Highly constrained linear oligopeptides containing heterocyclic α-amino carboxylic acids

Article abstract of DOI:10.1002/hlca.201300062

Two spiroheterocyclic 2H-azirin-3-amines, 1f and 1g, were shown to be useful synthons for the dipeptides N-(4-aminotetrahydro-2H-pyran-4-yl)prolinate (Thp-Pro) and the corresponding thiopyran derivative, Tht-Pro, respectively. By coupling of 4-bromobenzoic acid with 1f or 1g and saponification, followed by repeating the coupling and saponification steps, oligopeptides of type 4-BrBz-(Thp-Pro)_n-OMe and 4-BrBz-(Tht-Pro)_n-OMe were prepared, and their conformations were evaluated in solution by NMR techniques and in the crystalline state by X-ray crystallography. All of these sterically highly congested oligopeptides adopt fairly rigid helical conformations. It is interesting to note that the hexapeptide with Thp forms a 3₁₀-helix, whereas the Tht analog has a β-bend ribbon spiral confirmation. Copyright

Full text of DOI:10.1002/hlca.201300062

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Highly Constrained Linear Oligopeptides Containing Heterocyclic a-Amino
Carboxylic Acids
by Svetlana A. Stoykova1), Anthony Linden, and Heinz Heimgartner*
Organisch-Chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ 41446354282; fax: þ 41446356812; e-mail: heimgart@oci.uzh.ch)
Dedicated to Professor John A. Robinson on the occasion of his 60th birthday
Two spiroheterocyclic 2H-azirin-3-amines, 1f and 1g, were shown to be useful synthons for the
dipeptides N-(4-aminotetrahydro-2H-pyran-4-yl)prolinate (Thp-Pro) and the corresponding thiopyran
derivative, Tht-Pro, respectively. By coupling of 4-bromobenzoic acid with 1f or 1g and saponification,
followed by repeating the coupling and saponification steps, oligopeptides of type 4-BrBz-(Thp-Pro)n-
OMe and 4-BrBz-(Tht-Pro)n-OMe were prepared, and their conformations were evaluated in solution by
NMR techniques and in the crystalline state by X-ray crystallography. All of these sterically highly
congested oligopeptides adopt fairly rigid helical conformations. It is interesting to note that the
hexapeptide with Thp forms a 310-helix, whereas the Tht analog has a b-bend ribbon spiral confirmation.
1. Introduction. – In the last few decades, it has been shown that 2H-azirin-3-amines
(¼ 3-amino-2H-azirines) of type 1 (Fig. 1) can be successfully used in peptide synthesis
as synthons for a,a-disubstituted a-amino acids (¼2,2-disubstituted glycines) [2]. The
reaction sequence for the introduction of such amino acids into peptides, i.e., the azirine
coupling with N-protected amino acids or peptides, followed by selective hydrolysis of
the terminal amide function and coupling with the next amino component, is called the
ꢂazirine/oxazolone methodꢃ, and has been used for the preparation of peptaibols and
peptaibol segments [3]. Thus, the reaction of a peptide acid with the Aib synthon 1a
leads to a peptide amide with a backbone extended by a 2-methylalanine (a-
aminoisobutyric acid ¼ 2-amino-2-methylpropanoic acid; Aib) moiety. After the
selective acid-catalyzed hydrolysis of the terminal amide bond, the extended peptide
acid is obtained, which can then be used for a further ꢂazirine couplingꢃ or for segment
condensation. This strategy has been employed extensively in the synthesis of linear
oligopeptides [3][4], endothiopeptides [5], cyclic peptides [6], and cyclic depsipeptides
[7] containing 2,2-disubstituted gycines, in particular Aib. It has also been shown that
the ꢂazirine/oxazolone methodꢃ can be used under solid-phase conditions [8].
Furthermore, heterospirocyclic 2H-azirin-3-amines of type 1b and 1c were prepared
and used as synthons for heterocyclic a-amino acids [9]. Incorporated in tripeptides of
the type Z-Aib-Xaa-Aib-N(Ph)Me [9b], Z-Phe-Xaa-Val-OMe, and H-Asp-d-Ala-
1
)
Part of the Ph.D. thesis of S. A. S., Universitꢀt Zꢁrich, 2004; presented in part at the 25th
International Symposium on the Organic Chemistry of Sulfur (ISOCS 25), Czestochowa, Poland,
2012 [1].
ꢄ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 96 (2013)
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Fig. 1. 2H-Azirin-3-amines as synthons for Aib, heterocyclic a-amino carboxylic acids, Aib-Pro, and
heterocyclic dipeptides
Xaa-OMe [10], they behave like Aib and some other a,a-disubstituted a-amino acids
that are known to stabilize secondary structures such as b-turns and helical
conformations [11].
The first representative of a novel type of 2H-azirin-3-amines, namely methyl N-
(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate (1d), has been found to be suitable as a
dipeptide synthon for the sequence Aib-Pro [3c][4b] (for analogs, see [12]). This
building block has been used to prepare peptaibol antibiotics Trichovirin I 1B [3c],
Zervamicin II-2 [3d], and Hypomurocins A1, A3, and A5 [3e][3g], as well as in the
synthesis of endothiopeptides with a C-terminal Aib-Pro unit [5a – 5c][5e]. Further-
more, repeated coupling of benzoic acids with 1d led to Aib-Pro oligopeptides [13]. We
also reported the synthesis of methyl N-(1-aza-5-oxaspiro[2.4]oct-1-en-2-yl)-l-proli-
nate (1e) [9c], its homolog 1f [14], and the S-analog 1g [15] as examples of
heterospirocyclic N-(2H-azirin-3-yl)-l-prolinates. It has been shown that these novel
amino azirines can be used as dipeptide synthons, e.g., in the case of 1f, for the
dipeptide N-[(4-aminotetrahydro-2H-pyran-4-yl)carbonyl]-l-prolinate (Thp-Pro). As
a model, the nonapeptide Z-Ser(tBu)-Val-Thp-Pro-Aib-Leu-Thp-Pro-Leuol has been
prepared by a combination of the ꢂazirine/oxazolone methodꢃ and segment coupling
with HATU/HOBt [14].
Here, we report on the use of the amino-azirines 1f and 1g for the synthesis of the
sterically constrained oligopeptides 4-Br-C6H4CO-(Thp-Pro)n-OMe and 4-Br-
C6H4CO-(Tht-Pro)n-OMe by the ꢂazirine couplingꢃ, and the conformations of these
oligopeptides in the solid state and in solution were determined. It is well-known [16]
that peptides, which consist of consecutive (Aib-Pro) units, adopt a helical structure
called a b-bend ribbon, similar to a sequence of type-III b-turn conformations. This
finding was confirmed by our recently published results [13]. It was of interest to
establish whether the heteroatoms O and S in the heterocyclic a-amino acids Thp
(homoserine analog) and Tht (homocysteine analog), respectively, have an influence
on the folding of the peptide backbone.
2. Results and Discussion. – 2.1. Synthesis of Oligopeptides 4-BrꢀC6H4CO-(Thp-
Pro)n-OMe and 4-BrꢀC6H4CO-(Tht-Pro)n-OMe. The dipeptide synthon 1f was

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coupled with 4-bromobenzoic acid (4-Br-C6H4COOH) under standard conditions
(Scheme): to a solution of 4-BrC6H4COOH (1 mol-equiv.) in THFat 08, a solution of 1f
(1.1 mol-equiv.) was added. The mixture was stirred for 45 h at room temperature
(TLC control). Chromatographic purification gave the dipeptide derivative 4-Br-
C6H4CO-Thp-Pro-OMe (2a) in excellent yield (94%) as colorless crystals. Saponifi-
cation of the ester was performed with LiOH · H2O (4 mol-equiv.) in THF/MeOH/H2O
(3 :1:1) at room temperature, which furnished the corresponding dipeptide acid 3a in
98% yield. This product was sufficiently pure to be used in the next coupling reaction
with the building block 1f.
Scheme

