Bio-orthogonal phosphatidylserine conjugates for delivery and imaging applications

Article abstract of DOI:10.1021/jo8011336

(Chemical Equation Presented) The syntheses of phosphatidylserine (PS) conjugates are described, including fluorescent derivatives for potential cellular delivery and bioimaging applications. Installation of terminal functional groups (amine, thiol, or alkyne) onto the sn-2 chain provides reactive sites for bio-orthogonal conjugation of cargo with suitably protected PS derivatives. An amine-containing PS forms amide bonds with peptidic cargo, a thiol derivative is designed for conjugation to cargo that contain α-halo carbonyls or Michael acceptors, and the terminal alkyne PS analogue permits "click" conjugation with any azide-tagged molecule. This latter conjugation method is quite versatile as it can be performed without PS headgroup protection, in aqueous media, and with acid-labile cargo.

Full text of DOI:10.1021/jo8011336

Bio-orthogonal Phosphatidylserine Conjugates for Delivery and
Imaging Applications
Andrew J. Lampkins, Edward J. O’Neil, and Bradley D. Smith*
Department of Chemistry and Biochemistry and Walther Cancer Research Center, 251 Nieuwland Science
Hall, UniVersity of Notre Dame, Notre Dame, Indiana 46556
smith.115@nd.edu
ReceiVed May 23, 2008
The syntheses of phosphatidylserine (PS) conjugates are described, including fluorescent derivatives for
potential cellular delivery and bioimaging applications. Installation of terminal functional groups (amine,
thiol, or alkyne) onto the sn-2 chain provides reactive sites for bio-orthogonal conjugation of cargo with
suitably protected PS derivatives. An amine-containing PS forms amide bonds with peptidic cargo, a
thiol derivative is designed for conjugation to cargo that contain R-halo carbonyls or Michael acceptors,
and the terminal alkyne PS analogue permits “click” conjugation with any azide-tagged molecule. This
latter conjugation method is quite versatile as it can be performed without PS headgroup protection, in
aqueous media, and with acid-labile cargo.
Introduction
that vitamin receptors are used to deliver conjugates of folate,
B12, biotin, etc.6
Phosphatidylserine (PS) is emerging as a biologically im-
portant phospholipid that is associated with a wide range of
cell signaling events.1 There is good evidence that the plasma
membranes of most mammalian cells contain an active transport
system that pumps PS to the inner membrane surface.2 There
is also strong evidence that macrophages can engulf cells,3
lipososomes,4 and viruses5 that have surface-exposed PS. We
wish to determine if these endogenous PS transport systems
can deliver PS conjugates into cells, and whether they can be
used for delivery and imaging applications in the same way
This report describes our entry into this project, that is, the
preparation of appropriate PS conjugates for cell delivery
experiments. We decided that the cargo should not be attached
to the PS headgroup because this will likely interfere with the
molecular recognition, and that a more prudent point of
attachment was the terminus of one of the phospholipid acyl
chains.7,8 Therefore, the synthetic challenge was to devise mild
and general methods of attaching various types of molecular
cargo to the end of a PS acyl chain. The methods had to be
compatible with the PS headgroup, which contains both nu-
cleophilic amine and electrophilic carboxyl sites.
(6) (a) Lu, Y. J.; Low, P. S. AdV. Drug DeliVery ReV. 2002, 54, 675–693.
(b) Ojima, I. Acc. Chem. Res. 2008, 41, 108–119. (c) Russell-Jones, G.;
McTavish, K.; McEwan, J.; Rice, J.; Nowotnik, D. J. Inorg. Biochem. 2004, 98,
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Science 2008, 319, 210–213. (d) Zwaal, R. R. A.; Comfurius, P.; Bevers, E. M.
Cell. Mol. Life Sci. 2005, 62, 971–988.
(7) For examples of PS derivatives with fluorescent labels contained within
their sn-2 chains, see: (a) Heikinheimo, L.; Somerharju, P. Biochim. Biophys.
Acta 2002, 1591, 75–85. (b) Martin, O. C.; Pagano, R. E. Anal. Biochem. 1986,
159, 101–108. (c) Pomorski, T.; Herrmann, A.; Zimmermann, B.; Zachowski,
A.; Muller, P. Chem. Phys. Lipids 1995, 77, 139–146. (d) Pomorski, T.; Holthuis,
J. C. M.; Herrmann, A.; van Meer, G. J. Cell Sci. 2004, 117, 805–813. (e)
Pomorski, T.; Muller, P.; Zimmermann, B.; Burger, K.; Devaux, P. F.; Herrmann,
A. J. Cell Sci. 1996, 109, 687–698. (f) Tanhuanpaa, K.; Cheng, K. H.; Anttonen,
K.; Virtanen, J. A.; Somerharju, P. Biophys. J. 2001, 81, 1501–1510. (g)
Tanhuanpaa, K.; Somerharju, P. J. Biol. Chem. 1999, 274, 35359–35366.
