Scaling the Amphiphilic Character and Antimicrobial Activity of Gramicidin S by Dihydroxylation or Ketal Formation

Article abstract of DOI:10.1021/acs.joc.7b02177

The acid lability of aliphatic ketals, which often serve as protection groups for 1,2-diols, is influenced by their local structural environment. The acetonide of the protected amino acid cis-dihydroxyproline (Dyp) is a typical protecting group cleavable by traces of TFA. The tricyclic acetonide of the dipeptide d-Hot-Tap is resistant to TFA and thus can serve as a bioorthogonal modification of bioactive peptides. With the aim of improving antimicrobial activity and hemolytic properties, we use these reactivity differences to scale the membrane affinity of the decapeptide Gramicidin S cyclo(d-Phe-Pro-Val-Orn-Leu-)₂ (GS). The cis-dihydroxylated amino acids are used to increase the polarity of GS or obversely decrease the polarity by stereoselective ketal formation with an aliphatic ketone. While Dyp (GS mimetic 15) has only minimal influence on the biological properties of GS, d-Hot-Tap at the position of d-Phe1-Pro2 eradicates the biological activity (GS mimetic 16). The acid-stable ketals 17-19 are bioorthogonal modifications which reconstitute the biological activity of GS. We describe an improved synthesis of orthogonally protected Fmoc-Dyp-acetonide (9) and of several Fmoc-d-Hot-Tap-ketals for solid-phase peptide synthesis.

Full text of DOI:10.1021/acs.joc.7b02177

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Article
Scaling the amphiphilic character and antimicrobial activity
of Gramicidin S by dihydroxylation or ketal formation
Christoph Priem, André Wuttke, Marina Berditsch, Anne S. Ulrich, and Armin Geyer
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02177 • Publication Date (Web): 27 Oct 2017
Downloaded from http://pubs.acs.org on October 27, 2017
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Scaling the amphiphilic character and
antimicrobial activity of Gramicidin S by
dihydroxylation or ketal formation
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Christoph Priem†, André Wuttke†, Marina Berditsch#, Anne S. Ulrich#, Armin Geyer†
†PhilippsꢀUniversität Marburg, HansꢀMeerweinꢀStraße 4, 35032 Marburg, Germany
#Karlsruhe Institute of Technology, FritzꢀHaberꢀWeg 6, 76131 Karlsruhe, Germany
Corresponding Author
*Eꢀmail: geyer@staff.uniꢀmarburg.de. Phone: (+49) 6421 2822030
Abstract: The acid lability of aliphatic ketals, which often serve as protection groups for 1,2ꢀ
diols, is influenced by their local structural environment. The acetonide of the protected
amino acid cisꢀdihydroxyproline (Dyp) is a typical protecting group cleavable by traces of
TFA. The tricyclic acetonide of the dipeptide DꢀHot=Tap is resistant to TFA and thus can
serve as a bioorthogonal modification of bioactive peptides. With the aim of improving
antimicrobial activity and hemolytic properties, we use these reactivity differences to scale the
membrane affinity of the decapeptide Gramicidin S cyclo(DꢀPheꢀProꢀValꢀOrnꢀLeuꢀ)2 (GS).
The cisꢀdihydroxylated amino acids are used to increase the polarity of GS or obversely
decrease the polarity by stereoselective ketal formation with an aliphatic ketone. While Dyp
(GS mimetic 15) has only minimal influence on the biological properties of GS,
DꢀHot=Tap at
the position of ꢀPhe1ꢀPro2 eradicates the biological activity (GS mimetic 16). The acidꢀ
D
stable ketals 17ꢀ19 are bioorthogonal modifications which reconstitute the biological activity
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of GS. We describe an improved synthesis of orthogonally protected FmocꢀDypꢀacetonide (9)
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and of several FmocꢀDꢀHot=Tapꢀketals for solidꢀphase peptide synthesis.
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Introduction: Gramicidin S (GS) is a potent antimicrobial peptide which was first isolated
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from Aneurinibacillus migulanus (formerly Bacillus brevis) in the 1940s by GAUSE and
1,2
B
RAZHNIKOVA
.
The cyclic decapeptide with the primary structure cyclo(DꢀPheꢀProꢀValꢀ
OrnꢀLeuꢀ)2 assumes a C2ꢀsymmetric antiparallel βꢀsheet with two hydrogen bonded pairs of
amino acids (Val/Leu) flanking the two central Orn residues which are not involved in
D
hydrogen bonds.3 Two ꢀPheꢀPro II´ βꢀturns cap this secondary structure on both sides.4 Its
amphiphilic character with the aliphatic Val und Leu sidechains oriented to one side and the
cationic Orn sidechains to the opposite direction forms the basis for the antimicrobial activity
of GS towards a wide range of Gramꢀpositive and Gramꢀnegative bacteria by interacting with
the lipid bilayer, lowering its integrity, ultimately causing cell death.5ꢀ8 Bacterial resistance
develops slower against antibiotics targeting the cell membrane than against other modes of
action but the unwanted hemolytic activity restricts the use of GS beyond topical
applications.9,10 In spite of the many efforts and numerous GS analogs which were
synthesized in the past 40 years, it was not possible to separate and suppress this unspecific
membrane activity in a structureꢀbased approach. Several investigations report ring
contractions, ring expansions, mutations in the βꢀsheet and in the βꢀturn region, natural or
nonꢀnatural amino acids and their influence on the antimicrobial and hemolytic activity of
GS.10ꢀ20 Here, we describe a novel approach to the problem of unspecific membrane affinity
of GS: We systematically scale its membrane affinity by linking the peptide with different
types of membrane anchors in a modular way. We introduce Dyp at the position of Pro as
smallest possible dihydroxylation of GS. Dyp only minimally alters the bioactivity of GS and
its cisꢀdiol group is a useful bioorthogonal linker for the subsequent ligation to boronic esters
or benzoboroxoles in aqueous solution by dynamic covalent chemistry.21 A further mutation
of GS described here is the dihydroxylated dipeptide
DꢀHot=Tap
(Dꢀ
Hydroxythreonine=Thiaproline with the “=” representing the two covalent ring connections)
which allows the attachment of a number of different functionalities because it can form an
exceptionally stable ketal. This manuscript focuses on alkyl chains of varying length to keep
the structure activity relationship as simple as possible. The unique properties of the bicyclic
βꢀturn mimic
D
ꢀHot=Tap were described previously by our group.22 The
ꢀPhe residue and is, in contrast to Dyp, able to
D,Lꢀdipeptide has its
diol function at the former position of the
D
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form ketals that are remarkably stable under physiological conditions. This offers a novel way
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of introducing functionality to DꢀHot=Tap modified GS derivatives used as templates (Figure
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1) and gives access to several unique GS analogs modified with alkyl ketones. They show
different biological profiles, encompassing antimicrobial and hemolytic activity, by simple
manipulation of polarity and amphiphilic character.
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Figure 1. Two different types of dihydroxylation are shown here. Cisꢀdihydroxylation of
dehydroproline (Dhp) yields dihydroxyproline (Dyp). The bicyclic
several steps from the pentose ꢀribose. Hydroxyproline (Hyp) and the dipeptide
are shown as monohydroxylated structural analogs of Dyp and ꢀHot=Tap, respectively.
DꢀHot=Tap is obtained in
D
DꢀThrꢀPro
D
Results and Discussion: The synthesis of FmocꢀDypꢀacetonide was performed according to
literature procedures and optimized as shown in Scheme 1.23ꢀ25 Commercially available Hyp
was protected and activated in four steps to 4, followed by regioselective elimination to the
protected Dhp derivative 5.
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Scheme 1. Dyp-building block synthesisa
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a Reagents and conditions: (i) CbzCl, 1
N NaOH/THF 1:1, 16 h, RT; (ii) BnBr, K2CO3, NaI,
DMF, 16 h, RT; (iii) MsCl, NEt3, DMAP, DCM, 2 h, RT; (iv) Ph2Se2, NaBH4, tertꢀbutanol,
3 h, 80 °C; (v) H2O2, pyridine, DCM, 5 h, RT; (vi) K2OsO4•2H2O, NMO•H2O, tertꢀ
butanol/H2O 7:3, 21 h, RT; (vii) PTSA, 2,2ꢀdimethoxypropane, 4 h, RT; (viii) H2 (10 bar),
Pd/C, EtOAc, RT; (ix) FmocCl, 10% Na2CO3aq/1,4ꢀdioxane 1:1, 16 h, RT.
Diastereoselective dihydroxylation and reprotection yielded FmocꢀDypꢀacetonide (9) in an
overall yield of 30% over nine steps which could be directly used in solidꢀphase peptide
synthesis as described below.
Ketals are expected to easily hydrolyze under acidic conditions, but the tricyclic ring system
of the O7,O8ꢀacetonideꢀprotected N3ꢀ
D
ꢀHot=TapꢀOMe 10a is surprisingly stable against
acids, necessitating an alternative protecting group for the synthesis of oligomeric
DꢀHot=Tap
peptides.22 In the present approach, this acid stability was made a virtue of necessity to install
alkyl chains of different lengths and topology as shown in Scheme 2. The dipeptide precursor
10a was first deprotected by recurrent treatment with pure TFA to obtain diol 11 which was
reketalized with 2ꢀdecanone, 2ꢀpentadecanone, and 8ꢀpentadecanone to obtain the ketals 10b-
d, respectively. Surprisingly, the formations of ketals 10b and 10c yielded a single
diastereomer. These three azides were transformed to Fmocꢀprotected dipeptide building
blocks 14b-d, suitable for SPPS, in a sequence of reduction, saponification and acylation with
FmocCl. The dimethylketal 10a was transformed into the Fmocꢀprotected dipeptide 14a using
the same synthetic transformations.
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Scheme 2. D
-Hot=Tap-building block synthesisa
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aReagents and conditions; (i) Me3SnOH, DCE, 80 °C; (ii) H2 (1 bar), Pd/C, MeOH, RT; (iii)
FmocꢀOSu, DIPEA, 1,4ꢀdioxane/H2O 4:1; (iv) 95% TFAaq; (v) dimethylketal, CSA.
The configuration of the quaternary carbon of the asymmetric ketals 10b and 10c was
determined by NMR spectroscopy. The crystal structure of 10a was used as a template for the
configurational assignment (Figure 2). The proR methyl group of 10a is close to the ring
protons H7 and H8 of the valerolactam, while the proS methyl group is closer to H3 on the
other ring face. ROE (rotating frame NOE) contacts differentiate the diastereotopic methyl
groups in solution. The lowꢀfield methyl shows ROE contacts to H7 and H8 while the methyl
group at higher field shows a ROE contact to H3. The rigid 5,6,5ꢀtricyclic ring system of all
DꢀHot=Tap ketals shows this ROE pattern. The stereocenter at the ketals 10b and 10c is Sꢀ
configurated in both cases.
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Figure 2: Determination of the configuration at the ketal stereocentre of 10b and 10c.
(A) Crystal structure of 10a. The distances important for the NMRꢀbased analysis of
asymmetric ketals are highlighted. (B) Stereoconfiguration and atom numbering of 10b and
10c. (C) Expansion from the ROESY spectrum (500 MHz, 300 K, CDCl3) of 10b (upper
right) and 10c (lower right). The methyl group shows ROE contacts to H7 and H8 while the
methylene groups are close to H3.
The synthesis of the linear precursors of the GS analogs was performed by standard peptide
chemistry on 2ꢀCTCꢀresin with Fmocꢀprotected building blocks. The resin was treated with
1% TFA in DCM to cleave the peptide from the resin without fragmentation of the Bocꢀ
protecting group on the side chain of Orn. The crude peptide was cyclized using HATU/HOAt
and DIPEA in DMF under high dilution conditions to favor the intramolecular condensation.
The Boc protecting group on the side chain of Orn was removed with TFA/DCM (1:6) and
the final purification was done by semiꢀpreparative RPꢀHPLC to obtain the GS analogs 15-19
(Figure 3).
