(αMe)Hyv: Chemo-enzymatic synthesis, and preparation and preferred conformation of model depsipeptides

Article abstract of DOI:10.1039/b107691b

By a chemo-enzymatic approach we performed a large-scale, stereoselective synthesis of the C^α-methylated α-hydroxy acid L-(αMe)Hyv. We also prepared model depsipeptides based on this sterically demanding residue in combination with the α-amino

Full text of DOI:10.1039/b107691b

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(ꢀMe)Hyv: chemo-enzymatic synthesis, and preparation and
preferred conformation of model depsipeptides†
Cristina Peggion,a Alessandra Barazza,a Fernando Formaggio,a Marco Crisma,a
Claudio Toniolo,*a Marzia Villa,b Claudia Tomasini,b Herbert Mayrhofer,c Peter Pöchlauer,c
Bernard Kapteind and Quirinus B. Broxtermand
2
a Biopolymer Research Centre, CNR, Department of Organic Chemistry, University of Padova,
35131 Padova, Italy. E-mail: claudio.toniolo@unipd.it; Fax: (ϩ39)049-827-5239;
Tel: (ϩ39)049-827-5247
b Department of Chemistry “G. Ciamician”, University of Bologna, 40126 Bologna, Italy
c DSM Fine Chemicals Austria, P.O. Box 933, 4021 Linz, Austria
d DSM Fine Chemicals, Advanced Synthesis and Catalysis, P.O. Box 18, 6160 MD Geleen,
The Netherlands
Received (in Cambridge, UK) 24th August 2001, Accepted 3rd January 2002
First published as an Advance Article on the web 29th January 2002
By a chemo-enzymatic approach we performed a large-scale, stereoselective synthesis of the Cα-methylated α-hydroxy
acid -(αMe)Hyv. We also prepared model depsipeptides based on this sterically demanding residue in combination
with the α-amino acids -Ala, -Val, and Aib. From solution (FT-IR absorption and 1H NMR) and crystal-state
(X-ray diffraction) conformational analyses we found that -(αMe)Hyv forces depsipeptides to fold into right-handed
β-turn/helical structures by analogy with the reported propensity of -(αMe)Val, its α-amino acid counterpart.
(a variant of the 310-helix) characterized by the (Aib-Hib)n
Introduction
sequence, where Hib (α-hydroxyisobutyric acid) is the rigid,
Increasing effort is currently being devoted to the synthesis and
Cα-methylated α-hydroxy acid related to Aib.8,9
conformational analysis of a variety of cyclic and linear com-
In this paper we describe a large-scale, chemo-enzymatic
pounds characterized by the presence of α-hydroxy acids. More
synthesis of - (or S)-(αMe)Hyv (Cα-methyl, Cα-hydroxyiso-
specifically, these compounds include oligo- and poly-esters as
valeric acid or 2-hydroxy-2,3-dimethylbutanoic acid),30–32 the
biodegradable and biocompatible materials,1–3 and depsipep-
α-hydroxy analogue of (αMe)Val and the second member of the
tides and depsiproteins (in which amide and ester groups are
family of 3D-structurally restricted, Cα-methylated α-hydroxy
concomitantly present in the main chain) to mimic naturally-
acids, the preferred conformation of which has already been
occurring ion carriers4,5 or to check the influence of specific
investigated. We have incorporated (αMe)Hyv either in position
H-bonds on peptide or protein bioactivity and conformation
1 or in an internal position of a large set of depsipeptides using
with little effect on other structural parameters.6–16
specific solution methods which allowed us to overcome the
severe problems generated by its extreme steric bulkiness. A
solution and crystal-state conformational investigation clearly
showed that -(αMe)Hyv, in analogy to its α-amino counter-
part -(αMe)Val, supports right-handed β-turns and helical
structures of depsipeptides.
Experimental
Chemo-enzymatic synthesis of L-(S)-ꢀ-hydroxyisovaleric acid
13.8 g (160 mmol) of isopropyl methyl ketone were dissolved in
48 mL of methyl tert-butyl ether and the solution was cooled to
In recent years we have focused on the study of the 3D-
structure and applications of peptides based on conformationally
constrained α-amino acids, in particular those characterized by
Cα-methylation. For example, Aib (α-aminoisobutyric acid)17,18
and (αMe)Val (Cα-methyl valine)18–22 are among the strongest
known inducers of β-turn23–25 and 310/α-helical26 conform-
ations. These and related Cα-methylated α-amino acids have
been shown to represent excellent tools for the construction of
rigid spacers,27 templates28 and catalysts.29 Sometime ago, we
expanded the arsenal of structurally restricted building blocks
by designing and synthesizing a β-bend ribbon spiral structure
0 ЊC. Then, 30 mL of an aqueous HbHNL (hydroxynitrile lyase
from Hevea brasilensis) enzyme solution32 (5300 units mLϪ1)
were mixed with 34 mL of water and the pH of the solution was
adjusted to 4.0. This enzyme preparation was added to the
ketone solution and 7.6 g of sorbitol were added. The reaction
mixture was vigorously stirred for 10 min until a stable emul-
sion had formed. Freshly prepared hydrogen cyanide (30 mL,
760 mmol) was added and the reaction vessel was tightly sealed.