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After the addition of 1f (1.1 mol-equiv.) to a solution of 3a in THF at 08, the
mixture was stirred for 47 h at room temperature, and the chromatographic purification
of the crude product gave 4-Br-C6H4CO-(Thp-Pro)2-OMe (4a) in 88% yield as
colorless crystals. Hydrolysis of the latter with LiOH · H2O (4 mol-equiv.) in THF/
MeOH/H2O (3 :1:1) led to the corresponding tetrapeptide acid 5a in 91% yield, which
was then used in a third coupling reaction with 1f to afford the desired hexapeptide
derivative 6a. After column chromatography, 4-Br-C6H4CO-(Thp-Pro)3-OMe (6a),
containing three consecutive (Thp-Pro) units, was obtained in 86% yield. Its structure
was established on the basis of the spectroscopic data and by an X-ray crystal-structure
determination (Fig. 2, see Chapt. 2.2.). Saponification of 6a with LiOH · H2O (4 mol-
equiv.) in THF/MeOH/H2O (3 :1:1) gave the corresponding hexapeptide acid 7a in
96% yield, which was used directly in the next coupling reaction to give the octapeptide
4-Br-C6H4CO-(Thp-Pro)4-OMe (8). The peptide methyl ester 8, which consists of four
consecutive Thp-Pro units, was obtained in 78% yield.
The synthesis of the hexapeptide 4-Br-C6H4CO-(Tht-Pro)3-OMe (6b) was per-
formed in analogy to that of 6a (Scheme). The coupling of 1g with 4-Br-C6H4COOH
gave 2b (45%), saponification of which with LiOH · H2O (3 mol-equiv.) under the
usual conditions led to the dipeptide acid 3b in 90% yield, which was then coupled with
2 mol-equiv. of 1g (120 h, room temperature) to give the tetrapeptide 4b in 73% yield
after chromatographic purification. Hydrolysis of 4b with LiOH · H2O (3 mol-equiv.)
furnished the tetrapeptide acid 5b in 99% yield. The last coupling reaction with 1g was
again carried out in THF (116 h, room temperature). The product 6b gave, after
column chromatography, the hexapeptide 4-Br-C6H4CO-(Ttp-Pro)3-OMe in 73%
yield. The structure of the latter was also established by X-ray crystallography (Fig. 3).
2.2. Conformational Studies. 2.2.1. Hexapeptide Ester 6a and Octapeptide Ester 8.
Suitable crystals for the X-ray crystal-structure determination of 4-Br-C6H4CO-(Thp-
Pro)3-OMe (6a) were obtained from CHCl3/hexane by slow evaporation of the solvent.
The molecular structure is depicted in Fig. 2. There are two symmetry-independent
molecules (A and B) of 6a, and five CHCl3 and three H2O molecules in the asymmetric
unit. Both peptide molecules are of the same stereoisomer and have similar helical
conformations. The crystals are enantiomerically pure, and the absolute configuration
of the molecule has been determined independently by the diffraction experiment as
(3S,9S,15S). One five-membered ring in each of molecules A and B is disordered over
two envelope conformations, where the atoms C(39) and C(91), respectively, occupy
two alternate positions, which correspond to the envelope flap lying on opposite sides
of the ring plane. The major conformation occurs in ca. 55% of the type A molecules
and 53% of those of type B. Additionally, the Thp ring at C(78) in molecule B is
disordered over two chair conformations, where the O-atom and its two neighboring C-
atoms in the ring occupy two alternate positions, which correspond with inversion of the
chair. The major conformation occurs in ca. 79% of the type B molecules. Three of the
five CHCl3 and all H2O molecules are disordered over several orientations and were
omitted from the model (see Exper. Part).
Amide groups in both symmetry-independent peptide molecules act as donors for
H-bonds (Table 1). For each molecule, two of the interactions are intramolecular H-
bonds, which form a regular pattern along the peptide chain and serve to maintain a
fairly rigid helical conformation of the peptide. The most relevant torsion angles for the

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Helvetica Chimica Acta – Vol. 96 (2013)
Fig. 2. ORTEP Plot [17] of the molecular structures of the symmetry-independent molecules A and B of
6a (disorder conformations a; with 50% probability ellipsoids; arbitrary numbering of atoms; H-atoms
bonded to C-atoms and solvent molecules omitted for clarity)
peptide backbone of the two molecules A and B are compiled in Table 2. Structures of
both molecules are stabilized by two 1 4 intramolecular H-bonds between the NH
groups of the Thp3 and Thp5 residues, and the C¼O groups of the 4-Br-C6H4CO group
and of the Pro2 residue, respectively. For instance, in molecule A, N(7)ꢀH and
N(13)ꢀH interact with the amide O(14)- and O(20)-atoms, respectively, that are seven
atoms further along the peptide backbone. Each of these interactions has the graph set
motif [18] of S(10). Molecule B has an identical pattern of intramolecular interactions.
In molecule A, N(19)ꢀH, which is unable to form an intramolecular interaction
because of its position in the backbone, forms bifurcated intermolecular H-bonds with

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Fig. 3. ORTEP Plot [17] of the molecular structures of the symmetry-independent molecules A and B of
6b (disorder conformation a of molecule B; with 50% probability ellipsoids; arbitrary numbering of
atoms; H-atoms bonded to C-atoms and solvent molecules omitted for clarity)
the ester carbonyl O(62)-atom and the amide O(68)-atom near the middle five-
membered ring of an adjacent molecule B. In turn, molecule B forms the identical type
of bifurcated intermolecular H-bonds with a further molecule A. These interactions
link both of the symmetry-independent peptide molecules into extended ··· A ··· B ··· A
··· B ··· chains which run parallel to the [110] direction. The binary graph set motifs for
the intermolecular H-bonds are C22(40) and C22(28) for the interaction with the ester and
amide O-atoms, respectively. Due to the omission of the H2O molecules from the

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model, the involvement of these moieties in the H-bonding interactions could not be
assessed.
Table 1. Intra- and Intermolecular H-Bonds in 6a
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(7)ꢀH(7) ··· O(14)
N(13)ꢀH(13) ··· O(20)
N(19)ꢀH(19) ··· O(68’)
N(19)ꢀH(19) ··· O(62’)
N(67)ꢀH(67) ··· O(74)
N(73)ꢀH(73) ··· O(80)
N(79)ꢀH(79) ··· O(8)
N(79)ꢀH(79) ··· O(2)
0.88
0.88
0.88
0.88
0.88
0.88
0.88
0.88
2.35
2.22
2.34
2.49
2.21
2.16
2.46
2.43
3.140(5)
2.982(4)
3.090(4)
2.995(4)
3.033(4)
2.980(5)
3.195(5)
3.014(5)
149
145
144
117
156
156
142
124
a) Primed atoms refer to the molecule in the symmetry-related position ꢀ 1 þ x, ꢀ 1 þ y, z.
Table 2. Selected Main-Chain Torsion Angles [8] of the Two Symmetry-Independent Molecules of 6a
Molecule A
Thp(1)
Pro(2)
Thp(3)
Pro(4)
Thp(5)
Pro(6)
f
y
w
ꢀ 51.0(5)
ꢀ 44.8(5)
ꢀ 168.5(3)
ꢀ 64.0(5)
ꢀ 31.8(5)
175.5(3)
ꢀ 53.5(5)
ꢀ 48.6(5)
ꢀ 167.5(4)
ꢀ 71.3(5)
ꢀ 27.8(6)
ꢀ 174.9(4)
ꢀ 61.4(5)
ꢀ 43.5(5)
ꢀ 162.7(4)
ꢀ 55.2(7)
ꢀ 45.0(7)
ꢀ 174.1(4)
Molecule B
Thp(1’)
Pro(2’)
Thp(3’)
Pro(4’)
Thp(5’)
Pro(6’)
f
y
w
ꢀ 49.5(5)
ꢀ 37.1(5)
179.5(3)
ꢀ 69.3(5)
ꢀ 26.7(6)
ꢀ 176.4(4)
ꢀ 59.5(5)
ꢀ 34.5(5)
ꢀ 166.0(4)
ꢀ 68.7(5)
ꢀ 23.0(6)
179.0(4)
ꢀ 64.0(5)
ꢀ 45.5(5)
ꢀ 166.2(4)
ꢀ 54.8(5)
ꢀ 44.9(6)
ꢀ 175.3(3)
The possibility of a relationship between the conformation adopted by a peptide
molecule and the puckering of the pyrrolidine ring was also considered [19a]. The
pyrrolidine ring of proline residues has two preferred conformations that are
energetically equivalent [19b]. The four-atom fragment C(d)ꢀNꢀC(a)ꢀC(b) is
essentially planar, and the two conformations are distinguished by the direction in
which the C(g)-atom deviates from this plane. In the C(g)-exo conformation, the C(g)-
atom and the C¼O group are on opposite sides of the plane. The conformation is
characterized by negative side-chain torsion angles, c1 and c3, of the pyrrolidine ring
and positive ones for c2 and c4 (Fig. 4). In the C(g)-endo conformation, the C(g)-atom
and the C¼O group are on the same side of the above mentioned plane, and the signs of
the side-chain torsion angles of the pyrrolidine ring are reversed. Intramolecularly H-

