Fluorous linker facilitated synthesis of teichoic acid fragments

Article abstract of DOI:10.1021/ol2033652

The use of perfluorooctylpropylsulfonylethanol as a new phosphate protecting group and fluorous linker is evaluated in the stepwise solution phase synthesis of a number of biologically relevant (carbohydrate substituted) glycerol teichoic acid fragments.

Full text of DOI:10.1021/ol2033652

ORGANIC
LETTERS
2012
Vol. 14, No. 3
848–851
Fluorous Linker Facilitated Synthesis of
Teichoic Acid Fragments
Wouter F. J. Hogendorf, Lucien N. Lameijer, Thomas J. M. Beenakker, Herman S.
ꢀ
Overkleeft, Dmitri V. Filippov, Jeroen D. C. Codee,* and Gijsbert A. Van der Marel*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden,
The Netherlands
jcodee@chem.leidenuniv.nl; marel_g@chem.leidenuniv.nl
Received December 16, 2011
ABSTRACT
The use of perfluorooctylpropylsulfonylethanol as a new phosphate protecting group and fluorous linker is evaluated in the stepwise solution
phase synthesis of a number of biologically relevant (carbohydrate substituted) glycerol teichoic acid fragments. Teichoic acid fragments, up to
the dodecamer level, were assembled by means of phosphoramidite chemistry, using a relatively small excess of the building blocks and a
repetitive efficient purification procedure of the protected intermediates by fluorous solid phase extraction (F-SPE).
The synthesis of fragments of biopolymers, and func-
tionalized derivatives thereof, is essential for progress in
the life sciences and the development of ever more effi-
cient synthetic procedures for their assembly, which con-
tinues to be an active field of research. The generation of
biopolymers is pursued via solution phase or (automated)
solid phase approaches, both of which have advantages
and disadvantages. Solution phase synthesis protocols
are labor- and time-consuming due to the intermediate
isolation and purification steps but can be executed on a
small and large scale employing a stoichiometric amount
or small excess of reagents. (Automated) solid phase
synthesis strategies are time and labor efficient by post-
poning product purification to the final stage of the
synthesis. They do, however, require a relatively large
excess of reagents (including expensive building blocks)
and are mostly executed on a relatively small scale.1
Alternative synthetic strategies have recently emerged
that are based on the use of soluble supports. In an ideal
situation, these strategies combine the “best of both
worlds”: the soluble support allows the application of
(a relatively small) excess of reagents to drive the reac-
tions to completion; it also enables the rapid isolation and
purification of intermediates and is readily adapted to
different reaction scales. Several supports have been
recommended over the years, including polyethylene
glycol polymers (PEG),2 lipophilic tails,3 and ionic
tags.4 With the advent of fluorous solid phase extraction
(F-SPE) methodologies, a technique known as light
fluorous synthesis5 has become popular for the construc-
tion of biopolymers, especially in the area of carbohy-
drate chemistry.5e,6 Applications in the assembly of oligo-
peptides5e have further been reported, but the use of
fluorous chemistry in oligonucleotide synthesis has been
restricted to tagging techniques,7 in which a fluorous
(2) Toy, P. H.; Janda, K. D. Acc. Chem. Res. 2000, 33, 546–554.
(3) Encinas, L.; Chiara, J. L. Eur. J. Org. Chem. 2009, 2163–2173. See
also references cited in this article.
(4) He, X.; Chan, T. H. Synthesis 2006, 1645–1651.
(5) (a) Handbook of Fluorous Chemistry; Gladysz, J. A., Curran, D. P.,
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Horvath, I. T., Eds.; Wiley-VCH: Weinheim, 2004. (b) Curran, D. P.
Aldrichimica Acta 2006, 39, 3–9. (c) Zhang, W.; Curran, D. P. Tetra-
hedron 2006, 62, 11837–11865. (d) Zhang, W.; Cai, C. Chem. Commun.
2008, 5686–5694. For reviews on fluorous-linker facilitated biopolymer
syntheses, see: (e) Zhang, W. Chem. Rev. 2009, 109, 749–795. (f) Zhang,
W. Green Chem. 2009, 11, 911–920.
(6) (a) Manzoni, L; Castelli, R. Org. Lett. 2006, 8, 955–957. (b)
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Carrel, F. R.; Geyer, K.; Codee, J. D. C.; Seeberger, P. H. Org. Lett.
2007, 9, 2285–2288. (c) Carrel, F. R.; Seeberger, P. H. J. Org. Chem.
2008, 73, 2058–2065. (d) Cheng, Y.; Guo, A.-L.; Guo, D.-S. Curr. Org.
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Chem. 2010, 14, 977–999. (e) Codee, J. D. C.; Ali, A.; Overkleeft, H. S.;
Van der Marel, G. A. C. R. Chim. 2011, 14, 178–193.
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37, 257–263. (b) Pearson, W. H.; Berry, D. A.; Stoy, P.; Jung, K.-Y.;
Sercel, A. D. J. Org. Chem. 2005, 70, 7114–7122. (c) Beller, C.;
Bannwarth, W. Helv. Chim. Acta 2005, 88, 171–179.
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S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311. (c) Reese, C. B. Org.
Biomol. Chem. 2005, 3, 3851–3868.
r
10.1021/ol2033652
Published on Web 01/20/2012
2012 American Chemical Society

