Photodimerisation of glycothymidines in solution and in micelles

Article abstract of DOI:10.1039/c1cc13246f

Glycothymidines were designed and synthesized as a new class of functional glycomimetics in which a photochemical [2+2] cycloaddition of the thymine moiety induces structural changes of carbohydrate presentation. To test if photodimerisation of these glycothymidines is feasible within an array of molecules, the photochemical reaction was investigated using NMR and NMR diffusion experiments in solution as well as in the supramolecular context of detergent micelles that mimic cellular membranes.

Full text of DOI:10.1039/c1cc13246f

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COMMUNICATION
Photodimerisation of glycothymidines in solution and in micelleswz
¨
Kirsten Schwekendiek, Hauke Kobarg, Lena Daumlechner, Frank D. Sonnichsen and
Thisbe K. Lindhorst*
Received 1st June 2011, Accepted 28th June 2011
DOI: 10.1039/c1cc13246f
Glycothymidines were designed and synthesized as a new class
of functional glycomimetics in which a photochemical [2+2]
cycloaddition of the thymine moiety induces structural changes
of carbohydrate presentation. To test if photodimerisation of
these glycothymidines is feasible within an array of molecules,
the photochemical reaction was investigated using NMR and
NMR diffusion experiments in solution as well as in the supra-
molecular context of detergent micelles that mimic cellular
membranes.
Whereas glycothymidine monomer 4 absorbs at 267 nm, the
cyclobutane dimer derived from 4 shows maximal absorption
at 240 nm. In the 1H NMR spectra of 5, seven triplets are
resolved for the anomeric protons of the ribose rings (H-10) in
the range between 5.5 and 6.5 ppm, reflecting the formation of
at least four of the six possible diastereomeric products (cf. ESIz
and Scheme 2). The NMR spectrum of the monomer 4 shows a
single triplet for H-10 at 6.32 ppm. The dimeric products could
be easily separated from the monomeric starting material by
chromatography. The data demonstrate that this new class of
functional glycomimetics readily undergo the desired photo-
dimerisation reaction.
Accessibility, density, and conformation of cell surface carbo-
hydrates play essential roles in molecular recognition processes
such as in cell/bacterial adhesion.1 For their study, functional
glycomimetics are of great interest as tools for examining
structural details that influence interaction with carbo-
hydrates. In this context, it has been our goal to design
glycomimetics that feature a photosensitive functional group
to control carbohydrate presentation on surfaces or in other
model systems via irradiation.2
20-Deoxythymidine was selected as a bio-compatible scaffold
molecule for the design of photoswitchable glycothymidines.
Like thymidine, glycothymidines should undergo a photo-
chemical [2+2] cycloaddition reaction, thus leading to a
conformational change within an assembly of glycothymidine
molecules.3
As this glycothymidine was designed to mimic structural
changes within the glycocalyx, which may be utilized to affect
density, availability, and presentation mode of surface-
exposed carbohydrates, it was important to investigate whether
photodimerisation occurs in a supramolecular context. Hence,
the glycothymidine was furnished with a hydrophobic moiety,
yielding an amphiphilic glycothymidine that associates with
membranes or membrane mimics. To study the photochemical
reactivity in a supramolecular context, micelle-forming detergents
where utilized as a simplified (‘‘minimal’’) model membrane,
because these are amenable to NMR spectroscopy.
Synthesis of an amphiphilic glycothymidine started with
nucleoside 1, which was converted into its 50-azidodeoxy
derivative 6 employing an Appel reaction with sodium
azide (Scheme 3).7 Then, N-alkylation with the bromoethyl
mannoside 2 led to the desired azido-functionalized glyco-
thymidine 7, which was O-acetylated and then converted into
the fully protected isothiocyanate 9 in an aza-Wittig-type
reaction. Usually, isothiocyanates can be converted with
amines into thiourea derivatives in high yields. Here, 9 reacted
with octylamine to furnish 10. After de-O-acetylation, the
unprotected, amphiphilic thiourea derivative 11 was isolated.
Photodimerisation of 11 proceeded analogously to the
The preparation of glycothymidines was easily achieved by
a Williamson type synthesis with bromoalkyl glycosides such
as 2 (Scheme 1).4 It led to chemoselective N-alkylation of
unprotected 20-deoxythymidine. Deacetylation according to
Zemple
´
n5 gave glycothymidine 4 in high yield.
The photodimerisation of glycothymidine 4 in solution was
investigated, using acetone as triplet sensitizer. Upon irradiation
with light of 295 nm wavelength 4 undergoes a [2+2] cyclo-
addition (Scheme 2), resulting in six different stereo- and
regioisomers.6 Conversion of the monomer into its dimeric
cycloaddition product was clearly detected by the change of
the respective UV/Vis spectra of the isomeric product mixture 5.
Otto Diels Institute of Organic Chemistry,
Christiana Albertina University of Kiel, Otto-Hahn-Platz 3-4,
D-24098 Kiel, Germany. E-mail: tklind@oc.uni-kiel.de;
Fax: +49 431 880 7410; Tel: +49 431 8802023
w This article is part of the ChemComm ‘Glycochemistry and glyco-
biology’ web themed issue.
Scheme 1 Chemoselective N-alkylation of unprotected 20-deoxy-
thymidine 1 with bromoalkyl mannoside 2.
z Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13246f
c
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Chem. Commun., 2011, 47, 9399–9401 9399