After the emulsion had been stirred for another 15 min, the
reaction mixture was dissolved in 100 mL of organic solvent
and 5 g of Celite were added. The mixture was stirred for 10
min and after filtration the phases were separated. The aqueous
layer was extracted twice with methyl tert-butyl ether. The
combined organic phases were dried over anhydrous sodium
† Electronic supplementary information (ESI) available: analytical
data. See http://www.rsc.org/suppdata/p2/b1/b107691b/
644
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b107691b

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sulfate and the solvent was removed. The yield of the cyano-
hydrin was 77%. For the determination of the enantiomeric
excess, a sample of the residual was analysed by gas chrom-
atography using a 25 m × 0.32 mm Chirasil-Dex-CB capillary
column. The experimentally determined enantiomeric excess
was 82%.
T = 293 K, space group P21, Z = 6, Dc = 1.133 g cmϪ3, µ =
0.705 mmϪ1 (Cu-Kα), 4335 reflections measured, 4158 unique
(Rint = 0.068) which were used in all calculations, final R value
0.064 [on F ≥ 4σ(F)].
Crystallographic data for Ac-L-(ꢀMe)Hyv-(Aib)4-OMe
The (S)-cyanohydrin was hydrolysed using concentrated
hydrochloric acid according to the literature procedure31 (yield
63%). To improve the enantiomeric excess of (S)-α-hydroxy-
isovaleric acid, the crude acid was crystallized as its diastereo-
meric salt with (S)-α-phenylethylamine using a 9 : 1 mixture
of ethyl acetate–ethanol.30 The enantiomeric excess of the
(S)-α-hydroxyacid was analysed by gas chromatography on its
derivative 1,3-dioxolan-4-one using a 25 m × 0.32 mm Chirasil-
Dex-CB capillary column and found to be 93%.
C25H44N4O8, M = 528.6. Triclinic, a = 8.281(2), b = 9.539(3), c =
10.417(3) Å, α = 99.18(5), β = 96.34(4), γ = 109.03(5)Њ, U =
756.2(4) Å3, T = 293 K, space group P1, Z = 1, Dc = 1.161 g
cmϪ3, µ = 0.714 mmϪ1 (Cu-Kα), 2258 reflections measured, 2255
unique which were used in all calculations, final R value 0.081
[on F ≥ 4σ(F)].
Crystallographic data for Ac-(Aib)5-OMe
C23H41N5O7, M = 499.6. Monoclinic, a = 18.687(4), b = 8.326(2),
c = 19.443(4) Å, β = 105.99(7)Њ, U = 2908(1) Å3, T = 293 K,
space group P21/n, Z = 4, Dc = 1.141 g cmϪ3, µ = 0.700 mmϪ1
(Cu-Kα), 5107 reflections measured, 4306 unique (Rint = 0.076)
which were used in all calculations, final R value 0.083 [on
F ≥ 4σ(F)].
Details of the procedure for the most challenging depsi-
peptide coupling reaction are given below.
Synthesis of Boc-L-Ala-L-(ꢀMe)Hyv-OBzl
To a stirred solution of H--(αMe)Hyv-OBzl (1.3 mmol, 287
mg) in dry CH2Cl2 (3 mL) were added scandium triflate (0.78
mmol, 384 mg), Boc--Ala-OH (3.9 mmol, 738 mg) and 4-
(dimethylamino)pyridine (DMAP) (3.9 mmol, 476 mg), and
the mixture was cooled to Ϫ10 ЊC for 30 min. Then, N-ethyl-
NЈ-[3-(dimethylamino)propyl]carbodiimide (EDC) (3.9 mmol,
748 mg) was added and stirring was continued for 30 min.
The resulting mixture was allowed to warm up to room tem-
perature over a period of 16 h, stirred at 40 ЊC for an add-
itional 8 h and at room temperature for 40 h. The reaction
mixture was diluted with CH2Cl2 (15 mL). The organic layer
was washed with 0.1 M HCl (2 × 10 mL), an aqueous satur-
ated solution of NaHCO3 (2 × 10 mL) and brine (1 × 10
mL), dried over sodium sulfate, and concentrated. A pure
product was obtained after silica gel chromatography (a
cyclohexane–ethyl acetate 95 : 5 mixture as eluant) in 75%
yield (383 mg).
