Polyfluorinated amino acids for sensitive 19F NMR-based screening and kinetic measurements

Article abstract of DOI:10.1021/ja069128s

Two novel series of polyfluorinated amino acids (PFAs) were designed and synthesized according to a very short and scalable synthetic sequence. The advantages and limitations of these moieties for screening purposes are presented and discussed. The potent

Full text of DOI:10.1021/ja069128s

Published on Web 04/07/2007
Polyfluorinated Amino Acids for Sensitive 19F NMR-Based
Screening and Kinetic Measurements
Gianluca Papeo,* Patrizia Giordano, Maria Gabriella Brasca, Ferdinando Buzzo,
Dannica Caronni, Franco Ciprandi, Nicola Mongelli, Marina Veronesi,
Anna Vulpetti,† and Claudio Dalvit
Contribution from the Chemistry Department, NerViano Medical Sciences, Viale Pasteur 10,
20014 NerViano, Milano, Italy
Received December 20, 2006; E-mail: gianluca.papeo@nervianoms.com
Abstract: Two novel series of polyfluorinated amino acids (PFAs) were designed and synthesized according
to a very short and scalable synthetic sequence. The advantages and limitations of these moieties for
screening purposes are presented and discussed. The potential applications of these PFAs were tested
with their incorporation into small arginine-containing peptides that represent suitable substrates for the
enzyme trypsin. The enzymatic reactions were monitored by 19F NMR spectroscopy, using the 3-FABS
(three fluorine atoms for biochemical screening) technique. The high sensitivity achieved with these PFAs
permits a reduction in substrate concentration required for 3-FABS. This is relevant in the utilization of
3-FABS in fragment-based screening for identification of small scaffolds that bind weakly to the receptor
of interest. The large dispersion of 19F isotropic chemical shifts allows the simultaneous measurement of
the efficiency of the different substrates, thus identifying the best substrate for screening purposes.
Furthermore, the knowledge of KM and Kcat for the different substrates allows the identification of the structural
motifs responsible for the binding affinity to the receptor and those affecting the chemical steps in enzymatic
catalysis. This enables the construction of suitable pharmacophores that can be used for designing
nonpeptidic inhibitors with high affinity for the enzyme or molecules that mimic the transition state. The
novel PFAs can also find useful application in the FAXS (fluorine chemical shift anisotropy and exchange
for screening) experiment, a 19F-based competition binding assay for the detection of molecules that inhibit
the interaction between two proteins.
Introduction
culminated with the 3-FABS (three fluorine atoms for bio-
chemical screening) technique,17 a versatile NMR-based bio-
NMR-based screening for the identification of potential drug
candidates has gained a key role in the drug discovery process.1,2
As more refined versions of the original techniques become avail-
able, their throughput starts to approach more conventional high-
throughput screening (HTS) procedures. A number of review
articles covering this topic have been recently published.3-16
Our long-lasting interest in exploiting 19F NMR for screening
chemical assay. According to this methodology, a CF3-tagged
substrate for a given enzymatic reaction allows for the identi-
fication of inhibitors, along with accurate measurement of their
IC50, via 19F NMR spectroscopy. This methodology was
subsequently applied with success to the screening of a focused
library against caspases18 and of plant extracts against HIV-1
protease19 and prolyl oligopeptidase.20 However, a weak point
† Current address: Novartis Institute for Biomedical Research, CH-4002,
Basel Switzerland.
(1) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996,
274, 1531-1534.
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S. B.; Meadows, R. P.; Steinman, D. H.; Carrera, G. M., Jr.; Marcotte, P.
M.; Severin, J.; Walter, K.; Smith, H.; Gubbins, E.; Simmer, R.; Holzman,
T. F.; Morgan, D. W.; Davidsen, S. K.; Summers, J. B.; Fesik, S. W. J.
Am. Chem. Soc. 1997, 119, 5818-5827.
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Manan, N. Prog. Nucl. Magn. Reson. Spectrosc. 2004, 44, 225-256.
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2002, 1, 211-219. (b) Pellecchia, M.; Becattini, B.; Crowell, K. J.;
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Expert Opin. Ther. Targets 2004, 8, 597-611.
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187-231.
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9
10.1021/ja069128s CCC: $37.00 © 2007 American Chemical Society
J. AM. CHEM. SOC. 2007, 129, 5665-5672
5665

A R T I C L E S
Papeo et al.
Scheme 1. Synthesis of the Intermediate Primary Amines 4-6a
a Conditions and reagents: (a) [bis(trifluoroacetoxy)iodo]benzene, py-
ridine, DMF, H2O, room temp, 70%; (b) DCM, TFA, room temp, quant
(5), 98% (6).
Figure 1. Chemical structures of polyfluorinated glycine (PFG) and
polyfluorinated amino acids (PFAs).
of the originally formulated technique proved to be the low
sensitivity compared to that of other fluorescence-based21 and
radioactive-based22 procedures. In order to overcome this
problem, two possible strategies could be envisaged: (1) use
of cryogenic probe technology23 optimized to 19F detection24
and (2) use of substrates containing magnetically equivalent
multiple CF3- moieties.24,25 As more magnetically equivalent
fluorine atoms are present in the substrate the throughput
increases, less amount of enzyme is required, and weaker
inhibitors can be identified. This last point is particularly relevant
in a fragment-based screening approach performed with 3-FABS.
Fragments are low molecular weight molecules (typically <250
Da) that display a high binding efficiency index (BEI ) (-log
KD)/MW or BEI ) (-log IC50)/MW with MW expressed in
kDa),26-28 but due to their small size have a weak affinity for
the receptor. The detection of these weak affinity molecules
can be achieved by performing the assay with a substrate
concentration [S] that is comparable or smaller than its KM (for
a substrate competitive inhibitor). Recently, we synthesized PFG
(polyfluorinated glycine) (1, Figure 1).25 This unnatural amino
acid has been used to cap different small arginine-containing
peptides. 19F NMR studies of the trypsin-induced cleavage of
the aforementioned peptides were performed at unprecedented
low enzyme concentration. Increasing the distance between the
cleavage site and the fluorinated moiety by systematic elongation
of the peptide chain allowed us to unveil the limit of PFG 1.
