The Bip method, based on the induced circular dichroism of a flexible biphenyl probe in terminally protected -Bip-Xaa*-dipeptides, for assignment of the absolute configuration of β-amino acids

Article abstract of DOI:10.1021/ja800059d

An induced axial chirality of the biphenyl core of the Bip (2′,1′:1,2;1″,2″:3,4-dibenzcyclohepta-1, 3-diene-6-amino-6-carboxylic acid) residue in the terminally protected dipeptides Boc-Bip-β-Xaa*-OMe (β-Xaa* = L-β³-HAla, L-β³-HVal,

Full text of DOI:10.1021/ja800059d

Published on Web 04/10/2008
The Bip Method, Based on the Induced Circular Dichroism of
a Flexible Biphenyl Probe in Terminally Protected -Bip-Xaa*-
Dipeptides, for Assignment of the Absolute Configuration of
ꢀ-Amino Acids
Laurence Dutot,† Karen Wright,† Anne Gaucher,† Michel Wakselman,†
Jean-Paul Mazaleyrat,*,† Marta De Zotti,‡ Cristina Peggion,‡ Fernando Formaggio,‡
and Claudio Toniolo*,‡
ILV, UMR CNRS 8180, UniVersity of Versailles, 78035 Versailles, France, and Institute of
Biomolecular Chemistry, PadoVa Unit, CNR, Department of Chemistry, UniVersity of PadoVa,
35131 PadoVa, Italy
Received January 4, 2008; E-mail: claudio.toniolo@unipd.it
Abstract: An induced axial chirality of the biphenyl core of the Bip (2′,1′:1,2;1′′,2′′:3,4-dibenzcyclohepta-
1,3-diene-6-amino-6-carboxylic acid) residue in the terminally protected dipeptides Boc-Bip-ꢀ-Xaa*-OMe
(ꢀ-Xaa* ) L-ꢀ3-HAla, L-ꢀ3-HVal, L-ꢀ3-HLeu, L-ꢀ3-HPro, trans-(1S,2S)-ACHC, trans-(1R,2R)-ACHC, trans-
(1S,2S)-ACPC, trans-(1R,2R)-ACPC) resulted in an induced circular dichroism, revealing the usefulness
of the Bip method for a reliable and fast assignment of the absolute configuration of chiral ꢀ-amino acids.
Remarkably, the Bip method was also applied to the unique spin-labeled, cyclic, ꢀ-amino acids cis/trans-
ꢀ-TOAC and trans-POAC. In particular, this study allowed the assignment of the unknown absolute
configurations of the enantiomers of the latter compound.
Introduction
tage of the configurationally flexible biphenyl chromophore for
which induction of axial chirality by interaction with chiral
Within the past decade and stimulated by the early findings
of Seebach1a and Gellman,1b short oligomers of ꢀ-amino acids
(ꢀ-peptides) have attracted considerable attention because of
their propensities to adopt a variety of stable helical
secondary structures (mainly 2.612, 2.710/12, and 314 helices),2
different from those typically adopted by oligomers of protein
R-amino acids and CR-tetrasubstituted R-amino acids (3.613-
or R-helix and 310-helix, respectively).3 Furthermore, ꢀ-pep-
tides offer the considerable advantage of combined folding
tendency and resistance to proteolytic degradation in vitro and
in vivo, a useful starting point which has led to the successful
design, synthesis, and evaluation of functional mimics of
biologically active natural peptides and proteins.2,4 Accordingly,
robust synthetic methods for the production of a variety of
ꢀ-amino acids as well as for the determination of their
enantiomeric purity have been investigated.2,5
auxiliaries is well documented and has been exploited in molec-
ular recognition studies as well as in asymmetric catalysis.
Central-to-axial transfer of chirality results in an induced circular
dichroism (ICD) of the biphenyl chromophore, which has been
used as a tool for the determination of the absolute configuration
of chiral alcohols and carboxylic acids as well as R-amino acids.6
We have previously reported that in peptides as short as dimers
an axial chirality can be induced in the biphenyl moiety of 2′,1′:
1,2;1′′,2′′:3,4-dibenzcyclohepta-1,3-diene-6-amino-6-carboxy-
lic acid (Bip), a conformationally labile, atropoisomeric, CR-
tetrasubstituted R-amino acid.7 Unequal populations of -Bip-
Xaa*- dipeptide diastereoisomeric conformers [Xaa* ) Ala, Val,
1
Leu, (RMe)Val, and (RMe)Leu] were detected by CD and H
NMR techniques. The magnitude of this effect is particularly
remarkable when Xaa* is positioned at the C-terminus of Bip.
Moreover, the signs of the ICD bands correlate with the R-amino
acid absolute configuration. More specifically, in the NR-
protected R-amino esters Boc-Bip-Xaa*-OMe (Boc, tert-buty-
loxycarbonyl; OMe, methoxy), the C-terminal L-Xaa* and D-
Xaa* residues preferentially induce a negative and a positive
Cotton effect at 250 nm, and a P and a M torsion in the biphenyl
chromophore, respectively. This ICD phenomenon represents
However, development of methods allowing a reliable and
fast assignment of the absolute configuration of ꢀ-amino acids
still remains a challenge. An interesting approach takes advan-
† University of Versailles.