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NH2
D-Phe1
D-Phe1
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O
O
H
H
H
N
N
N
N
N
O
O
N
H
H
O
O
N
GS
15
N
9
O
O
O
H
H
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N
N
N
N
OH
H
H
O
O
O
OH
Pro2
Dyp2
H2N
D-Hot1
R1
H
O
R2
N
16 R1 = R2 = CH3
O
17 R1 = CH3, R2 = C8H17
18 R1 = CH3, R2 = C13H27
19 R1 = R2 = C7H15
O
16-19
N
S
O
Tap2
Figure 3: The parent peptide Gramicidin S (GS) and the five analogs containing the
dihydroxylated amino acid Dyp (15) or the turn mimetic ꢀHot=Tap (16-19).
D
The complete homoꢀ and heteronuclear assignment of 15 was achieved by TOCSY, HSQC,
1
HMBC, and ROESY spectra (600 MHz, MeOHꢀd3, 300K). The H NMR spectrum of 15
1
shows a decapeptide signal set in the H NMR spectrum caused by the loss of C2ꢀsymmetry.
The NH resonances and all other signals of the two ValꢀOrnꢀLeuꢀDꢀPhe tetrapeptide
sequences appear as pairs of signals with only minor chemical shift differences. The close to
identical coupling constants and NOE pattern of both molecular halves yield twice as much
spectroscopic information for 15 compared to the parent peptide GS and confirmed that the
dihydroxylation has a negligible influence on the overall structure. Because the NH
3
temperature coefficients and the JNHꢀHα coupling constants of the two pentapeptide moieties
of 15 were unmeasurably different in the 1H NMR, we discuss only one value per two amino
acids in the following. The temperature coefficients of the NH protons of Val, Orn, Leu and
ꢀPhe were ꢀ3.1, ꢀ7.5, ꢀ3.1 and ꢀ8.9 ppb Kꢀ1. The small temperature dependence of Val and
D
Leu confirm the expected intramolecular hydrogenꢀbonded pair Leu and Val as described for
GS.16 The JNHꢀ CH
α
coupling constants for Val, Orn Leu and
D
ꢀPhe were 9.2, 9.5, 9.5 and
3
3.6 Hz, respectively. The large values occurring for the extended βꢀsheet structure and the
small values occurring for ꢀPhe at the i+1 position of the βꢀturn. Comparison with
D
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temperature coefficients and coupling constants of wild type GS indicated a similar structure.
Only the puckering of the pyrrolidine ring of Dyp differs to natural GS. In contrast to the
pyrrolidine ring of Pro, which prefers the Cγꢀendoꢀpucker, the Cγꢀexoꢀpucker is favored by
Hydroxyꢀ and Dihydroxyproline in the single amino acid or dipeptides.26 Interestingly,
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conformational analysis of the Dyp in 15 showed a Cβꢀexoꢀpucker. The coupling constant
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3
3J
α
β
of < 1 Hz indicates a dihedral angle of about 90°. J
γ
δ
of 7.9 and 8.3 Hz
CHꢀ CH
CHꢀ CH
suggests a dihedral angle of approximately 30° and 150° between the γꢀ and the diastereotopic
δꢀprotons. This leads to Cβꢀexoꢀpucker as single possible ring conformation (Figure 4).
Figure 4. (A) The preferred rotamer along αCꢀβC bond explains the small coupling constant
of < 1 Hz. (B) Rotamer along γCꢀδC axis, with coupling constant of 7.9 and 8.3 Hz. (C)
Proposed Cβꢀexo ring conformation of the pyrrolidine ring of Dyp.
In contrast to 15, conformational analysis of the DꢀHot=Tap modified GS analog 16 indicated
greater influence of substituting the βꢀturn on the overall structure. The coupling constants of
Val (10.0 and 7.9 Hz), Orn (9.0 and 9.6 Hz) and Leu (7.5 and 8.2 Hz) differ slightly from wild
type GS, but indicate a similar extended βꢀsheet structure. Nevertheless, temperature
coefficients partly differ significantly from wild type GS. The biggest difference showed
Val3, Orn9 and Leu10 with values of ꢀ8.5, ꢀ2.0 and ꢀ7.0 ppb Kꢀ1, in comparison to wild type
GS with values of ꢀ2.5, ꢀ7.3 and ꢀ2.9 ppb Kꢀ1. This is caused by a higher rigidity of the
bicyclic ring system of the DꢀHot=Tap building block in 16, which results in a different twist
of the βꢀsheet and a consequential change of solvent exposure of the amino acids as
consequence (Figure 5).
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Figure 5: Structural relevant data from GS analog 16 compared to wild type GS. (A) JNHꢀαCH
coupling constants of βꢀsheet amide protons of 16 and wild type GS and the corresponding
1
expansion from H NMR spectrum (600 MHz, 300 K, MeOHꢀd3) of 16. (B) Temperature
dependence of amide protons in 16 and wild type GS as well as the corresponding expansion
from the 1H NMR spectrum of 16 in the temperature range between 280 and 310K.
CD studies of compound 15-19 were carried out in methanol from 195 nm to 260 nm to
investigate the relationship between structure and activity. As we expected, CD spectra of
Dyp modified GS analog 15 showed an identical behavior to wild type GS. CD spectra of
compound 16-19 showed, beside significantly lower intensities, a trough around 214 nm,
similar to wild type GS. Nevertheless, the minimum at 206 nm is not as distinct in the spectra
of 16-19. It was reported, that the origin of the through at 206 nm was the II´ βꢀturn and βꢀ
sheet structure of GS.27,28 Therefore, these findings can be explained with the substitution of
the whole βꢀturn with the D
ꢀHot=Tap building block in 16-19.29 This is consistent with the
NMR data and our proposed twisted βꢀsheet structure of compounds 16-19 (Figure 6).
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Figure 6: CD spectra of GS analogs 15 (red), 16 (green), 17 (blue), 18 (yellow), 19 (purple)
and wild type GS (black) in methanol from 195 nm to 260 nm at 27°C.
All peptides were tested against several Gramꢀpositive and ꢀnegative bacteria (minimum
inhibitory concentration) and investigated on their hemolytic activity (HC90) as described
earlier.8,30 The antimicrobial activity of the Dyp modified GS analog 15 is slightly lower.
Nevertheless the selectivity index (HC90/MIC) is higher for most bacteria tested. This may be
explained by the increased polarity of the two additional hydroxy groups and the altered
amphiphilic character of the GS derivative, both properties essential for the antimicrobial
activity.5 Interestingly, the dimethylketal modified GS analog 16 was inactive and has no
hemolytic activity. Enhanced polarity and decreased amphiphilic character, as well as polar
residues at the
D
ꢀPhe position are supposed to cause these findings.31,32 GS analog 18
modified with a C13ꢀalkyl chain, as well as analog 19 modified with two C7ꢀalkylꢀchains
restored the activity and showed a drastic increase of hemolytic activity and a lower activity
against gramꢀpositive bacteria tested, as well as no activity against the Gramꢀnegative bacteria
E. coli. For 18, this can be explained with the length of the C13 alkyl chain, which enhanced
the hemolytic activity, lowering the selectivity index. The two shortened alkyl chains in 19
have almost the same effect caused by the orientation of one alkyl chain to the polar residues,
lowering the amphiphilic character. Nevertheless, GS analog 17, modified with a C8ꢀalkyl
chain, showed comparable activities against all Gramꢀpositive bacteria, as well as slightly
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lowered activity against all Gramꢀnegative bacteria. Even hemolytic activity was significantly
lowered yielding an improved selectivity index compared to GS. The antimicrobial and
hemolytic activity of all compounds is summarized in Table 1.
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Table 1: Minimum inhibitory concentration [µg/mL] with parenthesized selectivity index
(HC90/MIC) against several bacteria and concentration causing 90% hemolysis (HC90, µg/mL)
of GS and analogs 15-19.
Aa
32 (0.38)
64 (1.39)
Ba
64 (0.19)
128 (0.66) 4 (21.25)
Cb
4 (3.00)
Db
2 (6.00)
16 (5.31)
Eb
8 (1.50)
32 (2.66)
HC90
12
85
GS
15
16
17
18
19
>256 (0.78) >256 (0.78) 256 (0.78) 128 (1.56) >256 (0.78) >200
64 (0.59)
>256 (0.02)
>256 (0.02)
128 (0.30)
64 (0.09) 16 (0.38)
128 (0.04) 8 (0.63)
4 (9.50)
4 (9.50)
8 (0.75)
8 (0.63)
8 (4.75)
16 (0.38)
8 (0.63)
38
6
5
a Gramꢀnegative bacteria. b Gramꢀpositive bacteria. A: E. coli, B: P. aeruginosa, C: S. aureus,
D: S. epidermidis, E: E. faecalis.
Conclusion: The synthesis of precursors of dihydroxylated amino acids and their
incorporation in the macrocyclic peptide GS are presented here. The dipeptide DꢀHot=Tap
offered the opportunity to incorporate one or two aliphatic chains by the formation of a ketal
of extraordinary stability against acidolysis. We observed that the dihydroxylation leads to an
enhanced biological profile of GS derivative 15 with slightly decreased antimicrobial activity
and concurrent significant decrease of hemolytic activity, yielding an increasedselectivity
index. We described a straightforward way to manipulate the polarity and amphiphilic
character of GS analogs by formation of different stable ketals via DꢀHot=Tap modification,
restoring as well as enhancing the selectivity index of those GS analogs. Dihydroxylation of
GS created the opportunity to synthesize different analogs with increased biological profiles
by ketalisation with various aliphatic ketones of different alkyl chain lengths and topology.
Experimental Section:
General information. Materials and reagents were of commercially available grade and used
without additional purification. Compound 10a was synthesized according to our former
publication.22 Solvents were used after distillation. Column chromatography was performed
by using Merck silica gel (0.040ꢀ0.063 mm 230ꢀ400 mesh) TLC was performed on Merck
silica gel 60 F254 aluminum plates with visualization with UV, ninhydrin or permanganate. 1H,
13C, TOCSY, COSY, HSQC, HMBC and ROESY spectra were recorded on Bruker AV II
300, Bruker DRXꢀ500, Bruker AV III 500 or Bruker AV II 600. Chemical shifts are reported
in ppm using the signal of solvent (DMSOꢀd6: 1H: 2.50 ppm, 13C: 39.52 ppm; CDCl3: 1H: 7.26
1
ppm, 13C: 77.16; MeOHꢀd3: H: 3.31 ppm, 13C: 49.00 ppm) as reference. Diastereotopic
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groups are differentiated by l (low field) and h (high field) signal. HSQC spectra were
recorded from 0 to 100 ppm in the C dimension. Mass spectra (ESI+) were acquired on a
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Thermo Fisher Scientific LTQꢀFT with an ion trap detector. Analytical HPLC was performed
on a Thermo Scientific Dionex UltiMate 3000 system with a ACE UltraCore 2.5 SuperC18
column (150 x 2.1 mm, 2.5 µm, 95 Ǻ) with an diode array detector. As eluent with a flow rate
of 0.45 mL/min were used the following solvents: H2O + 0.1% TFA, B: ACN + 0.085% TFA.
