies and
the standard sialidase assay which is based on the unselective
substrate 2¢-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid
bDivision of Physical Biochemistry, National Institute of Medical Research,
Mill Hill, London, UK, NW7 1AA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05384a
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5625–5629 | 5625
©

View Article Online
Fig. 1 Influenza neuraminidase inhibitors and target compounds 3a–d.
(MUNANA). The cross-reactivity of oseltamivir with other
sialidases is known to be low,15 i.e. an assay employing 3d or 3e
might even allow the presence of mixtures of sialidase activities in
test samples. Moreover, we hope that such compounds will help to
identify irregular distributions of neuraminidase (‘neuraminidase
patches’) on the viral surface, again without involvement of
antibodies or the influenza HA. It is known that the functional
balance between NA and HA is an important factor for the
replicative fitness of the virus.19
The synthesis of target conjugates 3a–e has been achieved
through a strategy introduced by us for inhibitors 2, all containing
the phospha-oseltamivir motif and inhibiting influenza NA in
the subnanomolar range.13,16 Protected monoester 4 is readily
available via our Hunsdiecker–Barton approach13,20 from the
‘acetamido-azide’, the precursor of oseltamivir in the industrial
synthesis (Scheme 1).21 w-Azido-hexyltriflate 9 was synthesised
from commercially available hexanediol through standard pro-
cedures. In brief, mono-silylation of the diol gave 5 which was
tosylated to give 6. Substitution of the tosylate using sodium
azide gave 7 which was deprotected to yield azido alcohol 822
Both 8 and commercially available 1-hexyne-6-ol were activated
with triflic anhydride and lutidine to give triflates 9 and 10,
respectively.
Monomethyl phosphonate 4 was alkylated with the w-
azidohexyl- and w-ethynylbutyltriflates 9 and 10, respectively, to
give the corresponding mixed diesters 11 and 12 in good yield.
Mixtures of diastereoisomers were typically obtained which were
used as such. Cleavage of the methyl esters 11 and 12 followed
by deprotection of the amine under acidic conditions resulted in
‘clickable’ inhibitors 3a and 3b.
5626 | Org. Biomol. Chem., 2011, 9, 5625–5629
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Scheme 2 Synthesis of a dimeric inhibitor via ‘click’-chemistry.
Scheme 1 Synthesis of ‘clickable’ inhibitors 3a and 3b.
To demonstrate the suitability of the approach for the produc-
tion of phospha-oseltamivir multimers, we ‘clicked’ azide 11 with
1,4-diethynylbenzene under Sharpless/Huisgen-conditions to give
protected dimer 13 which was readily deprotected to give dimeric
inhibitor 3c (Scheme 2).
Scheme 3 Synthesis of conjugates of phospha-oseltamivir and reporter
For (+)-biotin conjugate 3d, azide 11 was reduced with trimethyl
phosphine followed by hydrolysis of the aza-ylide. The resulting
amine was then coupled to (+)-biotin using benzotriazol-1-yl-
oxypyrrolidinephosphonium hexafluorophosphate (PyBOP) as
condensing agent. Protected conjugate 14 was deprotected with
sodium iodide and then triflic acid to yield 3d in 64% yield over 2
steps.
The same set of coupling and deprotection reactions was applied
for the fluorescein conjugate 3e (via protected 15) which was
obtained in a comparable yield of 75% (Scheme 3).
To confirm binding, compounds 3a–e were tested for inhibition
of the neuraminidase isolated from the influenza virus X31
(H3N2)23 and compared to benchmark inhibitors oseltamivir
carboxylate 1 and zanamivir.24 As expected, all 5 compounds are
potent inhibitors of the hydrolysis of 2¢-(4-methylumbelliferryl)-
a-D-neuraminic acid (MUNANA) by this neuraminidase (Table
1). This confirms that the ‘clickable’ derivatives 3a and 3b are
attractive candidates for the generation of multivalent phospha-
groups biotin and fluorescein.
oseltamivir-based inhibitors and phospha-oseltamivir-coated sur-
faces. Interestingly, dimeric inhibitor 3c exhibits are a marked
improvement compared to the monomer and certainly belongs
to the most powerful influenza NA inhibitors ever synthesized.