Helvetica Chimica Acta – Vol. 96 (2013)
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bonded conformations of (Aib-Pro)n sequences have been analyzed theoretically, and
the obtained results revealed that the pyrrolidine ring puckering has a pronounced
effect on the energies. Only C(g)-endo-puckered Pro residues can be accommodated in
1 4 H-bonded regular structures [19c]. On the other hand, the experimental results
obtained by Toniolo et al. have shown that both, C(g)-endo and C(g)-exo pyrrolidine
ring puckerings, are observed in the heptapeptide 4-Br-C6H4CO-Aib-(Aib-Pro)3-OMe
and in the nonapeptide 4-Br-C6H4CO-Aib-(Aib-Pro)4-OMe, which adopt a b-bend-
ribbon structure [16e]. The same result was obtained in our recent study on ArCO-
(Aib-Pro)n-X peptides [13]. In the case of the hexapeptide ester 4-Br-C6H4CO-(Thp-
Pro)3-OMe (6a), C(g)-endo and C(g)-exo pyrrolidine ring conformations are observed
in both molecules A and B.
Fig. 4. Side-chain torsion angles of the Pro residue
Unfortunately, no suitable crystals for X-ray crystallography could be obtained
from the octapeptide 8.
The conformations of the two peptides 6a and 8 in solution were studied by NMR
spectroscopy. An easily obtainable information is the dependence of the NH shifts on
the polarity of the solvent or on the temperature: NH groups involved in intra-
molecular H-bonds show a very low dependence, whereas the chemical shifts of
solvent-exposed NH groups are influenced more significantly [20] (see also
[9b][13][21]). Therefore, for the two peptides, 6a and 8, the involvement of the NH
groups in intramolecular H-bonds was evaluated on the basis of the solvent dependence
of d(NH) in CDCl3/(D6)DMSO (solvent-titration experiment [22]), and the temper-
ature dependence was determined in the range of 265 – 314 K. The assignments of all
1H- and 13C-NMR signals were accomplished by means of HSQC and HMBC
techniques. As shown in Figs. 5 and 6 for the hexapeptide 6a and the octapeptide 8, two
and three NH groups, respectively, are nearly unaffected by an increase of the
proportion of (D6)DMSO in the range of 0 – 12% and by varying the temperature. We
presume that these signals emanate from two and three amide NH groups, respectively,
which are involved in intramolecular H-bonds, forming two b-turns (as in the crystalline
state of 6a), and three b-turns in the case of 8. In contrast, Dd values for the benzamide
NH groups in the solvent titration experiment are 0.85 and 0.689 ppm for 6a and 8,
respectively, which are typical values for solvent-exposed NH groups. These values
correspond to the temperature coefficients Dd/DT, which are ꢀ 12.79 ꢁ 10ꢀ3 and
ꢀ 9.06 ꢁ 10ꢀ3 ppm/K for 6a and 8, respectively (Table 3).
2.2.2. Hexapeptide Ester 6b. Suitable crystals of 4-Br-C6H4CO-(Tht-Pro)3-OMe
(6b) for the X-ray crystal-structure determination were obtained from CH2Cl2/MeOH/
hexane by slow evaporation of the solvent. The molecular structure is shown in Fig. 3.
The compound in the crystal is enantiomerically pure, and the absolute configuration of

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Fig. 5. Dependence of the chemical shifts of the NH resonances of 6a as a function of a) the (D6)DMSO
concentration (% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
Fig. 6. Dependence of the chemical shifts of the NH resonances of 8 as a function of a) the (D6)DMSO
concentration (% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
Table 3. Temperature Coefficients of the Amide NH of the Peptides 6a and 8 in the Range of 265 – 314 K
Peptide
ꢀ Dd/DT [ppm/K]
NH(Thp(1))
NH(Thp(3))
NH(Thp(5))
NH(Thp(7))
6a
8
ꢀ 12.79 ꢁ 10ꢀ3
ꢀ 1.92 ꢁ 10ꢀ3
ꢀ 2.62 ꢁ 10ꢀ3
ꢀ
ꢀ 9.06 ꢁ 10ꢀ3
ꢀ 1.13 ꢁ 10ꢀ3
ꢀ 2.31 ꢁ 10ꢀ3
ꢀ 0.6 ꢁ 10ꢀ3
the molecule has been determined independently by the diffraction experiment. The
molecule has the expected (3S,9S,15S)-configuration. The asymmetric unit contains
two symmetry-independent peptide molecules, A and B, two fully occupied sites for
MeOH molecules, and one additional MeOH site, which is only 43% occupied. The two
independent peptide molecules have similar backbone conformations; the most
relevant main-chain torsion angles for molecules A and B are compiled in Table 4. The
main differences between the molecules are reversed chair puckering in two of the
heterocyclic six-membered rings (rings containing S(35)/S(95) and S(43)/(S103)), plus

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Table 4. Selected Main-Chain Torsion Angles [8] of the Two Symmetry-Independent Molecules of 6b
Molecule A
Tht(1)
Pro(2)
Tht(3)
Pro(4)
Tht(5)
Pro(6)
f
y
w
ꢀ 48.3(5)
ꢀ 37.0(6)
þ 174.1(4)
ꢀ 67.3(5)
ꢀ 20.4(6)
ꢀ 173.7(4)
ꢀ 51.5(5)
ꢀ 33.6(6)
ꢀ 163.3(4)
ꢀ 69.0(5)
ꢀ 4.5(6)
58.3(5)
45.7(5)
173.1(4)
ꢀ 60.2(6)
148.7(4)
161.8(4)
ꢀ 177.7(4)
Molecule B
Tht(1’)
Pro(2’)
Tht(3’)
Pro(4’)
Tht(5’)
Pro(6’)
f
y
w
ꢀ 51.9(6)
ꢀ 35.3(6)
ꢀ 179.3(3)
ꢀ 66.0(5)
ꢀ 9.8(6)
178.0(4)
ꢀ 40.5(6)
ꢀ 47.0(6)
ꢀ 170.1(4)
ꢀ 71.4(6)
ꢀ 0.6(7)
61.7(6)
43.3(6)
170.6(4)
ꢀ 63.3(6)
147.4(4)
167.8(4)
ꢀ 171.5(4)
some small conformational differences in some of the five-membered rings and a slight
pivot of the bromophenyl ring about its axis (238). In peptide molecule A, both C(g)-
endo and C(g)-exo pyrrolidine ring puckerings are observed (Fig. 4). The first five-
membered ring has a half-chair conformation twisted on C(22)ꢀC(23), the middle five-
membered ring has an envelope conformation with C(31) as the envelope flap, and the
third five-membered ring has a half-chair conformation twisted on C(38)ꢀC(39), but
distorted towards an envelope conformation. In peptide molecule B, the first five-
membered ring has a similar half-chair conformation, twisted on C(82)ꢀC(83), but
distorted towards an envelope conformation. The middle five-membered ring is
disordered over two conformations resulting from alternate flipping of the position of
one C-atom in the ring. This ring has a half-chair conformation twisted on
C(91a)ꢀC(92) for the major conformation (60%) and twisted on C(90)ꢀC(91b) for
the minor conformation. The third five-membered ring in molecule B has an envelope
conformation with C(99) as the envelope flap.
Each peptide molecule 6b forms two intramolecular H-bonds, plus one intermo-
lecular H-bond, which links the peptide molecules into extended chains (Table 5).
Each peptide molecule also accepts one H-bond from a MeOH molecule. In peptide
molecule A, the NH groups of the Tht3 and Tht5 residues, i.e., N(7)ꢀH and N(13)ꢀH,
form 1 4 intramolecular H-bonds with the amide O-atoms O(14) of Pro2 and O(20)
of the 4-Br-C6H4CO group, respectively, that are seven atoms further along the peptide
backbone. Each of these interactions has the graph set motif of S(10). This serves to
maintain a fairly rigid helical conformation of the peptide. The NH group of Tht1,
N(19)ꢀH, which is unable to form an intramolecular interaction because of its position
in the backbone, forms an intermolecular H-bond with the amide O(8’)-atom near the
middle of a neighboring peptide molecule A. These interactions link the A molecules
into extended chains which run parallel to the y-axis and have a graph set motif of
C(14). Peptide molecule A also accepts an intermolecular H-bond from the OH group

