Peptoidic amino- and guanidinium-carrier systems: Targeted drug delivery into the cell cytosol or the nucleus

Article abstract of DOI:10.1021/jm070603m

Efficient drug delivery is essential for many therapeutic applications. Some cell-penetrating peptides, peptide mimetics, and peptoids express transport function that, however, lack in most cases specific intracellular destination. In this study, carrier-peptoids with either amino or guanidinium side chains, were investigated with regard to their cellular uptake, toxicity, and intracellular localization. Transport specifically to the cytosol or to the nuclei was observed, thus providing a powerful tool for targeted drug delivery.

Full text of DOI:10.1021/jm070603m

376
J. Med. Chem. 2008, 51, 376–379
Peptoidic Amino- and Guanidinium-Carrier
Systems: Targeted Drug Delivery into the
Cell Cytosol or the Nucleus
Figure 1. Structures of the backbone of an R-peptide (1), ꢀ-peptide
(2), and a peptoid (3).
Tina Schröder, Nicole Niemeier,† Sergii Afonin,†
Anne S. Ulrich,† Harald F. Krug,‡ and Stefan Bräse*
aggregation.3,14,15 In contrast to both R- and ꢀ-peptides, the side
chains of peptoids are attached to the nitrogen atom instead of
the carbon, therefore, they lack hydrogen-bonding potential,
which prevents backbone-driven aggregation and thus increases
bioavailability.3,14,15
UniVersity of Karlsruhe (TH), Institute of Organic Chemistry,
Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
ReceiVed May 24, 2007
The use of peptoids as effective, water-soluble nontoxic
molecular transporters for intracellular drug delivery or as
molecular probes for bioconjugation has also been reported.16–21
Peptoids with guanidinium head groups attached to alkyl chains
have also been used to mimic peptide-hormones, antibiotics,
and receptor–ligands.16–20
The comparison of oligo(poly)-lysine (Lys)n and oligo(poly)-
arginine (Arg)n peptides has also been under investigation.3,22–24
It was demonstrated that (Arg)n shows faster uptake than (Lys)n.
Moreover, it seems that major penetration pathways may differ
for (Arg)n and (Lys)n.23,25 Notably, if CPPs are constructed with
a single type of cationic side chain, cellular uptake efficiency
usually decreases in the following order: Arg > Lys > Orn >
His.
Here we translate those findings to peptoid molecules and
compare the carrier potency of peptoids with amino side chains
(amino-peptoids) against peptoids with guanidinium side chains
(guanidinium-peptoids). Syntheses, toxicity, and cellular uptake
are reported. We observe transport to the cytosol or to the nuclei,
depending on the choice of the side-chain functionality. To our
knowledge, the possibility of determining the intracellular
destination without only additional localization signal is reported
for the first time, thus providing a powerful tool for targeted
drug delivery.
Abstract: Efficient drug delivery is essential for many therapeutic
applications. Some cell-penetrating peptides, peptide mimetics, and
peptoids express transport function that, however, lack in most cases
specific intracellular destination. In this study, carrier-peptoids with
either amino or guanidinium side chains, were investigated with regard
to their cellular uptake, toxicity, and intracellular localization. Transport
specifically to the cytosol or to the nuclei was observed, thus providing
a powerful tool for targeted drug delivery.
It has been well-known for several decades that some peptides
with basic amino acid residues are taken up rapidly by cells in
culture.1–9 Initial assays suggested that these peptides could
directly traverse the plasma membrane by an unknown mech-
anism, independent of classical receptor-mediated pathways.
Some of these peptides were discovered as basic domains
responsible for the translocation of naturally transduced proteins
and were, therefore, referred to as protein transduction domains
(PTDsa).10–12 They can transport into the cell covalently attached
cargo molecules of diverse chemical nature (oligonucleotides,
proteins, fluorophores, PNAs, and even liposomes or nanopar-
ticles). Many peptides composed of R-amino acids (1, Figure
1) with the same properties were discovered or designed since
then and are described as a functional group by the term cell-
penetrating peptides (CPPs).10–12
The most important structural features for cellular uptake
efficiency of CPPs appear to be the short size (i), high content
of cationic residues (ii), and variable spacing between the
charges (iii), while the backbone conformation does not seem
to play a critical role.10–12 However, the bioavailability of CPPs
is usually low due to in vivo proteolysis. Therefore, short peptide
mimetics with modified backbones, carrying basic functionalities
such as amino or guanidinium groups, may serve as a valuable
alternative to the CPPs because of their enhanced stability in
vivo.
