Cα-Methyl, Cα-n-Propylglycine Homo-oligomers

Article abstract of DOI:10.1021/ma030327v

A series of N^α-protected, monodispersed homo-oligopeptide esters to the octamer level from L-C^α-methyl, C ^α-n-propylglycine [or C^α-methylnorvaline, (αMe)Nva] has been synthesized by solution methods and fully ch

Full text of DOI:10.1021/ma030327v

8164
Macromolecules 2003, 36, 8164-8170
CR-Methyl, CR-n-Propylglycine Homo-oligomers
F er n a n d o F or m a ggio,* Ma r co Cr ism a , a n d Cla u d io Ton iolo
Institute of Biomolecular Chemistry, CNR, Department of Organic Chemistry, University of Padova,
35131 Padova, Italy
Qu ir in u s B. Br oxter m a n a n d Ber n a r d Ka p tein
DSM Research, Life Sciences, Advanced Synthesis and Catalysis, P.O. Box 18,
6160 MD Geleen, The Netherlands
Ca th er in e Cor bier
Laboratory of Crystallography, ESA 7036, Universite´ Henry Poincare´-Nancy I,
54506 Vandoeuvre-le`s-Nancy, France
Mich ele Sa via n o, P a squ a le P a lla d in o, a n d Ettor e Ben ed etti
Institute of Biostructure and Bioimaging, CNR, Interuniversity Research Center on Bioactive Peptides,
University of Naples “Federico II”, 80134 Naples, Italy
Received J une 13, 2003; Revised Manuscript Received August 4, 2003
ABSTRACT: A series of NR-protected, monodispersed homo-oligopeptide esters to the octamer level from
L-CR-methyl, CR-n-propylglycine [or CR-methylnorvaline, (RMe)Nva] has been synthesized by solution
methods and fully characterized. The preferred conformation of these homo-oligomers in solution has
been assessed by FT-IR absorption and 1H NMR techniques. Moreover, the molecular structures of the
homotrimer and homotetramer have been determined in the crystal state by X-ray diffraction. The obtained
results strongly support the view that right-handed, single or multiple, and consecutive â bends are
preferentially adopted by the conformationally restricted L-(RMe)Nva homo-oligomers. In particular, 310
helices are formed by the longest homo-oligomers. It is our contention that the [(RMe)Nva]n peptides
represent the best available choice among CR-tetrasubstituted R-amino acid-based homo-oligomers for
the construction of relatively easy to make, rigid foldamers with a well-defined screw-sense bias.
In tr od u ction
The experimental results obtained convincingly sup-
port our view that the Aib (R-amino isobutyric acid) and
Iva (isovaline) residues are strong â-turn13-15 and
310-helix formers (depending on the peptide main-chain
length) and are much more efficient than their
CR-unmethylated parent amino acids. However, in terms
of the screw-sense bias, Aib is achiral, thereby forming
isoenergetic, equally probable, enantiomeric, right- and
left-handed homo-oligomeric 310-helices. Peptides based
on Aib and protein (L-) amino acids can indeed provide
helical structures with a preferred (right-handed) screw
sense; however, they tend to fold into mixed 310-/R-
helical structures, which makes it difficult to determine
their end-to-end distance precisely. Iva is chiral, but the
small difference (one carbon atom only) between the two
aliphatic substituents on its CR atom does allow the
formation of only a relatively limited, although sizable,
excess of one 310-helix screw sense over the other in its
homo-oligomers. (Likewise, with protein amino acids,
the L residue gives a predominantly right-handed
helix.16,17) However, the homopeptides from the Câ-
branched, CR-methylated R-amino acid (RMe)Val (CR-
methyl valine) are folded in rigid 310-helical structures
(at least if very polar and protic solvents are avoided)18,19
with a strong screw-sense bias.20 However, with the
coupling methods available to date, the high steric
hindrance of the (RMe)Val residue, particularly at its
nucleophilic amino function, prevents one from prepar-
ing a homo-oligomeric series in good yield and in a
relatively short time.20
A proper understanding of the nature of electron- and
energy-transfer processes depends heavily upon our
ability to design and synthesize conformationally con-
strained structures, the intercomponent geometry (rigid
but precisely tunable) of which would be well defined.
To this end, we have recently reported a few studies on
the exploitation of short 310-helical peptides as spacers.1-4
The 310 helix, first predicted to be a reasonably stable
peptide secondary structure more than 60 years ago, has
only recently attracted the attention of structural
biochemists.5-7 It represents the third principal long-
range 3D structure occurring in globular proteins and
has been described at atomic resolution in model pep-
tides and in peptaibol antibiotics. Its promising role as
a spacer for spectroscopic and electrochemical applica-
tions is just emerging.
In this connection, the conformational preferences of
homo-oligomers based on CR-methylated R-amino acids
with a linear, saturated aliphatic side chain of increas-
ing length have particularly attracted our attention.8-12
The work reported in this article was aimed at
searching for an acceptable compromise between the 3D
structural and reactivity properties required by a homo-
* To whom correspondence should be addressed. E-mail:
fernando.formaggio@unipd.it. Tel (+39) 049-827-5277. Fax: (+39)
049-827-5239.
