Isotopically labelled and unlabelled β-peptides with geminal dimethyl substitution in 2-position of each residue: Synthesis and NMR investigation in solution and in the solid state

Article abstract of DOI:10.1002/1522-2675(200209)85:9<2877::AID-HLCA2877>3.0.CO;2-W

The preparation of (S)-β2.2.3-amino acids with two Me groups in the α-position and the side chains of Ala, Val, and Leu in the β-position (double methylation of Boc-β-HAla-OMe, Boc-β-Val-OMe, and Boc-β-Leu-OMe, Scheme 2) is described. These β-amino acids and unlabelled as well as specifically ¹³C- and ¹⁵N-labelled 2,2-dimethyl-3-amino acid (β^2.2-HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give protected (N-Boc, C-OMe), partially protected (N-Boc/C-OH, N-H/C-OMe), and unprotected β^2.2- and β^2.2.3.-hexapeptides, and β^2.2- and β^2.2.3-heptapeptides 1-7. NMR Analyses in solution (Tables 1 and 2, and Figs. 2-4) and in the solid state (2D-MAS NMR measurements of the fully labelled Boc-(β^2.2-HAib)₆-OMe ([¹³ C₃₀, ¹⁵N₆]-1e; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac-(β^2.2-HAib)₇-OMe (4) and of a labelled derivative ([¹³C₄,¹⁵N₂]-5; Figs. 7-11, and 19), a molecular-modeling study (Figs. 13-15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i) there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted peptide chains in solution; ii) there are distinct conformational preferences of the individual β^2.2- and β^2.2.3-amino acid residues: close to eclipsing around the C(O)-C(Me₂(CHR)) bond (τ_1.2), almost perfect staggering around the C(2)-C(3) ethane bond (τ_2.3), and antiperiplanar arrangement of H(C3) and H(N) (τ_3,N; Fig. 12) in the solid state; iii) the β^2.2-peptides may be part of a turn structure with a ten-membered H-bonded ring; iv) the main structure present in the solid state of F₃CCO(β^2.2-HAib)₇-OMe is a nonfolded chain (> 30 A between the termini and > 20 A between the N-terminus and the CH₂ group of residue 5) with all C=O bonds in a parallel alignment (± 10°). With these structural parameters, a simple modelling was performed producing three (maybe four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick-zack and crankshaft-type arrangement of the peptide bonds), and (possibly) a fourth with meander-like winding (D - G in Figs. 17 and 18).

Full text of DOI:10.1002/1522-2675(200209)85:9<2877::AID-HLCA2877>3.0.CO;2-W

Helvetica Chimica Acta Vol. 85 (2002)
2877
Isotopically Labelled and Unlabelled b-Peptides with Geminal Dimethyl
Substitution in 2-Position of Each Residue: Synthesis and NMR Investigation
in Solution and in the Solid State
by Dieter Seebach*, Thierry Sifferlen1), Daniel J. Bierbaum2), Magnus Rueping2), Bernhard Jaun,
and Bernd Schweizer
Laboratorium f¸r Organische Chemie der Eidgenˆssischen Technischen Hochschule, ETH-Hˆnggerberg, HCI,
Wolfgang-Pauli-Strasse 10, CH-8093 Z¸rich
and
Jacob Schaefer*, Anil K. Mehta, and Robert D. O×Connor
Faculty of Arts and Sciences, Department of Chemistry, Washington University, Campus Box 1134,
One Brookings Drive, Saint Louis, MO 63130-4899, USA
and
Beat H. Meier*, Matthias Ernst, and Alice Gl‰ttli2)3)
Laboratorium f¸r Physikalische Chemie der Eidgenˆssischen Technischen Hochschule, ETH-Hˆnggerberg,
HCI, Wolfgang-Pauli-Strasse 10, CH-8093 Z¸rich
Dedicated to Professor Albert I. Meyers, Colorado State University, on the occasion of his 70th birthday
The preparation of (S)-b2,2,3-amino acids with two Me groups in the a-position and the side chains of Ala,
Val, and Leu in the b-position (double methylation of Boc-b-HAla-OMe, Boc-b-Val-OMe, and Boc-b-Leu-
OMe, Scheme 2) is described. These b-amino acids and unlabelled as well as specifically 13C- and 15N-labelled
2,2-dimethyl-3-amino acid (b2,2-HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give
protected (N-Boc, C-OMe), partially protected (N-Boc/C-OH, N-H/C-OMe), and unprotected b2,2- and b2,2,3
hexapeptides, and b2,2- and b2,2,3-heptapeptides 1 7. NMR Analyses in solution (Tables 1 and 2, and Figs. 2 4)
and in the solid state (2D-MAS NMR measurements of the fully labelled Boc-(b2,2-HAib)6-OMe ([13C30 15N6]-
-
,
1e; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac-(b2,2-HAib)7-
OMe (4) and of a labelled derivative ([13C4,15N2]-5; Figs. 7 11, and 19), a molecular-modeling study (Figs. 13
15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i)
there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted
peptide chains in solution; ii) there are distinct conformational preferences of the individual b2,2- and b2,2,3-amino
acid residues: close to eclipsing around the C(O)ÀC(Me2(CHR)) bond (t1,2), almost perfect staggering around
the C(2)ÀC(3) ethane bond (t2,3), and antiperiplanar arrangement of H(C3) and H(N) (t3,N; Fig. 12) in the
solid state; iii) the b2,2-peptides may be part of a turn structure with a ten-membered H-bonded ring; iv) the
main structure present in the solid state of F3CCO(b2,2-HAib)7-OMe is a nonfolded chain (> 30 ä between the
termini and >20 ä between the N-terminus and the CH2 group of residue 5) with all CˆO bonds in a parallel
1
)
)
)
Postdoctoral research at ETH Z¸rich (1998 2000) financed by the Swiss National Science Foundation,
Grant No. 20-50674.97/1.
Part of the projected Ph.D. Theses of D. J. B. and A. G., and of the Dissertation No. 14677 by M. R., ETH-
Z¸rich.
2
3
Gruppe f¸r Informatikgest¸tzte Chemie.

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Helvetica Chimica Acta Vol. 85 (2002)
alignment (Æ108). With these structural parameters, a simple modelling was performed producing three (maybe
four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick-zack and
crankshaft-type arrangement of the peptide bonds), and (possibly) a fourth with meander-like winding (D
in Figs. 17 and 18).
G
1. Introduction. The influence of geminally disubstitued b-amino acid residues
(Fig. 1) [1 6] on the secondary structure (i.e., folding pattern) of b-peptides was first
discussed in 1996, when we showed by circular-dichroism (CD) spectroscopy that a b3-
HAib residue in the central positon of a b3-heptapeptide −destroys× the 314-helical
structure present in MeOH solutions of corresponding b3-hexa- and b3-heptapeptides
lacking the geminal dimethyl groups [5]. Detailed analyses revealed that the b-peptidic
314 helix does not tolerate non-H atoms4) or groups in axial positions due to steric
hindrance5) in the ca. 5-ä pitches [3][6][7]. Most disturbingly, the CD spectra of b-
hexapeptides consisting of b2,2,3-trisubstituted amino acid moieties6) were essentially
superimposable [3] on those of b-peptides, which clearly form 314 helices [4 8].
Without structural assignments and with no synthetic details, these CD spectra have
been published [3], and the CD −puzzle× has led to an elaborate molecular dynamics
(MD) study (with the GROMOS simulation package) and a theoretical calculation
(according to the so-called matrix method [9]) of the CD spectrum of a b-hexapeptide
built from trisubstituted b-amino acids7) in MeOH, including a comparison with the
corresponding simple b3-hexapeptide, the conclusion being that there is no preferred
conformation (secondary structure) in MeOH solution of the chain carrying three
substituents on each b-amino acid, in spite of very similar, distinct measured and
calculated CD spectra [10]. On the other hand, CHARM m23.1 and ab initio
calculations by Hofmann and co-workers of structures with simple geminally dimethyl-
substituted b2,2- and b3,3-amino acid residues8) led to helical and b-strand-type
secondary structures in the gas phase [11].
To obtain more experimental data about b-peptides with geminally-disubstituted
residues, we have now synthesized derivatives 1 5 of specifically 13C- and 15N-labelled
achiral b2,2-HAib oligomers (for solid-state NMR analysis), and we describe here, in
full detail, the preparation of the chiral precursors and the assembly of b2,2,3
-
hexapeptides 6 and 7, with and without terminal protection, as well as the results of
extensive solution and solid-state-NMR investigations of these compounds9).
4
)
)
)
Fluorine has, so far, not been tested; work on the synthesis of 3-amino-2-fluoro-acids and the derived b-
peptides is underway in the group of D.S.
Like in a-peptides and proteins, geminal disubstitution of residues should also prevent pleated-sheet
formation in b-peptides (see the discussion in [3]).
Even more irritating is that a b-peptide consisting of five (S)- and one (R)-3-amino-2,2-dimethylbutanoic
acid building blocks (see 6c, below) still gave rise to the same CD pattern [3], while incorporation of one
−wrong× or d-b3-amino acid residue into the center of a chain consisting of six l-b3-amino acid residues
caused the characterisitic CD pattern of a 314 helix to disappear [5].
5
6
7
8
9
)
)
)
Cf. the molecular formula 7 (below).
Cf. the molecular formulae 1 6 (below) and Fig. 1.
As mentioned above, the CD spectra have been published previously in this journal [3].