For the experiments, a fluorescently labeled homohexameric
peptoid with amino-group carrying side chains was compared
with two peptoids carrying guanidinium side chains, assembled
as a homopenta- or hexamer.
The building blocks were prepared starting from 1,6-
diaminohexane following a slightly modified procedure of
Bradley et al.15a and Liskamp et al. (for the reaction scheme,
see Supporting Information).18a,21 The protected and function-
alized building blocks were assembled on the solid-phase
(Scheme 1). The reactions were carried out using Rink amide
resin 4 as a result of its stability and ease of the first coupling
step. The synthesis of the backbone was completed by coupling
a fluorophore. The labels, 5(6)-carboxyfluorescein (Figure 2,
9a, Fluo, λabs ) 492 nm, λem ) 517 nm) or rhodamine-B (Figure
2, 9b, Rhod, λabs ) 550 nm, λem ) 580 nm), were attached to
peptoids prior to cleavage from the solid support. A spacer (6-
aminohexanoic acid) was used to prevent interactions of the
marker with the peptoid moiety (Scheme 1). To isolate the
amino-peptoid Fluo-{6,6,6,6,6,6}-NH2 10, it was simultaneously
deprotected and cleaved from the resin by using trifluoroacetic
acid (TFA)/triisopropyl silane (TIS; Scheme 2). The molecule
was precipitated in cold diethylether, filtered off, and dried in
vacuo.21 It was characterized by mass spectrometry, UV/vis,
and IR. Before the guanidinium-peptoids Rhod-{6G,6G,6G,6G,6G}-
NH2 11 and Fluo-{6G,6G,6G,6G,6G,6G}-NH2 12 could be cleaved
from the solid support, they had to be orthogonally deprotected
Proteolytically stable ꢀ-peptides (2, Figure 1), for example,
have been under intensive investigation during the past years13
and were shown to be efficiently internalized by cultured
mammalian cells.3
Peptoids (oligo-N-alkylglycines; 3, Figure 1) are stable against
proteases, like ꢀ-peptides, but usually are less prone to
* To whom correspondence should be addressed. Phone: (+)49 (0)721
608 2902. Fax: (+)49 (0)721 608 8581. E-mail: braese@ioc.uka.de.
†
Forschungszentrum Karlsruhe, IBG-1 and ITG, 76344 Eggenstein-
Leopoldshafen, Germany.
‡
Empa, Materials-Biology Interactions Laboratory, Lerchenfeldstr. 5,
9041 St. Gallen, Switzerland.
aAbbreviations: DAPI, 4′,6-diamidino-2-phenylindol; DBU, 1,8-diaza-
bicyclo[5.4.0]undecen; DIPEA, diisopropyl-ethyl amine; CPPs, cell-penetrating
peptides; FACS, fluorescence-assisted cell sorting; PG, protection group; PTDs,
protein transduction domains; TFA, trifluoroacetic acid; TIS, triisopropyl silane.
10.1021/jm070603m CCC: $40.75
2008 American Chemical Society
Published on Web 01/24/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 377
Scheme 1. Solid Phase Synthesis of Peptoids, Attachment of the
Spacer (6-Aminohexanoidc Acid), and Labeling with the
Fluorophore 5(6)-Carboxyfluorescein 9a or Rhodamine-B 9ba
Scheme 3. Deprotection, Formation of the Guanidinium Groups,
and Cleavage from the Solid Support of Guanidinium-peptoid
Rhod-{6G,6G,6G,6G,6G}-NH2 11a
a Reagents and conditions: (a) 2-mercaptoethanol/DBU (0.3 M in DMF),
3 times 45 min; (b) 10.0 equiv 1H-pyrazole-1-carboxamidine hydrochloride,
10.0 equiv DIPEA, DMF, rt, 24 h; (c) TFA/TIS (95:5), rt, 3 h.
