Role of secondary structure in the asymmetric acylation reaction catalyzed by peptides based on chiral Cα-tetrasubstituted α-amino acids

Article abstract of DOI:10.1021/jo040107v

In a recent series of papers, Miller and co-workers were able to show that His(π-Me)-based, terminally protected peptides are potent catalysts of the asymmetric acyl transfer reaction, useful for the kinetic resolution of alcohols. In a structure-supporting solvent, one of the most active compounds, an Aib-containing tetrapeptide, is folded in a doubly intramolecularly H-bonded β-hairpin motif incorporating a type-II′ β-turn conformation. In this work, we have expanded the study of the Miller tetrapeptide by examining a set of analogues and shorter sequences (dipeptide amides), characterized by chiral C^α-tetrasubstituted α-amino acids of diverging bulkiness and optical configuration. Peptide synthesis in solution, conformational analysis by FT-IR absorption and ¹H NMR techniques, and screening of catalytic activity as well have been performed. Our results confirm the close relationship between the β-hairpin 3D-structure and the catalytic activity of the peptides. A tetrapeptide analogue slightly more selective than the Miller compound has been found. However, the terminally protected, industrially more appealing, dipeptide amides are poorly effective.

Full text of DOI:10.1021/jo040107v

Role of Secon d a r y Str u ctu r e in th e Asym m etr ic Acyla tion
Rea ction Ca ta lyzed by P ep tid es Ba sed on Ch ir a l
Cr-Tetr a su bstitu ted r-Am in o Acid s
Fernando Formaggio,† Alessandra Barazza,† Andrea Bertocco,† Claudio Toniolo,*,†
Quirinus B. Broxterman,‡ Bernard Kaptein,‡ Elena Brasola,§ Paolo Pengo,§
Lucia Pasquato,§,| and Paolo Scrimin§
Department of Chemistry, University of Padova, and ICB, CNR, 35131 Padova, Italy, DSM Pharma
Chemicals, Advanced Synthesis and Catalysis, P.O. Box 18, 6160 MD Geleen, The Netherlands, and
Department of Chemistry, University of Padova, and ITM, CNR, 35131 Padova, Italy
claudio.toniolo@unipd.it
Received J anuary 12, 2004
In a recent series of papers, Miller and co-workers were able to show that His(π-Me)-based,
terminally protected peptides are potent catalysts of the asymmetric acyl transfer reaction, useful
for the kinetic resolution of alcohols. In a structure-supporting solvent, one of the most active
compounds, an Aib-containing tetrapeptide, is folded in a doubly intramolecularly H-bonded
â-hairpin motif incorporating a type-II′ â-turn conformation. In this work, we have expanded the
study of the Miller tetrapeptide by examining a set of analogues and shorter sequences (dipeptide
amides), characterized by chiral CR-tetrasubstituted R-amino acids of diverging bulkiness and optical
1
configuration. Peptide synthesis in solution, conformational analysis by FT-IR absorption and H
NMR techniques, and screening of catalytic activity as well have been performed. Our results confirm
the close relationship between the â-hairpin 3D-structure and the catalytic activity of the peptides.
A tetrapeptide analogue slightly more selective than the Miller compound has been found. However,
the terminally protected, industrially more appealing, dipeptide amides are poorly effective.
In tr od u ction
sistent with a preferential transition-state stabilization
governed by catalyst-substrate secondary contacts.
Significant improvements were recently achieved in
the development of peptide-based, chiral, nucleophilic
catalysts for the kinetic resolution of racemic alcohols by
means of enantioselective acyl transfer reactions (eq 1).1
A large body of results from Miller’s laboratory2 clearly
demonstrated that terminally protected, His(π-Me)-based
peptides as small as tripeptide amides and tetra-, penta-,
and octapeptide esters can be effectively exploited for this
purpose. From conformational investigations it was
proposed that rigidified â-hairpin-forming peptide plat-
forms are an absolute prerequisite for significant catalytic
activity and enantioselectivity. These catalysts, active as
monomers, show enantioselective rate accelerations con-
We focused on the Miller’s prototypical, terminally
protected, tetrapeptide catalyst Boc-L-His(π-Me)-D-Pro-
Aib-L-Phe-OMe 1′, which causes the reaction of the (R,R)
alcohol enantiomer (eq 1) to proceed remarkably faster
than that of the (S,S) enantiomer (krel ) 28).2a,d In a
structure-supporting solvent this peptide adopts a â-hair-
pin conformation characterized by [His(π-Me)]CdO‚‚‚H-
N(Phe) and [His(π-Me)]N-H‚‚‚OdC(Phe) intramolecular
H-bonds. The onset of a type-II′ â-turn3 in the -D-Pro-
Aib- sequence of this compound is not unexpected, as it
is known to typically occur in a -D-L-amino acid stereo-
* To whom correspondence should be addressed.
† University of Padova, ICB.
‡ DSM Pharma Chemicals.
§ University of Padova, ITM.
| Present address: Department of Chemical Sciences, University of
Trieste, 34127 Trieste, Italy.
(1) (a) Hoveyda, A. H. Chem. Biol. 1999, 6, R305-R308. (b) J arvo,
E. R.; Miller, S. J . Tetrahedron 2002, 58, 2481-2495.
10.1021/jo040107v CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/01/2004
J . Org. Chem. 2004, 69, 3849-3856
3849

Formaggio et al.
sequence3 (Aib, being achiral, can explore backbone
torsion angles appropriate for either a D- or an L-residue).
specific R-amino amidase to achieve enantioselective
resolution.
The Cbz NR-protected R-amino acids were obtained by
reacting the free amino acid with the Cbz 1-oxysuccin-
imido ester7 in acetonitrile/water in the presence of
triethylamine (TEA) or in acetonitrile in the presence of
tetramethylammonium hydroxide. In the difficult cou-
pling steps between two sterically demanding residues
the EDC [1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide] method with the efficient additive HOAt (7-aza-
1-hydroxy-1,2,3-benzotriazole)8 in methylene chloride in
the presence of NMM (N-methylmorpholine) was used.
The reaction yields ranged from moderate to good.
Removal of the Cbz NR-protecting group was performed
by catalytic hydrogenation. Boc-L-His(π-Me)-OH was a
commercially available product. Before use we have
verified its identity and purity by mono- and bidimen-
sional NMR experiments.9
The aim of the present investigation was 2-fold: (i) As
subtle perturbations in the sequence of these small
peptide catalysts were shown to lead to dramatic differ-
ences in enantioselectivity,1 we decided to synthesize and
compare the conformational and catalytic properties of
compound 1′ with those of its tetrapeptide analogues 2-4
characterized at position 3 of the sequence by an Aib f
L-Iva (2) or an Aib f L-(RMe)Val (3) replacement or at
position 4 of the sequence by an L-Phe f L-(RMe)Phg (4)
replacement, respectively. (ii) As extremely short peptides
(e.g., dipeptides) are particularly attractive for industrial
applications, we also prepared and examined the termi-
nally protected dipeptide amides 5-10 (still long enough
to fold in an intramolecularly H-bonded â-turn conforma-
tion) by preserving the nucleophilic His(π-Me) residue
at position 1 followed by either an L- or a D-(RMe)Val,
(RMe)Phe or (RMe)Phg residue.
