Ring-extended gramicidin S analogs Containing cis δ-sugar amino acid turn mimetics with varying ring size

Article abstract of DOI:10.1002/hlca.201200497

This article presents a series of ring-extended gramicidin S derivatives, 9-14, that have four ornithine residues as polar protonated side chains and one modified turn region containing a mono-functionalized cis-δ-oxetane, δ-furanoid, or δ-pyranoid sugar

Full text of DOI:10.1002/hlca.201200497

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Ring-Extended Gramicidin S Analogs Containing cis d-Sugar Amino Acid
Turn Mimetics with Varying Ring Size
by Annemiek D. Knijnenburga), Emile Spalburgb), Albert J. de Neelingb),
Roos H. Mars-Groenendijkc), Daan Noortc), Gijsbert M. Grotenbrega), Gijs A. van der Marela),
Herman S. Overkleefta), and Mark Overhand*a)
a) Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, NL-2300 RA Leiden
(fax: þ31715274307; e-mail: overhand@chem.leidenuniv.nl)
b) National Institute of Public Health and the Environment, Laboratory for Infectious Diseases,
P.O. Box 1, NL-3720 BA Bilthoven
c) TNO, Prins Maurits Laboratory, P.O. Box 45, NL-2280 AA Rijswijk
Dedicated to Prof. Dieter Seebach on the occasion of his 75th birthday
This article presents a series of ring-extended gramicidin S derivatives, 9 – 14, that have four
ornithine residues as polar protonated side chains and one modified turn region containing a mono-
functionalized cis-d-oxetane, d-furanoid, or d-pyranoid sugar amino acid residue. Of the GS analogs
evaluated, we identified compound 7, which contains the mono-benzyloxy cis-d-pyranoid sugar amino
acid, as having a better biological profile than the clinically applied topical antibiotic gramicidin S.
Introduction. – The cationic antimicrobial peptide gramicidin S (GS; cyclo(Pro-
Val-Orn-Leu-d-Phe)2) adopts a rigid cyclic b-hairpin structure in which the two d-Phe-
Pro residues form two-residue turns, and the NH and C¼O of opposing Val and Leu
residues of the strand regions form four H-bonds (Fig. 1). The bacterial membrane-
disrupting properties of GS is attributed to the amphiphilic nature of the cyclic b-
hairpin structure wherein the hydrophobic side chains of the Leu and Val amino acid
residues, and the protonated Orn side chains comprise the apolar and polar face of the
molecule, respectively (Fig. 1) [1]. As GS also disrupts other biomembranes, its clinical
application is limited to topical infections, and for this reason many synthetic studies [2]
have been conducted aimed to obtain derivatives with improved therapeutic properties.
One interesting approach in this respect is extension of the macrocyclic ring via the
designed incorporation [3] of four additional amino acid residues in order to finetune
the hydrophobicity of the cyclic b-hairpin structure [4]. This strategy is exemplified by
tetradecameric analogs 1 and 8 (Fig. 1). Compound 1 has four cationic charges and six
apolar residues in the strand regions. The replacement of one l-Orn residue with the
corresponding d-amino acid residue provides compound 8 with a diminished hydro-
phobicity, remarkably without dramatically changing the overall cyclic b-hairpin
structure [5].
Over the years, we have mainly focused on influencing the conformational behavior
of GS via the replacement of one of the d-Phe-Pro turn regions with conformationally
constrained w-amino acids [6]. In particular, we applied sugar-derived amino acid
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 1. The structure of the cyclic decamer antibiotic gramicidin S (GS): top view of its X-ray structure with
dashed lines indicating the four intramolecular H-bonds (side chains omitted for clarity), and side view
showing the hydrophobic face of the molecule formed by the side chains of the Val and Leu residues and
the hydrophilic face formed by the two Orn side chains, together with the structures of the tetradecameric
GS derivatives 1 and 8, and their turn-modified derivatives 2 – 7 and 9 – 14
building blocks, generally referred to as sugar amino acids (SAAs) [7], as these type of
compounds are conformationally biased and can be readily functionalized to obtain a
specific substitution pattern on the sugar core. Recently, we reported on the synthesis
and biological activity of ring-extended GS derivatives containing cis disubstituted
furanoid sugar amino acids [5a][8]. The two OH functionalities on the furanoid sugar
core in these compounds were used to introduce functionality in order to alter the
hydrophobic character of the molecules. As a follow-up of the latter study, we here
present the synthesis, conformational analysis, and biological evaluation, using human

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erythrocytes and a standard set of bacterial strains, of a series of ring-extended GS
derivatives containing mono-functionalized cis-d-oxetane, d-furanoid, and d-pyranoid
SAA turn mimetics (i.e., compounds 2 – 7 and 9 – 14, Fig. 1).
Results and Discussion. – Syntheses. The syntheses of the desired cis-d-oxetane, d-
furanoid-, and d-pyranoid sugar azido acid building blocks 15, 16a/16b, and 18a/b,
generally following previously described procedures, are outlined in Scheme 1. The
mono-benzyloxy-cis-d-oxetane sugar azido acid 15 [6a] is obtained from d-arabinose
via adaptation of two literature procedures featuring a ring-contraction reaction. d-
Furanoid sugar azido acid 16a is available starting from d-mannitol [6g]. We could
shorten the synthetic route towards 16a/16b by converting d-glucono-1,5-lactone to the
known [9a] dihydroxy furanoid 17a, treating the corresponding Ts derivative 17b with
NaN3 in DMF [9b], to obtain 16b, after saponification of the methyl ester with LiOH in
80% yield, over two steps. Mono-benzylation in the presence of excess NaH provided
compound 16a, albeit in modest yield. Compounds 18a/18b were obtained following
our literature procedure [6a] starting from commercially available 3,4,6-tri-O-acetyl-d-
glucal involving opening of a cyclic sulfate as key step.
Scheme 1. Overview of the Synthetic Strategy towards d-Sugar Azido Acids 15, 16a/b and 18a/b
i) NaN3, DMF, 85, 08, 85%. ii) Excess NaH, benzyl bromide (BnBr), DMF; 36%.
Peptides 2 – 7 and 9 – 14 were synthesized by using a Fmoc-based peptide coupling
protocol, and the corresponding building blocks 15, 16a/16b, or 18a/18b. The synthetic
route is exemplified for the synthesis of peptides 2 and 3 in Scheme 2. Thus, acid-labile
HMPB-BHA resin was functionalized to obtain pentapeptide 19. Coupling of sugar
azido acid 15 was accomplished with the coupling reagent HCTU in the presence of

Helvetica Chimica Acta – Vol. 95 (2012)
2547
EtNiPr2 to give 20. After azido reduction on resin using a solution of aqueous Me3P, the
peptide sequence was subsequently elongated uneventfully by automated solid-phase
chemistry to obtain the full-length immobilized linear sequence 21. The peptide was
released from the resin under mild acidic conditions using 1% TFA and cyclized under
high dilute conditions using PyBOP/HOBt. The (tert-butoxy)carbonyl (Boc) groups
were removed using strong acidic conditions to provide peptide 3 in 20% overall yield
after preparative HPLC purification. Peptide 3 was treated with Pd-black under H2 in
dioxane in the presence of aqueous HCl to obtain, after preparative HPLC purification,
peptide 2 in 80% yield. Similarly, the peptides 5, 7, and 9, 10, and 12 and 14 were
obtained. As the removal of the benzyl (Bn) functionality, upon hydrogenation of the
entire peptide, to provide the hydroxylated derivatives, did not proceed well in the case
of the furanoid and pyranoid derivatives, building blocks 16b and 18b were used to
obtain 4, 11, and 6, 13, respectively.
Scheme 2
i) Coupling of sugar amino acid (SAA) 15, 2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylami-
nium hexafluorophosphate (HCTU), EtNiPr2, DMF, 16 h. ii) Me3P (1m in 9 :1 THF/H2O, 16 equiv.),
16 h. iii) Standard sequential Fmoc solid-phase peptide synthesis (SPPS) (see Exper. Part for the details).
iv) 1% CF3COOH (TFA) in CH2Cl2. v) (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP), 1-hydroxybenzotriazole (HOBt), EtNiPr2, DMF. vi) TFA/triisopropylsilane
(TIS)/H2O 95 :2.5 :2.5. vii) H2, Pd-black in 1,4-dioxane/aq. HCl.
NMR Analysis. The secondary structures of the peptides were analyzed by
comparing the coupling constants of the amide H-atoms (3J(HNa)) and chemical-shift
perturbations of the a-H-atoms (DdHa) of peptides 2 – 7 and 9 – 14 with those of
peptides 1 and 8 with well-established cyclic b-hairpin structures in solution [5][10].
These values were extracted from the 600-MHz 1H-NMR spectra of the peptides in the
solvent CD3OD. The peptide residues were readily assigned using TOCSY and
cROESY experiments. Because of the similarity between the non-benzylated and the

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3
benzylated peptides, only the J(NH,Ha) and Dd(Ha) values of the non-benzylated
analogs 2, 4, 6, 9, 11, and 13, as well as the peptides 1 and 8 are presented in Figs. 2 and 3.
3
The J(NH,Ha) values of the Val, Orn, and Leu residues are generally above 7 Hz,
which indicates that these residues are part of an extended strand conformation. The d-
Phe H-atoms have coupling constants around 3 Hz, which is a typical value for a residue
that is part of a turn conformation. This confirms that the peptides adopt cyclic b-
hairpin structures in MeOH. The chemical-shift perturbation of the peptides also
corroborated the overall b-strand character of the Val, Orn, and Leu residues, as these
values are above 0.1 ppm. The d-Phe and Pro residues show negative perturbations
which is characteristic of a b-turn region [10].
3
3
Fig. 2. a) J(NH,Ha) Values of peptides 1, 2, 4, and 6 containing l-ornithines; b) J(NH,Ha) values of
peptides 8, 9, 11, and 13 having one d-ornithine. Numbering of amino acids N ! C: cyclo(Val(1)-Orn(2)-
Leu(3)-Orn(4)-Val(5)-SAA-Leu(8)-Orn(9)-Val(10)-Orn(11)-Leu(12)-d-Phe(13)-Pro(14)).
3
When focusing on the details, it should be noted that the J(NH,Ha) values of the
Val(10) of the oxetane and furanoid containing peptides are markedly lower than the
values obtained with the pyranoid-containing peptides (as is illustrated in Fig. 2,a, for
peptides 2, 4, and 6). This slight deviation of the 3J(NH,Ha) value of one of the strand
residues may indicate a partial conformational flexibility and confirm [6a] that the
pyranoid sugar amino acid residue is a better replacement of the d-Phe-Pro than the
oxetane and furanoid sugar amino acids with respect to maintaining b-strand integrity.
Antimicrobial and Hemolytic Activity. The antimicrobial activity of peptides 1 – 14
and GS was investigated using our standard panel of Gram-positive and Gram-negative
bacteria (Table). The toxicity of the peptides was evaluated by performing a hemolytic
assay using human erythrocytes (Table). The reversed-phase HPLC retention times as