Whether this effect can be attributed to statistical rebinding
leading, for instance, to a lower koff-rate or other effects remains
to be investigated but it will be interesting to see its performance
in plaque reduction assays.
Biotin-conjugate 3d and fluorescein-conjugate 3e are equally
strong inhibitors. The strong binding of 3d prompted us to look
at the impact of binding on the fluorescence of 3d (Fig. 2).
The binding of NA to the fluorescein conjugate 3e quenches
the fluorescence and shifts the emission maximum to a longer
wavelength (Fig. 2a and 2b). The time course for the quenching
is consistent with an association rate constant of 2 ¥ 106 M-1 s-1
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5625–5629 | 5627
©

View Article Online
Table 1 Inhibition constants (Ki [nM]) for target phosphonatesa
3a
3b
3c
3d
3e
Oseltamivir
Zanamivir
0.234 0.036
0.224 0.037
0.040 0.010
0.241 0.052
0.162 0.029
0.130 0.032
0.268 0.039
a Inhibition of MUNANA hydrolysis catalysed by the neuraminidase from influenza virus X31 (H3N2); inhibition constants were determined as described
in the Supporting Information; KM for MUNANA = 20.2 3.3 mM.
Fig. 2 (a) Decrease in fluorescence intensity (510 nm) of 3e over time upon binding to influenza virus NA. The binding experiment was carried out at
5
◦C and concentrations of 3e and NA were comparable (9 nM and 12 nM, respectively). (b) Red-shift of fluorescence maximum of 3e upon binding to
influenza NA. Top line: unbound 3e. Bottom line: 3e bound to NA. (c) Fluorescence titration of 3e with influenza virus NA, resulting in a dissociation
constant Kd of 0.23 0.05 nM.
measured (at 37 ◦C) using the methods described in Collins et
Notes and References
al.25 Fluorescence titration of 3d with influenza NA resulted in
1 WHO, Global Alert and Resistance (GAR)/Influenza, http://www.
who.int/csr/disease/influenza/en/.
a dissociation constant Kd of 0.23 0.05 nM which is in good
agreement with the value determined by MUNANA hydrolysis
(Fig. 2c and Table 1). The changes in fluorescence are rather large
and more than adequate for binding studies, given that fluorescein
is not generally considered an environmentally sensitive probe. 3e
can therefore be used for the determination of dissociation con-
stants by fluorescence-based displacement titration. In addition,
these effects suggest new opportunities for direct quantification of
influenza neuraminidases that are independent of calorimetry or
hydrolysis of substrates such as MUNANA.
We conclude that this project has resulted in conjugatable
compounds based on the oseltamivir motif which display selective,
high-affinity binding to influenza neuraminidase. The potential of
the approach has been demonstrated through the synthesis of
dimeric inhibitor 3c and 2 diagnostic tools containing reporter
groups widely used in biological research. The binding of one
of them, fluorescein conjugate 3e, to influenza NA has been
investigated in a quantitative fashion and an excellent correlation
between the fluorescence titration and the MUNANA-based assay
has been found. It can safely be expected that both compounds
will find various applications in influenza research for instance
quantification of NA, virus counting or detection of NA clusters
on the virus surface to name only a few.
2 Health Protection Agency, HPA National Influenza Report,
http://www.hpa.org.uk.
3 M. von Itzstein, Nat. Rev. Drug Discovery, 2007, 6, 967–
974.
4 E. De Clerq, Nat. Rev. Drug Discovery, 2006, 5, 1015–
1025.
5 J. Rich, Design and Synthesis of Sialidase Inhibitors for Influenza Virus
Infections, Elsevier, Amsterdam, 2007.