Iva, (RMe)Val, (RMe)Phe, and (RMe)Phg are CR-tetra-
substituted, chiral R-amino acids, members of the same
family the parent compound of which is Aib.4 They all
share with Aib the propensity for helical backbone torsion
angles,4d,e,5 appropriate for the i + 2 corner of a (more or
less distorted) â-turn. However, while L-(RMe)Val exhib-
its an extremely strong bias for right-handed helical
torsion angles, L-Iva, L-(RMe)Phe, and L-(RMe)Phg were
more or less frequently shown to fold in the left-handed
helical structure.
Initially, we discovered a substantial discrepancy
1
between the H NMR spectra of tetrapeptides 1 and 1′.
It involved the signal of the characteristic CH2 proton of
the His(π-Me) imidazole ring, which for 1 was seen at
8.48 ppm in CDCl3 solution, whereas it was reported at
7.40 for 1′ 2a,d (and tested on an authentic sample kindly
provided by Dr. S. J . Miller). It turned out that the factor
responsible for this anomalous behavior was the proto-
nation of the imidazole moiety that took place during our
synthetic protocol (peptide 1). Indeed, after deprotonation
of peptide 1 by treatment with TEA, its NMR spectrum
matched well that published for 1′. Therefore, before
testing the catalytic activity of our peptides 1-10, all of
them were treated with 3 equiv of TEA for 20 min, the
solutions were evaporated several times with acetonitrile,
and the peptides were liophilized.
Resu lts a n d Discu ssion
P ep tid e Syn th esis a n d Ch a r a cter iza tion . For the
large-scale production of the enantiomerically pure L-Iva,
L- and D-(RMe)Val, L- and D-(RMe)Phe, and L- and
D-(RMe)Phg an economically attractive and generally
applicable chemo-enzymatic synthesis was developed by
the DSM group a few years ago.6 It involves a combina-
tion of organic synthesis for the preparation of the
racemic R-amino amide followed by the use of a broadly
Solu tion Con for m a tion a l An a lysis. Among the tet-
rapeptides with a replacement at position 3 we have
investigated in detail the preferred conformation of the
most sterically hindered, prototypical, L-(RMe)Val-con-
taining 3 in the structure supporting solvent CDCl3 by
1
FT-IR absorption and H NMR techniques. In the FT-IR
absorption spectrum (amide A region) two intense bands
are seen (Figure 1A). From an inspection of the relative
areas of the free (3437 cm-1) to the broad H-bonded
(3363-3355 cm-1) N-H stretching band10 it is evident
that two NH groups of the peptide are H-bonded while
only one NH group is free (as the overall spectral pattern
does not change upon variation of peptide concentration
from 1.0 to 0.1 mM, we can safely assume that these
H-bonds are of the intramolecular type).
(2) (a) Miller, S. J .; Copeland, G. T.; Papaioannou, N.; Horstmann,
T. E.; Ruel, E. M. J . Am. Chem. Soc. 1998, 120, 1629-1630. (b)
Copeland, G. T.; J arvo, E. R.; Miller, S. J . J . Org. Chem. 1998, 63,
6784-6785. (c) Copeland, G. T.; Miller, S. J . J . Am. Chem. Soc. 1999,
121, 4306-4307. (d) J arvo, E. R.; Copeland, G. T.; Papaioannou, N.;
Bonitatebus, P. J ., J r.; Miller, S. J . J . Am. Chem. Soc. 1999, 121,
11638-11643. (e) Harris, R. F.; Nation, A. J .; Copeland, G. T.; Miller,
S. J . J . Am. Chem. Soc. 2000, 122, 11270-11271. (f) Blank, J . T.;
Guerin, D. J .; Miller, S. J . Org. Lett. 2000, 2, 1247-1249. (g) J arvo, E.
R.; Vasbinder, M. M.; Miller, S. J . Tetrahedron 2000, 56, 9773-9779.
(h) Vasbinder, M. M.; J arvo, E. R.; Miller, S. J . Angew. Chem., Int.
Ed. 2001, 40, 2824-2827. (i) Copeland, G. T.; Miller, S. J . J . Am. Chem.
Soc. 2001, 123, 6496-6502. (j) J arvo, E. R.; Evans, C. A.; Copeland,
G. T.; Miller, S. J . J . Org. Chem. 2001, 66, 5522-5527.
(6) (a) Kruizinga, W. H.; Bolster, J .; Kellogg, R. M.; Kamphuis, J .;
Boesten, W. H. J .; Meijer, E. M.; Schoemaker, H. E. J . Org. Chem.
1988, 53, 1826-1827. (b) Sonke, T.; Kaptein, B.; Boesten, W. H. J .;
Broxterman, Q. B.; Kamphuis, J .; Formaggio, F.; Toniolo, C.; Rutjes,
F. P. J . T.; Schoemaker, H. E. In Stereoselective Biocatalysis; Patel, R.
N., Ed.; Dekker: New York, 2000; pp 23-58.
(7) Frankel, M.; Ladkany, D.; Gilon, C.; Wolman, Y. Tetrahedron
Lett. 1966, 7, 4765-4768.
(8) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-4398.
(9) Serdijn, J .; Bloemhoff, W.; Kerling, K. E. T.; Havinga, E. Recl.
Trav. Chim. Pays-Bas 1984, 103, 50-54.
(10) (a) Mizushima, S.; Shimanouchi, T.; Tsuboi, M.; Souda, R. J .
Am. Chem. Soc. 1952, 74, 270-271. (b) Cung, M. T.; Marraud, M.; Ne´el,
J . Ann. Chim. (Paris) 1972, 7, 183-209. (c) Palumbo, M.; Da Rin, S.;
Bonora, G. M.; Toniolo, C. Makromol. Chem. 1976, 177, 1477-1492.
(d) Pulla Rao, C.; Nagaraj, R.; Rao, C. N. R.; Balaram, P. Biochemistry
1980, 19, 425-431. (e) Bonora, G. M.; Mapelli, C.; Toniolo, C.;
Wilkening, R. R.; Stevens, E. S. Int. J . Biol. Macromol. 1984, 6, 179-
188.
(3) (a) Venkatachalam, C. M. Biopolymers 1968, 6, 1425-1436. (b)
Toniolo, C. C. R. C. Crit. Rev. Biochem. 1980, 9, 1-44. (c) Rose, G. D.;
Gierasch, L. M.; Smith, J . A. Adv. Protein Chem. 1985, 37, 1-109. (d)
Haque, T. S.; Little, J . C.; Gellman, S. H. J . Am. Chem. Soc. 1996,
118, 6975-6985.
(4) (a) Marshall, G. R. In Intra-Science Chemistry Report; Kharasch,
N., Ed.; Gordon and Breach: New York, 1971; pp 305-316. (b) Karle,
I. L.; Balaram, P. Biochemistry 1990, 29, 6747-6756. (c) Toniolo, C.;
Benedetti, E. Macromolecules 1991, 24, 4004-4009. (d) Toniolo, C.;
Crisma, M.; Formaggio, F.; Valle, G.; Cavicchioni, G.; Pre´cigoux, G.;
Aubry, A.; Kamphuis, J . Biopolymers 1993, 33, 1061-1072. (e) Toniolo,
C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers (Pept. Sci.)
2001, 60, 396-419.
(5) Mossel, E.; Formaggio, F.; Valle, G.; Crisma, M.; Toniolo, C.; Doi,
M.; Ishida, T.; Broxterman, Q. B.; Kamphuis, J . Lett. Pept. Sci. 1998,
5, 223-225.
3850 J . Org. Chem., Vol. 69, No. 11, 2004

Enantioselective Peptide Catalysts
1
F IGURE 2. Plot of NH proton chemical shifts in the H NMR
spectra of peptide 3 as a function of increasing percentages of
DMSO (v/v) added to the CDCl3 solution (concentration: 1
mM).