This proved to be the minuscule chemical shifts difference
between the signals attributed to the substrate and the product
of the enzymatic reaction.
substrates containing these amino acids were successfully
performed. Although we have shown their application only to
the 3-FABS experiments, these novel fluorinated amino acids
can now find useful application also in the FAXS (fluorine
chemical shift anisotropy and exchange for screening) experi-
ment,29 a 19F-based competition binding assay, for the detection
in particular of molecules that inhibit the interaction between
two proteins.
Results and Discussion
Synthesis of Polyfluorinated Amino Acids. NR-Fmoc-PFAs
were secured by performing a mono- or double-reductive
amination reaction on the corresponding primary amines. Thus,
the suitable intermediates 4-6 were prepared either by Hofmann
amide degradation (4) from NR-Fmoc-glutamine30 or by Boc
protective group removal (5, 6)31 from NR-Fmoc-Nδ-Boc-
ornithine and NR-Fmoc-Nꢀ-Boc-lysine (Scheme 1).
Reductive amination reactions performed at room temperature
in a mixture of DMF/TFA 10:1 as the solvent32 employing a
large excess of 3,5-bis(trifluoromethyl)benzaldehyde and sodium
triacetoxyborohydride33 (Scheme 2) delivered, after treatment
with hydrochloric acid, the corresponding salts of PFAs 7-9
(Scheme 2).
Alternatively, PFAs 10-12 were smoothly secured by
performing the reductive amination reaction as before, this time
in the presence of a stoichiometric amount of the fluorinated
aldehyde. Compounds 10-12 were subsequently protected as
their Boc derivatives 13-15 (Scheme 3).
The synthetic methodology proved to be robust and therefore
scalable (a 9 g batch of compound 15 was prepared).
Synthesis of Peptides Containing Polyfluorinated Amino
Acids. The subsequent synthetic campaign was then focused
on the preparation of a handful of PFAs-containing peptides
encompassing an arginine residue. These peptides were designed
As the main drawback of PFG 1 lies on its mandatory
positioning at the N-terminus of a given peptide, two novel series
of polyfluorinated amino acids (PFAs) (2, 3, Figure 1) engi-
neered to be placed wherever desired along the peptide backbone
were designed. 3-FABS experiments on potential trypsin
(19) Frutos, S.; Tarrago´, T.; Giralt, E. Bioorg. Med. Chem. Lett. 2006, 16, 2677-
2681.
(29) (a) Dalvit, C.; Flocco, M.; Veronesi, M.; Stockman, B. J. Comb. Chem.
High Throughput Screening 2002, 5, 605-611. (b) Dalvit, C.; Fagerness,
P. E.; Hadden, D. T. A.; Sarver, R. W.; Stockman, B. J. J. Am. Chem. Soc.
2003, 125, 7696-7703.
(20) Tarrago´, T.; Frutos, S.; Rodriguez-Mias, R. A.; Giralt, E. ChemBioChem
2006, 7, 827-833.
(21) Eggeling, C.; Brand, L.; Ullmann, D.; Ja¨ger, S. Drug DiscoVery Today
2003, 8, 632-641.
(30) (a) de Mace´do, P.; Marrano, C.; Keillor, J. W. Bioorg. Med. Chem. 2002,
10, 355-360. (b) Sakura, N.; Itoh, T.; Uchida, Y.; Ohki, K.; Okimura, K.;
Chiba, K.; Sato, Y.; Sawanishi, H. Bull. Chem. Soc. Jpn. 2004, 77, 1915-
1924.
(22) Cook, N. D. Drug DiscoVery Today 1996, 1, 287-294.
(23) Kovacs, H.; Moskau, D.; Spraul, M. Prog. Nucl. Magn. Reson. Spectrosc.
2005, 46, 131-155.
(31) Spectroscopic data are in agreement with those reported in the literature:
(a) Varghese, J. PCT Int. Appl. 2003, 204, WO 2003045378. (b) Banwell,
M. G.; McRae, K. J. J. Org. Chem. 2001, 66, 6768-6774. (c) Boyle, W.
J., Jr.; Sifniades, S.; Van Peppen, J. F. J. Org. Chem. 1979, 44, 4841-
4847.
(32) Traquandi, G.; Brasca, M. G.; D’Alessio, R.; Polucci, P.; Roletto, F.;
Vulpetti, A.; Pevarello, P.; Panzeri, A.; Quartieri, F.; Ferguson, R.; Vianello,
P.; Fancelli, D. PCT Int. Appl. 2004, 226, WO 2004104007.
(33) Abdel-Magid, A. F.; Maryanoff, C. A. ACS Symp. Ser. 1996, 641, 201-
216.
(24) Dalvit, C.; Mongelli, N.; Papeo, G.; Giordano, P.; Veronesi, M.; Moskau,
D.; Ku¨mmerle, R. J. Am. Chem. Soc. 2005, 127, 13380-13385.
(25) Dalvit, C.; Papeo, G.; Mongelli, N.; Giordano, P.; Saccardo, B.; Costa, A.;
Veronesi, M.; Ko, S. Y. Drug DeV. Res. 2005, 64, 105-113.
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9
5666 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Polyfluorinated AA for 19F NMR-Based Screening
A R T I C L E S
Scheme 2. Synthesis of Polyfluorinated Amino Acids 7-9a
a Conditions and reagents: (a) 9 equiv of NaBH(AcO)3, 6 equiv of 3,5
bis(trifluoromethyl)benzaldehyde, DMF, TFA, room temp, 3.5 h; (b) 4 N
HCl, dioxane, room temp, 48% (7), 75% (8), 65% (9) over two steps.
Figure 3. 19F spectra of 10 µM of peptides 17, 18, and 19 in the presence
of 1 nM trypsin. The reaction was quenched with the addition of 80 µM
leupeptin. Spectra were acquired with 64 transients corresponding to a 3
min recording time. Samples were in 50 mM Tris, 0.001% Triton X-100,
8% D2O for the lock signal at pH 7.5 (lower trace) and pH 1.5 (middle and
upper traces). The increased noise level in the upper spectrum is due to the
strong resolution enhancement (lb ) -3 and gf ) 0.3) applied to the data
for resolving the two signals. S and P refer to the substrate and the cleaved
substrate, respectively. The incubation time and the speed of the reaction
are reported.
and exploiting well-precedented Fmoc chemistry37,38 (Scheme
4).