Helvetica Chimica Acta Vol. 85 (2002)
2879
Fig. 1. Geminally disubstituted b-amino-acid residues. Structural information on the cyclopropane and
cyclohexane derivatives has been obtained from single-crystal X-ray analysis; oligomers consisting of the 2,2-
dimethylene residues fold to ribbon-type arrangements of eight-membered H-bonded rings [2], those with 2,2-
pentamethylene substitution form a ten-membered H-bonded turn. No detailed structural information is
available for the b-HAib oligomers and for those consisting of trisubstituted b-amino acid residues, except that
the latter ones give rise to a pattern in the CD spectrum [3] identical to that of the nonmethylated analogs
[4 6].
2. Synthesis of the Achiral b2,2-Peptides 1 5.
The numerous methods for
preparing geminally-disubstituted b-amino acids have been collected in a review article
that covers the literature up to 1999 [12]10). For the large-scale preparation of the
unlabelled Boc-protected methyl 3-amino-2,2-dimethylpropanoate (Boc-b2,2-HAib-
OMe; 1a), we used the method described previously (double methylation of Boc-b-
HGly-OMe) [1]; we also followed procedures for the coupling and fragment coupling
to oligomers 1, 2, and 3 b f reported previously [1]11). The isotopically labelled
compounds [13C]-1a and [13C,15N]-1a were purchased12) and shown by NMR spectro-
scopy to contain >99% of the label(s) at the indicated positions. The fully 15N- and 13C-
labelled b2,2-HAib derivatives [13C5,15N]-1a and [13C30,15N6]-1e had been prepared by us
previously [1].
For application of the rotational-echo double-resonance (REDOR) NMR mea-
surements in the solid state (vide infra, Sect. 5.2), which provide distances between
magnetic nuclei, b2,2-heptapeptides 4 and 5 were prepared, with positioning of the 19F,
15N, and 13C labels in such a way that various intramolecular distances would result,
which could be compared with various models of the b2,2-peptide backbone (in these
models the distances between certain nuclei would be different, vide infra, Sect. 6). To
clearly distinguish between intra- and intermolecular distances by this method, the
10
)
For recent papers on b-amino acids and b-peptides with geminal disubstitution, see [13]. For cryptophycin
analogs containing b2,2-amino-acid residues and for human chymase inhibitiors containing b2,2,3-amino acid
residues, see [14].
The b2,2-HAib derivatives 1a, b, c, e, and 2a, b, and 3, which have been characterized or used in our
previous work [1], are not included in the Exper. Part of the present paper.
Isotec Inc., 3858 Benner Road, Miamisburg, OH 45342, USA.
11
12
)
)

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Helvetica Chimica Acta Vol. 85 (2002)
labelled materials [1,3,5,7-13C4, 1,5-15N2]-5 and [1,3,4,6-13C4, 1,4-15N2]-5 had to be diluted
with unlabelled material to form solid solutions13), and, for this purpose, we also
synthesized the b2,2-heptapeptide ester 4 with an N-terminal Ac group.
The assembly of the heptamers is outlined in Scheme 1. For peptide formation, the
Boc-b2,2-HAib methyl ester or the corresponding oligomers containing two, three, or
four b2,2-HAib units were deprotected at the N-terminus (with TFA14) in CH2Cl2) or at
the C-terminus (with NaOH in MeOH/H2O), and coupled with EDC/HOBt/Et3N14)
13
)
)
For an example of solid solutions formed by the dinitrobenzoates of ethyl (S)-3-hydroxybutanoate and
ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate, see [15].
14
TFA ˆ CF3COOH; EDC ˆ 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; HOBt ˆ 1-
Hydroxy-1H-benzotriazole; TFAA ˆ Trifluoroacetic acid anhydride; DIPEA ˆ H¸nig base ˆ EtN(i-Pr)2;
for years, the common coupling reagent HATU was thought to exist as uronium salt (N,N,N,N-
tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate [16]); however, X-ray crystallo-
graphic analysis revealed its true structure as guanidinium salt (N-form) [17].

Helvetica Chimica Acta Vol. 85 (2002)
2881
O
O
O
Boc
Boc
Boc
N
H
N
N
H
OMe
OMe
H
6
3
6a
6b
O
O
O
N
H
N
N
6c
OMe
H
H
3
2
O
O
O
7a R1 = Me, R2 = Boc
7b R1 = H, R2 = Boc
7c R1 = H, R2 = H·TFA
R2
OR1
N
N
N
H
H
H
Boc
(S,S)-8-10 (l or syn configuration)
(2R,3S)-8-10 (u or anti configuration)
NH
O
(S)
R
OMe
O
8 R = Me, 9 R = Me2CH, 10 R = Me2CHCH2
Me
11 R1 = Me, R2 = Boc
12 R1 = Me, R2 = H·TFA
13 R1 = H, R2 = Boc
R2
OR1
n
N
H
a n = 1
b n = 2
c n = 4
14 R1 = Me, R2 = Boc
15 R1 = Me, R2 = H·TFA
16 R1 = H, R2 = Boc
O
O
R2
OR1
N
N
H
H
n
a n = 1, b n = 2
O
O
O
17 R = Boc
R
N
N
H
N
18 R = H·TFA
OMe
H
H
2
(CHCl3, room temperature, overnight); the CF3CO ( ! 5) and the MeCO group
( ! 4) were introduced with the corresponding anhydrides14) and H¸nig×s base14). All
fully protected Boc-(b2,2-HAib)n-OMe compounds were chromatographed, the Boc-
(b2,2-HAib)n-OH acids and the TFA salts of the N-deprotected esters were obtained as
crude products in high purity (NMR analysis) and used as such for the coupling steps
(for characterizations of the intermediates and of the heptapeptides, see the Exper.
Part).

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Scheme 1. Assembly of b2,2-HAib-Oligomers from Labelled and Unlabelled b2,2-HAib Building Blocks
a) NaOH, MeOH/H2O, 08 ! r.t. b) TFA/CH2Cl2, 08 ! r.t. c) EDC, HOBt, Et3N, CHCl3, 08 ! r.t. d) TFAA,
DIPEA, CH2Cl2 (for abbreviations, see Footnote 14).
3. Synthesis of the b2,2,3-Peptides 6 and 7. For the b-hexapeptides 6 and 7 consisting
of trisubstituted b2,2,3-residues, we had to develop an easy preparative access to the
corresponding 3-amino-2,2-dimethyl-alkanoate building blocks 11 13 and 19 22
(Scheme 2). Of the many methods available [12][13], we chose the methylation of

Helvetica Chimica Acta Vol. 85 (2002)
2883
Scheme 2. Preparation of the N-Boc- or C-OMe-Protected b-Amino-Acids H-b-HAla(aMe2)-OH, H-b-
HVal(aMe2)-OH, and H-b-HLeu(aMe2)-OH for Solution Synthesis of b2,2,3-Peptides
a) 10n NaOH, CF3CH2OH, 408. b) TFA, CH2Cl2 (for abbreviations, see Footnote 14).
doubly lithiated b-amino acid derivatives Boc-NH-CHR-CH2-CO2Me [18], which we
had used before for the preparation of like- and unlike-2-methyl-3-amino-alkanoates 8,
9, and 10 [7][18][19]. The protected b-amino acids, in turn, are readily prepared by
Arndt-Eistert homologation15) of the a-amino acid derivatives Boc-Ala-OH, Boc-Val-
OH, and Boc-Leu-OH, with rearranging decomposition of the intermediate diazo
ketones in MeOH. The methylation of the doubly lithiated methyl N-Boc-b-amino-
alkanoates with MeIwas known to give mixtures of diastereoisomers of l and u
configuration16). At first, we were confident that we could use these mixtures without
separation of the stereoisomers for a subsequent second methylation to give the 2,2-
dimethyl derivatives 11a, 19, and 21, because, from both isomers, the same enolate A
15
)
)
For a review article with numerous original references, see [20].
Addition of Li salts or of the co-solvent DMPU (−N,N'-dimethylpropylene urea×) was found to give rise to
high diastereoselectivities, which depend also upon the nature of the protecting group on the N-atom
[18][21].
16