X-Ray crystal structure determinations
Colourless crystals (0.4 × 0.3 × 0.3 mm, 0.6 × 0.5 × 0.4 mm,
0.4 × 0.3 × 0.1 mm and 0.30 × 0.15 × 0.08 mm, respectively)
of H--(αMe)Hyv--Val-OMe, Ac--(αMe)Hyv--Val-OMe,
Ac--(αMe)Hyv-(Aib)4-OMe and Ac-(Aib)5-OMe were grown
from methanol solution (slow evaporation), methanol solution
(slow evaporation), acetonitrile solution (slow evaporation) and
a chloroform–ethyl acetate solvent mixture–petroleum ether
(vapour diffusion), respectively. Data collection was performed
on a Philips PW1100 four-circle diffractometer. The four struc-
tures were solved by direct methods, using the SHELXS 9733
program. Refinement was performed using the SHELXL 9734
program. H-atoms were calculated at idealized positions and
refined as riding. Fractional atomic coordinates, tables of hydro-
gen atoms coordinates, anisotropic displacement parameters,
bond lengths, bond angles, and torsion angles for the four struc-
tures are available from the Cambridge Crystallographic Data
Centre.‡
FT-IR absorption spectra
FT-IR absorption spectra were recorded using a Perkin-Elmer
model 1720X FT-IR spectrophotometer (Norwalk, CT), nitro-
gen flushed, equipped with a sample-shuttle device, at 2 cmϪ1
nominal resolution, averaging 100 scans. Solvent (baseline)
spectra were obtained under the same conditions. Cells with
path lengths of 0.1, 1.0 and 10 mm (with CaF2 windows) were
Results and discussion
Synthesis and characterization
2
used. Spectrograde [2H]chloroform (99.8% H) was purchased
-(S)-(αMe)Hyv was produced on a large scale via acid
hydrolysis of the corresponding (S)-cyanohydrin,31 which was
in turn synthesized from isopropyl methyl ketone and hydrogen
cyanide according to the procedure of Griengl et al.32 This
chemo-enzymatic method yielded the (S)-cyanohydrin with
82% enantiomeric excess. It is evident that it is difficult for the
enzyme (hydroxynitrile lyase from Hevea brasilensis, HbHNL)
used in this synthetic step to clearly stereodifferentiate between
the methyl and the isopropyl groups in the ketone substrate.
To increase the enantiopurity to a more acceptable degree
(93%), the (S)-hydroxy acid was further purified by preferential
crystallisation of its diastereomeric salt with (S)-α-phenyl-
ethylamine according to the method of Mori et al.30
from Merck (Darmstadt, Germany).
1H NMR spectra
1H NMR spectra were recorded with a Bruker model AM
400 spectrometer (Karlsruhe, Germany). Measurements were
carried out in [2H]chloroform (99.96% 2H; Merck) and in
[2H6]DMSO ([2H6]dimethyl sulfoxide) (99.96% 2H6; Fluka,
Buchs, Switzerland) with tetramethylsilane as the internal
standard.
Crystallographic data for H-L-(ꢀMe)Hyv-L-Val-OMe (OMe,
methoxy)
Peptide synthesis was performed in solution by established
procedures. The Z (benzyloxycarbonyl)/OMe protected -Val
homo-oligomers (from dimer to tetramer) were prepared in 75–
91% yield using Z--Val-OH,35 H--Val-OMe36 and the EDC–
1-hydroxy-1,2,3-benzotriazole (HOBt) method.37 Removal of
the Z N α-protection was achieved by catalytic hydrogenation.
Acetylation of H-(-Val)4-OMe and H-(Aib)5-OMe38 was
achieved with an excess of acetic anhydride.
C12H23NO4, M = 245.3. Monoclinic, a = 5.900(2), b = 10.151(3),
c = 12.088(3) Å, β = 100.64(3)Њ, U = 711.5(4) Å3, T = 293 K,
space group P21, Z = 2, Dc = 1.145 g cmϪ3, µ = 0.698 mmϪ1
(Cu-Kα), 1179 reflections measured, 1126 unique (Rint = 0.014)
which were used in all calculations, final R value 0.043 [on F ≥
4σ(F)].
Crystallographic data for Ac-L-(ꢀMe)Hyv-L-Val-OMe
(Ac, acetyl)
‡ CCDC reference numbers 172174–172177. See http://www.rsc.org/
suppdata/p2/b1/b107691b/ for crystallographic files in .cif or other
electronic format.