Semi preparative HPLC was performed on a Thermo Scientific Dionex UltiMate 3000 with a
MachereyꢀNagel VP Nucleodur C18 Gravity column with a flow rate of 15.0 mL/min or an
ACE 5 SuperC18 column with a flow rate of 7.0 mL/min. CD spectra were acquired on a
JASCO Jꢀ810 with an HELMA 110ꢀQS quartz cuvette (path length 0.1 cm), a scanning rate of
10 nm/min, data pitch of 0.5 response of 2 s and a temperature of 300K
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Resin loading: 2ꢀChlortritylchlorid resin (1.42 mmol/g, 200ꢀ400 mesh) was loaded by adding
FmocꢀLeuꢀOH (1.0 eq) and DIPEA (6.0 eq) in DMF and stirring for 1 h. After washing three
times with DMF, the resin was treated twice with DCM/MeOH/DIPEA (80:15:5) for
respectively 30 min. The resin was washed three times with DMF, MeOH and DCM. The
loading was determined by UVꢀVis spectroscopy at 289 and 300 nm after cleavage of
protecting group with 20% piperidine in DMF for 20 min using the following equation.
mmol
g
E ∙ V[mL]
B ꢀ
ꢁ =
mL
mmol ∙ cm
ε ꢂ
ꢃ ∙ d[cm] ∙ mꢄꢅꢆꢇꢈ[g]
B = Resin loading, E = Extinction, V = Total volume, ε = extinctioncoefficient, d = layer thickness, mresin = resin mass.
Manual peptide synthesis: The manual peptide synthesis was performed using the following
protocol.
1)
2)
3)
4)
Stirring in DMF for 20 min
2 x cleavage of the Fmoc protection group with 25% piperidine in DMF for 10 min
3 x washing with DMF, MeOH and DCM
Coupling with FmocꢀAAꢀOH (3.0 eq), HOBt (3.0 eq), HBTU (3.0 eq) and DIPEA
(8.0 eq) in DMF for 60 min
5)
3 x washing with DMF, MeOH and DCM
Steps 2ꢀ5 were performed as long as the peptide were fully assembled
Automated peptide synthesis: Peptides were synthesized by a microwave assisted peptide
synthesizer (Liberty Blue, CEM) using the following cycles of deprotection and coupling.
1)
Fmoc deprotection: T = 50 °C, Pmicrowave = 30 W, t = 210 s with 20% piperidine in
DMF, 3.00 mL/deprotection
2)
AA coupling: T = 50 °C, Pmicrowave = 30 W, t = 600 s with FmocꢀAAꢀOH (0.2
DMF, 5.0 eq, 2.5 mL/coupling), DIC (0.5 in DMF, 5.00 eq, 1.0 mL/coupling) and
Oxyma (1.0 in DMF, 5.0 eq, 0.5 mL/coupling)
M in
M
M
Resin cleavage: Resin cleavage was performed with 1% TFA in DCM to keep the sidechain
protecting groups intact. Coevaporation with toluene and lyophilisation from H2O/MeCN
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(4:1) afforded the sidechain protected, linear peptides as white solid and was used without
further purification.
Peptide cyclisation: To a solution of HATU (3.0 eq) and HOAt (3.0 eq) in DMF (90 mL)
was added DIPEA (15.0 eq). After cooling to 0 °C the linear peptide dissolved in DMF (10
mL) was added dropwise over 1 h. After stirring overnight at RT, the solvent was removed
under reduced pressure.
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Final deprotection: The cyclized peptide was in dissolved in DCM (6 mL) and TFA (1 mL)
was added. After stirring 1 h at RT, the volume was halved by removing the solvent under
reduced pressure. Toluene (10 mL) was added and the solvent removed under reduced
pressure. Purification by semiꢀpreparative RPꢀHPLC and lyophilisation gave the GS analogs
as white solid (Purity >95% in all cases).
Biological Studies: Minimum inhibitory concentration was tested by double dilution of
studied peptides in the MuellerꢀHinton broth as described earlier.8 Exponentially growing
control strains of Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228 and
Enterococcus faecalis ATCC 29212 were used for inoculation of the 96ꢀwell microtiter plates
to reach 5x105 colony forming units/mL. After incubation of plates for 22 h at 37°C bacterial
growth was examined as respiration activity, when the redox indicator resazurin (20 µL of an
aqueous 80 µM solution per well) was added and incubated for another 2 h at 37 °C. Positive
values of the differences in the absorbance of resorufin at 570 nm and resazurin at 600 nm
indicated bacterial growth and allowed the determination of the lowest peptide concentrations
that inhibit bacterial growth. All results were obtained from two independent experiments,
and each was performed in triplicate.
The hemolytic activity of all peptides was investigated using human erythrocytes as described
earlier.30 Human red blood cells in citrateꢀphosphateꢀdextrose buffer was obtained from the
blood transfusion department of the municipal hospital in Karlsruhe, washed twice in isotonic
TrisꢀHCl washꢀbuffer (pH 7.6, RT), and then diluted to 0.5 % of haematocrit in TrisꢀHCl
reactionꢀbuffer (pH 7.6, 37 °C). Studied peptides were diluted 1:1 in TrisꢀHCl reactionꢀbuffer
to an end volume of 200 ꢁL. Then the 200 ꢁL of erythrocyte suspension were added to start
the haemolytic reaction (37 °C, paused agitation, 30 minutes). After centrifugation, the
haemoglobin concentration in supernatant was characterized in a spectrophotometer at 540
nm. The haemolytic concentrations (HC90) were calculated as the percentage haemolysis
compared to 100% induced by 0.1% Triton Xꢀ100 in two independent experiments.
(2S,4R)-1-((Benzyloxy)carbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (1)33
Hydroxyproline (6.56 g, 50.0 mmol, 1.0 eq) was dissolved in 1 n NaOH (50 mL) and THF
(50 mL). Then a solution of CbzꢀCl (8.92 mL, 62.5 mmol, 1.25 eq) in 1 n NaOH (50 mL) and
THF (50 mL) was added dropwise at 0 °C about 1.5 h). The mixture was stirred overnight at
RT. After dilution with H2O (50 mL), the solution was washed with EtOAc (3 x 125 mL). 2 n
HCl was added to the aqueous layer until pH = 3. The aqueous layer was extracted with
EtOAc (4 x 125 mL). The combined organic layers were washed with brine (100 mL), dried
over MgSO4. The solvent was removed under reduced pressure 1 was obtained as white foam
(12.2 g, 46.0 mmol, 92%).1H NMR (300 MHz, 300 K, DMSOꢀd6): δ 7.37ꢀ7.32 (m, 5H, Harom),
5.13ꢀ5.04 (m, 3H, OH, PhCH2O), 4.31ꢀ4.18 (m, 2H, Hα, Hγ), 3.50ꢀ3.36 (m, 2H, Hδ), 2.23ꢀ
13
2.11 (m, 1H, Hβ), 2.00ꢀ1.87 (m, 1H, Hβ); C NMR (75 MHz, 300 K, DMSOꢀd6): δ 173.9,
173.5 (CO2H), 154.2, 153.9 (NCO2), 136.8, 136.8, 128.4, 128.2, 127.8, 127.6, 127.4, 127.0
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(Carom.), 68.5, 67.7 (Cγ), 66.0 (PhCH2Oꢀ), 59.0, 57.4 (Cα), 55.0, 54.5 (Cδ), 37.9 (Cβ). HRMS
(ESIꢀICR) m/z: [M + Na]+ Calcd for C13H15NO5Na 288.0842; Found 288.0844.
Dibenzyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (2)34
7
1 (12.2 g, 46.0 mmol, 1.0 eq) was dissolved in DMF (90 mL) and K2CO3 (12.7 g, 92.0 mmol,
2.0 eq), NaI (689 mg, 4.60 mmol, 0.1 eq) and BnBr (16.4 mL, 138 mmol, 3.0 eq) were added.
The solution was stirred at RT for 16 h. After dilution with H2O (250 mL), the aqueous layer
was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with H2O
(2 x 100 mL), brine (100 mL), dried over MgSO4, filtered and the solvent removed under
reduced pressure. The crude product was purified by column chromatography over silica gel
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1
(DCMꢀDCM/MeOH 20:1). 2 was obtained as pale yellow oil (12.8 g, 36.0 mmol, 78%). H
NMR (500 MHz, 300 K, DMSOꢀd6): δ 7.37ꢀ7.24 (m, 10H, Harom), 5.18ꢀ4.99 (m, 5H, OH,
PhCH2Oꢀ), 4.41 (t, 3J = 8.0 Hz, 1H, Hαc/t), 4.37 (t, 3J = 8.0 Hz, 1H, Hαc/t), 4.29 (bs, 1H, Hγ),
3.53ꢀ3.46 (m, 1H, Hδ), 3.42ꢀ3.37 (m, 1H, Hδ), 2.25ꢀ2.14 (m, 1H, Hβ), 2.02ꢀ1.90 (m, 1H, Hβ);
13C NMR (125 MHz, 300 K, DMSOꢀd6): δ 172.3, 171.9 (CO2H), 154.3, 153.7 (NCO2), 136.7,
136.6, 135.9, 135.7 (Carom), 128.4, 128.3, 128.0, 128.0, 127.8, 127.8, 127.7, 127.6, 127.5,
127.3 (Carom), 68.5, 67.7 (Cγ), 66.1, 66.0, 65.9 (PhCH2Oꢀ), 58.0, 57.5 (Cα), 55.0, 54.5 (Cδ),
37.8 (Cβ). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for C20H21NO5Na 378.1312; found
378.1313.
Dibenzyl (2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (3)
2 (12.4 g, 35.0 mmol, 1.0 eq) was dissolved in DCM (130 mL). After cooling to 0 °C, DMAP
(1.28 g, 10.5 mmol, 0.3 eq), NEt3 (6.70 mL, 48.8 mmol, 1.4 eq) and MsCl (3.78 mL,
48.8 mmol, 1.4 eq) were added. The solution was stirred at RT for 2 h. H2O (25 mL) was
added and the layers separated. The aqueous layer was extracted with DCM (3 x 30 mL). The
combined organic layers were washed with brine (50 mL), dried over MgSO4, filtered oder
the solvent removed under reduced pressure. 3 was obtained as yellow, viscous oil (15.5 g,
31.4 mmol, 90%). 1H NMR (500 MHz, 300 K, DMSOꢀd6): δ 7.39ꢀ 7.26 (m, 10 H, Harom), 5.30
3
3
(bs, 1H, Hγ), 5.20ꢀ5.00 PhCH2Oꢀ), 4.50 (t, J = 8.07 Hz,1H, Hαcis/trans), 4.45 (t, J = 8.07 Hz,
1H, Hαcis/trans), 3.79ꢀ 3.75 (m, 1H, Hδ), 3.73ꢀ 3.68 (m, 1H, Hδ), 3.25 (s, 3H, CH3), 2.66ꢀ2.58
(m, 1H, Hβ), 2.35ꢀ2.25 (m, 1H, Hβ). 13C NMR (125 MHz, 300 K, DMSOꢀd6): δ 171.4, 171.0
(CHCO2), 153.8, 153.4 (NHCO2), 136.4, 136.2, 135.7, 135.5 (Carom.), 128.4, 128.3, 128.1,
128.0, 127.9, 127.8, 127.7, 127.5, 127.4 (Carom.), 79.2, 78.6 (Cγ), 66.5, 66.5, 66.3, 66.2
(PhCH2Oꢀ), 57.3, 56.9 (Cα), 52.8, 52.4 (Cδ), 37.7 (ꢀSCH3), 36.6, 35.6 (Cβ). HRMS (ESIꢀ
ICR) m/z: [M + Na]+: Calcd for C21H23NO7SNa 456.1087; Ffound 456.1087.