10.1021/ma030327v CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/18/2003

Macromolecules, Vol. 36, No. 21, 2003
[L-(RMe)Nva]n Homopeptides 8165
oligopeptide series from a CR-methylated R-amino acid
to be an attractive spacer system. On the basis of the
results obtained, it is our contention that the (RMe)-
Nva (CR-methyl norvaline) homo-oligomeric series does
represent the best choice currently available for a set
of relatively easy to prepare, rigid, strongly 310-helix
screw-sense-biased peptide spacers. Here we describe
the synthesis, full chemical characterization, and con-
formational analysis in the crystal state (by X-ray
diffraction) and in solution (by FT-IR absorption and
1H NMR techniques) of the Z-[L-(RMe)Nva]n-OtBu (Z,
benzyloxycarbonyl; OtBu, tert-butoxy; n ) 2-6, 8)
homopeptide series.
115 °C (from EtOAc/PE). TLC Rf I 0.95, Rf II 0.95, Rf III 0.30;
20
[R]D -7.2° (c 0.4, methanol). IR absorption (KBr): νmax 3439,
3345, 3303, 1720, 1671, 1531 cm-1. 1H NMR (250 MHz, CDCl3,
10 mM): δ 7.35 (m, 5H, Z phenyl CH), 7.10 (br, 1H, NH), 6.91
(br, 1H, NH), 5.72 (br, 1H, NH), 5.08 (2d, 2H, Z CH2), 2.32-
2.05 (m, 2H, â-CH2), 1.78-1.63 (m, 4H, 2 â-CH2), 1.58 (s, 3H,
â-CH3), 1.54 (s, 3H, â-CH3), 1.51 (s, 3H, â-CH3), 1.47 (s, 9H,
OtBu 3 CH3), 1.34-1.03 (m, 6H, 3 γ-CH2), 0.89 (m, 9H, 3
δ-CH3).
Z-[L-(rMe)Nva ]4-OtBu . To a solution of Z-L-(RMe)Nva-OH
(3.48 g, 13.1 mmol) in anhydrous CH2Cl2 cooled to 0 °C, HOAt
(1.79 g, 13.1 mmol) and EDC‚HCl (2.52 g, 13.1 mmol) were
added. When a clear solution formed, H-[L-(RMe)Nva]3-OtBu
[obtained by catalytic hydrogenation of the corresponding Z
derivative (5.97 g, 10.9 mmol) in anhydrous CH2Cl2] and 1
equiv of NMM were added. The reaction was stirred at room
temperature for 6 days. Then, the solvent was removed in
vacuo, and the residue was dissolved in EtOAc. The solution
was washed with 10% KHSO4, H2O, 5% NaHCO3, and H2O,
dried over Na2SO4, and evaporated to dryness. The product
was isolated by flash chromatography (eluant EtOAc-PE
3:7): yield 73%, mp 148-150 °C (from EtOAc/PE). TLC Rf I
Exp er im en ta l Section
Syn th esis of P ep tid es. The synthesis and characterization
of the amino acid derivative Z-L-(RMe)Nva-OH21 have already
been described. Newly synthesized homopeptides are as fol-
lows.
Z-L-(rMe)Nva -OtBu . Isobutylene (55 mL) was slowly
bubbled into a solution of Z-L-(RMe)Nva-OH (10 g, 37.7 mmol)
in anhydrous CH2Cl2 (110 mL). The solution was cooled to -60
°C. Concentrated H2SO4 (0.37 mL) was added, and the pres-
sure-resistant reaction flask was hermetically sealed. After
keeping the solution at room temperature for 7 days, it was
poured into a 5% NaHCO3 solution (200 mL). The organic
solvent was removed under reduced pressure, and the aqueous
phase was extracted with ethyl acetate (EtOAc). The organic
layer was washed with 10% KHSO4, H2O, 5% NaHCO3, and
H2O, dried over Na2SO4, filtered, and evaporated to dryness:
yield 87%, oil. TLC (silica gel plates 60F-254 Merck): Rf I
(CHCl3-ethanol 9:1) 0.95, Rf II (1-butanol-acetic acid-water
20
0.95, Rf II 0.95, Rf III 0.35; [R]D 8.4° (c 0.5, methanol). IR
absorption (KBr): νmax 3428, 3334, 1732, 1705, 1672, 1527 cm-1
.
1H NMR (250 MHz, CDCl3, 10 mM): δ 7.36 (m, 5H, Z phenyl
CH), 7.12 (br, 1H, NH), 7.09 (br, 1H, NH), 6.62 (br, 1H, NH),
5.33 (br, 1H, NH), 5.10 (2d, 2H, Z CH2), 1.97-0.64 (m, 8H, 4
â-CH2), 1.59 (s, 6H, 2 â-CH3), 1.53 (s, 3H, â-CH3), 1.51 (s, 3H,
â-CH3) 1.46 (s, 9H, OtBu 3 CH3), 1.60-1.12 (m, 8H, 4 γ-CH2),
0.92 (m, 12H, 4 δ-CH3).
Z-[L-(rMe)Nva ]5-OtBu . To a solution of Z-L-(RMe)Nva-OH
(2.18 g, 8.2 mmol) in anhydrous CH2Cl2 cooled to 0 °C, HOAt
(1.11 g, 8.2 mmol) and EDC‚HCl (1.57 g, 8.2 mmol) were added.
When a clear solution formed, H-[L-(RMe)Nva]4-OtBu [obtained
by catalytic hydrogenation of the corresponding Z derivative
(4.50 g, 6.8 mmol) in MeOH] and 1 equiv of NMM were added.