F IGURE 3. Histogram showing the NH proton chemical shift
1
variations in the H NMR spectrum of peptide 3 as a function
of concentration (from 10 to 1 mM) in CDCl3 solution.
(RMe)Val N(3)H proton which exhibits a remarkable
downfield shift upon addition of DMSO; type ii includes
the [His(π-Me)]N(1)H and Phe N(4)H protons displaying
a behavior typical of shielded protons (absence or modest
sensitivity of chemical shift to solvent composition). In
Figure 3, the shifts experienced by the NH protons of
peptide 3 upon dilution from 10 to 1 mM concentration
are reported. Again, the chemical shift of the (RMe)Val
N(3)H proton is the most sensitive to the environmental
effect.
1
Taken together, these H NMR results agree well with
F IGURE 1. (Top) FT-IR absorption spectra (3500-3200 cm-1
region) and (bottom) inverted second derivative spectra of
peptides 3 (A), 4 (B), and 6 (C) in CDCl3 solution (concentra-
tion: 1 mM).
the FT-IR absorption data discussed above, allowing us
to conclude that in CDCl3 solution the terminally pro-
tected tetrapeptide 3 is highly folded in a conformation
stabilized by two intramolecular H-bonds involving the
His N(1)H and Phe N(3)H groups as donors. Interest-
ingly, the His N(1)H proton resonates at a unusually low
field (6.6 ppm) for an urethane NH proton in CDCl3. This
finding is in full agreement with the proposed involve-
ment of this NH group in a H-bond. In addition, the
partial ROESY spectrum shown in Figure 4 clearly
To obtain more detailed information on the conforma-
tional preference of 3 in CDCl3 solution we performed a
1
400 MHz H NMR analysis. The delineation of inacces-
sible (or intramolecularly H-bonded) NH groups was
carried out by monitoring the solvent dependence of NH
proton chemical shifts upon adding increasing amount
of the H-bonding acceptor solvent DMSO to the CDCl3
solution.11 In the titration experiment (Figure 2) two
types of NH protons were observed: type i includes the
(11) (a) Kopple, K. D.; Ohnishi, M.; Go, A. Biochemistry 1969, 8,
4087-4095. (b) Martin, D.; Hauthal, H. G. Dimethyl Sulphoxide; Van
Nostrand Reinhold: Wokingham, U.K., 1975.
J . Org. Chem, Vol. 69, No. 11, 2004 3851

Formaggio et al.
CHART 1
to a remarkable extent, but the occurrence of the ad-
ditional intramolecular H-bond stabilizing the â-hairpin
platform is much reduced.
Tetr a - a n d Dip ep tid es a s En a n tioselective Acy-
la tion Ca ta lysts. The peptides reported in Chart 1 were
used as catalysts in the model reaction (eq 1) using 5 mol
% of catalyst and 8.8 equiv of acetic anhydride in toluene
at 25 °C. The reaction course was monitored using GC
by sampling the reaction mixture at appropriate time
intervals. Each sample was quenched with methanol, and
subsequently, the extent of conversion, i.e. the concentra-
tions of products (both enantiomers) and reagents (both
enantiomers), was determined. The enantioselections
were calculated according to the method of Kagan12 (krel
) k(R,R)/k(S,S)). As the reactions are pseudo-first-order, the
ratio between the rate constants as well as the enantio-
meric excess do not depend on the extent of conversion.
Evaluation of racemic 2-acetamidocyclohexanol in the
asymmetric acetylation reaction in the presence of the
terminally protected tetrapeptide ester catalyst 1 (syn-
thesized in our laboratory) showed an excellent enanti-
oselection (krel ) 28) (Table 1), thereby exactly reproduc-
ing the published results of Miller et al.2a,d In our hands
the same peptide prepared in Miller’s laboratory and
shipped to us (1′) exhibited a modestly lower stereodif-
ferentiation (krel ) 22). The two other tetrapeptides
examined in this work with replacement at position 3 (2
and 3) are both significantly active. In particular, re-
placement of the Aib[3] residue with L-Iva (analogue 2),
with its linear but bulkier side chain, is characterized
by a selectivity (krel ) 23) slightly decreased as compared
to that of 1. In addition, the enhanced steric hindrance
slows down significantly (35-45%) the transacylation
reaction of both substrate enantiomers. The negative
reactivity trend is even more marked with analogue 3,
with its bulkier, â-branched L-(RMe)Val residue at posi-
tion 3. Interestingly, however, this analogue is more,
although not dramatically, selective (krel ) 33) than the
Aib-containing prototypical tetrapeptide 1.
F IGURE 4. Section of the ROESY spectrum of peptide 3 in
CDCl3 solution (concentration 1 mM). The arrow indicates the
[dRN(i, i + 1)] cross-peak typical of a type-II′ â-turn conforma-
tion.
F IGURE 5. Preferred conformation of peptide 3 in CDCl3
solution.
demonstrates the occurrence of a NOE interaction be-
tween the D-Pro RC(2)H and the (RMe)Val N(3)H protons,
strongly indicative of a type-II′ â-turn conformation. On
the basis of all these data, we therefore propose for
tetrapeptide 3 the â-hairpin structure depicted in Figure
5. It is gratifying that our conformational conclusions
match beautifully those published by Miller and co-
workers for the Aib[3] parent tetrapeptide 1′.2a,d
We have also extended our FT-IR absorption confor-
mational analysis in CDCl3 solution to tetrapeptide 4,
with a replacement at position 4 (Figure 1B), and to the
terminally protected dipeptide amides 5-10 (for a rep-
resentative example, the D-(RMe)Val[2] dipeptide amide
6, see Figure 1C). It is evident that in the L-(RMe)Phg[4]
tetrapeptide 4 as well as in the very short dipeptides,
despite the presence in the latter of a CR-tetrasubstituted
R-amino acid at position 2, the population of intramo-
lecularly H-bonded conformers is remarkably smaller
than that in tetrapeptide 3.
As compared to 1, replacement of the proteinogenic
L-Phe with the CR-tetrasubstituted L-(RMe)Phg residue
at position 4 (tetrapeptide 4) is accompanied not only by
a significantly decreased selectivity (krel ) 10) but by a
reduced reactivity as well. These results, together with
the decreased population of the double intramolecularly
H-bonded conformation for this peptide (see above),
indicate that the overall conformational constraint plays
a key role in the enantioselectivity of the catalyst.
In addition, the results of our mono- and two-dimen-
sional NMR analyses (in particular, the positions of the
NH proton chemical shifts and their variations upon
DMSO addition, and the NOE effects) on tetrapeptide 4
(not shown) unambiguously indicate that the intramo-
lecularly H-bonded type II′ â-turn conformer still forms
(12) Kagan, H. B.; Fiand, J . C. Top. Stereochem. 1988, 18, 249-
330.