NMR Data. The 19F NMR spectra of peptides 17-19 showed
a single sharp and intense signal at 20 °C (Figure 2). The spectra
indicate the absence of additional peaks originating from
conformers in slow exchange.
Fast rotation around three rotatable bonds averages the
chemical shifts of the four CF3 signals resulting in one signal
originating from 12 fluorine atoms. Chemical shifts for these
signals are reported in Table 1.
Figure 2. 19F NMR spectra of the three peptides 17, 18, and 19 at 10 µM
concentration. The samples were in 50 mM Tris pH 7.5, 0.001% Triton
X-100, and 8% D2O for the lock signal. A total number of 64 transients
were acquired for each spectrum corresponding to a 3 min recording time.
Peptides 17-19 were subsequently tested in the aforemen-
tioned trypsin-based assay, performed either at 800 pM or 1
nM enzyme concentration. All the peptides underwent the
enzymatic reaction, but with different velocities (Figure 3). The
velocity was obtained experimentally by measuring the integral
of the 19F product signal divided by the incubation time of the
reaction.
The chemical shift differences for the signals of the substrate
and cleaved substrate for peptides 17-19 at pH 7.5 are small,
as is evident from Table 1. An improvement in the separation
of the signals for peptides 18 and 19 was achieved by lowering
the pH of the solution to pH 1.5, as is shown in Table 1 and
Figure 3.
Table 1. 19F Isotropic Chemical Shifts of Substrate Signals
(
δS − δP
)
(δS − δP)
at pH 1.5b
a
peptide
δS
at pH 7.5b
17
18
19
20
21
22
12.812
12.806
12.793
12.904
12.834
12.785
6.6
4.3
2.3
4.7
14
5.3
17.2
-3.1
10.1
a Chemical shift of substrate signal in ppm at pH 7.5. b Chemical shift
difference of substrate and product signals in Hz.
to test PFAs as potential 19F NMR tools in a trypsin-based assay.
The placement of a glutamic residue at their N-termini has been
deliberately chosen in order to increase the solubility of the
peptides in aqueous medium. With the use of a Rink apparatus,34
pentapeptides 17-22 were prepared by anchoring the growing
peptide on a commercially available Rink amide AM resin35,36
Polyfluorinated amino acids 2 have some potential drawbacks
that could limit their applications. (i) The fluorinated moiety is
(37) (a) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748-5749.
For a review, see: (b) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res.
1990, 35, 161-214.
(38) (a) Stewart, J.; Young, J. Solid Phase Peptide Synthesis; Pierce Chemical
Company: Rockford, IL 1984. (b) Atherthon, E.; Sheppard, R. C. Solid
Phase Peptide Synthesis: A Practical Approach; IRL Press: Oxford, 1989.
(c) Kates, S. A., Albericio, F., Eds. Solid-Phase Synthesis. A Practical
Guide; Marcel Dekker: New York, 2000. (d) Chan, W. C., White, P. D.,
Eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Oxford
University Press: Oxford, 2000.
(34) Rink Solid Phase Washer; Rink Think Engineering: Bubendorf, Switzerland.
(35) Bernatowicz, M. S.; Daniels, S. B.; Ko¨ster, H. Tetrahedron Lett. 1989, 30,
4645-4648.
(36) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205-
208.
9
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A R T I C L E S
Papeo et al.
Scheme 3. Synthesis of Polyfluorinated Amino Acids 13-15a
a Conditions and reagents: (a) 3 equiv of NaBH(AcO)3, 1.2 equiv of 3,5-bis(trifluoromethyl)benzaldehyde, DMF, TFA, room temp, 1 h, 43% (10), 55%
(11), 65% (12); (b) (Boc)2O, NaHCO3(aq), AcOEt or DCM, room temp, 3 h, 83% (13), 98% (14), 98% (15).
Scheme 4. Synthesis of Peptides 17-22
rather hydrophobic, and therefore, insertion of this amino acid
into a peptide with low water solubility could result in a substrate
with poor solubility. (ii) The large size of the PFA can interfere
with the binding of the peptide to the enzyme resulting in a
weak affinity substrate for the desired target. (iii) The chemical
shift dispersion of the substrate and product fluorine signals
tends to be small. This is due to the averaging of the chemical
shift originating from rapid rotation around three bonds. The
small chemical shift dispersion requires the use of high-field
spectrometers or the use of shift reagents or dramatic pH changes
of the medium (Figure 3). However, these two last methods
are applicable only in an end-point format assay after reaction
quenching, thus excluding their utilization in online kinetic
measurements.
19. It is also interesting to note that at pH 7.5 the 19F signals of
all the products shift in the same direction similarly to what is
observed with other substrates for trypsin and other prote-
ases.17a,b,19 The fluorinated amino acids are at the N-terminal with
respect to the cleavage site. Therefore, the observed shift for the
products is probably due to the negative charge of the carboxylic
An approach for overcoming these limitations is the use of
the amino acids 3. Although they suffer from a 2-fold reduction
in sensitivity when compared to the amino acids 2, they do not
have the drawbacks associated with the latter. The fluorine
spectrum of the three combined substrates 20-22 (Figure 4,
lower trace) displays three well-separated sharp resonances
originating from the six fluorine atoms of the three substrates
SA (20), SB (21), and SC (22). Upon addition of 440 pM trypsin
and after an incubation time of 260 min, three novel signals of
different intensity appear in the spectrum (Figure 4, upper trace)
originating from the three cleaved peptides PA, PB, and PC.
Figure 4. 19F NMR spectra of the three substrates 20-22 acquired after 4
min (lower trace) and after 260 min (upper trace) from the addition of
trypsin. The concentrations of the substrates and of the enzyme were 20
µM and 440 pM, respectively. A total number of 84 transients were recorded
for each spectrum corresponding to a 4 min acquisition time. The SA, SB,
SC, PA, PB, and PC labels refer to the 19F signals of the substrates 20, 21,
and 22 and their corresponding enzymatically cleaved peptides, respectively.