(8) For a discussion of the membrane dynamics of phospholipids with probes
in their sn-2 chains, see: Devaux, P. F.; Fellmann, P.; Herve, P. Chem. Phys.
Lipids 2002, 116, 115–134.
(2) (a) Daleke, D. L. J. Lipid Res. 2003, 44, 233–242. (b) Darland-Ransom,
M.; Wang, X.; Sun, C. L.; Mapes, J.; Gengyo-Ando, K.; Mitani, S.; Xue, D.
Science 2008, 320, 528–531. (c) Chen, C. Y.; Ingram, M. F.; Rosal, P. H.;
Graham, T. R. J. Cell Biol. 1999, 147, 1223–1236. (d) Paulusma, C. C.; Elferink,
R. Biochim. Biophys. Acta 2005, 1741, 11–24. (e) Daleke, D. L. J. Biol. Chem.
2007, 282, 821–825. (f) Graham, T. R. Trends Cell Biol. 2004, 14, 670–677.
(3) (a) Bratton, D. L.; Henson, P. M. Curr. Biol. 2007, 18, R76-R79. (b)
Ravichandran, K. S.; Lorenz, U. Nat. ReV. Immunol. 2007, 7, 964–974.
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M.; Negishi, Y.; Aramaki, Y. FEBS Lett. 2007, 581, 325–330.
(5) Mercer, J.; Helenius, A. Science 2008, 320, 531–535.
10.1021/jo8011336 CCC: $40.75
Published on Web 07/11/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 6053–6058 6053

Lampkins et al.
SCHEME 1
SCHEME 2
The de novo synthesis of PS analogues has not been
investigated in great detail. The few reported routes are lengthy,9
target-specific,10 or plagued with stereochemical complica-
tions,11 thus they are unsuited for the rapid preparation of PS
scaffolds. Here we describe the preparation of three classes of
PS derivatives that are capable of efficient, bio-orthogonal
conjugation to the end of the sn-2 acyl chain (Scheme 1). First,
a primary amine version (PS-amine) allows formation of amide
bonds to peptidic cargo. Second, a thiol group (PS-thiol) can
be utilized for selective addition to R-halo carbonyls or conjugate
addition to Michael acceptors. Finally, a terminal alkyne (PS-
alkyne) provides direct access to “click” conjugation with any
functionalized azide. The latter two methods exploit orthogonal
reactivity patterns, which means that the conjugation can be
achieved in the presence of the unprotected PS headgroup.
hydroxyl group with an appropriately functionalized fatty acid.
This potentially troublesome acylation was carried out in
consistently high yield by using ultrasonication and glass beads
according to the procedure of Hajdu (Scheme 2).14
Initially, we attempted an enzyme-mediated transphosphati-
dylation of the PC headgroup to the unprotected PS using
phospholipase D (PLD) and excess serine.15 However, in our
hands, this approach gave consistently poor yields (0-5%) of
desired product even when the most active16 commercial PLD
(obtained from Streptomyces sp.) was employed. In most cases,
the corresponding phosphatidic acid (PA) hydrolysis products
(3a-c) were obtained in very high yield. Therefore, we decided
to take advantage of this outcome and synthetically convert
3a-c into the doubly protected PS derivatives 4a-c, which can
be subsequently deprotected using mild acid (vide infra). This
synthetic route is slightly longer, but it has several important
advantages. Compared to unprotected PS derivatives, which
aggregate extensively in organic solvents,11b,16 the protected PS
compounds (4a-c) are much easier to purify and characterize
by NMR spectroscopy.
Results and Discussion
Synthesis of Protected PS from PC Precursors. The general
synthetic strategy is to make the more tractable zwitterionic
phosphatidylcholine (PC) precursor and then convert the head-
group to PS. The PC derivatives (2a,12 2b,13 and 2c14) were
prepared from readily available lyso-PC (1) by reacting the sn-2
Conjugation Reactions with PS-amine. The PS-amine
precursor 4c has two orthogonally protected amines, and
selective deprotection of the N-Fmoc amine allows for coupling
with amine-reactive cargo (i.e., polypeptides, activated esters,
acid chlorides, etc.). The utility of this methodology is show-
(9) (a) Kazi, A. B.; Hajdu, J. Tetrahedron Lett. 1992, 33, 2291–2294. (b)
Qiu, X. X.; Ong, S. W.; Bernal, C.; Rhee, D.; Pidgeon, C. J. Org. Chem. 1994,
59, 537–543.