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Helvetica Chimica Acta Vol. 85 (2002)
Scheme 3. Coupling Steps Leading to the b2,2,3-Hexapeptides 6 with Three Me Groups on Each Residue
a) 10n NaOH, CF3CH2OH, 408. b) TFA, CH2Cl2. c) EDC, HOBt, Et3N, CHCl3 (for abbreviations, see Footnote
14).
(Scheme 2)17) should be generated. This was indeed the case with the butanoic acid
derivative Boc-NH-CHMe-CH2-CO2Me ( ! (l-8 ‡ u-8) ! A (R ˆ Me) ! 11a), so that
the oligomers 6, 11b 13b, c, 14a 16a, 16b, 17, and 18 could be prepared, with the same
methods as for the simple b2,2-HAib derivatives described in the previous section (see
formulae 1 5 and Scheme 1), except that the saponification of the C-terminal methyl
ester groups required much more drastic conditions (up to 100 equiv. NaOH in
CF3CH2OH at 40 508; see Exper. Part for details); a schematic presentation of the
assembly of the Boc-b2,2,3-hexapeptide esters 6 is given in Scheme 3. When we tried to
apply the same procedure of introducing the second Me group to the l/u mixtures of the
monomethylated b-HVal and b-HLeu derivatives 9 and 10, only one of the
diastereoisomers was actually methylated to give the desired products 19 and 21,
while the other diasteroisomer was recovered in very pure form; configurational
assignment of the diastereoisomers by comparison with literature data [7] revealed that
the u form had reacted, and the l form had remained unchanged18). To avoid tricky
chromatographic separation of the mono- and dimethylated compounds l-9/19 and
l-10/21, we separated the monomethylated l/u isomers of 9 and 10, and used the pure
u-forms for the second methylation, which occurred in 75 80% yield (Scheme 2). The
reason for the large rate difference for proton abstraction from the two diastereo-
isomers is obviously steric hindrance by the neighboring i-Pr and i-Bu groups R in the
intermediate mono-Li derivative l-B, but not in u-B (Scheme 2).
With the Boc-b2,2,3-amino acid esters 19 and 21 available, we could proceed with the
solution synthesis
by stepwise and by fragment coupling
of the hexapeptide
17
)
)
The structure of A in Scheme 2 is presented as a Li enolate / Li azacarboxylate with complexation of Li on
O and N, because this is the type of structure observed in crystal structures of lithiated carboxylic acid
amides [22].
18
NMR Analysis of the crude product mixtures showed that, within detection limits, there was no trace of
the u forms left.

Helvetica Chimica Acta Vol. 85 (2002)
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Scheme 4. Synthesis of the b2,2,3-Hexapeptide 7 with Ala, Val, and Leu Side Chains in the 3-Position
O
O
c)
Boc
20
12a
+
N
H
N
OR
H
23 R = Me
24 R = H
a)
O
O
O
d)
R2
OR1
24
22
+
N
N
H
N
H
H
25 R1 = Me, R2 = Boc
26 R1 = Me, R2 = H·TFA
27 R1 = H, R2 = Boc
a) b)
d)
a)
b)
27
26
7a
7b
7c
+
a), b) and c) as in Scheme 3. d) HATU/EtN(i-Pr)2, CHCl3 (for abbreviations, see Footnote 14).
derivatives 7 (by using the intermediate Boc-protected acids 20, 24, and 27, and the
methyl ester TFA salts 22 and 26) through the fully protected di- and tripeptides 23 and
25, respectively (see Scheme 4). Again, drastic conditions were required for saponi-
fication of methyl ester groups, and, for the last two coupling steps, activation by
HATU/H¸nig base14) rather than by EDC/HOBt/Et3N was necessary for achieving high
yields of 79 and 75%, respectively. For characterization of the final products and of the
intermediates involved, we refer to the Exper. Part.
The b-peptides 4 6 with their regular structures containing only one amino acid
residue of a kind (without −floppy side chains×) had been chosen for the present study in
the hope that we would obtain crystalline samples for single-crystal X-ray structure
analysis. Unfortunately, none of them met our expectations, so far, inspite of numerous
crystallization attempts by various co-workers over a period of several years. The b-
hexapeptide 7 with three different residues was prepared for an NMR analysis in
solution (see Sect. 4).
In contrast to previously prepared b2- and b3-peptides with aliphatic side chains on
the amino acid residues, which become insoluble19) in H2O and in most organic
solvents with increasing chain lengths [4][5][7], the compounds described here are well
soluble in organic media (not in H2O!).
4. NMR Measurements with the b2,2- and b2,2,3-Hexapeptide 4, 6a, and 7c in
Solution. The 400-MHz 1H-NMR spectrum of the N-acetyl-b2,2-heptapeptide methyl
ester 4 in CDCl3 is shown in Fig. 2. The most remarkable feature of the spectrum is the
This is not true for the −mixed× b2/b3-peptides, which are nonpolar compounds well-soluble in organic
19
)
media [7][23].

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 2. 400-MHz-1H-NMR Spectrum of the N-Acetyl-b2,2-HAib-heptapeptide methyl ester 4 in CDCl3. Note the
sharp signal for all Me2C H-atoms and the rather small coupling constant J(H(N)H(CH2)) (for details, see
Exper. Part).
single signal, actually a singlet, from all 14 geminal Me groups at 1.19 ppm; the CH2
groups give rise to a group of signals between 3.29 and 3.35 ppm; the NH H-atoms
appear as five signals between 6.5 and 7.3 ppm. There is, of course, no chance of
deriving a structure from this spectrum!
The 500-MHz 1H-NMR spectrum of the N-Boc-b2,2,3-hexapeptide methyl ester 6a is
shown in Fig. 3. In this case, the 18 backbone Me groups produce 14 clearly separated
signals (theoretically 12 singlets and 6 doublets) between 1.08 and 1.30 ppm (see the
inset in Fig. 3); the CH H-atoms give rise to three multiplets at 3.53, 3.81, and 3.98 ppm,
with the one at lowest field as a doublet (ca. 9 Hz) of quadruplet (ca. 7 Hz), the NH
doublets appear at 6.20, 7.10, 8.18, 8.30, 8.39, and 8.40 ppm (J ˆ 8.2 to 9.4 Hz). A
detailed NMR analysis leading to a solution structure does not appear to be
impossible, especially with a higher-field spectrometer.
In the b2,2,3-hexapeptide derivatives 7, containing the side chains of Val, Ala, and
Leu, the chance of arriving at full assignment of all signals and, thus, of determining the
structure was much larger. Hence, it was of interest to examine the secondary structure

Helvetica Chimica Acta Vol. 85 (2002)
2887
Fig. 3. 500-MHz- 1H-NMR Spectrum of the N-Boc-(b2,2-HAib(b-Me))6-OMe 6a consisting of (S)-residues. The
inset is an enlargement of the region containing the signals from the 18 MeC H-atoms (except for t-Bu). The NH
signals are doublets of ca. 8.8 Hz. For details, see the accompanying text and the Exper. Part.
of the b2,2,3-hexapeptide 7c by means of high-resolution NMR spectroscopy in MeOH.
The 1H-NMR spectrum of the fully deprotected hexapeptide 7c, recorded at 500 MHz,
showed, indeed, good separation of the signals and allowed complete assignment of all
resonances (including each of the 22 Me signals). By DQF-COSY, TOCSY, HSQC, and
HMBC techniques, the amino acid spin systems were identified (Fig. 4).
The sequence assignment was then established by HMBC measurements and
dN,a(Me)(i, i À 1) sequential NOEs (Table 1). The NH H-atoms of residues 2 6 show
coupling constants J(NH,HÀC(b)) in the range of 8 10 Hz, which correspond to an
antiperiplanar arrangement of NH and HÀC(b). To obtain more information about
the three-dimensional structure in solution, ROESY spectra at different mixing times
were acquired, and NOEs were extracted from the ROESY spectra with a mixing time
of 100 ms (Table 2). Integration of the cross-peak volumes followed by calibration
allowed the classification of the NOEs in three distance categories: strong, medium,
and weak.
Qualitative inspection of the NOEs reveals that only intraresidual and sequential
NOEs are present, indicating an extended or unstructured conformation of 7c in
solution. The NOE data also give no evidence for a 314-helical structure as suggested
by the CD measurements; in fact, there are no NOEs related to those observed for
any of the helical or turn structures found in b-peptides (without cyclic residues) so
far.

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 4. 1H-NMR Spectra of b2,2,3-hexapeptide 7c in MeOH solution. Assignments of the H-atoms in the spectra
are indicated by numbers 1 6, which correspond to residues 1 6 (residues 2 and 5 ˆ b-HAla, residues 4 and
5 ˆ b-HVal, residues 3 and 6 ˆ b-HLeu).