3852 J . Org. Chem., Vol. 69, No. 11, 2004

Enantioselective Peptide Catalysts
TABLE 1. Selectivities in th e Kin etic Resolu tion s of th e Secon d a r y Alcoh ol (()-2-Aceta m id ocycloh exa n ol w ith
Ca ta lysts 1-10
a
a
catalyst
k
(R,R) (min-1
)
k
(S,S) (min-1
)
k
(R,R)/k(S,S) (krel)
1′
1
2
3
4
5
6
7
8
Boc-L-His(π-Me)-D-Pro-Aib-L-Phe-OMeb
Boc-L-His(π-Me)-D-Pro-Aib-L-Phe-OMec
Boc-L-His(π-Me)-D-Pro-L-Iva-L-Phe-OMe
Boc-L-His(π-Me)-D-Pro-L-(RMe)Val-L-Phe-OMe
Boc-L-His(π-Me)-D-Pro-Aib-L-(RMe)Phg-OMe
Boc-L-His(π-Me)-L-(RMe)Val-NHt-Bu
Boc-L-His(π-Me)-D-(RMe)Val-NHt-Bu
Boc-L-His(π-Me)-L-(RMe)Phe-NHt-Bu
Boc-L-His(π-Me)-D-(RMe)Phe-NHt-Bu
Boc-L-His(π-Me)-L-(RMe)Phg-NHt-Bu
Boc-L-His(π-Me)-D-(RMe)Phg-NHt-Bu
0.0470
0.0334
0.0184
0.0098
0.0142
0.0045
0.0018
0.0035
0.0028
0.0025
0.0034
0.0021
0.0012
0.0008
0.0003
0.0014
0.0019
0.0036
0.0029
0.0041
0.0019
0.0037
22
28
23
33
10
2.3
0.5
1.2
0.7
1.3
0.9
9
10
a
b
Average values of two or three independent experiments. Peptide synthesized in Dr. S. J . Miller’s laboratory. c Peptide synthesized
in our laboratory.
Finally, all terminally protected dipeptide amides
investigated (5-10) (Table 1) are very poorly efficient as
catalysts. Even more important is the observation that
all of them exhibit an extremely modest enantiomeric
selectivity. However, it is worth noting that all sets of
diastereomeric peptides (5 vs 6; 7 vs 8; 9 vs 10) show
opposite enantioselectivities.
N-H‚‚‚OdC(i + 3) intramolecular H-bond. These authors
also demonstrated that: (i) the Aib residue at position i
+ 2 of the â-turn can be replaced by other achiral
members of its class, e.g. CR,R-di-n-butylglycine or 1-ami-
nocyclohexane-1-carboxylic acid, with only a limited
detrimental effect on resolution efficiency,2j and (ii) the
Phe residue at position i + 3 of the â-turn cannot be
replaced by the CR-tetrasubstituted residues 1-amino-
cyclopentane-1-carboxylic acid or 1-aminocyclohexane-1-
carboxylic acid without an almost complete loss of
enantioselectivity.2j
Con clu sion
Incorporation of noncoded, CR-tetrasubstituted R-amino
acids4 into peptides drastically reduces their conforma-
tional freedom by stabilizing specific secondary structures
such as â-turns,3 and 310- and R-helices.13 Peptides based
on this class of residues may represent unique foldam-
ers14 and can be exploited as rigidified molecular spacers
and templates for the design of mini-receptors, spectro-
scopic and electrochemical molecular rulers, and cata-
lysts.15
Miller and co-workers2 recently expanded this field by
showing that terminally protected peptides can be used
as catalysts for the kinetic resolution of racemic alcohols.
As most of these peptides are characterized by an Aib
residue, the prototype of CR-tetrasubstituted R-amino
acids,4 they are strongly biased toward a â-turn confor-
mation in solution which decreases catalyst flexibility.
Indeed, all of the tripeptide amides and the tetra- and
pentapeptide esters examined showing catalytic activity
share the common feature of a â-hairpin conformation,
in which a type-II′ â-turn, generated by a -D-Pro-Aib-
(i + 1, i + 2) sequence, is further stabilized by an (i)
In the present work, we have further extended the
investigation on the Miller tetrapeptide 1′ by synthesiz-
ing and studying two tetrapeptide analogues (2 and 3)
with the achiral Aib residue replaced by a chiral amino
acid of its class with different side-chain bulkiness and
helical screw-sense propensity [L-(RMe)Val is remarkably
bulkier and more strongly biased toward the right-
handed helical structure, appropriate for the i + 2
position of a type-II′ â-turn, than L-Iva].4d,e We found that
the transacylation reactivity is very sensitive to the steric
hindrance of the residue at position i + 2, rapidly
decreasing from Aib to Iva and from Iva to (RMe)Val.
However, both analogues are remarkably stereoselective
with the L-(RMe)Val-based peptide catalyst 3 being even
more effective than 1 (or 1′). Also, our conformational
1
analysis by FT-IR absorption and H NMR lent credence
to the close correlation between catalyst stereoselection
efficiency and a â-hairpin, rigidified structure (for ana-
logue 3 see Figure 5), as proposed by Miller and co-
workers.2
In the capture of the acylating reagent by the His(π-
Me) nucleophilic side chain these authors invoked the
occurrence of two diastereomeric transition states of
diverging stability on the basis of differential H-bonding
interactions of alcohol substrate enantiomers with the
chiral environment generated by the highly folded tet-
rapeptide catalyst.2a,d In this connection, the important
role played by the Pro-Xxx secondary amide bond was
also demonstrated.2h Our findings on tetrapeptides 1-3
fit nicely into this reaction scheme and unambiguously
confirm that â-turn formation (and its stabilization via
â-hairpin) in the peptide catalyst is a prerequisite for
excellent reactivity and stereoselectivity and that en-
hanced steric hindrance at position i+2 of the â-turn is
not detrimental, or it might be even beneficial, for the
stereoselection governed by the ancillary H-bonding
(13) (a) Toniolo, C.; Benedetti, E. Trends Biochem. Sci. 1991, 16,
350-353. (b) Bolin, K. A.; Millhauser, G. L. Acc. Chem. Res. 1999, 32,
1027-1033. (c) Pal, L.; Basu, G.; Chakrabarti, P. Proteins: Struct.
Funct. Genet. 2002, 48, 571-579.
(14) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180.
(15) (a) Scarso, A.; Scheffer, U.; Go¨bel, M.; Broxterman, Q. B.;
Kaptein, B.; Formaggio, F.; Toniolo, C.; Scrimin, P. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 5144-5149. (b) Formaggio, F.; Bonchio, M.;
Crisma, M.; Peggion, C.; Mezzato, S.; Polese, A.; Barazza, A.; Antonello,
S.; Maran, F.; Broxterman, Q. B.; Kaptein, B.; Kamphuis, J .; Vitale,
R. M.; Saviano, M.; Benedetti, E.; Toniolo, C. Chem. Eur. J . 2002, 8,
85-93. (c) Bianco, A.; Corvaja, C.; Crisma, M.; Guldi, D. M.; Maggini,
M.; Sartori, E.; Toniolo, C. Chem. Eur. J . 2002, 8, 1544-1553. (d)
Pispisa, B.; Palleschi, A.; Stella, L.; Venanzi, M.; Mazzuca, C.;
Formaggio, F.; Toniolo, C.; Broxterman, Q. B. J . Phys. Chem. 2002,
B106, 5733-5738. (e) Hanson, P.; Millhauser, G. L.; Formaggio, F.;
Crisma, M.; Toniolo, C. J . Am. Chem. Soc. 1996, 118, 7618-7625. (f)
Antonello, S.; Formaggio, F.; Moretto, A.; Toniolo, C.; Maran, F. J . Am.
Chem. Soc. 2003, 125, 2874-2875. (g) Voyer, N.; Lamothe, J . Tetra-
hedron 1995, 51, 9241-9284. (h) Andrews, M. J . I.; Tabor, A. B.
Tetrahedron 1999, 55, 11711-11743.