(10) Kazi, A. B.; Shidmand, S.; Hajdu, J. J. Org. Chem. 1999, 64, 9337–
9347.
(15) For selected examples of transphosphatidylation of PC substrates to their
PS analogues using PLD, see: (a) Brachwitz, H.; Olke, M.; Bergmann, J.; Langen,
P. Bioorg. Med. Chem. Lett. 1997, 7, 1739–1742. (b) Comfurius, P.; Zwaal,
R. F. A. Biochim. Biophys. Acta 1977, 488, 36–42. (c) Diaz, C.; Balasubramanian,
K.; Schroit, A. J. Bioconjugate Chem. 1998, 9, 250–254. (d) Picq, M.; Huang,
Y.; Lagarde, M.; Doutheau, A.; Nemoz, G. J. Med. Chem. 2002, 45, 1678–
1685. (e) Wang, P.; Schuster, M.; Wang, Y. F.; Wong, C. H. J. Am. Chem. Soc.
1993, 115, 10487–10491.
(11) (a) Delfino, J. M.; Stankovic, C. J.; Schreiber, S. L.; Richards, F. M.
Tetrahedron Lett. 1987, 28, 2323–2326. (b) Loffredo, W. M.; Tsai, M. D. Bioorg.
Chem. 1990, 18, 78–84.
(12) O’Neil, E. J.; DiVittorio, K. M.; Smith, B. D. Org. Lett. 2007, 9, 199–
202.
(13) Halter, M.; Nogata, Y.; Dannenberger, O.; Sasaki, T.; Vogel, V.
Langmuir 2004, 20, 2416–2423.
(16) Banerjee, M.; Drummond, D. C.; Srivastava, A.; Daleke, D.; Lentz, B. R.
Biochemistry 2002, 41, 7751–7762.
(14) Rosseto, R.; Hajdu, J. Tetrahedron Lett. 2005, 46, 2941–2944.
6054 J. Org. Chem. Vol. 73, No. 16, 2008

Bio-orthogonal PS Conjugates
SCHEME 3
SCHEME 4
cased by the preparation of the biotin-PS conjugate 6 (Scheme
3). Treatment of 4c with DBU selectively liberates the terminal
primary amine of the sn-2 acyl chain allowing for in situ amide
bond conjugation to afford protected biotin conjugate 5.
Subsequent headgroup deprotection with TFA cleanly gives the
desired PS derivative 6 as a single TLC spot that is stained by
both Dittmer’s17 and ninhydrin reagents (indicating the presence
of phosphate and primary amine, respectively). Although
straightforward, this amide conjugation protocol has several
potential limitations. First, a reactive acylating agent is required
for conjugation. Thus, problems in chemoselectivity may result
when polyfunctionalized substrates are used. Also, this method
requires TFA deprotection of the PS headgroup after cargo
attachment to the lipid scaffold. Therefore, this conjugation
method is not suitable for acid-labile cargo.
Conjugation Reactions with PS-thiol. The PS-thiol precur-
sor 4b was prepared and used to react with thiol-active groups.
Examples of electrophilic functional groups that react prefer-
entially with thiols (even in the presence of free amines and
carboxylates) include R-halo carbonyls and maleimides, both
of which are commonly found in commercially available
bioconjugation precursors. Scheme 4 illustrates a two-step, one-
pot thiol conjugation protocol. Treatment of PS-thiol precursor
4b with TFA, in the presence of (i-Pr)3SiH, affords the globally
deprotected PS-thiol intermediate, which is immediately reacted
with either R-bromoacetophenone or N-ethylmaleimide (NEM)
to afford conjugate 7 or 8, respectively. Both targets were
prepared in moderate yield, and ultrasonication was necessary
for optimal reactivity, as this apparently deaggregates the
deprotected PS derivatives. Of note, the freshly generated PS-
thiol loses its reactivity upon chromatographic purification or
prolonged storage. Thus, it should be used immediately in situ
after generation.
Conjugation Reactions with PS-alkyne. PS-alkyne deriva-
tive 4a was specifically constructed as a substrate for the well-
known Cu(I)-catalyzed cycloaddition reaction with suitably
functionalized azides.18 This method of click bioconjugation is
attractive because it is completely atom economical, tolerant
of virtually all other chemical functionality, and compatible with
aqueous reaction conditions.