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Table 1. 1H- and 13C-NMR Chemical Shifts of the b2,2,3-Hexapeptide 7c
b-Amino NH
CˆO MeÀC(a) HÀC(b) HÀC(g), 2 HÀC (g), HÀC (d), MeÀC (e)
acid
(J(NH, Hb) [Hz])
MeÀC(g)
MeÀC (d)
Val1
177.59 1.352/1.341 3.16
24.82/22.77 65.06
2.17
28.72
1.110/0.968
22.11/16.26
Ala2
Leu3
Val4
Ala5
Leu6
8.53
(8.86)
178.81 1.312/1.233 3.86
23.48/25.78 54.31
1.116
17.06
7.60
(9.72)
178.89 1.315/1.246 4.09
24.35/24.19 55.46
1.41/1.31
41.77
1.50
26.44
0.917/0.880
23.00/24.14
8.19
(9.35)
178.86 1.318/1.196 3.82
23.60/26.77 62.44
2.05
30.62
0.914/0.799
21.67/17.61
8.42
(8.61)
178.92 1.231/1.295 3.80
25.79/23.43 54.38
1.096
22.21
7.11
(9.84)
180.24 1.160/1.188 4.20
22.81/24.10 54.49
1.47/1.25
40.87
1.51
26.46
0.915/0.918
21.67/24.09
Table 2. NOEs in the ROESY Spectra (300 ms) of 7c in MeOH (w ˆ weak, m ˆ medium, s ˆ strong)
Atom
Residue
Atom
Residue
NOE
Atom
Residue
Atom
Residue
NOE
b
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
g
1
1
1
1
1
2
1
3
2
1
1
3
2
1
1
1
2
2
2
3
2
3
3
3
3
2
3
3
3
3
3
3
2
3
m
m
m
w
m
m
m
w
m
w
w
w
s
m
m
w
m
s
m
m
m
m
s
b
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
e
3
3
4
3
4
3
3
4
4
4
4
4
4
3
4
4
5
4
4
4
5
4
4
5
5
6
5
5
6
6
5
6
5
6
m
m
m
w
m
s
b
a(Me)
d
NH
NH
NH
NH
NH
NH
NH
NH
b
b
b
b
b
d
g
g
a(Me)
d
g
g
a(Me)
a(Me)
g
d
s
NH
NH
NH
NH
NH
NH
NH
NH
NH
b
b
d
w
m
m
s
b
d
b
g
g
b
a(Me)
a(Me)
g
b
s
a(Me)
a(Me)
b
d
m
m
m
w
m
w
s
g
a(Me)
d
g
g
d
g
d
a(Me)
a(Me)
NH
NH
NH
NH
NH
NH
NH
b
b
b
g
b
g
a(Me)
a(Me)
NH
NH
NH
NH
NH
NH
NH
NH
NH
b
b
s
b
g
m
w
w
m
m
m
s
m
w
w
w
w
m
w
d
d
g
d
a(Me)
a(Me)
s
g
m
w
w
w
m
s
m
s
w
s
NH
NH
NH
NH
NH
NH
NH
NH
b
b
d
g
e
a(Me)
a(Me)
a(Me)
a(Me)
e
d
b
g
b
g
g
b
a(Me)
g
e
b
g
e
b
e
b

Fig. 5. Two-dimensional 15N-exchange spectra of the fully 13C- and 15N-labelled hexapeptide 1e. The spectra were recorded with an initial cross-polarization period from
the protons to enhance signal intensity, and with proton-driven spin diffusion as a mixing sequence. The spinning frequency was set to 1000 Hz and stabilized to within
5 Hz. The spectrum with a mixing time of tm ˆ 0 s (a) shows, as expected, no cross-peaks, while the spectrum with a mixing time of tm ˆ 1 s (b) shows weak cross-peaks
on the first and second side diagonal. Part c shows five simulated quasi-equilibrium two-dimensional exchange spectra with different angles b between the two axially
symmetric N chemical-shielding tensors. For an angle b ˆ 08, as expected, no off-diagonal intensity is observed, while, for all other angles, significant off-diagonal cross-
peaks are found.

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5. Solid-State NMR Investigations. 5.1. Magic-Angle Spinning (MAS) Double
Resonance of the Fully 15N- and 13C-Labelled Hexapeptide 1e. Two-dimensional
exchange spectra [24] under slow magic-angle spinning (MAS) conditions allow
determination of the relative orientation of chemical-shielding tensors in a molecule
[25]. Such an experiment follows the general scheme of two-dimensional NMR
spectroscopy. During the time t1, the polarization is frequency-labelled with the
resonance frequency of the first spin, then transferred to a second spin, and during
t2 detected with the resonance frequency of the second spin [26]. Under MAS,
we obtain a side-band spectrum of [13C30,15N6]-1e, which is characteristic for the
relative orientation of the two chemical-shielding tensors. Fig. 5 shows two-dimensional
15N-exchange spectra of the fully labelled hexapeptide 1e under slow magic-angle
spinning (MAS) (nr ˆ 1000 Hz) for mixing times tm ˆ 0 (Fig. 5,a) and tm ˆ 1 s
(Fig. 5,b).
As expected, the spectrum with a vanishing mixing time (Figr. 5, a) shows no cross-
peaks and two sets of diagonal-peak families spaced by the spinning frequency. The
weaker side-band family with the centerband at À1.54 kHz arises from the N-terminal
Boc-protected amino group. All the other five amide N-atoms contribute to the
stronger side-band family with the center band at 0 Hz. The full width at half height of
the lines is ca. 140 Hz, indicating a strong structural similarity between the five amide
N-atoms. The spectrum with a mixing time (Fig. 5,b) shows only weak cross-peaks
along the first and second side diagonals. Numerical simulations of the tensor-
correlation spectra show that such a spectrum is a clear indication that the symmetry
axes of the two axially symmetric tensors are parallel or antiparallel to within 108, as
can be seen from Fig. 5,c. Assuming that the symmetry axis of the chemical-shielding
tensors is aligned along the NÀH bond direction, this implies that the NÀH bonds are
parallel or antiparallel to within 108. Very similar experimental results were obtained
for 13C 2D exchange spectra of [13C30,15N6]-1e by measuring the relative orientation of
the CˆO chemical-shielding tensors (data not shown). At a mixing time of tm ˆ 100 ms,
intense cross-peaks between the CˆO C-atom and all other C-atoms could be seen due
to the fast polarization transfer across the one-bond dipolar couplings. However, there
were only weak cross-peaks visible along the first side diagonal within the side-band
family of the CˆO tensor even at mixing times as long as tm ˆ 1 s. Numerical
simulations of the tensor-correlation spectra indicate that the two tensors are parallel or
antiparallel to within less than 108. Since the CˆO tensor is not axially symmetric, this
implies that the two peptide planes are either parallel or antiparallel to within 108, and
that the NÀH bond as well as the CˆO bond are also parallel or antiparallel to within
108.
5.2. REDOR and TEDOR-REDOR Spectra. As mentioned in Sect. 2, this solid-
state NMR technique requires −dilution× of the specifically labelled compound with the
unlabelled one to form a so-called solid solution. While this is no problem with the
isotopic labels 13C and 15N, CF 3 in the heptapeptide derivatives 5 had to be −diluted×
with CH313). To our surprise, solutions containing 16 50% of compound 4 (with N-Ac)
and 84 50% of compound 5 (with N-CF3CO) gave, upon solvent evaporation, solid
samples, in which the two compounds appeared to have been separated from each
other: upon heating in a melting-point tube, there was melting between 156 and 1608,
and resolidification and melting again above 1668 (see Fig. 6); the samples obtained

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from the melt upon solidification did not show the two-stage melting behavior any
more. Thus, it looks like the difference between CH3CO and CF3CO in a peptide of
molecular weight 734 is sufficient for a phase separation! This demixing was confirmed
by initial REDOR measurements, and the solid solutions used in the subsequent
experiments had to be prepared carefully (see Exper. Part).
Fig. 6. Melting behavior of mixtures of the N-acetyl- and N-trifluoroacetyl-b2,2-heptapeptide methyl esters 4 and 5.
As an example, the 33 :67 mixture melts between 157 and 1608, solidifies, and melts again between 173 and 1768.
The mixtures were prepared by precipitation with hexane from solutions in CH2Cl2.
The cross-polarization magic-angle-spinning (CPMAS) 50.3-MHz 13C-NMR spec-
trum of [1,3,5,7-13C4, 1,5-15N2]-5, (R ˆ COCF3), diluted tenfold by 4, shows peaks near
180, 45, and 25 ppm (Fig. 7,a), which are due to 13C-labelled and natural-abundance
CˆO, CH2, and Me C-atoms, respectively. Shift resolution of site differences within the
13
b-heptapeptide is poor. The 15N ! C TEDOR spectrum of this blend is simpler but
not any better resolved (Fig. 7,b). Nevertheless, an expansion of the CˆO C-atom
region of the rotational-echo double-resonance (REDOR) spectrum of the same
labelled version of 5, but now diluted 50-fold by 4, shows the presence of two
overlapping peaks (Fig. 8,c). The low-field peak of this pair is not observed in spectra
of [1,3,4,6-13C4, 1,4-15N2]-5 (R ˆ COCF3 (not shown)), a result that allows an
assignment of the low-field peak (178 ppm) to the terminal labelled CˆO C-atom in
residue 7 (see formula in Fig. 7).
The combination of transferred-echo double-resonance (TEDOR) with REDOR
13
within the same pulse sequence is called TEDOR-REDOR. The 15N ! C{19F}
TEDOR-REDOR full-echo spectrum of the CH2 C-atom region of [1,3,5,7-13C4, 1,5-
15N2]-5 (R ˆ COCF3), diluted 50-fold by 4, has only a single, relatively broad peak
centered near 45 ppm, with just a hint of a shoulder toward low field (Fig. 8,d). Only