C14H25NO4, M = 287.4. Monoclinic, a = 11.287(3), b =
17.451(4), c = 13.315(3) Å, β = 105.59(8)Њ, U = 2526(1) Å3,
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651
645

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The -(αMe)Hyv residue was easily incorporated (55–89%
yield) at the N-terminus of a peptide chain, i.e. of (H--Val)1–3
-
OMe and H-(Aib)4-OMe,38 without protection of the α-hydroxy
function, using the EDC–1-hydroxy-7-aza-1,2,3-benzotriazole
(HOAt) method.39 Acetylation of the O-terminal -(αMe)Hyv
residue in the depsipeptides was achieved in 55–87% yield using
an excess of acetic anhydride in the presence of DMAP.40
Incorporation of an -(αMe)Hyv residue in an internal pos-
ition of the peptide chain proved to be very challenging. In our
hands, all classical methods for C-activation of N α-protected
α-amino acids, including EDC–HOBt,37 EDC–HOAt,39 acyl
fluoride41 and symmetrical anhydride (in the presence/absence
of DMAP) failed. Eventually, we succeeded in the preparation
of Boc--Ala--(αMe)Hyv-OBzl (Boc, tert-butyloxycarbonyl;
OBzl, benzyloxy) in 75% yield using Boc--Ala-OH42 and a
recently published general procedure for acylation of hindered
tertiary alcohols which involves EDC and the unique combin-
ation of catalysts scandium triflate–DMAP43,44 (see Experi-
mental section). It is worth noting that Z--Ala-OH did not
survive the scandium triflate treatment, affording in part Z--
Ala-OBzl. The H--(αMe)Hyv-OBzl derivative was obtained in
95% yield from H--(αMe)Hyv-OH and benzyl bromide in the
presence of triethylamine. Selective cleavage of the C-terminal
benzyl ester function of the fully protected didepsipeptide
by catalytic hydrogenation proceeded smoothly. Finally, the
resulting N α-protected didepsipeptide free acid Boc--Ala--
(αMe)Hyv-OH was satisfactorily coupled to H--Val-OMe36 by
the EDC–HOBt procedure to afford the tridepsipeptide Boc--
Ala--(αMe)Hyv--Val-OMe with an internal -(αMe)Hyv
residue. If lengthening of the tridepsipeptide chain from the
N-terminus is required, methods are available for the selective
deprotection of the Boc group in the presence of an ester from a
tertiary alcohol [such as that characterizing the -Ala--(αMe)-
Hyv sequence].45
Fig. 1 FT-IR absorption spectra (3500–3200 cmϪ1 region) of Ac--
(αMe)Hyv-(-Val)3-OMe (A) and Ac-(-Val)4-OMe (B) in CDCl3
solution. Peptide concentrations: 1 × 10Ϫ3 mol dmϪ3 (I) and 1 × 10Ϫ4
mol dmϪ3 (II).
The final compounds and intermediate sequences were
obtained in a chromatographically homogeneous state and
were characterized by melting point determination (if solid),
polarimetry, mass spectrometry, and solid-state IR absorption
(Table 1). Additional analytical data (thin-layer chromato-
Fig. 2 FT-IR absorption spectra (3500–3200 cmϪ1 region) of Ac--
(αMe)Hyv-(Aib)4-OMe (A) and Ac-(Aib)5-OMe (B) in CDCl3 solution.
1
graphy Rf values in three different solvent systems and H/13C
Peptide concentration: 1 × 10Ϫ3 mol dmϪ3
.
NMR results) have been deposited as electronic supplementary
information.
Solution conformational analysis
The preferred conformation adopted by the -(αMe)Hyv con-
taining depsipeptides was assessed in the structure supporting
solvent CDCl3 by FT-IR absorption and 1H NMR techniques.
Figs. 1 and 2 illustrate the FT-IR absorption spectra in the
informative N–H stretching region, while Figs. 3 and 4 show the
1H NMR titrations of NH proton chemical shifts.
The FT-IR absorption curve of Ac--(αMe)Hyv-(-Val)3-
OMe (Fig. 1) is characterized by two bands in the 3445–
3425 cmϪ1 region (free, solvated NH groups) and one band of
comparable intensity at 3385 cmϪ1 (weakly H-bonded NH
groups).46,47 A 10-times dilution does not alter the spectral
pattern. In striking contrast, the FT-IR spectrum of the related
Ac-(-Val)4-OMe peptide (Fig. 1) changes dramatically from
1 × 10Ϫ3 to 1 × 10Ϫ4 mol dmϪ3 concentration, showing, in par-
ticular, a remarkable decrease in the intensity of the 3275 cmϪ1
band (associated with the extremely strongly H-bonded NH
groups typical of the β-sheet conformation) with a concomitant
significant increase of the band assigned to free NH groups at
3430 cmϪ1. From this analysis it is evident that the -(αMe)-
Hyv residue disrupts the strongly self-associated species formed
by the -Val homo-peptide, without being able, however,
to promote a substantial folding in the molecule. Indeed,
it is reasonable to associate the band at 3385 cmϪ1 exhibited
by the -(αMe)Hyv/-Val depsipeptide with intramolecularly
H-bonded, fully extended (C5) conformers.48
Fig. 3 Plot of NH proton chemical shifts in the 1H NMR spectrum of
Ac--(αMe)Hyv-(-Val)3-OMe as a function of increasing percentages
of DMSO (v/v) added to the CDCl3 solution. Peptide concentration:
1 × 10Ϫ3 mol dmϪ3
.
Fig. 2 shows that a -(αMe)Hyv residue can be hosted in an
(Aib)n homo-peptide chain without a marked perturbation of
its highly folded conformation. Indeed, the intense band at 3345
cmϪ1 of Ac-(Aib)5-OMe, typical of intramolecularly H-bonded,
helical peptides,46,49 is still largely preserved in the depsipeptide
analogue Ac--(αMe)Hyv-(Aib)4-OMe. However, the presence
of a band at 3385 cmϪ1 in the spectrum of the latter indicates
the co-existence of fully-extended forms to some degree.