‡ University of Padova.
(1) (a) Seebach, D.; Matthews, J. L. J. Chem. Soc., Chem. Commun. 1997,
2015–2022. (b) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173–180.
(2) (a) For leading review articles, see : Cheng, R. P.; Gellman, S. H.;
DeGrado, W. F. Chem. ReV. 2001, 101, 3219–3232. (b) Seebach, D.;
Beck, A. K.; Bierbaum, B. J. Chem. BiodiV. 2004, 1, 1111–1239.
(3) (a) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747–6756. (b)
Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16, 350–353.
(4) Gademann, K.; Hintermann, T.; Schreiber, J. V. Current Med. Chem.
1999, 6, 905–925.
(6) (a) Superchi, S.; Casarini, D.; Laurita, A.; Bavoso, A.; Rosini, C.
Angew. Chem. 2001, 113, 465–468. Angew. Chem. Int. Engl. Ed. 2001,
40, 451–454. (b) Superchi, S.; Bisaccia, R.; Casarini, D.; Laurita, A.;
Rosini, C. J. Am. Chem. Soc. 2006, 128, 6893–6902.
(7) (a) Mazaleyrat, J.-P.; Gaucher, A.; Šavrda, J.; Wakselman, M.
Tetrahedron: Asymmetry 1997, 8, 619–631. (b) Formaggio, F.; Crisma,
M.; Toniolo, C.; Tchertanov, L.; Guilhem, J.; Mazaleyrat, J.-P.;
Gaucher, A.; Wakselman, M. Tetrahedron 2000, 56, 8721–8734.
(5) Juaristi, E. EnantioselectiVe Synthesis of ꢀ-Amino Acids; Wiley-VCH:
New York, 1997, and references therein.
9
5986 J. AM. CHEM. SOC. 2008, 130, 5986–5992
10.1021/ja800059d CCC: $40.75
2008 American Chemical Society

Absolute Configuration by the Bip Method
A R T I C L E S
Figure 1. Conformational equilibrium of the Boc-Bip-Xaa*-OMe dipeptides. The abbreviations ACHC, ACPC, ꢀ-TOAC, and POAC stand for 2-amino-
cyclohexanecarboxylic acid, 2-amino-cyclopentanecarboxylic acid, 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine-3-carboxylic acid, and 4-amino-1-oxyl-
2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid, respectively.
Infrared Absorption. The FT-IR absorption spectra were
recorded using a Perkin-Elmer model 1720 X FT-IR spectropho-
tometer, nitrogen-flushed, equipped with a sample-shuttle device,
at 2 cm-1 nominal resolution, averaging 100 scans. Cells with path
lengths of 0.1, 1.0, and 10 mm (with CaF2 windows) were used.
Spectrograde deuterochloroform (99.8% d) was purchased from
Aldrich (St Louis, MO). Solvent (baseline) spectra were recorded
under the same conditions.
the basis for the Bip method, an easy and fast configurational
assignment of chiral R-amino acids.8 In the present paper, we
wish to report the full set of our results relative to the extension
of the Bip method to ꢀ-amino acids (ꢀ-Xaa*): L-ꢀ3-HAla, L-ꢀ3-
HVal, L-ꢀ3-HLeu, L-ꢀ3-HPro, L-ꢀ3-HPhe, trans (1R,2R)/(1S,2S)-
ACHC, trans (1R,2R)/(1S,2S)-ACPC (Figure 1),9 as well as the
unique spin-labeled, cyclic, chiral ꢀ 2,3-amino acids synthesized
and/or resolved in our groups, cis/trans-ꢀ-TOAC10 and
trans-POAC.11,12
Nuclear Magnetic Resonance. The 1H NMR and 13C NMR
spectra were recorded on a Bruker WM300 spectrometer operating
at 300 and 77 MHz, respectively, the solvent CDCl3 (1H: δ ) 7.27
ppm; 13C: δ ) 77.00 ppm) or CD3OD (1H: δ ) 3.31 ppm) being
used as internal standard. Spectrograde deuterochloroform (99.5%
d) and methyl alcohol d4 (99.8% d) were purchased from Euriso-
top (Saclay, France).
Experimental Section
Synthesis of Dipeptides. A new synthesis of Boc-Bip-OH,7a as
well as syntheses of all new dipeptides discussed in this work, are
reported in the Supporting Information.
Circular Dichroism. The CD spectra were obtained on a Jasco
(Tokyo, Japan) J-715 spectropolarimeter. Cylindrical fused quartz
cells (Hellma) of 0.1 mm path length were used. The values are
expressed in terms of [θ]T, total molar ellipticity (deg ·cm2 ·dmol-1).
Spectrograde methanol (MeOH) (Fluka, Buchs, Switzerland) was
used as solvent.