J . Org. Chem, Vol. 69, No. 11, 2004 3853

Formaggio et al.
calibration. Melting points are uncorrected. Analytical thin-
layer chromatography (TLC) was performed by using silica gel
plates in the following solvent systems: (I) chloroform-EtOH
9:1; (II) 1-BuOH-AcOH-water 6:2:2; (III) toluene-EtOH 7:1.
The compounds were revealed either with the aid of a UV lamp
or with the hypochlorite-starch-iodide chromatic reaction (a
single spot was observed in each case). The solid-state FT-IR
absorption spectra were recorded in KBr pellets. Unless
1
otherwise specified, all H NMR spectra were recorded at 250
MHz in CDCl3 or in CDCl3-DMSO-d6 solutions. Chemical
shifts are reported in ppm (δ units) downfield of internal TMS.
Gen er a l P r oced u r e for th e Syn th esis of Nr-P r otected
r-Am in oa cyl ter t-Bu tyla m id es. To a solution of the Cbz NR-
protected R-amino acid (4.15 mmol) in CH2Cl2 (30 mL), cooled
to 0 °C, was added EDC‚HCl (4.18 mmol). After 15 min, NH2
t-Bu (20.9 mmol) was added, and the solution was stirred at
rt for 4 d. Then, the solvent was removed in vacuo and the
residue dissolved in EtOAc. The solution was washed with 10%
KHSO4, water, 5% NaHCO3, and water, dried over Na2SO4,
and evaporated to dryness. The product was crystallized from
EtOAc-PE (petroleum ether).
F IGURE 6. Transition state proposed for the fast reacting
enantiomer (a ) and the conformer obtained by rotation of the
CR-C′ bond of the L-His(π-Me) residue in the presence of a
weaker H-bonding motif (b).
interaction between the peptide catalyst and the racemic
alcohol substrate (Figure 6a).
Furthermore, our results on tetrapeptide 4 indirectly
support the contention that a â-hairpin template (not just
a â-turn template) is strictly required for a peptide
catalyst to exhibit significantly different rates in the
acylation of the alcohol enantiomers. Indeed, as already
reported by J arvo et al.,2j incorporation at position 4 of
the â-turn of a CR-tetrasubstituted R-amino acid, e.g.
L-(RMe)Phg, with its propensity for a more or less
extended structure, weaker than that of L-Phe,4e,5 seems
to reduce the population of conformers with the [His(π-
Me)]N-H‚‚‚OdC(Phe) intramolecular H-bond, thereby
enhancing their conformational freedom and concomi-
tantly decreasing peptide catalyst stereoselectivity. A
weaker H-bonding stabilizing motif implies the existence
of an equilibrium where a significant population of a
conformer (Figure 6b) is present in which the catalytic
subunit does not reside on the same face of the recogni-
tion site for the substrate.
In search of a shorter, industrially more attractive,
peptide catalyst of this asymmetric acylation reaction we
have also synthesized and investigated the terminally
protected dipeptide amides 5-10. In these compounds,
long enough to generate an intramolecularly H-bonded
â-turn but too short for â-hairpin formation, the nucleo-
philic His(π-Me) residue at position 1 is followed by a
chiral CR-tetrasubstituted R-amino acid. Again, we have
noted a close relationship between peptide catalyst
conformation and stereoselectivity in that, unfortunately,
the population of â-turn conformers is quite limited in
these dipeptide amides and their stereoselectivity is poor.
Taken together, our experimental findings underscore
the points that a rigid, â-hairpin secondary structure is
an element of paramount importance characterizing the
platform required by a peptide to be an efficient stereo-
selective catalyst and that, in this connection, chiral CR-
tetrasubstituted R-amino acids may play a fundamental
role.
Cbz-L-(rMe)Va l-NH t-Bu : yield 81%; mp 116-117 °C;
[R]20 8.1 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.50; IR
D
(KBr) νmax 3418, 3299, 1719, 1660, 1536 cm-1; 1H NMR (CDCl3)
δ 7.34 (5H, m), 6.26 (1H, s), 5.33 (1H, s), 5.09 (2H, m), 2.30
(1H, m), 1.42 (3H, s), 1.30 (9H, s), 0.91 (6H, m); MS (ESI-TOF)
m/z calcd for C18H29N2O3 321.2173, found 321.2210 [M + H]+.
Cbz-D-(rMe)Va l-NH t-Bu : yield 85%; mp 115-117 °C;
[R]20 -8.1 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.50;
D
IR (KBr) νmax 3418, 3299, 1718, 1659, 1535 cm-1
;
1H NMR
(CDCl3) δ 7.34 (5H, m), 6.26 (1H, s), 5.32 (1H, s), 5.09 (2H,
m), 2.29 (1H, m), 1.42 (3H, s), 1.30 (9H, s), 0.91 (6H, m); MS
(ESI-TOF) m/z calcd for C18H29N2O3 321.2173, found 321.2201
[M + H]+.
Cbz-L-(rMe)P h e-NH t-Bu : yield 63%; mp 119-120 °C;
[R]20 -51.2 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.65;
D
IR (KBr) νmax 3282, 1722, 1664, 1658, 1542, 1514 cm-1
;
1H
NMR (CDCl3) δ 7.40-7.08 (10H, m), 5.83 (1H, s), 5.27 (1H, s),
5.14 (2H, s), 3.27 (1H, d, J 13.8 Hz), 3.13 (1H, d, J 13.8 Hz),
1.48 (3H, s), 1.31 (9H, s); MS (ESI-TOF) m/z calcd for
C
22H29N2O3 369.2173, found 369.2201 [M + H]+.
Cbz-D-(rMe)P h e-NH t-Bu : yield 68%; mp 120-121 °C;
[R]20 52.8 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.65;
D
IR (KBr) νmax 3280, 1721, 1659, 1540, 1516 cm-1 1H NMR
;
(CDCl3) δ 7.36-7.07 (10H, m), 5.82 (1H, s), 5.27 (1H, s), 5.12
(2H, s), 3.27 (1H, d, J 13.8 Hz), 3.13 (1H, d, J 13.8 Hz), 1.48
(3H, s), 1.28 (9H, s); MS (ESI-TOF) m/z calcd for C22H29N2O3
369.2173, found 369.2240 [M + H]+.
Cbz-L-(rMe)P h g-NH t-Bu : yield 75%; mp 103-104 °C;
[R]20 15.8 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.65;
D
IR (KBr) νmax 3260, 1719, 1670, 1527 cm-1; H NMR (CDCl3)
1
δ 7.37-7.21 (10H, m), 6.57 (1H, s), 5.24 (1H, s), 4.96 (2H, m),
1.85 (3H, s), 1.16 (9H, s); MS (ESI-TOF) m/z calcd for
C
21H27N2O3 355.2016, found 355.2055 [M + H]+.
Cbz-D-(rMe)P h g-NH t-Bu : yield 84%; mp 104-105 °C;
[R]20 -14.7 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.65;
D
IR (KBr) νmax 3258, 1717, 1668, 1526 cm-1; H NMR (CDCl3)
1
δ 7.37-7.21 (10H, m), 6.55 (1H, s), 5.23 (1H, s), 4.96 (2H, m),
1.85 (3H, s), 1.16 (9H, s); MS (ESI-TOF) m/z calcd for
C
21H27N2O3 355.2016, found 355.2062 [M + H]+.