646
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651

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Table 1 Physical properties and analytical data for the newly synthesized derivatives and peptides
MS (m/z)e
Recrystallization
[α]2D0
Calculated Observed Species
IR (KBr) ν/cmϪ1 g
c
Compound
Yield (%) Mp/ЊCa
solventb
H--(αMe)Hyv-OBzl
Boc--Ala--(αMe)Hyv-OBzl
95
75
Oil
Oil
—
—
Ϫ6.2
Ϫ37.1
245.115
416.205
245.117
416.214f
[M ϩ Na]ϩ 1729h
[M ϩ Na]ϩ 3382, 1746, 1717,
1503h
Boc--Ala--(αMe)Hyv-OH
99
Oil
Oil
—
Ϫ34.4
Ϫ67.3
Ϫ48.8
Ϫ72.4
Ϫ96.4
Ϫ28.8
Ϫ49.6
Ϫ71.6
Ϫ36.0
Ϫ62.2
304.176
417.260
246.170
345.238
444.307
288.180
387.249
486.317
365.207
464.276
304.190f
417.278
246.181
345.255
444.308
288.188
387.256
486.330
365.216
464.282
563.355
471.322
487.317
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
[M ϩ H]ϩ
3320, 1748, 1720,
1510h
Boc--Ala--(αMe)Hyv--Val-OMe 83
—
3326, 1745, 1693,
1524h
3414, 3329, 1749,
1655, 1514
3362, 3283, 1750,
1645, 1536
3300, 1747, 1641,
1546
3340, 3322, 1741,
1662, 1535
3312, 1751, 1650,
1544
3292, 1744, 1642,
1534
3300, 1743, 1691,
1652, 1538
3291, 1744, 1693,
1643, 1540
3283, 1743, 1704,
1641, 1539
3279, 1746, 1637,
1546
3450, 3396, 3316,
1729, 1672, 1652,
1524
3399, 3355, 3307,
1739, 1730, 1687,
1658, 1521
3434, 3305, 1733,
1666, 1538
H--(αMe)Hyv--Val-OMe
H--(αMe)Hyv--(Val)2-OMe
H--(αMe)Hyv--(Val)3-OMe
Ac--(αMe)Hyv--Val-OMe
Ac--(αMe)Hyv--(Val)2-OMe
Ac--(αMe)Hyv--(Val)3-OMe
Z--(Val)2-OMe
70
78
79
55
68
87
91
90
75
75
55
72–74
CH2Cl2–PE
184–185 EtOAc–PE
220–221 EtOAc–PE
56–58
EtOAc–PE
126–127 EtOAc–PE
200–202 EtOAc–PE
106–107 EtOAc–PE
212–214 EtOAc–PE
267–268 EtOAc–PE
310–312 MeOH–PE
208–209 CH2Cl2–PE
Z--(Val)3-OMe
Z--(Val)4-OMe
Ϫ80.0d 563.344
Ϫ130.6d 471.312
Ac--(Val)4-OMe
H--(αMe)Hyv-(Aib)4-OMe
Ϫ2.8
ϩ18.8
—
487.313
529.323
500.308
Ac--(αMe)Hyv-(Aib)4-OMe
73
44
203–204 CH2Cl2–PE
278–279 CH2Cl2–PE
529.337
500.317
[M ϩ H]ϩ
[M ϩ H]ϩ
Ac-(Aib)5-OMe
a Determined on a Leitz model Laborlux apparatus (Wetzlar, Germany). b PE, petroleum ether bp 40–60 ЊC; EtOAc, ethyl acetate; MeOH, methanol;
DE, diethyl ether. c Determined on a Perkin-Elmer model 241 polarimeter (Norwalk, CT) equipped with a Haake model L thermostat (Karlsruhe,
Germany); c = 0.5 (MeOH); reported in units of 10Ϫ1 deg cm2 gϪ1 d c = 0.5 (TFE). e Determined on a PerSeptive Biosystems model Mariner API-
.
TOF mass spectrometer. A 1% HCOOH in a H2O–CH3CN 1 : 1 solvent mixture was used for dissolving and injecting the samples. f The reported
m/z peak shows little intensity since the largest peak observed corresponds to the [M ϩ H Ϫ Boc]ϩ fragment. g Determined in KBr pellets on a
Perkin-Elmer model 580 B spectrophotometer equipped with a Perkin-Elmer model 3600 IR data station and a model 660 printer (only bands in
the 3500–3200 cmϪ1 and 1800–1500 cmϪ1 regions are reported). h Determined as a film.
using the solvent dependence of the NH chemical shifts by
adding increasing amounts of the H-bonding acceptor DMSO
to the CDCl3 solution.50,51 Unambiguous assignments for all
NH proton signals were obtained by ROESY and TOCSY
experiments.
From an inspection of Fig. 3 it is clear that all three NH
protons of Ac--(αMe)Hyv-(-Val)3-OMe are sensitive to the
addition of DMSO, although this phenomenon is less signifi-
cant for the NH2 proton. These results, taken together with the
corresponding FT-IR absorption data, strongly support the
view that the predominant 3D-structure of this tetradepsi-
peptide in CDCl3 solution is not characterized by intramole-
cularly H-bonded, folded forms. Rather, it is reasonable to
assume that an intramolecularly H-bonded, fully extended (C5)
conformer, involving the Val2 residue, might in part populate
the equilibrium mixture.