(8) (a) Mazaleyrat, J.-P.; Wright, K.; Gaucher, A.; Toulemonde, N.;
Wakselman, M.; Oancea, S.; Peggion, C.; Formaggio, F.; Setnicˇka,
V.; Keiderling, T. A.; Toniolo, C. J. Am. Chem. Soc. 2004, 126, 12874–
12879. (b) Mazaleyrat, J.-P.; Wright, K.; Gaucher, A.; Toulemonde,
N.; Dutot, L.; Wakselman, M.; Broxterman, Q. B.; Kaptein, B.;
Oancea, S.; Peggion, C.; Crisma, M.; Formaggio, F.; Toniolo, C. Chem.
Eur. J. 2005, 11, 6921–6929.
Results and Discussion
(9) Preliminary reports of this work have been published: (a) in which
evolution of the 1H NMR spectra of Boc-Bip-L-ꢀ3-HAla-OMe in
CD3OD as a function of temperature is presented: Dutot, L.; Gaucher,
A.; Wright, K.; Wakselman, M.; Mazaleyrat, J.-P.; Oancea, S.;
Peggion, C.; Formaggio, F.; Toniolo, C. Tetrahedron: Asymmetry 2006,
17, 363–371. (b) Dutot, L.; Gaucher, A.; Wright, K.; Wakselman, M.;
Mazaleyrat, J.-P.; Peggion, C.; Formaggio, F.; Toniolo, C. In Peptides
2006, Proceedings of the 4th International and 29th European Peptide
Symposium, Rolka, K., Rekowski, P., Silberring, J., Eds.; Kenes
International: Geneva, Switzerland, 2007; pp 76–77. (c) Wright, K.;
Dutot, L.; Gaucher, A.; Wakselman, M.; Mazaleyrat, J.-P.; Peggion,
C.; Formaggio, F.; Toniolo, C. In Peptides for Youth, Proceedings of
the 20th American Peptide Symposium, in press [abstract in Biopoly-
mers (Pept. Sci.) 2007, 88, 535].
(10) (a) Wright, K.; Crisma, M.; Toniolo, C.; Török, R.; Péter, A.;
Wakselman, M.; Mazaleyrat, J.-P. Tetrahedron Lett. 2003, 44, 3381–
3384. (b) Wright, K.; de Castries, A.; Sarciaux, M.; Formaggio, F.;
Toniolo, C.; Toffoletti, A.; Wakselman, M.; Mazaleyrat, J.-P. Tetra-
hedron Lett. 2005, 46, 5573–5576. (c) Wright, K.; Sarciaux, M.; de
Castries, A.; Wakselman, M.; Mazaleyrat, J.-P.; Toffoletti, A.; Crisma,
M.; Formaggio, F.; Toniolo, C. Eur. J. Org. Chem. 2007, 3133–3144.
Synthesis. The NR-protected amino acid derivative Boc-Bip-
OH was readily prepared in a different way than previously
reported,7a by bisalkylation of ethyl isocyanoacetate with 2,2′-
bis(bromomethyl)-1,1′-biphenyl under the solid–liquid phase
transfer conditions developed by Kotha and Brahmachary,13 with
potassium carbonate as a base and tetra-n-butylammonium
hydrogen sulfate (TBAHS) as the catalyst, in acetonitrile at
(11) Tominaga, M.; Barbosa, S. R.; Poletti, E. F.; Zukerman-Schpector,
J.; Marchetto, R.; Schreier, S.; Paiva, A. C. M.; Nakaie, C. R. Chem.
Pharm. Bull. 2001, 49, 1027–1029.
(12) (a) Wright, K.; Formaggio, F.; Toniolo, C.; Török, R.; Péter, A.;
Wakselman, M.; Mazaleyrat, J.-P. Tetrahedron Lett. 2003, 44, 4183–
4186. (b) Péter, A.; Török, R.; Wright, K.; Wakselman, M.; Maza-
leyrat, J.-P. J. Chromatogr. A 2003, 1021, 1–10. (c) Wright, K.; Dutot,
L.; Wakselman, M.; Mazaleyrat, J.-P. Crisma, M.; Formaggio, F.;
Toniolo, C. Tetrahedron 2008, in press.
(13) Kotha, S.; Brahmachary, E. J. Org. Chem. 2000, 65, 1359–1365.
9
J. AM. CHEM. SOC. VOL. 130, NO. 18, 2008 5987

A R T I C L E S
Dutot et al.
Table 1. Chemical Shifts (δ) of the -COOCH3 Singlet at 293 and
233 K (Two Diastereoisomers) and Calculated Diastereoisomeric
Ratios (dr) for the Dipeptides Boc-Bip-ꢀ-Xaa*-OMe in CD3OD
Solution
δ-COOCH3 (ppm)
dipeptides
293 K
233 K
dr
Boc-Bip-L-ꢀ3-HAla-OMe9b
Boc-Bip-L-ꢀ3-HVal-OMe
Boc-Bip-L-ꢀ3-HLeu-OMe
Boc-Bip-L-ꢀ3-HPhe-OMe
Boc-Bip-L-ꢀ3-HPro-OMe
Boc-Bip-(1S,2S)-ACPC-OMeb
Boc-Bip-(1S,2S)-ACHC-OMec
3.69
3.69
3.68
3.68
3.67
3.69
3.69
3.72 and 3.65
3.75 and 3.63
3.79 and 3.63
3.69 and 3.68a
3.68 and 3.64
3.74 and 3.64
3.74 and 3.62
68:32
72:28
61:39
∼60:40a
64:36
62:38
74:26
a ∆δ (-COOCH3) between the two diastereoisomers was too small
(shoulder) to allow calculation of the corresponding dr with accuracy.
b Or Boc-Bip-(1R,2R)-ACPC-OMe. c Or Boc-Bip-(1R,2R)-ACHC-OMe.