a Reagents and conditions: (a) 20% piperidine in DMF, 3 × 2 min; (b)
3.00 equiv monomer, 2.00 equiv PyBrOP, 4.00 equiv DIPEA, CH2Cl2, rt,
24 h; (c) 3.00 equiv Fmoc-aminohexanoic acid, 2.00 equiv PyBrOP, 4.00
equiv DIPEA in CH2Cl2, rt, 24 h; (d) 3.00 equiv 5(6)-carboxyfluorescein,
3.00 equiv HOBt, 3.00 equiv DIC in DMF/CH2Cl2 (1:1), rt, 5 h; (e) 3.00
equiv rhodamine-B, CH2Cl2, rt, 4 d.21
Scheme 4. Deprotection, Formation of the Guanidinium Groups,
and Cleavage from the Solid Support of the
Guanidinium-peptoid Fluo-{6G,6G,6G,6G,6G,6G}-NH2 12a
Figure 2. Structures of the fluorophores 5(6)-carboxyfluorescein 9a
and rhodamine-B 9b.
a Reaction conditions are identical to those described in Scheme 3.
Scheme 2. Cleavage of Amino-peptoid Fluo-{6,6,6,6,6,6}-NH2
10a
Furthermore, as can be seen from Figure 3, the amino-peptoid
10 accumulates preferentially in the cytosol (cytosolic staining),
whereas the fluorescence from guanidinium-peptoids 11 and 12
is mainly associated with the cell nuclei and even with the
nucleoli (Figure 3 and SI). We did not observe any profound
differences in either uptake rate or in intracellular distribution
between 11 and 12. Therefore, in our case, the attached
fluorophore had no influence on the diverse destinations inside
the cell in contrast to previously reported examples.3,22–24 Only
a slight difference is noted in the intensities due to the lower
light extinction coefficient of 5(6)-carboxyfluorescein 9a (for
more information, e.g., the uptake in tabular form, see SI).
Thus, a differential transport to the cytosol (amino-peptoid
10) and to the nucleus (guanidinium-peptoids 11 and 12, Figure
3) could be observed. Note that the guanidinium-peptoids do
not exclusively accumulate in the nucleous, but preferentially.
The results for the uptake efficiency confirm previous findings
with (Arg)n and (Lys)n peptides23,24 and can be explained
analogously, suggesting an uptake by macropinocytosis for the
amino-peptoid and direct membrane transduction for the guani-
dinium-peptoids.
To verify that the synthesized peptoids could be used for in
vivo applications and to check that the faster uptake of carriers
11 and 12 is not due to cell membrane damage or perforation,
their toxicity toward mammalian cells was investigated. To do
so, the peptoids were tested in different established cytotoxicity
assays. For an independent cross-check, two tests based on
different detection principles were carried out. The WST-1 test
detects only the viable cells by examination of the intracellular
reduction potential, whereas the LDH test detects exclusively
dead cells.21,28–30
a Reagents and conditions: (a) TFA/TIS (95:5), rt, 3 h.
and the free amino side chains were transformed to guanidinium
groups. Finally, the guanidinium-peptoids were cleaved from
the resin (Schemes 3 and 4), isolated, and characterized
following the procedure of 10. All peptoids were purified by
HPLC (purity >95%; for method see SI).
To test the cellular uptake of the peptoids, FACS analysis
was performed. The uptake was measured as increase in the
number of fluorescent cells. The uptake of the molecules after
incubation for 10, 30, and 60 min and at three concentrations
(50, 100, 200 µM) was tested on the adherent cell lines from
human epithelial lung cancer (A549)26 and normal human
endothelia (ECV304).27 For the results of the ECV304 cells,
see SI. A clear concentration and time-dependent increase in
peptoid-associated fluorescence was detected for both cell lines
(Figure 4). For the highest concentration of peptoids (200 µM)
after 10 min incubation already 90% of the cells could be
detected by FACS for 11 and 12, while only 32% of the cells
were detected if incubated with 10. Thus, uptake rates for
guanidinium-peptoids are higher than for the amino-peptoid.