In both parts A and B of Fig. 4 two classes of NH protons
were observed. Class (i) [NH2 proton for Ac--(αMe)Hyv-
(Aib)4-OMe, and NH1 and NH2 protons for Ac-(Aib)5-OMe]
includes protons whose chemical shifts are remarkably sensitive
to the addition of DMSO. Class (ii) (NH3 to NH5 protons of
both compounds) includes those displaying behaviour charac-
teristic of shielded protons (extremely modest sensitivity of
chemical shifts to solvent composition). These 1H NMR results
are in agreement with the FT-IR absorption data discussed
above, allowing us to conclude that both Ac-(Aib)5-OMe46,49
Fig. 4 Plot of NH proton chemical shifts in the 1H NMR spectrum of
Ac--(αMe)Hyv-(Aib)4-OMe (A) and Ac-(Aib)5-OMe (B) as a function
of increasing percentages of DMSO (v/v) added to the CDCl3 solution.
Peptide concentration: 1 × 10Ϫ3 mol dmϪ3
.
More detailed information on the conformational prefer-
ences of the -(αMe)Hyv depsipeptides in CDCl3 solution was
extracted from a 400 MHz 1H NMR investigation (Figs. 3 and
4). The delineation of inaccessible (presumably intramole-
1
cularly H-bonded) NH groups by H NMR was performed by
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651
647

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and its related depsipeptide Ac--(αMe)Hyv-(Aib)4-OMe
are largely folded in a 310-helical conformation where the first
two residues in the main chain are not involved in the intra-
molecularly H-bonded scheme.
Crystal-state conformational analysis
By X-ray diffraction we determined the molecular and crystal
structures of the didepsipeptides H--(αMe)Hyv--Val-OMe
and Ac--(αMe)Hyv--Val-OMe, the pentadepsipeptide Ac--
(αMe)Hyv-(Aib)4-OMe and the related pentapeptide Ac-(Aib)5-
OMe. The molecular structures with the atomic numbering
schemes are illustrated in Figs. 5–8. Backbone and side-chain
Fig. 5 X-Ray diffraction structure of H--(αMe)Hyv--Val-OMe with
numbering of the atoms. The intramolecular H-bond is indicated by a
dashed line.
torsion angles52 are given in Table 2. In Table 3 the intra-
and intermolecular H-bond parameters are listed. The X-ray
diffraction structures of two other Aib homo-pentapeptides,
namely Tos-(Aib)5-OMe (Tos, tosyl)53 and Z-(Aib)5-OtBu
(OtBu, tert-butoxy)54 have already been reported.
Bond lengths and bond angles are in general agreement with
previously reported values for the geometry of the ester55 and
amide56 groups, the peptide unit,57,58 and the Aib residue.59,60
In H--(αMe)Hyv--Val-OMe (Fig. 5) the ψ1 angle is close
to the cis conformation, thereby preventing the occurrence of
the intramolecularly H-bonded O01–H01 ؒ ؒ ؒ O1᎐C1 form
᎐
(oxy-analogue of the C5 conformation),24 but favouring the
formation of a different pseudocyclic C5 species involving the
(peptide) N2–H2 ؒ ؒ ؒ O01 (alcohol) intramolecular H-bond.
The C-terminal Val residue is semi-extended.
The only relevant backbone difference seen among the three
independent molecules in the asymmetric unit of Ac--
(αMe)Hyv--Val-OMe (Fig. 6) is found in the conformation of
the Val2 residue, right-handed (distorted) helical in molecules A
and B, but semi-extended in molecule C. In all three molecules
the conformation of the -(αMe)Hyv residue is right-handed
helical.
Fig.
6
X-Ray diffraction structures of the three independent
molecules in the asymmetric unit of Ac--(αMe)Hyv--Val-OMe with
numbering of the atoms.
The pentadepsipeptide Ac--(αMe)Hyv-(Aib)4-OMe is folded
in a right-handed 310-helical structure26 stabilized by three 1
4
C᎐O ؒ ؒ ؒ H–N intramolecular H-bonds (Fig. 7). All O ؒ ؒ ؒ N
᎐
distances are well within the accepted range for such H-
bonds.61–63 Interestingly, one of the H-bond acceptors is the
acetoxy ester carbonyl. The usual inversion of the handed-
ness of the C-terminal helical residue with respect to that of
the preceding ones64 is also found in this 310-helical peptide
ester. Overall, the conformation adopted by the penta-
peptide Ac-(Aib)5-OMe (Fig. 8) strictly parallels those pub-
lished for Tos-(Aib)5-OMe53 and Z-(Aib)5-OtBu54 and that
discussed above for Ac--(αMe)Hyv-(Aib)4-OMe. These find-
ings are strongly in favour of the conclusion that neither the
nature of the N- and C-protecting groups nor the Aib
(αMe)Hyv replacement are effective in inducing a significant
alteration in the global architecture of the -(Aib)5- homo-
peptide sequence.
Fig. 7 X-Ray diffraction structure of Ac--(αMe)Hyv-(Aib)4-OMe
with numbering of the atoms. The three intramolecular H-bonds are
indicated by dashed lines.