6 C. U. Kim, W. Lew, M. A. Williams, H. Liu, L. Zhang, S. Swaminathan,
N. Bischofberger, M. S. Chen, D. B. Mendel, C. Y. Tai, W. G. Laver and
R. C. Stevens, J. Am. Chem. Soc., 1997, 119, 681–690.
7 M. von Itzstein, W.-Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B. Jin,
T. V. Phan, M. L. Smythe, H. F. White, S. W. Oliver, P. M. Colman, J.
N. Varghese, D. M. Ryan, J. M. Woods, R. C. Bethell, V. J. Hotham, J.
M. Cameron and C. R. Penn, Nature, 1993, 363, 418–423.
8 P. Chand, P. L. Kotian, A. Dehghani, Y. El-Kattan, T.-H. Lin, T. L.
Hutchison, Y. S. Babu, S. Bantia, A. J. Elliott and J. A. Montgomery,
J. Med. Chem., 2001, 44, 4379–4392.
9 W. P. Burmeister, B. Henrissat, C. Bosso, S. Cusack and R. W. H.
Ruigrok, Structure, 1993, 1, 19–26.
10 J. N. Varghese, W. G. Laver and P. M. Colman, Nature, 1983, 303,
35–40.
11 J. N. Varghese, J. L. McKimm-Breschkin, J. B. Caldwell, A. A. Kortt
and P. M. Colman, Proteins: Struct., Funct., Genet., 1992, 14, 327–
332.
12 P. Bossart-Whitaker, M. Carson, Y. S. Babu, C. D. Smith, W. G. Laver
and G. M. Air, J. Mol. Biol., 1993, 232, 1069–1083.
13 B. Carbain, P. J. Collins, L. Callum, S. R. Martin, A. J. Hay, J. McCauley
and H. Streicher, ChemMedChem, 2009, 4, 335–337.
14 H. Busse and H. Streicher, Bioorg. Med. Chem., 2006, 14, 1047–
1057.
Acknowledgements
15 K. Hata, K. Koseki, K. Yamaguchi, S. Moriya, Y. Suzuki, S.
Yingsakmongkon, G. Hirai, M. Sodeoka, M. von Itzstein and T.
Miyagi, Antimicrob. Agents Chemother., 2008, 52, 3484–3491.
We thank F. Hoffmann-La Roche Ltd for the donation of valuable
starting material.
5628 | Org. Biomol. Chem., 2011, 9, 5625–5629
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
16 B. Carbain, S. R. Martin, P. J. Collins, P. B. Hitchcock and H. Streicher,
Org. Biomol. Chem., 2009, 7, 2570–2575.
17 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
18 S. J. F. Macdonald, R. Cameron, D. A. Demaine, R. J. Fenton, G.
Foster, D. Gower, J. N. Hamblin, S. Hamilton, G. J. Hart, A. P. Hill,
G. G. A. Inglis, B. Jin, H. T. Jones, D. B. McConnell, J. McKimm-
Breschkin, G. Mills, V. Nguyen, I. J. Owens, N. Parry, S. E. Shanahan,
D. Smith, K. G. Watson, W.-Y. Wu and S. P. Tucker, J. Med. Chem.,
2005, 48, 2964–2971.
19 R. Wagner, M. Matrosovich and H.-D. Klenk, Rev. Med. Virol., 2002,
12, 159–166.
20 B. Carbain, P. B. Hitchcock and H. Streicher, Tetrahedron Lett., 2010,
51, 2717–2719.
21 S. Abrecht, M. C. Federspiel, H. Estermann, R. Fischer,
M. Karpf, H.-J. Mair, T. Oberhauser, G. Rimmler,
R. Trussardi and U. Zutter, Chimia, 2007, 61, 93–
99.
22 G. Mantovani, V. Ladmiral, L. Tao and D. M. Haddleton, Chem.
Commun., 2005, 2089–2091.
23 The strain is
a standard in influenza research, http://www.
uniprot.org/taxonomy/132504.
24 M. A. Rameix-Welti, F. Agou, P. Buchy, S. Mardy, J. T. Aubin, M.
Veron, S. van der Werf and N. Naffakh, Antimicrob. Agents Chemother.,
2006, 50, 3809–3815.
25 P. J. Collins, L. F. Haire, Y. P. Lin, J. Liu, R. J. Russel, P. A. Walker, J. J.
Skehel, S. R. Martin, A. J. Hay and S. J. Gamblin, Nature, 2008, 453,
1258–1262.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5625–5629 | 5629
©