Electrophilic S-trifluoromethylation of cysteine side chains in α- and β-peptides: Isolation of trifluoromethylated sandostatin (octreotide) derivatives

Article abstract of DOI:10.1002/hlca.200890217

The new electrophilic trifluoromethylating 1-(trifluoromethyl)-benziodoxole reagents A and B (Scheme 1) have been used to selectively attach CF₃ groups to the S-atom of cysteine side chains of α- and β-peptides (up to 13-residues-long; products 7-14). Other functional groups in the substrates (amino, amido, carbamate, carboxylate, hydroxy, phenyl) are not attacked by these soft reagents. Depending on the conditions, the indole ring of a Trp residue may also be trifluoromethylated (in the 2-position). The products are purified by chromatography, and identified by ¹H-, ¹³C-, and ¹⁹F-NMR spectroscopy, by CD spectroscopy, and by high-resolution mass spectrometry. The CF₃ groups, thus introduced, may be replaced by H (Na/NH₃), an overall Cys/Ala conversion. The importance of trifluoromethylations in medicinal chemistry and possible applications of the method (spin-labelling, imaging, PET) are discussed.

Full text of DOI:10.1002/hlca.200890217

Helvetica Chimica Acta – Vol. 91 (2008)
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Electrophilic S-Trifluoromethylation of Cysteine Side Chains in
a- and b-Peptides: Isolation of Trifluoromethylated Sandostatinꢀ
(Octreotide) Derivatives
by Stefania Caponea)1), Iris Kieltschb)2), Oliver Flçgela)3), Gerald Lelaisa)4), Antonio Togni*b), and
Dieter Seebach*a)
a) Laboratorium fꢀr Organische Chemie, Departement Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich, Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(phone: þ 41-44-632-2990; fax: þ 41-44-632-1144; e-mail: seebach@org.chem.ethz.ch)
b) Laboratorium fꢀr Anorganische Chemie, Departement Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich Hçnggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(fax: þ 41-44-632-1310; e-mail: togni@inorg.chem.ethz.ch)
Dedicated to Duilio Arigoni on the occasion of his 80th birthday
The new electrophilic trifluoromethylating 1-(trifluoromethyl)-benziodoxole reagents A and B
(Scheme 1) have been used to selectively attach CF3 groups to the S-atom of cysteine side chains of a-
and b-peptides (up to 13-residues-long; products 7 – 14). Other functional groups in the substrates
(amino, amido, carbamate, carboxylate, hydroxy, phenyl) are not attacked by these soft reagents.
Depending on the conditions, the indole ring of a Trp residue may also be trifluoromethylated (in the 2-
position). The products are purified by chromatography, and identified by 1H-, 13C-, and 19F-NMR
spectroscopy, by CD spectroscopy, and by high-resolution mass spectrometry. The CF3 groups, thus
introduced, may be replaced by H (Na/NH3), an overall Cys/Ala conversion. The importance of
trifluoromethylations in medicinal chemistry and possible applications of the method (spin-labelling,
imaging, PET) are discussed.
1. Introduction. – In one of our groups, there is a long-standing interest in the
modification of peptides: naphthylselenation [1], dehydration of serine moieties [2],
C¼O ! C¼S thionylation [3], electrochemical decarboxylation [4], Li-enolate
formation and reactions with electrophiles [5], and homologation of amino-acid
residues [6][7]. In continuation of this tradition, we have now investigated the
conversion of CH2ꢀSꢀH groups of cysteine side chains in peptides to CH2ꢀSꢀCF3
groups, by using a method [8][9] that has been recently introduced by another group of
co-authors of the present article5). The method uses the hypervalent iodine reagents A
1
)
Postdoctoral Research Fellow at ETH-Zꢀrich, 2006 – 2008, financed by the Swiss National Science
Foundation (Project No. 200020-109065 and 200020-117586).
Part of the Ph.D. Thesis of I. K., ETH-Zꢀrich, 2008, Dissertation No. 17990.
Postdoctoral Research Fellow at ETH-Zꢀrich, 2004 – 2006, financed by the Deutsche Forschungs-
gemeinschaft (DFG), by the Swiss National Science Foundation (Project No. 200020-100182), and by
Novartis Pharma AG, Basel.
2
3
)
)
4
5
)
)
Part of the Ph.D. Thesis of G. L., ETH-Zꢀrich, 2000 – 2004, Dissertation No. 15429.
Both our groups have a history of preparing and investigating organo-fluoro compounds [8 – 11].
ꢁ 2008 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Helvetica Chimica Acta – Vol. 91 (2008)
and B with a CF3 group attached to the I-atom. They are capable of transferring CF3 to
nucleophilic centers of substrate molecules. This electrophilic trifluoromethylation
reaction is remarkably mild and selective. Examples are shown in Scheme 1: soft
nucleophiles (with high-lying HOMO) such as the anions of b-keto esters, or of a-nitro
esters, silyl enol ethers, electron-rich aromatic systems (phenol, pyrrol, indole),
phosphanes, and thiols are preferred over harder nucleophiles such as amino or OH
groups to an extent that the reactions can be carried out in MeOH/H2O (at low
temperature) [8][9]. The CF3ꢀI s* orbital (LUMO) in these molecules must be low-
lying. The new method provides an umpolung of the nucleophilic trifluoromethylation
with Me3SiCF3/Fꢀ (Ruppert – Prakash reagent) [12].
Scheme 1. Molecular Formulae of the CF3-Substituted Benziodoxoles A and B, and of the Products
Obtained with Various Nucleophiles. A and B correspond to CFþ3 synthons, while Me3SiCF3 corresponds
to a CFꢀ3 synthon.
1.1 Preparation of CF3ꢀS Derivatives. Reagents A and B are readily prepared from
inexpensive starting materials, and the I-containing reduced species can be recovered
after delivery of the CF3 fragment [8][9]. It has already been shown that these
hypervalent iodine(III)ꢀCF3 reagents can be used for S-trifluoromethylation of
cysteine esters (with or without N-protection) in high yields; the reaction is carried out
in MeOH at dry-ice temperature in the absence of base [9]. Before invention of the
reagents A and B, SCF3 substituents were introduced into organic molecules by
functional-group interconversions under rather drastic conditions [13], by direct
transfer of the SCF3 moiety [14], or by the delivery of CF3 to a S-atom by a
nucleophilic, or, more often, a radical reaction [15][16]. The hitherto less common
electrophilic S-trifluoromethylation with (trifluoromethyl)dibenzothio-, -seleno-, or
-tellurophenium salts (Umemoto and Ishihara [17]) requires preformed sodium
thiolates, and the product is accompanied by a considerable amount of disulfide. In
particular, for the direct preparation of S-(trifluoromethyl)-cysteine derivatives,
reaction conditions involving t-BuO2H [15][18] or liquid NH3 [19] had to be employed.
In contrast, with reagent A essentially no disulfide by-product is formed. As outlined
above, the SH ! SCF3 transformation exhibits a remarkably high functional-group

Helvetica Chimica Acta – Vol. 91 (2008)
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selectivity: amino, hydroxy, carboxy, amido, thioacetal, and alkinyl groups do not
interfere.
Here, we present applications of the electrophilic trifluoromethylation of cysteine
side chains in a- and b-peptides consisting of up to 13 amino-acid residues with a variety
of other side-chain functional groups.
1.2. Why Trifluoromethylation of Cysteine Side Chains? The introduction of F-
atoms, in particular CF3 groups, into drug candidates constitutes an important tool of
the lead-finding process of medicinal chemists [20]. The reduced polarity and the
increased metabolic stability brought about by the CF3 moiety can lead to higher
biological activity and improved bioavailability of a potential drug. Very often, when
CH3 is replaced by CF3, there is no more P450-dependent hydroxylation of neighboring
sites, and the lipophilicity rises considerably (Hansch parameter p of SCH3 0.62, of
SCF3 1.58 in the ꢂ3-substituted phenoxy acetic acidꢃ system) [21]. Thus, trifluorome-
thylation of a cysteine residue, not involved in a disulfide bridge, of a peptide or a
protein may greatly alter its properties. Furthermore, conversion of a cystine group,
CH2ꢀSꢀSꢀCH2, to a [CH2ꢀSꢀCF3/CF3ꢀSꢀCH2] moiety, while removing a
structure-stabilizing covalent bond, may create a hydrophobic pocket within the
peptide or protein. As a first example of this kind of structural change, we chose the
cyclic disulfide derivative Sandostatinꢄ (octreotide) [22]6).
2. Preparation of the Peptides for Trifluoromethylation. – As simple peptidic
substrates for this reaction, we chose the a-dipeptides R-Phe-Cys-OMe and R-Cys-
Ala-OMe (R ¼ Boc or H), which we generated in situ from the corresponding
disulfides (cystine derivatives) 1 and 2 (Scheme 2) by reduction with Et3P in aqueous
MeOH. As analogous b-dipeptide substrates Boc-b3hPhe-b3hCys-OH (3) and Boc-
b3hCys-b3hAla-OH (4) were chosen (Scheme 3). All six dipeptides were prepared by
coupling appropriately protected amino acids with HATU in CH2Cl2 solution as
outlined in Schemes 2 and 3, and as described in detail in the Exper. Part.
Two more complex peptides 5 and 6 with the functional groups in the side chains of
Lys, Asp, Trp, and Thr, and without terminal protection were also prepared for
trifluoromethylation reactions. The b-tridecapeptide 5 was prepared by solid-phase
peptide synthesis (SPPS) on Wang resin, using the Fmoc strategy [26]. For the last
coupling step, Boc-protected b3hCys(Trt)-OH was used. After cleavage from the resin
and removal of the protecting groups [27], the linear peptide was purified by
preparative reversed-phase HPLC to provide the CF3COOH (TFA) salt of 5. The a-
octapeptide derivative 6 is the ring-opened form of the drug Sandostatinꢄ (octreotide)
marketed by Novartis6). A sample of the cyclic disulfide octreotide acetate7) was
dissolved in degassed phosphate buffer and treated with 1,4-dithiothreitol (DTT) to
cleave the disulfide bond. The resulting solution was diluted in aqueous TFA and
directly subjected to reversed-phase HPLC purification, to afford the dithiol 6 as TFA
salt.
6
)
)
For recent discussions of the pharmacological properties of this drug, see [23 – 25].
As provided by Novartis Pharma AG, Basel.
7

