Synthesis and biological evaluation of novel gramicidin s analogues

Article abstract of DOI:10.1002/ejoc.200900460

The synthesis of three new analogues of the cyclic cationic antimicrobial peptide Gramicidin S is described. These derivatives contain a modified turn region in which the ^DPhe-Pro motif has been replaced by a constrained furanoid sugar amino ac

Full text of DOI:10.1002/ejoc.200900460

FULL PAPER
DOI: 10.1002/ejoc.200900460
Synthesis and Biological Evaluation of Novel Gramicidin S Analogues
Adriaan Willem Tuin,[a] Dimitrios Konstantinos Palachanis,[a] Annelies Buizert,[a]
Gijsbert Marnix Grotenbreg,[a][‡] Emile Spalburg,[b] Albert J. de Neeling,[b]
Roos H. Mars-Groenendijk,[c] Daan Noort,[c] Gijsbert A. van der Marel,[a]
Herman S. Overkleeft,[a] and Mark Overhand*[a]
Keywords: Antibiotics / Peptidomimetics / Sugar amino acid / Conformation analysis
The synthesis of three new analogues of the cyclic cationic
antimicrobial peptide Gramicidin S is described. These de-
rivatives contain a modified turn region in which the DPhe-
Pro motif has been replaced by a constrained furanoid sugar
amino acid or a flexible linear aminoethoxy acetic acid moi-
ety. Structural analysis revealed conformational changes in
the modified turn region compared to GS. The biological pro-
file of these compounds however resembles that of Gramici-
din S and previously described analogues.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Gramicidin S (GS 1) is a cationic antimicrobial peptide[1] this respect, an interesting finding underscoring the mode
(CAP) that was first isolated from Bacillus brevis by Gause of action of GS is that enantiomeric GS is equally active as
and Brazhnikova.[2] GS is active against a wide range of the natural compound.[4] Clearly, such would not be the
bacteria, both Gram-positive and Gram-negative strains, case when a specific molecule, for instance an integrated
and as such represents an attractive lead compound for the membrane protein, would be the main target of GS.
development of new antibiotic strategies. Unfortunately, GS
itself is highly hemolytic, and its clinical use is therefore
limited to topical applications.[2] Both bacterial and hemo-
lytical activities are rooted in its molecular mode of ac-
tion.[1a] GS acts on the cell membrane and disrupts the in-
tegrity of the lipid bilayer, with cell lysis as a result. Here
lies the difficulty in GS-inspired research towards new anti-
biotic strategies: although highly different in nature and
function, the bacterial cell is not that different from an
erythrocyte from the point of view of the cell membrane.
That GS is at all considered as an attractive starting point
is based on the same molecular mode of action: unlike most
other antibiotic compound classes GS does not target a spe-
cific molecule (protein, DNA or RNA) within the bacterial
cell and thus the development of resistance towards GS and
related CAPs is commonly thought to be less likely.[3] In
Research on GS analogues with improved medicinal
properties is mainly aimed at the development of analogues
that have bactericidal properties inherent to GS, while not
being active against erythrocytes. In practice, this entails the
development of compounds able to distinguish between a
mammalian cell membrane and that of a bacterial cell. This
is a daunting task, firstly because, as said before, there is
not much difference between the two types of lipid bilayers
(and not much information on this issue is available). And
secondly, there is little known about the exact mode of ac-
tion by which GS inserts into a lipid bilayer.[5] Contempo-
rary research on new and improved GS analogues therefore
takes a three-pronged approach, namely: (1) the design and
synthesis of new GS derivatives, (2) the in-depth analysis
of the secondary structure these compounds may adopt in
(aqueous) solution, and (3) the assessment of these com-
pounds on their activities on both bacterial strains and
erythrocytes. Recent years have further witnessed ap-
proaches to study the conformational behavior of GS in the
context of membrane fractions, but no conclusive results
have emerged from these studies yet.[6] Current state-of-the-
art dictates that, whereas the conformational behavior of
GS[7] (and that of quite a few synthetic analogues)[8] in
aqueous solution is known in detail, it is not known
whether, and to what extent, this behavior holds true for
the conformational behavior (and therefore mode of action)
of GS in lipid membranes. X-ray diffraction studies on GS
assemblies further give conflicting results,[7d] and in any
[a] Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden
University,
P.O. Box 9502, 2300 RA Leiden, The Netherlands
Fax: +31-71-527-4307
E-mail: overhand@chem.leidenuniv.nl
[b] Institute of Public Health and the Environment, Research Lab-
oratory for Infectious Diseases,
P.O. Box 1, 3720 BA Bilthoven, The Netherlands
[c] TNO Defense, Security and Safety, Business unit Biological and
Chemical Protection,
P.O. Box, 45, 2280 AA Rijswijk, The Netherlands
[‡] Current address: Immunology Programme and Departments of
Microbiology and Biological Sciences, National University of
Singapore,
Singapore.