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 3. a) Dd(Ha) Values of peptides 1, 2, 4, and 6 having l-ornithines; b) Dd(Ha) values of the peptides 8,
9, 11, and 13 having one d-ornithine. Dd(Ha) ¼ d(Ha) ꢀ d(Ha) random coil. The d(Ha) values of the
amino acid residues in GS are not significantly affected when using MeOH instead of H2O as solvent
system. For the Orn residues, the random coil value of Lys was used.
a measure of peptide hydrophobicity [11] show that the presence of a d-Orn residue in
the sequence provides a more hydrophilic peptide series (i.e., 8 – 14) than the series
with only l-Orn residues (i.e., 1 – 7; Table). Peptides 9 – 14, generally with low
hemolytic activities, only displayed modest antimicrobial activities against the Gram-
positive strain S. epidermidis. In contrast, analogs 2 – 7 display antimicrobial activities
against all tested strains.
Notable is the higher activity of 2 – 7 against Gram-negative bacteria compared to
GS and peptide 1. Peptides 2, 5, and 7 have potent and broad-spectrum antibacterial
activities. Peptide 7 has a slightly better hemolytic value than GS.
To give a complete overview, we also included the properties of the known [4]
compounds 22 and 26, as well as the previously reported [5a][8] compounds 23 – 25 and
27 – 29 (Table and Fig. 4).
Conclusions. – A study is presented in which subtle changes in hydrophobicity were
introduced in a series of cyclic ring-extended GS analogs by replacement of one of the
two-residue turns with sugar amino acid residues varying in ring size and having a
hydroxy or benzyloxy functionality combined with a single l/d-ornithine substitution in
one of the strands. To this end, peptides 2 – 7 and 9 – 14 were prepared, and their
conformational and biological properties were compared with those of GS and the
known peptides 1 and 8. According to NMR, the peptides 2 – 7 and 9 – 14 have similar

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Table. Antimicrobial Activities (MICs) against a Standard Set of Gram-Positive and Gram-Negative
bacteria, Retention Times, and Hemolytic Activities of GS and of the Peptides 1 – 14. Included are also the
data of the previously published [5a][8] peptides 22 – 29 (22 is the reference compound GS14, 26 is the
reference compound GS14K4; for structures, see Fig. 4).
Analogs Retention S.
S.
E.
B.
P.
E.
100%
timea)
aureusb) epidermidisb) faecalisb) cereusb) aeruginosac) colic) Hemolysisd)
GS
1
2
3
4
5
6
7
8
8.38
7.67
6.63
6.67
6.71
7.30
6.67
7.42
6.25
5.76
6.34
5.79
6.31
5.56
6.15
7.26
6.23
6.28
5.70
5.95
5.67
5.33
4.98
4
32
8
8
16
4
32
4
64
> 64
32
> 64
32
> 64
> 64
> 64
4
4
8
64
8
2
2
1
2
8
4
8
2
4
16
4
16
4
64
8
64
4
4
4
8
8
8
32
4
16
16
8
32
8
64
> 64
64
> 64
64
> 64
> 64
32
8
8
8
64
4
32
2
16
16
8
64
64
4
16
16
8
32
8
> 64
64
32
> 64
64
> 64
> 64
> 64
16
16
16
> 64
64
64
32
32
2
8
16
8
16
16
64
64
16
31.5
3.9
31.5
62.5
31.5
31.5
31.5
62.5
62.5
32
8
16
64
16
> 64
16
> 64
32
32
2
4
4
16
8
8
9
250
125
10
11
12
13
14
22
23
24
25
26
27
28
29
64 > 500
16 125
> 64 > 500
32
> 64
16
16
8
64
32
500
3.9
15.6
15.6
31.3
500
500
500
16
32
> 64
16
> 64
8
16
32
64
> 64
64 > 500
a) Retention times from RP-HPLC in min (see Exper. Part). b) Gram-positive bacteria, MIC in mg/l.
c) Gram-negative bacteria, MIC in mg/l. d) Hemolytic activity (100% lysis of the erythrocytes) in mm.
conformational properties as the peptides 1 and 8 in the solvent MeOH, which is in
agreement with an overall cyclic b-hairpin conformation. The peptides 9 – 14 having a
d-ornithine substitution are largely biologically inactive. Based on their HPLC
retention times under controlled conditions, this finding is in agreement with the
general trend that hydrophilic peptides of this type are not antimicrobial [4b][12]. The
more hydrophobic peptides 2 – 7 exhibit antimicrobial activities against both Gram-
positive and Gram-negative bacteria. The best compound in this series is peptide 7,
containing the cis-mono-benzyloxy-pyranoid sugar amino acid residue, having potent
and broad spectrum antibacterial activity within the small set of bacterial strains tested,
and a somewhat better hemolytic value than GS. This finding seems to confirm [6a] that
the cis-mono-benzyloxy-pyranoid sugar amino acid residue is a good, slightly less
hydrophobic mimic of the d-Phe-Pro two-residue turn. It should be noted that the
differences in hemolytic values are not large indicating that the peptides 2, 5, and the
previously reported compound 25 also have interesting biological properties.

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 4. Structures of the known tetradecameric gramicidin S derivatives GS14 and GS14K4, 24 and 26,
respectively, and the previously published analogs with a disubstituted furanoid SAA modified b-turn, 23 –
25 and 27 – 29
We are grateful to Kees Erkelens and Fons Lefeber for assistance in recording the NMR spectra. This
work was supported by a grant from the Leiden Institute of Chemistry.
Experimental Part
General. Light petroleum ether (PE) with a boiling range of 40 – 608 was used. All other solvents
used under anh. conditions were stored over 4-ꢂ molecular sieves except for MeOH which was stored
over 3-ꢂ molecular sieves. Solvents used for workup and silica-gel column chromatography (CC) were of
technical grade and distilled before use. All other solvents were used without further purification.
Reactions were monitored by TLC. The linear peptides were cleaved from resin, cyclized, and purified by
RP-HPLC (Gilson GX-281) with a prep. Gemini C18 column (Phenomenex, 5-mm particle size) or semi-
prep. (250 ꢁ 21.2 mm, 5-mm particle size). The applied eluents were A: 0.1 % aq. TFA, B: MeCN. The
linear peptides and cyclized peptides were analyzed with LC/MS (detection simultaneously at 214 and
254 nm) equipped with an anal. C18 column (4.6 mm ꢁ 250 mm, 5-mm particle size). The applied eluents
were A: H2O, B: MeCN, and C: 1.0% aq. TFA. HR-MS: direct injection (2 ml of a 2 mm soln. in H2O/
MeCN 50 :50 (v/v) and 0.1% HCOOH) on a mass spectrometer Thermo Finnigan LTQ Orbitrap
equipped with an electrospray ion source in positive-ion mode (source voltage, 3.5 kV; sheath gas flow,
10; cap. temp., 523 K) with resolution R ¼ 60000 at m/z 400 (mass range m/z 150 – 2000) and dioctyl
phthalate (m/z 391.28428) as lock mass. Hemolytic curves were analyzed with Graphpad Prism version
5.01 for Windows, GraphPad Software, San Diego California USA. Optical rotations were measured on a

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Helvetica Chimica Acta – Vol. 95 (2012)
Propol automatic polarimeter (sodium D-line, l 589 nm). Specific rotations [a]D are given in degrees per
centimeter and the concentration c is given in mg/ml in the specific solvent.
NMR Spectroscopy. 1H- and 13C-APT NMR for all intermediates were recorded on a Bruker AV-400
(400 and 100 MHz, resp.) or a Bruker DMX 600. The spectra of the peptides 2 – 7 and 9 – 14 were
recorded on a Bruker DMX 600 equipped with a pulsed-field gradient accessory and a cryo-probe. For
the cROESY spectra (200-ms mixing time), the peptides were dissolved in CD3OD. Standard DQF-
COSY (512c ꢁ 2084c) and TOCSY (400c ꢁ 2048c) spectra were recorded using presaturation for solvent
suppression. cROESY spectra (400c ꢁ 2048c, tmix 180 ms) were recorded using the presat solvent
suppression. All spectra were recorded in phase-sensitive mode, using either the TPPI or states-TPPI for
quadrature detection in the indirect dimension. Homonuclear coupling constants were determined from
the corresponding 1H spectra.
Synthesis of Methyl 2,5-Anhydro-3-deoxy-d-ribo-hexonate (17a). Following the procedure described
in [9a]: d-glucono-1,5-lactone (40 g, 225 mmol) was added in portions during 10 – 15 min to Ac2O
(100 ml, 1 mol) containing a few drops of 60% aq. HClO4. The mixture was kept for 1 h at r.t., and the
clear soln. was subsequently concentrated in vacuo. The crude product was dissolved in CH2Cl2 (300 ml)
and cooled to 08, and Et3N (48 ml, 1,5 mol) was added in one portion. The yellow soln. was kept for
15 min at 08, and subsequently washed with 2m HCl and with H2O, dried (Na2SO4) and concentrated in
vacuo. The residue was purified by CC (AcOEt/PE 1:3) to give 2,4,6-tri-O-acetyl-3-deoxy-d-erythro-hex-
2-enono-1,5-lactone (¼(2R*,3S*)-2-[(acetyloxy)methyl]-6-oxo-3,6-dihydro-2H-pyran-3,5-diyl diacetate;
65 g, 225 mmol, 100%). Colorless syrup. The spectroscopic data were in agreement with those reported in
[9a]. 1H-NMR (400 MHz, CDCl3): 6.45 (d, J ¼ 4.2, HꢀC(3)); 5.63 (dd, J ¼ 5.8, 4.2, HꢀC(4)); 4.77 (dd, J ¼
10.3, 4.6, HꢀC(5)); 4.36 (ddd, J ¼ 34.8, 12.3, 4.6, CH2(6)); 2.27 (s, Ac); 2.14 (s, Ac); 2.11 (s, Ac). 13C-NMR
(101 MHz, CDCl3): 170.4, 169.7, 168.1 (3 CO); 157.2 (C(1)); 139.6 (C(2)); 126.1 (C(3)); 77.8 (C(5)); 64.2
(C(4)); 62.0 (C(6)); 20.7, 20.6, 20.3 (3 Ac).
2,4,6-Tri-O-acetyl-3-deoxy-d-erythro-hex-2-enono-1,5-lactone (57.2 g, 200 mmol) was dissolved in
AcOEt (200 ml) and hydrogenated for 16 h at 65 bar in the presence of 10% Pd/C. Filtration and
concentration gave 2,4,6-tri-O-acetyl-3-deoxy-d-arabino-hexono-1,5-lactone (¼(2R,3S,5S)-2-[(acetyl-
oxy)methyl]-6-oxotetrahydro-2H-pyran-3,5-diyl diacetate; 52.7 g, 183 mmol, 92%). Colorless syrup. The
spectroscopic data were in agreement with those reported in [9a]. 1H-NMR (400 MHz, CDCl3): 5.58 (dd,
J ¼ 12.4, 7.1, HꢀC(2)); 5.19 (td, J ¼ 6.1, 3.2, HꢀC(4)); 4.65 – 4.60 (m, HꢀC(5)); 4.36 (dd, J ¼ 12.3, 3.6,
HꢀC(6)); 4.27 (dd, J ¼ 12.3, 5.0, HꢀC(6’)); 2.44 (ddd, J ¼ 14.3, 12.4, 6.2, HꢀC(3)); 2.38 – 2.26 (m,
HꢀC(3’)); 2.19 (s, Ac); 2.14 (s, Ac); 2.11 (s, Ac). 13C-NMR (101 MHz, CDCl3): 170.3, 169.7, 167.3 (3 CO);
77.3 (C(5)); 65.7 (C(4)); 64.0 (C(2)); 62.7 (C(6)); 30.5 (C(3)); 20.9, 20.7, 20.6 (3 Ac).
2,4,6-Tri-O-acetyl-3-deoxy-d-arabino-hexono-1,5-lactone (18.48 g, 64.11 mmol in MeOH (100 ml))
was treated with MeONa (0.02n of 5.56m MeONa in MeOH). The mixture was stirred for 3 h, after
which it was acidified with Dowex Hþ (pH 5). The mixture was heated for 16 h at 508. The resin was
removed by filteration, and the mixture was concentrated in vacuo. The crude residue was dissolved in
acetone, and H2SO4 (1 ml) and MgSO4 (10 g) were added. After completion (Rf (AcOEt) 0.6), the
mixture was neutralized with Et3N and filtered over Celiteꢃ. The mixture was concentrated and sub-
jected to a flash CC (AcOEt/PE 1:1) to yield 3-deoxy-5,6-O-isopropylidene-d-arabino-hexono-1,4-
lactone (¼(3R*,5R*)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxydihydrofuran-2(3H)-one; 9.816 g,
48.55 mmol, 76%). The spectroscopic data were in agreement with those reported in [9c]. 1H-NMR
(400 MHz, MeOD): 4.56 (dd, J ¼ 10.7, 8.7, HꢀC(2)); 4.44 – 4.39 (m, HꢀC(4)); 4.26 (dd, J ¼ 11.9, 5.6,
HꢀC(5)); 4.13 (dd, J ¼ 8.6, 6.7, HꢀC(6)); 3.84 (dd, J ¼ 8.7, 5.4, HꢀC(6’)); 2.69 – 2.63 (m, HꢀC(3)); 1.98 (q,
J ¼ 10.4, HꢀC(3’)); 1.42 (s, Me); 1.35 (s, Me). 13C-NMR (101 MHz, MeOD): 178.5 (CO); 111.1 (Me2C¼);
77.7 (C(5)); 77.3 (C(4)); 68.8 (C(2)); 66.8 (C(6)); 34.1 (C(3)); 26.6 (Me); 25.2 (Me).
3-Deoxy-5,6-O-isopropylidene-d-arabino-hexono-1,4-lactone (9.78 g, 48.37 mmol) was co-evapo-
rated with toluene and dissolved in pyridine (50 ml). The mixture was cooled to 08 and MsCl (2 equiv.
7.49 ml) was added dropwise. After 1 h, the reaction was completed (Rf (AcOEt) 0.8). Ice and H2O were
added, keeping the temp. at 08, which caused the product to precipitate. The solids were removed by
filteration, the filtrate washed with H2O and Et2O, and dried to give 2-O-mesyl-3-deoxy-5,6-O-
isopropylidene-d-arabino-hexono-1,4-lactone (¼(3R*,5R*)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-oxote-
trahydrofuran-3-yl methanesulfonate; 7.02 g, 25.03 mmol, 76%). The spectroscopic data were in