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Fig. 7. CPMAS 13C-NMR Spectrum (a) and 15N ! 13C TEDOR spectrum (b) of a homogeneous blend of 1 part
[13C,15N,19F]-labelled b-heptapeptide [1,3,5,7-13C4, 1,5-15N2]-5 (R ˆ COCF3) and 10 parts unlabelled b-heptapep-
tide 4. The blend was precipitated from CH2Cl2/pentane. The TEDOR selection was adjusted so that only 13C
directly bonded to 15N was observed.
the labelled CH2 C-atoms in residues 1 and 5 are directly bonded to 15N and so
contribute to this spectrum. The corresponding REDOR difference is also a single
peak, but the center of this peak is shifted downfield from the center of the full-echo
peak by several ppm (Fig. 8,b and d). Curve fitting with overlapping Gaussian peaks
results in a deconvolution that is consistent with a much stronger 13C, 19F dipolar
coupling (larger REDOR difference) for the weaker, low-field full-echo peak. The
1
same strong 13C{19F} dephasing was observed following a H-19F-13C double-cross-
polarization preparation of carbon magnetization (not shown). Thus, we assign this
low-field peak (48 ppm) to the labelled CH2 C-atom in residue 1 (orange in the formula
in Fig. 7), which is close to the CF3 group, and the stronger, high-field full-echo peak

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Fig. 8. Deconvolution of 13C{19F} REDOR spectra (a and c) and 15N ! 13C{19F} TEDOR-REDOR spectra (b and
d) of a homogeneous blend of 1 part [13C,15N,19F]-labelled b-heptapeptide [1,3,5,7-13C4, 1,5-15N2]-5 (R ˆ COCF3;
see formula in Fig. 7) and 50 parts unlabelled b-heptapeptide 4. The REDOR differences are shown in a and b
and the full echoes in c and d. An intensive low-field peak (red) near 180 ppm in the REDOR spectra was only
observed in samples containing a 13C-labelled CˆO C-atom in residue 7.
(46 ppm) to the more distant labelled CH2 C-atom in residue 5 (green). We attribute
the differences in full-echo intensities after 128 rotor cycles to differences in T2 values
for the end and middle of the b-heptapeptide. The 13C{19F} DS/S0 for the CH2 C-label in
residue 1 suggests a ca. 5-ä distance to the terminal CF3 (Fig. 9), which is plausible for
a three-bond distance [27].
The 13C{19F} DS/S0 for the CˆO C-label in residue 7 (Fig. 10) and the CH2 C-label in
residue 5 (Fig. 11) is between 6 and 10% after 25 ms of dipolar evolution. For
dephasing as small as this, the possibility exists of contributions to the REDOR
difference from natural-abundance 13C proximate to 19F and with the same shift as one
of the labels. The small but discernable shift resolution of the terminal CˆO C-atom in
residue 7 (see Fig. 8,c) allowed estimation of this natural-abundance contribution to
the dephasing. We determined that the 13C{19F} DS/S0 (for the low-field peak at
178 ppm) for a homogeneous blend of 1 part of 5 (no C-labels) and 10 parts of 4 was
12 Æ 2% after 128 rotor cycles, and for a blend of 1 part of 5 and 50 parts of 4, only 2%.
Thus, some 70 80% of the dephasing of Figs. 10 and 11 (solid lines) is due to b-
heptapeptide intramolecular 13C, 19F coupling. This means that a fraction of the b-
heptapeptides exist in a bent or kinked conformation that brings the two ends of the
peptide close to one another. The dephasing simulations [28] shown in the insets to

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Fig. 9. 13C{19F}-Dephasing (DS/S0) for the labelled CH2 C-atom in residue 1 (orange) of the b-heptapeptide
sample of Fig. 7. The solid and dotted lines show the calculated dephasing, assuming a 5-ä distance from the 13
C
label to the center of the F3 triangle and a 13C ! C(F3) vector parallel (08) or perpendicular (908) to the plane of
the three F-atoms.
Figs. 10 and 11 suggest that ca. 5 10% of the b-heptapeptides are kinked and that 90
95% are fully extended in the solid state.
6. Discussion and Conclusions. 6.1 Solution Structures. As pointed out in Sect. 4,
the NMR spectra in MeOH solution could either not be analyzed in detail (4 and 6a) or
led to the conclusion that there is no folded secondary structure, which would produce
long- or medium-range inter-residual NOEs, on the NMR time scale (7c). This
conclusion about the structure of the b-hexapeptide 7c (carrying three side chains on
each residue) is in agreement with the results of long (100 ns) MD simulations,
performed in MeOH at 298 and 340 K, by which a myriad of different conformers was
found [10]20). The preferred conformations around the C(Me2)ÀCO (t1,2),
C(HR)ÀC(Me2) (t2,3), C(HR)ÀN(HCO) (t3,N), and C(O)ÀN(H) (t1,N) bonds in
the backbone of b-hexapeptide 7c are shown in Figs. 12 15; besides the peptide bond
(t1,N) the N(H)ÀC(HR) bond has a single conformation at 298 and 340 K in five amino
acid residues of 7c with N(H) and HC(R) in an antiperiplanar disposition (t3,N
,
20
)
This is in sharp contrast to the situation with geminally disubstituted a-peptides, such as oligomers of Aib
or Iva, which form helices with as few as three residues! For a structural analysis (NMR and X-ray) of
oligomers consisting of Iva residues, Boc(NH-CMeEt-CO)n-OMe, see [29] and refs. cit. therein, as well as
papers by Toniolo, Balaram, and Karle with their respective co-workers [30].

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Fig. 10. 13C{19F}-Dephasing (DS/S0) for the labelled CˆO C-atom in residue 7 (red) of the b-heptapeptide
(shown in Fig. 7). The solid line in the inset shows the calculated dephasing, assuming a Gaussian distribution of
distances (centered at 6.7 ä with a 5-ä width) from the 13C label to the CF3-dephasing center for 6% of the
labelled b-heptapeptides, and a separation of more than 30 ä for 94% of the labelled b-heptapeptides.
Fig. 11. 13C{19F}-Dephasing (DS/S0) for the labelled CH2 C-atom in residue 5 (green) of the b-heptapeptide
(formula in Fig. 7). The solid line in the inset shows the calculated dephasing assuming a Gaussian distribution
of distances (centered at 7.7 ä with a 3.6-ä width) from the 13C label to the CF3 dephasing center for 11% of the
labelled b-heptapeptides, and a separation of more than 20 ä for 89% of the labelled b-heptapeptides.

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Fig. 12. Definition of the dihedral angles t in the back bone of b2,2- and b2,2,3-peptides and preferred angles t1,2 , t2,3
,
t1,N, and t3,N found by MD studies of 7c with the GROMOS simulation package (cf. Figs. 13 16) [10]. The atoms
between which the dihedral angles are defined are printed in bold in the Newman projections. For t1,2 see
Fig. 13, for t2,3 see Fig. 14, for t3,N see Fig. 15. The torsion angle t1,N around the amide (peptide) bond is close to
1808 in all conformations of all residues! ap ˆ antiperiplanar, sc ˆ synclinal, ac ˆ anticlinal.
Fig. 15), in agreement with the large coupling constant between these two H-atoms in
the NMR spectrum of 7c21).
6.2 Solid-State Structures. For proposing a solid-state structure of [13C30,15N6]-1e and
5, we feel like pupils, having to determine n unknowns with fewer than n equations.
Disregarding the <10% turn content22) of 5 and assuming that the b2,2-hexa- and
the b2,2-heptapeptide derivatives [13C30,15N6]-1e and 5 have the same backbone
arrangements, we consider the following structural features: i) all CˆO bonds are
parallel (or antiparallel), ii) the distance between the termini of 5 is >30 ä; iii) the
distance between the N-terminus of 5 and the 13CH2 of residue 5 is >20 ä; iv) the
21
)
)
We also observed these large coupling constants in the NMR spectrum of the b-peptide 6a (see Sect. 4 and
Fig. 3) with three Me groups on each residue.
b2,2-Amino acid residues can be part of a turn formed by a ten-membered H-bonded ring [2]. It is probably
possible to have such a turn or kink in part of the molecules in the solid state of 5 and arrive at a distance
of ca. 7 ä between the N-terminus and the 13CO of residue 7.
22

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Fig. 13. Distribution of dihedral angles C(HR)ÀC(Me2)ÀC(O)ÀN(H) (t1,2) of the residues 1 5 in the b2,2,3
-
hexapeptide 7c observed in 100-ns MD simulations in MeOH at 25 and 678. The dihedral angles were calculated
for over 200 000 structures extracted at 0.5-ps intervals from the simulations [10]. x-Axis: angles [8], y-axis:
number of conformations having a given dihedral angle in the corresponding amino acid residue 1 5. Blue:
298 K, red: 340 K. As can be seen, t1,2 is ca. 2008 in the two b-HVal(Me2) and ca. 3008 in the other three residues
at room temperature. At higher temperature, other t1,2 angles are found (30, 80, 100, 160, and 2708). The most
prominent angles are shown as Newman projections in Fig. 12. Clearly, there is a rather flat conformational
energy profile around t1,2 , with less sharp preferences than found for t2,3, t3,N, and t1,N (cf. Figs. 12 and 14 16).