All the signals of the substrates and products have different
isotropic chemical shifts, and the chemical shift dispersion is
significantly larger when compared to that of the peptides 17-
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5668 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Polyfluorinated AA for 19F NMR-Based Screening
A R T I C L E S
Table 2. Kinetic Parameters for the Hydrolysis of Polyfluorinated
Peptides by Trypsin at 20 °C
Kcat
KM
M)
Kcat/KM
∆∆GESq
(kcal/mol)
1
1
peptide
(s-
)
(
µ
(s-1 M-
)
20
21
22
32 ( 5
28 ( 2
30 ( 2
805 ( 145
186 ( 6
80 ( 6
0.4 × 105
1.5 × 105
3.75 × 105
(-0.77)a
(-0.53)b
a,b Difference in free energy between peptides 21 and 20, 22 and 21,
respectively.
From these data it is evident that Kcat is the same for the
three peptides, whereas KM ranges from 80 µM for SC to 805
µM for SA. The identity of Kcat for the three peptides indicates
that the chemical steps subsequent to the enzyme-substrate
complex formation are not influenced by changes in size of the
fluorinated amino acid. The additional binding energy origi-
nating from the added CH2 groups is used to bind the substrate,
i.e., decrease KM. Therefore, the different catalytic enzyme
efficiencies (Kcat/KM) with the three substrates are simply due
to their different KM. In other words, the substrate specificity
merely involves discrimination in binding of the substrates to
their ground states. The ratio Kcat/KM is related to the free energy
difference between the free reactants (enzyme and substrate)
and the transition state complex (ESq). Table 2 reports the
difference in transition state energies (∆∆GESq) derived from
the experimental values of Kcat/KM according to the equation39,40
Figure 5. Plot of the enzymatic velocity (product/min) as a function of
the substrate concentration. The reactions were performed in Eppendorf
vials in the presence of 50 mM Tris pH 7.5, 0.001% Triton X-100, and 1
nM trypsin and quenched after a defined delay with 80 µM leupeptin. After
the quenching of the reaction, the solutions were transferred to 5 mm tubes
and 19F spectra were recorded. Curves A, B, and C correspond to substrates
SA, SB, and SC, respectively. The reaction speed was calculated by dividing
the normalized integral of the product 19F signal (the sum of the substrate
and product 19F signal integrals correspond to the concentration of the
peptide used in the experiments) with the incubation time. Data were
analyzed with nonlinear least-squares methods in the Origin 5.0 software
package. The best fit of the experimental data provides the value of KM
and Kcat according to the Henri-Michaelis-Menten equation.
(Kcat/K )
M (n+2)CH2
∆∆GESq ) -RT ln
(Kcat/K )
M 2CH2
group. The small chemical shift difference of 0.019 ppm
between the 19F signals of 17 and 19 is amplified to 0.119 ppm
for 20 and 22. The large chemical shift dispersion in the spectra
of Figure 4 allows a direct comparison of the efficiency of the
substrates. Analysis of these spectra identifies substrate SC as
the most efficient substrate with the following ranking order in
efficiency: SC > SB > SA. The possibility of monitoring
simultaneously the kinetics of different substrates is important
in the optimization process for the identification of the best
substrate. The selected substrate can then be used in the assay
or used for generating a potent inhibitor by replacing the amino
acid that is modified by the enzyme. In addition, the knowledge
of the KM and Kcat for the different substrates allows the
identification of the structural motifs responsible for the binding
affinity to the receptor and those affecting the chemical steps
in enzymatic catalysis. This enables the construction of a
suitable pharmacophore that can be used for designing non-
peptidic inhibitors with high affinity for the enzyme or molecules
that mimic the transition state. The simultaneous detection of
different substrates is also useful for monitoring enzymatic
reaction cascades where the fluorinated product becomes the
substrate of another enzyme of the cascade or where different
fluorinated substrates are used in a multiple screening run.17b
Titration experiments on the individual peptides as a function
of the peptide concentration were performed in order to gain
more detailed insight into the kinetics of these substrates and
to understand the differences in catalytic enzyme efficiency.
Figure 5 shows the 3-FABS results for the three peptides with
the plot of the enzymatic velocity as a function of the substrate
concentration.
where n is 1 or 2.
The addition of the first CH2 group results in an incremental
Gibbs free energy of 0.77 kcal/mol, whereas the addition of
the second CH2 provides a supplementary smaller improvement
of 0.53 kcal/mol. The same ranking order in efficiency was
observed for substrates 17-19. In addition, the relative mag-
nitude changes in the initial reaction rate are also very similar
with V19/V18 ) 1.4, V22/V21 ) 1.6 and V19/V17 ) 3.3, V22/V20
)
3.9. This allows the general statement that the shorter the spacer
between the fluorinated moiety and the backbone, the slower
the rate of the reaction. Docking studies were performed in order
to gain insight into the binding of these substrates to trypsin.
The three peptidic ligands were docked into the trypsin crystal
structure (entry 2PTC in the Protein Data Bank, 1.90 Å
resolution).41 The results were evaluated in term of total
estimated binding energy. As expected, the arginine side chain
of all the three ligands is located into the S1 binding pocket,
which is occupied by a lysine in the 2PTC crystal structure.
The long lysine and arginine side chains extend in the S1 pocket
interacting with the carboxylic oxygens of Asp189 side chain.
Through this S1 pocket interaction, trypsin favors cleavage at
the amide bond next to lysine or arginine amino acids.
Figure 6 shows the top-ranked pose of ligand SB (21) docked
into trypsin. This proposed binding mode highlights a wide
hydrogen-bonding network: between the side chain of the ligand
arginine and Asp189 and between the carbonyl oxygen of the
(39) Fersht, A. Enzyme Structure and Mechanism; W.H. Freeman and Com-
pany: New York, 1985.
(40) Copeland, R. A. Enzymes; Wiley-VCH Inc.: New York, 2000.
(41) Marquart, M.; Walter, J.; Deisenhofer, J.; Bode, W.; Huber, R. Acta
Crystallogr., Sect. B 1983, 39, 480-490.
Table 2 reports the kinetic parameters for the three peptides
extracted from the experimental data shown in Figure 5.
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J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5669

A R T I C L E S
Papeo et al.