Gen er a l P r oced u r e for th e Syn th esis of Nr-P r otected
P ep tid yl Meth yl Ester s a n d ter t-Bu tyla m id es. To a solu-
tion of Cbz (or Boc) NR-protected R-amino acid (4.45 mmol) in
CH2Cl2 (30 mL), cooled to 0 °C, were added HOAt (5.56 mmol)
and EDC‚HCl (5.56 mmol). After 10 min, the C-protected (or
tert-butylamide) R-amino acid or peptide hydrochloride (5.56
Exp er im en ta l Section
Gen er a l P r oced u r es. General methods for the synthesis
of peptides containing CR-tetrasubstituted R-amino acids were
as described previously.16 High-resolution mass spectra were
obtained by electrospray ionization (ESI) on a Perseptive
Biosystems Mariner API-TOF spectrometer; a 1 nM solution
of neurotensin, angiotensin I and bradykinin in acetonitrile/
water 1:1 mixture, containing 1% formic acid, was used for
(16) (a) Formaggio, F.; Broxterman, Q. B.; Toniolo, C. In Houben-
Weyl, Methods of Organic Synthesis; Goodman, M., Felix, P., Moroder,
L., Toniolo, C., Eds.; Thieme: Stuttgart, Germany, 2003; Vol. E22c,
pp 292-310. (b) Peggion, C.; Fiammengo, R.; Mossel, E.; Broxterman,
Q. B.; Kaptein, B.; Kamphuis, J .; Formaggio, F.; Crisma, M.; Toniolo,
C. Tetrahedron 2000, 56, 3589-3601.
3854 J . Org. Chem., Vol. 69, No. 11, 2004

Enantioselective Peptide Catalysts
mmol) and NMM (11.13 mmol) were added and the solution
was stirred at rt for 3 d. Then, the solvent was removed in
vacuo and the residue dissolved in EtOAc. The solution was
washed with 10% KHSO4, water, 5% NaHCO3, and water,
dried over Na2SO4, and evaporated to dryness.
Cbz-D-P r o-L-(rMe)Va l-L-P h e-OMe: yield 89%; mp 85-86
°C (EtOAc-PE); [R]20 23.2 (c 0.5, MeOH); Rf(I) 0.95, Rf(II)
D
0.95, Rf(III) 0.40; IR (KBr) νmax 3321, 1734, 1691, 1660, 1540
cm-1; 1H NMR (CDCl3) δ 7.35-7.18 (11H, m), 6.86 (1H, s), 5.13
(2H, s), 4.84 (1H, m), 4.25 (1H, m), 3.68 (3H, s), 3.67-3.49 (2H,
m), 3.14 (2H, m), 2.27-1.77 (5H, m), 1.43 (3H, s), 0.74 (3H, d,
J 6.9 Hz), 0.68 (3H, d, J 6.0 Hz); MS (ESI-TOF) m/z calcd for
Cbz-Aib-L-P h e-OMe (lit.17a): yield 89%; mp 93-94 °C
(EtOAc-PE); [R]20 2.0 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95,
D
Rf(III) 0.65; IR (KBr) νmax 3425, 1740, 1661, 1538 cm-1
;
1H
C
29H38N3O6 524.2756, found 524.2794 [M + H]+.
NMR (CDCl3) δ 7.35-7.07 (10H, m), 6.63 (1H, br d), 5.24 (1H,
s), 5.07 (2H, s), 4.82 (1H, m), 3.71 (3H, s), 3.10 (2H, m), 1.47
(6H, s); MS (ESI-TOF) m/z calcd for C22H27N2O5 399.1914,
found 399.1959 [M + H]+.
Boc-L-His(π-Me)-D-P r o-L-(rMe)Va l-L-P h e-OMe (3): yield
47% [after purification by flash chromatography (9:1, CH2Cl2-
EtOH) followed by reversed-phase (prepacked C18 cartridge)
chromatography (CH3CN-H2O gradient)]; mp 93-94 °C; [R]20
D
Cbz-D-P r o-Aib-L-P h e-OMe: yield 71%; mp 65-70 °C (waxy
-34.0 (c 0.5, MeOH); Rf(I) 0.65, Rf(II) 0.60, Rf(III) 0.10; IR
solid); [R]20 29.4 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III)
1
(KBr) νmax 3350, 1743, 1684, 1639, 1516 cm-1; H NMR (400
D
0.35; IR (KBr) νmax 3325, 1745, 1682, 1535 cm-1
;
1H NMR
MHz, CDCl3) δ 7.49 (1H, s), 7.31-7.19 (6H, m), 7.07 (1H, d, J
8.7 Hz), 6.63 (1H, d, J 8.8 Hz), 6.42 (1H, s), 4.97 (1H, m), 4.53
(1H, m), 4.25 (1H, m), 3.96 (3H, s), 3.72 (3H, s), 3.61-3.49 (2H,
m), 3.25-2.83 (4H, m), 2.11-1.87 (5H, m), 1.45 (3H, s), 1.41
(9H, s), 0.84 (3H, d, J 6.8 Hz), 0.73 (3H, d, J 6.8 Hz); MS (ESI-
TOF) m/z calcd for C33H49N6O7 641.3657, found 641.3705 [M
+ H]+.
(CDCl3) δ 7.34-7.12 (11H, m), 6.84 (1H, br d), 5.13 (2H, m),
4.80 (1H, m), 4.18 (1H, m), 3.68 (3H, s), 3.52 (2H, m), 3.11
(2H, m), 2.24-1.86 (4H, m), 1.45 (3H, s), 1.36 (3H, s); MS (ESI-
TOF) m/z calcd for C27H34N3O6 496.2442, found 496.2489 [M
+ H]+.
Boc-L-His(π-Me)-D-P r o-Aib-L-P h e-OMe (1) (lit.2a,d): yield
72% [after purification by flash chromatography (95:5, CH2-
Cbz-Aib-L-(rMe)P h g-OMe: yield 81% [after purification by
flash chromatography (99:1, CH2Cl2-EtOH)]; oil; [R]20D +29.8
(c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.50; IR (film) νmax
3312, 1729, 1676, 1514 cm-1; 1H NMR (CDCl3) δ 7.53 (1H, s),
7.42-7.26 (10H, m), 5.35 (1H, s), 5.10 (2H, s), 3.68 (3H, s),
1.98 (1H, s), 1.53 (6H, s); MS (ESI-TOF) m/z calcd for
Cl2-EtOH)]; mp 100-103 °C (waxy solid); [R]20 -28.4 (c 0.5,
D
MeOH); Rf(I) 0.25, Rf(II) 0.55, Rf(III) 0.05; IR (KBr) νmax 3338,
1
1743, 1679, 1639, 1511 cm-1. H NMR (CDCl3) δ 7.39 (1H, s),
7.29-7.15 (7H, m), 6.69 (1H, s), 6.52 (1H, s), 6.01 (1H, d, J
8.0 Hz), 4.90 (1H, m), 4.54 (1H, m), 4.22 (1H, m), 3.88 (3H, s),
3.69 (3H, s), 3.61-3.51 (2H, m), 3.20-2.91 (4H, m), 2.13-2.04
(4H, m), 1.52 (3H, s), 1.41 (9H, s), 1.33 (3H, s); MS (ESI-TOF)
m/z calcd for C31H45N6O7 613.3344, found 613.3303 [M + H]+.
Cbz-L-Iva -L-P h e-OMe (lit.17b): yield 88%; mp 95-96 °C
(EtOAc-PE); [R]20D -15.6 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95,
Rf(III) 0.55; IR (KBr) νmax 3329, 3319, 1751, 1692, 1662, 1537,
C
22H27N2O5 399.1914, found 399.1958 [M + H]+.