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case it is debatable whether such artificial assemblies reflect
With respect to the latter, of specific interest is the re-
those possibly found in lipid bilayers as pore entities. The cently reported structure of 2a,[12] in which one of the two
approach taken in this study is therefore as outlined above, β-turn dipeptides is replaced by a so-called sugar amino
namely: (1) modify GS at specific positions, (2) evaluate acid (SAA) dipeptide isoster 3a.[13] SAAs have attracted
the structure in solution, and (3) measure their biological some interest in recent years due to their ease of access, the
activities.
conformational control they may give to a target peptide,
At the basis of the study presented here is the solution their (putative) protease stability and the number of func-
structure of GS itself, together with that of a number of tional groups that are inherent to the parent carbohydrate
modified structures that have recently appeared in the lit- and that opens ways for further functionalization.[14] Rather
erature.[7,8] GS is a cyclic decapeptide with sequence (Pro- surprisingly, the secondary structure of 2a and 2b, that con-
Val-Orn-Leu-DPhe-)2 and has C2 symmetry. In aqueous tains one copy of SAA 3a and 3b respectively, deviate from
solution, and as measured by proton NMR,[7b] it adopts GS in that the leucine carbonyl adjacent to the SAA amine
an antiparallel β-sheet with the two Val-Orn-Leu stretches is flipped (in comparison to GS) out of the ring, and is not
aligning as two β-strands and the two DPhe-Pro dipeptides involved in hydrogen bonding.[14] Rather, there appears to
as two type II’ β-turn. The cyclic β-hairpin structure is sta- be a hydrogen bond between the SAA-NH and one of the
bilized (see Figure 1) by four interstrand hydrogen bonds oxygen functionalities (C3-OH) attached to the SAA furan
and the molecule adopts a slight twist.[7c] This overall con- core. As a result, the solution structures of 2a and 2b devi-
formational assembly gives GS its amphiphilic nature that ate from that of GS in that, although the β-sheet character
is thought to be the basis for its lytic properties.[9] Here the is to a large extent intact, one of the β-turn regions is drasti-
two basic ornithine residues point to one side of the mole- cally altered and the overall molecule has a much more pro-
cule and the four hydrophobic Val/Leu residues are situated nounced twist around the β-strand. Interestingly, both bac-
at the opposite side. GS analogues of varying nature have terial and hemolytical activities of GS and 2b (R = Bn) are
been reported, including extended homologues,[9] deriva- comparable and a tentative conclusion from these studies
tives featuring different α-amino acids[10] and derivatives in- may be that some conformational freedom in the amphi-
corporating amino acid analogues.[11] Modifications may be philic structure is allowed, and possibly may be capitalized
thus brought to both β-strand and β-turn regions.
upon in the development of species-specific GS analogues.
Figure 1. Gramicidin S and analogues thereof in which the DPhe-Pro motif (highlighted in GS) has been replaced by different dipeptide
isosters.