Shown in Scheme 5 are the straightforward syntheses of
several examples of azide-tagged cargo. Azides 11, 12,19 and
1320 all react smoothly with PS-alkyne precursor 4a to give
the corresponding nitrobenzofurazan- (NBD) (24), biotin- (23),
and pyrene-functionalized (22) triazoles in good yields (Scheme
6). This conjugation reaction was optimized by using 50 mol
% of CuSO4, sodium ascorbate, and a biphasic CHCl3/H2O
(18) For recent reviews on “click chemistry” and biological applications,
see: (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001,
40, 2004–2021. (b) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2002, 67, 3057–3064. (c) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H.
Eur. J. Org. Chem. 2005, 5, 1–68. (d) Baskin, J. M.; Bertozzi, C. R. QSAR
Comb. Sci. 2007, 26, 1211–1219. (e) Moses, J. E.; Moorhouse, A. D. Chem.
Soc. ReV. 2007, 36, 1249–1262. (f) Laughlin, S. T.; Baskin, J. M.; Amacher,
S. L.; Bertozzi, C. R. Science 2008, 320, 664–667.
(19) Lin, P. C.; Ueng, S. H.; Tseng, M. C.; Ko, J. L.; Huang, K. T.; Yu,
S. C.; Adak, A. K.; Chen, Y. J.; Lin, C. C. Angew. Chem., Int. Ed. 2006, 45,
4286–4290.
(17) Dittmer, J. C.; Lester, R. L. J. Lipid Res. 1964, 5, 126–127.
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Lampkins et al.
SCHEME 5
SCHEME 6
solvent system. The products were isolated in high purity by
simple liquid extraction followed by flash chromatography.
Removal of the acid-labile protecting groups with TFA cleanly
affords the corresponding PS conjugates (25, 26, and 27) in
high yield.
We envision that, in some cases, the conjugated molecular
cargo may be sensitive to the acidic conditions required for PS
headgroup deprotection, so we evaluated the click conjugation
chemistry with the deprotected PS alkyne 28 (Scheme 7).
Initially, very low yields (<5%) were obtained when the
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Bio-orthogonal PS Conjugates
SCHEME 7
previously described click conditions were employed. However,
consistently higher product yields (45-90%) were obtained
when the reactions were carried out with ultrasonication. In fact,
25, 26, and 27 were also prepared directly from 28 using this
methodology in similar yields as reported in Scheme 6. Removal
of copper salts from these unprotected PS reaction products is
necessary,21 in contrast to their protected derivatives, and is
efficiently accomplished by treatment of the crude reaction
mixtures with silica-thiol resin and simple filtration.
Scheme 7 highlights additional examples that were prepared
directly from the deprotected PS-alkyne 28. Ester 29 was the
first derivative whose synthesis required this direct methodology.
While ester-azide 16 successfully underwent cycyloaddition with
protected PS-alkyne precursor 4a (reaction not shown, 81%
yield), subsequent TFA-mediated PS headgroup deprotection
resulted in ester cleavage,22 rendering 29 a previously unat-
tainable target. Labile amino acid derivative 21 was also directly
“clicked” with PS scaffold 28 resulting in D-ala-D-ala PS
conjugate 30, demonstrating that this method is ideal for acid-
sensitive cargo. A final example is the production of fluorescent
coumarin conjugate 32, which is suitable for microscopic
imaging studies. In summary, direct click conjugation is a robust,
bio-orthogonal method for preparing PS conjugates from
potentially any azide-functionalized cargo, including molecules
that are acid-labile.
Conclusions
Synthetic PS precursors with reactive amine, thiol, and alkyne
groups at the end of the sn-2 acyl chain are prepared and
conjugated with a wide variety of cargo molecules. Direct
reaction of alkyne 28 with azide-functionalized cargo is the
broadest in scope and most applicable to widespread production
of PS conjugates, although all of the conjugation methods have
utility depending on the exact conditions. The products of this
versatile and mild synthetic chemistry allow us to broadly
investigate PS-mediated cellular delivery, and the results will
be presented in due course.
Experimental Section
Each general method of PS conjugation is illustrated below with
one example. Detailed experimental procedures and spectral data
for all products are provided in the Supporting Information along
with chemical structures showing the countercation for each anionic
phospholipid.
General Procedure for PLD Hydrolysis of PC Derivative.