building block is employed at the end of a solid phase
oligonucleotide synthesis to discriminate the target
full length oligomers from unwanted, capped deletion
sequences.
nitrogen protecting group, it turned out to be too base
labile in use as a hydroxyl protecting group (as in 2). Thus,
for the fluorous version of the Msc carbonate, we incor-
porated an extra methylene moiety between the fluorous
part and the sulfonyl group to provide extra insulation for
the C8F17 tail, giving the [1H,1H,2H,2H,3H,3H]-per-
fluoro-octylpropylsulfonylethoxycarbonyl (F-Psc, 3).12b
Based on these findings we decided to explore the F-Pse
group 4 as a fluorous linker and phosphate protecting
group. With this linker, synthesized as reported pre-
viously, we set out to establish the scope and limitations
of fluorous teichoic acid synthesis. As depicted in Scheme
1, we elongated F-Pse alcohol 5 in a stepwise manner with
glycerol phosphoramidite 6.10a Each elongation cycle
consisted of four steps: (1) reaction of the alcohol with
phosphoramidite 6 under the agency of dicyanoimidazole
Teichoic acids are biopolymers comprised of repeating
alditol phosphate residues, seemingly randomly deco-
rated with carbohydrate and/or D-alanyl substituents.
They are prime constituents of the Gram-positive bacter-
ial cell wall and, as such, interact with their surroundings
and play an important role in immunology.8 We have
recently reported on both the solution and solid phase
synthesis of teichoic acid fragments of Enterococcus
faecalis,9 built up from repeating glycerol phosphate
residues. Using the former technique we assembled a
kojibiose containing glycerol phosphate hexamer, and
with the latter technique, we generated a small library of
teichoic acid fragments, varying in both size and sub-
stitution pattern.10 We here report on a fluorous phase
synthesis strategy to assemble teichoic acid structures.
To this end, we implemented a new fluorous phos-
phate protective group and developed synthetic proto-
cols compatible with F-SPE extraction techniques. We
show that a light fluorous synthesis strategy is an effi-
cient approach to assemble medium sized teichoic acid
fragments.
Our first objective was the selection of a suitable light
fluorous phosphate protecting group. To date only one
such protecting group has been reported, which has been
applied in the synthesis of a disaccharide.11 We have
recently reported on the use of fluorous sulfonylethyl-
based groups to protect both amino and hydroxyl func-
tions, in the form of a carbamate and carbonate respec-
tively (see Figure 1).12 We deemed this group suitable to
protect phosphate functions since it can be removed at the
end of the synthesis by base catalyzed β-elimination. The
effective use of the 2-(methylsulfonyl)-ethyl (MSc) group
in solid phase oligonucleotide synthesis bodes well for this
approach. Whereas the use of the fluorous version of
the MSc group, the [1H,1H,2H,2H]-perfluorodecylsulfo-
nylethoxycarbonyl (F-Msc, 1), worked very well as a
Figure 1. Fluorous versions of the MSc type protecting group.
Scheme 1. Synthesis of Glycerolphosphate Dodecamer 19
(8) (a) Fischer, W. Adv. Microb. Physiol. 1988, 29, 233–302. (b)
Naumova, I. B.; Shashkov, A. S.; Tul’skaya, E. M.; Streshinskaya,
G. M.; Kozlova, Y. I.; Potekhina, N. V.; Evtushenko, L. I.; Stackebrandt,
E. FEMS Microbiol. Rev. 2001, 25, 269–283. (c) Neuhaus, F. C.; Baddiley,
J. Microbiol. Mol. Biol. Rev. 2003, 67, 686–723. (d) Weidenmaier, C.;
Peschel, A. Nat. Rev. Microbiol. 2008, 6, 276–287.
(9) (a) Theilacker, C.; Kaczynski, Z.; Kropec, A.; Fabretti, F.; Sange,
T.; Holst, O.; Huebner, J. Infect. Immun. 2006, 74, 5703–5712. (b)
Theilacker, C.; Kaczynski, Z.; Kropec, A.; Sava, I.; Ye, L.; Bychowska,
A.; Holst, O.; Huebner, J. Plos One 2011, 6, e17839. (c) Theilacker, C.;
ꢀ
Kropec, A.; Hammer, F.; Sava, I.; Wobser, D.; Sakinc, T.; Codee,
(DCI); (2) oxidation of the intermediate phosphite
with I2; (3) removal of the DMT protecting group using
dichloroacetic acid (DCA) in the presence of triethylsi-
lane (TES); and (4) F-SPE purification. It proved to be
essential that, before each F-SPE purification, the crude
reaction mixtures were partitioned between MeCN/water
(80/20) and hexane to remove most of the 4,40-dimethox-
ytriphenylmethane and excess TES, since we found that
these could not be separated from the target compounds
by F-SPE when present in relatively large amounts.
Using this protocol we were able to rapidly and efficiently
J. D. C.; Hogendorf, W. F. J.; Van der Marel, G. A.; Huebner, J.
J. Infect. Dis., in press.
(10) (a) Hogendorf, W. F. J.; Van den Bos, L. J.; Overkleeft, H. S.;
ꢀ
Codee, J. D. C.; van der Marel, G. A. Bioorg. Med. Chem. 2010,
18, 3668–3678. (b) Hogendorf, W. F. J.; Meeuwenoord, N.; Overkleeft,
H. S.; Filippov, D. V.; Laverde, D.; Kropec, A.; Huebner, J.; Van
ꢀ
der Marel, G. A.; Codee, J. D. C. Chem. Commun. 2011, 47, 8961–
8963.
(11) Liu, L.; Pohl, N. L. B. Org. Lett. 2011, 13, 1824–1827.
(12) (a) De Visser, P. C.; Van Helden, M.; Filippov, D. V.; Van der
Marel, G. A.; Drijfhout, J. W.; Van Boom, J. H.; Noort, D.; Overkleeft,
H. S. Tetrahedron Lett. 2003, 44, 9013–9016. (b) Ali, A.; Van den Berg,
R. J. B. H. N.; Overkleeft, H. S.; Filippov, D. V.; Van der Marel, G. A.;
ꢀ
Codee, J. D. C. Tetrahedron Lett. 2009, 50, 2185–2188.
Org. Lett., Vol. 14, No. 3, 2012
849