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Scheme 2. Preparation of the Disulfanediyl-Bridged Dipeptide Diesters 1 and 2. All starting materials
and reagents were used as purchased.
a) 2-(7-Aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2,6-di-
methylpyridine (DMP), CH2Cl2/OCHNMe2 (DMF), 12 h. b) CF3CO2H (TFA), CH2Cl2, 3 h.
Scheme 3. Preparation of the Boc-b-Dipeptides 3 and 4. The starting materials were purchased or
prepared according to known procedures (see references in the Exper. Part). For abbreviations, see
Scheme 2.
a) HATU, Et3N, CH2Cl2, 12 h. b) NaOH, MeOH, 508, 12 h. c) Na/NH3, t-BuOH, THF, ꢀ 808, 10 min.
3. Trifluoromethylations of Peptide Cysteine Side Chains to Give Derivatives 7–11. –
First, the cysteine derivatives 1 and 2 were cleaved to the corresponding peptide thiols
by treating solutions in MeOH/H2O 9 :1 with 2 equiv. of Et3P at room temperature.
Subsequent addition at ꢀ 788 of 2.3 equiv. of reagent A (dissolved in MeOH), warming
to room temperature, and removing the solvent in vacuo gave the crude products 7a
and 7b, and 8a and 8b, which were purified by chromatography and isolated in yields
ranging between 50 and 60%. Likewise, the reactions (in MeOH, ꢀ 788 !þ 258) of the
b-dipeptides 3 and 4 with 1.1 equiv. of reagent A provided the trifluoromethylated
products 9 and 10, respectively, in essentially quantitative yields. The b-tridecapeptide 5
(containing, besides the SH group, six unprotected functional groups, plus the twelve
amide groups) was subjected to a slight excess of trifluoromethylating reagent A under
the same conditions (degassed MeOH solution, inert atmosphere, ꢀ 788 to þ 258), to
give a 40% yield of its CF3 derivative 11 (after preparative RP-HPLC purification) on a
5-mm scale (for details and characterization of 11 see the Exper. Part).

Helvetica Chimica Acta – Vol. 91 (2008)
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4. Trifluoromethylation of the Octreotide Derivative 6. – From the reaction (under
various condition) of disulfanyl derivative 6 with reagent A or B, we were able to isolate
three products: the octreotide 12 with a single CF3 group in position 2 of the d-Trp
residue, the open-chain peptide derivatives 13 with two SꢀCF3 groups, and 14 with
trifluoromethylated indole ring and two SꢀCF3 groups. The three compounds were
separated by preparative RP-HPLC, and characterized by NMR and CD spectroscopy,
and by mass spectrometry (LC/MS). Under the standard conditions, reagent A (2.9
equiv.) produced 12, 13, and 14 in a ratio of ca. 1:6 :6 in a total yield (after purification)
of 34% (þ 3% octreotide). As can be seen from the analytical HPLC trace of the
crude-product mixture in Fig. 1,a, a number of minor by-products is also formed.
Notably, octreotide itself is not trifluoromethylated by reagent A. With 2.1 equiv. of
reagent B and disulfanyl derivative 6, the mono-trifluoromethylated octreotide 12 and
octreotide, the product of disulfide coupling, are formed (ratio 1:3, combined yield
50% after RP-HPLC separation/purification). The reaction with reagent B (the
ꢂharderꢃ one, as compared to A) is much cleaner (see Fig. 1, b). If the SH groups of 6
are deprotonated by addition of 3 equiv. of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), the only product detectable by NMR analysis in the reaction with the
benziodoxole derivative B is the bis(trifluoromethyl) derivative 13 – a remarkable
switch from attack on the indole ring to reaction at the S-atom (CF3 transfer from the
hard reagent B is charge-controlled).

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Helvetica Chimica Acta – Vol. 91 (2008)
The CD spectra of octreotide, and of its derivatives 6, 12, 13, and 14 are shown in
Fig. 2. As it had to be expected, the [F3C-Trp]-octreotide 12 gives rise to a pattern
similar to that of octreotide itself, with a weak positive Cotton effect between 250 and
220 nm, and a stronger negative one between 205 and 200 nm. Why the depth of the
short-wavelength peak observed for the trifluoromethylated compound is so much
smaller is not clear at this point. The open-chain starting material, i.e., disulfanyl
derivative 6, and the tris(trifluoromethylated), likewise open-chain product 14 give rise
to negative-only spectra between 250 and 195 nm with very similar shapes. Again, the
spectrum of lower intensity belongs to the CF3 derivative. There is a resemblance of the
CD spectra of these two compounds (minima near 220 and 205 nm) with those
considered characteristic of a-helices (minima at 220 and 210 nm)8). Interestingly, it
has been shown by X-ray and NMR investigations of octreotide that the molecule can
adopt both, antiparallel b-sheet and 310-helix-like secondary structures, in which the C-
terminal residues fold to a ꢂhelical ensembleꢃ [29].
8
)
For typical CD curves of a-helices, b-sheets, and random coils, see textbooks of peptide and protein
chemistry, for instance, Fig. 5.12 in [28].

Helvetica Chimica Acta – Vol. 91 (2008)
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Detailed structural analysis of the new octreotide derivatives and their affinities for
somatostatin receptors are under investigation and will be published in a forthcoming
article.
The electrophilic trifluoromethylation described here may be used for an overall
reductive removal of the SH group in a cysteine side chain: we have treated the
trifluoromethylated compound 10 under Birch-reduction conditions (Na in liq. NH3),
which led to a quantitative yield of diastereoisomerically pure N-Boc-b3hAla-b3hAla-
OH, as established by NMR comparison with an authentic sample (Scheme 4 and
Fig. 3).
5. Conclusions and Outlook. – We have shown that the new electrophilic
trifluoromethylating reagents A and B can be used for remarkably selective trans-
formations of peptides. The reactions can be carried out in the protic solvents MeOH
and H2O, and the primary goal of attack is the SH group of cysteine. Amino groups of
lysine residues, free N-terminal amino groups, Boc-carbamoyl groups, amide groups in

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Fig. 1. HPLC Traces of the crude product from reaction of the dithiol 6 with a) reagent A (2.9 equiv. of A
in MeOH at ꢀ 788, 12 h; 34%) and b) reagent B (2.1 equiv. of B in MeOH at ꢀ 788, 12 h; 50%).
Chromatographic conditions: C18 column (Macherey-Nagel 250 mm ꢁ 4.6 mm ꢁ 300 ꢅ, 5 m; 5 – 95%
MeCN in H2O/0.1% TFA (1 ml/min) in 45 min: linear gradient). The intensive peaks at 24.6 min (a) and
19.4 min (b) correspond to 2-(2-iodophenyl)propan-2-ol and 2-iodobenzoic acid, the products formed
from A and B, resp., after the electrophilic CF3 group has been ꢂdeliveredꢃ.
the chain, carboxylate groups of aspartic acid, OH groups of threonine, and benzene
rings of phenylalanine side chains do not appear to be able to compete for the
electrophilic CF3 group of the reagents A and B. By preliminary optimization
experiments, it was demonstrated that, depending on the conditions and the reagent
used, only the indole ring of tryptophan competes with the SH groups, but at higher pH

Helvetica Chimica Acta – Vol. 91 (2008)
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Fig. 2. Normalized CD spectra in MeOH (0.2 mm) of Octreotide (blue), and of its derivatives 6 (black),
12 (red), and 14 (green). Solutions were prepared by weighing the peptide on a mg-precision balance and
dissolving it in a volumetric flask.
Scheme 4
(cf. addition of DBU) the generated thiolate wins. Independent work by one of our
groups has shown that ortho-/para-trifluoromethylation of phenol groups (cf. tyrosine)
occurs more slowly than electrophilic attack at an indole ring, an effect, which should
be pH-dependent.
To the peptide chemist the transformations described herein may look like standard
reactions: very small scales, LC/MS detection, preparative HPLC separation, and
purification, lyophilization, HR-MS characterization, and CD and NMR spectra. For
the preparative chemist, the reported experiments must be considered preliminary and
mainly analytical. Thus, future experiments will have to address upscaling and further
optimization of selectivity of the trifluoromethylation of peptides.
What could this reaction be useful for? One aspect has been discussed in the
introduction: CF3-modified compounds are interesting drug candidates. Another
application could be the overall replacement of the cysteine SH group by other groups
X, (cf. Scheme 4), for instance through the nucleophilic or radical processes involving
dehydro-alanine moieties, as outlined in Scheme 5 [30]. The CF3S group should be an

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Fig. 3. Analysis of the reductive cleavage of 10. NMR Spectra of a) Boc-b3hAla-b3hAla-OH, b) of the
epimer Boc-(R)-b3hAla-b3hAla-OH, c) crude product of the reaction of 10 with Na/NH3, d) mixture
obtained by adding Boc-(R)-b3hAla-b3hAla-OH to the crude reaction mixture from 10 and Na/NH3.
excellent leaving group, if we consider the increased stability of CF3Sꢀ as compared to
alkyl – Sꢀ (the pKa value of an alkyl-thiol is ca. 7, the pKa value of F3CSꢀH could well
be 5 to 10 units lower)9). Indeed, in a preliminary experiment we have found that, by
treating compound 8a with t-BuOK in MeOD, CF3SH is eliminated from the cysteine
residue, giving rise to a dehydroalanine residue. Of course, the SeH group of
selenocysteine (Sec) is expected to be an even ꢂbetter targetꢃ for the trifluoromethylat-
ing reagents A and B, and in corresponding CF3Se derivatives CF3Seꢀ will be an even
better leaving group than CF3Sꢀ.
The most attractive use of the trifluoromethylation reaction is in the fields of
biochemistry, biology, and medicine. It is a well-known technology to use surface
cysteines in proteins for attaching all kinds of groups [32], either through disulfide
9
)
See pKa tables in [31] or Evans pKa Table. Examples are CH3/CF3CH2OH (DpKa 4), CH3/CF3CO2H
(DpKa 5), CH3/CF3SO3H (DpKa 11).

