Fluorination of mammalian cell surfaces via the sialic acid biosynthetic pathway

Article abstract of DOI:10.1016/j.bmcl.2008.09.010

Metabolic oligosaccharide engineering has been employed to introduce fluorine-containing groups onto mammalian cell surfaces. Incubation of HeLa, Jurkat, and HL60 cells in culture with fluorinated sialic acid and mannosamine analogues resulted in cell-sur

Full text of DOI:10.1016/j.bmcl.2008.09.010

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5945–5947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorination of mammalian cell surfaces via the sialic acid biosynthetic pathway
Laila Dafik , Marc d’Alarcao b,*, Krishna Kumar
a
a,c,*
a
Department of Chemistry, Tufts University, Medford, 62 Talbot Avenue, MA 02155-5813, USA
Department of Chemistry, San José State University, San José, CA 95192-0101, USA
Cancer Center, Tufts Medical Center, Boston, MA 02110, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Metabolic oligosaccharide engineering has been employed to introduce fluorine-containing groups onto
mammalian cell surfaces. Incubation of HeLa, Jurkat, and HL60 cells in culture with fluorinated sialic acid
and mannosamine analogues resulted in cell-surface presentation of fluorinated glycans. Metabolic con-
version of fluorinated precursors was detected and quantified by DMB-derivatization and HPLC ESI-MS
analysis. Between 7% and 72% of total membrane-associated sialosides were fluorinated, depending on
the precursor used and the cell type. Fluorination of mammalian cell surfaces provides a means for intro-
ducing a bioorthogonal surface for modulating noncovalent interactions such as those involved in cell
adhesion.
Received 21 June 2008
Revised 28 August 2008
Accepted 2 September 2008
Available online 6 September 2008
Keywords:
Glycoengineering
Sialic acid
Fluorination
Cell adhesion
Ó 2008 Elsevier Ltd. All rights reserved.
The ability to decorate the exterior of living cells with cova-
lently attached chemical entities not normally found on cell sur-
faces provides a means for low background detection and highly
specific chemical modification of the modified cells. Metabolic gly-
iodo-, thio-, methylthio-, and methylsulfonyl- in place of the 9-po-
sition hydroxyl group of sialic acid. Since none of these functional
groups is normally found on cell surfaces, their installation imbues
the engineered cells with covalent or noncovalent chemical prop-
erties distinct from those normally found on cells. This technique
has been effective in modulating several biological phenomena
6
coengineering has proven to be a very successful tactic for attach-
ing unnatural functionalities to cells.1
–3
In this approach, a
1
0,19,22
8,9
18,23
synthetic monosaccharide similar in structure to a natural precur-
sor in a biosynthesis pathway for a cell-surface glycan, but bearing
an unnatural functional group, is incubated with cells. If the mod-
ified monosaccharide enters the cell and is processed by the bio-
synthetic enzymes similarly to the natural precursor, then the
resulting cell-surface glycan bears the unnatural functional group.
The sialic acid pathway has been extensively used for metabolic
glycoengineering of cell surfaces because of its tolerance of precur-
sors with unnatural N-acyl groups. Both unnaturally acylated neu-
including cell adhesion,
differentiation, viral infection,
1
2
and immunogenicity. Recently our laboratory has extended this
strategy to permit presentation of fluoroalkyl groups on the glyco-
1
9
calyx of cultured mammalian cells. Fluorination of cell surfaces
in vivo imparts several new attributes to the cells because: (1) flu-
orocarbons provide a means of generating interaction interfaces
that are simultaneously hydrophilic and lipophobic, a feature that
has been valuable in modulating interaction in other engineered
2
4,25
biomolecules;
and (2) fluorine is virtually absent in soft tissue.
4
–6
7–14
raminic acids
and mannosamines
are processed by the
Accordingly, fluorination of cells results in reduced adhesion to
normal (unfluorinated) extracellular matrix biomolecules,19 and
may provide a means of patterning cells on surfaces via altered
noncovalent interactions. Also, since tumor cells typically express
pathway and have been used to present unnatural functional
groups on the surfaces of cells, both in culture and in live ro-
1
5–17
dents.
fully
A wide range of unnatural groups have been success-
installed onto cell-surface sialoconjugates via
x
elevated levels of sialic acids, especially as sialyl Lewis and sialyl
glycoengineering, including chain-extended N-acyl groups such
Lewisa epitopes, on their glycocalyx compared with non-trans-
4
12,13
18
26,27
as N-propanoyl-, N-butanoyl-,
and N-pentanoyl-, as well as
formed cells,
treatment with fluorinated precursors in the sia-
6
6,19
N-acyl groups comprising fluoromethyl-, trifluoromethyl-,
lic acid pathway may allow low-background detection and imaging
of tumors by fluorine MRI.
keto-, azido-, thio-, succinato-,6 and aryl
2
0
21
11
5,12
moieties at the
5
-position of sialic acid, and amino-, acetamido-, succinatamido-,
The present study examines the scope and limitations for fluo-
rination of cells using the sialic acid biosynthetic pathway, and re-
ports the adhesion of labeled cells to the matrix biomolecule
fibronectin. A panel of unnatural mannosamine and sialic acid ana-
logues with differing fluoroalkyl groups (Scheme 1) was synthe-
sized and incubated with cells. Biosynthetic conversion and
*
Corresponding authors. Tel.: +1 4089244962; fax: +1 4089244945 (M.D.), tel.:
+1 6176275651; fax: +1 6176273443 (K.K.).
E-mail addresses: marc.dalarcao@science.sjsu.edu (M. d’Alarcao), krishna.
kumar@tufts.edu (K. Kumar).
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.010