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Novel Gramicidin S Analogues
Incidentally, the hydrophobic nature of the turn region is tion of the tritylated alcohol under slightly acidic condi-
much more important: earlier studies proved that the analo- tions (13 Ǟ 14, 70%) and oxidation of the primary alcohol
gous compound 2a, without a benzyl group at a position to the carboxylic acid under classical TEMPO conditions
corresponding to DPhe in the GS structure, has no activity (14 Ǟ 15, 80%) followed by Boc removal and diazotransfer
at all, even though its solution structure is highly similar to using freshly prepared triflic azide afforded SAA 4 in 77%
that of 2b.[12,14]
yield.
The subject of this paper is to further study to what ex-
Azidoethoxy acetic acid (AAA) 5 was synthesized as fol-
tent the SAA design in 3 is amenable to modification. Three lows (Scheme 2). Commercially available (S)-glycidol 16
analogues are presented here, of which the first (compound was selectively azidolyzed with NaN3 in wet tBuOH under
7, see Figure 1) lacks the C3-OH group involved in hydro- Lewis acid catalysis according to the literature procedure,[15]
gen bonding and the other two (8 and 9, see Figure 1) are affording diol 18 in 49% yield. Tin-ketal formation followed
rendered more flexible through the incorporation of linear by selective alkylation towards the primary benzyl ether
dipeptide isosters. The synthesis of these molecules, and the yielded 20 in 62% yield. Alkylation using tert-butyl bromo-
SAA building blocks on which they are based, are pre- acetate (20 Ǟ 22, 64% yield) and acidic deprotection re-
sented. Further, their conformational behavior in solution sulted in formation of 5 in 72% yield. The stereoisomeric 6
and their bactericidal/hemolytical activities are discussed, was obtained following the same sequence of reactions
taking into consideration the data known on GS and ana- starting from (R)-glycidol in comparable overall yield.
logues 2a and 2b.
The synthesis of GS analogues 7–9 is exemplified by the
The synthesis of SAA 4 commenced from the known in- synthesis of 7 and is depicted in Scheme 3. Commercially
termediate 10[14] of which the primary alcohol was selec- available polystyrene based resin, preloaded with Fmoc-
tively protected using trityl chloride in pyridine to give com- Leu, attached via the hyper acid labile benzhydrilamine/4-
pound 11 in 68% yield (Scheme 1). Conversion of the azide hydroxymethyl-3-(methoxyphenoxy)butanoic acid linker
group in 11 into the more compatible Boc-protective group was elongated with ornithine, valine, proline, -phenylala-
yielded compound 12 in 93% yield. Next, Barton deoxygen- nine, leucine, ornithine and valine using repetitive cycles of
ation using thiocarbonyldiimidazole, AIBN and Bu3SnH standard Fmoc-based SPPS as depicted in Scheme 3. Cou-
gave deoxygenated compound 13 in 74% yield. Deprotec- pling of SAA 4 was performed with a 1.5-fold excess of
Scheme 1. Reagents and conditions: (i) TrtCl (1.5 equiv.), pyridine, 16 h, 0 °C, 68%. (ii) a: PMe3 (1  soln); toluene, 2 equiv., 0 °C, THF,
1 h; then H2O 2 equiv., 6 h, b: Boc2O (1.5 equiv.), DiPEA (2 equiv.) 16 h, 93% over two steps. (iii) a: 1,1-thiocarbodiimidazole (4 equiv.),
90 °C, toluene, 5 h, b: AIBN (0.2 equiv.), Bu3SnH (4 equiv.), 80 °C, toluene, 4 h, 74% over two steps. (iv) pTsOH (0.5 equiv.), 40 °C, 10%
MeOH/DCM 6 h, 70%. (v) TEMPO (cat.), KBr (0.1 equiv.), NaOCl (2 equiv.), ACN, 80%. (vi) a: 50% TFA/DCM, b: TfN3 (5 equiv.),
CuSO4 (0.1 equiv.), K2CO3 (3 equiv.), MeOH/H2O, 77% over two steps.