378 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Letters
overall higher toxicity and was the only one where viability of
the cells dropped below 50% (at concentrations equal to and
above 150 µM). To examine whether the observed toxicity for
the highest concentrations could be associated with the coupled
fluorophore, the viability tests were performed with the fluo-
rophore-free peptoids {6G,6G,6G,6G,6G}-NH2 (11 w/o) and
{6,6,6,6,6,6}-NH2 (10 w/o; see SI). It can be seen (Figure 5)
that such an assumption was right, as after a 48 h incubation
time with the same high concentrations of peptoids, indeed the
cell viability was restored or even improved. Performing the
WST-1 test on the ECV304 cell line as well as an LDH assay
on the A549 cells, very similar results are obtained for all the
tested peptoids (see SI).
In conclusion, the synthesis of guanidinylated peptoids and
biological studies of these carriers are presented compared to a
peptoid with amino side chains. We have investigated these
efficient carrier systems with respect to their toxicity, cellular
uptake, and destination within the cells. It is demonstrated here
that both systems did not show any significant toxicity. The
guanidinium-peptoids caused a decrease in the viability of
sensitive cell lines only at high concentrations and after long
incubation times, and this effect seems to be due to the coupled
fluorophore. All peptoids translocated into the cells and are,
therefore, useful as carriers for drug delivery. However, certain
differences in the uptake related to the cationic moiety are
present. First, the amino-peptoid requires longer times to
complete translocation into the cell, while the uptake rate for
the guanidinium-peptoids is much faster, presumably due to a
different translocation mechanism.23–25 Second, intracellular
accumulation of the peptoids is different; the amino-peptoid
resides in the cytosol, while the guanidinium-peptoids ac-
cumulate preferentially in the nucleus. Thus, the uptake rate
and destination of the carriers can be tuned by just modifying
the side chain functionality and does not require any additional
leading motif. Low cytotoxicity and high stability makes the
molecules based on the peptoidic backbone attractive candidates
for in vivo intracellular drug delivery. Further applications are
in progress.
Figure 3. Fluorescence microscopy of the A549 cells incubated with
the purified peptoids 10, 11, and 12. Concentration of peptoids, 10 µM;
coincubation time, 24 h. The guanidinium-peptoids 11 and 12 show
higher uptake rates and accumulate in the cellular nucleoli. For images
with other concentrations and incubation times, see SI.
Figure 4. FACS analysis of the uptake of peptoids 10, 11, and 12.
The results are given as the amount of fluorescent A549 cells in percent
as a function of incubation time for the different concentrations of
peptoids.
Peptoid-carriers selectively deliver the cargo either to the
cytosol (amino-peptoid) or to the cell nucleous (guanidinium-
peptoid) were synthesized and shown to be a promising tool
for the use in drug delivery.
Acknowledgment. This work has been supported by the
DFG (CFN, E 1.1, E 1.2) and the LGF BW (fellowship to T.S.).
Supporting Information Available: Detailed experimental
procedures, analytical data, and results of the biological tests/assays
are available. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Frankel, A. D.; Pabo, C. O. Cellular uptake of the tat protein from
human immunodeficiency virus. Cell 1988, 55, 1189–1193.
(2) Green, M.; Loewenstein, P. M. Autonomous functional domains of
chemically synthesized human immunodeficiency virus tat trans-
activator protein. Cell 1988, 55, 1179–1188.
(3) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.;
Steinman, L.; Rothbard, J. B. The design, synthesis, and evaluation
of molecules that enable or enhance cellular uptake: peptoid molecular
transporters. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003–13008.
(4) Fawell, S.; Seery, J.; Daikh, Y.; Moore, C.; Chen, L. L.; Pepinsky,
B.; Barsoum, J. Tat-mediated delivery of heterologous proteins into
cells. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 664–668.
Figure 5. Viability test (WST-1) of A549 cells incubated for 48 h in
the presence of fluorescently-labelled peptoids 10, 11, and 12 and
respective fluorophore-free analogues 10 w/o and 11 w/o. The decrease
of viability at high concentrations is due to the coupled fluorophore.