Helvetica Chimica Acta – Vol. 91 (2008)
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Scheme 5
formation or by elimination and addition (cf. Scheme 5). This has been applied for
glycoconjugation, for introduction of N3 or HCꢂC groups (cf. ꢂclickꢃ chemistry [33]),
for attachment of fluorophores, and for many other transformations (ꢂpost-transla-
tional mimickingꢃ [34]). If the trifluoromethylation of such surface SH groups in
proteins and enzymes were possible (Scheme 6), without drastically altering their
physiological properties, three important applications could be envisioned. i) The CF3
group will provide a unique identification of the species by 19F-NMR spectroscopy;
there is no interference of 19F-resonance with any other magnetic nucleus in the
molecule, which would allow facile NMR imaging in cells, in tissues, and in plants or
animals. ii) The CF3 group could be used as a spin label, exploiting nuclear Overhauser
effects for probing its neighborhood within a complex molecule. iii) Carried out with a
18F-labelled CF3 group, the reaction would allow for a quick, one-step introduction of
the positron-emitting isotope, and thus for positron-emission tomography (PET)10).
For these purposes [36], the phenol ring of tyrosine or the indole ring of tryptophan at
the surface of a protein are also possible targets of trifluoromethylation by reagents of
type A or B11). Work along the lines discussed in the previous paragraphs is under-way
in our laboratories or is being pursued in collaboration with specialists in the
corresponding fields.
Scheme 6. Possible Trifluoromethylation of Cysteine Side Chains at the ꢀSurfaceꢁ of Proteins
10
)
)
For an example and for leading references, see an article entitled: ꢀFirst 18F-Labeled Tracer Suitable
for Routine Clinical Imaging of sst Receptor-Expressing Tumors Using PETꢁ [35].
The reactivity, and thus the selectivity, of these reagents could be modified by putting electron-
donating or -withdrawing substituents in various positions of their benzene rings.
11

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Helvetica Chimica Acta – Vol. 91 (2008)
Experimental Part
1. General. Abbreviations: Bn: benzyl; Boc: (tert-butoxy)carbonyl; DTT: dithiothreitol; HATU: 2-
(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TFA: CF3COOH; FC:
flash chromatography; h.v.: high vacuum (0.01 – 0.1 Torr); HR-MALDI: high-resolution matrix-assisted
laser desorption ionization; RP: reversed-phase; TLC: thin layer chromatography; SPPS: solid-phase
peptide synthesis.
Materials and Methods. Solvents for reactions, workup procedures, and/or chromatography were
distilled (AcOEt, MeOH (Sikkon, anh. CaSO4), CH2Cl2 (P2O5), Et2O (KOH and filtered through Al2O3
(Alumina Woelm N, activity I)), THF (Na)), or used in p.a. quality (Fluka, Aldrich). All other reagents
were used as received from Fluka or Aldrich. Wang resins were purchased from Novabiochem. TLC:
silica gel Merck 60 F254 plates (0.2-mm layer) and FC on Merck Kieselgel 60 (230 – 400 mesh). Anal. RP-
HPLC: Merck/Hitachi HPLC system (LaChrom, L-7150 pump, UV detector L-7400, interface D-7000);
column: Nucleosil 100-5 C18 (250 ꢁ 4 mm, Macherey-Nagel). HPLC Analyses: with a linear gradient of
A (0.1% TFA in H2O) and B (MeCN) at a flow rate of 1 ml/min with UV detection at 220 nm. Retention
time (tR) in min. Prep. RP-HPLC: Merck/Hitachi HPLC system (LaChrom, pump type L-7150, UV
detector L-7400, interface D-7000, HPLC manager D-7000); column: Nucleosil 100-7 C18 (250 ꢁ 21 mm,
Macherey-Nagel). Crude products were purified with a gradient of A (0.1% TFA in H2O) and B (MeCN)
at a flow rate of 10 ml/min with UV detection at 220 nm, and then lyophilized. UV-Grade TFA (> 99%
GC) was used for RP-HPLC. Lyophilization: Hetosicc cooling condenser with a h.v. pump. CD Spectra:
Jasco J-710 spectropolarimeter from 190 – 250 nm at r.t. in 1-mm pathlength cells. The optical system was
flushed with N2 at a flow rate of ca. 10 l/min. Parameters: band width 1.0 nm, resolution 0.2 ꢃ 1 nm,
sensitivity 100 mdeg, response 0.5 s, speed 50 nm/min, 5 accumulations. All spectra were corrected for the
corresponding solvent spectrum. The molar ellipticity [q] in deg cm2 molꢀ1 (l in nm) is calculated for the
corresponding peptide and normalized, taking into account the mass of TFA for each free amino group.
Smoothing was done by Jasco software. Solvents: MeOH (HPLC grade). M.p.: in open-end glass cap.
tubes on a Bꢂchi 510 apparatus, uncorrected. [a]2D0: Jasco PIP-370 polarimeter (1.0-dm cell); solvent
MeOH, unless otherwise specified. IR Spectra: Perkin-Elmer Spectrum-100 spectrometer; in cmꢀ1. NMR
Spectra: Bruker DPX-500 (1H: 500 MHz, 13C: 125 MHz), DRX-400 (1H: 400 MHz, 13C: 100 MHz, 19F:
376 MHz), DPX-400 (1H: 400 MHz, 13C: 100 MHz, 19F: 376 MHz), ARX-300 (1H: 300 MHz, 13C:
75 MHz), or Varian Mercury-300BB (1H: 300 MHz, 13C: 75 MHz, 19F: 282 MHz); chemical shifts d in
ppm; J values in Hz; all spectra recorded in CD3OD, unless otherwise specified; CFCl3 external standard
for 19F-NMR. MS: IonSpecUltima 4.7-T-FT Ion Cyclotron Resonance (ICR; HR-MALDI, in 2,5-
dihydroxybenzoic acid matrix) spectrometer; in m/z. LC/MS was performed on a Thermo Separation
Products (TSP) HPLC system connected to a PDA detector (UV6000LB, TSP) and to an ion-trap mass-
spectrometry detector (LCQdeca, Finnigan). Linear gradients of 5 – 60% MeCN in H2O/0.1% HCOOH
(0.2 ml/min) on a Waters Atlantis dC18-3 3 ꢁ 100 mm column were used.
Preparation and characterization of Boc-(R)-b3hCys(Bn)-OMe [37][38], Boc-(R)-b3h-Cys(Bn)-
OH [38][39], and Boc-(S)-b3hAla-OBn [7][40] has been previously reported.
2. Boc Deprotection: General Procedure 1 (GP 1). GP 1a: The Boc-protected a-peptide was
dissolved in CH2Cl2 (0.5m) and cooled to 08. An equal volume of TFA was added, and the mixture was
allowed to warm slowly to r.t. and stirred for further 3 h. Concentration under reduced pressure and co-
evaporation with hexane of the residue under h.v. yielded the crude TFA salt, which was lyophilized from
dioxane or MeCN/H2O 3 :7 (containing 0.1% TFA), identified by NMR, and used without further
purification.
GP 1b: The Boc-protected b-amino acid was dissolved in CH2Cl2 (0.5m) and cooled to 08. An equal
volume of TFAwas added, and the mixture was allowed to warm slowly to r.t. and stirred for further 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. Peptide Coupling: General Procedure 2 (GP 2). GP 2a: The HCl salt of the a-amino acid was
dissolved in CH2Cl2/DMF 1:1 (v/v, 0.1m) and cooled to 08. This soln. was treated successively with
collidine (2.7 equiv.), a soln. of the Boc-protected a-amino acid (1.1 equiv.) in CH2Cl2 (0.25m), and
HATU (1.1 equiv.). The mixture was allowed to warm to r.t. and stirred for 12 h. Subsequent dilution