Scheme 2. Reagents and conditions: (i) NaN3 (1.2 equiv.) LiBF4 (0.2 equiv.), tBuOH/H2O (5:1), 49% 18; 44% 19; (ii) a: Bu2SnO
(1.5 equiv.) 100 °C, toluene, 3 h, b: BnBr (2 equiv.) 100 °C, toluene, 16 h, 62% 20; 28% 21; (iii) NaH (60% suspension in mineral oil,
1.2 equiv.), tert-butyl bromoacetate (2 equiv.), 0 °C to room temp. DMF, 16 h, 64% 22; 40% 23; (iv) TFA/DCM/TIS, 50:50:1. 72% 5;
86% 6.
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M. Overhand et al.
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Scheme 3. Reagents and conditions: (i) 8 cycles of a: 20% pip/NMP, 3ϫ5 min; b: Fmoc-AA-OH (5 equiv.), HCTU (5 equiv.), DiPEA
(10 equiv.) 90 min. (ii) a: 20% pip/NMP, 3ϫ5 min. b: 4, 5 or 6 (1.5 equiv.), HCTU (1.5 equiv.), DiPEA (3 equiv.), 16 h. (iii) a: PMe3 (1 
in 9:1 THF/H2O, 25 equiv.), 16 h. b: 1% TFA/DCM. (iv) a: PyBOP (5 equiv.), HOBt (5 equiv.), DiPEA (10 equiv.), 16 h. b: 50% TFA/
DCM, 1 h.
SAA with respect to the resin-loading and the coupling
Structural analysis of the newly synthesized analogues
time was extended to 16 h affording 25. On-resin Staud- was performed by extensive 1D and 2D NMR experiments.
1
inger reduction using excess PMe3 in wet THF[16] followed From the H NMR, all the NH-coupling constants could
by acidic cleavage using 1% TFA/DCM yielded linear pro- be determined and compared to those obtained from native
tected nonamer 26. Cyclization under dilute conditions GS (1) and 2b (Figure 2, A). The presence of a β-turn con-
(10 m in DMF) using PyBOP/HOBt and DiPEA and formation around -Phe5 was evidenced in all analogues by
acidic deprotection (50% TFA/DCM) afforded GS ana- its small coupling constant[17] (3JNH-Hα Ͻ 4 Hz) and a nega-
logue 7 in 20% after HPLC purification. Analogues 8 and tive value for the chemical shift perturbation of the α-pro-
9 were prepared in a similar fashion.
ton of the -Phe and Pro residues.[18]
3
Figure 2. A: JNH-Hα of GS, 2b and 7–9; B: chemical shift perturbation of GS, 2b and 7–9.
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Novel Gramicidin S Analogues
(Figure 2, B). For 7, the remaining coupling constants a minimal inhibitory concentration (MIC) of 16 µg/mL.
are similar to both native GS and 2b, indicating a compar- The GS analogues 7, 8 and 9 are potent against gram-posi-
able overall β-sheet structure[19] (8.5 Hz Ͻ 3JNH-Hα Ͻ 9 Hz), tive strains with MIC values between 8 and 32 µg/mL.
except Val2 and Orn8 which are less evident. The coupling Compound 8 is about as potent as GS, 7 and 9 are about
constants and chemical shift perturbation of 8 and 9 indi- half as potent.
cate a similar structure of these two analogues.
The hemolytic activity of the GS analogues was deter-
A close inspection of the amide region in the NOE spec- mined and the results are plotted in Figure 4. Analogues 7
trum of 7 (see Figure 3) reveals, besides numerous sequen- and 8 are about as toxic towards human erythrocytes as GS
tial NOEs, several interstrand NOEs indicating the presence and 2b both showing 100% hemolysis at 125 µ peptide
of a β-sheet character in this analogue. Interestingly, NH- concentration. Compound 9 shows a reduced hemolytical
NH NOE cross correlations were found between the amide activity with only 90% lysis at a peptide concentration of
protons of SAA and Leu9 (A, in Figure 3), the SAA and 500 µ.