Figure 6. Best docking pose of ligand SB (21) docked into trypsin. The surface is colored according to the force-field charge assigned to trypsin (left); the
hydrogen-bonding interactions between trypsin and ligand are highlighted (right).
data ESI(+) were obtained on a Waters Q-Tof Ultima directly connected
with an Agilent micro-HPLC 1100 as previously described.43 1H NMR
spectroscopy was performed on a Mercury VX 400 operating at 400.45
MHz equipped with a 5 mm double-resonance probe (1H {15N-31P}
ID_PFG Varian) or on an INOVA 500 operating at 499.76 MHz. 19F
NMR spectroscopy was performed at 20 °C on a Varian Inova 600
MHz NMR spectrometer operating at a 19F Larmor frequency of 564
MHz. Full details of the HPLC/MS instrumentation and both the
analytical and the purification methods used in this work can be found
in the Supporting Information.
ligand arginine and Ser195 and Gly193 of trypsin. The carbonyl
oxygens of the alanine and glutamic acid flanking the PFA are
also involved in hydrogen bond interactions with Gln192 and
Gly216 residues. One difference among the three ligands is that
only ligand SA (20) is not capable of making the hydrogen bond
+
between the NH2 group of the PFA and the carbonyl oxygen
of the backbone of Asn97. This is due to the shorter methylenic
side chains of the PFA moiety (two methylene units in ligand
SA vs three and four in ligands SB and SC, respectively). This
might explain the lower affinity of compound SA against trypsin.
2. General Procedure for the Double-Reductive Amination
Reaction. 2.1. (S)-4-[Bis-(3,5-bis-trifluoromethyl-benzyl)-amino]-2-
(9H-fluoren-9-ylmethoxycarbonylamino)-butyric Acid Trifluoro-
acetate (7). To a solution of 4 (82 mg, 0.18 mmol) in DMF (5 mL),
stirred at room temperature, sodium triacetoxyborohydride (337 mg,
1.6 mmol) and 3,5-bis(trifluoromethyl)benzaldehyde (0.18 mL, 1.08
mmol) were subsequently added. After 5 min, trifluoroacetic acid (0.5
mL) was added, and the reaction mixture was stirred at room
temperature for 3.5 h. The reaction mixture was then evaporated to
dryness in vacuo, taken up in dichloromethane, and the organic phase
was washed with water and brine, dried over Na2SO4, and concentrated.
The crude material was then purified by flash chromatography
(cyclohexane/AcOEt 50/50, AcOEt, and then AcOEt/MeOH 80/20) to
afford compound 7 (100 mg, 61% yield) as a white solid: mp 152 °C,
[R]D +1.2 (c 1, MeOH). 1H NMR (400 MHz, DMSO-d6): δ ppm 7.73-
7.92 (m, 8 H), 7.65 (d, J ) 7.93 Hz, 2 H), 7.25-7.46 (m, 4 H), 6.91
(s, 1 H), 4.12-4.32 (m, 3 H), 3.69-3.95 (m, 5 H), 2.73 (t, J ) 6.58
Hz, 2 H), 1.82-2.14 (m, 2 H). HRMS (ES+): m/z calcd for
C37H29F12N2O4, 793.1930 [M + H]+; found, 793.1957. 19F NMR
(DMSO-d6): 11.84 ppm.
Conclusion
In summary, we have presented the design and synthesis of
novel PFAs, their insertion into short peptides, and their
utilization in the 3-FABS experiments. Kinetics parameters
together with the NMR properties of these different moieties
have also been characterized in detail and compared. The novel
amino acids can be used for tagging whichever peptide
substrates wherever desired in the sequence. This results in
numerous and diverse applications of the 3-FABS experiments
within different drug discovery projects. In addition, their use
can be extended to the FAXS-NMR-based binding assay. A
short peptide that represents the amino acid sequence of a protein
that is recognized by another protein can be tagged with one of
these novel PFAs. The peptide is then used as a spy molecule
in the FAXS experiments for the detection of inhibitors of
protein-protein interactions.
2.2. (S)-5-[Bis-(3,5-bis-trifluoromethyl-benzyl)-amino]-2-(9H-fluo-
ren-9-ylmethoxycarbonylamino)-pentanoic Acid Trifluoroacetate
(8). With the use of the procedure described above, starting from 5
(125 mg, 0.27 mmol), compound 8 was obtained (151 mg, 61% yield)
Materials and Methods
1. General Considerations. Amino acid derivatives (all in their L
configuration), PyBOP, and Rink amide AM resin (200-400 mesh,
loading 0.68 mmol/g) were purchased from Novabiochem, Switzerland.
All reagents were obtained from commercial suppliers and used as
received. The abbreviations used for both the amino acids and the
protecting groups are based on recommendations of the JCBN.42 For a
complete glossary of terms see the Supporting Information.
Flash chromatography was performed on silica gel (Merck grade
9385, 60 Å). Melting points were determined in open glass capillaries
with a Buchi 535 melting point apparatus and are uncorrected. Optical
rotations were recorded on a Perkin-Elmer 241 polarimeter in a 1 dm
cell at ambient temperature with a sodium lamp (589 nm). Exact mass
1
as a white solid: mp 141 °C, [R]D +3.0 (c 1, MeOH). H NMR (400
MHz, DMSO-d6): δ ppm 7.77-7.93 (m, 8 H), 7.65 (d, J ) 7.68 Hz,
2 H), 7.35-7.42 (m, 2 H), 7.24-7.33 (m, 2 H), 6.77-7.08 (br s, 1 H),
4.14-4.34 (m, 3 H), 3.70-3.87 (m, 5 H), 2.62 (t, J ) 6.43 Hz, 2 H),
1.73-1.86 (m, 1 H), 1.47-1.72 (m, 3 H). HRMS (ES+): m/z calcd
for C38H31F12N2O4, 807.2087 [M + H]+; found, 807.2093. 19F NMR
(DMSO-d6): 11.83 ppm.
2.3. (S)-6-[Bis-(3,5-bis-trifluoromethyl-benzyl)-amino]-2-(9H-fluo-
ren-9-ylmethoxycarbonylamino)-hexanoic Acid Trifluoroacetate (9).
With the use of the procedure described above, starting from 6 (128
(42) IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Eur.
J. Biochem. 1984, 138, 9-37.
(43) Colombo, M.; Riccardi-Sirtori, F.; Rizzo, V. Rapid Commun. Mass
Spectrom. 2004, 18, 511-517.