Phospholipase D (ca. 0.5 mg, from Streptomyces chromofoscus)
was added to a stirring solution of PC 2a (1.52 mmol) in a biphasic
buffer/CHCl3 (140/100 mL) system, and the reaction was allowed
to stir overnight at 40 °C. The layers were separated, and the
aqueous layer was extracted with 2:1 CHCl3:MeOH (×3). All
organic layers were combined and washed with water (×3), dried
over Na2SO4, and concentrated to dryness. The residue was purified
using column chromatography (65:25:4 CHCl3:MeOH:H2O eluent
system) to afford the PA product 3a (91% yield).
General Procedure for the Conjugation of PS-amine with
NHS Ester. To a solution of protected amine 4c (0.04 mmol) in
CHCl3 (10 mL) was added DBU (0.67 mmol), and the solution
was stirred for 30 min at ambient temperature. A solution of biotin
NHS ester (0.06 mmol, in 10 mL DMF) was then added, and the
mixture was allowed to stir for another 16 h. The reaction was
then concentrated under reduced pressure, and the crude residue
(20) McMinn, D. L.; Greenberg, M. M. J. Am. Chem. Soc. 1998, 120, 3289–
3294.
(21) The unprotected PS headgroup is known to complex divalent metal
cations, especially Cu(II); see: Hanshaw, R. G.; O’Neil, E. J.; Foley, M.;
Carpenter, R. T.; Smith, B. D. J. Mater. Chem. 2005, 15, 2707–2713.
(22) Distal ester cleavage was confirmed by TLC analysis of the reaction
mixture after stirring in 1:3 TFA/CH2Cl2 for 12 h. Two distinct spots were
observed; one was visualized using Dittmer’s and ninhydrin stains (signifying
the deprotected PS headgroup), and the other was intensely UV-active and
matched a co-spot of 1-pyrenemethanol.
J. Org. Chem. Vol. 73, No. 16, 2008 6057

Lampkins et al.
was purified using flash chromatography (65:30:5 CHCl3:MeOH:
H2O eluent system) to give the conjugated amide 5 in 67% yield.
The PS headgroup was then deprotected by dissolving in CH2Cl2/
TFA (2:1) and allowing it to stir at rt for 12 h. The reaction was
then concentrated to a crude residue which was taken up in 2:1
CHCl3:MeOH and washed with saturated NaHCO3 and water. The
organic layer was then concentrated and purified using flash
chromatography (65:25:4 CHCl3:MeOH:H2O eluent system) to
afford the PS-biotin conjugate 6 in 85% yield.
General Procedure for the Conjugation of PS-thiol with
Maleimide or r-Halo Ketone. A solution of protected thiol 4b
(0.110 mmol) and (i-Pr)3SiH (0.220 mmol) in CH2Cl2 (6 mL) and
TFA (2 mL) was allowed to stir at ambient temperature for 12 h.
The reaction was then concentrated to a crude residue which was
taken up in 2:1 CHCl3:MeOH and washed with saturated NaHCO3
and water. The organic layer was then concentrated to dryness.
The residue was taken up in dry CHCl3 (3 mL), R-bromoacetophe-
none (0.110 mmol) and Cs2CO3 (0.240 mmol) were added, and
the resulting suspension was placed in a sealed vial and warmed
in an ultrasonicating water bath at 40 °C for 12 h. The reaction
mixture was loaded directly onto a silica column, which was eluted
with CHCl3, then 65:25:4 CHCl3:MeOH:H2O to afford the conju-
gated product 7 in 42% yield.
16 (0.091 mmol), CuSO4 ·5 H2O (0.038 mmol), and sodium
ascorbate (0.076 mmol) in 1:1 CHCl3/H2O (10 mL) was placed in
a sealed vial and warmed in an ultrasonicating water bath at 40 °C
for 3 h. The organic layer was then separated and the aqueous layer
extracted with 2:1 CHCl3:MeOH (×3). All organics were combined,
dried over Na2SO4, concentrated, and purified using flash chroma-
tography (0-20% MeOH in CHCl3 then 65:25:4 CHCl3:MeOH:
H2O). The fractions that contained product were combined, and
thiol-derived silica gel was added followed by vigorous stirring
for 30 min. The resin was then removed by suction filtration, and
the filtrate was concentrated to afford conjugated product 29 in
55% yield.
Acknowledgment. We thank the Walther Cancer Research
Center, the National Institutes of Health, and the University of
Notre Dame for their generous support.
Supporting Information Available: Detailed experimental
1
procedures, spectral data, copies of H and 13C NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
General Procedure for the Conjugation of PS-alkyne with
Functionalized Azide. A mixture of alkyne 28 (0.076 mmol), azide
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