The reaction was stirred at room temperature for 8 days. Then,
the solvent was removed in vacuo, and the residue was
dissolved in EtOAc. The solution was washed with 10%
KHSO4, H2O, 5% NaHCO3, and H2O, dried over Na2SO4, and
evaporated to dryness. The product was isolated by flash
chromatography (eluant CH2Cl2-EtOH (ethanol) 95:5): yield
38%, mp 207-209 °C (from CH2Cl2/EtOH). TLC Rf I 0.95, Rf -
II 0.95, Rf III 0.35; [R]D20 -4.0° (c 0.3, methanol). IR absorption
(KBr): νmax 3424, 3329, 1728, 1698, 1673, 1665, 1533 cm-1. 1H
NMR (250 MHz, CDCl3, 10 mM): δ 7.37 (m, 5H, Z phenyl CH),
7.31 (br, 1H, NH), 7.22 (br, 1H, NH), 7.12 (br, 1H, NH), 6.33
(br, 1H, NH), 5.11 (br, 1H, NH), 5.10 (2d, 2H, Z CH2), 1.46 (s,
9H, OtBu 3 CH3), 1.90-1.10 (m, 35H, 5 â-CH3, 5 â-CH2 and 5
γ-CH2), 0.91 (m, 15H, 5 δ-CH3).
3:1:1) 0.95, Rf III (toluene-ethanol 7:1) 0.85; [R]20 -4.1° (c
D
0.3, methanol). IR absorption (film): νmax 3418, 3361, 1719
cm-1 1H NMR (250 MHz, CDCl3, 10 mM): δ 7.34 (m, 5H, Z
.
phenyl CH), 5.69 (br, 1H, NH), 5.07 (s, 2H, Z CH2), 1.67 (dt,
1H, â-CH2), 1.54 (s, 3H, â-CH3), 1.45 (s, 9H, OtBu 3 CH3),
1.40-1.10 (m, 3H, 1 â-CH2 and γ-CH2), 0.89 (dd, 3H, δ-CH3).
Z-[L-(rMe)Nva ]2-OtBu . To a solution of Z-L-(RMe)Nva-OH
(8.65 g, 32.6 mmol) in anhydrous CH2Cl2 cooled to 0 °C, 7-aza-
1-hydroxy-1,2,3-benzotriazole (HOAt) (4.44 g, 32.6 mmol) and
N-ethyl,N′-(3-dimethylamino)propylcarbodiimide (EDC) hy-
drochloride (6.26 g, 32.6 mmol) were added. When a clear
solution formed, H-L-(RMe)Nva-OtBu [obtained by catalytic
hydrogenation of the corresponding Z derivative (9.52 g, 29.6
mmol) in anhydrous CH2Cl2] and 1 equiv of N-methylmorpho-
line (NMM) were added, and the reaction was stirred at room
temperature for 7 days. Then, the solvent was removed in
vacuo, and the residue was dissolved in EtOAc. The solution
was washed with 10% KHSO4, H2O, 5% NaHCO3, and H2O,
dried over Na2SO4, and evaporated to dryness. The product
was isolated by flash chromatography [eluant EtOAc-PE
(petroleum ether) 3:7]: yield 71%, mp 71-73 °C (from EtOAc/
Z-[L-(rMe)Nva ]6-OtBu . Z-L-(RMe)Nva-OH (0.103 g, 0.39
mmol) and pyridine (73 µL, 0.74 mmol) were dissolved in
anhydrous CH2Cl2 (3 mL). The solution was cooled to 0 °C,
and cyanuric fluoride (63 µL, 0.74 mmol) was added. After
stirring the reaction mixture for 3 h ice water was added and
the organic layer was separated, washed with cool water, and
concentrated to dryness to give Z-L-(RMe)Nva-F as an oil. To
a solution of crude Z-L-(RMe)Nva-F in anhydrous CH2Cl2, H-[L-
(RMe)Nva]5-OtBu [obtained by catalytic hydrogenation of the
corresponding Z derivative (0.1 g, 0.13 mmol) in MeOH] and
1 equiv of NMM were added. The reaction was stirred at room
temperature for 14 days. Then, the solvent was removed in
vacuo, and the residue was dissolved in EtOAc. The solution
was washed with 10% KHSO4, H2O, 5% NaHCO3, and H2O,
dried over Na2SO4, and evaporated to dryness. The product
was isolated by flash chromatography (eluant CH2Cl2-EtOH
95:5): yield 33%, mp 81-82 °C (from CH2Cl2/EtOH). TLC Rf I
20
PE). TLC Rf I 0.95, Rf II 0.95, Rf III 0.40; [R]D -0.9° (c 0.4,
methanol). IR absorption (KBr): νmax 3391, 3332, 1726, 1669,
1499 cm-1 1H NMR (250 MHz, CDCl3, 10 mM ): δ 7.36 (m,
.
5H, Z phenyl CH), 6.93 (br, 1H, NH), 5.84 (br, 1H, NH), 5.08
(s, 2H, Z CH2), 2.32 (dt, 1H, â-CH2), 2.15 (m, 1H, â-CH2), 1.68
(dt, 2H, â-CH2), 1.56 (s, 3H, â-CH3), 1.52 (s, 3H, â-CH3), 1.47
(s, 9H, OtBu 3 CH3), 1.28-1.14 (m, 4H, 2 γ-CH2), 0.87 (dd,
6H, 2 δ-CH3).