Scheme 2. Synthesis of Glucosamine Containing Hexamers 28a and 28b
generate protected oligoglycerolphosphates up to the
dodecamer level. Although the relative fluorous con-
tent in compounds 7ꢀ18 significantly decreases with
increasing oligomer length,13 this had little or no effect
on the purification efficiency and a single fluorous
silica column (2 or 4 g) was sufficient to purify all
oligomers (0.1ꢀ0.25 mmol). We did observe that a
larger excess of 6 was required to push the coupling
reactions to completion when going for larger oligo-
mers. Up to 4 equiv of the phosphoramidite were
required in the final coupling step to the dodecamer
(see Scheme 1). We argue that for longer oligomers the
automated solid phase approach becomes competitive,
and therefore, we did not explore the synthesis of
longer oligomers.
Deprotection of the fully protected dodecamer started
with removal of the F-Pse and cyanoethyl groups using
25% aqueous ammonia solution. In a model deprotection
experiment (see Supporting Information) we observed
that the F-Pse group at the terminal phosphotriester
was selectively cleaved with respect to the cyanoethyl
group. Elimination of the remaining cyanoethyl group
on the obtained phosphodiester required slightly elevated
temperatures (40 °C) and prolonged reaction times
(typically overnight) to ensure complete unmasking to
the target phosphomonoester. Applying this protocol to
fully deprotected dodecamer 19 in 86% yield over the last
two steps.
Having established that F-Pse alcohol 5 can be used for
the efficient assembly of glycerol phosphate teichoic
acids, we next investigated the incorporation of glycosyl
substituents in the TA chains through the assembly of two
teichoic acid hexamers. TA 28a carries a GlcNAc residue,
as present in TA chains of Staphylococcus aureus,14
whereas a positively charged glucosamine is grafted on
hexamer 28b, a structural element found in several Strep-
tomyces species.15 The required glucosaminyl glycerol
phosphoramidites were obtained as depicted in Scheme
2. Triol 20 was benzylated to give intermediate 2116 from
which both building blocks 24a and 24b were assembled.
Reduction of the azide functionality in 21 and subsequent
acetylation gave N-acetyl glucosamine derivative 22a,
while protection with a benzyloxycarboxyl group led to
22b. Both glucosaminyl glycerol building blocks were
then transformed into the required phosphoramidites
24a and 24b following a well-established sequence of
reactions, involving deallylation, dimethoxytritylation,
desilylation, and phosphitylation. With building blocks
24a/b the target hexamers 28a/b were assembled starting
from fluorous pentamer 11. Thus, condensation of 11 and
(14) Morath, S.; Geyer, A.; Hartung, T. J. Exp. Med. 2001, 193, 393–
397.
dodecamer 18, and ensuing hydrogenolysis of the par-
tially protected oligomer and gel filtration, led to 30 mg of
(15) (a) Tul’skaya, E. M.; Vylegzhanina, K. S.; Streshinskaya, G. M.;
Shashkov, A. S.; Naumova, I. B. Biochim. Biophys. Acta 1991, 1074,
237–242. (b) Kozlova, Y. I.; Streshinskaya, G. M.; Shashkov, A. S.;
Evtushenko, L. I.; Naumova, I. B. Biochem. (Mosc.) 1999, 64, 671–677.
(16) Figueroa-Perez, I.; Stadelmaier, A.; Morath, S.; Hartung, T.;
Schmidt, R. R. Tetrahedron: Asymmetry 2005, 16, 493–506.
(13) The relative fluorine content ranges from 37% in compound 7 to
7% in hexaGlcNHAc-glycerol phosphate 38.
850
Org. Lett., Vol. 14, No. 3, 2012