the Val2 amide protons (B in Figure 3) and between the
NH protons of Val2 and Leu9 similar to those found for 2a
and 2b which is indicative for the same altered orientation
of the SAA amide proton. These results clearly indicate that
a hydrogen bond between the SAA amide and the hydroxy
group is not the determining factor for the observed re-
orientation of the Leu - SAA amide bond in 2a and 2b. A
stabilizing effect of this hydrogen bond can of course not
be ruled out. It does, however, implicate the involvement of
conformational restrictions imposed by the furanoid ring as
major factor in the amide re-orientation. This conclusion is
substantiated by results obtained from flexible analogues 8
and 9 where the NOE spectra lack the NOE cross corre-
lation signals between the AAA and Val2 amide protons.
Figure 4. Hemolytical activity of GS and analogues thereof.
Conclusions
Three novel dipeptide isosters were successfully synthe-
sized and used to replace the -Phe/Pro turn motif of
Gramicidin S. A cyclic SAA was designed without a hy-
droxy group on C3 that could possibly function as a hydro-
gen-bond-accepting moiety that could stabilize the re-orien-
tated backbone conformation found in GS analogue 2a and
2b. Two stereoisomeric acyclic dipeptide isosters were also
synthesized and incorporated into GS. All three GS ana-
logues were analyzed by means of 1D- and 2D-NMR ex-
periments to investigate their structural conformation in or-
Figure 3. Turn region of 7 and part of the amide region of the
NOE spectrum of 7.
Next, the newly synthesized analogues were assessed for ganic solvent. In the case of 7, containing the cyclic SAA
their antibacterial activity and compared to GS and 2b. The 4, the backbone showed a similar re-orientation as was
results are depicted in Table 1. Both the Gram-negative bac- found in 2a and 2b. The backbone conformations of the
terial strains used in this assay, E. coli and P. aeruginosa, acyclic dipeptide isoster containing GS analogues 8 and 9
were largely unaffected by the presence of any of the pep- were not distorted and resembled the conformation found
tides. Compound 8 was most effective against E. coli with in native GS.
Table 1. MIC values for 1, 2b and 7–9.
Gram pos.
Staph. aureus
Gram pos.
Staph. epidermis
Gram pos.
Entrerococ. faecalis E. coli
Gram neg.
Gram neg.
P. auruginosa
Gram pos.
Bacillus ereus
1
2b
7
8
9
8
8
8
16
8
8
8
Ͼ64
32
64
16
32
Ͼ64
Ͼ64
Ͼ64
64
8
8
16
8
8
16
16
8
32
32
16
32
16
Ͼ64
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prior to measurements with a calibration mixture (Thermo Finni-
gan). 1H- and 13C-NMR spectra were measured on a Joel JNM-
FX-200 (200/50 MHz). Chemical shifts are given in ppm (δ) relative
to TMS (δ = 0 ppm) or MeOD (δ = 3.30 ppm) and coupling con-
stants are given in Hz. Optical rotations were measured on a propol
automatic polarimeter.
The biological evaluation revealed that 7 was similarly
active as 2b and slightly less active compared to GS in both
the bacterial and hemolytical assays. Replacing the β-turn
motif in GS with a flexible dipeptide isoster resulted in ana-
logues with a similar (8) or slightly decreased (9) activity
towards both bacterial strains as well as erythrocytes de-
pending on the stereochemistry of the aromatic moiety in
the dipeptide isoster. Compound 8 shows a slight reduction
in toxicity against human erythrocytes compared to GS.
However, a similar decrease in potency was observed in the
antibacterial assay. The stereoisomeric analogue 9 pos-
sessed reduced bactericidal potency combined with a
marked decrease in hemolytical activity. These findings
indicate that the distorted backbone conformation of GS,
imposed by the cyclic nature of furanoid SAAs 2b and 4, is
a determining factor in the decreased antibacterial activity.
However, the presence of a linear flexible turn moiety as in
compounds 8 and 9 is more detrimental with respect to the
antibacterial activity. Although the precise correlation be-
tween the increased conformational freedom in the modi-
fied turn region in 8 and 9 and the decreased biological
activity remains unidentified, these results demonstrate the
potential of peptide isosters as elements to replace second-
ary structural elements in biologically active peptides in an
attempt to optimize the behavior in biological surroundings.