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Table 5. Intra- and Intermolecular H-Bonds in 6b
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(7)ꢀH(7) ··· O(14)
0.88
0.88
0.88
0.88
0.88
0.88
0.84
0.84
2.12
2.25
2.21
2.11
2.03
2.08
2.10
1.99
2.999(4)
3.128(5)
3.056(4)
2.988(5)
2.889(5)
2.933(5)
2.834(7)
2.734(7)
173
173
161
174
163
165
146
148
N(13)ꢀH(13) ··· O(20)
N(19)ꢀH(19) ··· O(8’)
N(67)ꢀH(67) ··· O(74)
N(73)ꢀH(73) ··· O(80)
N(79)ꢀH(79) ··· O(68’)
O(112)ꢀH(112) ··· O(5’)
O(114)ꢀH(114) ··· O(65)
a) Primed atoms refer to the molecule in the symmetry-related position x, ꢀ 1 þ y, z.
of one of the fully occupied MeOH molecules. Peptide molecule B is involved in an
identical pattern of intra- and intermolecular H-bonds, so that, overall, two
independent parallel chains of molecules run through the structure.
1
A hint about the conformation of 6b in solution was obtained from H-NMR
spectroscopy. The involvement of the NH groups in intramolecular H-bonds was
evaluated on the basis of the solvent dependence of d(NH) values in CDCl3/
1
(D6)DMSO (solvent-titration experiment); the assignments of all H- and 13C-NMR
signals were carried out by using HSQC and HMBC techniques. As shown in Fig. 7, two
NH groups of the hexapeptide 6a are nearly unaffected by an increase of concentration
of (D6)DMSO. Therefore, we propose that these signals emanate from the two NH
groups of Tht3 and Tht5, which are involved in two intramolecular H-bonds, forming
two b-turns (as in the crystalline state). The Dd value of the remaining NH group of
Tht1 is 0.85 ppm, which is typical for a solvent-exposed NH group.
Fig. 7. Dependence of the chemical shifts of the NH
resonances of 6b as a function of the (D6)DMSO
concentration (% v/v) in CDCl3
3. Conclusions. – The presented results show that the new spiroheterocyclic N-(2H-
azirin-3-yl)prolinates 1f and 1g can be prepared according to the previously reported
protocols. In the reactions with PhCOSH, 4-Br-C6H4COOH, and N-protected a-amino
acids, both azirines behave like other 2H-azirin-3-amines (¼ 3-amino-2H-azirines) that
have been extensively used as building blocks for a,a-disubstituted glycines in peptide

Helvetica Chimica Acta – Vol. 96 (2013)
1725
synthesis [14][15]. They have been shown to be useful synthons for the dipeptides N-
[(4-aminotetrahydro-2H-pyran-4-yl)carbonyl]-l-proline (Thp-Pro) and N-[(4-amino-
tetrahydro-2H-thiopyran-4-yl)carbonyl]-l-proline (Tht-Pro), respectively. As models
for sterically congested oligopeptides, 4-Br-C6H4CO-(Thp-Pro)n-OMe and 4-Br-
C6H4CO-(Tht-Pro)n-OMe were prepared via repeated ꢂazirine couplingꢃ analogous to
the synthesis of ArCO-(Aib-Pro)n-OMe peptides with the amino-azirine 1d [13].
By X-ray crystallography and NMR techniques, it has been shown that the
examined hexapeptides 6a and 6b adopt fairly rigid helical conformations with some
significant differences, namely in the values of the y torsion angle for the Pro2 and the
Pro4 residues in the peptide backbone. In the case of 6a, containing consecutive (Thp-
Pro) units, this torsion angle has the values of ꢀ 31.8(5) (Pro2) and ꢀ 27.7(6)8 (Pro4)
(molecule A), and ꢀ 26.7(6) (Pro2) and ꢀ 23.0(6)8 (Pro4) (molecule B). The
corresponding values in the case of 6b, which consists of consecutive (Tht-Pro) units,
are ꢀ 20.4(6) (Pro2) and ꢀ 4.5(6)8 (Pro4) (molecule A), and ꢀ 9.8(6) (Pro2) and
ꢀ 0.6(7)8 (Pro4) (molecule B). It is worth mentioning that the values obtained in the
case of 6a could be compared with the values of a regular 310-helix (y ¼ ꢀ308), whereas
those obtained in the case of 6b are much closer to the values obtained in the case of
the b-bend ribbon spiral [16e].
We thank the analytical sections of our institute for spectra and elemental analyses, and the Swiss
National Science Foundation and F. Hoffmann-La Roche AG, Basel, for financial support.
Experimental Part
1. General. Solvents were purified by standard procedures. TLC: Merck TLC aluminum sheets, silica
gel 60 F254. Column chromatography (CC): Uetikon-Chemie, silica gel C-560 (0.04 – 0.063 mm). M.p.:
Bꢀchi Melting Point B-450 apparatus; uncorrected. IR Spectra: Perkin-Elmer, Spectrum one FT-IR
spectrophotometer; in KBr unless otherwise stated; ˜n in cmꢀ1. NMR Spectra: Bruker AC-300 (1H, 13C,
DEPT) at 300 and 75 MHz, resp., or Bruker DRX-600 (1H, 13C, HSQC, HMBC, COSY) at 600 and
150 MHz, resp., in CDCl3 at 300 K, unless otherwise stated; d in ppm refer to residual CHCl3 (1H,
7.27 ppm) and to CDCl3 (13C, 77.0 ppm); coupling constants J in Hz; 13C-signal multiplicities from DEPT
spectra. MS: Finnigan SSQ-700 (CI with NH3), or Finningan TSQ-700 instrument (ESI); m/z (rel. %).
2. Synthesis of Oligopeptides 4-Br-C6H4CO-(Thp-Pro)n-OMe. General Procedure 1 (GP 1). To a
soln. of 4-bromobenzoic acid or the corresponding peptide acid in dry THF was added the azirine 1f [14]
(1.1 equiv.), and the mixture was stirred at r.t. After completion of the reaction (TLC), the soln. was
concentrated in vacuo, and the residue was purified by CC (SiO2).
General Procedure 2 (GP 2). To a soln. of the corresponding peptide methyl ester in THF/MeOH/
H2O 3 :1:1, LiOH · H2O (4 mol-equiv.) was added. The mixture was stirred at r.t. After completion of the
reaction (TLC), 1m HCl was added until pH 1 was reached, and the org. solvent was evaporated. The
residue was extracted with CH2Cl2 or AcOEt, the combined org. phase was dried (MgSO4), evaporated,
and the residue was dried under h.v.
Methyl 1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-prolinate (4-
Br-C6H4CO-Thp-Pro-OMe; 2a). According to GP 1, with 4-Br-C6H4COOH (169 mg, 0.845 mmol) in dry
THF (10 ml) and 1f (220 mg, 0.925 mmol); stirring for 45 h. The obtained precipitate was collected by
filtration, washed with AcOEt/hexane 1:1, and dried under h.v.: 348 mg (94%) of 2a. Colorless crystals.
M.p. 2238. IR: 3333m, 2956m, 2874w, 1743s, 1654s, 1616s, 1590w, 1528m, 1483m, 1421w, 1361w, 1301w,
1284w, 1208w, 1164m, 1106m, 1069w, 1011m, 939w, 847m, 758s, 614w. 1H-NMR: 8.17 (s, NH); 7.78 – 7.75
(m, 2 arom. H); 7.63 – 7.59 (m, 2 arom. H); 4.59 – 4.56 (m, CH(a)(Pro)); 4.02 – 3.95 (m, 1 H of
CH2(d)(Pro)); 3.83 – 3.81 (m, 2 H); 3.74 – 3.68 (m, 2 H of CH2(2), CH2(6)(Thp), MeO); 3.39 – 3.31 (m,
1 H of CH2(d)(Pro)); 2.63 – 2.55 (m, 1 H); 2.20 – 1.85 (m, 7 H). 13C-NMR: 173.9, 171.6 (2s, 2 C¼O); 166.8