80 °C. The resulting 2′,1′:1,2;1′′,2′′:3,4-dibenzcyclohepta-1,3-
diene-6-isocyano-6-carboxylic acid ethyl ester was not isolated,
but rather the crude reaction mixture was submitted to acid
hydrolysis to afford directly the R-amino ester H-Bip-OEt (OEt,
ethoxy)7a in 61% yield, from which Boc-Bip-OEt7a (72%) and
then the desired compound Boc-Bip-OH7a (85%) were obtained.
The syntheses and characterizations of the terminally pro-
tected dipeptides Boc-Bip-R-Xaa*-OMe [R-Xaa* ) L/D-Ala,
L/D-Val, L/D-Leu, L/D-Phe, L-(RMe)Val, L-(RMe)Leu] have been
previously reported.7,8 The Boc-Bip-ꢀ-Xaa*-OMe9b dipeptides
(ꢀ-Xaa* shown in Figure 1) were prepared in solution by
coupling of Boc-Bip-OH with the amino ester hydrochlorides
HCl·H-ꢀ-Xaa*-OMe or with H-ꢀ-Xaa*-NHC6H11. Full details
are given in the Supporting Information.
Figure 2. Far-UV CD spectra in MeOH solution of (A) Boc-Bip-L-ꢀ3-
HVal-OMe and Boc-Bip-L-Val-OMe, and (B) Boc-Bip-L-ꢀ3-HLeu-OMe and
Boc-Bip-L-Leu-OMe. Peptide concentration: 1 mM.
1H NMR Analysis. The chirality transfer phenomenon in the
Boc-Bip-Xaa*-OMe dipeptides could be observed by 1H NMR
in CD3OD for both ꢀ3-Xaa* and ꢀ2,3-Xaa* amino esters, as well
as previously reported8 for R-Xaa*-containing dipeptide esters.
Indeed, two sets of signals, each corresponding to the presence
of one diastereoisomeric conformer, exchanging slowly on the
NMR time scale, are seen at low temperature (233 K) with
unequal populations, while a single set of sharp signals is
observed at 333 K under fast-interconverting conditions with
coalescence occurring at about 293 K, as expected from the
rotational energy barrier of 14 kcal mol-1 along the 1–1′ bond
of the biphenyl moiety.7 The diastereoisomeric ratio (dr) was
determined at 233 K by integration of the singlet corresponding
to the carboxylic ester methyl group, which generally appears
at different chemical shifts for the two stereoisomers (Table 1).
the A band,14 followed by an even more intense absorption at
ca. 210–215 nm (C band). In the CD spectra of biphenyl-based
chiral molecules a negative maximum corresponding to the A
band is associated with a P torsion of the CAr-CAr bond.15 We
performed a CD analysis in MeOH solution of all of the Boc/
OMe terminally protected -Bip-ꢀ-Xaa*-dipeptides listed in
Figure 1 and compared them with the corresponding -Bip-R-
Xaa*-dipeptides. The spectra show that the ICD resulting from
the induced axial chirality in the biphenyl core of the Bip residue
gives a clear information on the ꢀ-Xaa* configuration for both
ꢀ3- and cyclic ꢀ2,3-amino acids. Taking all data together, the
most relevant points are as follows:
(i) A comparison between the CD spectra of Boc-Bip-L-ꢀ3-
HVal-OMe and Boc-Bip-L-Val-OMe, as well as Boc-Bip-L-ꢀ3-
HLeu-OMe and Boc-Bip-L-Leu-OMe, shows that in these
dipeptides, as previously observed for Boc-Bip-L-ꢀ3-HAla-OMe
and Boc-Bip-L-Ala-OMe,8b,9b a negative sign of the Bip Cotton
effect at 250 nm is preferentially biased by both the R-Xaa*
and ꢀ-Xaa* amino acids of the L configuration (Figure 2 and
Table 2). In the case of ꢀ-Xaa*-containing dipeptides, the CD
signal is weaker but still quite informative. Only Boc-Bip-L-
ꢀ3-HPhe-OMe (spectrum not shown) exhibits a positive Cotton
effect for the A band (Table 2), probably resulting from a
coupling of the aromatic chromophore present in the amino acid
Significantly high dr values (ranging from 60:40 to 74:26)
are observed at 233 K in CD3OD for all combinations of Bip
with both ꢀ3- and cyclic ꢀ2,3-amino acids. However, it is in-
teresting to note that the dr values for ꢀ3-HAla, ꢀ3-HVal, and
ꢀ3-HLeu are always lower than those for the corresponding
R-amino acids Ala (81:19), Val (77:23), and Leu (89:11),
respectively,8b except for ꢀ3-HPhe versus Phe (58:42)8b (in this
last case both dipeptides show relatively low dr values). As
expected, the dipeptides Boc-Bip-(1R,2R)-ACPC-OMe and Boc-
Bip-(1R,2R)-ACHC-OMe exhibit identical 1H NMR spectra and
dr values as Boc-Bip-(1S,2S)-ACPC-OMe and Boc-Bip-(1S,2S)-
ACHC-OMe, respectively, clearly indicating that enantiomeric
ꢀ-amino acids induce an opposite chirality of the Bip pro-
atropoisomeric residue.