Dibenzyl (2S,4S)-4-(phenylselanyl)pyrrolidine-1,2-dicarboxylate (4)24
Ph2Se2 (6.55 g, 21.0 mmol, 0.6 eq) was dissolved in tertꢀbutanol (100 mL). NaBH4 (1.63 g,
43.0 mmol, 1.2 eq) was added in several portions. The mixture was refluxed until the yellow
solution became colorless. Then, 3 (15.2 g, 35.0 mmol, 1.0 eq) in tertꢀbutanol (120 mL) was
added and the solution was refluxed for 3 h. EtOAc (400 mL) was added and the organic layer
was washed with H2O (3 x 500 mL). The organic layer was washed with brine (200 mL),
dried over MgSO4, dried and the solvent removed under reduced pressure. The crude product
was purified by column chromatography over silica gel (DCMꢀDCM/MeOH 100:1). 4 was
1
obtained as pale yellow oil (13.7 g, 27.7 mmol, 79%). H NMR (500 MHz, 300 K, DMSOꢀ
3
d6): δ 7.53ꢀ7.24 (m, 15H, Harom), 5.20ꢀ4.99 (m, 6H, PhCH2O), 4.47 (t, J = 7.38 Hz, 1H,
3
Hαc/t), 4.41 (t, J = 7.38 Hz, 1H, Hαc/t), 4.00ꢀ3.85 (m, 2H, Hγ, Hδ), 3.36ꢀ3.29 (m, 1H, Hδ),
13
2.82ꢀ2.73 (m, 1H, Hβ), 2.00ꢀ1.90 (m, 1H, Hβ). C NMR (125 MHz, 300 K, DMSOꢀd6): δ
171.5, 171.1 (CHCO2), 153.6, 153.2 (NCO2), 136.6, 136.4, 135.7, 135.5, 133.6, 129.3, 128.4,
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128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3 (Carom.), 66.3, 66.2, 66.2, 66.0
(PhCH2Oꢀ), 58.3, 58.8 (Cα), 52.6, 53.2 (Cδ), 36.3, 37.3 (Cβ), 36.5, 35.2 (Cγ), HRMS (ESIꢀ
ICR) m/z: [M + Na]+ Calcd for C26H25NO4SeNa 518.0843; Found 518.0839.
Dibenzyl (S)-2,5-dihydro-1H-pyrrole-1,2-dicarboxylate (5)24
9
4 (13.7 g, 27.7 mmol, 1.0 eq) was dissolved in DCM (210 mL). Pyridine (3.13 mL,
38.8 mmol, 1.4 eq) was added and the solution cooled to 0 °C. After addition of H2O2
(50 wt% in H2O, 3.93 mL, 69.3 mmol, 2.5 eq) the mixture was stirred at RT for 5 h. The
solvent was removed under reduced pressure und the residue resolved in EtOAc (100 mL).
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The organic layer was washed with 0.1
N HCl (50 mL), sat. NaHCO3 (50 mL) and brine
(50 mL), dried over MgSO4, filtered and the solvent removed under reduced pressure. The
crude product was purified by column chromatography over silica gel (DCMꢀDCM/MeOH
1
100:1) 5was obtained as colorless oil (7.70 g, 22.8 mmol, 82%). H NMR (500 MHz, 300 K,
DMSOꢀd6): δ 7.39ꢀ7.26 (m, 10 H, Harom), 6.14ꢀ6.09 (m, 1H, Hβ), 5.89ꢀ5.84 (m, 1H, Hγ), 5.16ꢀ
5.03 (m, 5H, Hα, PhCH2O), 4.24ꢀ4.18 (m, 2H, Hδ). 13C NMR (125 MHz, 300 K, DMSOꢀd6):
δ 169.7, 169.4 (CHCO2), 153.5, 153.1 (NCO2), 136.7, 136.5, 135.7, 135.6 (Carom.), 129.8,
129.4 (Cβ), 128.4, 128.3, 128.1, 128.0, 127.8, 127.7, 127.7, 127.6, 127.4, 127.3 (Carom.),
124.6, 124.3 (Cγ), 66.4, 66.2 (PhCH2Oꢀ), 66.2, 66.2 (Cα), 66.1, 65.9 (PHCH2Oꢀ), 53.8, 53.28
(Cδ). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for C20H19NO4Na 360.1206; Found 360.1204.
Dibenzyl (2S,3R,4S)-3,4-dihydroxypyrrolidine-1,2-dicarboxylate (6)25
5 (2.00 g, 5.93 mmol, 1.0 eq) was dissolved in tertꢀbutanol (70 mL) and H2O (30 mL). Then
NMO•H2O (800 mg, 5.93 mmol, 1.0 eq) and K2OsO4•2H2O (44.0 mg, 120 µmol, 2 mol%)
was added, the mixture was stirred at RT for 21 h. 5% Na2SO3aq (50 mL) was added and the
solution stirred for additional 30 min. The aqueous layer was extracted with EtOAc
(3 x 100 mL). The combined organic layers were washed with 0.1
N HCl (100 mL) and brine
(100 mL), dried over MgSO4, filtered and the solvent removed under reduced pressure. The
crude product was purified by column chromatography over silica gel (DCM/MeOH 20:1) 6
1
was obtained as pale yellow, viscous oil (1.56g, 4.23 mmol, 71%). H NMR (500 MHz, 300
K, DMSOꢀd6): δ 7.29ꢀ7.23 (m, 10H, Harom), 5.56 (d, 3J = 5.9 Hz, 1H, OH), 5.17ꢀ4.98 (m, 5H,
OH, PhCH2Oꢀ), 4.09ꢀ4.01 (m, 3H, Hα,Hβ,Hγ), 3.56ꢀ3.50 (m, 1H, Hδ), 3.34ꢀ3.32 (m, 1H, Hδ).
13C NMR (125 MHz, 300 K, DMSOꢀd6): δ 171.2, 170.7 (CHCO2), 154.2, 153.7 (NCO2),
136.7, 136.5, 135.9, 135.7, 128.4, 128.3, 127.8, 127.6, 127.5, 127.3 (Carom.), 75.5, 74.6 (Cγ),
69.9, 69.2 (Cβ), 66.2, 66.1, 66.1, 66.0 (PhCH2Oꢀ), 64.6, 64.1 (Cα), 51.2, 50.8 (Cδ). HRMS
(ESIꢀICR) m/z: [M + Na]+ Calcd for C20H21NO6Na 394.1261; Found 394.1259.
Dibenzyl
(3aR,4S,6aS)-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-4,5-
dicarboxylate (7)
6 (3.80 g, 10.2 mmol, 1.0 eq) was dissolved in 2,2ꢀdimethoxypropane (50 mL). After addition
of PTSA (350 mg, 2.05 mmol, 0.2 eq) the mixture was stirred for 4 h at RT. Sat. NaHCO3
(50 mL) was added and the mixture stirred for additional 15 min. The aqueous layer was
extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine
(50 mL), dried over MgSO4, filtered and the solvent removed under reduced pressure. 7 was
1
obtained as pale yellow oil (4.10 g, 9.96 mmol, 97%). H NMR (500 MHz, 300 K, DMSOꢀ
d6): δ 7.39ꢀ7.24 (m, 10H, Harom), 5.18ꢀ5.04 (m, 4H, PhCH2O), 4.88ꢀ4.79 (m, 2H, Hβ,Hγ), 4.42
(s, 1H, Hα), 3.76ꢀ3.51 (m, 2H, Hδ), 1.36 (s, 3H, CH3), 1.26 (s, 3H, CH3). 13C NMR (125
MHz, 300 K, DMSOꢀd6): δ 169.4, 169.2 (CHCO2), 154.5, 153.9 (NCO2), 136.5, 136.4, 135.4,
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135.3, 128.4, 128.4, 128.3, 128.2, 128.2, 127.9, 127.9, 127.8, 127.8, 127.3, 127.2 (Carom.),
111.6 (ꢀC(CH3)2), 82.7, 81.6 (Cβ), 79.0, 78.0 (Cγ), 66.7, 66.6, 66.4 (PhCH2Oꢀ), 66.2, 65.9
(Cα), 52.0, 51.6 (Cδ), 26.5 (CH3), 24.6 (CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for
C23H25NO6Na 434.1574, Found 434.1574.
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(3aR,4S,6aS)-2,2-Dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyrrole-4-carboxylic acid (8)
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7 (4.10 g, 9.46 mmol) was dissolved in EtOAc (60 mL) and Pd/C (10 wt%, 410 mg) was
added. After 19 h at RT with 9 bar H2ꢀatmosphere, MeOH (60 mL) was added and the
suspension was filtered. The solvent was removed under reduced pressure. 8 was obtained as
1
3
colorless solid (1.52 g, 7.76 mmol, 82%). H NMR (500 MHz, 300 K, D2O) δ 5.20 (d, J =
3
3
2
5.8 Hz, 1H, Hβ), 5.12 (dd, J = 4.5 Hz, J = 5.8 Hz, 1H, Hγ), 4.34 (s, 1H, Hα), 3.67 (d, J =
13.6 Hz, 1H, Hδ), 3.57 (dd, 2J = 13.6 Hz, 3J = 4.5 Hz, 1H, Hδ), 1.58 (s, 3H, CH3), 1.42 (s, 3H,
CH3). 13C NMR (125 MHz, 300 K, D2O): δ 169.7 (CO2H), 112.7 (CMe2), 82.1 (Cβ), 77.9
(Cγ), 50.3 (Cδ), 66.9 (Cα), 24.9 (CH3), 22.8 (CH3), HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd
for C8H13NO4Na 210.0737; Found 210.0738.
3aR,4S,6aS)-5-(((9H-Fluoren-9-yl)methoxy)carbonyl)-2,2-dimethyltetrahydro-4H-
[1,3]dioxolo[4,5-c]pyrrole-4-carboxylic acid (9)35
8 (1.82 g, 8.12 mmol, 1.0 eq) was dissolved in 10% Na2CO3aq (12.5 mL) and FmocCl (2.31 g,
8.93 mmol, 1.1 eq) was added in 1,4ꢀDioxane (12.5 mL). After stirring for 16 h, the mixture
was washed with Et2O (3 x 25 mL). 1
N HCl was added until pH = 6 und extracted with
EtOAc (6 x 25 mL). The combined organic layers were dried over MgSO4, filtered and the
solvent was removed under reduced pressure. 9 was obtained as pale yellow solid (2.80 g,
1
6.82 mmol, 84%). H NMR (500 MHz, 300 K, DMSOꢀd6): δ 7.90ꢀ7.28 (m, 8H, Harom), 4.84ꢀ
4.66 (m, 2H, Hβ, Hγ), 4.34ꢀ4.06 (m, 4H, Hα, CO2CH2CH), 3.57ꢀ3.51 (m, 2H, Hδ), 1.38 (s,
3H, CH3), 1.34 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.23 (s, 3H, CH3), 13C NMR (125 MHz, 300
K, DMSOꢀd6): δ 170.3 (CHO2), 154.8, 154.4 (NCO2), 143.9, 143.7, 140.7, 140.6, 127.6
127.1, 125.5, 125.4, 125.2, 125.1, 120.1, 120.0, (Carom.), 110.4, 110.3 (CMe2), 84.1, 83.0 (Cβ),
78.8, 77.9 (Cγ), 67.9, 67.8, 66.4, 66.3 (CO2CH2CH), 52.1, 51.8 (Cδ), 46.6, 46.6 (Cα), 26.7,
24.6, 24.6 (CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for C23H23NO6Na 432.1418;
Found 432.1416.
Methyl
(3R,6R,7S,8S,8aS)-6-azido-7,8-dihydroxy-5-oxohexahydro-5H-thiazolo[3,2-
a]pyridine-3-carboxylate (11)
10a (617 mg, 1.88 mmol, 1.0 eq) was dissolved in 95% TFAaq (10 mL). After stirring for 1 h,
the solvent was removed under reduced pressure. This procedure was repeated four times. The
crude product was purified by column chromatography over silica gel (DCM/DCEtOAc 1:3).
11 was obtained as white solid (439 mg, 1.52 mmol, 81%).1H NMR (500 MHz, 300 K,
3
3
DMSOꢀd6): δ 5.59ꢀ5.50 (m, 2H, 7ꢀOH, 8ꢀOH), 5.09 (dd, J = 7.4 Hz, J = 4.9 Hz, 1H, H3),
3
5.02 (d, J = 4.3 Hz, 1H, H8a), 4.20ꢀ4.14 (m, 2H, H6, H7), 4.12ꢀ4.06 (m, 1H, H8), 3.68 (s,
2
3
3H, COOCH3), 3.32 (dd, 2J = 11.6 Hz, 3J = 7.5 Hz, 1H, H2), 3.03 (dd, J = 11.5 Hz, J = 4.9
13
Hz, 1H, H2). C NMR (125 MHz, 300 K, DMSOꢀd6): δ 170.0 (COOCH3), 164.9 (C5), 69.7
(C7), 66.4 (C8), 65.4 (C8a), 61.0 (C3), 60.4 (C6), 52.4 (COOCH3), 30.9 (C2). HRMS (ESIꢀ
ICR) m/z: [M + Na]+ Calcd for C9H12N4O5SNa: 311.0421, Found; 311.0424.