Z-[L-(rMe)Nva ]3-OtBu . To a solution of Z-L-(RMe)Nva-OH
(5.86 g, 22.1 mmol) in anhydrous CH2Cl2 cooled to 0 °C, HOAt
(3 g, 22.1 mmol) and EDC‚HCl (4.24 g, 22.1 mmol) were added.
When a clear solution formed, H-[L-(RMe)Nva]2-OtBu [obtained
by catalytic hydrogenation of the corresponding Z derivative
(9.60 g, 22.1 mmol) in anhydrous CH2Cl2] and 1 equiv of NMM
were added, and the reaction was stirred at room temperature
for 6 days. Then, the solvent was removed in vacuo, and the
residue was dissolved in EtOAc. The solution was washed with
10% KHSO4, H2O, 5% NaHCO3, and H2O, dried over Na2SO4,
and evaporated to dryness. The product was isolated by flash
chromatography (eluant EtOAc-PE 4:6): yield 57%, mp 114-
20
0.85, Rf II 0.95, Rf III 0.30; [R]D -4.4° (c 0.3, methanol). IR
absorption (KBr): νmax 3426, 3320, 1724, 1704, 1662, 1532 cm-1
.
1H NMR (250 MHz, CDCl3, 10 mM): δ 7.47 (br, 1H, NH), 7.40
(br, 1H, NH), 7.32 (m, 5H, Z phenyl CH), 7.25 (br, 1H, NH),
7.19 (br, 1H, NH), 6.28 (br, 1H, NH), 5.32 (br, 1H, NH), 5.05
(s, 2H, Z CH2), 1.41 (s, 9H, OtBu 3 CH3), 1.90-1.10 (m, 42H,
6 â-CH3, 6 â-CH2 and 6 γ-CH2), 0.87 (m, 18H, 6 δ-CH3).

8166 Formaggio et al.
Macromolecules, Vol. 36, No. 21, 2003
Ta ble 1. Cr ysta l Da ta a n d Diffr a ction P a r a m eter s for th e Z-[L-(rMe)Nva ]n -OtBu (n ) 3, 4) Hom o-oligom er s
Z-[L-(RMe)Nva]4-OtBu
parameter
Z-[L-(RMe)Nva]3-OtBu
dihydrate
mol formula
C
547.7
orthorhombic
P212121
30H49N3O6
C
696.9
monoclinic
P21
36H60N4O7‚2H2O
mol wt, amu
crystal system
space group
Z, unit cell
a (Å)
4
2
11.927(2)
10.671(2)
b (Å)
18.192(2)
15.804(5)
c (Å)
15.201(2)
13.787(2)
â (deg)
90
112.39(1)
2149.8(8)
1.077
Cu KR (1.54178 Å)
θ/2θ
1-70
4056
V (Å3)
3298.3(8)
1.103
CuKR (1.54178 Å)
θ/2θ
1-70
3513
d (calcd), g/cm-3
radiation
data collection method
θ range, deg
indep refls
obs refls
goodness of fit
solved by
3076[I>2σ(I)]
3591[I > 2σ(I)]
0.939
1.675
SIR9722
SIR9722
refined by
SHELXL9723
R1 ) 0.0572, wR2 ) 0.1912
R1 ) 0.0637, wR2 ) 0.2082
0.532/-0.316
293
SHELXL9723
R1 ) 0.0696, wR2 ) 0.1989
R1 ) 0.0749, wR2 ) 0.2086
0.542/-0.345
293
final R indices [I > 2σ(I)]
R indices all data
∆F, e/Å3
temp, K
crystallization solvent
EtOAc/PEa
CH3CN/H2O
a
EtOAc, ethyl acetate; PE, petroleum ether.
5(4H)-Oxa zolon e fr om Z-[L-(rMe)Nva ]4-OH. Z-[L-(RMe)-
Nva]4-OH (0.09 g, 0.15 mmol), obtained by treating Z-[L-
(RMe)Nva]4-OtBu with CH2Cl2-TFA (trifluoroacetic acid), 1:1
was dissolved in anhydrous CH2Cl2. Then, EDC‚HCl (0.021 g,
0.17 mmol) was added. The reaction was stirred at room
temperature for 3 h. Then, the solvent was removed in vacuo,
and the residue was dissolved in EtOAc. The solution was
washed with 10% KHSO4, H2O, 5% NaHCO3, and H2O, dried
over Na2SO4, and evaporated to dryness: yield 95%, oil. TLC
Solvent (baseline) spectra were obtained under the same
conditions.
1
1H Nu clea r Ma gn etic Reson a n ce. The H NMR spectra
for conformational analysis were recorded with a Bruker model
AM 400 spectrometer. Measurements were carried out in
deuterochloroform (99.96% d; Acros Organics) and dimethyl-
d6 sulfoxide (Me2SO) (99.96% d6; Acros Organics) with tet-
ramethylsilane as the internal standard. The free radical
TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxy) was purchased
from Sigma.
X-r a y Diffr a ction . Colorless single crystals of the tripep-
tide Z-[L-(RMe)Nva]3-OtBu and the tetrapeptide Z-[L-(RMe)-
Nva]4-OtBu dihydrate were grown by slow evaporation at room
temperature from the solvents reported in Table 1. Data
collection was carried out on a CAD4 Enraf-Nonius X-ray
diffractometer at the Institute of Biostructure and Bioimaging,
CNR, Naples. Unit-cell determinations were carried out by
least-squares refinement of the setting angles of 25 high-angle
reflections that were accurately centered. No significant varia-
tion was observed in the intensities of the standard reflections
monitored at regular intervals during data collection, thus
implying electronic and crystal stability. Lorentz and polariza-
tion corrections were applied to the intensities, but no absorp-
tion correction was made. Crystallographic data for the two
compounds are listed in Table 1.