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Fig. 14. Distribution of dihedral angles N(H)ÀC(HR)ÀC(Me2)ÀCO (t2,3) of the residues 1 6 in the b2,2,3
-
hexapeptide 7c observed in 100-ns MD simulations in MeOH at 25 and 678 [10]. For details and definitions, see
caption of Fig. 13, and for Newman projections of the preferred conformations, see Fig. 12. All t1,2 angles (ca. 60,
180, and 3008) are those of staggered ethane moieties, and, at room temperature, the b2,2,3-amino acid residues
with Ala and Leu side chains (No. 2, 3, 5 and 6) are mostly in an ap conformation (ca. 1808), while the residues
(No. 1 and 4) with Val side chains are in a (À)-sc conformation (ca. 3008). At 678, all residues show a shift of the
conformational equilibrium towards sc (−gauche×), while b2,2,3-HVal(Me2) (No. 1) is mainly in the ap (−trans×)
conformation.

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Fig. 15. Dihedral angle C(O)ÀN(H)ÀC(HR)ÀC(Me2) (t3,N) of the residues 2 6 in the b2,2,3-hexapeptide 7c
observed in 100-ns MD simulations in MeOH at 25 and 678 [10]. For details and definitions, see caption of
Fig. 13, and, for presentations (Newman and −flying wedge× formulae), see Fig. 12. Clearly, the angle t3,N is fixed
near 2458, i.e. (À)-ac, with the CˆO O-atom and the C(R,CMe2)H H-atom in a 1,5-coplanar arrangement (no
Newman strain! [52]) and the NH H-atom staggering between the two large groups R and C(Me2(CO)).

Helvetica Chimica Acta Vol. 85 (2002)
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amide group is planar, placing C(Me2), C(O), O, N, H, and C(H2) in one plane; v) the
corresponding dihedral angles OˆCÀNÀH and C(Me2)ÀC(O)ÀN(H)ÀC(H2) are
ca. 1808; and vi) the ethane bond C(H2)ÀC(Me2) has a staggered, rather than an
eclipsed conformation. With these structural features, there are still numerous
possibilities for the backbone arrangements of 5.
To obtain more structural information, a search in the Cambridge file23) for the
substructures of (C3)CÀC(O)ÀN(H)ÀC(H2)ÀC(C3)24) was undertaken; the results
are collected in Fig. 16. Not surprisingly, the structural features defined in iv vi above
are confirmed. In addition, there is a preference for a dihedral angle near 1808 of
HÀNÀCÀH (Fig. 16,c; structural feature vii; cf. t3,N, Figs. 12 and 15).
Simple computer modelling for the N-acetyl-b-heptapeptide ester 4, with the
structural prerequisites i and iv vii above, produced a number of arrangements of b2,2-
peptide chains, including wide helical [11] and meander-type structures. The four
structures D G, which are also compatible with the conclusions ii and iii from the
REDOR spectra, are shown schematically in Fig. 17 and as rendered 3D presentations
in Fig. 18. All models have the CˆO groups pointing in the same direction, and they are
all unable to form sheets due to steric hindrance (by Me groups) of intermolecular H-
bonding25). The fully extended conformation D (in-line amide and CÀC bonds) has a
length well above 30 ä (as deduced from the REDOR measurements), and it is the
only one in which H(N) and H(CH) are not antiperiplanar (t3,N); the pairs of geminal
Me groups are equivalent in this conformation. Backbone arrangement E is the one
found in b-peptide sheets [4][19][32] with parallel displaced amide planes. In F, the
peptide bonds are parallel to the chain axis. In the somewhat short model G, there is a
meander-type winding of the chain, which consists of elements found in cyclo-b-
tripeptides [33], and also in the b-peptide 31 or 314 helix [7]. There is no way, at this
point26), to distinguish between the models, or to be sure that there are not alternative
arrangements27).
In summary, the solution- and solid-state-NMR investigations of the labelled and
unlabelled b-peptides with geminal dimethyl substitution in the 2-position, combined
with simple modelling, Cambridge file searches, and MD calculations [10], have
provided intriguing details about the structure and dynamic behavior of this type of b-
peptides. Unlike in a-peptides20), the geminal disubstitution leads to a much more
flexible structure of b-peptides in solution, with no evidence for folding to a helix. Like
geminally disubstituted a-amino acid residues, the b2,2-analogs may be part of a turn
23
)
)
Cambridge Structural Data Base, CSD ConQuest software; search of July 2002.
There are quite a few more structures in the CSD now, as compared to the early eighties, when Dunitz
et al. performed their classical analysis of amide and ester crystal structures [31].
24
25
)
With antiparallel CˆO groups, it is difficult to have staggering of the ethane moieties and to arrive at large
enough distances (conditions ii, iii, and vi).
26
27
)
)
Powder X-ray measurements with 1f and 5 produced no suitable diffraction patterns.
When the coordinates of the models D, E, and F, as shown in Fig. 18, are loaded into the program
SPARTAN SGIVersion 5.1.3 with constraints for parallel C ˆO bonds and subjected to MD or semi-
empirical energy minimization, the models D, E, and F interchange, depending on the method of
optimization. Furthermore, all three models D, E, and F wind up with a flat curvature rather than as
straight chains. This is mainly caused by an enlargement of the angle MeÀC(Me)ÀC(O) above 1098 (cf.
Fig. 16), with simultaneous slight rotation around the C(Me2)ÀC(O) bond, such that the distance between
the NH H-atom and the neighboring Me group increases.

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Fig. 16. Results of a CSD search for the substructures of C. C(2) was chosen to have any three C substituents.
The angles t are as in Fig. 12. Conditions for structures included: R value <0.07 ä, no errors, no disorder,
polymers allowed, ionic species allowed. a1) Angles t1,2 vary widely from 0 to Æ 1808 (cf. the results of the MD
study in Fig. 13); a2) as −we rotate× around the C(1)ÀC(2) bond, there is widening of the C(1)ÀC(2)ÀC angle
from tetrahedral to ca. 1148, as we approach coplanarity of the NH H-atom with a C substituent at C(2). b) The
sp3Àsp3 bond between C(2) and C(3) is staggered (t2,3 angles Æ 60 and 1808). c) The quaternary C(2)-atom is
out-of-plane with respect to the amide plane, i.e., one H(3) and the H(N) are approximately antiperiplanar
(ap), avoiding O, C(C3) 1,5-repulsion (Newman strain [52]; cf. the MD conclusion, Fig. 15): t3,N angles are
centered near Æ 1008.