Methyl
(2S,3aS,4R,7R,9aS,9bS)-4-azido-2-methyl-2-octyl-5-oxohexahydro-5H-
[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylate (10b)
2ꢀDekanone (231 µL, 1.21 mmol, 5.0 eq) was dissolved in MeOH (5 mL) and HC(OMe)3
(2.5 mL). After PPTS (30.5 mg, 121 µmol, 0.5 eq) was added, the solution was stirred
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overnight at RT. The reaction was quenched by the addition of NEt3 (40 µL) and the solvent
was removed under reduced pressure. The residue was dissolved in toluene (3 mL) and added
to a solution of the diol 11 (70.0 mg, 243 µmol, 1.0 eq) in MeOH (5 mL). After addition of
CSA (28.1 mg, 121 µmol, 0.5 eq), the mixture was stirred at 65 °C for 2.5 h. The reaction was
quenched by the addition of NEt3 (50 µL) and the solvent removed under reduced pressure.
The crude product was purified by column chromatography over silica gel (DCM/MeOH
1
60:1). The ketal 10b was obtained as pale yellow solid (60.0 mg, 141 µmol, 58%). H NMR
10
11
12
13
14
15
16
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23
24
25
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50
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56
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58
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60
3
3
3
(500 MHz, 300 K, CDCl3): δ 5.34 (dd, J = 6.6 Hz, J = 0.9 Hz, 1H, H3), 4.80 (dd, J = 7.7
Hz, J = 3.6 Hz, 1H, H7), 4.75 (d, J = 1.8 Hz, 1H, H8a), 4.54 (dd, J = 7.7 Hz, 3J = 1.8 Hz,
3
3
3
3
2
3
1H, H8), 3.78 (s, 3H, COOCH3), 3.73 (d, J = 3.5 Hz, 1H, H6), 3.38 (dd, J = 11.2 Hz, J =
6.5 Hz, 1H, H2l), 3.25 (dd, J = 11.2 Hz, J = 1.0 Hz, 1H, H2h), 1.69ꢀ1.62 (m, 2H,
2
3
CCH2(CH2)6CH3), 1.34 (s, 3H, CCH3), 1.32ꢀ1.19 (m, 12H, CCH2(CH2)6CH3), 0.88 (t, 3J = 7.0
13
Hz, 3H, CCH2(CH2)6CH3). C NMR (125 MHz, 300 K, CDCl3): δ 169.5 (COOCH3), 163.8
(C5), 111.8 (CCH3), 76.5 (C8), 75.7 (C7), 61.8 (C3), 60.2 (C8a), 59.9 (C6), 53.1 (COOCH3),
38.8 (CCH2(CH2)6CH3), 32.5 (C2), 31.9 (CCH2(CH2)6CH3), 29.3 (CCH2(CH2)6CH3), 24.2
(CCH3), 22.9 (CCH2(CH2)6CH3), 22.6 (CCH2(CH2)6CH3), 14.0 (CCH2(CH2)6CH3). HRMS
(ESIꢀICR) m/z: [M + Na]+ Calcd for C19H30N4O5SNa 449.1829; Found 449.1835.
Methyl
(2S,3aS,4R,7R,9aS,9bS)-4-azido-2-methyl-5-oxo-2-tridecylhexahydro-5H-
[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylate (10c)
2ꢀPentadekanone (589 mg, 2.60 mmol, 3.0 eq) was dissolved in MeOH (7 mL) and
HC(OMe)3 (3.5 mL). After PPTS (65.4 mg, 260 µmol, 0.3 eq) was added, the solution was
stirred 5 h at RT. The reaction was quenched by the addition of NEt3 (60 µL) and the solvent
was removed under reduced pressure. The residue was dissolved in Toluene (6 mL) and added
to a solution of the diol 11 (250 mg, 867 µmol, 1.0 eq) in MeOH (9 mL). After addition of
CSA (60.4 mg, 260 µmol, 0.3 eq), the mixture was stirred at 65 °C for 2.5 h. The reaction was
quenched by the addition of NEt3 (50 µL) and the solvent removed under reduced pressure.
The crude product was purified by column chromatography over silica gel (DCM/MeOH 70:1
ꢀ 50:1). The ketal 10c was obtained as white solid (241 mg, 485 µmol, 55%). 1H NMR (500
MHz, 300 K, CDCl3): δ 5.34 (d, 3J = 6.3 Hz, 1H, H3), 4.80 (dd, 3J = 7.6 Hz, 3J = 3.5 Hz, 1H,
3
H7), 4.75 (d, 3J = 1.4 Hz, 1H, H8a), 4.54 (dd, J = 7.7 Hz, 3J = 1.6 Hz, 1H, H8), 3.78 (s, 3H,
3
2
3
COOCH3), 3.73 (d, J = 3.5 Hz, 1H, H6), 3.38 (dd, J = 11.2 Hz, J = 6.5 Hz, 1H, H2l), 3.24
(d, J = 11.2 Hz, 1H, H2h), 1.69ꢀ1.62 (m, 2H, CCH2(CH2)11CH3), 1.34 (s, 3H, CCH3), 1.32ꢀ
2
1.20 (m, 22H, CCH2(CH2)11CH3), 0.88 (t, 3J = 6.9 Hz, 3H, CCH2(CH2)11CH3). 13C NMR (125
MHz, 300 K, CDCl3): δ 169.5 (COOCH3), 163.9 (C5), 111.9 (CCH3), 76.5 (C8), 75.7 (C7),
61.8 (C3), 60.2 (C8a), 59.9 (C6), 53.0 (COOCH3), 38.8 (CCH2(CH2)11CH3), 32.4 (C2), 31.8
(CCH2(CH2)11CH3), 29.6 (CCH2(CH2)11CH3), 24.2 (CCH3), 22.6 (CCH2(CH2)11CH3), 14.1
(CCH2(CH2)11CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for C24H40N4O5SNa 519.2612;
Found 519.2611.
Methyl (3aS,4R,7R,9aS,9bS)-4-azido-2,2-diheptyl-5-oxohexahydro-5H-[1,3]dioxolo[4,5-
c]thiazolo[3,2-a]pyridine-7-carboxylate (10d)
8ꢀPentadekanone (274 mg, 1.21 mmol, 5.0 eq) was dissolved in MeOH (5 mL) and
HC(OMe)3 (2.5 mL). After PPTS (30.5 mg, 121 µmol, 0.5 eq) was added, the solution was
stirred overnight at RT. The reaction was quenched by the addition of NEt3 (50 µL) and the
solvent was removed under reduced pressure. The residue was dissolved in Toluene (3 mL)
and added to a solution of the diol 11 (70.0 mg, 243 µmol, 1.0 eq) in MeOH (5 mL). After
addition of CSA (28.1 mg, 121 µmol, 0.5 eq), the mixture was stirred at 65 °C for 2.5 h. The
reaction was quenched by the addition of NEt3 (50 µL) and the solved removed under reduced
pressure. The crude product was purified by column chromatography over silica gel
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(DCM/MeOH 50:1). The ketal 10d was obtained as white solid (80.4 mg, 162 µmol, 67%). 1H
NMR (500 MHz, 300 K, CDCl3): δ 5.34 (d, J = 6.3 Hz, 1H, H3), 4.80 (dd, J = 7.6 Hz, J =
3
3
3
3
3
3
3.5 Hz, 1H, H7), 4.75 (d, J = 1.4 Hz, 1H, H8a), 4.54 (dd, J = 7.7 Hz, J = 1.6 Hz, 1H, H8),
3.78 (s, 3H, COOCH3), 3.73 (d, 3J = 3.5 Hz, 1H, H6), 3.38 (dd, 2J = 11.2 Hz, 3J = 6.5 Hz, 1H,
H2l), 3.24 (d, J = 11.2 Hz, 1H, H2h), 1.69ꢀ1.62 (m, 2H, CCH2(CH2)11CH3), 1.34 (s, 3H,
2
3
CCH3), 1.32ꢀ1.20 (m, 22H, CCH2(CH2)11CH3), 0.88 (t, J = 6.9 Hz, 3H, CCH2(CH2)11CH3).
13C NMR (125 MHz, 300 K, CDCl3): δ 169.5 (COOCH3), 163.9 (C5), 111.9 (CCH3), 76.5
(C8), 75.7 (C7), 61.8 (C3), 60.2 (C8a), 59.9 (C6), 53.0 (COOCH3), 38.8 (CCH2(CH2)11CH3),
32.4 (C2), 31.8 (CCH2(CH2)11CH3), 29.6 (CCH2(CH2)11CH3), 24.2 (CCH3), 22.6
(CCH2(CH2)11CH3), 14.1 (CCH2(CH2)11CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for
C24H40N4O5SNa 519.2612; Found 519.2611.
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(3aS,4R,7R,9aS,9bS)-4-Azido-2,2-dimethyl-5-oxohexahydro-5H-[1,3]dioxolo[4,5-
c]thiazolo[3,2-a]pyridine-7-carboxylic acid (12a)
To a solution of 10a (215 mg, 655 µmol, 1.0 eq) in DCE (10 mL) was added Me3SnOH
(592 mg, 3.28 mmol, 5.0 eq). The mixture was stirred at 80°C until complete conversion.
Then, EtOAc was added and the organic layer was washed with 0.2
N HCl and brine. The
organic layer was dried over MgSO4, filtered and the solvent removed under reduced
1
pressure. 12a was obtained as pale yellow solid (201 mg, 639 µmol, 98%). H NMR (500
3
3
MHz, 300 K, DMSOꢀd6): δ 13.15 (bs, 1H, COOH), 5.15 (dd, J = 6.2 Hz, J = 1.5 Hz, 1H,
H3), 4.94 (d, 3J = 1.9 Hz, 1H, H8a), 4.76 (dd, 3J = 7.6 Hz, 3J = 3.4 Hz, 1H, H7), 4.51 (dd, 3J =
3
3
2
3
7.5 Hz, J = 1.9 Hz, 1H, H8), 4.24 (d, J = 3.4 Hz, 1H, H6), 3.19 (dd, J = 11.2 Hz, J = 6.2
2
3
Hz, 1H, H2), 3.15 (dd, J = 11.2 Hz, J = 1.6 Hz, 1H, H2), 1.30 (s, 3H, CCH3), 1.28 (s, 3H,
CCH3). 13C NMR (125 MHz, 300 K, DMSOꢀd6): δ 170.6 (COOH), 163.8 (C5), 109.2
(C(CH3)2), 76.9 (C8), 76.0 (C7), 61.7 (C3), 59.7 (C8a), 59.4 (C6), 32.3 (C2), 26.1 (CCH3),
HRMS (ESIꢀICR) m/z: [M ꢀ H]ꢀ Calcd for C11H13N4O5S 313.0612; Found 313.0614.
(3aS,4R,7R,9aS,9bS)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-5-
oxohexahydro-5H-[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylic acid (14a)
Pd/C (10 wt%) was added to a solution of 12a (135 mg, 430 µmol, 1.0 eq) in MeOH (15 mL).