1726
Helvetica Chimica Acta – Vol. 96 (2013)
(s, ArC¼O); 132.8 (s, 1 arom. C); 132.1, 129.4 (2d, 4 arom. CH); 126.9 (s, 1 arom. C); 64.9, 63.4 (2t, C(2),
C(6)(Thp)); 61.0 (d, CH(a)(Pro)); 57.4 (s, C(4)(Thp)); 52.3 (q, MeO); 48.0 (t, CH2(d)(Pro)); 32.6, 32.4,
27.9, 26.0 (4t, 4 CH2). ESI-MS: 463 (100, [M(81Br) þ Na]þ), 461 (96, [M(79Br) þ Na]þ), 383 (63).
1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-proline
(4-Br-
C6H4CO-Thp-Pro-OH; 3a). According to GP 2, with 2a (319 mg, 0.726 mmol) in 10 ml THF/MeOH/
H2O, LiOH · H2O (122 mg, 2.904 mmol); stirring for 23 h: 302 mg (98%) of 3a. The product was
sufficiently pure to be used in the next step without further purification. M.p. 2278. IR: 3350m, 2977m,
2876w, 1736s, 1617s, 1592m, 1530s, 1482m, 1442m, 1422s, 1354w, 1311m, 1273w, 1250w, 1210w, 1165s,
1097m, 1071m, 1025w, 1013m, 972w, 943m, 895w, 850m, 777m, 757m, 710w, 651w, 605m. 1H-NMR
((D6)DMSO): 12.18 (s, OH); 8.59 (s, NH); 7.86 – 7.83 (m, 2 arom. H); 7.72 – 7.69 (m, 2 arom. H); 4.25 –
4.22 (m, CH(a)(Pro)); 3.83 – 3.65 (m, 4 H); 3.48 – 3.60 (m, 1 H); 3.20 – 3.15 (m, 1 H); 2.38 – 2.25 (m,
1 H); 2.10 – 1.69 (m, 7 H). 13C-NMR ((D6)DMSO): 173.8, 170.3 (2s, 2 C¼O); 164.9 (s, ArC¼O); 133.2 (s,
1 arom. C); 131.5, 129.9 (2d, 4 arom. CH); 125.3 (s, 1 arom. C); 63.6, 62.5 (2t, C(2), C(6)(Thp)); 60.3 (d,
CH(a)(Pro)); 56.6 (s, C(4)(Thp)); 47.3 (t, CH2(d)(Pro)); 31.8, 31.7, 27.5, 25.4 (4t, 4 CH2). ESI-MS: 449
(86, [M(81Br) þ Na]þ), 447 (100, [M(79Br) þ Na]þ).
Methyl 1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-prol-
yl]amino}-3,4,5,6-tetrahydro-2H-pyran-4-yl)carbonyl]-l-prolinate (4-Br-C6H4CO-(Thp-Pro)2-OMe; 4a).
According to GP 1, with 3a (250 mg, 0.588 mmol) in dry THF (12 ml) and 1f (154 mg, 0.647 mmol),
stirring for 47 h, CC (CH2Cl2/MeOH 100 :1 ! 20 :1): 390 mg (88%) of 4a. M.p. 194.28. IR: 3319m,
2955m, 2870w, 1744m, 1646s, 1590w, 1530m, 1482m, 1442w, 1404m, 1301w, 1279w, 1247w, 1207w, 1162m,
1143w, 1107m, 1070w, 1030w, 938w, 845m, 804w, 760m. 1H-NMR: 7.89 (s, NH); 7.73 – 7.70 (m, 2 arom. H);
7.62 (s, NH); 7.60 – 7.57 (m, 2 arom. H); 4.59 – 4.55 (m, CH(a)(Pro)); 4.19 – 4.18 (m, CH(a)(Pro)); 4.01 –
3.96 (m, 2 H of 2 CH2(d)(Pro)); 3.93 – 3.61 (m, 8 H); 3.56 (s, MeO); 3.32 – 3.29 (m, 1 H of CH2(d)(Pro));
3.29 – 3.28 (m, 1 H of CH2(d)(Pro)); 2.70 – 2.64 (m, 1 H); 2.24 – 1.99 (m, 9 H); 1.98 – 1.71 (m, 6 H).
13C-NMR: 173.0, 171.4, 171.3, 171.0 (4s, 4 C¼O); 166.3 (s, ArC¼O); 132.2 (d, 2 arom. CH); 131.6 (s, 1
arom. C); 129.1 (d, 2 arom. CH); 127.2 (s, 1 arom. C); 64.4, 63.7, 63.2, 62.7 (4t, 2 C(2), 2 C(6)(Thp)); 62.6,
60.6 (2d, 2 CH(a)(Pro)); 57.7, 56.9 (2s, 2 C(4)(Thp)); 51.9 (q, MeO); 48.4, 47.5 (2t, 2 CH(d)(Pro)); 32.5,
32.0, 31.5, 31.3 (4t, 2 C(3), 2 C(5)(Thp)); 28.4, 27.8 (2t, 2 CH(b)(Pro)); 26.0, 25.6 (2t, 2 CH(g)(Pro)).
ESI-MS: 703 (9, [M(81Br) þ K]þ), 701 (7, [M(79Br) þ K]þ), 687 (26, [M(81Br) þ Na]þ), 685 (25,
[M(79Br) þ Na]þ), 665 (17, [M(81Br) þ H]þ), 663 (15, [M(79Br) þ H]þ), 536 (100, [M(81Br) ꢀ (Pro-
OMe) þ H]þ), 534 (98, [M(79Br) ꢀ (Pro-OMe) þ H]þ), 409 (45, [M(81Br) ꢀ (Thp-Pro-OMe) þ H]þ), 407
(48, [M(79Br) ꢀ (Thp-Pro-OMe) þ H]þ), 312 (14, [M(81Br) ꢀ (Pro-Thp-Pro-OMe) þ H]þ), 310 (14,
[M(79Br) ꢀ (Pro-Thp-Pro-OMe) þ H]þ), 284 (6, [M(81Br) ꢀ (Pro-Thp-Pro-OMe) ꢀ CO]þ), 282 (6,
[M(79Br) ꢀ (Pro-Thp-Pro-OMe) ꢀ CO]þ), 130 (17, [(Pro-OMe) þ H]þ).
1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-prolyl]ami-
no}-3,4,5,6-tetrahydro-2H-pyran-4-yl)carbonyl]-l-proline (4-Br-C6H4CO-(Thp-Pro)2-OH; 5a). Accord-
ing to GP 2, with 4a (282 mg, 0.425 mmol) in 20 ml of THF/MeOH/H2O and LiOH · H2O (71.3 mg,
1.7 mmol), stirring for 25 h: 251 mg (91%) of 5a. The isolated peptide acid was sufficiently pure to be
used in the next step without further purification. M.p. 1988. IR: 3350m, 2955m, 2849w, 1746s, 1652s,
1617s, 1590m, 1525m, 1482s, 1411m, 1356w, 1299w, 1282w, 1248w, 1206w, 1162s, 1108m, 1070m, 1011m,
940w, 898w, 847m, 780w. 1H-NMR ((D6)DMSO): 12.14 (br. s, OH); 8.85 (s, NH); 7.88 – 7.85 (m, 2 arom.
H); 7.79 (s, NH); 7.76 – 7.73 (m, 2 arom. H); 4.52 – 4.50 (m, CH(a)(Pro)); 4.18 – 4.14 (m, CH(a)(Pro));
3.76 – 3.48 (m, 10 H); 3.42 – 3.24 (m, 2 H); 3.24 – 1.88 (m, 9 H); 1.82 – 1.65 (m, 6 H). 13C-NMR
((D6)DMSO): 173.8, 170.9, 170.6, 170.2 (4s, 4 C¼O); 165.7 (s, ArC¼O); 137.7 (s, 1 arom. C); 131.6, 129.9
(2d, 4 arom. CH); 125.8 (s, 1 arom. C); 63.2, 63.1, 63.1, 62.2 (4t, 2 C(2), 2 C(6)(Thp)); 61.5, 60.3 (2d,
2 CH(a)(Pro)); 57.1, 55.9 (2s, 2 C(4)(Thp)); 47.9, 46.9 (2t, 2 CH2(d)(Pro)); 31.9, 31.8, 31.7, 31.0 (4t,
2 C(3), 2 C(5)(Thp)); 28.2, 27.5 (2t, 2 CH2(b)(Pro)); 25.4, 25.4 (2t, 2 CH2(g)(Pro)). ESI-MS: 673 (96,
[M(81Br) þ Na]þ), 671 (100, [M(79Br) þ Na]þ), 391 (20).
Methyl 1-{[4-({1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-
l-prolyl]amino}-3,4,5,6-tetrahydro-2H-pyran-4-yl)carbonyl]-l-prolyl}amino)-3,4,5,6-tetrahydro-2H-pyr-
an-4-yl]carbonyl}-l-prolinate (4-Br-C6H4CO-(Thp-Pro)3-OMe; 6a). According to GP 1, with 5a (205 mg,
0.316 mmol) in dry THF (12 ml) and 1f (83 mg, 0.348 mmol), stirring for 25 h, CC (CH2Cl2/MeOH
80 :1 ! 20 :1): 241 mg (86%) of 6a. M.p. 334.58. IR: 3274m, 2956m, 2875w, 1747m, 1637s, 1590w, 1536s,