The results are confirmed by the LDH assay (data shown in SI).
As can be seen from Figure 5, over a prolonged incubation
time (48 h), the viability of A549 cells in the presence of the
two peptoids 10 and 12 did slightly decrease only for
concentrations above 100 µM and 150 µM, respectively.
Interestingly, the shorter guanidinium-peptoid 11 showed an
(5) Pepinsky, R. B.; Androphy, E. J.; Corina, K.; Brown, R.; Barsoum, J.
Specific inhibition of a human papillomavirus E2 trans-activator by
intracellular delivery of its repressor. DNA Cell Biol. 1994, 13, 1011–
1019.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 379
(6) Vives, E.; Charneau, P.; van Rietschoten, J.; Rochat, H.; Bahraoui, E.
Effects of the Tat basic domain on human immunodeficiency virus
type 1 transactivation, using chemically synthesized Tat protein and
Tat peptides. J. Virol. 1994, 68, 3343–3353.
(7) Vives, E.; Brodin, P.; Lebleu, B. A truncated HIV-1 Tat protein basic
domain rapidly translocates through the plasma membrane and
accumulates in the cell nucleus. J. Biol. Chem. 1997, 272, 16010–
16017.
(18) (a) Kruijtzer, J. A. W.; Hofmemeyer, L. J. F.; Wigger, H.; Versluis,
C.; Liskamp, R. M. J. Solid-phase syntheses of peptoids using Fmoc-
protected N-substituted glycines: The synthesis of (retro)peptoids of
leu-enkephalin and substance P. Chem.sEur. J. 1998, 4, 1570–1580.
(b) Kruijtzer, J. A. W.; Nijenhuis, W. A. J.; Wanders, N.; Willem,
H.; Liskamp, R. M. J.; Adan, R. A. Peptoid-peptide hybrids as potent
novel melanocortin receptor ligands. J. Med. Chem. 2005, 48, 4224–
4230.
(8) Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A. The third
helix of the Antennapedia homeodomain translocates through biologi-
cal membranes. J. Biol. Chem. 1994, 269, 10444–10450.
(9) Elliott, G.; Ohare, P. Intercellular trafficking and protein delivery by
a herpesvirus structural protein. Cell 1997, 88, 223–233.
(10) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Cell-
penetrating peptides. Trends Pharmacol. Sci. 2000, 21, 99–103.
(11) Jeang, K. T.; Xiao, H.; Rich, E. A. Multifaceted activities of the HIV-1
transactivator of transcription, Tat. J. Biol. Chem. 1999, 274, 28837–
28840.
(12) Nagahara, H.; Vocero-Akbani, A. M.; Snyder, E. L.; Ho, A.; Latham,
D. G.; Lissy, N. A.; Becker-Hapak, M.; Ezhevsky, S. A.; Dowdy,
S. F. Transduction of full-length TAT fusion proteins into mammalian
cells: TAT-p27 Kip1 induces cell migration. Nat. Med. 1998, 4, 1449–
1452.
(13) Rueping, M.; Mahajan, Y. R.; Jaun, B.; Seebach, D. Design, synthesis
and structural investigations of a ꢀ-peptide forming a 314-helix
stabilized by electrostatic interactions. Chem.sEur. J. 2004, 10, 1607–
1615.
(14) (a) Figliozzi, G. M.; Goldsmith, R.; Ng, S.; Banville, S. C.; Zucker-
mann, R. N. Synthesis of N-substituted glycine peptoid libraries.
Methods Enzymol. 1996, 267, 437–447. (b) Miller, S. M.; Simon, R. J.;
Ng, S.; Zuckermann, R. N.; Kerr, J. M.; Moos, W. H. Comparison of
the proteolytic susceptibilities of homologous L-amino acid, D-amino
acid, and N-substituted glycine peptide and peptoid oligomers. Drug
DeV. Res. 1995, 35, 20–32. (c) Simon, R. J.; Kania, R. S.; Zuckermann,
R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.;
Rosenberg, S. Peptoids: A modular approach to drug discovery. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 9367–9371.