Helvetica Chimica Acta – Vol. 95 (2012)
2553
agreement with those reported in [9d]. 1H-NMR (600 MHz, CDCl3, T¼ 303 K): 5.38 (t, J ¼ 9.5, HꢀC(2));
4.39 (dt, J ¼ 9.2, 6.3, HꢀC(4)); 4.21 (dd, J ¼ 11.2, 6.3, HꢀC(5)); 4.14 (dd, J ¼ 8.8, 6.6, HꢀC(6)); 3.90 (dd,
J ¼ 9.0, 4.6, HꢀC(6’)); 3.27 (s, MeS); 2.90 (ddd, J ¼ 14.5, 8.9, 5.9, HꢀC(3)); 2.39 (dt, J ¼ 13.2, 9.8,
HꢀC(3’)); 1.45 (s, Me); 1.36 (s, Me).
A suspension of 2-O-mesyl-3-deoxy-5,6-O-isopropylidene-d-arabino-hexono-1,4-lactone (7.02 g,
25.03 mmol) in H2O (50 ml) and TFA (0.5 ml) was boiled for 3 h. The soln. was concentrated, and the
residue was dissolved in MeOH (100 ml), and Amberlite IR120 Hþ was added. The soln. was stirred for
3 h. The Amberlite was filtered off, and the mixture was neutralized with Et3N and concentrated. The
methyl ester was purified with CC (AcOEt 100%) to yield 17a (2.46 g, 13.94 mmol 59%). Colorless oil.
1
The spectroscopic data were in agreement with those reported in [9a]. H-NMR (400 MHz, MeOD):
4.68 – 4.61 (m, HꢀC(1)); 4.27 (dt, J ¼ 5.7, 2.8, HꢀC(3)); 3.91 (td, J ¼ 5.0, 2.9, HꢀC(4)); 3.75 (s, Me); 3.58 –
3.56 (m, CH2(5)); 2.29 – 2.09 (m, CH2(2)). 13C-NMR (101 MHz, MeOD): 175.3 (CO); 89.5 (C(4)); 77.3
(C(1)); 73.2 (C(3)); 63.5 (C(5)); 52.9 (Me); 39.7 (C(2)).
2,5-Anhydro-6-azido-3-deoxy-d-ribo-hexonic Acid (16b). Following the procedure in [9b]: com-
pound 17a (419 mg, 2.38 mmol) was coevaporated with toluene (3 ꢁ 50 ml) and dissolved in CH2Cl2
(100 ml), and Et3N (1.2 equiv., 397 ml), TsCl (1.1 equiv., 499 mg), and DMAP (¼4-(dimethylamino)pyr-
idine; 10 mg) were added, and the mixture was stirred until completion (Rf (AcOEt/PE 1:1) 0.50). The
mixture was washed with 1m HCl, sat. aq. NaHCO3, and brine. The org. layer was dried (Na2SO4) and
concentrated. The residue was subjected to a CC (AcOEt/PE 1:4) to yield 17b (566 mg, 1.71 mmol,
72%). Slightly yellow oil.
Compound 17b (1.65 g, 1.55 mmol) was dissolved in DMF (50 ml), and NaN3 was added (5 equiv.,
1.62 g). The mixture was heated for 16 h at 858. DMF was evaporated. The residue was subjected to CC
(AcOEt/PE 1:4) and yielded methyl 2,5-anhydro-6-azido-3,6-dideoxy-d-ribo-hexonate as a colorless oil
(264 mg, 1.33 mmol, 85%). The spectroscopic data were in agreement with those reported in [9b].
1H-NMR (400 MHz, CDCl3): 4.71 (t, J ¼ 7.7, HꢀC(2)); 4.35 (dd, J ¼ 8.1, 4.7, HꢀC(4)); 4.05 (td, J ¼ 5.4, 3.2,
HꢀC(5)); 3.77 (s, MeO); 3.45 (qd, J ¼ 12.8, 5.4, CH2(6)); 2.29 (dd, J ¼ 7.8, 4.8, CH2(3)); 1.86 (br. s, OH).
13C-NMR (101 MHz, CDCl3): 172.31 (CO); 85.7 (C(5)); 76.6 (C(2)); 73.5 (C(4)); 52.6 (C(6)); 52.3
(MeO); 38.7 (C(3)).
2,5-Anhydro-6-azido-3,6-dideoxy-d-ribo-hexonate (1.31 mmol) was dissolved in THF/H2O 7:1
(16 ml) and stirred for 3 h. The solvent was evaporated, and the residue was dissolved in H2O (50 ml) and
excess Amberlyte Hþ. The mixture was stirred for 2 h, and the resin was removed by filtration. The filtrate
was evaporated in vacuo to yielding 16b (1.24 mmol, 232 mg, 95%). Rf (AcOEt/PE 1:1, 1% AcOH) 0.1.
i
1
[a]2D0 ¼ þ39.6 (c ¼ 0.5, PrOH). H-NMR (400 MHz, CDCl3): 4.73 (t, J ¼ 7.6, HꢀC(2)); 4.37 (dd, J ¼ 9.7,
5.4, HꢀC(4)); 4.08 (q, J ¼ 4.3, HꢀC(5)); 3.61 (qd, J ¼ 13.0, 4.3, CH2(6)); 2.38 (dd, J ¼ 7.6, 5.5, CH2(3)).
13C-NMR (101 MHz, CDCl3): 173.2 (CO); 85.0 (C(4)); 76.2 (C(1)); 72.5 (C(3)); 51.9 (C(5)); 38.8 (C(2)).
HR-ESI-MS: 188.0665 ([M þ H]þ, C6H10N3Oþ4 ; calc. 188.0666).
2,5-Anhydro-6-azido-4-O-benzyl-3,6-dideoxy-d-ribo-hexonic Acid (16a). Compound 16b (133 mg,
0.71 mmol) was dissolved in DMF and cooled to 08, and NaH was added (2.5 equiv., 71 mg).
Subsequently, BnBr was added (1.1 equiv., 92 ml). The soln. was stirred for 3 h at 08, and product
formation was observed by TLC (Rf (AcOEt, 1% AcOH) 0.57). The reaction was quenched with H2O,
and the volatiles were evaporated. The mixture was stirred with DOWEX Hþ for 1 h in H2O. The resin
was removed by filtration, and the solvent was evaporated. The crude product was subjected to CC
(AcOEt/PE 1:4, 1% AcOH) and the product was collected as a colorless oil (71 mg, 0.25 mmol, 36%).
Note: this reaction is prone for epimerization, and keeping the soln. at 08 is essential. The spectroscopic
data were in agreement with those reported in [6d]. 1H-NMR (400 MHz, CDCl3): 7.48 – 7.28 (m, 5 arom.
H); 4.71 (t, J ¼ 7.8, HꢀC(2)); 4.57 (d, J ¼ 11.7, 1 H of PhCH2); 4.49 (d, J ¼ 11.7, 1 H of PhCH2); 4.20 (dd,
J ¼ 7.9, 4.1, HꢀC(4)); 4.05 (dd, J ¼ 5.8, 3.1, HꢀC(3)); 3.52 – 3.42 (m, CH2(6)); 2.58 – 2.35 (m, HꢀC(3));
2.35 – 2.10 (m, HꢀC(3’)). 13C-NMR (101 MHz, CDCl3): 175.7 (CO); 137.2 (1 arom. C); 128.6, 128.1, 127.7
(5 arom. CH); 83.6 (C(5)); 79.5 (C(4)); 76.5 (C(2)); 71.7 (PhCH2); 52.3 (C(6)); 36.0 (C(3)).
General Peptide Synthesis. a) Stepwise Elongation for L-O-V-O-L: Fmoc-Leucine preloaded HMPB-
BHA resin (1.24 g, 0.81 mmol/g, 1 mmol) was submitted to four cycles of automated solid-phase synthesis
using the Fmoc-based solid-phase peptide synthesis (SPPS) protocols with the building blocks in the
order: Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-OH, and Fmoc-Leu-OH. The amino acids