The two structures were solved by direct methods using the
SIR 97 program.22 The solution with the best figure of merit
revealed the coordinates of all non-H atoms. Refinements were
performed by full-matrix least-squares procedures with the
SHELXL 97 program.23 All non-H atoms were refined aniso-
tropically. H atoms were calculated, and during the refinement
they were allowed to ride on their carrying atoms, with Uiso
set equal to 1.2 times the Ueq of the attached atom. The
scattering factors for all atomic species were calculated from
Cromer and Waber.24 CCDC-216284 and CCDC-216283 con-
tain the supplementary crystallographic data for Z-[L-(RMe)-
Nva]3-OtBu and Z-[L-(RMe)Nva]4-OtBu dihydrate, respectively.
These data can be obtained free of charge at www.ccdc.ca-
m.ac.uk/conts/retrieving.html (or from the Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
U.K.; fax: (+44) 1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
20
Rf I 0.85, Rf II 0.95, Rf III 0.30; [R]D -5.0° (c 0.5, methanol).
IR absorption (film): νmax 3330, 1816, 1707, 1671, 1526 cm-1
.
1H NMR (250 MHz, CDCl3, 10 mM): δ 7.37 (s, 5H, Z phenyl
CH), 7.21 (br, 1H, NH), 6.58 (br, 1H, NH), 5.40 (br, 1H, NH),
5.11 (2d, 2H, Z CH2), 1.85-0.63 (m, 8H, 4 â-CH2), 1.50 (s, 3H,
â-CH3), 1.48 (s, 3H, â-CH3), 1.39 (s, 6H, 2 â-CH3), 1.33-1.05
(m, 8H, 4 γ-CH2), 0.96-0.83 (m, 12H, 4 δ-CH3).
Z-[L-(rMe)Nva ]8-OtBu . The 5(4H)-oxazolone from Z-[L-
(RMe)Nva]4-OH (0.084 g, 0.14 mmol) was dissolved in CH3-
CN, and H-[L-(RMe)Nva]4-OtBu [obtained by catalytic hydro-
genation of the corresponding Z derivative (0.10 g, 0.15 mmol)
in MeOH] was added. The reaction was stirred under reflux
for 12 days. The solvent was removed in vacuo, and the residue
was dissolved in EtOAc. The solution was washed with 10%
KHSO4, H2O, 5% NaHCO3, and H2O, dried over Na2SO4, and
evaporated to dryness. The product was isolated by flash
chromatography (eluant EtOAc-PE 3:7): yield 18%, mp 225-
226 °C (from EtOAc/PE). TLC Rf I 0.25, Rf II 0.95, Rf III 0.25;
20
[R]D 3.4° (c 0.3, methanol). IR absorption (KBr): νmax 3425,
1
3314, 1703, 1659, 1534 cm-1. H NMR (250 MHz, CDCl3, 10
mM): δ 7.68 (br, 1H, NH), 7.56 (br, 1H, NH), 7.52 (br, 1H,
NH), 7.44 (br, 1H, NH), 7.38 (s, 5H, Z phenyl CH), 7.33 (br,
1H, NH), 7.23 (br, 1H, NH), 6.32 (br, 1H, NH), 5.18 (br, 1H,
NH), 5.14 (2d, 2H, Z CH2), 1.79-0.64 (m, 16H, 8 â-CH2), 1.59-
1.41 (m, 24H, 8 â-CH3), 1.45 (s, 9H, OtBu 3 CH3), 1.40-1.28
(m, 16H, 8 γ-CH2), 1.02-0.81 (m, 24H, 8 δ-CH3).
In fr a r ed Absor p tion . The solid-state infrared absorption
spectra (KBr disk technique) were recorded with a Perkin-
Elmer model 580 B spectrophotometer equipped with a Perkin-
Elmer model 3600 IR data station and a model 660 printer.
The solution spectra were recorded using a Perkin-Elmer
model 1720 X FT-IR spectrophotometer, nitrogen-flushed and
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 Sigma.
Resu lts a n d Discu ssion
P ep tid e Syn th esis. For the large-scale preparation
of enantiomerically pure H-L-(RMe)Nva-OH, an eco-

Macromolecules, Vol. 36, No. 21, 2003
[L-(RMe)Nva]n Homopeptides 8167
nomically attractive and generally applicable chemoen-
zymatic synthesis was developed by DSM Research.25-29
Racemic H-(RMe)Nva-NH2 was prepared in 61% total
yield by a partial Strecker synthesis using 2-pentanone
and mild acid hydrolysis of the R-amino nitrile inter-
mediate. H-D,L-(RMe)Nva-NH2 was resolved using the
R-amino amidase from M. neoaurum ATCC 25795. At
51% conversion, H-L-(RMe)Nva-OH was obtained with
an ee >95%, the remaining H-D-(RMe)Nva-NH2 having
an ee >99.5%. This procedure resulted in a remarkably
high enantiomeric ratio of the specificity constants
(E = 232) for this enzymatic resolution.30 The same
resolution was also performed using the recently devel-
oped R-amino amidase from Ochrobactrum anthropi
NCIMB 40321.31 However, this enzyme proved to be less
selective, resulting in E = 97.