(19) Yoo, B.; Kirshenbaum, K. Protease-mediated ligation of abiotic
oligomers. J. Am. Chem. Soc. 2005, 127, 17132–17133.
(20) Seurynck-Servoss, S. L.; Dohm, M. T.; Barron, A. E. Effects of
including an N-terminal insertion region and arginine-mimetic side
chains in helical peptoid analogues of lung surfactant protein B.
Biochemistry 2006, 45, 11809–11818.
(21) Schröder, T.; Schmitz, K.; Niemeier, N.; Balaban, T. S.; Krug, H. F.;
Schepers, U.; Bräse, S. Solid-phase synthesis, bioconjugation, and
toxicology of novel cationic oligopeptoids for cellular drug delivery.
Bioconjugate Chem. 2007, 18, 342–354.
(22) Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C. G.; Rothbard,
J. B. Polyarginine enters cells more efficiently than other polycationic
homopolymers. J. Pept. Res. 2000, 56, 318–325.
(23) Zaro, J. L.; Shen, W.-C. Cytosolic delivery of a p16-peptide oligoargi-
nine conjugate for inhibiting proliferation of MCF7 cells. J. Controlled
Release 2005, 108, 409–417.
(24) Sakai, N.; Matile, S. Anion-mediated transfer of polyarginine across
liquid and bilayer membranes. J. Am. Chem. Soc. 2003, 125, 14348–
14356.
(25) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender,
P. A. Role of membrane potential and hydrogen bonding in the
mechanism of translocation of guanidinium-rich peptides into cells.
J. Am. Chem. Soc. 2004, 126, 9506–9507.
(26) Lieber, M.; Smith, B.; Nelson-Rees, A. W.; Todaro, G. A continuous
tumor-cell line from a human lung carcinoma with properties of type
II alveolar epithelial cells. Int. J. Cancer 1976, 17, 62–70.
(27) Takahashi, K.; Sawasaki, Y.; Hata, J.; Mukai, K.; Goto, T. Spontaneous
transformation and immortalization of human endothelial cells. In Vitro
Cell. DeV. Biol. 1990, 26, 265–274.
(28) Berridge, M. V.; Herst, P. M.; Tan, A. S. Tetrazolium dyes as tools
in cell biology: New insights into their cellular reduction. Biotechnol.
Annu. ReV. 2005, 11, 127–152.
(15) (a) Peretto, I.; Sanchez-Martin, R. M.; Wang, X. H.; Ellard, J.; Mittoo,
S.; Bradley, M. Cell penetrable peptoid carrier vehicles: synthesis and
evaluation. Chem. Commun. 2003, 2312–2313. (b) Fara, M. A.; Diaz-
Mochon, J. J.; Bradley, M. Microwave-assisted coupling with DIC/
HOBt for the synthesis of difficult peptoids and fluorescently labeled
peptides-a gentle heat goes a long way. Tetrahedron Lett. 2006, 47,
1011–1014.
(16) Holder, J. R.; Bauzo, R. M.; Xiang, Z.; Scott, J.; Haskell-Luevano,
C. Design and pharmacology of peptoids and peptide-peptoid hybrids
based on the melanocortin agonists core tetrapeptide sequence. Bioorg.
Med. Chem. Lett. 2003, 13, 4505–4509.
(29) Berridge, M. V.; Tan, A. S. Characterization of the cellular reduction
of 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT):
Subcellular localization, substrate dependence, and involvement of
mitochondrial electron transport in MTT reduction. Arch. Biochem.
Biophys. 1993, 303, 474–482.
(30) Wörle-Knirsch, J. M.; Pulskamp, K.; Krug, H. F. Oops they did it
again! Carbon nanotubes hoax scientists in viability assays. Nano Lett.
2006, 6, 1261–1268.
(17) Uno, T.; Beausoleil, E.; Goldsmith, R. A.; Levine, B. H.; Zuckermann,
R. N. New submonomers for poly N-substituted glycines (peptoids).
Tetrahedron Lett. 1999, 40, 1475–1478.
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