2554
Helvetica Chimica Acta – Vol. 95 (2012)
were coupled with 3 equiv. HCTU and 3 equiv. of amino acid in 30 min. The resin was air-dried (1.57 g;
loading 0.64 mmol/g).
b) General Incorporation of Sugar Amino Acid (SAA): a) The resin was washed with MeOH (2 ꢁ
10 ml), NMP (N-methylpyrrolidin-2-one; 2 ꢁ 10 ml), CH2Cl2 (2 ꢁ 10 ml); b) coupling of SAA to resin
(for conditions: see exper. procedure for analogs 2 – 7, 9 – 14); c) washing with NMP (2 ꢁ 10 ml) and
CH2Cl2 (2 ꢁ 10 ml).
c) Reduction of N3: The resins were capped with Ac2O (5 equiv.), EtNiPr2 (5 equiv.) in 4 ml of NMP
for 10 min. The resins were subsequently washed with 1,4-dioxane (3 ꢁ 10 ml) and taken up in 1,4-
dioxane (10 ml) to which Me3P (16 equiv., 1m in THF), pre-mixed with H2O (0.6 equiv.), was added. The
resin was shaken for 24 h; the reduction of the N3 was monitored with the Kaiser test.
d) Automated SPPS Elongation. Azide-reduced resins were subjected to seven cycles of SPPS with
the use of commercially available building blocks in the following order: Fmoc-Val-OH, Fmoc-l-
Orn(Boc)-OH/Fmoc-d-Orn(Boc)-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Leu-OH, Fmoc-
Pro-OH, and Fmoc-d-Phe-OH, and subsequent Fmoc deprotection.
e) Cleavage from Resin. The peptides were released from the resin by mild acidic cleavage (4 ꢁ
10 min, 10 ml 1% TFA in CH2Cl2). The fractions were collected and co-evaporated with toluene (3 ꢁ
50 ml) to give the crude linear peptides which were immediately cyclized without further purification.
f) Cyclization. To a soln. of HOBt (5 equiv.), PyBOP (5 equiv.), and EtNiPr2 (15 equiv.) in DMF
(160 ml) were dropwise added the crude peptides in DMF (20 ml) during 16 h using the syringe pump.
The solvent was removed under reduced pressure, and the resulting mixture was applied to a Sephadexꢃ
size-exclusion column (50.0 mm ꢁ 1500 mm) and eluted with MeOH.
g) Deprotection: The Boc groups were removed by addition of TFA/TIS/H2O 95 :2.5 :2.5 (10 ml),
and subsequently the peptides 2 – 7 and 9 – 14 were purified by prep. or semi-prep. RP-HPLC.
cyclo[(3-OH)SAA4-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,20R,21R,24S,27S,30S,33S,36S,39R,44aS)-6,27,33-Tris(3-aminopropyl)-39-benzyl-
tetratetracontahydro-20-hydroxy-9,24,36-tris(2-methylpropyl)-1,4,7,10,13,16,22,25,28,31,34,37,40-tride-
caoxo-3,15,30-tri(propan-2-yl)-19,21-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaaza-
cyclodotetracontin-12-yl]propan-1-aminium Trifluoroacetate; 2). Peptide 3 (20.65 mg, 10.26 mmol) was
hydrogenated under H2 in 1,4-dioxane (1 ml) and aq. HCl (0.7m, 1 ml) with Pd-black (20 mg). The
mixture was filtered over Celiteꢃ yielding the crude peptide, which was purified by RP-HPLC (linear
gradient of 38 – 47%, 3 column volumes (CVs)) to give 2 (15.71 mg, 80%). White amorphous powder.
LC/MS: tR 4.75 min; linear gradient 10 ! 90% B in 13.5 min; m/z 1467.3 ([M þ H]þ). 1H-NMR
(600 MHz, CD3OD): 8.98 (d, J ¼ 9.4, NH(Val5)); 8.93 (br. s, NH(d-Phe13)); 8.73 (d, J ¼ 9.1, NH(Leu3));
8.65 (d, J ¼ 9.0, NH(Leu8), NH(Orn9), NH(Leu12)); 8.62 (d, J ¼ 8.9, NH(Orn4)); 8.53 (d, J ¼ 8.8,
NH(Orn11)); 8.44 (d, J ¼ 9.2, NH(Orn2)); 8.01 (t, J ¼ 5.6, NH(SAA)); 7.97 (br. s, NH2(Orn)); 7.93 (d, J ¼
5.7, NH(Val10)); 7.75 (d, J ¼ 8.6, NH(Val1)); 7.36 – 7.22 (m, 5 arom. H); 5.10 (d, J ¼ 7.3, Ha(Orn11)); 5.03 –
5.01 (m, Ha(Orn2), Ha(Orn9)); 4.97 – 4.91 (m, Ha(Orn4)); 4.80 – 4.74 (m, 1 H); 4.72 (d, J ¼ 6.4, 1 H);
4.70 – 4.61 (m, Ha(Leu3), Ha(Leu12)); 4.52 (dt, J ¼ 10.9, 4.4, Ha(d-Phe13)); 4.41 (dd, J ¼ 9.3, 7.2,
Ha(Leu8)); 4.37 (d, J ¼ 6.7, Ha(Pro14)); 4.25 – 4.19 (m, Ha(Val5), Ha(Val10)); 4.16 (br. s, 1 H); 4.12 (t, J ¼
8.6, Ha(Val1)); 3.72 (dd, J ¼ 12.8, 5.3, 1 H); 3.57 – 3.50 (m, 2 H); 3.13 – 3.00 (m, 2 H); 3.00 – 2.86 (m, 8 H);
2.49 (q, J ¼ 8.9, 1 H); 2.30 (dd, J ¼ 14.8, 6.9, 1 H); 2.13 – 2.03 (m, 1 H); 2.03 – 1.92 (m, 3 H); 1.88 – 1.41 (m,
23 H); 1.42 – 1.33 (m, 1 H); 1.05 – 0.75 (m, 36 H). 13C-NMR (151 MHz, CD3OH): 174.6; 174.1; 174.0;
173.9; 173.7; 173.5; 173.4; 173.4; 172.9; 172.7; 172.6; 137.0; 130.5; 129.8; 128.6; 88.7; 85.4; 70.7; 62.1; 60.8;
59.1; 56.1; 53.7; 53.6; 53.6; 53.4; 52.7; 52.4; 51.8; 49.7; 48.0; 44.3; 42.2; 41.5; 41.4; 41.0; 40.8; 40.7; 37.4; 34.2;
31.9; 30.8; 30.7; 30.4; 30.3; 25.9; 25.9; 25.7; 25.7; 25.3; 25.1; 24.8; 24.6; 24.1; 23.3; 23.2; 23.2; 22.6; 21.9;
20.2; 19.9; 19.7; 19.6; 19.4; 19.0. HR-ESI-MS: 1466.9474 ([M þ H]þ, C72H124N17O1þ5 ; calc. 1466.9437).
cyclo[(3-OBn)SAA4-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12R,15S,19R,24S,27S,30S,39R,44aS)-6,27,33-Tris(3-aminopropyl)-39-benzyl-20-(benzyloxy)-
tetratetracontahydro-9,24,36-tris(2-methylpropyl)-1,4,7,10,13,16,22,25,28,31,34,37,40-tridecaoxo-3,15,30-
tri(propan-2-yl)-19,21-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacyclodotetracon-
tin-12-yl]propan-1-aminium Trifluoroacetate; 3). SAA 15 (105.3 mg, 2 equiv.) was pre-activated with
HBTU (3 equiv., 3 ml of 0.2m HBTU in NMP) and EtNiPr2 (6 equiv., 199 ml) in 10 ml of NMP, and
subsequently coupled to the resin 19 (498 mg, 0.2 mmol, 0.402 mmol/g) for 4 h (mini-cleavage from resin:

Helvetica Chimica Acta – Vol. 95 (2012)
2555
LC/MS: tR 6.21 min, linear gradient 10 ! 90% B in 13.5 min; m/z 817.33 ([M þ H]þ)). Resin 20
(0.1 mmol) was subjected to steps c – g, and the Boc-deprotected peptide was purified by RP-HPLC
(linear gradient of 36 – 45%, 3 CV) and yielded 3 (40.6 mg, 20%). White amorphous powder. LC/MS: tR
6.13 min, linear gradient 10 ! 90% B in 13.5 min; m/z 1557.3 ([M þ H]þ). 1H-NMR (600 MHz, CD3OH):
8.85 (d, J ¼ 3.3, NH(d-Phe13)); 8.66 (d, J ¼ 8.7, NH(Val5)); 8.55 (d, J ¼ 10.0, NH(Leu12)); 8.53 (d, J ¼ 8.8,
NH(Leu5)); 8.53 (d, J ¼ 8.9, NH(Orn11)); 8.49 (d, J ¼ 8.2, NH(Orn9)); 8.47 (d, J ¼ 10.0, NH(Val10)); 8.40
(d, J ¼ 8.3, NH(Orn2), NH(Orn4)); 8.13 (t, J ¼ 5.5, NH(SAA4)); 8.06 (d, J ¼ 6.2, NH(Leu8)); 7.93 (br. s,
NH2(Orn)); 7.83 (br. s, NH2(Orn)); 7.78 (d, J ¼ 8.2, NH(Val1)); 7.40 – 7.21 (m, 10 H); 4.93 – 4.83 (m,
Ha(Orn2), Ha(Orn9), Ha(Orn11)); 4.79 – 4.77 (m, Ha(Orn4)); 4.62 – 4.54 (m, Ha(Leu3), Ha(Leu12), 1 H of
PhCH2, Ha(d-Phe13)); 4.43 (d, J ¼ 11.6, 1 H of PhCH2); 4.36 – 4.34 (m, Ha(Pro14), Ha(Val10)); 4.32 – 4.28
(m, Ha(Leu8)); 4.25 – 4.20 (m, Ha(Val5)); 4.05 (t, J ¼ 8.4, Ha(Val1)); 3.72 – 3.69 (m, 1 H); 3.50 – 3.47 (m,
1 H); 3.41 – 3.39 (m, 1 H); 3.07 – 3.02 (m, 2 H); 2.99 – 2.89 (m, 8 H); 2.78 – 2.76 (m, 1 H); 2.55 (q, J ¼ 8.3,
1 H); 2.29 – 2.24 (m, 1 H); 2.10 – 1.87 (m, 3 H); 1.87 – 1.45 (m, 23 H); 1.39 – 1.35 (m, 1 H); 0.98 (d, J ¼ 4.9,
3 H); 0.97 (d, J ¼ 5.2, 3 H); 0.94 – 0.85 (m, 27 H); 0.83 (d, J ¼ 6.1, 3 H). 13C-NMR (151 MHz, CD3OH):
174.7; 174.5; 174.1; 173.8; 173.6; 173.5; 173.1; 172.9; 172.9; 172.7; 138.5; 137.1; 130.5; 129.8; 129.7; 129.3;
128.9; 128.6; 86.3; 83.2; 77.4; 72.4; 62.0; 61.1; 60.9; 59.6; 55.9; 54.2; 53.8; 53.6; 53.3; 52.9; 51.9; 48.1; 43.6;
42.3; 42.2; 41.6; 40.9; 40.9; 40.7; 40.6; 37.6; 33.7; 31.6; 30.9; 30.8; 30.7; 30.6; 30.4; 30.0; 25.9; 25.9; 25.8;
25.3; 25.2; 24.8; 24.7; 24.1; 23.5; 23.2; 23.0; 22.4; 21.8; 20.0; 20.0; 19.8; 19.7; 19.5; 19.1. HR-ESI-MS:
1556.9925 ([M þ H]þ, C79H130N17O1þ5 ; calc. 1556.9927).
cyclo[(3-OH)SAA5-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12R,15S,19R,20R,22R,25S,28S,31S,40R,45aS)-6,28,34-Tris(3-aminopropyl)-40-benzyltetrate-
tracontahydro-20-hydroxy-9,25,37-tris(2-methylpropyl)-1,4,7,10,13,16,23,26,29,32,35,38,41-tridecaoxo-
3,15,31-tri(propan-2-yl)-1 H-19,22-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacy-
clotritetracontin-12-yl]propan-1-aminium Trifluoroacetate; 4). SAA 16a (28.1 mg, 1.5 equiv.) was pre-
activated with HCTU (1.4 equiv., 57.9 mg) and EtNiPr2 (2.8 equiv., 46 ml) in 10 ml of NMP, and
subsequently coupled to the resin 19 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (mini-cleavage: LC/MS tR
5.14 min, linear gradient 10 ! 90% B in 13.5 min; m/z 741.33 ([M þ H]þ)). The resin (0.1 mmol) was
subjected to steps c – g, and Boc-deprotected peptide was purified by RP-HPLC (linear gradient of 38 –
47%, 3 CV) and yielded 4 (12.0 mg, 6.2 mmol, 6.2%). White amorphous powder. LC/MS: tR 5.81 min,
linear gradient 10 ! 90% B in 13.5 min; m/z 1482.3 ([M þ H]þ). 1H-NMR (600 MHz, CD3OH): 9.05 (d,
J ¼ 9.6, NH(Val5)); 8.94 (d, J ¼ 3.1, NH(d-Phe13)); 8.78 (d, J ¼ 9.1, NH(Leu3)); 8.66 (d, J ¼ 8.4,
NH(Orn9)); 8.65 (d, J ¼ 7.5, NH(Leu12)); 8.62 (d, J ¼ 9.6, NH(Leu8)); 8.54 (d, J ¼ 8.4, NH(Orn11)); 8.48
(br. s, 1 H of NH2(Orn)); 8.45 (d, J ¼ 8.5, NH(Orn4)); 8.43 (d, J ¼ 8.0, NH(Orn2)); 8.28 (t, J ¼ 6.2,
NH(SAA5)); 8.01 (d, J ¼ 6.4, NH(Val10)); 7.92 (br. s, NH2(Orn)); 7.76 (d, J ¼ 8.6, NH(Val1)); 7.37 – 7.18
(m, 5 H); 5.12 – 5.08 (m, Ha(Orn4), Ha(Orn11)); 5.08 – 5.00 (m, Ha(Orn2), Ha(Orn9)): 4.68 – 4.65 (m,
Ha(Leu3), Ha(Leu12)); 4.57 – 4.48 (m, Ha(d-Phe13)); 4.42 (dd, J ¼ 10.8, 5.4, 1 H); 4.38 – 4.35 (m,
Ha(Pro14), Ha(Leu8)); 4.27 – 4.15 (m, Ha(Val10)); 4.11 – 4.09 (m, Ha(Val1), Ha(Val5)); 4.07 – 4.05 (m,
1 H); 3.91 (dd, J ¼ 13.9, 7.0, 1 H); 3.73 (dd, J ¼ 12.5, 5.4, 1 H); 3.10 – 3.05 (m, 3 H); 3.00 – 2.83 (m, 8 H);
2.48 (d, J ¼ 8.1, 1 H); 2.30 (dd, J ¼ 15.0, 6.9, 1 H); 2.09 (dd, J ¼ 11.8, 5.6, 1 H); 2.05 – 1.28 (m, 32 H); 1.08 –
0.70 (m, 36 H). 13C-NMR (151 MHz, CD3OH): 175.2; 175.2; 174.3; 173.9; 173.7; 173.7; 173.7; 173.6;
173.5; 173.4; 172.9; 172.8; 172.6; 163.2; 163.0; 137.0; 130.5; 129.8; 128.6; 89.5; 79.1; 73.6; 62.1; 60.9; 60.6;
59.4; 56.1; 54.1; 53.4; 53.2; 52.7; 52.4; 51.8; 49.7; 47.9; 44.6; 42.2; 42.1; 41.9; 41.4; 40.7; 37.4; 34.3; 31.8; 31.7;
30.7; 30.5; 29.6; 25.9; 25.7; 25.4; 25.2; 24.8; 24.6; 24.3; 23.5; 23.2; 23.1; 22.5; 21.8; 19.9; 19.7; 19.7; 19.3;
19.3. HR-ESI-MS: 1480.9633 ([M þ H]þ, C73H126N17O1þ5 ; calc. 1480.9614).
cyclo[(3-OBn)SAA5-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12R,15S,19R,20R,22R,25S,28S,31S,40R,45aS)-6,28,34-Tris(3-aminopropyl)-40-benzyl-20-
(benzyloxy)tetratetracontahydro-9,25,37-tris(2-methylpropyl)-1,4,7,10,13,16,23,26,29,32,35,38,41-tride-
caoxo-3,15,31-tri(propan-2-yl)-1H-19,22-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tride-
caazacyclotritetracontin-12-yl]propan-1-aminium Trifluoroacetate; 5). SAA 16b (33.2 mg, 1.2 equiv.) was
pre-activated with HCTU (1.2 equiv., 50 mg) and EtNiPr2 (3 equiv., 60 ml) in 10 ml of NMP, and
subsequently coupled to the resin 19 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (mini-cleavage: LC/MS: tR
6.31 min, linear gradient 10 ! 90% B in 13.5 min; m/z 831.53 ([M þ H]þ)). The resin (0.05 mmol) was
subjected to steps c – g, and Boc-deprotected peptide was purified by RP-HPLC (linear gradient of 43 –

2556
Helvetica Chimica Acta – Vol. 95 (2012)
52%, 3 CV) to yield 5 (11.84 mg, 5.84 mmol, 12%). White amorphous powder. LC/MS: tR 6.39 min, linear
gradient 10 ! 90% B in 13.5 min; m/z 1571.5 ([M þ H]þ). 1H-NMR (600 MHz, CD3OH): 9.04 (d, J ¼ 9.6,
NH(Val5)); 8.94 (s, NH(d-Phe13)); 8.76 (d, J ¼ 9.0, NH(Leu3)); 8.65 (d, J ¼ 7.6, NH(Orn11)); 8.64 (d, J ¼
7.7, NH(Leu12)); 8.63 (d, J ¼ 9.3, NH(Val10)); 8.55 (d, J ¼ 8.1, NH(Orn9)); 8.43 (d, J ¼ 9.2, NH(Orn2),
NH(Orn4)); 8.33 (t, J ¼ 5.9, NH(SAA)); 8.01 (d, J ¼ 6.3, NH(Leu8)); 7.92 (br. s, NH2(Orn)); 7.77 (d, J ¼
8.6, NH(Val1)); 7.41 – 7.20 (m, 10 H); 5.14 – 5.12 (m, Ha(Orn4)); 5.09 – 4.99 (m, Ha(Orn2), Ha(Orn11));
4.88 (m, Ha(Orn9)); 4.72 – 4.61 (m, Ha(Leu3), Ha(Leu12)); 4.57 – 4.50 (m, Ha(d-Phe13)); 4.39 – 4.37 (m,
Ha(Pro), Ha(Val10), 1 H of PhCH2); 4.28 (s, 1 H); 4.19 (dt, J ¼ 10.9, 5.5, Ha(Leu8)); 4.15 – 4.05 (m,
Ha(Val1), Ha(Val5), 1 H of PhCH2); 3.94 (dd, J ¼ 13.8, 7.0, 1 H); 3.73 (dd, J ¼ 12.8, 5.3, 1 H); 3.15 – 3.01
(m, 3 H); 3.00 – 2.78 (m, 8 H); 2.49 (q, J ¼ 8.9, 1 H); 2.37 – 2.24 (m, 2 H); 2.05 – 1.25 (m, 32 H); 1.04 – 0.71
(m, 36 H). 13C-NMR (151 MHz, CD3OH): 175.2; 174.9; 174.3; 174.0; 173.7; 173.7; 173.6; 173.6; 173.5;
173.4; 172.8; 172.8; 172.6; 163.3; 163.0; 139.3; 137.0; 130.5; 129.8; 129.6; 129.0; 128.9; 128.6; 87.2; 81.8;
79.4; 72.2; 62.0; 60.9; 60.6; 59.4; 56.1; 54.2; 53.3; 53.1; 52.7; 52.4; 51.8; 49.8; 47.9; 44.7; 42.6; 42.2; 41.4;
41.0; 40.8; 40.8; 40.7; 39.3; 37.4; 34.3; 31.8; 31.8; 30.7; 30.7; 30.5; 29.5; 25.9; 25.7; 25.4; 25.3; 25.1; 24.8; 24.6;
24.3; 23.5; 23.2; 23.1; 22.4; 21.8; 20.0; 19.7; 19.7; 19.6; 19.3; 19.3. HR-ESI-MS: 11571.0108 ([M þ H]þ,
C80H132N17Oþ15 ; calc. 1571.0083).
cyclo[(3-OH)SAA6-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12R,15S,19R,20R,23R,26S,29S,32S,41R,46aS)-6,29,35-Tris(3-aminopropyl)-41-benzylhexate-
tracontahydro-20-hydroxy-9,26,38-tris(2-methylpropyl)-1,4,7,10,13,16,24,27,30,33,36,39,42-tridecaoxo-
3,15,32-tri(propan-2-yl)-19,23-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacyclote-
tratetracontin-12-yl]propan-1-aminium trifluoroacetate; 6). SAA 18a (62.4 mg, 1.55 equiv.) was pre-
activated with HCTU (1.45 equiv., 120 mg) and EtNiPr2 (2.9 equiv., 96 ml) in 10 ml of NMP, and
subsequently coupled to the resin 19 (400 mg, 0.2 mmol, 0.5 mmol/g) for 4 h (mini-cleavage LC/MS: tR
5.28 min, linear gradient 10 ! 90% B in 13.5 min; m/z 755.33 ([M þ H]þ)). The resin (0.1 mmol) was
subjected to steps c – g, and Boc-deprotected peptide was purified by RP-HPLC (linear gradient of 37 –
46%, 3 CV) to yield 6 (1.8 mg, 0.9 mmol, 1%). White amorphous powder. LC/MS: tR 5.77 min, linear
gradient 10 ! 90% B in 13.5 min; m/z 1495.3 ([M þ H]þ). 1H-NMR (600 MHz, CD3OH): 8.92 (d, J ¼ 3.2,
NH(d-Phe13)); 8.74 (d, J ¼ 8.9, NH(Leu8)); 8.63 (d, J ¼ 8.3, NH(Orn11)); 8.62 (d, J ¼ 8.5, NH(Leu12)); 8.61
(d, J ¼ 9.3, NH(Val5), NH(Val10)); 8.53 (d, J ¼ 8.3, NH(Orn4)); 8.42 (t, J ¼ 9.0, NH(Orn2), NH(Orn9));
8.09 (d, J ¼ 8.5, NH(Leu3), NH(SAA6)); 7.77 (d, J ¼ 8.5, NH(Val1)); 7.38 – 7.21 (m, 5 H); 5.11 – 5.10 (m,
Ha(Orn9)); 5.04 – 5.01 (m, Ha(Orn2), Ha(Orn11)); 4.87 – 4.85 (m, Ha(Orn4)); 4.67 – 4.64 (m, Ha(Leu8),
Ha(Leu12)); 4.53 (dd, J ¼ 10.7, 4.6, Ha(d-Phe13)); 4.50 – 4.40 (m, Ha(Leu3), 1 H of PhCH2); 4.39 – 4.32 (m,
Ha(Pro14), Ha(Val10)); 4.15 (t, J ¼ 9.4, Ha(Val5)); 4.11 (t, J ¼ 8.4, Ha(Val1)); 3.94 – 3.90 (m, 2 H); 3.73 (t,
J ¼ 9.0, 1 H); 3.37 – 3.18 (m, 3 H); 3.12 – 3.01 (m, 2 H); 3.01 – 2.78 (m, 8 H); 2.50 (dd, J ¼ 17.2, 8.2, 1 H);
2.30 (dd, J ¼ 14.6, 7.0, 1 H); 2.14 – 1.90 (m, 6 H); 1.86 – 1.44 (m, 26 H); 1.43 – 1.30 (m, 1 H); 1.18 – 1.12 (m,
1 H); 1.06 – 0.71 (m, 36 H). 13C-NMR (151 MHz, CD3OH): 175.2; 174.3; 174.2; 174.1; 174.0; 173.7; 173.5;
172.9; 172.6; 163.2; 137.0; 130.5; 129.8; 128.6; 81.7; 75.8; 66.1; 62.0; 60.9; 59.4; 56.1; 54.1; 53.4; 52.7; 52.6;
52.4; 51.8; 48.0; 42.3; 42.1; 41.0; 40.9; 40.7; 40.1; 37.4; 34.2; 32.5; 31.9; 31.8; 30.9; 30.7; 29.8; 29.6; 25.9;
25.9; 25.7; 25.3; 24.9; 24.6; 24.3; 23.4; 23.2; 23.1; 22.5; 21.9; 20.0; 19.8; 19.7; 19.6; 19.3. HR-ESI-MS:
747.9920 ([M þ H]2þ, C74H129N17O1þ5 ; calc. 747.9922).
cyclo[(3-OBn)SAA6-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-Orn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12R,15S,19R,20R,23R,26S,29S,32S,41R,46aS)-6,29,35-Tris(3-aminopropyl)-41-benzyl-20-
(benzyloxy)hexatetracontahydro-9,26,38-tris(2-methylpropyl)-1,4,7,10,13,16,24,27,30,33,36,39,42-tride-
caoxo-3,15,32-tri(propan-2-yl)-19,23-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaaza-
cyclotetratetracontin-12-yl]propan-1-aminium trifluoroacetate; 7). SAA 18b (44 mg, 1.5 equiv.) was pre-
activated with HCTU (1. 5 equiv., 63 mg) and EtNiPr2 (3 equiv., 50 ml) in 10 ml of NMP, and subsequently
coupled to the resin 19 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (LC/MS: tR 6.68 min, linear gradient 10 !
90% B in 13.5 min; m/z 845.33 ([M þ H]þ)). The resin (0.1 mmol) was subjected to steps c – g, and Boc-
deprotected peptide was purified by RP-HPLC (linear gradient of 43 – 52%, 3 CV) to yield 7 (14.3 mg,
7.0 mmol, 7%). White amorphous powder. LC/MS: tR 6.51 min, linear gradient 10 ! 90% B in 13.5 min;
1
m/z 1585.4 ([M þ H]þ). H-NMR (600 MHz, CD3OH): 8.92 (d, J ¼ 3.3, NH(d-Phe13)); 8.73 (d, J ¼ 8.9,
NH(Leu3)); 8.63 (d, J ¼ 8.9, NH(Val10), NH(Leu12)); 8.61 (d, J ¼ 9.2, NH(Orn11), NH(Val5)); 8.53 (d, J ¼
8.2, NH(Orn9)); 8.42 (d, J ¼ 9.2, NH(Orn2), NH(Orn4)); 8.11 (d, J ¼ 8.4, NH(Leu8)); 7.92 (t, J ¼ 6.1,