Then, the preparation and full characterization of the
terminally protected, monodispersed homo-oligomeric
series Z-[L-(RMe)Nva]n-OtBu (n ) 1-6, 8) were per-
formed. Z-L-(RMe)Nva-OtBu was obtained by esterifi-
cation of the corresponding Z-protected amino acid with
isobutylene in anhydrous methylene chloride in the
presence of a catalytic amount of sulfuric acid. The
synthesis from dimer to hexamer was carried out step-
by-step in solution, beginning from the C-terminal
R-amino tert-butyl ester. L-(RMe)Nva-L-(RMe)Nva pep-
tide bond formation was achieved either by the EDC/
HOAt32 or by the acyl fluoride33 C-activation procedure
in the presence of a tertiary amine (NMM). Z-L-(RMe)-
Nva-F was prepared in situ from the N-protected amino
acid and cyanuric fluoride in pyridine. The synthesis of
the homo-octamer was performed by the 4 + 4 segment
condensation strategy using the 5(4H)-oxazolone from
Z-[L-(RMe)Nva]4-OH as the electrophilic C-activated
component.34-36 The latter derivative was synthesized
from the N-protected tetrapeptide free acid, prepared
in turn by mild acidolysis of Z-[L-(RMe)Nva]4-OtBu with
a diluted TFA solution, using EDC in methylene chlo-
ride. Removal of the Z N-protecting group was achieved
by catalytic hydrogenation. Using these methodologies,
the L-(RMe)Nva-L-(RMe)Nva peptide bonds were formed
in variable yields, generally decreasing with increasing
peptide size.
F igu r e 1. FT-IR absorption spectra in the N-H stretching
region of the Z-[L-(RMe)Nva]n-OtBu (n ) 2-6, 8) homo-
oligomers in CDCl3 solution. Peptide concentration, 1 mM.
interpreted as arising almost exclusively from intra-
molecular N-H‚‚‚OdC interactions.
The present FT-IR absorption analysis has provided
evidence that intramolecular H bonding typical of folded
conformations is the predominant feature of the termi-
nally protected, longer L-(RMe)Nva homo-oligomers in
CDCl3 solution.
To get more detailed information on the preferred
conformation in CDCl3 of Z-[L-(RMe)Nva]8-OtBu, the
longest and most significant homopeptide of this series,
1
we carried out a 400-MHz H NMR investigation.
The NH proton resonances were assigned by means
of a 2D ROESY experiment beginning from the ure-
thane N(1)H proton at higher field and by analogy with
the results obtained with homo-octamers from other CR-
tetrasubstituted R-amino acids. An analysis of the
spectra as a function of peptide concentration (not
shown) indicates that a 6-fold dilution (from 6 to 1 mM)
produces a variation, albeit small (∆ppm ) 0.023, to
higher fields), of the chemical shift of the N(1)H proton.
For the N(2)H-N(8)H protons, the concentration effect
is even less significant or negligible. In agreement with
the FT-IR absorption results discussed above, we con-
clude that at 6 mM the modest self-association phe-
nomenon involves the urethane N(1)H group as the
main H-bonding donor.39,41
In the absence of self-association (peptide concentra-
tion, 1 mM), the delineation of inaccessible (or intra-
molecular H-bonded) NH protons was carried out with
the use of the solvent (Me2SO)42 dependence of NH
proton chemical shifts and free-radical (TEMPO)43
-induced line broadening of NH proton resonances.
Figure 2 (parts a and b) graphically describes the results
obtained. Two classes of NH protons were clearly
observed. The first class [N(1)H and N(2)H protons]
includes protons whose chemical shifts are sensitive to
the addition of the strong H-bonding-acceptor solvent
Me2SO44 and whose resonances significantly broaden
upon the addition of TEMPO. Interestingly, the sensi-
tivity of the N(1)H proton is significantly higher than
that of the N(2)H proton. The second class [N(3)H-
N(8)H protons] includes those displaying a behavior that
is characteristic of shielded protons (relative insensitiv-
ity of chemical shifts to solvent composition and of line
widths to the presence of paramagnetic agent TEMPO).
The present 1H NMR data support the view that at
low concentration (1 mM) in CDCl3 solution the N(3)H-
Solu t ion Con for m a t ion . The preferred confor-
mations of the Z/OtBu-protected L-(RMe)Nva homo-
oligomers were investigated in a structure-support-
ing solvent (CDCl3) by FT-IR absorption and 1H
NMR over the peptide concentration range from 10-
0.1 mM.
The FT-IR absorption spectra in the N-H stretching
(amide A) region (peptide concentration, 1 mM) are
illustrated in Figure 1. The curves are characterized by
bands at 3436-3425 cm-1 (free, solvated NH groups),
3398-3395 cm-1 (weakly H-bonded NH groups of fully
extended conformations),37 and 3368-3328 cm-1 (strongly
H-bonded NH groups of folded conformations).38-40 The
band at 3398-3395 cm-1 is the only one observed below
3400 cm-1 for the dimer; it is still one of the dominant
features in the spectrum of the trimer, but it is almost
completely absent from the spectrum of the tetramer.