Cbz-D-P r o-Aib-L-(rMe)P h g-OMe: yield 82% [after puri-
fication by flash chromatography (98:2, CH2Cl2-EtOH)]; mp
57-58 °C (EtOAc-PE); [R]20D +52.8 (c 0.5, MeOH); Rf(I) 0.95,
Rf(II) 0.90, Rf(III) 0.35; IR (KBr) νmax 3318, 1742, 1686, 1515
cm-1; 1H NMR (CDCl3) δ 7.67 (1H, s), 7.44-7.25 (10H, m), 7.01
(1H, s), 5.05 (2H, s), 4.25 (1H, m), 4.25 (1H, m), 3.66 (3H, s),
3.59-3.36 (2H, m), 2.26-1.79 (4H, m), 1.97 (3H, s), 1.50 (3H,
s), 1.46 (3H, s); MS (ESI-TOF) m/z calcd for C27H34N3O6
496.2442, found 496.2501 [M + H]+.
1
1521 cm-1; H NMR (CDCl3) δ 7.34-7.08 (10H, m), 6.45 (1H,
d, J 7.2 Hz), 5.56 (1H, s), 5.06 (2H, s), 4.87 (1H, m), 3.71 (3H,
s), 3.11 (2H, m), 1.68 (2H, m), 1.44 (3H, s), 0.76 (3H, t, J 7.1
Hz); MS (ESI-TOF) m/z calcd for C23H29N2O5 413.2071, found
413.2121 [M + H]+.
Boc-L-His(π-Me)-D-P r o-Aib-L-(rMe)P h g-OMe (4): yield
39% [after purification by flash chromatography (94:6, CH2-
Cl2-EtOH) followed by reversed-phase (prepacked C18 car-
tridge) chromatography (CH3CN-H2O gradient)]; mp 103-104
°C; [R]20D +6.6 (c 0.5, MeOH); Rf(I) 0.35, Rf(II) 0.55, Rf(III) 0.05;
Cbz-D-P r o-L-Iva -L-P h e-OMe: yield 89% [after purification
by flash chromatography] (98:2, CH2Cl2-EtOH)]; mp 73-74
°C; [R]20D 21.8 (c 0.5, MeOH); Rf(I) 0.95, Rf(II) 0.95, Rf(III) 0.40;
1
IR (KBr) νmax 3332, 1746, 1684, 1500 cm-1; H NMR (CDCl3)
IR (KBr) νmax 3402, 3346, 1738, 1679, 1642, 1509 cm-1 1H
;
δ 7.34-7.12 (10H, m), 6.94 (1H, s), 6.87 (1H, d, J 5.9 Hz), 5.12
(2H, s), 4.83 (1H, m), 4.22 (1H, m), 3.69 (3H, s), 3.55-3.38 (2H,
m), 3.18 (1H, dd, J 5.7, 14.0 Hz), 3.04 (1H, dd, J 6.7, 14.0 Hz),
2.32-1.76 (6H, m), 1.43 (3H, s), 0.65 (3H, br t); MS (ESI-TOF)
m/z calcd for C28H36N3O6 510.2598, found 510.2637 [M + H]+.
Boc-L-His(π-Me)-D-P r o-L-Iva -L-P h e-OMe (2): yield 61%
[after purification by flash chromatography (93:7, CH2Cl2-
NMR (400 MHz, CDCl3) δ 7.63 (1H, s), 7.48-7.24 (6H, m), 7.18
(1H, s), 7.06 (1H, s), 5.93 (1H, d, J 5.0 Hz), 4.47 (1H, m), 4.35
(1H, m), 3.76 (3H, s), 3.69 (3H, s), 3.60-3.38 (2H, m), 3.05-
2.80 (2H, m), 2.08-1.87 (4H, m), 2.02 (3H, s), 1.57 (3H, s), 1.50
(3H, s), 1.42 (9H, s); MS (ESI-TOF) m/z calcd for C31H45N6O7
613.3344, found 613.3338 [M + H]+.
EtOH)]; mp 67-68 °C; [R]20 -25.6 (c 0.5, MeOH); Rf(I) 0.40,
D
Boc-L-His(π-Me)-L-(rMe)Va l-NH t-Bu (5): yield 63% [after
Rf(II) 0.25, Rf(III) 0.05; IR (KBr) νmax 3336, 1745, 1688, 1639,
purification by flash chromatography (95:5, CH2Cl2-MeOH)];
1507 cm-1 1H NMR (CDCl3) δ 7.46 (1H, s), 7.21-7.12 (6H,
;
mp 192-193 °C; [R]20 -23.6 (c 0.5, MeOH); Rf(I) 0.40, Rf(II)
D
m), 6.76 (1H, s), 6.69 (1H, s), 6.04 (1H, d, J 7.1 Hz), 4.81 (1H,
m), 4.51 (1H, m), 4.04 (1H, m), 3.62 (3H, s), 3.59 (3H, s), 3.41-
3.36 (2H, m), 3.14-2.75 (4H, m), 2.02-1.52 (6H, m), 1.38 (3H,
s), 1.34 (9H, s), 0.58 (3H, t, J 7.2 Hz); MS (ESI-TOF) m/z calcd
for C32H47N6O7 627.3501, found 627.3548 [M + H]+.
0.35, Rf(III) 0.10; IR (KBr) νmax 3337, 1703, 1675, 1523 cm-1
;
1H NMR (CDCl3) δ 7.45 (1H, s), 6.89 (1H, s), 6.57 (1H, s), 6.32
(1H, s), 5.03 (1H, d, J 5.5 Hz), 4.15 (1H, m), 3.62 (3H, s), 3.11
(1H, dd, J 5.7, 16.0 Hz), 2.96 (1H, dd, J 7.8, 16.0 Hz), 2.14
(1H, m), 1.66 (3H, s), 1.44 (9H, s), 1.33 (9H, s), 0.88 (3H, d, J
4.4 Hz), 0.86 (3H, d, J 4.4 Hz); MS (ESI-TOF) m/z calcd for
Cbz-L-(rMe)Va l-L-P h e-OMe (lit.17b): yield 88%; mp 98-
99 °C (CHCl3-PE); [R]20 -10.0 (c 0.5, MeOH); Rf(I) 0.95, Rf-
D
C
22H40N5O4 438.3075, found 438.3022 [M + H]+.
(II) 0.95, Rf(III) 0.55; IR (KBr) νmax 3402, 3303, 1746, 1721,
1665, 1539 cm-1. 1H NMR (CDCl3) δ 7.35-7.12 (10H, m), 6.85
(1H, br d), 5.22 (1H, s), 5.07 (2H, s), 4.87 (1H, m), 3.69 (3H, s),
3.15 (1H, dd, J 5.8, 14.3 Hz), 3.06 (1H, dd, J 7.1, 14.3 Hz),
2.12 (1H, m), 1.41 (3H, s), 0.87 (3H, d, J 6.8 Hz), 0.77 (3H, d,
J 6.8 Hz); MS (ESI-TOF) m/z calcd for C24H31N2O5 427.2227,
found 427.2252 [M + H]+.
Boc-L-His(π-Me)-D-(rMe)Va l-NH t-Bu (6): yield 41% [af-
ter purification by flash chromatography (95:5, CH2Cl2-
MeOH)]; mp 81-82 °C; [R]20 -2.0 (c 0.5, MeOH); Rf(I) 0.40,
D
Rf(II) 0.35, Rf(III) 0.10; IR (KBr) νmax 3355, 3314, 1686, 1511
1
cm-1; H NMR (CDCl3) δ 7.49 (1H, s), 6.84 (1H, s), 6.63 (1H,
s), 6.35 (1H, s), 5.27 (1H, d, J 5.1 Hz), 4.15 (1H, m), 3.59 (3H,
s), 3.04 (2H, m), 2.25 (1H, m), 1.42 (9H, s), 1.38 (3H, s), 1.31
(9H, s), 0.84 (6H, m); MS (ESI-TOF) m/z calcd for C22H40N5O4
438.3075, found 438.3046 [M + H]+.