After stirring overnight under H2ꢀatmosphere, the suspension was filtered and the solvent was
removed under reduced pressure. The residue was dissolved in 1,4ꢀDioxane/H2O (4:1, 10 mL)
and FmocꢀOSu (189 mg, 559 µmol, 1.3 eq) and DIPEA (293 µL, 1.72 mmol, 4.0 eq) were
added at 0°C. The mixture was stirred at RT for 4.5 h. After the solvent was removed under
reduced pressure, the crude 13a was dissolved in EtOAc (20 mL) and 0.5
N HCl (5 mL) was
added. The aqueous layer was separated and extracted with EtOAc (20 mL) three times. The
combined organic layers were dried over MgSO4, filtrated and the solvent removed under
reduced pressure. The crude product was purified by column chromatography over silica gel
1
(DCM/MeOH 5:1). 14a was obtained as pale yellow solid (88.0 mg, 172 µmol, 40%). H
3
NMR (500 MHz, 300 K, DMSOꢀd6): δ 7.89 (d, J = 7.5 Hz, 2H, FmocꢀHarom.), 7.83ꢀ7.79 (m,
3
3
2H, FmocꢀHarom.), 7.42 (t, J = 7.5 Hz, 2H, FmocꢀHarom.), 7.33 (t, J = 7.4 Hz, 2H Fmocꢀ
3
Harom.), 7.14 (d, J = 8.1 Hz, 1H, FmocꢀNH), 5.07 (s, 1H, H8a), 5.02ꢀ4.96 (m, 1H, H3), 4.65
(dd, J = 7.4 Hz, J = 2.9 Hz, 1H, H7), 4.55ꢀ4.46 (m, 2H, H6, H8), 4.34ꢀ4.21 (m, 3H, Fmocꢀ
CH, FmocꢀCH2), 3.18ꢀ3.10 (m, 2H, H2), 1.28 (s, 3H, CCH3), 1.27 (s, 3H, CCH3). C NMR
3
3
13
(125 MHz, 300 K, DMSOꢀd6): δ 172.8 (COOH), 164.7 (C5), 143.8 (FmocꢀCarom.,q), 143.7
(FmocꢀCarom.,q), 140.7 (FmocꢀCarom.,q), 127.6 (FmocꢀCarom.), 127.1 (FmocꢀCarom.), 127.0
(FmocꢀCarom.), 125.6 (FmocꢀCarom.), 125.5 (FmocꢀCarom.), 120.0 (FmocꢀCarom.), 108.6
(C(CH3)2), 76.8 (C8), 74.8 (C7), 66.0 (FmocꢀCH2), 62.5 (C3), 59.8 (C8a), 53.5 (C6), 46.5
(FmocꢀCH), 32.5 (C2), 26.2 (CCH3), 24.2 (CCH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd
for C26H26N2O7SNa 533.1353; fFound 533.1346.
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(2S,3aS,4R,7R,9aS,9bS)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-methyl-2-
octyl-5-oxohexahydro-5H-[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylic
(14b)
acid
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7
8
To a solution of corresponding ketal 10b (60.0 mg, 141 µmol, 1.0 eq) in DCE (10 mL) was
added Me3SnOH (114 mg, 633 µmol, 4.5 eq). The mixture was stirred at 80°C until complete
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conversion. Then, EtOAc was added and the organic layer was washed with 0.2
N HCl and
brine. The organic layer was dried over MgSO4, filtered and the solvent removed under
reduced pressure. The crude 12b was dissolved in MeOH (10 mL) was added Pd/C (10 wt%).
After stirring overnight under H2ꢀatmosphere, the suspension was filtered and the solvent was
removed under reduced pressure. The crude 13b was dissolved in 1,4ꢀDioxane/H2O (4:1,
7.5 mL) and FmocꢀOSu (61.7 mg, 183 µmol, 1.3 eq) and DIPEA (95.9 µL, 564 µmol, 4.0 eq)
were added at 0°C. The mixture was stirred at RT overnight. The solution was diluted with
EtOAc (20 mL) and 0.2
N HCl (7 mL) was added. The aqueous layer was separated and
extracted with EtOAc (10 mL) three times. The combined organic layers were dried over
MgSO4, filtrated and the solvent removed under reduced pressure. The crude product was
purified by column chromatography over silica gel (DCM/MeOH 10:1). The Fmoc protected
build block 14b was obtained as pale yellow solid (58.3 mg, 96.0 µmol, 68%). 1H NMR (500
3
MHz, 300 K, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H, FmocꢀHarom.), 7.65ꢀ7.61 (m, 2H, Fmocꢀ
3
3
Harom.), 7.40 (t, J = 7.5 Hz, 2H, FmocꢀHarom.), 7.34ꢀ7.29 (m, 2H, FmocꢀHarom.), 5.91 (d, J =
3
3
7.2 Hz, 1H, FmocꢀNH), 5.29 (d, J = 6.4 Hz, 1H, H3), 4.87 (d, J = 1.5 Hz, 1H, H8a), 4.84
(dd, 3J = 7.6 Hz, 3J = 3.4 Hz, 1H, H7), 4.56 (dd, J = 7.6 Hz, 3J = 1.8 Hz, 1H, H8), 4.53 (dd,
3
3J = 7.3 Hz, J = 3.3 Hz, 1H, H6), 4.48ꢀ4.34 (m, 2H, FmocꢀCH2), 4.25 (t, J = 7.2 Hz, 1H,
3
3
FmocꢀCH), 3.40 (dd, J = 11.2 Hz, J = 6.6 Hz, 1H, H2t), 3.32 (d, J = 11.1 Hz, 1H, H2h),
2
3
2
1.69ꢀ1.54 (m, 2H, CCH2(CH2)6CH3), 1.32 (s, 3H, CCH3), 1.30ꢀ1.15 (m, 12H,
3
13
CCH2(CH2)5CH3), 0.89 (t, J = 6.9 Hz, 3H, CCH2(CH2)5CH3). C NMR (125 MHz, 300 K,
CDCl3): δ 171.9 (COOH), 166.0 (C5), 156.5 (FmocꢀC=O), 143.9 (FmocꢀCarom.,q), 143.8
(FmocꢀCarom.,q), 141.4 (FmocꢀCarom.,q), 127.9 (FmocꢀCarom.), 127.3 (FmocꢀCarom.), 127.2
(FmocꢀCarom.), 125.4 (FmocꢀCarom.), 125.3 (FmocꢀCarom.), 120.1 (FmocꢀCarom.), 111.5 (CCH3),
76.8 (C8), 74.1 (C7), 67.8 (FmocꢀCH2), 62.1 (C3), 60.7 (C8a), 54.0 (C6), 47.2 (FmocꢀCH),
39.0 (CCH2(CH2)5CH3), 32.2 (C2), 32.0 (CCH2(CH2)5CH3), 29.8 (CCH2(CH2)5CH3), 29.6
(CCH2(CH2)5CH3), 29.4 (CCH2(CH2)5CH3), 23.3 (CCH3), 22.8 (CCH2(CH2)5CH3), 22.7
(CCH2(CH2)5CH3), 14.3 (CCH2(CH2)5CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for
C33H40N2O7SNa 631.2448; Found 631.2464.
(2S,3aS,4R,7R,9aS,9bS)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-methyl-5-
oxo-2-tridecylhexahydro-5H-[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylic acid
(14c)
To a solution of corresponding ketal 10c (240 mg, 483 µmol, 1.0 eq) in DCE (10 mL) was
added Me3SnOH (306 mg, 1.69 mmol, 3.5 eq). The mixture was stirred at 80°C until
complete conversion. Then, EtOAc was added and the organic layer was washed with 0.2
N
HCl and brine. The organic layer was dried over MgSO4, filtered and the solvent removed
under reduced pressure. The crude 12c was dissolved in MeOH (15 mL) was added Pd/C (10
wt%). After stirring overnight under H2ꢀatmosphere, the suspension was filtered and the
solvent was removed under reduced pressure. The crude 13c was dissolved in 1,4ꢀ
Dioxane/H2O (4:1, 10 mL) and FmocꢀOSu (212 mg, 628 µmol, 1.3 eq) and DIPEA (329 µL,
1.93 mmol, 4.0 eq) were added at 0°C. The mixture was stirred at RT for overnight. The
solution was diluted with EtOAc (30 mL) and H2O (5 mL) was added. The aqueous layer was
separated, diluted with 1 n HCl to pH = 2 and extracted with EtOAc (10 mL) three times. The
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combined organic layers were dried over MgSO4, filtrated and the solvent removed under
reduced pressure. The crude product was purified by column chromatography over silica gel
(DCM/MeOH 10:1). The Fmoc protected build block 14c was obtained as pale yellow solid
1
3
(125mg, 184 µmol, 38%). H NMR (500 MHz, 300 K, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H,
FmocꢀHarom.), 7.66ꢀ7.60 (m, 2H, FmocꢀHarom.), 7.40 (t, 3J = 7.4 Hz, 2H, FmocꢀHarom.), 7.31 (t,
3J = 7.3 Hz, 2H, FmocꢀHarom.), 5.91 (d, 3J = 6.2 Hz, 1H, FmocꢀNH), 5.28 (d, 3J = 5.9 Hz, 1H,
3
3
H3), 4.88 (s, 1H, H8a), 4.83 (dd, J = 7.4 Hz, J = 2.8 Hz, 1H, H7), 4.63ꢀ4.48 (m, 2H, H6,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
H8), 4.47ꢀ4.31 (m, 2H, FmocꢀCH2), 4.24 (t, J = 7.2 Hz, 1H, FmocꢀCH), 3.41ꢀ3.27 (m, 2H,
H2), 1.69ꢀ1.54 (m, 2H, CCH2(CH2)11CH3), 1.31 (s, 3H, CCH3), 1.31ꢀ1.17 (m, 22H,
CCH2(CH2)11CH3), 0.87 (t, 3J = 6.9 Hz, 3H, CCH2(CH2)11CH3). 13C NMR (125 MHz, 300 K,
CDCl3: δ 172.1 (COOH), 166.0 (C5), 156.5 (FmocꢀC=O), 144.0 (FmocꢀCarom.,q), 143.8
(FmocꢀCarom.,q), 141.4 (FmocꢀCarom.,q), 127.9 (FmocꢀCarom.), 127.3 (FmocꢀCarom.), 127.2
(FmocꢀCarom.), 125.4 (FmocꢀCarom.), 125.3 (FmocꢀCarom.), 120.1 (FmocꢀCarom.), 111.5 (CCH3),
76.7 (C8), 74.1 (C7), 67.8 (FmocꢀCH2), 62.2 (C3), 60.7 (C8a), 54.0 (C6), 47.2 (FmocꢀCH),
39.0 (CCH2(CH2)11CH3), 32.3 (C2), 32.1 (CCH2(CH2)11CH3), 29.9 (CCH2(CH2)11CH3), 29.8
(CCH2(CH2)11CH3), 29.7 (CCH2(CH2)11CH3), 29.5 (CCH2(CH2)11CH3), 23.3 (CCH3), 22.8
(CCH2(CH2)11CH3), 22.7 (CCH2(CH2)11CH3), 14.3 (CCH2(CH2)11CH3). HRMS (ESIꢀICR)
m/z: [M + Na]+ Calcd for C38H50N2O7SNa 701.3231; Found 701.3237.
(3aS,4R,7R,9aS,9bS)-4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2,2-diheptyl-5-
oxohexahydro-5H-[1,3]dioxolo[4,5-c]thiazolo[3,2-a]pyridine-7-carboxylic acid (14d)
To a solution of corresponding ketal 10d (71.6 mg, 144 µmol, 1.0 eq) in DCE (10 mL) was
added Me3SnOH (117 mg, 649 µmol, 4.5 eq). The mixture was stirred at 80°C until complete
conversion. Then, EtOAc was added and the organic layer was washed with 0.2
N HCl and
brine. The organic layer was dried over MgSO4, filtered and the solvent removed under
reduced pressure. The crude 12d was dissolved in MeOH (15 mL) was added Pd/C (10 wt%).