The intensity of the lowest-frequency band relative to
those of the higher-frequency bands increases signifi-
cantly as the main-chain length increases. We have also
been able to demonstrate that even at 10 mM self-
association via intermolecular N-H‚‚‚OdC H bonding
is negligible or modest for all oligomers (results not
shown). Therefore, the observed H bonding should be

8168 Formaggio et al.
Macromolecules, Vol. 36, No. 21, 2003
F igu r e 2. (a) Plot of NH proton chemical shifts in the 1H NMR
spectrum of the Z-[L-(RMe)Nva]8-OtBu homo-oligomer as a
function of increasing percentages of Me2SO with respect to
the CDCl3 solution (v/v). (b) Plot of the bandwidth of the NH
protons of the same homo-oligomer as a function of increasing
percentages of TEMPO with respect to CDCl3 (w/v).
F igu r e 4. X-ray diffraction structure of homotetramer Z-[L-
(RMe)Nva]4-OtBu with atom numbering. The two intra-
molecular H bonds are represented by dashed lines.
Ta ble 2. Selected Tor sion An gles (d eg) for th e
Z-[L-(rMe)Nva ]n -OtBu (n ) 3, 4) Hom o-oligom er s
torsion angle Z-[L-(RMe)Nva]3-OtBu
Z-[L-(RMe)Nva]4-OtBu
θ3,1
θ3,2
θ2
-101.6(5)
79.6(4)
112.7(10)
-66.6(10)
158.4(7)
94.3(4)
θ1
-177.4(3)
-166.5(3)
-62.0(4)
-27.6(4)
-175.8(3)
-64.2(4)
-29.5(4)
-178.6(3)
44.5(4)
-177.0(7)
-178.5(4)
-50.9(6)
-35.9(5)
-169.6(3)
-54.3(6)
-30.0(6)
-179.4(4)
-60.0(5)
-36.9(5)
-175.7(3)
49.7(5)
ωo
φ1
ψ1
ω1
φ2
ψ2
ω2
φ3
ψ3
ω3
φ4
51.3(3)a
174.7(3)b
ψ4
ω4
43.9 (4)c
174.1(4)d
178.6(6)
179.6(11)
63.7(6)
-171.9(6)
-68.0(8)
-172.6(15)
-61.6(5)
176.6(6)
1
ø1
60.9(4)
-169.0(3)
60.1(5)
-178.4(5)
-60.5(5)
-174.7(5)
2
ø1
F igu r e 3. X-ray diffraction structure of homotrimer Z-[L-
(RMe)Nva]3-OtBu with atom numbering. The intramolecular
H bond is represented by a dashed line.
1
ø2
2
ø2
1
ø3
2
ø3
1
N(8)H protons of the octamer are inaccessible to solvent
and perturbing agents and are therefore most probably
intramolecularly H-bonded. The intramolecular H-bond-
ing scheme of the octamer does not appear to change
upon self-association [involving the N(1)H proton as the
donor of the intermolecular H bond]. Because all NH
protons, beginning with the N(3)H proton of Z-[L-(RMe)-
Nva]8-OtBu, form intramolecular H bonds, we are
inclined to conclude that the structure predominantly
adopted in CDCl3 by these peptides is the 310 helix (a
series of consecutive type-III â bends) rather than the
R helix, which would require the NH protons involved
in the intramolecular H bonding to begin from the
N(4)H proton.5 These more detailed conclusions are in
full agreement with the preliminary indications ex-
tracted from the FT-IR absorption study discussed
above.
Cr ysta l-Sta te Con for m a tion . We determined by
X-ray diffraction the molecular and crystal structures
of two Z/OtBu-protected L-(RMe)Nva homo-oligomers,
namely, the trimer and the tetramer (the latter in its
dihydrated form). The molecular structures with the
atomic numbering schemes are shown in Figures 3 and
4, respectively. Protecting groups, backbone, and side-
ø4
2
ø4
a
d
N3-CR3-C′3-OT. b CR3-C′3-OT-CT1
.
c N4-CR4-C′4-OT. CR
-
4
C′4-OT-CT1
.
chain torsion angles45 are given in Table 2. In Table 3,
the intra- and intermolecular H-bond parameters are
listed. Figure 5 illustrates the packing mode of the
tetramer in the crystal.
Bond lengths and bond angles (deposited) are in
general agreement with previously reported values for
the geometry of the benzyloxycarbonylamino urethane46
and tert-butyl ester47 moieties and the peptide unit.48,49
All seven L-(RMe)Nva residues populate the helical
region (A or A*)50 of the conformational (φ, ψ) space.
The average values for the (φ, ψ) backbone torsion
angles of the (RMe)Nva residue are (55.1 and (36.4°,
close to those expected for a 310 helix.5 In both the trimer
and tetramer, the signs of the (φ, ψ) values of the
C-terminal residue are opposite with respect to those
of the preceding ones. This observation is quite common
in the X-ray diffraction structures of peptide esters that
are heavily based on CR-tetrasubstituted R-amino ac-
ids.10,51 The 1-2 sequence of the trimer is folded into a

Macromolecules, Vol. 36, No. 21, 2003
[L-(RMe)Nva]n Homopeptides 8169
Ta ble 3. In tr a - a n d In ter m olecu la r H-Bon d P a r a m eter s for th e Z-[L-(rMe)Nva ]n -OtBu (n ) 3, 4) Hom o-oligom er s
symmetry
operation
N‚‚‚O
distance, Å
C′dO‚‚‚N
angle, deg
peptide
donor
acceptor
Z-[L-(RMe)Nva]3-OtBu
N3H
N1H
O0
O3
x, y, z
3.083(3)
2.929(3)
131.0(2)
143.6(2)
-x + 2, y + 1/2, -z + 1/2
Z-[L-(RMe)Nva]4-OtBu
dihydrate
N3H
N4H
N1H
N1H
N2H
O0
O1
O3
Ow1
Ow1
O2
O4
Ow2
x, y, z
x, y, z
1 + x, y, z
x, y, z
x, y, z
x, y, z
-x, y + 1/2, -z + 2
1 + x, y, z
3.240(5)
2.855(4)
2.958(8)
3.159(9)
3.086(6)
2.796(6)
2.917(7)
2.722(8)
131.8(3)
136.8(3)
177.1(3)
O
O
O
w2H
w2H
w1H
168.8(4)
119.8(3)
The crystal packing mode of the tripeptide is charac-
terized by an intermolecular H bond (N1-H‚‚‚O3dC′3),
giving rise to rows of molecules H-bonded in a head-to-
tail fashion along the b direction. van der Waals
interactions link together rows of peptide molecules in
the a and c directions.