Helvetica Chimica Acta – Vol. 91 (2008)
2047
with CH2Cl2 was followed by thorough washing with 0.5m HCl, sat. aq. NaHCO3 (3ꢁ) and NaCl solns.
(1ꢁ). The org. phase was dried (MgSO4) and concentrated under reduced pressure. FC or
recrystallization yielded the pure peptide.
GP 2b: The TFA salt of the b-amino acid was dissolved in CH2Cl2 (0.5m) and cooled to 08. This soln.
was treated successively with Et3N (5 equiv.), a soln. of the Boc-protected b-amino acid (1 equiv.) in
CH2Cl2 (0.25m), and HATU (1.05 equiv.). The mixture was allowed to warm to r.t. and stirred for 12 h.
Subsequent dilution with CH2Cl2 was followed by thorough washing with 0.5m HCl, sat. aq. NaHCO3
(3ꢁ), and NaCl solns. (1ꢁ). The org. phase was dried (MgSO4) and concentrated under reduced
pressure. FC or crystallization yielded the pure peptide.
4. Deprotection of Cysteine Residue of b-Peptides: General Procedure 3 (GP 3). GP 3: A three-
necked round-bottomed flask was fitted with a dry ice condenser and two septum cups and flamed-dried
under N2 flow. The flask was then cooled to ꢀ 788 (acetone/CO2) and NH3 gas (25 ml) was condensed
into the flask via needle. The liq. NH3 was kept under N2 at ꢀ 788. Na (3.2 mmol, 74 mg) was added and,
once the metal had dissolved, the appropriate b-peptide (320 mg, 0.64 mmol) dissolved in THF (6 ml)
and t-BuOH (4% (v/v)) was added slowly by syringe. The temp. was kept at ꢀ 788, and the mixture was
stirred for 10 min at this temp. MeOH (2 ml) was slowly added, and after the blue color disappeared, the
mixture was allowed to warm to r.t., and the NH3 was evaporated under a stream of N2. H2O (5 ml) was
finally added, followed by AcOEt (5 ml), and the org. layer was washed with 1m HCl and brine. The aq.
layers were extracted with AcOEt (2 ꢁ 5 ml), the combined org. layers were dried (Na2SO4), filtered,
and the solvent was removed under reduced pressure to yield a white solid, which was used without
further purifications.
5. Trifluoromethylation of Peptide Fragments: General Procedure 4 (GP 4). GP 4a: Et3P (2 equiv., 1m
in THF) was added to a stirred soln. of a-peptide disulfide in degassed MeOH/H2O 9 :1 (v/v; 33 mm) at
r.t. After 2 h, the mixture was cooled to ꢀ 788, and a soln. of 1,3-dihydro-3,3-dimethyl-1-(trifluor-
omethyl)-1,2-benziodoxole (2.3 equiv.) in degassed MeOH (46 mm) was added dropwise. The slight
yellow mixture was allowed to warm to r.t. and stirred for 12 h. The soln. was evaporated to dryness under
vacuum, and the remaining oil was subjected to FC or HPLC purification.
GP 4b: The appropriate b-peptide was dissolved in degassed MeOH (0.15m) and cooled to ꢀ 788.
1,3-Dihydro-3,3-dimethyl-1-(trifluoromethyl)-1,2-benziodoxole (1.1 equiv.) dissolved in degassed MeOH
(0.3m) and cooled to ꢀ 788 was added, and the reaction was kept at ꢀ 788 under N2 atm for 2 h, then
allowed to warm to r.t., and stirred for 12 h. The solvent was removed under vacuum, and the crude
product was purified by RP-HPLC.
6. Preparation of [Boc-(S)-Phe-(R)-Cys-OMe]2 (1a) and [H-(S)-Phe-(R)-Cys-OMe]2 (1b).
(6S,9R,14R,17S)-Dimethyl 6,17-Dibenzyl-2,2,21,21-tetramethyl-4,7,16,19-tetraoxo-3,20-dioxa-11,12-di-
thia-5,8,15,18-tetraazadocosane-9,14-dicarboxylate ([Boc-(S)-Phe-(R)-Cys-OMe]2; 1a). [H-(R)-Cys-
OMe]2 (1.45 mmol, 389 mg) was coupled with Boc-(S)-Phe-OH (3.63 mmol, 965 mg) according to
GP 2a. Recrystallization from AcOEt/hexane yielded 1a (1.050 g, yield 95%). White solid [a]2D0 ¼ ꢀ42.3
(c ¼ 0.13, MeOH). IR: 3318w, 2975w, 1738m, 1686m, 1649s, 1521s, 1495m, 1454m, 1436m, 1390m, 1365m,
1293m, 1245s, 1164s, 1045m, 1022m, 927w, 865m, 753m, 699s, 642m. 1H-NMR (300 MHz): 1.38 (s, Me3C);
2.74 – 2.94 (m, CH2); 3.02 (dd, J ¼ 7.9, 14.0, 1 H, CH2); 3.12 (dd, J ¼ 5.2, 14.0, 1 H, CH2); 3.72 (s, MeO);
4.32 – 4.44 (m, CH); 4.70 – 4.80 (m, CH); 7.20 – 7.40 (m, 5 arom. H). 13C-NMR (75 MHz): 29.3 (Me3C);
39.7, 40.5 (CH2); 53.2 (CH); 53.3 (MeO); 57.3 (CH); 80.8 (Me3C); 128.0, 129.5, 130.5 (arom. CH); 138.6
(arom. C); 157.7, 172.2, 174.7 (CO). HR-MS (MALDI): 785.285 ([M þ Na]þ, C36H50N4NaO10S2þ ; calc.
785.286). The molecular peak (C36H50N4O10S2) was not observed.
(2S,5R,10R,13S)-Dimethyl 2,13-Diamino-3,12-dioxo-1,14-diphenyl-7,8-dithia-4,11-diazatetradecane-
5,10-dicarboxylate ([H-(S)-Phe-(R)-Cys-OMe]2; 1b). Dipeptide 1a (0.69 mmol, 525 mg) was Boc-
deprotected according to GP 1a. The residue was lyophilized from MeCN/H2O (30%, containing 0.1%
TFA) to give the TFA salt of 1b, which was used without further purifications (504 mg, yield 92%). Part
of this product was purified by RP-HPLC (C18; 5% of B for 5 min, then from 5 to 57% in 40 min; tR
27 min, 40% B). [a]D20 ¼ ꢀ58.5 (c ¼ 0.82, MeOH). IR: 2955m, 1740m, 1665s, 1522m, 1456m, 1437m,
1
1305m, 1176s, 1129s, 1023m, 835m, 798s, 747m, 721s, 700s. H-NMR (300 MHz): 3.00 – 3.13 (m, CH2);
3.19 – 3.28 (m, CH2); 3.74 (s, MeO); 4.21 (dd, J ¼ 5.9, 8.1, CH); 4.79 (dd, J ¼ 5.3, 8.2, CH). 13C-NMR
(75 MHz): 38.6, 40.3 (CH2); 53.2 (CH); 53.4 (MeO); 55.6 (CH); 128.9, 130.2, 130.7 (arom. CH); 135.5