(14) Suzuki, H. Electronic Absorption Spectra and Geometry of Organic
Molecules, Academic Press: New York, 1967.
(15) (a) Mislow, K.; Glass, M. A. W.; O’Brien, R. E.; Rutkin, P.; Steinberg,
D. H.; Weiss, J.; Djerassi, C. J. Am. Chem. Soc. 1962, 84, 1455–
1478. (b) Mislow, K.; Bunnenberg, E.; Records, R.; Wellman, K.;
Djerassi, C. J. Am. Chem. Soc. 1963, 85, 1342–1349. (c) Loncar-
Tomascovic, L.; Sarac-Arneri, R.; Hergold-Brundic, A.; Nagl, A.;
Mintas, M.; Sandström, J. HelV. Chim. Acta 2000, 83, 479–494.
CD Analysis. The biphenyl chromophore is known to exhibit
an intense electronic absorption near 240–250 nm, assigned to
9
5988 J. AM. CHEM. SOC. VOL. 130, NO. 18, 2008

Absolute Configuration by the Bip Method
A R T I C L E S
Table 2. Intensity and Sign of the A Band, and Absolute
Configuration of the Asymmetric Center(s) Correlated with the P or
M Torsion of the Biphenyl Core, for the Dipeptides
Boc-Bip-Xaa*-OMe in MeOH Solution (Peptide Concentration:
1mM)
Cotton effect (A band)
central
asymmetry
biphenyl
torsion
dipeptide derivative
intensitya
sign
Boc-Bip-L-ꢀ3-HAla-OMe17a
Boc-Bip-L-Ala-OMe16b
15
30
22
30
18
50
21
28
15
15
28
29
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(+)
(S)
P
P
P
P
P
P
P
M
P
(S)
Boc-Bip-L-ꢀ3-HVal-OMe
Boc-Bip-L-Val-OMe16
(R)b
(S)
(S)
(S)
(S)
Boc-Bip-L-ꢀ3-HLeu-OMe
Boc-Bip-L-Leu-OMe16b
Boc-Bip-L-ꢀ3-HPro-OMe
Boc-Bip-L-ꢀ3-HPhe-OMe
Boc-Bip-(1S,2S)-ACPC-OMe
Boc-Bip-(1R,2R)-ACPC-OMe
Boc-Bip-(1S,2S)-ACHC-OMe
Boc-Bip-(1R,2R)-ACHC-OMe
(S)
(1S,2S)
(1R,2R)
(1S,2S)
(1R,2R)
M
P
M
a Expressed by the total molar ellipticity [Θ]T
×
10-3
(deg·cm2 ·dmol-1). b Absolute configuration (R) according to the
accepted nomenclature, but corresponding to the same spatial arrange-
ment as (S)-ꢀ3-HAla, (S)-ꢀ3-HLeu, and (S)-L-ꢀ3-HPro.
Figure 4. Far-UV CD spectra in MeOH solution of Boc-Bip-L-ꢀ3-HAla-
OMe, Boc-Bip-L-ꢀ3-HVal-OMe, Boc-Bip-L-ꢀ3-HLeu-OMe, and Boc-Bip-
L-ꢀ3-HPro-OMe (A), and Boc-Bip-(1R,2R)-ACHC-OMe, Boc-Bip-(1S,2S)-
ACHC-OMe, Boc-Bip-(1R,2R)-ACPC-OMe, and Boc-Bip-(1S,2S)-ACPC-
OMe (B). Peptide concentration: 1 mM.
this phenomenon, but no inversion of the sign of the 250 nm
band is observed.
(iii) A negative sign for the strong Cotton effect of the A
band is seen for both series of terminally protected Bip
dipeptides containing L-ꢀ3-Xaa* amino acids (except for that
based on L-ꢀ3-HPhe, as mentioned above) or (1S,2S)-ꢀ2,3-cyclic
amino acids (ACHC, ACPC) (Figure 4 and Table 2), character-
ized by the same absolute configuration at their ꢀ-carbon atom.
Obviously, the CD spectra of the enantiomeric Boc-Bip-(1S,2S)-
ACHC-OMe and Boc-Bip-(1R,2R)-ACHC-OMe, as well as Boc-
Bip-(1S,2S)-ACPC-OMe and Boc-Bip-(1R,2R)-ACPC-OMe, are
mirror images (Figure 4B).