5
946
L. Dafik et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5945–5947
O
OH
CO R'
2
NH ·HCl
HN
R
HO
R
OH
HO
HO
HO
2
HO
O
RCO2H, HBTU
DMF
HO
O
sodium pyruvate
aldolase
H
N
O
OH
OH
HO
OH
HO
1
2
O
3 (R' = H)
MeOH, AcCl
4
(R' = Me)
R
Compound
CH3
−Me
Ac O, Py
2
Ac O, Py
2
a
CF3
b
c
d
OAc
O
CO Me
2
AcO
R
OAc
H
N
O
CF3
AcO
HN
R
OAc
AcO
O
AcO
AcO
OAc
O
CF3
e
f
5
6
F2
C
CF3
C
F2
C
CF3
g
F
2
Scheme 1. Synthesis of modified mannosamines and sialic acids.
expression on cell surface was measured by hydrolysis of the oligo-
of cellular membrane presentation of the unnatural sialic acids
(Table 1). The incorporation was confirmed by HPLC analysis and
the identities of the peaks were confirmed by co-injection of the
synthetic standards and LC ESI MS. Incubation with fluorinated
mannosamines 5b and 5d resulted in cells displaying fluorinated
sialic acids comprising 22% and 63% of their total cellular mem-
brane sialic acids. However, incubation with mannosamines 5e–
g, possessing longer fluoroalkyl chains, resulted in no detectable
cellular membrane fluorination.
2
8
saccharides and HPLC analysis of a fluorescent derivative, and
cell adhesion was measured to fibronectin-coated culture plates.
Mannosamine analogues 5a–g (Scheme 1) were prepared by
HBTU-mediated acylation of mannosamine with the corresponding
alkanoic acid, followed by peracetylation. Sialic acids analogues
6
a–g were prepared from the corresponding acylated mannosam-
ines 2a–g by sialic acid aldolase (EC 4.1.3.3) catalyzed condensa-
tion with pyruvate, followed by formation of the methyl esters
with methanolic HCl and peracetylation. The compounds were pre-
pared as the peracetylated derivatives to facilitate diffusion across
Incubation of cells with fluorinated sialic acids 6b, d, e, and f re-
sulted in higher levels of cellular membrane presentation of the
modified glycans than with the corresponding mannosamine, as
expected since sialic acid is later in the biosynthetic pathway than
mannosamine. This circumvents the most stringent enzyme in the
5
the cell membrane.
To determine if cells would process the fluorinated mannosam-
ines and display them on their cell-surface glycans, cultured
2
9
30
HL60, Jurkat, and HeLa cells were incubated with 5a–g and the
membrane-bound sialic acids were released by acid hydrolysis,
derivatized with the fluorogenic reagent 1,2-diamino-4,5-methy-
lene-dioxybenzene (DMB), and analyzed by HPLC, according to
pathway with respect to substrate tolerance. For example, incu-
bation of HL60 cells with 6d resulted in cells displaying the fluori-
nated sialic acid comprising 72% of their total cellular membrane
sialic acids (Table 1). Incubation of HL60 cells with 6f lead to mod-
ification of 7% of the cellular membrane sialic acids with the pen-
tafluoropentanoyl group, a modification not possible by incubation
with mannosamine 5f. Incubation with 6g led to no detectable cel-