648
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651

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Table 2 Selected torsion angles (deg)52 for the four X-ray diffraction structures solved in this work
Ac--(αMe)Hyv--Val-OMe
Torsion angle
H--(αMe)Hyv--Val-OMe Mol. A
Mol. B
Mol. C
Ac--(αMe)Hyv-(Aib)4-OMe
Ac-(Aib)5-OMe
ω0
φ1
ψ1
ω1
φ2
ψ2
ω2
φ3
ψ3
ω3
φ4
174.2(3)
Ϫ54.3(4)
Ϫ43.8(4)
Ϫ178.0(3)
Ϫ91.5(4)
Ϫ44.5(4)c
178.1(6)d
Ϫ178.2(4)
Ϫ54.8(4)
Ϫ45.0(4)
Ϫ177.0(4)
Ϫ98.5(4)
Ϫ48.7(5)e
Ϫ178.0(6)f
176.5(5)
Ϫ55.9(4)
Ϫ47.3(4)
179.7(3)
Ϫ95.1(4)
138.6(3)g
176.8(4)h
Ϫ167.1(7)
Ϫ55.6(7)
Ϫ48.5(8)
Ϫ169.8(6)
Ϫ55.3(9)
Ϫ36.5(9)
Ϫ172.7(6)
Ϫ58.0(9)
Ϫ34.8(9)
Ϫ173.8(6)
Ϫ61.2(8)
Ϫ35.4(9)
175.5(7)
Ϫ168.0(5)
Ϫ56.8(6)
Ϫ33.6(6)
Ϫ177.2(4)
Ϫ51.2(6)
Ϫ36.4(6)
Ϫ175.1(4)
Ϫ55.7(3)
Ϫ32.5(6)
Ϫ177.2(4)
Ϫ61.4(5)
Ϫ30.9(6)
177.3(4)
13.9(3)
173.7(2)
Ϫ66.3(4)
150.2(2)a
179.7(3)b
ψ4
ω4
φ5
47.2(10)
47.2(6)
ψ5
ω5
46.0(9)i
50.3(5)i
175.4(9)j
Ϫ57.8(8)
176.8(7)
172.7(4)j
1,1
χ11,2
Ϫ62.9(3)
62.4(3)
61.6(4)
Ϫ60.5(4)
173.1(3)
179.3(4)
Ϫ58.3(5)
173.6(3)
Ϫ59.7(4)
Ϫ173.7(4)
Ϫ53.5(5)
Ϫ60.9(5)
169.4(4)
Ϫ68.6(6)
170.0(6)
χ11,1
χ21,2
χ2
Ϫ65.3(4)
a N2–C2A–C2–OT. b C2A–C2–OT–CT. c N2–C2A–C2–OTA. d C2A–C2–OTA–CTA. e N4–C4A–C4–OTB. f C4A–C4–OTB–CTB. g N6–C6A–C6–
OTC. h C6A–C6–OTC–CTC. i N5–C5A–C5–OT. j C5A–C5–OT–CT.
Table 3 Intra- and intermolecular H-bond parameters for the four X-ray diffraction structures solved in this work
Distance/Å
Compound
Donor D–H
Acceptor A
Symmetry operations of A
D ؒ ؒ ؒ A
H ؒ ؒ ؒ A
Angle/deg D–H ؒ ؒ ؒ A
H--(αMe)Hyv--Val-OMe
Ac--(αMe)Hyv--Val-OMe
N2–H2
O01–H01
N2–H2
N4–H4
N6–H6
N3–H3
N4–H4
N5–H5
N2–H2
N3–H3
N4–H4
N5–H5
N1–H1
N2–H2
O01
O1
O5
O1
O3
O0
O1
O2
O4
O0
O1
O2
O4
O5
x, y, z
2.558(3)
2.771(3)
3.075(5)
2.999(4)
2.979(4)
3.147(9)
3.123(7)
3.083(8)
2.998(7)
3.029(5)
2.987(5)
3.012(5)
2.817(5)
3.198(5)
2.15
1.95
2.22
2.16
2.13
2.42
2.35
2.44
2.32
2.22
2.20
2.28
1.97
2.44
109
175
171
166
170
143
150
132
136
156
152
144
168
147
x ϩ 1, y, z
x ϩ 1, y, z ϩ 1
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z
x, y, z Ϫ 1
x, y, z
x, y, z
Ac--(αMe)Hyv-(Aib)4-OMe
Ac-(Aib)5-OMe
x, y, z
x Ϫ ½, Ϫy ϩ ½, z Ϫ ½
x Ϫ ½, Ϫy ϩ ½, z Ϫ ½
deviate from trans planarity by 10.2–12.9Њ. The methyl ester
group adopts a conformation with respect to the C-terminal
CA–N bond between the antiperiplanar and anticlinal con-
formations for molecules A and B of Ac--(αMe)Hyv--Val-
OMe, Ac--(αMe)Hyv-(Aib)4-OMe and Ac-(Aib)5-OMe, but
between the synperiplanar and synclinal conformations for H--
(αMe)Hyv--Val-OMe and molecule C of Ac--(αMe)Hyv--
1,1
Val-OMe.65 The -(αMe)Hyv isopropyl side chain (χ1 and
1,2
χ1 torsion angles) is found in the t, gϪ conformation in all
structures except in H--(αMe)Hyv--Val-OMe where it adopts
the gϩ, gϪ conformation. The same conclusions apply for the
1,1
1,2
-Val isopropyl side chain (χ2 and χ2 torsion angles) of the
didepsipeptide.66
The molecules of the O-unprotected didepsipeptide ester
H--(αMe)Hyv--Val-OMe pack in the unit cell via inter-
molecular (alcohol) O01–H01 ؒ ؒ ؒ O1᎐C1 (peptide) H-bonds,
᎐
forming rows in the a direction. The O ؒ ؒ ؒ O distance is
Fig. 8 X-Ray diffraction structure of Ac-(Aib)5-OMe with number-
ing of the atoms. The three intramolecular H-bonds are indicated by
dashed lines.