Figure 3. Far-UV CD spectra of Boc-Bip-L-ꢀ3-HLeu-OMe in MeOH, 1,4-
dioxane, THF, CH3CN, EtOH, TFE (2,2,2-trifluoroethanol), and CH2Cl2
solutions. Peptide concentration: 1 mM.
(iv) The enantiomeric dipeptides containing both O-acetyl
protected side-chain nitroxide ꢀ-amino acid derivatives cis- and
trans-ꢀ-TOAC(Ac) exhibit intense Cotton effects (Figure 5A).
Remarkably, the sign of the A band in the CD spectra of both
Boc-Bip-(3S,4R)-ꢀ-TOAC(Ac)-OMe and Boc-Bip-(3R,4R)-ꢀ-
TOAC(Ac)-OMe is positive, as that of Boc-Bip-(1R,2R)-ACHC-
OMe. Therefore, the sign of the Cotton effect at 250 nm is
determined by the absolute configuration of the carbon bearing
the nitrogen atom (4R, 4R, and 2R, respectively), not by that of
the carbon bearing the carboxyl group (3S, 3R, and 1R,
respectively). Again, it is worth noting that the CD spectra of
the two enantiomeric pairs Boc-Bip-(3R,4S)/(3S,4R)-ꢀ-TOA-
C(Ac)-OMe and Boc-Bip-(3S,4S)/(3R,4R)-ꢀ-TOAC(Ac)-OMe
are mirror images. In the same manner, as reported elsewhere,12c
the O-acetyl protected side-chain nitroxide-based dipeptides
Boc-Bip-(-)-POAC(Ac)-OMe and Boc-Bip-(+)-POAC(Ac)-
OMe show a strong Cotton effect at 250 nm, the sign of which
is the same as that of Boc-Bip-(1R,2R)-ACPC-OMe and Boc-
Bip-(1S,2S)-ACPC-OMe, respectively (Figure 5B). This finding
side chain with that of Bip,16 as reported for its Boc-Bip-L-
Phe-OMe analogue.8b
(ii) We also checked the role of the solvent on the ICD of
the Boc-Bip-ꢀ-Xaa*-OMe dipeptides. Figure 3 illustrates the
CD spectra of Boc-Bip-L-ꢀ3-HLeu-OMe in a variety of solvents.
A strong negative maximum at about 250 nm is seen in all
solvents tested with the following rank order for the ellipticity:
MeOH > 1,4-dioxane > THF > CH3CN ≈ EtOH > TFE >
CH2Cl2. A similar solvent rank order was previously observed
in the CD spectra of several Boc-Bip-R-Xaa*-OMe dipeptides,
the dr values of which, extracted from an NMR analysis, were
found to be higher in CD3OD than in CD3CN and CDCl3.8b
Therefore, the nature of solvent does play a remarkable role in
(16) (a) Beychok, S. In Poly-R-Amino Acids: Protein Models for Confor-
mational Studies, Fasman, G. D., Ed.; Dekker: New York, 1967; pp
293–337. (b) Goodman, M.; Toniolo, C. Biopolymers 1968, 6, 1673–
1689. (c) Woody, R. W. J. Polymer Sci., Macromol. ReV. 1977, 12,
181–320.
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Figure 7. Interaction CdO (i-2)· · ·O (i) (unfavorable) for Boc-Bip-L-Leu-
OMe and CdO (i-2)· · ·H-N (i) (favorable) for Boc-Bip-L-Leu-NH2 in the
postulated folded (nascent 310-helical) conformation (not depicted here).
Figure 8. CD spectra in MeOH solution of Boc-Bip-L-Leu-OMe and Boc-
Bip-L-Leu-NH2. Peptide concentration: 1 mM.
Figure 5. Far-UV CD spectra in MeOH solution of Boc-Bip-(3S,4R)-ꢀ-
TOAC(Ac)-OMe, Boc-Bip-(3R,4S)-ꢀ-TOAC(Ac)-OMe, Boc-Bip-(3R,4R)-
ꢀ-TOAC(Ac)-OMe, Boc-Bip-(3S,4S)-ꢀ-TOAC(Ac)-OMe, Boc-Bip-(1R,2R)-
ACHC-OMe, and Boc-Bip-(1S,2S)-ACHC-OMe (A), and Boc-Bip-(-)-
POAC(Ac)-OMe, Boc-Bip-(+)-POAC(Ac)-OMe, Boc-Bip-(1R,2R)-ACPC-
OMe, and Boc-Bip-(1S,2S)-ACPC-OMe (B).12c Peptide concentration: 1
mM.