After stirring overnight under H2ꢀatmosphere, the suspension was filtered and the solvent was
removed under reduced pressure. The crude 13d was dissolved in 1,4ꢀDioxane/H2O (4:1,
7.5 mL) and FmocꢀOSu (63.1 mg, 187 µmol, 1.3 eq) and DIPEA (98.0 µL, 579 µmol, 4.0 eq)
were added at 0°C. The mixture was stirred at RT for overnight. The solution was diluted with
EtOAc (20 mL) and 0.2
N HCl (7 mL) was added. The aqueous layer was separated and
extracted with EtOAc (10 mL) five times. The combined organic layers were dried over
MgSO4, filtrated and the solvent removed under reduced pressure. The crude product was
purified by column chromatography over silica gel (DCM/MeOH 10:1). The Fmoc protected
1
build block 14d was obtained as white solid (71.4 mg, 105 µmol, 73%). H NMR (500 MHz,
300 K, CDCl3): δ 7.76 (d, 7.5 Hz, 2H, FmocꢀHarom.), 7.67ꢀ7.61 (m, 2H, FmocꢀHarom.), 7.40 (t,
3J = 7.4 Hz, 2H, FmocꢀHarom.), 7.32 (t, 3J = 7.4 Hz, 2H, FmocꢀHarom.), 5.91 (d, 3J = 7.1 Hz, 1H,
3
3
3
FmocꢀNH), 5.28 (d, J = 6.4 Hz, 1H, H3), 4.86 (s, 1H, H8a), 4.80 (dd, J = 7.5 Hz, J = 3.2
3
Hz, 1H, H7), 4.58ꢀ4.49 (m, 2H, H6, H8), 4.47ꢀ4.33 (m, 2H, FmocꢀCH2), 4.25 (t, J = 7.2 Hz,
1H, FmocꢀCH), 3.39 (dd, 2J = 11.1 Hz, 3J = 6.7 Hz, 1H, H2l), 3.31 (d, 2J = 11.1 Hz, 1H, H2h),
1.66ꢀ1.49 (m, 4H, C(CH2(CH2)5CH3)2), 1.40ꢀ1.13 (m, 20H, C(CH2(CH2)5CH3)2), 0.89 (t, 3J =
6.9 Hz, 3H, CCH2(CH2)5CH3), 0.87 (t, J = 6.9 Hz, 3H, CCH2(CH2)5CH3). 13C NMR (125
3
MHz, 300 K, CDCl3): δ 172.0 (COOH), 166.1 (C5), 156.5 (FmocꢀC=O), 144.0 (Fmocꢀ
Carom.,q), 143.8 (FmocꢀCarom.,q), 141.4 (FmocꢀCarom.,q), 127.9 (FmocꢀCarom.), 127.3 (Fmocꢀ
Carom.), 127.2 (FmocꢀCarom.), 125.4 (FmocꢀCarom.), 125.3 (FmocꢀCarom.), 120.1 (FmocꢀCarom.),
113.7 (C(CH2(CH2)5CH3)2), 77.1 (C8), 74.4 (C7), 67.8 (FmocꢀCH2), 62.1 (C3), 60.8 (C8a),
54.2 (C6), 47.1 (FmocꢀCH), 36.3 (CCH2(CH2)5CH3), 36.0 (CCH2(CH2)5CH3), 32.2 (C2), 32.0
(C(CH2(CH2)5CH3)2), 31.9 (C(CH2(CH2)5CH3)2), 29.9 (C(CH2(CH2)5CH3)2), 29.8
(C(CH2(CH2)5CH3)2), 29.4 (C(CH2(CH2)5CH3)2), 29.3 (C(CH2(CH2)5CH3)2), 24.5
(C(CH2(CH2)5CH3)2), 23.1 (C(CH2(CH2)5CH3)2), 22.8 (C(CH2(CH2)5CH3)2), 14.3
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(CCH2(CH2)5CH3), 14.2 (CCH2(CH2)5CH3). HRMS (ESIꢀICR) m/z: [M + Na]+ Calcd for
4
5
C38H50N2O7SNa 701.3231; Found 701.3243.
6
7
8
9
Cyclo(D-Phe-Dyp-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-) (15)
The peptide 15 was synthesized using automated peptide synthesis in 0.1 mM scale.1H NMR
3
3
(600 MHz, 300 K, MeOHꢀd3): δ 8.89 (d, J = 3.5 Hz, 1H,
D
ꢀPhe6ꢀNH), 8.76 (d, J = 3.7 Hz,
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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31
32
33
34
35
36
37
38
39
40
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44
45
46
47
48
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1H,
D
ꢀPhe1ꢀNH), 8.75 (d, J = 9.5 Hz, 1H, Leu10ꢀNH), 8.71 (d, J = 9.5 Hz, 1H, Leu5ꢀNH),
8.70 (d, 3J = 9.5 Hz, 1H, Orn9ꢀNH), 8.68 (d, 3J = 9.4 Hz, 1H, Orn4ꢀNH), 7.71 (d, 3J = 9.2 Hz,
3
1H, Val3ꢀNH), 7.70 (d, J = 9.2 Hz, 1H, Val8ꢀNH), 7.35ꢀ7.20 (m, 10H, HAr), 4.98ꢀ4.95 (m,
2H, Orn4ꢀHα, Orn9ꢀHα), 4.69ꢀ4.61 (m, 2H, Leu5ꢀHα, Leu10ꢀHα), 4.54ꢀ4.47 (m, 2H, DꢀPhe1ꢀ
2
3
3
Hα,
D
ꢀPhe6ꢀHα), 4.34 (dd, J = 8.0, J = 1.7 Hz, 1H, Pro7ꢀHα), 4.17 (ddd, J = 8.0, 3J = 8.0,
3J = 4.0 Hz, 1H, Dyp2ꢀHβ), 4.15ꢀ4.12 (m, 2H, Val3ꢀHα, Val8ꢀHα), 4.11 (dd, J = 4.0,
3
3J = 0.8 Hz, 1H, Dyp2ꢀHβ), 4.03 (dd, 2J = 10.0, 3J = 7.9 Hz, 1H, Dyp2ꢀHδ), 3.77ꢀ3.67 (m, 1H,
Pro7ꢀHδ), 3.12ꢀ2.98 (m, 5H, Orn4ꢀHδ,
DꢀPhe1ꢀHβ, DꢀPhe6ꢀHβ, Orn9ꢀHδ), 2.96ꢀ2.82 (m, 3H,
2
3
Orn4ꢀHδ, ꢀPhe6ꢀHβ, Orn8ꢀHδ), 2.66 (dd, J = 10.0, J = 8.3 Hz, 1H, Dyp2ꢀHδ), 2.52ꢀ2.42
D
(m, 1H, Pro7ꢀHδ), 2.33ꢀ2.22 (m, 2H, Val3ꢀHβ, Val8ꢀHβ), 2.10ꢀ1.94 (m, 3H, Orn4ꢀHβ, Pro7ꢀ
Hβ, Orn9ꢀHβ), 1.80ꢀ1.66 (m, 6H, Orn4ꢀHγ, Pro7ꢀHβ, Pro7ꢀHγ, Orn9ꢀHγ), 1.65ꢀ1.57 (m, 3H,
Orn4ꢀHβ, Pro7ꢀHγ, Orn9ꢀHβ), 1.56ꢀ1.46 (m, 4H, Leu5ꢀHβ, Leu5ꢀHγ, Leu10ꢀHβ, Leu10ꢀHγ),
3
1.45ꢀ1.36 (m, 2H, Leu5ꢀHβ, Leu10ꢀHβ), 0.97 (d, J = 6.9 Hz, 3H, Val3ꢀHγ), 0.95 (d,
3J = 6.9 Hz, 3H, Val8ꢀHγ), 0.91ꢀ0.85 (m, 18H, Val3ꢀHγ, Leu5ꢀHδ, Val8ꢀHγ, Leu10ꢀHδ).
HRMS (ESIꢀICR) m/z: [M + 2H]2+ Calcd for C60H92N12O12H2 587.3552, Found 587.3552.
Cyclo(D D-Phe-Pro-Val-Orn-Leu-) (16)
-Hotp=Tap-Val-Orn-Leu-
1
The peptide 16 was synthesized using manual peptide synthesis in 0.1 mM scale. H NMR
(600 MHz, 300 K, MeOHꢀd3): δ 9.02 (sbr, 1H,
D
ꢀPhe6ꢀNH), 8.40 (d, 3J = 2.8 Hz, 1H,
D
ꢀHotp1ꢀ
3
3
NH), 8.36 (d, J = 9.0 Hz, 1H, Orn4ꢀNH), 8.03 (d, J = 8.0 Hz, 1H, Val8ꢀNH), 7.99 (d,
3
3
3J = 9.7 Hz, 1H, Orn9ꢀNH), 7.90 (d, J = 8.1 Hz, 1H, Leu10ꢀNH), 7.72 (d, J = 10.0 Hz, 1H,
Val3ꢀNH), 7.62 (d, 3J = 7.5 Hz, 1H, Leu5ꢀNH), 7.33ꢀ7.21 (m, 5H, HAr), 5.20 (s, 1H, ꢀHotp1ꢀ
H8a), 5.05ꢀ4.99 (m, 2H,
ꢀHotp1ꢀH6, Tap2ꢀH3), 4.72ꢀ4.65 (m, 3H, ꢀHotp1ꢀH7, ꢀHotp1ꢀH8,
Leuꢀ10ꢀHα), 4.54 (ddd, J = 10.9, J = 8.9, J = 4.1 Hz, 1H, Orn4ꢀHα), 4.50ꢀ4.40 (m, 3H,
D
D
D
D
3
3
3
3
3
Leu5ꢀHα,
DꢀPhe6ꢀHα, Orn9ꢀHα), 4.30 (dd, J = 8.2, J = 2.8 Hz, 1H, Pro7ꢀHα), 4.16 (t,
3
3
3J = 10.2 Hz, 1H, Val3ꢀHα), 3.85 (dd, J = 10.9, J = 8.2 Hz, 1H, Val8ꢀHα), 3.72 (ddd,
2J = 10.7, 3J = 7.9, J = 3.3 Hz, 1H, Pro6ꢀHδ), 3.50 (dd, 2J = 11.9, 3J = 7.7 Hz, 1H, Tap2ꢀH2),
3
2
3
3
3.09 (d, J = 12.1 Hz, 1H, TapꢀH2), 3.06 (dd, J = 12.8, J = 5.6 Hz, 1H,
DꢀPhe6ꢀHβ), 3.03ꢀ
2.89 (m, 5H, Orn4ꢀHδ, ꢀPhe6ꢀHβ, Orn9ꢀHδ), 2.71ꢀ2.62 (m, 1H, Pro7ꢀHδ), 2.41ꢀ2.31 (m, 1H,
D
Val8ꢀHβ), 2.10ꢀ1.95 (m, 3H, Val3ꢀHβ, Pro7ꢀHβ, Orn9ꢀHβ), 1.81ꢀ1.59 (m, 11H, Orn4ꢀHβ,
Orn4ꢀHγ, Pro7ꢀHβ, Pro7ꢀHγ, Orn9ꢀHβ, Orn9ꢀHγ, Leu10ꢀHβ, Leu10ꢀHγ), 1.58ꢀ1.51 (m, 2H,
Leu5ꢀHβ, Leu5ꢀHγ), 1.45ꢀ1.36 (m, 1H, Orn4ꢀHγ), 1.34 (s, 3H, CCH3), 1.33 (s, 3H, CCH3),
3
3
3
1.04 (d, J = 7.0 Hz, 3H, Val8ꢀHγ), 1.01 (d, J = 6.8 Hz, 3H, Val8ꢀHγ), 0.98 (d, J = 6.5 Hz,
3H, Leu10ꢀHδ), 0.94 (d, 3J = 6.5 Hz, 3H, Leu10ꢀHδ), 0.93ꢀ0.89 (m, 12H, Val3ꢀHγ, Leu5ꢀHδ).
HRMS (ESIꢀICR) m/z: [M + H]+ Calcd for C57H90N12O12SH 1167.6595; Found 1167.6632.