Helvetica Chimica Acta – Vol. 96 (2013)
1727
1483m, 1441w, 1406s, 1356w, 1301w, 1282w, 1247w, 1208w, 1184w, 1161m, 1140w, 1107m, 1071w, 1032w,
1012m, 932w, 840m, 802w, 761m. 1H-NMR: 8.41 (s, NH); 7.71 – 7.68 (m, 2 arom. H, NH); 7.59 – 7.57 (m, 2
arom. H, NH); 4.63 – 4.58 (m, CH(a)(Pro)); 4.41 – 4.31 (m, 2 CH(a)(Pro)); 3.98 – 3.67 (m, 12 H); 3.62 (s,
MeO); 3.59 – 3.25 (m, 6 H); 2.58 – 2.43 (m, 2 H); 2.24 – 2.19 (m, 5 H); 2.18 – 1.75 (m, 17 H). 13C-NMR:
173.3, 172.6, 171.9, 171.3, 171.2, 171.1 (6s, 6 C¼O); 167.2 (s, ArC¼O); 131.8 (d, 2 arom. CH); 131.5 (s, 1
arom. C); 128.8 (d, 2 arom. CH); 127.0 (s, 1 arom. C); 64.2, 64.0, 63.6, 62.9, 62.9, 62.3 (6t, 3 C(2),
3 C(6)(Thp)); 62.9, 62.3, 60.6 (3d, 3 CH(a)(Pro)); 57.0, 56.7, 56.4 (3s, 3 C(4)(Thp)); 51.6 (q, MeO); 48.1,
47.7, 47.6 (3t, 3 CH2(d)(Pro)); 32.1, 31.4, 31.1, 31.0, 30.7, 30.6 (6t, 3 C(3), 3 C(5)(Thp)); 29.0, 27.8, 27.6 (3t,
3 CH2(b)(Pro)); 25.7, 25.6, 23.4 (3t, 3 CH2(g)(Pro)). ESI-MS: 927 (22, [M(81Br) þ K]þ), 925 (18,
[M(79Br) þ K]þ), 911 (100, [M(81Br) þ Na]þ), 909 (98, [M(79Br) þ Na]þ), 889 (21, [M(81Br) þ H]þ), 887
(21, [M(79Br) þ H]þ), 760 (16, [M(81Br) ꢀ (Pro-OMe) þ H]þ), 758 (13, [M(79Br) ꢀ (Pro-OMe) þ H]þ),
536 (12, [M(81Br) ꢀ (Pro-Thp-Pro-OMe) þ H]þ), 534 (12, [M(79Br) ꢀ (Pro-Thp-Pro-OMe) þ H]þ).
Suitable crystals for the X-ray crystal-structure determination were grown from CHCl3/hexane by
slow evaporation of the solvent at r.t.
1-{[4-({1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-prol-
yl]amino}-3,4,5,6-tetrahydro-2H-pyran-4-yl)carbonyl]-l-prolyl}amino)-3,4,5,6-tetrahydro-2H-pyran-4-
yl]carbonyl}-l-proline (4-Br-C6H4CO-(Thp-Pro)3-OH; 7a). According to GP 2, with 6a (208 mg,
0.235 mmol) in 25 ml THF/MeOH/H2O and LiOH · H2O (39.4 mg, 0.939 mmol), stirring for 21 h:
198 mg (96%) of 7a. The isolated peptide acid was sufficiently pure to be used in the next step without
further purification. M.p. 3388. IR: 3313m, 2958m, 2875w, 1742m, 1643s, 1591w, 1531s, 1483w, 1443w,
1407s, 1387s, 1353w, 1303w, 1279w, 1246w, 1207w, 1141m, 1107s, 1073w, 1028w, 1011m, 937w, 843m, 803w,
760m, 605w. 1H-NMR ((D6)DMSO): 8.88 (s, NH); 7.89 – 7.86 (m, 2 arom. H); 7.83 (s, NH); 7.77 – 7.74 (m,
2 arom. H); 7.69 (s, NH); 4.48 – 4.45 (m, CH(a)(Pro)); 4.40 – 4.35 (m, CH(a)(Pro)); 4.21 – 4.17 (m,
CH(a)(Pro)); 3.73 – 3.55 (m, 18 H); 2.27 – 1.56 (m, 24 H). 13C-NMR ((D6)DMSO): 173.7, 171.9, 170.8,
170.8, 170.4, 170.0 (6s, 6 C¼O); 165.8 (s, ArC¼O); 132.3 (s, 1 arom. C); 131.5, 129.8 (2d, 4 arom. CH);
125.6 (s, 1 arom. C); 63.0, 63.0, 62.8, 62.6, 62.6, 62.1 (6t, 3 C(2), 3 C(6)(Thp)); 61.9, 61.2, 60.2 (3d,
3 CH(a)(Pro)); 56.8, 55.9, 55.8 (3s, 3 C(4)(Thp)); 47.8, 47.3, 47.0 (3t, 3 CH2(d)(Pro)); 31.7, 31.5, 31.4, 31.2,
31.1, 30.8 (6t, 3 C(3), 3 C(5)(Thp)); 28.5, 27.9, 27.4 (3t, 3 CH2(b)(Pro)); 25.5, 25.4, 25.1 (3t,
3 CH2(g)(Pro)). ESI-MS: 897 (100, [M(81Br) þ Na]þ), 895 (91, [M(79Br) þ Na]þ).
Methyl 1-({4-[(1-{[4-({1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}-
carbonyl)-l-prolyl]amino}-3,4,5,6-tetrahydro-2H-pyran-4-yl)carbonyl]-l-prolyl}amino)-3,4,5,6-tetrahy-
dro-2H-pyran-4-yl]carbonyl}-l-prolyl)amino]-3,4,5,6-tetrahydro-2H-pyran-4-yl}carbonyl)-l-prolinate
(4-Br-C6H4CO-(Thp-Pro)4-OMe; 8). According to GP 1, with 7a (143 mg, 0.164 mmol) in dry THF
(15 ml) and 1f (43 mg, 0.180 mmol), stirring for 72 h, CC (CH2Cl2/MeOH from 80 :1 to 20 :1): 143 mg
(78%) of 8. M.p. 334.58. IR: 3491m, 3307m, 2958m, 2869w, 1744m, 1644s, 1530s, 1482w, 1443w, 1407s,
1386w, 1301m, 1280w, 1246m, 1207w, 1162m, 1141w, 1106m, 1029w, 1011w, 925w, 841m, 804w, 760m.
1H-NMR: 8.13 (s, NH); 7.75 – 7.72 (m, 2 arom. H, NH); 7.65 (s, NH); 7.62 – 7.60 (m, 2 arom. H); 7.53 (s,
NH); 4.61 – 4.56 (m, CH(a)(Pro)); 4.44 – 4.34 (m, 2 CH(a)(Pro)); 4.23 – 4.21 (m, CH(a)(Pro)); 3.88 –
3.72 (m, 13 H); 3.63 – 3.51 (m, 10 H); 3.33 – 3.26 (m, 1 H); 2.60 – 2.37 (m, 3 H); 2.26 – 2.14 (m, 8 H);
2.09 – 1.65 (m, 22 H). 13C-NMR: 173.4, 173.3, 172.9, 172.1, 171.9, 171.7, 171.6, 171.5 (8s, 8 C¼O); 167.2 (s,
ArC¼O); 132.3 (d, 2 arom. CH); 131.6 (s, 1 arom. C); 129.2 (d, 2 arom. CH); 127.4 (s, 1 arom. C); 64.3,
64.2, 63.4, 63.0, 62.9, 62.9, 62.9, 62.4 (8t, 4 C(2), 4 C(6)(Thp)); 63.4, 62.7, 62.4, 60.8 (4d, 4 CH(a)(Pro));
57.6, 57.0, 56.9, 56.5 (4s, 4 C(4)(Thp)); 51.9 (q, MeO); 48.6, 48.0, 48.0, 47.9 (4t, 4 CH2(d)(Pro)); 32.1, 32.0,
31.1, 31.0, 31.0, 30.9, 29.6, 29.4 (8t, 4 C(3), 4 C(5)(Thp)); 25.5, 28.5, 28.4, 28.4 (4t, 4 CH2(b)(Pro)); 26.3,
26.0, 26.0, 25.9 (4t, 4 CH2(g)(Pro)). ESI-MS: 1135 (100, [M(81Br) þ Na]þ), 1133 (84, [M(79Br) þ Na]þ).
3. Synthesis of Oligopeptides 4-Br-C6H4CO-(Tht-Pro)n-OMe. General Procedure 3 (GP 3). To a soln.
of the peptide methyl ester in THF/MeOH/H2O 3 :1:1, LiOH · H2O (3 mol-equiv.) was added, and the
mixture was stirred at r.t. After completion of the reaction (TLC), 10% KHSO4 was added until pH 1
was reached, and the org. solvent was evaporated. The residue was extracted with AcOEt, the org. phases
were dried (MgSO4), the solvent was evaporated, and the residue was dried under h.v. and used without
further purification.
1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-thiopyran-4-yl}carbonyl)-l-proline (4-Br-
C6H4CO-Tht-Pro-OH; 3b). According to GP 3, with 2b [15] (190 mg, 0.42 mmol) and LiOH · H2O