(-)-(3S,4S) and (+)-(3R,4R). This result was achieved by
comparing the CD spectra of Boc-Bip-(-)-POAC-OMe and Boc-
Bip-(+)-POAC-OMe (not shown) with those of Boc-Bip-
(1R,2R)-ACPC-OMe and Boc-Bip-(1S,2S)-ACPC-OMe, respec-
tively (Figure 5B). Altogether, the “Bip method” appears to
allow the determination of the absolute configuration of spin-
labeled ꢀ-amino acids, even without the preliminary protection
of their side-chain nitroxide function. Application of this
methodology to POAC is of special interest since incorporation
of this labeled residue into peptides for structural investigations
has been reported.11
Relationship between ICD and Conformation of the Bip
Dipeptides. In the present study a few observations are pertinent
to the central-to-axial chirality transfer process in the -Bip-Xaa*-
dipeptides. In particular, we have noted that the magnitude of
the ICD is generally lower for the ꢀ3-Xaa* residues than for
their R-Xaa* counterparts for each pair Boc-Bip-L-Ala-OMe/
Boc-Bip-L-ꢀ3-HAla-OMe, Boc-Bip-L-Val-OMe/Boc-Bip-L-ꢀ3-
HVal-OMe, and Boc-Bip-L-Leu-OMe/Boc-Bip-L-ꢀ3-HLeu-OMe
(Table 2). However, in these three pairs, the number of bonds
separating the asymmetric center of Xaa* and the chiral axis of
Bip is just the same. Therefore, the factor playing the major
role in the induced axial chirality is apparently not a through-
bond coupling between the side chain of the asymmetric C(NH)
carbon atom of the Xaa* residue and the biphenyl group of the
Bip residue, but rather one has to consider a specific through-
space (conformational) coupling which could be responsible for
the chirality transfer.
Figure 6. Far-UV CD spectra in MeOH solution of Boc-Bip-(3S,4R)-ꢀ-
TOAC-OMe, Boc-Bip-(3R,4S)-ꢀ-TOAC-OMe, Boc-Bip-(3R,4R)-ꢀ-TOAC-
OMe, and Boc-Bip-(3S,4S)-ꢀ-TOAC-OMe, Boc-Bip-(1R,2R)-ACHC-OMe,
and Boc-Bip-(1S,2S)-ACHC-OMe. Peptide concentration: 1 mM.
has allowed us to assign the previously unknown (-)-(3S,4S)
and (+)-(3R,4R) absolute configurations to the enantiomers of
trans-POAC(Ac).
(v) Analogous conclusions were extracted from the CD
spectra of both enantiomers of the two dipeptides Boc-Bip-trans-
ꢀ-TOAC-OMe and Boc-Bip-cis-ꢀ-TOAC-OMe, containing side-
chain unprotected nitroxide moieties, as compared to the
corresponding enantiomers of Boc-Bip-ACHC-OMe (Figure 6).
Again, an extension of this study to the enantiomers of trans-
POAC allowed the assignment of their absolute configurations,
The most significant results of our previous conformational
studies in this area3b,7b,8 suggest that the central-to-axial
chirality transfer process in the Boc-Bip-R-Xaa*-OMe di-
peptides is likely to be related to their nascent 310-helical
secondary structure. This hypothesis is supported by a
comparison of the CD spectra for the dipeptide ester Boc-
Bip-L-Leu-OMe and the corresponding dipeptide amide Boc-
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Absolute Configuration by the Bip Method
A R T I C L E S
Figure 10. CD spectra in MeOH solution of Boc-Bip-(1S,2S)-ACPC-OMe
and Boc-Bip-(1S,2S)-ACPC-NHC6H11. Peptide concentration: 1 mM.
Figure 9. FT-IR absorption spectra (N-H stretching region) of Boc-Bip-
L-Leu-OMe (A) and Boc-Bip-L-Leu-NH2 (B) in CDCl3 solution. Peptide
concentration: 1 mM.
Bip-L-Leu-NH2 in which, in the case of a folded (nascent
310-helical) conformation, the interaction CdO (i-2) · · · O (i)
and CdO (i-2) · · · H-N (i), respectively, should change from
unfavorable to favorable (Figure 7). Indeed, the CD spectra
of the two dipeptides present a dramatic change of the Cotton
effect at 250 nm (A band), the sign of which inverts from
negative for the ester to positive for the amide (Figure 8).
The FT-IR absorption spectra for these two compounds in
CDCl3 solution, a solvent of relatively low polarity and known
to support the ordered secondary structure of peptides, also show
a dramatic change in the conformationally sensitive N-H
stretching (amide A) region (3470–3300 cm-1).17 For Boc-Bip-
L-Leu-OMe a single band is seen, centered at 3428 cm-1 and
assigned to the free urethane/amide NH groups (Figure 9A),
while for Boc-Bip-L-Leu-NH2 three intense bands are present
(Figure 9B). The bands at 3420 and 3481 cm-1 may be assigned
to the free urethane/secondary amide NH groups and the
C-terminal primary amide -NH2 group, respectively, while the
band at 3353 cm-1 is typical of intramolecularly H-bonded
amide NH groups. These results strongly support the view that
in Boc-Bip-L-Leu-NH2 a folded conformation, stabilized by an
intramolecular H-bond, is highly populated in CDCl3 solution.
On the basis of the length of the peptide and the known 3D-
structural properties of the Bip residue,7b,8 we believe that the
Figure 11. FT-IR absorption spectra (N-H stretching region) of Boc-
Bip-(1S,2S)-ACPC-OMe (A) and Boc-Bip-(1S,2S)-ACPC-NHC6H11 (B) in
CDCl3 solution. Peptide concentration: 1 mM.
folded conformation observed is a C10 (ꢀ-turn),18 the basic unit
of the 310-helix.
