spacing or chirality) the relative positions of the functional
groups. The success of these analogues in disrupting the Tat-
TAR interaction provides further evidence that the presenta-
tion of an arginine side chain in a cationic context is a
primary determinant of affinity and also suggests that other
peptidomimetics might have similar potency.
The study of â-peptides has accelerated over the past
decade, propelled by demonstrations that they can be
programmed to adopt protein-like secondary structures.10
These structures have given rise to a variety of biological
activities,11 and the protease resistance of â-peptides makes
them potentially attractive from a pharmaceutical stand-
point.12 During our previous work on membrane translocation
of analogues of the Tat 47-57 sequence,13 we noted that
â-peptide 1 is unstructured in aqueous solution. Because this
region of the native Tat protein adopts an extended confor-
mation,14 we hypothesized that flexible â-peptide 1 would
itself be competent to bind TAR.
column chromatography, with fraction-by-fraction HPLC
analysis, to eliminate small (e1%) amounts of starting
material Fmoc-Arg(Pmc) impurity. We have also detected
R-Lys impurity in Fmoc-â3HLys(Boc) and its oligomers,
albeit at lower levels. If chromatography is performed only
once on the building blocks, these impurities give rise in
highly redundant sequences to a population of R-Arg- and
R-Lys-containing contaminants, detectable by mass spec-
trometry but inseparable from the desired â-peptide by
HPLC. Peptides used for the studies described here were
synthesized using Fmoc-â3HArg(Pmc) and Fmoc-â3HLys-
(Boc) containing undetectable (<0.05%) amounts of Fmoc-
R-amino acid. Analogous control R-peptides 3 and 4 were
synthesized similarly from R-amino acids.
Electrophoretic mobility shift data (see Supporting Infor-
mation) showed binding of 1 to TAR RNA. However,
because partial dissociation of complexes during gel elec-
trophoresis has been reported to interfere with assessments
of peptoid-TAR binding,8a an alternative method for de-
termining Kd was required. We therefore developed a
fluorescence anisotropy (FA) assay for Tat-TAR binding.
When a fluorophore is excited by polarized light, the loss
of polarization in the emitted light (FA) can be correlated
with the mobility of the fluorophore. In our assay, wild-
type and bulge-deleted TAR RNA were labeled with
fluorescein. Binding of peptides 1-4 (MW ≈ 1400-1700)
to labeled wild-type or bulgeless TAR (MW ≈ 9700 or 8900)
increases the effective molecular weight of the fluorophore-
bearing complex, decreasing the effective fluorophore mobil-
ity and, hence, increasing FPA. Fluorescence anisotropy was
measured as a function of â-peptide concentration (Figure
1), and Kd was determined from these curves (Table 1).18
The syntheses of â-peptide 1 (as previously reported13)
and control â-peptide 2, in which all arginine side chains
have been replaced by lysine side chains, were carried out
by automated solid-phase methods15 from Fmoc-protected
â-substituted â-amino acids (“â3-amino acids”) obtained
enantiospecifically using Mu¨ller’s modification16 of See-
bach’s methodology.17 Fmoc-â3HArg(Pmc) obtained by
homologation, as previously reported,13 requires repeated
The affinity of Tat protein and Tat-derived peptides for
TAR is known to depend sensitively on assay conditions.
For example, electrophoresis-derived Kd values for extremely
similar peptides can differ by 2 orders of magnitude with
varying salt concentrations (70 mM NaCl vs 20 mM KCl)
and experimental conditions.6,19 Therefore, the difference in
Kd values for 3 between our assay and one reported
previously6 is not completely unexpected. The Kd and binding
mode of 3 to fluorescein-tagged TAR have been verified by
FRET to rhodamine-tagged 3 (Cao, H.; Rana, T. M.
Unpublished results).
(9) Calnan, B. J.; Tidor, B.; Biancalana, S.; Hudson, D.; Frankel, A. D.
Science (Washington, DC,) 1991, 252, 1167-1171.
(10) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001,
101, 3219-3232. Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr.
Med. Chem. 1999, 6, 905-925.
(11) (a) Werder, M.; Hauser, H.; Abele, S.; Seebach, D. HelV. Chim.
Acta 1999, 82, 1774-1783. (b) Gademann, K.; Ernst, M.; Hoyer, D.;
Seebach, D. Angew. Chem., Int. Ed. 1999, 38, 1223-1226. (c) Hamuro,
Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-
12201. (d) Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S.
H. Nature (London) 2000, 404, 565.
(12) Seebach, D.; Abele, S.; Schreiber, J. V.; Martinoni, B.; Nussbaum,
A. K.; Schild, H.; Schulz, H.; Hennecke, H.; Woessner, R.; Bitsch, F. Chimia
1998, 52, 734-739.
(13) Umezawa, N.; Gelman, M. A.; Haigis, M. C.; Raines, R. T.;
Gellman, S. H. J. Am. Chem. Soc. 2002, 124, 368-369.
(14) Gregoire, C.; Peloponese, J. M., Jr.; Esquieu, D.; Opi, S.; Campbell,
G.; Solomiac, M.; Lebrun, E.; Lebreton, J.; Loret, E. P. Biopolymers 2001,
62, 324-335.
(15) Synthesis and purification of 1 and 2 are discussed in Supporting
Information.
(16) Mu¨ller, A.; Vogt, C.; Sewald, N. Synthesis 1998, 6, 837-841.
(17) Guichard, G.; Abele, S.; Seebach, D. HelV. Chim. Acta 1998, 81,
187-206.
Because of this variation in measured Kd values, a standard
procedure in this field is to report dissociation constant ratios
(Krel), in which a value above 1 denotes stronger binding of
the analogue relative to the native peptide. The Krel value
for 1 relative to 3 is 0.072, indicating a significant diminution
of affinity when the backbone is altered to a â-peptide
backbone. Interestingly, alteration of the backbone to an
oligocarbamate gives Krel ) 0.69,6 and alteration to an
oligourea gives Krel ) 7.1,7 indicating that affinity of Tat
analogues for TAR cannot be explained as a simple function
of side chain spacing.
(18) More detailed discussion and experimental details are given in
Supporting Information.
(19) Calnan, B. J.; Biancalana, S.; Hudson, D.; Frankel, A. D. Genes
DeV. 1991, 5, 201-210.
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