2048
Helvetica Chimica Acta – Vol. 91 (2008)
(arom. C); 170.0, 171.8 (CO). HR-MS (MALDI): 563.199 (Mþ, C26H35N4O6S2þ ; calc. 563.199), 585.181
([M þ Na]þ).
7. Preparation of [Boc-(R)-Cys-(S)-Ala-OMe]2 (2a) and [H-(R)-Cys-(S)-Ala-OMe]2 (2b).
(2R,5S,10S,13R)-Dimethyl 5,10-Bis[(tert-butoxycarbonyl)amino]-2,13-dimethyl-7,8-dithia-3,12-diazate-
tradecanedioate ([Boc-(R)-Cys-(S)-Ala-OMe]2; 2a). H-(S)-Ala-OMe (5.0 mmol, 516 mg) was coupled
with [Boc-(R)-Cys-OH]2 (2.28 mmol, 1.0 g) according to GP 2a. Recrystallization from AcOEt/hexan
yielded 2a (795 mg, 57%). White solid. M.p. 151.7 – 154.6. [a]2D0 ¼ ꢀ91.0 (c ¼ 0.12, MeOH). 1H-NMR
(300 MHz): 1.40 (d, J ¼ 7.5, Me); 1.45 (s, Me3C); 2.90 (dd, J ¼ 9.6, 12.3, 1 H, CH2); 3.12 – 3.21 (m, 1 H,
CH2); 3.71 (s, MeO); 4.38 – 4.52 (m, 2 CH). 13C-NMR (75 MHz): 18.1 (Me); 29.3 (Me3C); 43.2 (CH2)12);
53.4 (MeO); 55.8 (CH); 81.5 (Me3C); 158.3, 173.5, 175.0 (CO). HR-MS (MALDI): 649.199 ([M þ K]þ),
633.224 ([M þ Na]þ, C24H42N4NaO10S2þ ; calc. 633.223), 611.241 (Mþ).
(2R,5S,10S,13R)-Dimethyl 5,10-Diamino-2,3-dimethyl-7,8-dithia-3,12-diazatetradecanedioate ([H-
(R)-Cys-(S)-Ala-OMe]2; 2b). Compound 2a (0.82 mmol, 500 mg) was Boc-deprotected according
GP 1a. Lyophilization from dioxane afforded 2b (337 mg, > 98%), used as such without further
purifications. White solid. [a]2D0 ¼ ꢀ121.9 (c ¼ 0.31, MeOH). IR: 2961w, 1720m, 1663s, 1556m, 1431m,
1383m, 1293m, 1178s, 1130s, 1058m, 974m, 833m, 800m, 719s, 637m. 1H-NMR (300 MHz): 1.43 (d, J ¼ 7.1,
Me); 3.10 (dd, J ¼ 8.8, 14.7, 1 H, CH2); 3.46 (dd, J ¼ 4.1, 14.7, 1 H, CH2); 3.72 (s, MeO); 4.26 – 4.37 (m,
CH); 4.41 – 4.56 (m, CH). 13C-NMR (75 MHz): 16.8 (Me); 38.8 (CH2); 49.26 (CH); 52.3 (CH); 52.5
(MeO); 168.1, 173.6 (CO). HR-MS (MALDI): 433.118 ([M þ Na]þ), 411.136 (Mþ, C14H27N4O6S2þ ; calc.
411.137).
8. Preparation of Boc-(S)-b3hPhe-(R)-b3hCys-OH (3). Boc-(R)-b3hCys(Bn)-OMe (2.18 mmol,
742 mg) was Boc-deprotected according to GP 1b and coupled with Boc-(S)-b3hPhe-OH (2.18 mmol,
611 mg) according to GP 2b. Filtration on silica gel (CH2Cl2/Et2O/hexan 0.5 :1:1) afforded Boc-(S)-
b3hPhe-(R)-b3hCys(Bn)-OMe (1.068 g, 98%). White solid. M.p. 146.9 – 147.7. [a]2D0 ¼ ꢀ18.7 (c ¼ 1.12,
MeOH). IR: 3346m, 3027w, 1733m, 1685s, 1641m, 1524s, 1436m, 1367m, 1345m, 1328m, 1310m, 1288m,
1272m, 1249m, 1213m, 1159s, 1139m, 1131m, 1098m, 1082m, 1072m, 1042m, 1032m, 1020s, 997m, 762m,
735m, 696s. 1H-NMR (300 MHz, CDCl3): 1.40 (s, Me3C); 2.21 (dd, J ¼ 5.8, 15.1, 1 H, CH2); 2.36 (dd, J ¼
4.9, 15.1, 1 H, CH2); 2.50 – 2.86 (m, 2 CH2, 1 H of CH2); 2.88 – 3.02 (m, 1 H, CH2); 3.67 (s, MeO); 3.73 (s,
PhCH2S); 3.98 – 4.13 (m, CH); 4.35 – 4.50 (m, CH); 5.40 – 5.55 (m, NH); 6.14 (d, J ¼ 8.8, NH); 7.15 – 7.40
(m, 10 arom. H). 13C-NMR (75 MHz, CDCl3): 28.3 (Me3C); 34.7, 36.1, 36.8, 38.8, 40.1 (CH2); 44.8, 49.4
(CH); 51.7 (MeO); 79.1 (Me3C); 126.3, 127.1, 128.4, 128.5, 128.8, 129.2 (arom. CH); 137.8, 138.0 (arom.
C); 155.3, 170.3, 171.6 (CO). HR-MS (MALDI): 539. 1970 ([M þ K]þ), 523.223 ([M þ Na]þ,
C27H36N2NaO5Sþ; calc. 523.224), 501.242 (Mþ).
Boc-(S)-b3hPhe-(R)-b3hCys(Bn)-OH.
Boc-(S)-b3hPhe-(R)-b3hCys(Bn)-OMe
(134 mg,
0.267 mmol) was dissolved in MeOH (4 ml) and 5n NaOH (4 ml) and the soln. was warmed to 508
and kept at this temp. for 12 h. The solvent was removed under vacuum, and the residue was diluted with
AcOEt and washed with 5n HCl. The org. phase was washed with brine, dried (Na2SO4), and the solvent
was removed under vacuum. The residue was dissolved in dioxane and lyophilized to afford Boc-(S)-
b3hPhe-(R)-b3hCys(Bn)-OH (130 mg, > 99%) and used without further purifications. White solid.
[a]2D0 ¼ ꢀ18.0 (c ¼ 0.2, MeOH). IR: 3307w, 2922w, 1717m, 1686m, 1666m, 1625m, 1516m, 1453m, 1408m,
1391m, 1366m, 1307m, 1274m, 1248m, 1210m, 1158s, 1081m, 1023m, 914m, 856m, 778m, 756m, 701s,
1
664m, 622m. H-NMR (300 MHz): 1.34 (s, Me3C); 2.34 (d, J ¼ 6.5, CH2); 2.45 – 2.78 (m, 2 CH2, 1 H of
CH2); 2.86 (dd, J ¼ 5.9, 13.5, 1 H, CH2); 3.75 (s, PhCH2S); 4.00 – 4.16 (m, CH); 4.35 – 4.48 (m, CH); 7.11 –
7.41 (m, 10 arom. H). 13C-NMR (75 MHz): 28.8 (Me3C); 36.2, 36.9, 38.9, 41.6, 41.9 (CH2); 47.0, 51.3 (CH);
80.0 (Me3C); 127.4, 128.0, 129.4 129.5 130.2, 130.6 (arom. CH); 139.8 (2 overlapped arom. C); 157.6,
173.0, 174.5 (CO). HR-MS (MALDI): 525.182 ([M þ K]þ), 509.207 ([M þ Na]þ, C26H34N2NaO5Sþ);
calc. 509.208).
(R)-3-{(S)-3-[(tert-Butoxycarbonyl)amino]-4-phenylbutanamido}-4-(sulfanyl)butanoic acid (Boc-
(S)-b3hPhe-(R)-b3hCys-OH; 3). Boc-(S)-b3hPhe-(R)-b3hCys(Bn)-OH was deprotected at the cysteine
side chain according to GP 3 to afford 3 (47 mg, > 99%). White solid, which was used without further
purifications. M.p. 143.2 (dec.). [a]2D0 ¼ ꢀ26.4 (c ¼ 0.16, MeOH). IR: 3287w, 2929w, 1680m, 1645m, 1555s,
12
)
The missing CH signal is overlapped by the signal of the solvent.

Helvetica Chimica Acta – Vol. 91 (2008)
2049
1410m, 1367m, 1249m, 1156s, 1114s, 1046m, 1024m, 756m, 736m, 699s, 661m. 1H-NMR (300 MHz): 1.34
(s, Me3C); 2.20 – 2.47 (m, CH2); 2.50 – 3.05 (m, 3 CH2); 3.98 – 4.18 (m, CH); 4.20 – 4.40 (m, CH); 7.10 –
7.30 (m, 5 arom. H). 13C-NMR (75 MHz): 28.8 (Me3C); 30.8, 38.0, 41.9, 42.0 (CH2); 50.2, 51.4 (CH);
80.1 (Me3C); 127.4, 129.4, 130.6 (arom. CH); 139.8 (arom. C); 157.6, 173.2, 174.5 (CO). HR-MS
(MALDI): 419.161 ([M þ Na]þ, C19H28N2NaO5Sþ; calc. 419.161), 397.179 (Mþ).
9. Preparation of Boc-(R)-b3hCys-(S)-b3hAla-OH (4). Boc-(R)-b3hCys(Bn)-(S)-b3hAla-OBn. Boc-
(S)-b3hAla-OBn (3.69 mmol, 1.08 g) was Boc-deprotected according to GP 1b and coupled with Boc-
(R)-b3hCys(Bn)-OH (3.69 mmol, 1.20 g) according to GP 2b. FC (Et2O/hexan 1:1.5) afforded Boc-(R)-
b3hCys(Bn)-(S)-b3hAla-OBn (918 mg, yield 50%). White solid. M.p. 123.0 – 124.3. [a]2D0 ¼ ꢀ15.7 (c ¼
0.42, MeOH). IR: 3353w, 3316w, 2980w, 1729s, 1720s, 1683s, 1635s, 1525s, 1495m, 1454m, 1433w,
1411w, 1391w, 1370w, 1362w, 1347m, 1301s, 1272m, 1242m, 1215m, 1197m, 1171s, 1159s, 1137s, 1065m,
1041m, 1022s, 996m, 970m, 911w, 875w, 777w, 755m, 745m, 696s, 689s. 1H-NMR (300 MHz): 1.14 (d, J ¼
6.8, Me); 1.43 (s, Me3C); 2.24 – 2.62 (m, 3 CH2); 3.73 (s, PhCH2S); 3.97 – 4.10 (m, CH); 4.20 – 4.33 (m,
CH); 5.07 (d, J ¼ 12.1, 1 H, PhCH2O); 5.12 (d, J ¼ 12.1, 1 H, PhCH2O); 7.18 – 7.40 (m, 10 arom. H).
13C-NMR (75 MHz): 20.1 (Me); 28.5 (Me3C); 36.3, 36.6, 41.1, 41.4 (CH2); 43.4 (CH)12); 67.0 (CH2); 79.8
(Me3C); 127.5, 128.7, 128.8, 128.9, 129.1, 129.7 (arom. CH); 137.0, 139.3 (arom. C); 157.0 (Me3CO); 171.7,
171.9 (CO). HR-MS (MALDI): 501.241 (Mþ), 523.223 ([M þ Na]þ, C27H36N2NaO5Sþ; calc. 523.224),
539.197 ([M þ K]þ).
(S)-3-{(R)-3-[(tert-Butoxycarbonyl)amino]-4-sulfanylbutanamido}butanoic acid (Boc-(R)-b3hCys-
(S)-b3hAla-OH; 4). Boc-(R)-b3hCys(Bn)-(S)-b3hAla-OBn (320 mg, 0.64 mmol) was deprotected at the
cysteine side chain according to GP 3 to yield 4 (206 mg, > 99%), which was used without further
purifications. White solid. M.p. 135 (dec.). [a]2D0 ¼ ꢀ8.2 (c ¼ 1.2, MeOH). IR: 3334w, 2976w, 1686s,
1688m, 1603w, 1524s, 1445w, 1423w, 1391w, 1367m, 1351w, 1275m, 1248m, 1164s, 1108w, 1060m, 1029m,
1
984w, 935w, 858w, 778w, 751w, 648m. H-NMR (300 MHz): 1.19 (d, J ¼ 6.5, Me); 1.43 (s, Me3C); 2.34 –
2.44 (m, CH2, 1 H of CH2); 2.53 (dd, J ¼ 15.6, J ¼ 6.2, 1 H, CH2); 2.60 – 2.65 (m, CH2); 3.87 – 4.00 (m,
CH); 4.17 – 4.30 (m, CH). 13C-NMR (75 MHz): 20.0 (Me); 28.4 (Me3C); 29.0, 40.3, 41.1 (CH2); 43.4, 51.8
(CH); 80.0 (Me3C); 157.4, 171.9, 174.4 (CO). HR-MS (MALDI): 343.130 ([M þ Na]þ, C13H24N2NaO5Sþ;
calc. 543.130), 321.148 (Mþ).
10. Preparation of b-tridecapeptide 5. CF3CO2H · H2N-b3hCys-b3hAla-b3hLeu-b3hLys-b3hLeu-
b3hPhe-b3hAsp-b3hVal-b3hPhe-b3hLys-b3hAla-b3hIle-b3hAsp-OH (5). Esterification of the Fmoc-
b3hAsp(t-Bu)-OH with Wang resin was performed according to [26] by the MSNT/MeIm method. The
resin was placed into a dried manual SPPS reactor, swelled in CH2Cl2 (20 ml/g resin) for 1 h, and washed
with CH2Cl2. In a separate dry round-bottomed flask equipped with magnetic stirrer, Fmoc-b3hAsp(t-
Bu)-OH (1,25 mmol, 532 mg, 5 equiv.) was dissolved in CH2Cl2 (4 ml), then MeIm (3.75 equiv.) and
MSNT (5 equiv.) were added under Ar. Stirring was continued until MSNTwas dissolved. Thereafter, the
soln. was transferred via syringe to the SPPS reactor containing the resin (269 mg, 0.93 mmol/g, 100 – 200
mesh) and mixed by Ar bubbling for 2 h. Subsequently, the resin was filtered, washed with DMF (5 ml,
5 ꢁ 1 min) and CH2Cl2 (5 ml, 5 ꢁ 1 min), and dried under h.v. for 24 h. The resin substitution was
determined by measuring the absorbance of the dibenzofulvene – piperidine adduct: two aliquots of the
Fmoc-amino acid resin were weighed exactly and suspended in piperidine (20%) in DMF, in volumetric
flasks (10 ml). After 30 – 40 min, the mixtures were transferred to a UV cell and piperidine (20%) to
another UV cell (blank), and the absorbance was measured at 290 nm. The concentrations (c1 and c2,
[mm]) of the benzofulvene – piperidine adduct in soln. were determined using a calibration curve [26].
The loading (Subst) was then calculated according to Eqn. 1:
Subst [mmol/g resin] ¼ c · V/{m(resin) · [c · V· (MW· 18)/1000]}
(MW¼ molecular weight of the Fmoc-protected amino acid)
(1)
The yield for the attachment to the resin (loading yield) was determined by Eqn. 2:
Loading yield ¼ [(Subst1 þ Subst2)/2]/Substtheor.
(2)