normal.67,68
The three molecules in the asymmetric unit of the fully
blocked didepsipeptide Ac--(αMe)Hyv--Val-OMe are linked
together in the crystal through intermolecular H-bonds
involving exclusively the peptide carbonyl groups as acceptors.
More specifically, the three H-bonds, (molecule A peptide)
All amide, peptide and ester groups of the four structures are
trans (ω torsion angles) with no deviation >7.3Њ from planarity,
except for the ω0 and ω1 torsion angles of Ac--(αMe)Hyv-
(Aib)4-OMe and the ω0 torsion angle of Ac-(Aib)5-OMe, which
N2–H2 ؒ ؒ ؒ O5᎐C5 (molecule C peptide), (molecule B peptide)
᎐
J. Chem. Soc., Perkin Trans. 2, 2002, 644–651
649

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20 C. Toniolo, M. Crisma, G. M. Bonora, B. Klajc, F. Lelj, P. Grimaldi,
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21 F. Formaggio, M. Pantano, G. Valle, M. Crisma, G. M. Bonora,
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34 G. M. Sheldrick, SHELXL 97. Program for Crystal Structure
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35 H. Schwarz, F. M. Bumpus and I. H. Page, J. Am. Chem. Soc., 1957,
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36 R. A. Boissonnas, S. Guttmann, R. L. Huguenin, P. A. Jaquenoaud
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N4–H4 ؒ ؒ ؒ O1᎐C1 (molecule A peptide), and (molecule C
᎐
peptide) N6–H6 ؒ ؒ ؒ O3᎐C3 (molecule B peptide) give rise to
᎐
B–A–C–B–A–C chains of molecules along the a,c direction.
The crystal packing mode for the pentadepsipeptide Ac-
-(αMe)Hyv-(Aib)4-OMe is characterized by (peptide) N2–
H2 ؒ ؒ ؒ O4᎐C4 (peptide) intermolecular H-bonds, generating
᎐
rows of molecules along the c direction. In the crystals of the
related pentapeptide Ac-(Aib)5-OMe two different types of inter-
molecular H-bonds link the molecules in a head-to-tail fashion
along the a,c direction, a strong (amide) N1–H1 ؒ ؒ ؒ O4᎐C4
᎐
(peptide) H-bond and a weak (peptide) N2–H2 ؒ ؒ ؒ O5᎐C5
᎐
(ester) H-bond.61–63
Conclusions
In this paper we have reported the stereospecific synthesis of
the α-hydroxy acid -(αMe)Hyv by a combined chemical and
enzymatic approach. Due to the extremely poor reactivity of
its hydroxy function, we successfully incorporated -(αMe)Hyv
in an internal position of the peptide chain only by taking
advantage of a recently proposed method for the acylation of
sterically hindered tertiary alcohols which concomitantly
exploits the Lewis acid scandium() triflate and the tertiary
amine DMAP as catalysts.43,44 For the same reason, addition
of -(αMe)Hyv at the N-terminus of a peptide chain is particu-
larly straightforward as this reaction does not even require
O-protection.
In the X-ray diffraction structures of all four molecules of
the two depsipeptides containing an O-acylated -(αMe)Hyv
residue that were solved in this work, this first chiral Cα-
tetrasubstituted α-hydroxy acid studied to date is right-handed
helical (with average φ, ψ torsion angles Ϫ55.1, Ϫ46.1Њ) as
expected on the basis of the well known gem-dialkyl effect.69
This structural property, making it ideally suited for the stab-
ilization of β-turn and 310/α-helices in depsipeptides, strictly
reflects the published propensity of the parent Hib α-hydroxy
acid.8,9 The conformational tendency of -(αMe)Hyv also
closely resembles that of the related chiral, Cα-tetrasubstituted
α-amino acid -(αMe)Val.18–22 It is also worth noting that
replacement with -(αMe)Hyv of a single residue in the inter-
molecular β-sheet forming sequence -(-Val)4- is sufficient to
disrupt this ordered self-associated secondary structure. Taken
together, these results support the view that the -(αMe)Hyv
residue represents an additional valuable tool for the design and
synthesis of conformationally constrained, folded depsipeptides.
43 H. Zhao, A. Pendri and R. B. Greenwald, J. Org. Chem., 1998, 63,
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