In the crystals of the tetrapeptide, the molecules
are connected through an intermolecular
H bond
(N1-H‚‚‚O3dC′3), which generates rows in a head-to-
tail arrangement along the c direction (Figure 5). These
rows are held together by four intermolecular H bonds
(N1-H‚‚‚Ow1, N2-H‚‚‚Ow1, Ow2-H‚‚‚O2dC′2, and Ow2
-
H‚‚‚O4dC′4) involving the peptide and the two water
molecules. An additional intermolecular H bond is
observed between the two water molecules. The crystal
structure is further stabilized by van der Waals inter-
actions between the hydrophobic groups.
Con clu sion s
In this paper, we have described the successful
solution-phase synthesis of the sterically hindered
L-(RMe)Nva homo-oligomers to the octamer level using
either the step-by-step or the segment condensation
approach. Furthermore, the results of the solution
conformational analysis, combined with those extracted
from the crystal-state X-ray diffraction study, also
reported here, definitely confirm our earlier, preliminary
findings21 that L-(RMe)Nva has a remarkable propensity
for â-bend and 310-helix formation. This conclusion
strictly parallels those already reported for other
CR-methylated R-amino acids.11,12 As for the relationship
between (RMe)Nva R-carbon chirality and the screw
sense of the bend/helix that is adopted by its peptides,
the available X-ray diffraction data support the conten-
tion that this structural property is analogous to that
exhibited by protein amino acids (i.e., L-amino acids fold
into right-handed bends/helices).
In our view, the L-(RMe)Nva homo-oligomeric series
is the best among those derived from CR-tetrasubsti-
tuted R-amino acids for application as a set of rigid,
foldameric spacers in physicochemical investigations for
the following reasons: (i) It exhibits a stronger bias,
compared to the L-Iva series,16,17 toward right-handed
bends and helical structures because the two alkyl
substituents on the CR atom differ by two carbons. (ii)
It is much easier to synthesize than the series based
on the more sterically demanding â-branched (RMe)-
Val residue.20
F igu r e 5. Packing mode of the molecules of Z-[L-(RMe)Nva]4-
OtBu dihydrate in the crystal state as viewed along the a
direction (black circles, oxygen atoms; patterned circles,
nitrogen atoms).
1 r 4 C′dO‚‚‚H-N intramolecularly H-bonded â
bend conformation of the helical (III) type.13-15 The
C′0dO0‚‚‚H-N3 intramolecular separation is within the
limits expected for such H bonds.52-54 The 1-3 sequence
of the tetramer adopts a regular, right-handed, incipient
310-helical structure, stabilized by two consecutive 1 r
4 C′dO‚‚‚H-N intramolecular H bonds. One of the two
N‚‚‚O distances, N3‚‚‚O0 [3.240(5) Å], is near the upper
acceptable limit.
In each of the two molecules, only one significant
deviation in the ω backbone torsion angles (|∆ω| > 10°)
from the ideal value of the trans-planar urethane,
peptide, and ester units (180°) is observed: the urethane
ω0 value of the trimer and the peptide ω1 value of the
tetramer, which differ by 13.5 and 10.4°, respectively.
The trans, trans arrangement of the θ1, ω0 torsion
angles of the Z-NH-urethane moiety (type-b conforma-
tion) found for both homo-oligomeric molecules is that
commonly reported for Z-protected peptides.46 The tert-
butyl ester conformation of both the trimer and tetramer
with respect to the preceding CR-N bond is intermediate
between the anticlinal and antiperiplanar conforma-
tions.55 In the seven L-(RMe)Nva residues of the two
compounds examined, the N-CR-Câ-Cγ (ø1) torsion
angle is either in the g+ conformation (three times) or
in the g- (three times) and t (once) conformations (i.e.,
no clear side-chain conformation bias is observed for this
parameter). Conversely, the CR-Câ-Cγ-Cδ (ø2) torsion
angles are all trans.
Su p p or tin g In for m a tion Ava ila ble: Tables of positional
parameters, bond distances, bond angles, and torsion angles
for the X-ray diffraction structures of Z-[L-(RMe)Nva]3-OtBu
and Z-[L-(RMe)Nva]4-OtBu. This material is available free of
charge via the Internet at http://pubs.acs.org.

8170 Formaggio et al.
Macromolecules, Vol. 36, No. 21, 2003
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R. N., Ed.; Marcel Dekker: New York, 1999; pp 23-58.
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