Altogether, the evidence for a ꢀ-turn conformation in Boc-
Bip-L-Leu-NH2, combined with the observed sign inversion of
the Cotton effect for this amide versus the corresponding ester
Boc-Bip-L-Leu-OMe, confirms the paramount importance of the
(nascent) 310-helical structure, induced by the conformational
properties of the Bip residue, in the central-to-axial chirality
transfer process and in the resulting ICD observed for the Boc-
Bip-R-Xaa*-OMe dipeptides.
(17) (a) Cung, M. T.; Marraud, M.; Néel, J. Ann. Chim. (Paris) 1972, 183–
209. (b) Palumbo, M.; Da Rin, S.; Bonora, G. M.; Toniolo, C.
Makromol. Chem. 1976, 177, 1477–1492. (c) Bonora, G. M.; Mapelli,
C.; Toniolo, C.; Wilkening, R. R.; Stevens, E. S. Int. J. Biol. Macromol.
1984, 6, 179–188. (d) Toniolo, C.; Bonora, G. M.; Barone, V.; Bavoso,
A.; Benedetti, E.; Di Blasio, B.; Grimaldi, P.; Lelj, F.; Pavone, V.;
Pedone, C. Macromolecules 1985, 18, 895–902.
As for the Boc-Bip-ꢀ-Xaa*-OMe dipeptide series, an analo-
gous process of chirality transfer related to the onset of a turn
(18) (a) Venkatachalam, C. M. Biopolymers 1968, 6, 1425–1436. (b)
Toniolo, C. CRC Crit. ReV. Biochem. 1980, 9, 1–44. (c) Rose, G. D.;
Gierasch, L. M.; Smith, P. J. AdV. Protein Chem. 1985, 37, 1–109.
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A R T I C L E S
Dutot et al.
(nascent helical) conformation may be expected, since Gellman
and co-workers have shown that for mixed R/ꢀ-peptides the
11-helical structure is favored by the presence of cyclic,
conformationally constrained, ꢀ-residues (typically ACPC) and
CR-tetrasubstituted R-residues (typically Aib).19 Figure 10 shows
clearly that, even for an NR-protected Boc-Bip-ꢀ-Xaa*-dipeptide
sequence with a rigidified ꢀ-amino acid (ACPC), the signs of
the biphenyl CD bands change on going from the ester to the
alkyl (cyclohexyl) amide functionality.
The corresponding FT-IR absorption spectra in the N-H
stretching region are reported in Figure 11. It is evident that a
band indicative of intramolecularly H-bonded (C11) species (at
3370 cm-1),17c although of modest intensity, appears only in
the spectrum of the alkyl amide dipeptide. We conclude that
the onset of an incipient helix in a Bip dipeptide alkylamide
based on a structurally restricted ꢀ-amino acid may have
profound consequences in the process of chirality transfer, as
observed for -Bip-R-Xaa*-dipeptides. Apparently however,
when in a R/ꢀ-dipeptide the rigid ACPC is replaced by a more
flexible ꢀ-amino acid, such as L-ꢀ3-HLeu, the variations of the
biphenyl CD bands from the ester to the alkylamide are less
clear-cut and more difficult to interpret (spectra not shown).
ꢀ3-HPro) or cyclic ꢀ2,3-Xaa*-OMe (ACHC, ACPC) amino
ester residues to the NR-Boc protected, pro-atropoisomeric,
CR-tetrasubstituted R-amino acid Bip has been revealed in
simple linear dipeptides, thus demonstrating that the Bip
residue may be used as a convenient CD probe for assignment
of the absolute configuration of not only R-amino acids as
previously reported,8 but ꢀ-amino acids as well. Interestingly,
we have also applied the Bip method to the spin-labeled,
cyclic, chiral ꢀ2,3-amino acids cis and trans ꢀ-TOAC and
even to the assignment of the unknown absolute configura-
tions of the trans-POAC enantiomers.
Taking our results together, we suggest that (i) MeOH is the
best solvent for the application of the Bip method to R- or
ꢀ-amino acid configurational issues for its excellent solubilizing
properties and high CD response and (ii) Boc-Bip-Xaa*-
dipeptide ester is the sequence of choice, as the NR-protecting
Boc moiety lacks any aromatic group potentially interfering with
the biphenyl CD band near 250 nm, and the C-terminal ester
functionality is devoid of any H-bonding donor and therefore
unable to contribute to the stability of dipeptide folded forms
which, in turn, may not be beneficial for an unambiguous
interpretation of the chirospectroscopic data.
Conclusions
Supporting Information Available: Details of the synthesis
of the dipeptides. This material is available free of charge via
the Internet at http://pubs.acs.org.
A remarkable central-to-axial induction of chirality from
the C-terminal ꢀ3-Xaa*-OMe (ꢀ3-HAla, ꢀ3-HVal, ꢀ3-HLeu,
(19) Schmitt, M. A.; Choi, S. H.; Guzei, I. A.; Gellman, S. H. J. Am. Chem.
Soc. 2005, 127, 13130–13131.
JA800059D
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