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D
E
F
G
amide plane,
C(2)–C(3) bond
N to C
36 Å
24 Å
35 Å
23 Å
34 Å
23Å
27 Å
18 Å
terminus
N-terminus
to residue 5
REDOR distance
N to C terminus >30 Å
N terminus to residue 5 > 20 Å
Fig. 17. Schematic representations of four possible back-bone structures D G of the b2,2-heptapeptide derivative
4 (and 5) in the solid state. The bold lines represent the amide planes CÀCOÀNHÀC, the thin lines the sp3Àsp3
C,C bonds. The distances between the N-terminal CH3 (CF3) Ac C-atom and the C-terminal CˆO C-atom
(CO2Me) or the CH2 C-atom of residue 5 in these models are given in the table and are compared with the
distances derived from REDOR measurements. Except for G, these models have chain lengths within the limits
from the REDOR NMR determination. For ball-and-stick models of the four conformations, see Fig. 18.
structure (ten-membered H-bounded ring; cf. Fig. 1 [1]22), and both prevent sheet
formation by steric hindrance of the NH H-bond donor; the models in Fig. 18 nicely
show the shielding of the NH H-atoms by the −parallel× C-CH3 groups, especially in E
G; it is also evident that the chains in Fig. 18 are still able to act as H-bond acceptors,
because access to the CˆO O-atom is not blocked a chain like E could be used for
−capping× a b-peptide sheet [19]. Thus, geminally dimethyl-substituted b2,2-peptide
sections may be useful components in our ongoing construction of more complex b-
peptides and in our search for tertiary structures of b-peptides.
We gratefully acknowledge the financial support by the Swiss National Science Foundation1) and by
Novartis. This work was also supported, in part, by NIH grant GM40634 (J. S.).
Experimental Part
1. General. Abbreviations: DIPA: (i-Pr)2NH, DIPEA: (i-Pr)2NEt, EDC: 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride, FC: flash chromatography, HATU: N,N,N',N'-tetramethyl-O-(7-azabenzo-
triazol-1-yl)uronium hexafluorophosphate14), HOBt: 1-hydroxy-1H-benzotriazole, h.v.: high vacuum, 0.01 0.1

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Fig. 18. Ball-and-stick models of four conformations of Ac-(b2,2-HAib)7-OMe, fulfilling (D F) or almost
fulfilling (G) the structural conditions i vii given in the accompanying text. For a schematic presentation of the
backbones, see Fig 17. The models were produced with the program MOLMOL 2.6 and rendered with POV-
Ray 3.5 for Mac OS.

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Torr. b-HXxx: b-homoamino acid [4][5], TFA: CF3CO2H, TFAA: (CF3CO)2O, TFE: CF3CH2OH. CHCl3
employed for the coupling reactions was filtered over Al2O3 (Alumina Woelm N and ICN Alumina B, Akt. I) to
remove EtOH. THF was freshly distilled over K/benzophenone under Ar before use. Et3N was distilled from
CaH2 and stored under Ar. DIPA and DIPEA were freshly distilled over CaH2. Solvents for chromatography
and workup were distilled from Sikkon (anh. CaSO4: Fluka; AcOEt, pentane), P2O5 (CH2Cl2) or FeSO4/KOH
(Et2O). Amino acid derivatives were purchased from Bachem, Senn, or Degussa. The labelled b-amino acids
were purchased from ISOTEC Inc. All other reagents were used as received from Fluka. The unlabelled b2,2
-
HAib derivatives up to the tripeptide were synthesized according to [1]. For the synthesis of the b3-amino acids
and the a-methyl b3-amino acids l-8/u-8, ent-l-8l/ent-u-8, u-9, u-10, see [4][5][7][18][19]. Flasks and stirring bars
for the alkylations were dried for ca. 12 h at 1208 and allowed to cool in a dessicator over silica gel. All indicated
temps. were monitored with an internal thermometer (Ebro TTX-690 digital thermometer). TLC: Merck silica
gel 60 F254 plates with UV, anisaldehyde (9.2 ml of anisaldehyde, 3.75 ml of AcOH, 12.5 ml of conc. H2SO4,
350 ml of EtOH) or KMnO4 soln. (0.5 g of KMnO4, 100 ml of 1m NaOH). FC: Fluka silica gel 60 (40 63 mm); at
ca. 0.3 bar. Anal. HPLC: Knauer HPLC system K 1000, pump type 64, EuroChrom 2000 integration package,
degaser, UV detector K 2000 (variable-wavelength monitor), Macherey-Nagel C18 column: Nucleosil 100-5 C18
(250 Â 4 mm). Prep. HPLC: Knauer HPLC system: pump type 64, programmer 50, UV detector (variable-
wavelength monitor), Macherey-Nagel C18 column: Nucleosil 100-7 C18 (250 Â 21 mm). M.p.: B¸chi 510
apparatus; uncorrected. Optical rotations: Perkin-Elmer 241 polarimeter (10 cm, 1-ml cell) at r.t. Circular
dichroism (CD) spectra: Jasco J-710, recording from 190 to 250 nm at 208; 1-mm rectangular cell; average of five
scans, corrected for the baseline; peptide concentration 0.2 mm in MeOH; molar ellipticity [q] in deg ¥ cm2 ¥
molÀ1 (l in nm); smoothing by Jasco software. IR Spectra: Perkin-Elmer 782 spectrophotometer. NMR Spectra:
Bruker AMX-500 (1H: 500 MHz, 13C: 125 MHz), AMX-400 (1H: 400 MHz, 13C: 100 MHz): chemical shifts d in
ppm downfield from internal Me4Si (ˆ0 ppm); J values in Hz; signals for rotamers are underlined; signals for
labelled C-atoms are in italics. MS: Hitachi-Perkin-Elmer RHU-6M (FAB), Finnigan MAT TSQ-7000 (ESI)
spectrometer and IonSpec Ultima FTMS-Spectrometer (HR-MALDI); in m/z (% of basis peak). Elemental
analyses were performed by the Microanalytical Laboratory of the Laboratorium f¸r Organische Chemie, ETH-
Z¸rich.
2. Boc Deprotection: General Procedure 1 (GP 1). Similarly to the procedures reported in [1][4][5][7], the
Boc-protected amino acid was dissolved in CH2Cl2 (0.23 0.8m) and cooled to 08. An equal volume of TFA was
added, and the mixture was allowed to warm to r.t., and stirring continued for further 1 to 1.5 h. Concentration
under reduced pressure, co-evaporation with CH2Cl2, and drying of the residue under h.v. yielded the crude TFA
salt, which was identified by NMR and used without further purification.
3. Methyl Ester Hydrolysis: General Procedure 2 (GP 2). GP 2a: Similarly to the procedure reported in
[34], a soln. of the protected amino acid or b-peptide in MeOH (1.2m) was treated with 1n NaOH (1.2 equiv.) at
08. The soln. was allowed to warm to r.t., and stirring was continued for 16 h at r.t. After dilution with H2O, the
soln. was adjusted to pH 2 with 1n NaHSO4 and extracted with AcOEt (3Â). The org. phase was washed with
sat. aq. NaCl soln., dried (MgSO4), and concentrated under reduced pressure. The acid was dried under h.v. and
used in the next step without further purification.
GP 2b: The protected amino acid or b-peptide was dissolved in TFE (1.2m), treated with 10n NaOH (20
30 equiv.), and heated at 408. After completion of the reaction (TLC), the mixture was diluted with H2O, and
the pH was adjusted to 2 3 with 1n HCl (08). After extraction with AcOEt, the org. phase was washed with sat.
aq. NaCl soln., dried (MgSO4), and concentrated under reduced pressure. The acid was dried under h.v. and
used in the next step without further purification.
4. Peptide Coupling with EDC: General Procedure 3 (GP 3). According to [1][35], the appropriate TFA
salt was dissolved in CHCl3 or CH2Cl2 (0.5m) and cooled to 08. This soln. was treated successively with Et3N,
HOBt (1.2 equiv.), a soln. of the Boc-protected fragment (1 equiv.) in CHCl3, CH2Cl2, or DMF (0.25m), and
EDC (1.2 equiv.). The mixture was allowed to warm to r.t., and stirring was continued for 15 h with exclusion of
light. Subsequent dilution with CHCl3 was followed by thorough washing with 1n HCl soln. (3Â) and sat. aq.
NaHCO3 (2Â) and NaCl solns. (1Â). The org. phase was dried (MgSO4) and then concentrated under reduced
pressure. FC yielded the pure peptide.
5. Preparation of the Trifluoroacetamide Compounds: General Procedure 4 (GP 4). Similarly to the
procedure reported in [36], a CH2Cl2 soln. of the TFA salt (0.1m) of the appropriate peptide was cooled to 08 and
treated with DIPEA (4 5 equiv.) and TFAA (1.5 equiv.). After 60 min at 08, the reaction soln. was allowed to
warm to r.t., and stirring was continued for another 30 min. Subsequent dilution with CHCl3 was followed by
thorough washing with 1n NaHSO4 soln. (3Â) and sat. aq. NaHCO3 (3Â), and NaCl solns. (2Â). The org. phase
was dried (MgSO4) and then concentrated under reduced pressure. FC yielded the pure trifluoroacetamide.