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Helvetica Chimica Acta – Vol. 96 (2013)
(52.5 mg, 1.252 mmol), 27 h: 167 mg (90%) of 3b. White foam. M.p. 2018. IR: 3348m, 2952m, 1736s,
1640s, 1615s, 1590w, 1526s, 1481s, 1408s, 1310m, 1278m, 1254w, 1217w, 1180m, 1150w, 1070m, 1010m,
912w, 843m, 757m, 710w, 658w, 607w. 1H-NMR ((D6)DMSO): 12.18 (br. s, OH); 8.42 (s, NH); 7.85 – 7.82
(m, 2 arom. H); 7.71 – 7.68 (m, 2 arom. H); 4.24 – 4.22 (m, CH(a)(Pro)); 3.74 – 3.71, 3.39 – 3.35 (2m,
CH2(d)(Pro)); 3.17 – 3.10 (m, 1 H); 2.99 – 2.91 (m, 1 H); 2.74 – 2.51 (m, 2 H); 2.43 – 2.25 (m, 3 H); 1.96 –
1.94 (m, 2 H); 1.92 – 1.76 (m, 3 H). 13C-NMR ((D6)DMSO): 173.5, 170.3 (2s, 2 C¼O); 164.7 (s, ArC¼O);
133.0 (s, 1 arom. C); 131.2, 129.7 (2d, 4 arom. CH); 125.1 (s, 1 arom. C); 60.1 (d, CH(a)(Pro)); 57.8 (s,
C(4)(Tht)); 47.1 (t, CH2(d)(Pro)); 32.6, 32.3 (2t, C(2), C(6)(Tht)); 27.2, 25.2 (2t, C(3), C(5)(Tht)); 23.3
(t, CH2(b)(Pro)); 22.3 (t, CH2(g)(Pro)). ESI-MS: 481 (13, [M(81Br) þ K]þ), 479 (20, [M(79Br) þ K]þ),
465 (100, [M(81Br) þ Na]þ), 463 (86, [M(79Br) þ Na]þ).
Methyl 1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-thiopyran-4-yl}carbonyl)-l-
prolyl]amino}-3,4,5,6-tetrahydro-2H-thiopyran-4-yl)carbonyl]-l-prolinate (4-Br-C6H4CO-(Tht-Pro)2-
OMe; 4b). According to GP 1, with 3b (167 mg, 0.378 mmol) and 1g (192 mg, 0.756 mmol), stirring
for 120 h, CC (CH2Cl2/MeOH 150 :1 ! 20 :1): 191 mg (73%) of 4b. White foam. M.p. 1558. IR: 3311m,
2950m, 2875w, 1743s, 1643s, 1531s, 1480m, 1402m, 1280m, 1255w, 1174m, 1103w, 1070w, 1040w, 1009m,
927w, 884w, 845w, 760m, 659w, 607w. 1H-NMR: 7.82 – 7.87 (m, 2 arom. H); 7.69 – 7.66 (m, 2 arom. H); 7.52
(br. s, NH); 7.42 (s, NH); 4.59 – 4.51, 4.39 – 4.34 (2m, 2 CH(a)(Pro)); 4.09 – 4.08 (m, 1 H of CH2(d)(Pro));
3.85 – 3.73 (m, 1 H of CH2(d)(Pro)); 3.69 (s, MeO); 3.23 – 3.09 (m, 2 H of 2 CH2(d)(Pro)); 2.83 – 2.51 (m,
12 H); 2.43 – 2.10 (m, 6 H); 1.87 – 1.74 (m, 6 H). 13C-NMR: 173.2, 171.6, 171.3, 170.9 (4s, 4 C¼O); 166.0 (s,
ArC¼O); 132.2 (d, 2 arom. CH); 131.3 (s, 1 arom. C); 129.3 (d, 2 arom. CH); 127.4 (s, 1 arom. C); 62.7,
60.9 (2d, 2 CH(a)(Pro)); 59.1, 58.3 (2s, 2 C(4)(Tht)); 51.7 (q, MeO); 48.1, 47.6 (2t, 2 CH2(d)(Pro)); 33.2,
32.9, 32.6, 32.5 (4t, 2 C(2), 2 C(6)(Tht)); 28.4, 27.8 (2t, 2 CH2(b)(Pro)); 26.1, 25.9 (2t, 2 CH2(g)(Pro));
24.0, 23.7, 23.5, 23.1 (4t, 2 C(3), 2 C(5)(Tht)). ESI-MS: 719 (100, [M(81Br) þ Na]þ), 717 (51, [M(79Br) þ
Na]þ).
1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-thiopyran-4-yl}carbonyl)-l-prolyl]-
amino}-3,4,5,6-tetrahydro-2H-thiopyran-4-yl)carbonyl]-l-proline (4-Br-C6H4CO-(Tht-Pro)2-OH; 5b).
According to GP 3, 4b (191 mg, 0.275 mmol) and LiOH · H2O (35 mg, 0.825 mmol), 24 h: 174 mg
(99%) of 5b. White foam. M.p. 2398. IR: 3305m, 2923m, 1735m, 1644s, 1528s, 1480s, 1400m, 1309w,
1279w, 1253w, 1179m, 1070m, 1010m, 910w, 844w, 758w. 1H-NMR: 7.76 – 7.73 (m, 2 arom. H); 7.69 (br. s,
NH); 7.62 – 7.59 (m, 2 arom. H); 7.44 (br. s, NH); 4.57 – 4.52, 4.48 – 4.44 (2m, 2 CH(a)(Pro)); 3.92 – 3.77
(m, 2 H of 2 CH2(d)(Pro)); 3.61 – 3.48 (m, 1 H of CH2(d)(Pro)); 3.21 – 3.13 (m, 1 H of CH2(d)(Pro));
2.99 – 2.68 (m, 4 H); 2.61 – 2.22 (m, 9 H); 2.18 – 2.05 (m, 4 H); 1.99 – 1.74 (m, 7 H). 13C-NMR: 173.5,
173.1, 172.0, 171.9 (4s, 4 C¼O); 166.6 (s, ArC¼O); 132.3 (d, 2 arom. CH); 131.4 (s, 1 arom. C); 129.1 (d, 2
arom. CH); 127.5 (s, 1 arom. C); 62.9, 62.0 (2d, 2 CH(a)(Pro)); 59.0, 58.3 (2s, 2 C(4)(Tht)); 48.5, 48.3 (2t,
2 CH2(d)(Pro)); 34.0, 33.9, 32.7, 32.2 (4t, 2 C(2), 2 C(6)(Tht)); 28.8, 28.1 (2t, 2 CH2(b)(Pro)); 26.2, 26.0
(2t, 2 CH2(g)(Pro)); 24.6, 24.0, 23.5, 23.0 (4t, 2 C(3), 2 C(5)(Tht)). ESI-MS: 705 (100, [M(81Br) þ Na]þ),
703 (85, [M(79Br) þ Na]þ).
Methyl 1-{[4-({1-[(4-{[1-({4-[(4-Bromobenzoyl)amino]-3,4,5,6-tetrahydro-2H-thiopyran-4-yl}car-
bonyl)-l-prolyl]amino}-3,4,5,6-tetrahydro-2H-thiopyran-4-yl)carbonyl]-l-prolyl}amino)-3,4,5,6-tetrahy-
dro-2H-thiopyran-4-yl]carbonyl}-l-prolinate (4-Br-C6H4CO-(Tht-Pro)3-OMe; 6b). According to GP 1,
with 5b (137 mg, 0.201 mmol) and 1g (102 mg, 0.402 mmol), stirring for 116 h, CC (CH2Cl2/MeOH
150 :1 ! 10 :1): 191 mg (73%) of 6b. White foam. M.p. 3168 (dec.). IR: 3475w, 3390w, 3310m, 2948m,
2871w, 1757s, 1639s, 1591m, 1567m, 1522s, 1482m, 1402s, 1314w, 1278m, 1257w, 1209w, 1194m, 1172m,
1153w, 1098w, 1071w, 1041w, 1011m, 994w, 967w, 928m, 910w, 882w, 850w, 803w, 761m, 727w, 682w, 662w.
1H-NMR ((D6)DMSO): 8.61 (s, NH) 7.88 – 7.85 (m, 2 arom. H); 7.81 (s, NH); 7.75 – 7.72 (m, 2 arom. H,
NH); 4.48 – 4.46 (m, 2 CH(a)(Pro)); 4.23 – 4.22 (m, CH(a)(Pro)); 3.59 – 3.56 (m, 2 H of 3 CH2(d)(Pro));
3.59 – 3.51 (m, 4 H of 3 CH2(d)(Pro), MeO); 2.79 – 2.75 (m, 6 H); 2.73 – 2.70 (m, 4 H); 2.49 – 2.23 (m,
15 H); 2.19 – 2.02 (m, 11 H). 13C-NMR ((D6)DMSO): 172.5, 172.0, 171.0, 170.8, 170.7, 170.0 (6s, 6 C¼O);
165.0 (s, ArC¼O); 132.6 (s, 1 arom. C); 131.4 (d, 2 arom. CH); 129.8 (d, 2 arom. CH); 126.9 (s, 1 arom. C);
61.4, 60.8, 60.1 (3d, 3 CH(a)(Pro)); 58.1, 57.3, 57.1 (3s, 3 C(4)(Tht)); 51.4 (q, MeO); 47.6, 47.2, 46.9 (3t,
3 CH2(d)(Pro)); 32.6, 32.5, 32.4, 32.3, 31.9, 31.8 (6t, 3 C(2), 3 C(6)(Tht)); 28.1, 27.9, 27.2 (3t,
3 CH2(b)(Pro)); 25.4, 25.3, 25.2 (3t, 3 CH2(g)(Pro)); 23.0, 22.9, 22.8, 22.7, 22.6, 22.5 (6t, 3 C(3),