lular membrane modification, suggesting that the size limit for the
fluoroacylated sialic acid to be processed by the biosynthetic path-
way had been exceeded.
2
8
Hara et al. Synthetic sialic acid standards 3a–g were also deriva-
tized to establish retention times. The results were similar for all
three cell types, with HL60 typically showing the greatest extent
Table 1
Presentation of modified sialic acid derivatives on cellular membranes of HL60 cells
treated with compounds 5 and 6
To rule out the possibility that the modified sialic acids detected
were due to contamination by the precursor with which the cells
were incubated rather than cellular membrane-derived, we per-
formed a pulse-chase experiment with two different analogues.
HL60 cells were incubated with compound 6d for 72 h, then 6b
was added at an equimolar concentration for 4 h prior to harvest-
ing the cells. Analysis of the membranes as described above
showed only 6d on the cellular membrane, with no detectable
6b. This result confirms that the detected modified sialic acids
are not the result of precursor contamination, and establishes that
flux through the biosynthetic pathway from fluorinated sialic acids
to cellular membrane glycans does not occur to any significant ex-
tent in 4 h.
Incubation
compound
N-Ac-Neu acida
Modified
sialic acid
Total sialic
acid
No. of CF
3
a
a
groups/cellb
None
1.00
1.10
0.90
1.00
0.30
0.29
0.35
0.84
1.49
1.20
—
1.00
1.70
1.10
1.20
0.80
0.91
1.24
1.14
1.61
1.20
—
—
5
5
5
5
6
6
6
6
6
a
b
c
d
b
d
e
f
0.60
0.20
0.20
0.50
0.62
0.89
0.30
0.12
—
7
2.0 Â 10
—
7
4.4 Â 10
7
4.5 Â 10
7
6.8 Â 10
7
3.9 Â 10
7
1.5 Â 10
g
—
The relative amounts were determined by DMB-labeling, integrated areas in HPLC,
and the cell count.
By comparing the HPLC peak fluorescence intensities of the
DMB derivatives of the cellular membrane-derived fluorinated sia-
lic acids with a standard curve generated from the synthetic stan-
dards, an estimate of total sialic acid and of the modified sialic acid
a
Values are normalized to N-acetylneuraminic acid (3–Me) in untreated cells.
Calculated number of CF groups based on total sialic acid as determined by the
3
b
DMB labeling standard curve.

L. Dafik et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5945–5947
5947
than HeLa or Jurkat cells, but all three cell types behaved qualita-
tively similarly. Cells with surfaces modified by trifluorobutanoyl
groups showed reduced adhesion to fibronectin. Studies are under-
way to elucidate the physical basis for this phenomenon.
Acknowledgments
We thank the Tufts MC GRASP center (D. Jefferson, S. Kwok and
D. Lee), D. Walt, D. Kaplan and D. Lee for the use of their tissue cul-
ture facilities. HL60 cells were a kind gift from R. Horstkorte (Cha-
2
9
rité – Universitätsmedizin, Berlin).
This work was partially
supported by the NIH (CA125033) and Tufts University.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.010.
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