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the molecules in a pulsed field gradient (PFG). The signal
intensity attenuation in a PFG NMR experiment is described
by the Stejskal–Tanner equation,10 assuming a single component
with a diffusion coefficient (D) is contributing to the signal
according to eqn (1):
2
2 2
I = I0eꢀg G d (D ꢀ d/3)D
(1)
Scheme 2 Photodimerisation of glycothymidine 4.
where g is the gyromagnetic ratio, G the amplitude of the
magnetic field gradient, d denotes the effective duration of the
gradient pulses and D is the effective diffusion time.
The peak areas of the series of 1H spectra of a diffusion
experiment were plotted against g2G2d2(D ꢀ d/3), and fitted to
a monoexponential decay to obtain the diffusion coefficient
(D). In the case of more than one differently diffusing species
contributing to the analyzed peak, a systematic deviation of
the monoexponential decay described by eqn (1) is observed.
For determination of the diffusion coefficient, the methyl
signals of the alkyl chains of the investigated compounds at
0.82 ppm (SDS) and 0.89 ppm (11) were used. At first, the
diffusion of the substances 4, 11, and deuterated SDS were
determined separately. These measurements expectedly show
that the micelle forming SDS diffuses an order of magnitude
slower than the glycothymidines 4 and 11 (Table 1). Also,
the molecular diffusion of the alkylated glycothymidine 11
(14 ꢁ 10ꢀ11 m2 sꢀ1) is about 2.5-times slower than that of the
non-alkylated species 4 (38 ꢁ 10ꢀ11 m2
s
ꢀ1). This large
decrease in diffusion can not be explained based on the
increase in the molecular size due to addition of the alkyl
chain alone. Rather, this slower diffusion originates from a
partial aggregation of the amphiphilic glycothymidine 11.
Another indication for the presence of aggregates are the
strongly broadened signals in the spectra.
Scheme 3 Synthesis and photodimerisation of amphiphilic glyco-
thymidine 11.
To investigate the incorporation of 11 into SDS micelles, the
diffusion PFG NMR spectra of 11 were recorded at two
different concentrations in SDS solutions (Table 1). The
analysis shows a large decrease in the diffusion constants
(5.7 ꢁ 10ꢀ11 m2 sꢀ1; 5.3 ꢁ 10ꢀ11 m2 sꢀ1). A comparison wꢀit1h
photoaddition reaction of 4, and led to the dimer 12 in
comparable yield (Scheme 3).
The amphiphilic glycothymidine derivative was incorporated
into well-defined SDS micelles,8 directly irradiated in the
NMR tube, and the samples characterized by NMR spectro-
scopy (Fig. 1).
the diffusion of the pure glycothymidine 11 (14 ꢁ 10ꢀ11 m2 s
)
and pure SDS (6.3 ꢁ 10ꢀ11 m2
s
ꢀ1) shows that the glyco-
thymidine 11 is incorporated quantitatively into the SDS
micelles.
In particular, a diffusion NMR experiment (DSTEBPLED)9
was used to determine diffusion constants (cf. suppl.
1
Subsequently, the micellar glycothymidine 11 was irradiated,
and re-examined by NMR. The successful dimerization inside
the micelles is evident by the appearance of multiple anomeric
ribose protons (H0-1) of the dimeric thymidine in the expected
area. The rate of dimerization inside the micelle is comparable
to that in the absence of SDS (Fig. 2). The determined
diffusion constants after dimerization are essentially unaltered
(Table 1).
informationz). This experiment comprises a series of H NMR
spectra in which the signal intensity is attenuated by motion of
To further corroborate the incorporation of the glyco-
thymidine into the SDS micelles as well as to demonstrate
that the carbohydrate moiety remains accessible, relaxation
broadening titrations with 12 mM Mn2+-EDTA solution were
performed. Addition of Mn2+-EDTA, followed by 1H–1H
gCOSY NMR experiments led to partial line broadening of
cross peaks of the ethyl mannoside (M, L) and ribose (R)