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Helvetica Chimica Acta – Vol. 91 (2008)
The loading was determined to be 0.67 mmol/g (72%), corresponding to 0.18 mmol of Fmoc-(S)-
b3hAsp-OH. The peptide resin was covered with DMF (5 ml), and unreacted OH groups were capped
using Ac2O (1 ml, 10 equiv.) and 4-(dimethylamino)pyridine (DMAP; 50 mg, 0.1 equiv.) for 2 h under Ar
bubbling. The resin was then washed with DMF (5 ml, 5 ꢁ 1 min) and CH2Cl2 (5 ml, 5 ꢁ 1 min). The
Fmoc group was removed by treating the resin with 20% piperidine in DMF (4 ml, 4 ꢁ 10 min) under Ar
bubbling. After filtration, the resin was washed with DMF (5 ml, 4 ꢁ 1 min). Solid-phase synthesis was
continued by sequential incorporation of Fmoc-protected amino acids. For each coupling step, the resin
was treated with a soln. of the Fmoc-protected amino acid (4 equiv.), HATU (3.7 equiv.), and EtN(i-Pr)2
(DIPEA; 8 equiv.) in DMF (5 ml) for 1 h. Monitoring of the coupling reaction was performed with the
TNBS test [41]. In the case of a positive TNBS test (indicating incomplete coupling), the suspension was
allowed to react further for 0.5 – 4 h, or, after filtration, the peptide – resin was treated again with the
same Fmoc-protected amino acid (1 – 3 equiv.) and with the coupling reagents. The resin was then filtered
and washed with DMF (5 ml, 5 ꢁ 1 min) prior to the following deprotection step. For the last coupling,
Boc-protected b3hCys(trityl)-OH [42] was used. The cleavage from the resin and the peptide
deprotection were performed according to [27]. The dry peptide – resin was suspended in a soln. of
TFA/H2O/ethane-1,2-dithiol (EDT)/triisopropylsilane (TIS) 94 :2.5 :2.5 :1 (10 ml) for 2.5 h. The resin
was removed by filtration, washed with TFA (2ꢁ), and the org. phase was concentrated under reduced
pressure. The resulting oily residue was treated with cold Et2O, and the formed precipitate was separated.
Part of the crude peptide (60 mg) was purified by prep. RP-HPLC (C-8, 50% B for 5 min then from 50 to
63% B in 20 min, flow rate: 17 ml/min, tR 15 min, 56% B) to afford 20 mg (33%) of 5 as TFA salt. White
solid. Anal. RP-HPLC (C-4, 2 – 95% B in 40 min, tR 23.4 min, 55% B): purity > 97%. 1H-NMR
(400 MHz): 0.80 – 1.10 (m, 9 Me); 1.11 – 1.23 (m, Me, 2 CH2); 1.24 – 1.80 (m, 7 CH2, 4 CH); 2.19 – 3.10 (m,
13 a-CH2, 7 CH2); 4.26 – 4.80 (m, 13 b-CH); 7.10 – 7.39 (m, 10 arom. H); 8.16 (d, J ¼ 8.7, NH); 8.29 (d, J ¼
9.6, NH); 8.33 – 8.50 (m, 2 NH); 8.59 (d, J ¼ 8.6, NH). HR-MS (MALDI): 1665.009 (Mþ,
C83H137N15O18Sþ; calc. 1665.006). For the CD spectrum, see Fig. 4.
Fig. 4. Normalized CD spectra of compounds 5 and 11 in MeOH (0.2 mm). Solutions were prepared by
weighing the peptides on a mg-precision balance and dissolving them in volumetric flasks.
11. Preparation of a-Octapeptide 6. CF3CO2H · H2N-(d)Phe-Cys-Phe-(d)Trp-Lys-Thr-Cys-Thr-ol
(6). Octreotide acetate (130 mg, 0.12 mmol) was dissolved in 10 ml of degassed phosphate buffer