(17) (a) Toniolo, C.; Crisma, M.; Valle, G.; Bonora, G. M.; Polinelli,
S.; Becker, E. L.; Freer, R. J .; Sudhanand; Balaji Rao, R.; Balaram,
P.; Sukumar, M. Pept. Res. 1989, 2, 275-281. (b) Formaggio, F.;
Pantano, M.; Crisma, M.; Toniolo, C.; Boesten, W. H. J .; Schoemaker,
H. E.; Kamphuis, J .; Becker, E. L. Bioorg. Med. Chem. Lett. 1993, 3,
953-956.
Boc-L-His(π-Me)-L-(rMe)P h e-NH t-Bu (7): yield 77%
[after purification by flash chromatography (9:1, CH2Cl2-
EtOH)]; mp 159-160 °C; [R]20D -55.8 (c 0.5, MeOH); Rf(I) 0.50,
Rf(II) 0.60, Rf(III) 0.10; IR (KBr) νmax 3415, 3283, 1691, 1657,
J . Org. Chem, Vol. 69, No. 11, 2004 3855

Formaggio et al.
1508 cm-1
;
1H NMR (CDCl3) δ 7.43 (1H, s), 7.27-7.25 (3H,
50 °C. When all reagent was dissolved, 100 µL of a 16.3 nM
stock solution of catalyst (0.0016 mmol of peptide) were added
at 25 °C. After 20 min, acetic anhydride (25 µL, 0.265 mmol,
8.8 equiv) was introduced. The reaction mixture was allowed
to stir at 25 °C. Aliquots of 50 µL were removed, quenched
with methanol, and directly tested by GC analysis as described
below.
Assa y of Con ver sion . The extent of reagent conversion
was determined using a Hewlett-Packard model 5890 gas
-chromatograph equipped with a 15 m wide bore (ØID ) 0.53
mm) AT-1701 column (Alltech). Conditions: isotherm temper-
ature, 150 °C; flow rate, 1 mL/min. Retention times: 15 min
(products), 18 min (reagents). Conversions were determined
in analogy to the method reported by Miller and co-workers.2a,d
Assa y of En a n tiom er ic P u r ity. Enantiomers of starting
material and product were separated by chiral GC employing
a Shimadzu model GC-14B gas chromatograph equipped with
a 30 m Chiraldex G-TA capillary column (Alltech). Condi-
tions: flow rate, 120 kPA; initial temperature, 128 °C (0-26
min); temperature ramp, 1 °C/min; final temperature, 135 °C.
Reaction times: 22 min [(R,R) product], 24 min [(S,S) product],
41 min [(R,R) reagent], 45 min [(S,S) reagent]. krel ) s
(selectivity) values were calculated according to the method
of Kagan.12
m), 7.10-7.07 (2H, m), 6.82 (1H, s), 6.79 (1H, s), 5.86 (1H, s),
4.99 (1H, d, J 3.9 Hz), 4.10 (1H, m), 3.59 (3H, s), 3.24 and
3.12 (2H, 2d, both J 13.6 Hz), 3.10 (1H, dd, J 6.0, 14.4 Hz),
2.91 (1H, dd, J 7.9, 14.4 Hz), 1.55 (3H, s), 1.38 (9H, s), 1.30
(9H, s); MS (ESI-TOF) m/z calcd for C26H40N5O4 486.3075,
found 486.3109 [M + H]+.
Boc-L-His(π-Me)-D-(rMe)P h e-NH t-Bu (8): yield 52%
[after purification by flash chromatography (9:1, CH2Cl2-
EtOH)]; oil; [R]20 -4.6 (c 0.5, MeOH); Rf(I) 0.50, Rf(II) 0.60,
D
Rf(III) 0.10; IR (KBr) νmax 3332, 3315, 1680, 1506 cm-1
;
1H
NMR (CDCl3) δ 7.38 (1H, s), 7.25-7.23 (3H, m), 7.10-7.07 (2H,
m), 6.78 (1H, s), 6.63 (1H, s), 6.06 (1H, s), 5.10 (1H, br s), 4.09
(1H, m), 3.58 (3H, s), 3.35 (1H, d, J 13.7 Hz), 3.08 (1H, d, J
13.7 Hz), 3.06 (1H, dd, J 6.0, 14.4 Hz), 2.84 (1H, dd, J 7.9,
14.4 Hz), 1.47 (3H, s), 1.37 (9H, s), 1.29 (9H, s); MS (ESI-TOF)
m/z calcd for C26H40N5O4 486.3075, found 486.3072 [M + H]+.
Boc-L-His(π-Me)-L-(rMe)P h g-NH t-Bu (9): yield 64%
[after purification by flash chromatography (9:1, CH2Cl2-
EtOH)]; oil; [R]20 -5.2 (c 0.5, MeOH); Rf(I) 0.55, Rf(II) 0.60,
D
Rf(III) 0.10; IR (KBr) νmax 3415, 3357, 1677, 1507 cm-1
;
1H
NMR (CDCl3) δ 7.88 (1H, s), 7.36-7.27 (7H, m), 6.83 (1H, s),
5.17 (1H, s), 4.30 (1H, m), 3.44 (3H, s), 2.99 (2H, d, J 6.5 Hz),
1.86 (3H, s), 1.44 (9H, s), 1.20 (9H, s); MS (ESI-TOF) m/z calcd
for C25H38N5O4 472.2918, found 472.2967 [M + H]+.
Boc-L-His(π-Me)-D-(rMe)P h g-NH t-Bu (10): yield 56%
[after purification by flash chromatography (9:1, CH2Cl2-
Ack n ow led gm en t. We are extremely grateful to Dr.
S. J . Miller (Boston College, Chestnut Hill, MA) for
providing us with a sample of peptide 1′ and for helpful
discussions. We are also indebted to Dr. Daniele Dal-
zoppo and Dr. Barbara Biondi for mass spectral analy-
ses.
EtOH)]; oil; [R]20 -26.6 (c 0.5, MeOH); Rf(I) 0.55, Rf(II) 0.60,
D
Rf(III) 0.10; IR (KBr) νmax 3416, 3358, 1685, 1680, 1506 cm-1
.
1H NMR (CDCl3) δ 7.80 (1H, s), 7.43 (1H, s), 7.36-7.24 (5H,
m), 6.78 (1H, s), 5.20 (1H, s), 5.15 (1H, d, J 8.4 Hz), 4.25 (1H,
m), 3.46 (3H, s), 2.96 (2H, m), 1.86 (3H, s), 1.44 (9H, s), 1.20
(9H, s); MS (ESI-TOF) m/z calcd for C25H38N5O4 472.2918,
found 472.2971 [M + H]+.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
all synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
Sta n d a r d Con d ition s for Resolu tion of th e Ra cem ic
Alcoh ol Su bstr a te. In a thermostated reaction vessel (()-
trans-2-acetamidocyclohexanol (5.0 mg, 0.03 mmol) was dis-
solved in 3.1 mL of toluene (distilled from Na) by heating at
J O040107V
3856 J . Org. Chem., Vol. 69, No. 11, 2004