9
5670 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Polyfluorinated AA for 19F NMR-Based Screening
A R T I C L E S
mg, 0.27 mmol), compound 9 was obtained (152 mg, 60% yield) as a
white solid: mp 121 °C, [R]D -2.3 (c 1, MeOH). 1H NMR (400 MHz,
DMSO-d6): δ ppm 7.78-7.9 (m, 8 H), 7.62 (d, J ) 7.72 Hz, 2 H),
7.32-7.38 (t, J ) 6.22 Hz, 2 H), 7.22-7.3 (t, J ) 6.54 Hz, 2 H),
6.62-6.72 (br s, 1 H), 4.15-4.23 (m, 3 H), 3.65-3.78 (m, 5 H), 2.58
(t, 2 H), 1.62-1.78 (m, 1 H), 1.43-1.58 (m, 3 H), 1.19-1.3 (m, 2 H).
HRMS (ES+): m/z calcd for C39H33F12N2O4, 821.2243 [M + H]+;
found, 821.2273. 19F NMR (DMSO-d6): 11.83 ppm.
3. Procedure for the Conversion of Compounds 7-9 into Their
Corresponding Hydrochloride Salts. Pure TFA salts 7-9 were
dissolved in dry dioxane (1 mmol in 16 mL) and exposed to a 4 N
solution of hydrochloric acid in dioxane (4 mL). The resulting solutions
were stirred at room temperature for 1 h and then evaporated to dryness.
The corresponding hydrochloride salts were subsequently used without
any further purification.
4. General Procedure for the Monoreductive Amination Reac-
tion. 4.1. (S)-4-(3,5-Bis-trifluoromethyl-benzylamino)-2-(9H-fluoren-
9-ylmethoxycarbonylamino)-butyric Acid Trifluoroacetate (10). To
a solution of 4 (177 mg, 0.39 mmol) in DMF (5 mL), stirred at room
temperature, sodium triacetoxyborohydride (290 mg, 1.4 mmol) and
3,5-bis(trifluoromethyl)benzaldehyde (0.07 mL, 0.43 mmol) were
subsequently added. After 20 min, TFA (0.5 mL) was added, and the
reaction mixture was stirred at room temperature for 1 h. The reaction
mixture was then poured into water (20 mL), and the pH was adjusted
to 6-7, using aqueous NaOH 32% w/w. The aqueous phase was then
extracted with AcOEt (2 × 20 mL). The combined organic phases were
washed with water (3 × 50 mL) and brine, dried over Na2SO4, and
evaporated to dryness. The crude material was finally purified by flash
chromatography (dichloromethane, dichloromethane/MeOH 95/5, dichlo-
romethane/MeOH 80/20) to afford compound 10 as a white solid (114
mg, 43% yield): mp> 158 °C (decomposition), [R]D +1.1 (c 1, MeOH).
1H NMR (400 MHz, DMSO-d6): δ ppm 8.09 (s, 2 H), 7.99 (s, 1 H),
7.89 (d, J ) 7.44 Hz, 2 H), 7.69 (d, J ) 7.93 Hz, 2 H), 7.42 (t, J )
7.32 Hz, 2 H), 7.24-7.37 (m, 3 H), 4.13-4.37 (m, 3 H), 3.88-4.08
(m, 3 H), 2.61-2.79 (m, 2 H), 1.85-2.00 (m, 1 H), 1.72-1.85 (m, 1
H). HRMS (ES+): m/z calcd for C28H25F6N2O4, 567.1713 [M + H]+;
found, 567.1693. 19F NMR (DMSO-d6): mixture of rotational isomers;
12.15 ppm (major), 12.02 ppm (minor). 19F NMR (50 mM Tris, 1 mM
DTT, 0.001% Triton X-100, pH ) 7.5): mixture of rotational isomers;
12.83 ppm (major), 12.81 ppm (minor).
4.2. (S)-5-(3,5-Bis-trifluoromethyl-benzylamino)-2-(9H-fluoren-
9-ylmethoxycarbonylamino)-pentanoic Acid Trifluoroacetate (11).
With the use of the procedure described above, starting from 5 (2.2 g,
4.7 mmol), compound 11 was obtained (1.8 g, 55% yield) as a white
solid: mp 162 °C, [R]D +7.3 (c 1, MeOH). 1H NMR (400 MHz,
DMSO-d6): δ ppm 8.06 (s, 2 H), 7.96 (s, 1 H), 7.89 (d, J ) 7.44 Hz,
2 H), 7.71 (d, J ) 7.44 Hz, 2 H), 7.42 (t, J ) 7.44 Hz, 2 H), 7.27-
7.37 (m, 3 H), 4.19-4.33 (m, 3 H), 3.84-3.94 (m, 3 H), 2.52-2.58
(m, 2 H), 1.73-1.83 (m, 1 H), 1.59-1.71 (m, 1 H), 1.48-1.59 (m, 2
H). HRMS (ES+): m/z calcd for C29H27F6N2O4, 581.1870 [M + H]+;
found, 581.1875. 19F NMR (50 mM Tris, 1 mM DTT, 0.001% Triton
X-100, pH ) 7.5): 12.78 ppm.
tert-butoxycarbonyl-amino]-2-(9H-fluoren-9-ylmethoxycarbonyl-
amino)-butyric Acid (13). To a stirred solution of compound 10 (200
mg, 0.29 mmol) in AcOEt (2 mL), saturated aqueous NaHCO3 (2 mL)
and di-tert-butyl dicarbonate (256 mg, 1.18 mmol) were added. The
biphasic system was swirled for 3 h at room temperature. The reaction
mixture was diluted with additional AcOEt; the organic phase was
separated, washed with water and then with brine. Finally, it was dried
over Na2SO4 and evaporated to dryness. The crude material was purified
by flash chromatography (dichloromethane, dichloromethane/MeOH
95/5, dichloromethane/MeOH 90/10) to afford compound 13 (160 mg,
83% yield) as a white foam: mp 178 °C, [R]D +5.8 (c 1, MeOH). 1H
NMR (400 MHz, DMSO-d6): δ ppm 8.01 (s, 1 H), 7.81-7.94 (m, 4
H), 7.68 (d, J ) 7.80 Hz, 2 H), 7.41 (t, J ) 7.44 Hz, 2 H), 7.23-7.35
(m, 3 H), 4.54 (br s, 2 H), 4.17-4.33 (m, 3 H), 3.74 (br s, 1 H), 3.32-
3.45 (m, 2 H), 1.91-2.05 (m, 1 H), 1.73-1.85 (m, 1 H), 1.28-1.45
(br s, 9 H). HRMS (ES+): m/z calcd for C33H33F6N2O6, 667.2237 [M
+ H]+; found, 667.2266. 19F NMR (50 mM Tris, 1 mM DTT, 0.001%
Triton X-100, pH ) 7.5): mixture of rotational isomers; 12.88 ppm
(major), 12.83 ppm (minor).