Helvetica Chimica Acta – Vol. 91 (2008)
2051
(100 mm, pH 7.5), and 1,4-dithio-dl-threitol (DTT; 278 mg, 1.8 mmol) was added, and the mixture was
stirred for 1 h. Then the soln. was diluted with H2O (containing 0.1%TFA) and purified by RP-HPLC.
Prep. RP-HPLC (C-8, from 5% to 70% B in 50 min, tR 32 min, 47% B) afforded 105 mg (70%) of 6 as a
TFA salt. White solid. Anal. RP-HPLC (C-8, 5 – 95% B in 40 min, tR 18 min, 46% B): purity > 98%. LC/
MS (C-18, 5% B for 2 min, then from 5 to 95% B in 20 min, tR 15 min, [M þ H]þ: 1022.3; ([M þ 2 H]þ/2):
512.8). 1H-NMR (300 MHz): 0.70 – 0.80 (m, 2 H); 1.16 (d, J ¼ 6.6, Me); 1.22 (d, J ¼ 6.3, Me); 1.27 – 1.47
(m, 4 H); 1.67 – 1.82 (m, 1 H); 2.60 – 2.75 (m, 2 H); 2.80 – 3.05 (m, 5 H); 3.07 – 3.27 (m, 3 H); 3.55 – 3.70
(m, 2 H); 3.80 – 3.90 (m, 1 H); 4.01 – 4.30 (m, 4 H); 4.33 – 4.46 (m, 2 H); 4.58 – 4.68 (m, 1 H); 4.70 – 4.83
(m, 2 H); 7.00 – 7.43 (m, 14 arom. H); 7.50 (d, J ¼ 7.7, arom. H); 7.67 (d, J ¼ 9.1, NH); 8.12 (d, J ¼ 8.5, NH);
8.23 (d, J ¼ 7.8, NH); 8.32 (d, J ¼ 8.0, NH); 8.39 (d, J ¼ 8.0, NH); 8.51 (d, J ¼ 5.8, NH); 10.35 (br. d, NH).
HR-MS (ESI): 1021.468 ([M þ H]þ), 1043.446 ([M þ Na]þ, C49H68N10NaO10Sþ2 ; calc. for 1043.445).
12. Trifluoromethylation of Peptide Cysteine Side Chain in a-Peptides. (S)-Methyl 2-{(S)-2-[(tert-
Butoxycarbonyl)amino]-3-phenylpropanamido}-3-[(trifluoromethyl)sulfanyl]propanoate (Boc-(S)-Phe-
(R)-Cys(CF3)-OMe; 7a). Compound 1a (76 mg, 0.1 mmol) was trifluoromethylated according to GP 4a.
FC (cyclohexanes/AcOEt 2 :1) afforded 7a (49.3 mg, 55%). White solid. Part of the peptide was further
purified by RP-HPLC (C-18, from 5% to 80% of B in 60 min; tR 55 min, 70% B). Anal. RP-HPLC (C-18,
from 5% to 95% of B in 40 min, flow rate 1 ml/min; tR 29 min, 70% B). M.p. 122.3 – 122.8. [a]2D0 ¼ ꢀ18.7
(c ¼ 0.12, MeOH). IR: 3324w, 2936w, 1742m, 1682m, 1665s, 1518s, 1438m, 1392m, 1368m, 1321m, 1297m,
1250m, 1218m, 1148s, 1106s, 1046m, 1027m, 934w, 863m, 796m, 756m, 698m, 642m. 1H-NMR (300 MHz):
1.40 (s, Me3C); 2.82 (dd, J ¼ 9.0, 13.8, 1 H, CH2); 3.09 (dd, J ¼ 5.8, 13.8, 1 H, CH2); 3.24 (dd, J ¼ 7.6, 14.3,
1 H, CH2); 3.45 (dd, J ¼ 5.6, 14.3, 1 H, CH2); 3.72 (s, MeO); 4.31 (dd, J ¼ 5.8, 9.0, CH); 4.66 (dd, J ¼ 5.6,
7.6, CH); 7.18 – 7.35 (m, 5 arom. H). 13C-NMR (63 MHz): 27.2 (Me3C); 30.2, 37.7 (CH2); 51.8 (MeO);
52.1, 55.9 (CH); 79.3 (Me3C); 126.3, 128.0, 129.0 (arom. CH); 131.0 (q, J ¼ 305.1, CF3); 137.0 (arom. C);
155.8, 169.7, 173.1 (CO). 19F-NMR (188 MHz): ꢀ 41.42 (s, CF3), ꢀ 75.19 (s, TFA). HR-MS (EI): 450.144
(Mþ, C19H25F3N2O5Sþ; calc. 450.143).
(S)-Methyl 2-[(S)-2-amino-3-phenylpropanamido]-3-[(trifluoromethyl)sulfanyl]propanoate (H-
(S)-Phe-(R)-Cys(CF3)-OMe; 7b). Compound 1b (55 mg, 0.07 mmol) was trifluoromethylated according
to GP 4a. FC (cyclohexanes/AcOEt 2 :1) afforded 7b (42 mg, 70%). White solid. Part of the product was
further purified by prep RP-HPLC (C-18, from 5% to 20% of B in 5 min, than from 20% to 70% of B in
45 min, tR 26 min, 43% B). Anal. RP-HPLC (C-18, from 5% to 40% of B in 30min, tR 28 min, 40% B).
[a]2D0 ¼ ꢀ15.2 (c ¼ 0.56, MeOH). IR: 2960w, 1744m, 1664s, 1530m, 1499m, 1456m, 1439m, 1319m, 1179s,
1104s, 837s, 798s, 756s, 721s, 700s. 1H-NMR (300 MHz): 3.05 (dd, J ¼ 8.2, 14.3, 1 H, CH2); 3.22 – 3.33 (m,
CH2); 3.48 (dd, J ¼ 5.5, 14.3, 1 H, CH2); 3.76 (s, MeO); 4.15 (dd, J ¼ 6.0, 8.2, CH); 4.75 (dd, J ¼ 7.7, 5.5,
CH); 7.26 – 7.42 (m, 5 arom. H). 13C-NMR (75 MHz): 30.1, 37.0 (CH2); 51.9 (Me); 52.2, 54.0 (CH); 127.4,
128.7, 129.1 (arom. CH); 130.8 (q, J ¼ 302.7, CF3); 133.8 (arom. C); 168.3, 169.3 (CO). 19F-NMR
(282 MHz): ꢀ 41.37 (s, SCF3), ꢀ 75.19 (s, TFA). HR-MS (ESI): 351.099 ([M þ H]þ, C14H18F3N2O3Sþ;
calc. 351.098).
(S)-Methyl 2-{(R)-2-[(tert-butoxycarbonyl)amino]-3-[(trifluoromethyl)sulfanyl]propanamido}pro-
panoate (Boc-(R)-Cys(CF3)-(S)-Ala-OMe; 8a). Compound 2a (60.9 mg, 0.1 mmol) was trifluorome-
thylated according GP 4a. FC (cyclohexanes/AcOEt 2 :1) afforded 8a (0.116 mmol, 58%). White solid.
[a]2D0 ¼ ꢀ110.0 (c ¼ 0.05, MeOH). IR: 3315m, 1747m, 1693m, 1657m, 1526m, 1447w, 1389w, 1369w,
1341m, 1293m, 1279m, 1251m, 1213m, 1146m, 1105s, 1050m, 1026m, 981m, 871m, 809w, 780w, 757m,
666m. 1H-NMR (300 MHz, CDCl3): 1.43 (d, J ¼ 7.2, Me); 1.48 (s, Me3C); 3.23 (dd, J ¼ 5.7, 14.1, 1 H, CH2);
3.32 (dd, J ¼ 5.7, 14.1, 1 H, CH2); 3.78 (s, MeO); 4.46 (m, CH); 4.57 (m, CH); 5.29 (d, J ¼ 8.4, NH); 6.87
(br. s, NH). 13C-NMR (75 MHz, CDCl3): 18.2 (CH3); 28.2 (Me3C); 31.3 (CH2); 48.3 (CH); 52.6 (MeO);
53.5 (CH); 81.1 (Me3C); 130.7 (q, J ¼ 306.4, CF3); 155.3, 169.1, 172.8 (CO). 19F-NMR (188 MHz, CDCl3):
ꢀ 41.2 (s, CF3). HR-MS (EI): 318.049 ([M ꢀ C4H9]þ, C13H21F3N2O5Sþ; calc. 318.049).
(S)-Methyl 2-{(R)-2-amino-3-[(trifluoromethyl)sulfanyl]propanamido}propanoate ((R)-Cys(CF3)-
(S)-Ala-OMe; 8b). Compound 2b (66 mg, 0.1 mmol) was trifluoromethylated according GP 4a to afford
1
8b (135 mg crude product, yield 92%, as estimated by H-NMR). Part of the product was purified by
prep. RP-HPLC (C-18, 5% of B for 5 min then from 5% to 70% of B in 55 min; tR 24 min, 28% B). Anal.
RP-HPLC (C-18, 5% of B for 5 min then from 5% to 40% of B in 30 min; tR 14 min, 21% B). White solid.
[a]2D0 ¼ ꢀ10.8 (c ¼ 0.41, MeOH). IR: 3334w, 2962m, 1732s, 1663s, 1599w, 1554w, 1479w, 1452w, 1375w,