24a/b and subsequent oxidation and removal of the
DMT-group gave crude 25a/b. Also in this case F-SPE
purification proceeded uneventfully and hexamers 25a/b
were obtained in 73% and 88% respectively. The hexam-
ers were then equipped with a hexanolamine spacer using
phosphoramidite 26 to give fully protected TA structures
27a/b in pure form after F-SPE. Global deprotection
using the deprotection scheme described above led to
both target hexamers 28a/b, the successful assembly of
which indicates that the fluorous synthesis strategy can
also be applied to substituted oligoglycerolphosphates.
As a final research goal we sought to explore the
assembly of more complex structures as exemplified by
the synthesis of teichoic acid fragment 39, which is char-
acterized by the [f6)-R-glucosamine-(1f2)-sn-glycerol-
1-phosphate-] repeating unit and is found in Spirilli-
planes yamanashiensis (Scheme 3).17 The synthesis of the
required GlcNHAc-glycerol phosphoramidite building
block 32 commenced with dimethoxytritylation of the
primary alcohol in 20 (Scheme 3). Subsequent desilyla-
tion and benzylation of the resulting triol gave the
fully protected GlcNHAc-glycerol 30, which was trans-
formed into required phosphoramidite 32 through azide
reduction and acetylation followed by a deallylationꢀ
phosphitylation reaction sequence. For the assembly of
hexamer 39, F-Pse linker 5 was elongated in a stepwise
manner with 32 using the chemistry described above. As
can be seen in Scheme 3 all elongation steps proceeded
efficiently. Although 32 is a more lipophilic building
block than the above-described glycerol phosphates, this
did not pose any problems in the purification of the
oligomers. Notably the single C8F17-tail sufficed for the
easy purification of fully protected hexamer 38, having
a molecular mass of ∼4.7 kD. Finally deprotection of
38 was accomplished by β-elimination of the F-Pse
linker and cyanoethyl groups and global debenzylation
led to 32 mg of target compound 39 in 76% yield.
Scheme 3. Synthesis of Complex Hexamer 39
protecting group. The strategy is especially useful for the
assembly of multimilligram quantities of medium sized TA
fragments, featuring 6ꢀ12 repeating units. As displayed by
the assembly of teichoic acid fragment 39, complex glycerol
phosphate building blocks can also be used, indicating that
our strategy is a valuable asset for the construction of various
classes of phosphate ester containing biomolecules.
Acknowledgment. The Netherlands Organization for
Scientific Research (NWO-CW) is kindly acknowledged
for financial support.
In conclusion, we have developed an efficient fluorous
synthesis strategy for the assembly of teichoic acid
fragments, based on the application of perfluorooctyl-
propylsulfonylethanol as a new fluorous phosphate
Supporting Information Available. Full experimental
procedures and copies of 1H, 13C, and 31P NMR spectra
for 7ꢀ19, 21ꢀ25, and 27ꢀ39. This material is available
free of charge via the Internet at http://pubs.acs.org.
(17) Shashkov, A. S.; Streshinskaya, G. M.; Evtushenko, L. I.;
Naumova, I. B. Carbohydr. Res. 2001, 336, 237–242.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 3, 2012
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