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6. Alkylation of the b2,3-Amino Acid Derivatives: General Procedure 5 (GP 5). According to [5], BuLi (2.2
equiv.) was added to a soln. of DIPA (2.2 equiv.) in THF (1.42m) at À788. After 1 h, a soln of the b2,3-amino acid
derivative (1 equiv.) in THF (0.40m) was added, and stirring was continued for 1.5 h at À788. MeI(4 equiv.) was
then added slowly, and the mixture was stirred for 5 h at this temp., subsequently hydrolyzed with a sat. NH4Cl
soln., diluted with Et2O, and extracted with sat. NaHCO3, NH4Cl, and NaCl solns. The org. layer was dried
(MgSO4) and then concentrated under reduced pressure. FC yielded the pure b2,2,3-amino acid.
7. Peptide Coupling with HATU: General Procedure 6 (GP 6). The appropriate TFA salt, HATU (1.2
equiv.) and the Boc-protected fragment (1 equiv.) were dissolved in CHCl3 (0.4m), and the soln. was treated
with DIPEA (5 equiv.). The resulting mixture was stirred under Ar at r.t., and the reaction was monitored by
TLC. After completion of the reaction, the mixture was diluted with CHCl3, followed by thorough washing with
1n HCl (4Â), sat. aq. NaHCO3 soln. (3Â), and sat. aq. NaCl soln. (1Â). The org. phase was dried (MgSO4) and
then concentrated under reduced pressure. FC yielded the pure peptide.
Boc-b2,2-HAib-b2,2-HAib(13CO)-OMe ([13C]-1b). Amino acid [13C]-1a (502 mg, 2.04 mmol, labelling ca.
99%, 1 equiv.) was Boc-deprotected according to GP 1, dissolved in CHCl3 (4.1 ml), and treated with Et3N
(1.28 ml, 9.18 mmol, 4.5 equiv.), HOBt (400 mg, 2.96 mmol, 1.45 equiv.), acid 3a (532 mg, 2.45 mmol, 1.2 equiv.)
dissolved in CHCl3 (8.2 ml), and EDC (626 mg, 3.26 mmol, 1.6 equiv.) according to GP 3. FC (Et2O/pentane
3 :2) yielded [13C]-1b (622 mg, 1.88 mmol, 92%). White powder. Rf (Et2O/pentane 3 :2) 0.31. 1H-NMR
3
(400 MHz, CDCl3): 1.18 1.19 (m, 4 Me); 1.42 (s, t-Bu); 3.21 (d, J(H,NH) ˆ 6.5, CH2); 3.33 3.36 (m, CH2);
3.71 (d, 3J(H,13C) ˆ 3.8, MeO); 5.20 (br., NH); 6.41 (br., NH). 13C-NMR (100 MHz, CDCl3): 23.13, 23.65, 28.38
(Me); 42.93, 43.38, 43.49 (C); 46.67, 48.89 (CH2); 52.15, 52.18 (Me); 79.02 (C); 83.10 (CH2); 156.41, 177.00,
177.61, 177.89 (C).
Boc-b2,2-HAib[13CH2,15N]-b2,2-HAib-b2,2-HAib[13CO]-OMe ([13C2,15N]-1c). Dipeptide [13C]-1b (812 mg,
2.45 mmol, 1 equiv.) was Boc-deprotected according to GP 1, dissolved in CHCl3 (4.5 ml), and treated with Et3N
(1.88 ml, 13.5 mmol, 5.5 equiv.), HOBt (463 mg, 3.43 mmol, 1.4 equiv.), acid [13C,15N]-3a (630 mg, 2.87 mmol, 1.2
equiv.) in CHCl3 (8 ml), and EDC (751 mg, 3.92 mmol, 1.6 equiv.) according to GP 3. FC (AcOEt/pentane 3 :2)
yielded [13C2,15N]-1c (961 mg, 2.22 mmol, 91%). White powder. Rf (AcOEt/pentane 3 :2) 0.22. IR (CHCl3):
3446m, 3007m, 2977m, 2933m, 2872w, 1703s, 1652s, 1514s, 1490s, 1472s, 1392m, 1368m, 1310m, 1166s, 1144s,
916w, 858w. 1H-NMR (400 MHz, CDCl3): 1.17 1.19 (m, 6 Me); 1.42 (s, t-Bu); 3.03 (d, 3J(H,NH) ˆ 5.7, 0.5 CH2);
3
3.31 3.39 (m, 2.5 CH2); 3.71 (d, J(H,13C) ˆ 3.8, MeO); 5.25 (br., 1J(H,15N) ˆ 91.1, NH); 6.47 (br., NH); 6.88
(br., NH). 13C-NMR (100 MHz, CDCl3): 23.10, 23.11, 23.62, 23.80, 28.40 (Me); 42.55, 42.90, 43.02, 43.39, 43.46
(C); 46.71, 47.36, 48.89, 49.00, 50.29 (CH2); 52.19, 52.22 (Me); 78.88 (C); 83.23 (CH2); 156.28, 156.54, 177.26,
177.56, 177.62, 177.90, 178.18 (C).
Boc-b2,2-HAib-b2,2-HAib-b2,2-HAib-b2,2-HAib-OMe (1d). Tripeptide 1c (3.71 g, 8.64 mmol, 1 equiv.) was
Boc-deprotected according to GP 1, dissolved in CH2Cl2 (25 ml), and treated with Et3N (6.0 ml, 43 mmol, 5
equiv.), HOBt (1.40 g, 10.3 mmol, 1.2 equiv.), 3a (1.91 g, 8.79 mmol, 1 equiv.) in CH2Cl2 (15 ml), and EDC
(1.98 g, 10.3 mmol, 1.2 equiv.) according to GP 3. FC (CH2Cl2/MeOH 40 :1) yielded 1d (3.38 g, 6.39 mmol,
74%). White powder. M.p. 135.0 136.08. Rf (CH2Cl2/MeOH 40 :1) 0.15. IR (CHCl3): 3451m, 3007m, 2972m,
2933w, 2872w, 1710s, 1649s, 1507s, 1474m, 1392w, 1367m, 1312m, 1158s, 990w. 1H-NMR (400 MHz, CDCl3): 1.18
(s, 2 Me); 1.19 (s, 6 Me); 1.42 (s, t-Bu); 3.20 (d, 3J(H,NH) ˆ 6.4, CH2); 3.30-3.35 (m, 3 CH2); 3.71 (s, MeO); 5.30
(br., NH); 6.50 (br., NH); 7.00 7.05 (br., 2 NH). 13C-NMR (100 MHz, CDCl3): 23.09, 23.61, 23.73, 23.75, 28.39
(Me); 42.35, 42.41, 43.15 (C); 46.72, 47.38, 47.41, 48.95 (CH2); 52.23 (Me); 78.82, 156.42, 177.20, 177.48, 177.68,
‡
‡
177.91 (C). FAB-MS: 551.3 (7.5, [M ‡ Na] ), 529.3 (100.0, M ), 429.2 (69.4), 399.2 (10.3), 231.2 (11.2). Anal.
calc. for C26H48N4O7 (528.69): C 59.07, H 9.15, N 10.60; found: C 59.19, H 9.12, N 10.48.
Boc-b2,2-HAib[15N,13CH2]-b2,2-HAib-b2,2-HAib[13CO]-b2,2-HAib-OMe ([1,3-13C2, 1-15N]-1d). Amino acid 1a
(150 mg, 648 mmol, 1.3 equiv.) was Boc-deprotected according to GP 1, dissolved in CHCl3 (1 ml), and treated
with Et3N (383 ml, 2.74 mmol, 5.5 equiv.), HOBt (105 mg, 773 mmol, 1.55 equiv.), [13C2,15N]-3c (209 mg,
499 mmol, 1 equiv.) in CHCl3 (2 ml), and EDC (172 mg, 898 mmol, 1.8 equiv.) according to GP 3. FC (CH2Cl2/
MeOH 40 :1) yielded [1,3-13C2, 1-15N]-1d (251 mg, 472 mmol, 95%). White powder. Rf (CH2Cl2/MeOH 40 :1)
0.15. 1H-NMR (500 MHz, CDCl3): 1.17 1.19 (m, 8 Me); 1.42 (s, t-Bu); 3.06 (d, 3J(H,NH) ˆ 5.8, 0.5 CH2); 3.30
3.35 (m, 3.5 CH2); 3.71 (s, MeO); 5.29 (br., 1J(15N,H) ˆ 91.1, NH); 6.48 (br., NH); 7.00 7.06 (br., 2 NH).
13C-NMR (125 MHz, CDCl3): 23.10, 23.63, 23.74, 23.77, 28.40 (Me); 42.19, 42.35, 42.58, 43.01, 43.16, 43.30 (C);
46.71, 47.39, 47.42, 48.90, 48.99, 50.30 (CH2); 52.24 (Me); 78.82(C); 83.23 (CH2); 156.31, 156.52, 177.20, 177.28,
177.48, 177.68, 177.77, 177.92(C).
Boc-b2,2-HAib-b2,2-HAib[15N,13CH2]-b2,2-HAib-b2,2-HAib[13CO]-OMe ([2,4-13C2, 2-15N]-1d). Tripeptide
[13C2,15N]-1c (206 mg, 476 mmol, 1 equiv.) was Boc-deprotected according to GP 1, dissolved in CHCl3 (1 ml),
and treated with Et3N (366 ml, 2.63 mmol, 5.5 equiv.), HOBt (100 mg, 740 mmol, 1.55 equiv.), 3a (135 mg,