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Helvetica Chimica Acta – Vol. 91 (2008)
1334w, 1231w, 1155w, 1107s, 1050w, 980w, 929w, 843w, 756w, 618w. 1H-NMR (400 MHz): 1.34 (d, J ¼ 7.3,
Me); 3.20 – 3.23 (m, CH2); 3.27 (dd, J ¼ 7.9, 14.8, 1 H, CH2); 3.49 (dd, J ¼ 5.3, 14.8, 1 H, CH2); 3.64 (s,
MeO); 4.08 (dd, J ¼ 5.3, 8.0, CH); 4.39 (dd, J ¼ 7.3, 14.4, CH). 13C-NMR (100 MHz): 17.2 (Me); 31.3
(CH2); 49.8 (CH); 52.9 (MeO); 53.4 (CH); 131.8 (q, J ¼ 306.0, CF3); 167.5, 173.9 (CO). 19F-NMR
(376 MHz): ꢀ 42.95 (s, CF3), ꢀ 75.20 (s, TFA). HR-MS (EI): 318.049 (Mþ ꢀ C4H9, C13H21F3N2O5S; calc.
318.049).
13. Trifluoromethylation of the Peptide Cysteine Side Chain in b-Peptides. (S)-3-{(R)-3-[(tert-But-
oxycarbonyl)amino]-4-phenylbutanamido}-4-[(trifluoromethyl)sulfanyl]butanoic Acid (Boc-(S)-
b3hPhe-(R)-b3hCys(CF3)-OH; 9). Compound 3 (31.8 mg, 0.08 mmol) was trifluoromethylated according
to GP 4b to give 9 (57 mg crude product, yield 90%, as estimated by 1H-NMR). The crude peptide was
dissolved in 30% MeCN in H2O (containing 0.1% TFA) and purified by prep. RP-HPLC (C-18, 30% of B
for 5 min, then from 30% to 70% of B in 40 min, tR 38 min, 62% B). Anal. RP-HPLC (C-18, 5% to 95%
of B in 40 min, tR 25 min, 60% B). [a]2D0 ¼ ꢀ2 (c ¼ 0.05, MeOH). IR: 3400w, 3293w, 2981w, 1721m, 1666s,
1632s, 1568m, 1512s, 1452w, 1411w, 1393w, 1367m, 1307m, 1271w, 1254w, 1208m, 1191w, 1147s, 1122s,
1
1104s, 1083s, 1025m, 854w, 779w, 757w, 703s, 656w. H-NMR (300 MHz): 1.34 (s, Me3C); 2.30 – 2.60 (m,
2 CH2); 2.63 – 2.90 (m, CH2); 3.05 – 3.18 (m, 1 H, CH2); 3.22 (dd, J ¼ 5.9, 13.5, 1 H, CH2); 4.02 – 4.20 (m,
PhCH2CH); 4.40 – 4.55 (m, HꢀC(3)); 7.15 – 7.35 (m, 5 arom. H). 13C-NMR (DMSO, 100 MHz): 28.1
(Me3C); 37.6, 37.7, 40.5 (CH2)13); 45.4, 49.2 (CH); 77.3 (Me3C); 125.7, 127.8 (arom. CH); 129.8 (q, J ¼ 275,
CF3); 131.2 (arom. CH); 138.7 (arom. C); 154.6 (CO); 169.9, 171.5 (CO). 19F-NMR (282 MHz): ꢀ 40.89
(s, CF3); ꢀ 75.22 (s, TFA). HR-MS (MALDI): 487.149 ([M þ Na]þ, C20H27F3N2NaO5Sþ; calc. 487.148).
The molecular peak (C36H50N4O10Sþ2 ) was not observed.
(S)-3-{(R)-3-[(tert-Butoxycarbonyl)amino]-4-[(trifluoromethyl)sulfanyl]butanamido}butanoic acid
(Boc-(R)-b3hCys(CF3)-(S)-b3hAla-OH; 10). Compound 4 (100 mg, 0.31 mmol) was trifluoromethylated
according to GP 4b to yield 4 (197 mg crude product, yield > 98%, as estimated by 1H-NMR). The crude
peptide was dissolved in 30% MeCN in H2O (containing 0.1% TFA) and purified by prep. RP-HPLC (C-
18, 30% for 5 min, then from 30% to 70% of B in 45 min; tR 29 min, 53% B). Anal. RP-HPLC (C-18, 5%
to 95% of B in 40 min; tR 21.3 min, 53% B). M.p. 165.6 – 166.5. [a]2D0 ¼ ꢀ18.2 (c ¼ 0.48, MeOH). IR:
3388w, 2983w, 2938w, 1720m, 1669s, 1621s, 1551m, 1513s, 1445w, 1419m, 1392w, 1369m, 1320w, 1295w,
1275m, 1251w, 1193m, 1156s, 1106s, 1069m, 1046m, 1032m, 952w, 877m, 845w, 781w, 756m, 664w.
1H-NMR (400 MHz): 1.19 (d, J ¼ 6.7, Me); 1.43 (s, Me3C); 2.38 (dd, J ¼ 7.1, 15.6, 1 H, CH2); 2.45 (d, J ¼
6.6, CH2); 2.53 (dd, J ¼ 6.2, 15.6, 1 H, CH2); 3.07 – 3.21 (m, CH2); 4.08 – 4.20 (m, CH); 4.21 – 4.28 (m,
CH). 13C-NMR (100 MHz): 20.2 (Me); 28.7 (Me3C); 34.8, 40.8, 41.3 (CH2); 43.7 (CH)12); 80.0 (Me3C);
132.6 (q, J ¼ 378.7, CF3); 157.4, 171.4, 174.6 (CO). 19F-NMR (282 MHz): ꢀ 40.95 (s, CF3); ꢀ 75.22 (s,
TFA). HR-MS (MALDI): 411.116 ([M þ Na]þ, C14H23F3N2NaO5Sþ; calc. 411.117).
CF3CO2H · H2N-b3hCys(CF3)-b3hAla-b3hLeu-b3hLys-b3hLeu-b3hPhe-b3hAsp-b3hVal-b3hPhe-b3hLys-
b3hAla-b3hIle-b3hAsp-OH (11). Compound 5 (10 mg, 5 mmol) was trifluoromethylated according to
GP 4b. The crude peptide was purified by prep. RP-HPLC (C-8, 50% B for 10 min, then from 50 to 95%
B in 45 min; tR 25 min, 64% B) to afford 11 as TFA salt (4.2 mg, 40%). White solid. Anal. RP-HPLC (C-
8, 5 – 95% B in 40 min; tR 30 min, 64% B): purity > 98%. LC/MS (C-18, 5% B for 2 min, then from 5 to
95% B in 20 min; tR 19.9 min; [M þ H]þ: 1733.7; ([M þ 2 H]þ/2): 868.9; ([M þ 3 H]þ/3): 580.1; for
1
[C50H65F3N10O10S2]). H-NMR (400 MHz): 0.80 – 1.10 (m, 8 Me); 1.11 – 1.23 (m, 2 Me, CH); 1.24 – 1.80
(m, 9 CH2, 3 CH); 2.19 – 3.10 (m, 20 CH2); 4.26 – 4.80 (m, 13 CH); 7.10 – 7.39 (m, 10 arom. H); 8.16 (d,
J ¼ 8.7, NH); 8.29 (d, J ¼ 9.6, NH); 8.33 – 8.50 (m, 2 NH); 8.59 (d, J ¼ 8.6, NH). 19F-NMR (282 MHz):
ꢀ 41.21 (s, CF3); ꢀ 75.22 (s, TFA). HR-MS (MALDI): 1732.998 ([M þ H]þ, C83H138N15O18Sþ; calc.
1732.993). For the CD spectrum, see Fig. 4.
14. Trifluoromethylation of 6. CF3CO2H · H2N-(d)Phe-[Cys-Phe-(d)-(2-CF3)Trp-Lys-Thr-Cys]-Thr-
ol (12), CF3CO2H · H2N-(d)Phe-Cys(CF3)-Phe-(d)-Trp-Lys-Thr-Cys(CF3)-Thr-ol (13), and CF3CO2H ·
H2N-(d)Phe-Cys(CF3)-Phe-(d)-(2-CF3)Trp-Lys-Thr-Cys(CF3)-Thr-ol (14). Compound 6 (17.6 mg,
14.1 mmol) was dissolved in degassed MeOH (7.05 mm) and cooled to ꢀ 788. 1,3-Dihydro-3,3-
dimethyl-1-(trifluoromethyl)-1,2-benziodoxole (2.9 equiv.) dissolved in degassed MeOH (1 ml) and
cooled to ꢀ 788 was added to this soln., and the mixture was kept at ꢀ 788 under N2 for 2 h, and was then
13
)
The missing CH2 signals are overlapped by the signal of the solvent.

Helvetica Chimica Acta – Vol. 91 (2008)
2053
allowed to warm to r.t. and stirred for 12 h. The solvent was removed under vacuum, and the crude
products were purified by prep. RP-HPLC (C-8, 5 – 70% B in 50 min) to afford 0.4 mg of 12 (tR 35 min,
50% B), 3 mg of 13 (tR 40 min, 57% B), and 3 mg of 14 (tR 41.5 min, 59% B) as TFA salts. White solids.
Data of 12. Anal. RP-HPLC (C-8, 5 – 95% B in 40 min, tR 18.3 min): purity > 98%. LC/MS (C-18,
5% B for 2 min, then from 5 to 95% B in 20 min; tR 15.5 min, [M þ H]þ: 1087.4; ([M þ 2 H]þ/2): 544.7; for
[C50H65F3N10O10S2]). 1H-NMR (300 MHz): 0.62 – 0.90 (m, 1 H); 1.00 – 1.40 (m, 7 H); 1.42 – 1.62 (m,
2 H); 1.83 – 2.22 (m, 3 H); 2.42 – 2.63 (m, 2 H); 2.65 – 3.05 (m, 6 H); 3.33 – 3.42 (m, 1 H); 3.50 – 3.63 (m,
3 H); 3.64 – 3.87 (m, 3 H); 3.90 – 4.07 (m, 2 H); 4.09 – 4.40 (m, 3 H); 4.50 – 4.60 (m, 1 H); 5.02 – 5.17 (m,
2 H); 6.95 – 7.50 (m, 14 arom. H). 19F-NMR (282 MHz): ꢀ 58.32 (s, CF 3); ꢀ 75.21 (s, TFA).
Data of 13. Anal. RP-HPLC (C-8, 5 – 95% B in 40 min; tR 21.3 min): purity > 95%. LC/MS (C-18,
5% B for 2 min, then from 5 to 95% B in 20 min; tR 16.3 min; [M þ H]þ: 1157.8; ([M þ 2 H]þ/2): 580.3; for
1
[C51H66F6N10O10S2]). H-NMR (300 MHz): 0.70 – 0.80 (m, 1 H); 1.14 (d, J ¼ 6.6, Me); 1.23 (d, J ¼ 6.3,
Me); 1.27 – 1.45 (m, 4 H); 1.67 – 1.82 (m, 1 H); 2.58 – 2.78 (m, 2 H); 2.78 – 3.25 (m, 8 H); 3.40 – 3.70 (m,
5 H); 3.80 – 3.90 (m, 1 H); 4.01 – 4.10 (m, 1 H); 4.11 – 4.28 (m, 2 H); 4.35 – 4.45 (m, 2 H); 4.55 – 4.65 (m,
2 H); 4.61 – 4.78 (m, 1 H); 7.00 – 7.43 (m, 14 arom. H); 7.52 (d, J ¼ 7.5, 1 arom. H). 19F-NMR (282 MHz):
ꢀ 41.41 (s, SCF 3); ꢀ 41.51 (s, SCF 3); ꢀ 75.21 (s, TFA).
Data of 14. Anal. RP-HPLC (C-8, 5 – 95% B in 40 min, tR 22.2 min): purity > 98%. LC/MS (C-18,
5% B for 2 min, then from 5 to 95% B in 20 min; tR 16.3 min; [M þ H]þ: 1225.8; ([M þ 2 H]þ/2): 615.5; for
[C52H65F9N10O10S2]). 1H-NMR (500 MHz): 0.68 (quint., J ¼ 7.5, 1 H, CH2); 1.16 (d, J ¼ 6.6, Me); 1.18 (d,
J ¼ 6.4, Me); 1.20 – 1.43 (m, 2 CH2); 1.62 – 1.72 (m, 1 H, CH2); 2.69 – 2.73 (m, CH2); 2.95 (d, J ¼ 7.5, CH2);
3.01 – 3.11 (m, 3 H, 3 CH2); 3.23 – 3.33 (m, 3 H, 3 CH2); 3.39 (dd, J ¼ 13.9, J ¼ 10.3, 1 H, CH2); 3.49 (dd,
J ¼ 13.5, 6.1, 1 H, CH2); 3.62 – 3.72 (m, CH2); 3.87 (td, J ¼ 6.3, 3.1, CH); 4.08 (qd, J ¼ 6.6, 3.1, CH); 4.13 –
4.18 (m, 2 CH); 4.26 (qd, J ¼ 6.4, 5.1, CH); 4.39 (dd, J ¼ 10.3, 5.7, CH); 4.44 (d, J ¼ 5.1, CH); 4.60 (t, J ¼
7.5, CH); 4.8 – 4.9 (m, CH); 4.92 (dd, J ¼ 7.5, 6.0, CH); 7.1 – 7.5 (m, 13 arom. H); 7.66 (d, J ¼ 8.1, arom. H).
19F-NMR (376 MHz): ꢀ 42.90 (s, CF3); ꢀ 42.96 (s, CF3); ꢀ 59.08 (s, CF3).
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