portions of glycothymidine 11, while cross peaks of the octyl
alkyl chain (A) remain unaffected (Fig. 3). Expectedly, similar
Fig. 1 Glycothymidine 11 could be incorporated into SDS micelles
and photodimerised within the micellelar structure.
c
9400 Chem. Commun., 2011, 47, 9399–9401
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Table 1 Overview of diffusion coefficients determined by PFG NMR
spectroscopy in D2O
D (m2 sꢀ1) (fitting errors)
after 230 min of irradiation
at 295 nm
D (m2 sꢀ1) (fitting errors)
before irradiation
Pure SDS 6.3 ꢁ 10ꢀ11 (ꢂ1.9 ꢁ 10ꢀ13
)
—
—
Pure 4
38 ꢁ 10ꢀ11 (ꢂ4.2 ꢁ 10ꢀ12
14 ꢁ 10ꢀ11 (ꢂ6.7 ꢁ 10ꢀ13
)
Pure 11
)
9.6 ꢁ 10ꢀ11 (ꢂ2.1 ꢁ 10ꢀ12
)
11 in SDS
solutiona
SDS
5.9 ꢁ 10ꢀ11 (ꢂ7.4 ꢁ 10ꢀ14
5.7 ꢁ 10ꢀ11 (ꢂ5.6 ꢁ 10ꢀ14
)
)
6.2 ꢁ 10ꢀ11 (ꢂ2.1 ꢁ 10ꢀ13
)
)
11
5.5 ꢁ 10ꢀ11 (ꢂ1.8 ꢁ 10ꢀ13
11 in SDS
solutionb
SDS
Fig. 3 Overlay of 1H–1H gCOSY experiments of glycothymidine 11
in SDS detergent micelles, in the absence (black contours), and in the
presence of 2.6 mM Mn2+-EDTA (red contours). Correlations are
labeled as follows: R: deoxyribose, M: mannose, A: octyl chain,
L: ethyl linker, SDS: cross peaks of the residual protons in the
deuterated SDS.
5.7 ꢁ 10ꢀ11 (ꢂ2.7 ꢁ 10ꢀ13
5.3 ꢁ 10ꢀ11 (ꢂ5.9 ꢁ 10ꢀ14
)
)
5.7 ꢁ 10ꢀ11 (ꢂ2.2 ꢁ 10ꢀ13
)
)
11
5.3 ꢁ 10ꢀ11 (ꢂ4.2 ꢁ 10ꢀ14
a
Concentration was chosen to give two molecules of 11 per SDS
b
micelle at an average. Concentration was chosen to give eight
molecules of 11 per SDS micelle at an average.
context, they were incorporated into SDS micelles, which serve
as a model system for the in vivo situation of the glycosylated
cell surface. Using 1D- and diffusion NMR experiments it is
demonstrated that glycothymidines undergo [2+2] cycloaddition
when incorporated into micelles, thus leading to an altered
pattern of surface-exposed carbohydrates. As a glyco-micelle
might be regarded as a minimal glycocalyx model, this
investigation represents the first step towards light switching
of glycosylated surfaces, and elucidation of the role of
conformational control in glycobiology.
H.K. acknowledges financial support by SFB 677 (DFG
collaborative network).
Fig. 2 Irradiation of glycothymidine 11 (pure) in D2O-solution (left
spectra) and with SDS micelles (right spectra), followed over time by
1D-1H NMR spectroscopy at 25 1C.
Notes and references
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results were obtained with the dimer in micellar solution (see
suppl. informationz). This implies that both the monomeric
and dimeric form of the investigated amphiphilic glycothymidine
are embedded into the SDS micelle via the lipophilic alkyl
chain, while the hydrophilic mannose is surface-exposed and
accessible to the paramagnetic relaxation agent, and thus
available for lectin binding.
4 J. Dahme
G. Noori, Carbohydr. Res., 1983, 116, 303–307.
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In conclusion, glycothymidines are easily obtained from
20-deoxythymidine and appropriate glycosides. They undergo
a photosensitized [2+2] dimerisation in solution, and are thus
in principle suited to test the influence of accessibility change
and conformational alterations on carbohydrate recognition
within a glycosylated surface. This is important in the context
of cell adhesion and microbial adhesion to cell surfaces,
e.g. lectin binding to the glycocalyx. To investigate if glyco-
thymidines can be used in such a more complex supramolecular
´
6 Y. Inaki, in CRC Handbook of Organic Photochemistry and
Photobiology, CRC Press, Boca Raton, FL, 2004, 104/1–104/34.
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¨
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c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9399–9401 9401