Helvetica Chimica Acta – Vol. 96 (2013)
1729
3 C(5)(Tht)). ESI-MS: 959 (100, [M(81Br) þ Na]þ), 957 (79, [M(79Br) þ Na]þ), 808 (4, [M(81Br) ꢀ (Pro-
OMe) þ H]þ), 806 (4, [M(79Br) ꢀ (Pro-OMe) þ H]þ).
Suitable crystals for the X-ray crystal-structure determination were grown from CH2Cl2/MeOH/
hexane by slow evaporation of the solvent.
4. Solvent and Temp. Dependence of the Chemical Shifts of the NH Groups. The peptides 6a, 6b, and 8
were dissolved in CDCl3 (ca. 0.2m), and the chemical shifts of the NH groups were determined at ca. 308.
Then, using a syringe, 2, 4, 6, 8, 10, and 12% (v/v) of (D6)DMSO were added, and, after each addition, the
chemical shifts were determined again. For the determination of the temp. dependence, the NH
absorption in CDCl3 soln. (ca. 0.2m) was recorded between 265 and 314 K in 7-K intervals.
5. X-Ray Crystal Structure Determinations of 6a and 6b (see Tables 1, 2, 4 – 6, and Figs. 2 and 3)2).
The determinations for 6a and 6b were accomplished on a Nonius KappaCCD area-detector
diffractometer [23] using graphite-monochromated MoKa radiation (l 0.71073 ꢅ) and an Oxford
Cryosystems Cryostream 700 cooler. Data reduction was performed with HKL Denzo and Scalepack
[24]. The intensities were corrected for Lorentz and polarization effects. A numerical absorption
correction [25] was applied in the case of 6a, and an absorption correction based on the multi-scan
method [26] was applied in the case of 6b. In all cases, equivalent reflections, other than Friedel pairs,
were merged. Data collection and refinement parameters are collected in Table 6. The structures were
solved by direct methods using SIR92 [27], which revealed the positions of all non-H atoms.
The asymmetric unit of 6a contains two symmetry-independent peptide, five CHCl3, and three H2O
molecules. The atomic coordinates were tested carefully for a relationship from a higher symmetry space
group using the program PLATON [28], but none could be found. Three of the five CHCl3 molecules and
all H2O molecules are disordered over several orientations, and attempts to model the disorder led to
unsatisfactory results. Therefore, the SQUEEZE routine [29] of the program PLATON was employed.
This procedure allowed omission of the disordered solvent molecules from the subsequent refinement
model (the ordered CHCl3 molecules were retained), and gave satisfactory refinement results, which
were better than any attempt to include the disordered solvent atoms in the model. The voids left in the
structure due to the omission of the disordered solvent molecules had a total volume of 1317 ꢅ3 per unit
cell. Most of this volume comprised a single void of 1069 ꢅ3 centered at 0, 0, 0.5, and there were two
smaller symmetry-related voids of 124 ꢅ3. There were no significant peaks of residual electron density to
be found in these voids. The electron count in the voids was calculated to be ca. 415 e, which corresponds
well with 408 e calculated for the six CHCl3 and six H2O molecules per unit cell that were omitted from
the model. One five-membered ring in each of molecules A and B is disordered over two envelope
conformations. Two positions were defined for the atoms C(39) and C(91), resp., and refinement of the
site occupation factors yielded values of 0.55(2) and 0.53(2), resp., for the major conformations of
molecules A and B. Additionally, the six-membered ring at C(78) in molecule B is disordered over two
chair conformations. Two positions were defined for O(3), C(2), and C(4) in this ring, and refinement of
the site occupation factor yielded a value of 0.79(1) for the major conformation. Pseudo-isotropic
restraints were applied to the atomic displacement parameters of some of the disordered atoms in this
ring. Bond-length restraints were applied to all bonds involving disordered atoms so as to maintain
reasonable geometry.
The asymmetric unit of 6b contains two symmetry-independent peptide molecules, two fully
occupied sites for MeOH molecules, plus one partially occupied MeOH site. The site occupation factors
for this latter molecule refined to a value of 0.43(1). The atomic coordinates of the molecules were tested
carefully for a relationship from a higher symmetry space group using the program PLATON [28], but
none could be found. One C-atom in the central five-membered ring of peptide molecule B is disordered
over two positions, and refinement of the site occupation factors yielded a value of 0.60(2) for the major
conformation. Bond-length restraints were applied to all bonds involving the disordered atom and to the
CꢀO bonds of the MeOH molecules. The two positions for the disordered atom were also restrained to
have similar pseudo-isotropic atomic displacement parameters.
2
)
CCDC-890142 and -890144 contain the supplementary crystallographic data for this article. These
data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.

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Helvetica Chimica Acta – Vol. 96 (2013)
Table 6. Crystallographic Data for Compounds 6a and 6b
6a
6b
Crystallized from
Empirical formula
Formula weight
Crystal color, habit
Crystal dimensions [mm]
Temp. [K]
hexane/CHCl3
CH2Cl2/MeOH/hexane
2 C41H55BrN6O11 · 5 CHCl3 · 3 H2O C41H55BrN6O8S3 · 1.21 MeOH
2426.57
colorless, plate
0.02 ꢁ 0.22 ꢁ 0.25
160(1)
974.78
colorless, prism
0.12 ꢁ 0.15 ꢁ 0.30
160(1)
Crystal system
Space group
monoclinic
P21
triclinic
P1
Z
2
2
Reflections for cell determination
284369
46166
2q Range for cell determination [8] 4 – 50
4 – 50
Unit cell parameters a [ꢅ]
18.2782(1)
9.3659(4)
12.9994(3)
19.2946(8)
79.668(2)
80.836(2)
87.877(2)
2281.5(2)
1.419
b [ꢅ]
c [ꢅ]
a [8]
b [8]
12.9913(1)
24.1243(2)
90
101.9790(2)
90
5603.75(7)
1.438
1.154
g [8]
V [ꢅ3]
Dx [g cmꢀ3
]
m(MoKa) [mmꢀ1
Scan type
]
1.101
f and w
50
w
2q(max) [8]
50
Transmission factors [min; max]
Total reflections measured
0.802; 0.962
120234
0.832; 0.879
32037
Symmetry-independent reflections 18888
15434
Reflections with I > 2s(I)
Reflections used in refinement
Parameters refined; restraints
Final R(F) [I > 2s(I) reflections]
wR(F2) (all data)
Weighting parameters [a; b]a)
Goodness of fit
16118
18885
1187; 35
0.0565
0.1550
12109
15433
1142; 36
0.0496
0.1253
0.1031; 1.3458
1.052
0.0616; 0.9994
1.015
Secondary extinction coefficient
Final Dmax/s
0.0023(3)
0.001
0.59; ꢀ 0.48
0.0076(7)
0.002
0.57; ꢀ 0.33
D1(max; min) [e ꢅꢀ3
]
a) wꢀ1 ¼ s2(F2o ) þ (aP)2 þ bP where P ¼ (Fo2 þ 2F2c )/3.
The non-H-atoms were refined anisotropically. All of the H-atoms were placed in geometrically
calculated positions and refined using a riding model where each H-atom was assigned a fixed isotropic
displacement parameter with a value equal to 1.2 Ueq of its parent atom (1.5 Ueq for the Me and OH
groups). In the case of 6b, the orientations of the OꢀH vectors were optimized to correspond with the
direction indicated by residual electron-density peaks. The OH H-atom of the partially occupied MeOH
molecule was not included in the model. The refinement of the structures was carried out on F2 using full-
matrix least-squares procedures [30], which minimized the function Sw(F2o – Fc2 )2. A correction for
secondary extinction was applied in both cases. Three and one reflection for 6a and 6b, resp., whose
intensities were considered to be extreme outliers, were omitted from the final refinement of each
structure. Refinement of the absolute structure parameter [31] yielded a value of 0.104(6) and 0.000(6),
resp., which confidently confirm that the refined model represents the true enantiomorphs.

Helvetica Chimica Acta – Vol. 96 (2013)
1731
Neutral atom-scattering factors for non-H-atoms were taken from [32a], and the scattering factors
for H-atoms were taken from [33]. Anomalous dispersion effects were included in Fc [34]; the values for
f’ and f’’ were those of [32b]. The values of the mass-attenuation coefficients are those of [32c]. All
calculations were performed using the SHELXL97 [30] program.
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Received January 29, 2013