5.2. (S)-5-[(3,5-Bis-trifluoromethyl-benzyl)-tert-butoxycarbonyl-
amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanoic Acid
(14). With the use of the procedure described above, starting from 11
(195 mg, 0.28 mmol), and using dichloromethane (2 mL) instead of
AcOEt, compound 14 was obtained (195 mg, 98% yield) as a white
1
foam: mp > 170 °C (decomposition), [R]D +5.0 (c 1, MeOH). H
NMR (400 MHz, DMSO-d6): δ ppm 8.00 (s, 1 H), 7.84-7.94 (m, 4
H), 7.69 (d, J ) 7.56 Hz, 2 H), 7.42 (t, J ) 7.28 Hz, 2 H), 7.27-7.37
(m, 3 H), 7.06 (br s, 1 H), 4.51 (br s, 2 H), 4.16-4.32 (m, 3 H), 3.78
(br s, 1 H), 3.17-3.33 (m, 2 H), 1.65-1.75 (m, 1 H), 1.48-1.62 (m,
3 H), 1.19-1.48 (br s, 9 H). HRMS (ES+): m/z calcd for C34H35F6N2O6,
681.2394 [M + H]+; found, 681.2425. 19F NMR (50 mM Tris, 1 mM
DTT, 0.001% Triton X-100, pH ) 7.5): 12.83 ppm.
5.3. (S)-6-[(3,5-Bis-trifluoromethyl-benzyl)-tert-butoxycarbonyl-
amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic Acid
(15). With the use of the procedure described above, starting from 12
(200 mg, 0.29 mmol), and using dichloromethane (2 mL) instead of
AcOEt, compound 15 was obtained (185 mg, 98% yield) as a white
foam: mp 68 °C, [R]D -4.1 (c 1, MeOH). 1H NMR (400 MHz, DMSO-
d6): δ ppm 8.02 (s, 1 H), 7.84-7.95 (m, 4 H), 7.72 (d, J ) 7.44 Hz,
2 H), 7.59 (d, J ) 8.41 Hz, 1 H), 7.42 (t, J ) 7.44 Hz, 2 H), 7.33 (d,
J ) 7.56 Hz, 2 H), 7.10 (br s, 1 H), 4.48-4.60 (m, 2 H), 4.17-4.36
(m, 3 H), 3.85-3.98 (m, 1 H), 3.14-3.28 (m, 2 H), 1.22-1.75 (m, 15
H). HRMS (ES+): m/z calcd for C35H37F6N2O6, 695.2550 [M + H]+;
found, 695.2574. 19F NMR (50 mM Tris, 1 mM DTT, 0.001% Triton
X-100, pH ) 7.5): 12.84 ppm.
6. General Procedure for the Solid-Phase Peptide Synthesis. The
peptide synthesis was performed on the solid phase starting from the
commercially available Rink amide AM resin, 4-(2′,4′-dimethoxyphen-
yl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-aminomethyl co-
poly(styrene-1% DVB). The resin was always preswelled with DCM
and DMA and accurately washed with DMA, NMP, and 2-propanol
after coupling, capping, and deprotection steps.
4.3. (S)-6-(3,5-Bis-trifluoromethyl-benzylamino)-2-(9H-fluoren-
9-ylmethoxycarbonylamino)-hexanoic Acid Trifluoroacetate (12).
With the use of the procedure described above, starting from 6 (2.8 g,
5.81 mmol), compound 12 was obtained (2.23 g, 54% yield) as a white
The synthesis of the supported tripeptide 16, Fmoc-Ala-Arg(Pbf)-
Ala-NH-Rink amide AM resin, was exploited on a Rink apparatus.34
The Fmoc deprotection was achieved with rapid (1 min) and multiple
(usually 14-15) treatments with the mixture of piperidine/DMA 20%.
For each elongation step a double coupling occurred (60 and 120 min,
respectively), using each time 2 equiv of the appropriate amino acid,
2 equiv of PyBOP, and 10-12 equiv of DIEA (Hunig’s base), in DMA
as solvent. A capping step usually followed: two 5-10 min reactions
with the mixture DMA/Ac2O/DIEA 80:15:5. After thorough drying,
4.05 g of peptide 16 was obtained (97% yield based on resin loading).
Six samples of 16, 160 mg each, were then transferred into a Quest
210 synthesizer44 for further elongation with the PFAs 7-9, 13-15
1
solid: mp > 160 °C (decomposition). H NMR (400 MHz, DMSO-
d6): δ ppm 8.04 (s, 2 H), 7.94 (s, 1 H), 7.89 (d, J ) 7.44 Hz, 2 H),
7.69 (d, J ) 6.95 Hz, 2 H), 7.43 (t, J ) 7.44 Hz, 2 H), 7.28-7.38 (m,
3 H), 4.17-4.33 (m, 3 H), 3.73-3.93 (m, 3 H), 2.43-2.52 (m, 2 H),
1.65-1.79 (m, 1 H), 1.51-1.63 (m, 1 H), 1.28-1.49 (m, 4 H). HRMS
(ES+): m/z calcd for C30H29F6N2O4, 595.2026 [M + H]+; found,
595.2047. 19F NMR (50 mM Tris, 1 mM DTT, 0.001% Triton X-100,
pH ) 7.5): 12.76 ppm.
5. General Procedure for the Introduction of the t-Butoxycar-
bonyl Protective Group. 5.1. (S)-4-[(3,5-Bis-trifluoromethyl-benzyl)-
(44) Quest 210; Argonaut Technologies: Foster City, CA.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5671

A R T I C L E S
Papeo et al.
and the Fmoc-Glu(OtBu)-OH. Double-coupling and capping steps were
exploited as described before, while the alternate Fmoc-deprotection
steps were performed with piperidine/DMA 20%, three times for 1, 5,
and 20 min, respectively. The six Fmoc-deprotected peptides were
finally cleaved from the resin as C-terminal amides by treatment with
a mixture of TFA/TIS/H2O 95/2.5/2.5,45 repeated twice for 60 and 30
min, respectively. After filtration, the resins were washed three times
with DCM, and the collected solutions were evaporated to dryness.