General Procedure for Peptide Synthesis. (a) Stepwise Elongation:
Fmoc-Leu-HMPB-BHA resin 6 (196 mg, 0.51 mmol/g, 0.1 mmol)
was submitted to seven cycles of Fmoc solid-phase synthesis with
the appropriate commercially available amino acid building blocks
Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-DPhe-
OH, as follows: a) deprotection with piperidine/NMP (1:4, v/v,
5 mL, 15 min); b) washing with NMP (5 mL, 3ϫ , 3 min); c) cou-
pling of the appropriate Fmoc amino acid (5 equiv., 0. 5 mmol) in
the presence of HCTU (5 equiv., 0.5 mmol, 206 mg) and DiPEA
(10 equiv., 1 mmol, 162 µL) which was preactivated for 2 min in
NMP (5 mL) and shaken for 90 min; d) washing with NMP (5 mL,
3ϫ , 3 min). Couplings were monitored for completion by the Kai-
ser test.[21] Finally, the N-terminal amine was liberated by Fmoc-
deprotection with piperidine/NMP (1:4, v/v, 5 mL, 15 min) fol-
lowed by washing with NMP (5 mL, 3ϫ , 3 min). Coupling of SAA
4 was performed as follows: To the resin bound peptide, a preacti-
vated solution of SAA 4 (1.5 equiv. 44 mg, 0.150 mmol), HCTU
(1.5 equiv., 62 mg, 0.150 mmol) and DiPEA (3.0 equiv., 74 µL,
0.45 mmol) in NMP (3 mL) was added and the resulting suspension
was shaken for 16 h. The resin was finally washed with NMP
(5 mL, 3ϫ , 3 min) to give the azide protected linear nonapeptide.
(b) On-Resin Staudinger Reduction: The resin-bound azide is
treated with a pre-mixed cocktail of H2O (0.5 mL) and PMe3
(3.5 mL, 1  in THF) and shaken for 16 h. The resin is washed
with methanol (4 mL, 3ϫ , 3 min) and DMF (4 mL, 3ϫ , 3 min.)
Experimental Section
General: PE with a boiling range of 40–60 °C was used. THF and
Et2O were distilled from LiAlH4 prior to use. DCM was distilled
from CaH2 prior to use. All other solvents used under anhydrous
conditions were stored over molecular sieves (4 Å) except for meth-
anol, which was stored over molecular sieves (3 Å). Solvents used
for work-up and column chromatography were of technical grade
and distilled before use. Unless stated otherwise, solvents were re-
moved by rotary evaporation under reduced pressure at 40 °C. Re-
actions were monitored by TLC-analysis using DC-fertigfolien
(Schleicher & Schuell, F1500, LS254) with detection by spraying
with 20% H2SO4 in EtOH, (NH4)6Mo7O24·4H2O (25 g/L) and
(NH4)4Ce(SO4)4·2H2O (10 g/L) in 10% sulfuric acid or by spraying
with a solution of ninhydrin (3 g/L) in EtOH/AcOH (20:1 v/v), fol-
lowed by charring at about 150 °C. Column chromatography was
performed on Fluka silica gel (0.04–0.063 mm). For LC/MS analy-
sis, an JASCO HPLC-system (detection simultaneously at 214 and
(c) Cleavage from the Resin: Resin-bound nonapeptide is repeatedly
treated with 1% TFA/DCM (4 mL). The filtrate was coevapporated
with toluene (3ϫ) and used as such in the cyclization step.
(d) Cyclization: The linear nonapeptide was taken up in DMF
(5 mL) and added dropwise over the course of one hour to a solu-
tion of benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexa-
fluorophosphate (PyBOP) (5 equiv., 270 mg, 0.5 mmol), HOBt
(5 equiv., 67 mg, 0.5 mmol) and DiPEA (15 equiv., 254 µL,
1.5 mmol) in DMF (70 mL) and stirred for 16 h. The solvent was
removed in vacuo and the resulting mixture was used without fur-
ther purification in the deprotection step.