Helvetica Chimica Acta – Vol. 95 (2012)
2557
NH(SAA)); 7.77 (d, J ¼ 8.5, NH(Val1)); 7.40 – 7.22 (m, 10 H); 5.09 (t, J ¼ 7.0, Ha(Orn4)); 5.04 – 5.02 (m,
Ha(Orn2), Ha(Orn11)); 4.87 – 4.83 (m, Ha(Orn9)); 4.73 – 4.60 (m, Ha(Leu3), Ha(Leu12), 1 H of PhCH2);
4.58 – 4.49 (m, Ha(d-Phe13), 1 H of PhCH2); 4.44 – 4.42 (m, Ha(Leu8)); 4.37 – 4.36 (m, Ha(Pro14),
Ha(Val10)); 4.15 (t, J ¼ 9.4, Ha(Val5)); 4.11 (t, J ¼ 8.5, Ha(Val1)); 3.96 – 3.87 (m, 2 H); 3.73 (dd, J ¼ 12.7,
5.3, 1 H); 3.46 (d, J ¼ 9.3, 1 H); 3.27 – 3.16 (m, 2 H); 3.16 – 3.01 (m, 3 H); 3.01 – 2.89 (m, 6 H); 2.84 (s,
2 H); 2.49 (t, J ¼ 8.5, 1 H); 2.38 – 2.24 (m, 2 H); 2.10 (dd, J ¼ 13.3, 2.7, 1 H); 2.08 – 1.90 (m, 4 H); 1.86 –
1.43 (m, 26 H); 1.41 – 1.25 (m, 1 H); 1.12 (d, J ¼ 14.7, 1 H); 1.03 – 0.74 (m, 36 H). 13C-NMR (151 MHz,
CD3OH): 175.2; 174.3; 174.1; 174.0; 174.0; 173.7; 173.7; 173.5; 173.4; 172.9; 172.8; 172.6; 139.9; 137.0;
130.5; 129.8; 129.5; 129.0; 128.9; 128.6; 80.1; 75.9; 73.5; 71.8; 62.0; 60.9; 60.9; 59.4; 56.1; 54.2; 53.4; 53.4;
52.7; 52.6; 52.5; 51.8; 49.7; 48.0; 42.3; 42.1; 41.0; 40.8; 40.2; 37.4; 34.1; 31.9; 31.8; 30.9; 30.8; 30.7; 29.6; 29.3;
29.0; 25.9; 25.9; 25.7; 25.2; 24.8; 24.6; 24.3; 23.4; 23.2; 23.1; 22.6; 21.9; 20.0; 19.8; 19.7; 19.6; 19.3. HR-ESI-
MS: 1585.0258 ([M þ H]þ, C81H134N17O1þ5 ; calc. 1585.0240).
cyclo[(3-OH)SAA4-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,24S,27S,30S,39R,44aS)-6,27,33-Tris(3-aminopropyl)-39-benzyltetratetraconta-
hydro-20-hydroxy-9,24,36-tris(2-methylpropyl)-1,4,7,10,13,16,22,25,28,31,34,37,40-tridecaoxo-3,15,30-
tri(propan-2-yl)-19,21-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacyclodotetracon-
tin-12-yl]propan-1-aminium Trifluoroacetate; 9). Peptide 10 (18.51 mg, 9.19 mmol) was hydrogenated
under H2 in 1,4-dioxane (1 ml) and aq. HCl (0.7m, 1 ml) with Pd-black (20 mg). The mixture was filtered
over Celiteꢃ to yield the crude peptide, which was purified by RP-HPLC (linear gradient of 31 – 40%, 3
CV) to give 9 (5.74 mg, 2.98 mmol, 32%). White amorphous powder. LC/MS: tR 4.96 min, linear gradient
10 ! 90% B in 13.5 min; m/z 1467.3 ([M þ H]þ). 1H-NMR (600 MHz, CD3OH): 8.81 (d, J ¼ 3.9, NH(d-
Phe13)); 8.55 (d, J ¼ 8.4, NH(Orn2)); 8.54 (d, J ¼ 7.7, NH(Orn9)); 8.45 (d, J ¼ 8.3, NH(Orn11)); 8.42 (d, J ¼
8.8, NH(Leu12)); 8.33 (d, J ¼ 8.3, NH(Leu8), NH(SAA4)); 8.30 (d, J ¼ 8.0, NH(Leu3)); 8.24 (d, J ¼ 8.2,
NH(Val5)); 7.98 (d, J ¼ 8.4, NH(Val10)); 7.94 (d, J ¼ 7.8, NH(Orn4)); 7.86 (d, J ¼ 8.0, NH(Val1)); 7.85 (br. s,
NH2(Orn)); 7.32 – 7.24 (m, 5 H); 4.82 – 4.78 (m, Ha(Orn11)); 4.72 (d, J ¼ 4.6, 1 H); 4.63 – 4.46 (m, Ha(d-
Phe13), Ha(Orn2), Ha(Orn4), Ha(Orn9), Ha(Leu3), Ha(Leu8), Ha(Leu12)); 4.41 – 4.34 (m, Ha(Pro14));
4.32 (t, J ¼ 7.4, Ha(Val5)); 4.26 (t, J ¼ 7.9, Ha(Val10)); 4.21 (t, J ¼ 4.3, 1 H); 4.06 – 3.95 (m, Ha(Val1)); 3.72 –
3.69 (m, 1 H); 3.65 (s, 1 H); 3.21 – 3.14 (m, 2 H); 3.06 (dd, J ¼ 12.8, 5.7, 1 H); 3.03 – 2.90 (m, 9 H); 2.63
(dd, J ¼ 17.9, 9.4, 1 H); 2.26 (td, J ¼ 13.6, 6.8, 1 H); 1.95 – 1.49 (m, 29 H); 1.44 – 1.35 (m, 2 H); 1.06 – 0.82
(m, 36 H). 13C-NMR (151 MHz, CD3OH): 175.3; 174.6; 174.3; 174.2; 174.0; 173.8; 173.6; 173.5; 173.5;
173.4; 172.8; 163.1; 162.9; 137.2; 130.5; 129.8; 128.5; 89.1; 86.1; 72.8; 64.6; 61.9; 61.3; 60.3; 55.7; 54.4; 54.1;
53.7; 53.4; 53.4; 52.9; 52.3; 49.7; 48.1; 43.2; 42.3; 41.9; 41.8; 40.9; 40.7; 40.5; 37.7; 32.3; 32.2; 31.5; 31.1; 30.7;
29.8; 29.6; 26.2; 26.0; 25.4; 25.2; 25.1; 24.8; 24.7; 23.8; 23.6; 23.0; 22.0; 21.7; 20.2; 19.9; 19.8; 19.7; 18.9;
18.8. HR-ESI-MS: 11466.9469 ([M þ H]þ, C72H124N17O1þ5 ; calc. 1466.9457).
cyclo[(3-OBn)SAA4-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,24S,27S,30S,39R,44aS)-6,27,33-Tris(3-aminopropyl)-39-benzyl-20-(benzyloxy)te-
tratetracontahydro-9,24,36-tris(2-methylpropyl)-1,4,7,10,13,16,22,25,28,31,34,37,40-tridecaoxo-3,15,30-tri-
(propan-2-yl)-19,21-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacyclodotetracon-
tin-12-yl]propan-1-aminium Trifluoroacetate; 10). SAA 15 (105.3 mg, 2 equiv.) was pre-activated with
HBTU (3 equiv., 3 ml of 0.2m HBTU in NMP) and EtNiPr2 (6 equiv., 199 ml) in 10 ml of NMP, and
subsequently coupled to the L-O-V-DO-L resin (498 mg, 0.2 mmol, 0.402 mmol/g) for 4 h (mini-cleavage:
LC/MS: tR 6.21 min, linear gradient 10 ! 90% B in 13.5 min; m/z 817.33 ([M þ H]þ)). The resin
(0.1 mmol) was subjected to c – g, and Boc-deprotected peptide was purified by RP-HPLC (linear
gradient of 34 – 43%, 3 CV) to give 10 (45.2 mg, 22.5 mmol, 23%). White amorphous powder. LC/MS: tR
5.90 min, linear gradient 10 ! 90% B in 13.5 min; m/z 1558.3 ([M þ H]þ). 1H-NMR (600 MHz,
CD3OH): 8.87 (d, J ¼ 3.8, NH(d-Phe13)); 8.60 (d, J ¼ 7.9, NH(Orn2)); 8.53 (d, J ¼ 6.9, NH(Orn9)); 8.47 (d,
J ¼ 8.4, NH(Leu12)); 8.46 (d, J ¼ 6.8, NH(Orn11)); 8.33 (d, J ¼ 9.6, NH(Leu3), NH(Leu8), NH(SAA4));
8.25 (d, J ¼ 8.5, NH(Val10)); 8.08 (d, J ¼ 8.6, NH(Val5)); 7.87 (d, J ¼ 8.0, NH(Val1)); 7.81 (d, J ¼ 7.8,
NH(Orn4)); 7.38 – 7.20 (m, 10 H); 4.71 – 4.33 (m, PhCH2, Ha(d-Phe13), Ha(Orn2), Ha(Orn4), Ha(Orn9),
Ha(Orn11), Ha(Leu3), Ha(Leu8), Ha(Leu12), Ha(Val10), Ha(Pro14)); 4.26 (t, J ¼ 8.2, Ha(Val5)); 4.15 (t, J ¼
4.2, 1 H); 4.05 – 3.99 (m, Ha(Val1)); 3.70 (d, J ¼ 7.1, 1 H); 3.16 – 3.04 (m, 2 H); 3.04 – 2.90 (m, 10 H); 2.58
(d, J ¼ 8.1, 1 H); 2.27 (dd, J ¼ 15.4, 6.8, 1 H); 2.06 – 1.48 (m, 30 H); 1.40 (dd, J ¼ 13.0, 6.6, 1 H); 1.00 – 0.88
(m, 36 H). 13C-NMR (151 MHz, CD3OH): 175.3; 174.6; 174.3; 174.1; 174.0; 173.9; 173.8; 173.6; 173.5;