The desired crude pentapeptides 17-22 were obtained as yellowish
solids by trituration with ether and afterward submitted to HPLC
purification as extensively described in the Supporting Information.
6.1. H-Glu-X1aa-Ala-Arg-Ala-NH2 Trifluoroacetate (17). Where
X1aa is compound 2 with n ) 2. Overall yield: 45%. Retention time:
9.869 min. MW (monoisotopic) calcd for C39H48F12N10O7: 996.35. MS
(ES+): 998.34 [M + H]+, 500.02 [M + 2H]2+/2. MS (ES-): 996.37
[M - H]-. 19F NMR (50 mM Tris, 1 mM DTT, 0.001% Triton X-100,
pH ) 7.5): 12.81 ppm.
6.2. H-Glu-X2aa-Ala-Arg-Ala-NH2 Trifluoroacetate (18). Where
X2aa is compound 2 with n ) 3. Overall yield: 67%. Retention time:
9.474 min. MW (monoisotopic) calcd for C40H50F12N10O7: 1010.37.
MS (ES+): 1012.36 [M + H]+, 507.00 [M + 2H]2+/2. 19F NMR (50
mM Tris, 1 mM DTT, 0.001% Triton X-100, pH ) 7.5): 12.80 ppm.
6.3. H-Glu-X3aa-Ala-Arg-Ala-NH2 Trifluoroacetate (19). Where
X3aa is compound 2 with n ) 4. Overall yield: 41%. Retention time:
9.503 min. MW (monoisotopic) calcd for C41H52F12N10O7: 1024.38.
MS (ES+): 1026.45 [M + H]+, 514.06 [M + 2H]2+/2. MS (ES-):
1024.46 [M - H]-. 19F NMR (50 mM Tris, 1 mM DTT, 0.001% Triton
X-100, pH ) 7.5): 12.79 ppm.
6.4. H-Glu-X4aa-Ala-Arg-Ala-NH2 (20). Where X4aa is compound
3 with n ) 2. Overall yield: 15%. Retention time: 0.876 min. MW
(monoisotopic) calcd for C30H44F6N10O7: 770.33. MS (ES+): 771.19
[M + H]+, 386.17 [M + 2H]2+/2. MS (ES-): 769.28 [M - H]-. 19F
NMR (50 mM Tris, 1 mM DTT, 0.001% Triton X-100, pH ) 7.5):
12.90 ppm.
6.5. H-Glu-X5aa-Ala-Arg-Ala-NH2 (21). Where X5aa is compound
3 with n ) 3. Overall yield: 25%. Retention time: 0.909 min. MW
(monoisotopic) calcd for C31H46F6N10O7: 784.35. MS (ES+): 785.24
[M + H]+, 393.17 [M + 2H]2+/2. MS (ES-): 783.35 [M - H]-. 19F
NMR (50 mM Tris, 1 mM DTT, 0.001% Triton X-100, pH ) 7.5):
12.83 ppm.
(monoisotopic) calcd for C32H48F6N10O7: 798.36. MS (ES+): 799.26
[M + H]+, 400.17 [M + 2H]2+/2. MS (ES-): 797.33 [M - H]-. 19F
NMR (50 mM Tris, 1 mM DTT, 0.001% Triton X-100, pH ) 7.5):
12.78 ppm.
7. NMR. The enzyme trypsin and the inhibitor leupeptin were
purchased from Roche Molecular Biochemical (cat. nos. 1418475 and
1017101, respectively). The enzymatic reactions were performed at
room temperature in 50 mM Tris pH 7.5, 0.001% Triton X-100, and
8% D2O for the lock signal. The concentration of substrates was 10
and 20 µM for 17-19 and 20-22, respectively. Reactions were
quenched after a defined delay with the addition of 80 µM leupeptin.
All NMR spectra were recorded at 20 °C with a Varian Inova 600
MHz NMR spectrometer operating at a 19F Larmor frequency of 564
1
MHz. A 5 mm probe with the inner coil tunable to either 19F or H
frequency was used. The instrument was equipped with a sample
management system (SMS) autosampler for automatic data collection.
The data were recorded without proton decoupling with an acquisition
time of 0.8 s and a relaxation delay of 2.8 s. Chemical shifts are
referenced to trifluoroacetic acid.
8. Docking Studies. The bound inhibitor present in the crystal
structure (entry 2PTC in the Protein Data Bank, 1.90 Å resolution)
was removed as well as all water molecules except one, buried deep in
the S1 pocket (corresponding to HOH-416). Polar hydrogens were
added to the protein, the lysine and arginine residues were positively
charged, the glutamic and aspartic acid residues were negatively
charged, while the histidines were defined as neutral. The docking
studies were performed with the program QXP,46 using the docking
algorithm, referred to as MCdock. During the docking the protein was
kept rigid; the HOH-416 water oxygen was instead treated as partially
flexible, while its hydrogens were treated as completely flexible. The
three peptidic ligands were docked as completely flexible. The docking
runs included 10 000 steps of Monte Carlo perturbation and final energy
minimization.
Acknowledgment. This work was supported by the Italian
Grant FIRB RBNE03LF7X of the Ministero dell’Istruzione,
dell’Universita` e della Ricerca.
Supporting Information Available: Glossary, HPLC/MS
instrumentation, and methods. This material is available free
of charge via the Internet at http://pubs.acs.org.
6.6. H-Glu-X6aa-Ala-Arg-Ala-NH2 (22). Where X6aa is compound
3 with n ) 4. Overall yield: 25%. Retention time: 0.95 min. MW
JA069128S
(45) Pearson, D. A.; Mary Blanchette, M.; Baker, M. L.; Guindon, C. A.
(46) McMartin, C.; Bohacek, R. S. J. Comput.-Aided Mol. Des. 1997, 11, 333-
Tetrahedron Lett. 1989, 30, 2739-2742.
344.
9
5672 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007