Cyclo(D-Hot[(7S,8S)-O-(2(S)-decyliden)]=Tap-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu–)
(17)
The peptide 17 was synthesized using automated peptide synthesis 0.1 mM scale.1H NMR
3
(600 MHz, 300 K, MeOHꢀd3): δ 9.00 (sbr, 1H,
D
ꢀPhe6ꢀNH), 8.39 (d, J = 6.0 Hz, 1H, Orn4ꢀ
NH), 8.03 (d, 3J = 8.0 Hz, 1H, Val8ꢀNH), 8.00 (sbr, 1H, Orn9ꢀNH), 7.90 (sbr, 1H, Leu10ꢀNH),
3
3
7.69 (d, J = 10.0 Hz, 1H, Val3ꢀNH), 7.62 (d, J = 7.4 Hz, 1H, Leu5ꢀNH), 7.33ꢀ7.22 (m, 5H,
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HAr), 5.24 (s, 1H,
DꢀHot1ꢀH8a), 5.01ꢀ4.99 (m, 1H,
DꢀHot1ꢀH6), 4.93ꢀ4.91 (m, 1H, Tap2ꢀH3),
4
5
6
7
8
9
4.67ꢀ4.64 (m, 2H,
D
ꢀHot1ꢀH7,
D
ꢀHot1ꢀH8), 4.57ꢀ4.38 (m, 5H, Orn4ꢀHα, Leu5ꢀHα, DꢀPhe6ꢀ
Hα, Orn9ꢀHα, Leu10ꢀHα), 4.31 (dd, 3J = 8.1, 3J = 2.4 Hz, 1H, Pro7ꢀHα), 4.17 (t, 3J = 10.1 Hz,
3
3
1H, Val3ꢀHα), 3.85 (dd, J = 10.8, J = 8.0 Hz, 1H, Val8ꢀHα), 3.75ꢀ3.68 (m, 1H, Pro7ꢀHδ),
3.49 (dd, 3J = 11.8, 3J = 7.7 Hz, 1H, Tap2ꢀH2), 3.11 (d, 3J = 11.8 Hz, 1H, Tap2ꢀH2), 3.06 (dd,
3
3J = 12.8, J = 5.7 Hz, 1H,
DꢀPhe6ꢀHβ), 3.00ꢀ2.85 (m, 5H, Orn4ꢀHδ,
DꢀPhe6ꢀHβ, Orn9ꢀHδ),
2.68ꢀ2.60 (m, 1H, Pro7ꢀHδ), 2.40ꢀ2.30 (m, 1H, Val8ꢀHβ), 2.07ꢀ1.94 (m, 3H, Val3ꢀHβ, Pro7ꢀ
Hβ, Orn9ꢀHβ), 1.80ꢀ1.46 (m, 16H, ꢀHot1ꢀCH2(CH2)6CH3, Orn4ꢀHβ, Orn4ꢀHγ, Leu5ꢀHβ,
Leu5ꢀHγ, Pro7ꢀHβ, Pro7ꢀHγ, Orn9ꢀHβ, Orn9ꢀHγ, Leu10ꢀHβ, Leu10ꢀHγ), 1.33ꢀ1.23 (m, 18H,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
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D
3
D
ꢀHot1ꢀCH2(CH2)6CH3, DꢀHot1ꢀCCH3, Orn4ꢀHγ), 1.04 (d, J = 6.9 Hz, Val8ꢀHγ), 1.02 (d,
3J = 6.9 Hz, Val8ꢀHγ), 1.04 (d, J = 6.9 Hz, Val8ꢀHγ), 0.98 (d, J = 6.5 Hz, Leu10ꢀHδ), 0.95
3
3
3
(d, J = 6.5 Hz, Leu10ꢀHδ), 0.92ꢀ0.86 (m, 15H,
DꢀHot1ꢀCH2(CH2)6CH3, Val3ꢀHγ, Leu5ꢀHδ).
HRMS (ESIꢀICR) m/z: [M + 2H]2+ Calcd for C64H104N12O12SH2 633.3881; Found 633.3883.
Cyclo(D-Hot[(7S,8S)-O-(2(S)-pentadecyliden)]=Tap-Val-Orn-Leu-D-Phe-Pro-Val-Orn-
Leu-) (18)
The peptide 18 was synthesized using automated peptide synthesis in 0.1 mM scale.1H NMR
(600 MHz, 300 K, MeOHꢀd3): δ 9.02 (sbr, 1H,
NH), 8.37 (sbr, 1H,
3
D
ꢀPheꢀNH), 8.38 (d, J = 8.9 Hz, 1H, Orn4ꢀ
D
ꢀHot1ꢀNH), 8.03 (d, 3J = 8.0 Hz, 1H, Val8ꢀNH), 7.99 (d, 3J = 9.6 Hz, 1H,
3
3
Orn9ꢀNH), 7.89 (d, J = 8.1 Hz, 1H, Leu10ꢀNH), 7.69 (d, J = 10.0 Hz, 1H, Val3ꢀNH), 7.61
3
(d, J = 7.4 Hz, 1H, Leu5ꢀNH), 7.35ꢀ7.20 (m, 5H, HAr), 5.23 (s, 1H,
(m, 2H, ꢀHot1ꢀH6, Tap2ꢀH3), 4.70ꢀ4.60 (m, 3H, ꢀHot1ꢀH7, ꢀHot1ꢀH8, Leu10ꢀHα), 4.57ꢀ
4.39 (m, 4H, Orn4ꢀHα, Leu5ꢀHα, ꢀPhe6ꢀHα, Orn9ꢀHα), 4.30 (dd, J = 8.2, J = 2.5 Hz, 1H,
DꢀHot1ꢀH8a), 4.99ꢀ4.92
D
D
D
3
3
D
3
3
3
Pro7ꢀHα), 4.16 (t, J = 10.2 Hz, Val3ꢀHα), 3.85 (dd, J = 10.9, J = 8.1 Hz, 1H, Val8ꢀHα),
3.74ꢀ3.68 (m, 1H, Pro7ꢀHδ), 3.49 (dd, 3J = 11.9, 3J = 7.7 Hz, 1H, Tap2ꢀH2), 3.11 (d, 3J = 12.0
Hz, 1H, Tap2ꢀH2), 3.06 (dd, 2J = 12.8, 3J = 5.7 Hz, 1H,
D
ꢀPhe6ꢀHβ), 3.00ꢀ2.85 (m, 5H, Orn4ꢀ
ꢀPhe6ꢀHβ, Orn9ꢀHδ), 2.76ꢀ2.59 (m, 1H, Pro7ꢀHδ), 2.40ꢀ2.30 (m, 1H, Val8ꢀHβ), 2.07ꢀ
1.92 (m, 3H, Val3ꢀHβ, Pro7ꢀHβ, Orn9ꢀHβ), 1.80ꢀ1.57 (m, 12H, Orn4ꢀHβ, Pro7ꢀHβ, Pro7ꢀHγ,
Orn9ꢀHβ, Orn9ꢀHγ, Leu10ꢀHβ, Leu10ꢀHγ), 1.56ꢀ1.47 (m, 6H, ꢀHot1ꢀCH2(CH2)11CH3, Orn4ꢀ
Hγ, Leu5ꢀHβ, Leu5ꢀHγ), 1.40ꢀ1.20 (m, 26H, ꢀHot1ꢀCCH3, ꢀHot1ꢀCH2(CH2)11CH3, Orn4ꢀ
Hγ), 1.04 (d, J = 6.9 Hz, 3H, Val8ꢀHγ), 1.01 (d, J = 6.8 Hz, 3H, Val8ꢀHγ), 0.98 (d,
3J = 6.5 Hz, 3H, Leu10ꢀHδ), 0.94 (d, 3J = 6.6 Hz, 3H, Leu10ꢀHδ), 0.92ꢀ0.87 (m, 15H,
ꢀHot1ꢀ
Hδ,
D
D
D
D
3
3
D
CH2(CH2)11CH3, Val3ꢀHγ, Leu5ꢀHδ). HRMS (ESIꢀICR) m/z: [M + H]+ Calcd for
C69H114N12O12SH 1335.8473; Found 1335.8524.
Cyclo(D-Hot[(7S,8S)-O-(2(S)-8-pentadecyliden)]=Tap-Val-Orn-Leu-D-Phe-Pro-Val-Orn-
Leu-) (19)
The peptide 19 was synthesized using automated peptide synthesis in 0.1 mM scale. 1H NMR
(600 MHz, 300 K, MeOHꢀd3): δ 9.00 (sbr, 1H, ꢀPhe6ꢀNH), 8.41 (sbr, 1H, Orn4ꢀNH), 8.03 (d,
3J = 8.1 Hz, 1H, Val8ꢀNH), 7.98 (sbr, 1H, Orn9ꢀNH), 7.92 (sbr, 1H, Leu10ꢀNH), 7.66 (d,
3J = 10.1 Hz, 1H, Val3ꢀNH), 7.61 (sbr, 1H, Leu5ꢀNH), 7.34ꢀ7.21 (m, 5H, HAr), 5.01 (s, 1H,
Hot1ꢀH8a), 5.01ꢀ4.92 (m, 2H, ꢀHot1ꢀH6, Tap2ꢀH3), 4.65ꢀ4.59 (m, 3H, ꢀHot1ꢀH7, ꢀHot1ꢀ
H8, Leu10ꢀHα), 4.57ꢀ4.39 (m, 4H, Orn4ꢀHα, Leu5ꢀHα, ꢀPhe6ꢀHα, Orn9ꢀHα), 4.31 (dd,
D
Dꢀ
D
D
D
D
3
3
3
3J = 8.7, J = 2.6 Hz, 1H, Pro7ꢀHα), 4.17 (t, J = 10.2 Hz, 1H, Val3ꢀHα), 3.85 (dd, J = 10.8,
3J = 8.3 Hz, 1H, Val8ꢀHα), 3.75ꢀ3.68 (m, 1H, ProꢀHδ), 3.49 (dd, J = 11.9, J = 7.7 Hz, 1H,
2
3
2
2
3
TapꢀH2), 3.12 (d, J = 12.2 Hz, 1H, Tap2ꢀH2), 3.06 (dd, J = 12.8, J = 5.7 Hz, 1H,
Hβ), 3.00ꢀ2.85 (m, 5H, Orn4ꢀHδ, ꢀPhe6ꢀHβ, Orn9ꢀHδ), 2.69ꢀ2.59 (m, 1H, Pro7ꢀHδ), 2.40ꢀ
2.30 (m, 1H, Val8ꢀHβ), 2.07ꢀ1.90 (m, 3H, Val3ꢀHβ, Pro7ꢀHβ, Orn9ꢀHβ), 1.80ꢀ1.35 (m, 20H,
ꢀHot1ꢀ(CH2(CH2)5CH3)2, Orn4ꢀHβ, Orn4ꢀHγ, Leu5ꢀHβ, Leu5ꢀHγ, Pro7ꢀHβ, Pro7ꢀHγ, Orn9ꢀ
Hγ, Leu10ꢀHβ, Leu10ꢀHγ), 1.30ꢀ1.20 (m, 20H, ꢀHot1ꢀ(CH2(CH2)5CH3)2), 1.03 (d,
DꢀPheꢀ
D
D
D
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3J = 7.2 Hz, 3H, Val8ꢀHγ), 1.01 (d, J = 7.1 Hz, 3H, Val8ꢀHγ), 0.99 (d, J = 6.3 Hz, 3H,
Leu10ꢀHδ), 0.95 (d, 3J = 6.2 Hz, 3H, Leu10ꢀHδ), 0.92ꢀ0.86 (m, 12H,
(CH2(CH2)5CH3)2), Val3ꢀHγ, Leu5ꢀHδ). HRMS (ESIꢀICR) m/z: [M + H]+ Calcd for
C69H114N12O12SH 1335.8473; Found 1335.8465.
DꢀHot1ꢀ
8
9
1
Supporting Information. H and 13C NMR spectra of the reported compounds, 2D NMR
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spectra, VT studies, HPLC data.
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