2558
Helvetica Chimica Acta – Vol. 95 (2012)
172.8; 172.7; 138.5; 137.2; 130.5; 129.8; 129.5; 129.2; 129.1; 128.6; 86.7; 84.0; 78.6; 72.3; 61.9; 61.3; 60.3;
59.9; 55.8; 54.7; 54.2; 53.6; 53.4; 53.2; 52.6; 52.2; 48.0; 43.0; 42.4; 41.9; 41.8; 40.9; 40.7; 40.7; 40.5; 37.6;
32.5; 32.2; 31.8; 31.4; 31.3; 30.7; 29.5; 26.2; 26.0; 26.0; 25.5; 25.2; 24.7; 24.6; 23.8; 23.7; 23.1; 23.0; 21.9;
21.6; 20.2; 19.9; 19.7; 18.9; 18.8. HR-ESI-MS: 1556.9934 ([M þ H]þ, C79H130N17O1þ5 ; calc. 1556.9927).
cyclo[(3-OH)SAA5-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,20R,22R,25S,28S,31S,40R,45aS)-6,28,34-Tris(3-aminopropyl)-40-benzyltetrate-
tracontahydro-20-hydroxy-9,25,37-tris(2-methylpropyl)-1,4,7,10,13,16,23,26,29,32,35,38,41-tridecaoxo-
3,15,31-tri(propan-2-yl)-1H-19,22-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaaza-
cyclotritetracontin-12-yl]propan-1-aminium Trifluoroacetate; 11). SAA 16a (30 mg, 1.6 equiv.) was pre-
activated with HCTU (1.6 equiv., 66 mg), and EtNiPr2 (3 equiv., 53 ml) in 10 ml of NMP, and
subsequently coupled to the L-O-V-DO-L resin (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (mini-cleavage:
LC/MS: tR 5.14 min, linear gradient 10 ! 90% B in 13.5 min; m/z 741.33 ([M þ H]þ)). The resin
(0.1 mmol) was subjected to steps c – g, and the Boc-deprotected peptide was purified by RP-HPLC
(linear gradient of 29 – 38%, 3 CV) to give 11 (9.11 mg, 4.70 mmol, 5%). White amorphous powder. LC/
1
MS: tR 4.89 min, linear gradient 10 ! 90% B in 13.5 min; m/z 1482.1 ([M þ H]þ). H-NMR (600 MHz,
CD3OH): 8.83 (d, J ¼ 2.7, NH(d-Phe13)); 8.55 (d, J ¼ 8.3, NH(Orn9)); 8.52 (d, J ¼ 8.6, NH(Orn2)); 8.49 (d,
J ¼ 8.4, NH(Orn11)); 8.46 (d, J ¼ 8.9, NH(Leu12)); 8.44 (t, J ¼ 4.7, NH(SAA)); 8.34 (d, J ¼ 8.3,
NH(Leu3)); 8.21 (d, J ¼ 7.7, NH(Val10)); 8.20 (d, J ¼ 8.3, NH(Leu8)); 8.14 (d, J ¼ 7.8, NH(Orn4)); 8.07
(d, J ¼ 8.5, NH(Val5)); 7.85 (br. s, NH2(Orn)); 7.82 (d, J ¼ 8.2, NH(Val1)); 7.47 – 7.14 (m, 5 H); 4.83 (m,
Ha(Orn11)); 4.67 (m, Ha(Orn4), Ha(Orn9)); 4.64 – 4.48 (m, Ha(d-Phe13), Ha(Orn2), Ha(Leu3), Ha(Leu8),
Ha(Leu12)); 4.42 – 4.33 (m, Ha(Pro14)); 4.29 (t, J ¼ 7.7, Ha(Val5), Ha(Val10)); 4.20 – 4.11 (m, 1 H); 4.06 (t,
J ¼ 8.4, Ha(Val1)); 4.02 (d, J ¼ 8.4, 1 H); 3.77 – 3.62 (m, 1 H); 3.60 (s, 1 H); 3.25 – 3.05 (m, 3 H); 3.02 – 2.85
(m, 8 H); 2.59 (d, J ¼ 8.3, 1 H); 2.28 – 1.50 (m, 33 H); 1.40 (q, J ¼ 6.2, 1 H); 1.09 – 0.76 (m,
36 H).13C-NMR (151 MHz, CD3OH): 175.5; 175.1; 174.6; 174.3; 174.1; 173.9; 173.7; 173.6; 173.5;
173.4; 173.3; 172.8; 137.2; 130.5; 129.8; 128.5; 89.2; 79.6; 74.3; 68.2; 62.0; 61.1; 60.2; 59.9; 55.8; 53.9; 53.8;
53.8; 53.3; 53.2; 53.0; 52.2; 49.7; 48.0; 43.7; 42.6; 42.3; 41.8; 40.8; 40.7; 40.6; 40.0; 37.6; 32.5; 31.8; 31.7;
31.0; 30.7; 30.1; 30.0; 26.2; 26.0; 25.9; 25.3; 25.1; 24.7; 23.9; 23.4; 23.0; 22.1; 20.0; 20.0; 20.0; 19.6; 18.7;
18.6. HR-ESI-MS: 1480.7266 ([M þ H]þ, C73H126N17O1þ5 ; calc. 1480.9614).
cyclo[(3-OBn)SAA5-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,20R,22R,25S,28S,31S,40R,45aS)-6,28,34-Tris(3-aminopropyl)-40-benzyl-20-
(benzyloxy)tetratetracontahydro-9,25,37-tris(2-methylpropyl)-1,4,7,10,13,16,23,26,29,32,35,38,41-tride-
caoxo-3,15,31-tri(propan-2-yl)-1 H-19,22-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tride-
caazacyclotritetracontin-12-yl]propan-1-aminium Trifluoroacetate; 12). SAA 16b (33.2 mg, 1.2 equiv.)
was pre-activated with HCTU (1.2 equiv., 50 mg) and EtNiPr2 (3 equiv., 60 ml) in 10 ml of NMP, and
subsequently coupled to the L-O-V-DO-L resin (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (mini-cleavage:
LC/MS: tR 6.31 min, linear gradient 10 ! 90% B in 13.5 min; m/z 831.53 ([M þ H]þ)). The resin
(0.05 mmol) was subjected to steps c – g, and the Boc-deprotected peptide was purified by RP-HPLC
(linear gradient of 36 – 45%, 3 CV) to give 12 (10.2 mg, 5.03 mmol, 10%). White amorphous powder. LC/
1
MS: tR 5.64 min, linear gradient 10 ! 90% B in 13.5 min; m/z 1572.4 ([M þ H]þ). H-NMR (600 MHz,
CD3OH): 8.84 (s, NH(d-Phe13)); 8.55 (d, J ¼ 7.8, NH(Orn9)); 8.53 (d, J ¼ 8.1, NH(Orn2)); 8.48 (d, J ¼ 7.7,
NH(Orn11)); 8.46 (d, J ¼ 9.7, NH(Leu12)); 8.43 (t, J ¼ 5.7, NH(SAA)); 8.35 (d, J ¼ 8.2, NH(Leu3)); 8.20
(d, J ¼ 8.0, NH(Val5)); 8.18 (d, J ¼ 8.3, NH(Leu8)); 8.14 (d, J ¼ 7.8, NH(Orn4)); 8.09 (d, J ¼ 8.5,
NH(Val10)); 7.87 (br. s, NH2(Orn)); 7.82 (d, J ¼ 8.2, NH(Val1)); 7.52 – 7.12 (m, 10 H); 4.86 – 4.83 (m,
Ha(Orn11); 4.74 – 4.65 (m, Ha(Orn4), Ha(Orn9)); 4.64 – 4.48 (m, Ha(Orn2), Ha(Leu3), Ha(Leu8),
Ha(Leu12), Ha(d-Phe13)); 4.40 – 4.34 (m, Ha(Pro13)); 4.32 – 4.29 (m, Ha(Val5), Ha(Val10)); 4.24 – 4.17
(m, 1 H); 4.06 (t, J ¼ 8.4, Ha(Val1)); 3.99 (d, J ¼ 4.8, 1 H); 3.75 – 3.62 (m, 2 H); 3.35 (s, 3 H); 3.13 – 3.05
(m, 3 H); 2.98 – 2.95 (m, 8 H); 2.65 – 2.58 (m, 1 H); 2.45 (dd, J ¼ 12.7, 6.8, 1 H); 2.26 (dd, J ¼ 15.1, 6.8,
1 H); 2.12 (dd, J ¼ 13.4, 6.7, 1 H); 2.08 – 1.49 (m, 29 H); 1.45 – 1.33 (m, 1 H); 1.21 (s, 1 H); 1.07 – 0.75 (m,
36 H). 13C-NMR (151 MHz, CD3OH): 175.2; 175.0; 174.6; 174.2; 174.1; 173.9; 173.7; 173.5; 173.4; 173.4;
173.3; 172.8; 163.0; 162.8; 139.3; 137.2; 130.5; 129.8; 129.5; 128.9; 128.9; 128.5; 86.9; 82.0; 79.8; 72.2; 62.0;
61.1; 60.2; 59.9; 55.8; 53.9; 53.8; 53.3; 53.2; 53.1; 52.2; 50.0; 49.7; 48.0; 43.7; 42.6; 42.3; 41.8; 40.8; 40.7;
40.6; 37.6; 37.1; 32.6; 31.8; 31.7; 31.2; 31.0; 30.9; 30.7; 30.1; 30.0; 26.2; 26.0; 25.9; 25.3; 25.1; 24.7; 23.9; 23.4;

Helvetica Chimica Acta – Vol. 95 (2012)
2559
23.1; 23.0; 22.1; 22.1; 20.0; 20.0; 19.6; 18.7; 18.5. HR-ESI-MS: 1571.0091 ([M þ H]þ, C80H132N17O1þ5 ; calc.
1571.0083).
cyclo[(3-OH)SAA6-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,20R,23R,26S,29S,32S,41R,46aS)-6,29,35-Tris(3-aminopropyl)-41-benzylhexate-
tracontahydro-20-hydroxy-9,26,38-tris(2-methylpropyl)-1,4,7,10,13,16,24,27,30,33,36,39,42-tridecaoxo-
3,15,32-tri(propan-2-yl)-19,23-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaazacyclote-
tratetracontin-12-yl]propan-1-aminium Trifluoroacetate; 13). SAA 18a (62.4 mg, 1.55 equiv.) was pre-
activated with HCTU (1.45 equiv., 120 mg) and EtNiPr2 (2.9 equiv., 96 ml) in 10 ml of NMP, and
subsequently coupled to the L-O-V-DO-L resin (400 mg, 0.2 mmol, 0.5 mmol/g) for 4 h (mini-cleavage:
LC/MS: tR 5.28 min, linear gradient 10 ! 90% B in 13.5 min; m/z 755.33 ([M þ H]þ)). The resin
(0.1 mmol) was subjected to steps c – g, and the Boc-deprotected peptide was purified by RP-HPLC
(linear gradient of 31 – 40%, 3 CV) to give 13 (34.0 mg, 17.4 mmol, 17%). White amorphous powder. LC/
1
MS: tR 5.09 min, linear gradient 10 ! 90% B in 13.5 min; m/z 1496.2 ([M þ H]þ). H-NMR (600 MHz,
CD3OH): 8.86 (d, J ¼ 3.5, NH(d-Phe13)); 8.66 (d, J ¼ 8.5, NH(Orn9)); 8.52 (d, J ¼ 9.1, NH(Orn11)); 8.51
(d, J ¼ 9.4, NH(Leu12)); 8.46 (d, J ¼ 8.7, NH(Orn4)); 8.44 (d, J ¼ 8.7, NH(Leu3)); 8.43 (d, J ¼ 8.7,
NH(Orn2)); 8.38 (d, J ¼ 8.9, NH(Val10)); 8.32 – 8.23 (m, (NH(SAA)); 8.14 (d, J ¼ 8.6, NH(Val5)); 7.88
(br. s, NH2(Orn)); 7.78 (d, J ¼ 8.4, NH(Val1)); 7.67 (d, J ¼ 6.7, NH(Leu8)); 7.37 – 7.20 (m, 5 H); 5.05 – 5.03
(m, Ha(Orn4)); 5.01 – 4.98 (m, Ha(Orn9)); 4.90 – 4.88 (m, Ha(Orn2), Ha(Orn11)); 4.68 – 4.66 (m,
Ha(Leu3)); 4.58 – 4.56 (m, Ha(Leu12), Ha(d-Phe13)); 4.51 – 4.42 (m, Ha(Val5), Ha(Leu8)); 4.40 – 4.38
(m, Ha(Pro14), Ha(Val10)); 4.16 (t, J ¼ 8.1, Ha(Val1)); 3.88 – 3.80 (m, 1 H); 3.72 – 3.69 (m, 1 H); 3.65 (s,
1 H); 3.61 – 3.56 (m, 1 H); 3.51 – 3.37 (m, 1 H); 3.26 – 3.16 (m, 2 H); 3.12 – 2.82 (m, 10 H); 2.60 (dd, J ¼
16.9, 8.7, 1 H); 2.37 – 2.17 (m, 2 H); 2.17 – 1.42 (m, 33 H); 1.45 – 1.30 (m, 1 H); 1.10 – 0.71 (m, 36 H).
13C-NMR (151 MHz, CD3OH): 174.6; 174.3; 174.0; 173.8; 173.6; 173.5; 173.3; 173.2; 173.0; 172.9; 163.2;
137.2; 130.5; 129.8; 128.6; 83.9; 77.2; 68.2; 62.0; 60.7; 59.2; 55.8; 54.1; 53.9; 53.5; 53.2; 53.1; 52.8; 52.3; 49.7;
48.0; 43.8; 42.4; 42.3; 42.0; 40.7; 37.7; 33.4; 33.0; 32.6; 32.2; 32.0; 30.7; 28.9; 26.3; 26.0; 25.8; 25.1; 24.8;
24.7; 24.5; 24.2; 23.1; 23.1; 23.0; 22.3; 20.3; 20.1; 19.8; 19.4; 18.7; 18.0. HR-ESI-MS: 1494.9780 ([M þ H]þ,
C74H128N17Oþ15 ; calc. 1494.9770).
cyclo[(3-OBn)SAA6-Leu-Orn-Val-Orn-Leu-dPhe-Pro-Val-Orn-Leu-dOrn-Val] TFA Salt (¼ 3-
[(3S,6S,9S,12S,15S,19R,20R,23R,26S,29S,32S,41R,46aS)-6,29,35-Tris(3-aminopropyl)-41-benzyl-20-
(benzyloxy)hexatetracontahydro-9,26,38-tris(2-methylpropyl)-1,4,7,10,13,16,24,27,30,33,36,39,42-tride-
caoxo-3,15,32-tri(propan-2-yl)-19,23-epoxypyrrolo[2,1-r][1,4,7,10,13,16,19,22,25,28,31,34,37]tridecaaza-
cyclotetratetracontin-12-yl]propan-1-aminium Trifluoroacetate; 14). SAA 18b (58 mg, 2 equiv.) was pre-
activated with HCTU (2 equiv., 83 mg) and EtNiPr2 (4 equiv., 66 ml) in 10 ml of NMP, and subsequently
coupled to the L-O-V-DO-L resin (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h (mini-cleavage: LC/MS: tR
6.68 min, linear gradient 10 ! 90% B in 13.5 min; m/z 845.33 ([M þ H]þ)). The resin (0.1 mmol) was
subjected to steps c – g, and Boc-deprotected peptide was purified by RP-HPLC (linear gradient of 31 –
40%, 3 CV) to give 14 (53.56 mg, 26.2 mmol, 26%). White amorphous powder. LC/MS: tR 5.25 min, linear
gradient 10 ! 90% B in 13.5 min; m/z 1585.4 ([M þ H]þ). 1H-NMR (750 MHz, CD3OH): 8.87 (d, J ¼ 3.6,
NH(d-Phe13)); 8.67 (d, J ¼ 8.5, NH(Orn9)); 8.52 (d, J ¼ 7.4, NH(Orn11), NH(Leu12)); 8.45 (d, J ¼ 6.1,
NH(Orn4)); 8.44 (d, J ¼ 8.5, NH(Leu3), NH(Orn2)); 8.37 (d, J ¼ 8.0, NH(Val10)); 8.27 – 8.20 (m,
NH(SAA)); 8.16 (d, J ¼ 8.7, NH(Val5)); 7.90 (br. s, NH2(Orn)); 7.77 (d, J ¼ 8.4, NH(Val1)); 7.69 (d, J ¼
6.4, NH(Leu8)); 7.36 – 7.21 (m, 10 H); 5.06 – 5.03 (m, Ha(Orn4)); 5.00 – 4.98 (m, Ha(Orn9)); 4.91 – 4.89 (m,
Ha(Orn2), Ha(Orn11)); 4.67 – 4.65 (m, Ha(Leu3)); 4.65 (d, J ¼ 11.4, 1 H); 4.60 – 4.58 (m, Ha(Leu12), Ha(d-
Phe13)); 4.57 – 4.51 (m, Ha(d-Phe13)); 4.46 – 4.43 (m, Ha(Val5)); 4.45 (d, J ¼ 11.4, 1 H); 4.43 – 4.36 (m,
Ha(Leu8), Ha(Pro14)); 4.17 (t, J ¼ 8.2, Ha(Val1)); 3.91 – 3.86 (m, 1 H); 3.72 – 3.69 (m, 1 H); 3.65 (s, 1 H);
3.64 – 3.55 (m, 1 H); 3.44 – 3.42 (m, 1 H); 3.40 – 3.34 (m, 1 H); 3.28 – 3.26 (m, 1 H); 3.07 (dd, J ¼ 12.7, 5.4,
1 H); 3.02 (s, 1 H); 2.98 – 2.88 (m, 7 H); 2.83 (br. s, 1 H); 2.59 – 2.57 (m, 1 H); 2.43 – 2.41 (m, 1 H); 2.32 –
2.22 (m, 1 H); 2.12 – 2.09 (m, 1 H); 2.06 – 1.86 (m, 5 H); 1.82 – 1.46 (m, 25 H); 1.40 – 1.33 (m, 1 H); 1.00 –
0.88 (m, 36 H).13C-NMR (189 MHz, CD3OH): 174.6; 174.2; 174.0; 173.8; 173.6; 173.5; 173.2; 173.2;
173.0; 172.9; 163.1; 162.9; 139.7; 137.2; 130.5; 129.8; 129.4; 129.0; 128.9; 128.5; 82.1; 77.3; 75.5; 71.8; 62.0;
60.8; 59.4; 59.3; 55.8; 54.2; 53.9; 53.6; 53.3; 53.1; 52.9; 52.3; 48.0; 43.8; 42.3; 42.3; 42.0; 40.8; 37.6; 33.3;
32.6; 32.0; 30.7; 29.4; 28.5; 26.3; 26.0; 25.8; 25.1; 24.8; 24.7; 24.5; 24.1; 23.1; 23.1; 23.0; 23.0; 22.3; 20.3;
20.1; 19.8; 19.4; 18.7; 18.0. HR-ESI-MS: 1585.0253 ([M þ H]þ, C81H134N17O1þ5 ; calc. 1585.0240).

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Helvetica Chimica Acta – Vol. 95 (2012)
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Received August 28, 2012