(e) Deprotection: The crude cyclised peptide was treated with 50%
TFA/DCM (10 mL) for 1 h before it was concentrated and purified
by HPLC purification.
254 nm)
equipped
with
an
analytical
C18
column
(2S,3R,4S,5R)-5-(Azidomethyl)-4-(benzyloxy)-2-(trityloxymethyl)-
tetrahydrofuran-3-ol (11): Diol 10 (1.11 g, 2.13 mmol) was dissolved
(4.6 mmϫ250 mm, 5 µ particle size) in combination with buffers
A: H2O, B: MeCN and C: 0.5% aq. TFA and coupled to a mass in pyridine (11 mL) and cooled to 0 °C. Trityl chloride (0.89 g,
instrument with a custom-made Electronspray Interface (ESI) was 3.2 mmol, 1.5 equiv.) was added at 0 °C. The reaction mixture was
used. For reversed-phase HPLC purification of the final com-
pounds, an automated HPLC system supplied with a semi-prepera-
tive C18 column (10.0 mmϫ250 mm, 5µ particle size) was used.
The applied buffers were A: H2O, B: MeCN and C: 1.0% aq. TFA.
High resolution mass spectra were recorded by direct injection
(2 µL of a 2 µ solution in water/acetonitrile; 50:50; v/v and 0.1%
formic acid) on a mass spectrometer (Thermo Finnigan LTQ Or-
bitrap) equipped with an electrospray ion source in positive mode
(source voltage 3.5 kV, sheath gas flow 10, capillary temperature
250 °C) with resolution R = 60000 at m/z 400 (mass range m/z =
150–2000) and dioctyl phthalate (m/z = 391.28428) as a “lock
mass”.[20] The high resolution mass spectrometer was calibrated
stirred for 18 h and when TLC analysis (3:20 v/v EtOAc in PE)
indicated complete conversion of the diol into a higher running
product, the reaction mixture was evaporated to dryness. The resi-
due was taken up in EtOAc, washed with satd. aq. NaHCO3 and
Brine, dried with Na2SO4, filtered and the solvents evaporated. Pu-
rification was performed by column chromatography (mixture of
eluents: EtOAc/PE, 0:1 Ǟ 3:20 v/v) and afforded compound 11
(0.75 g, 1.44 mmol, 68 % yield) as a yellow syrup. 1H NMR
(200 MHz, CDCl3): δ = 7.82–7.11 (m, 20 H, HAr), 4.69 (d, J =
11.8 Hz, 1 H, CH2Ph), 4.57 (d, J = 11.8 Hz, 1 H, CH2Ph), 4.30 (m,
1 H, CHOH), 4.17 (d, J = 4.9 Hz, 1 H, CHOTrt), 4.04 (d, J =
3.7 Hz, 1 H, CHOTrt), 3.84–3.76 (m, 1 H, CHOPh), 3.60–3.34 (m,
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Novel Gramicidin S Analogues
3 H, CH2N3, OCHCCH2OTrt, OCHCH2N3), 3.18 (d, J = 5.7 Hz, 17 mmol) was diluted in a mixture of DCM/MeOH (9:1 v/v,
1 H, CH2N3)2.36 (s, 1 H, OH) ppm. 13C NMR (50 MHz, CDCl3): 106 mL), heated up to 40 °C, pTsOH (10.2 mmol, 1.94 g) was
δ = 147.94 (CqAr), 142.05 (CqAr), 133.04 (CHAr), 132.55 (CHAr), added and the system was heated under reflux for 6 h. When TLC
132.46 (CHAr), 132.38 (CHAr), 132.23 (CHAr), 131.75 (CHAr), analysis (1:5 v/v EtOAc in PE) indicated completion, satd. aq.
131.68 (CHAr), 91.88 (CqTrt), 91.21 (CH2OBn), 86.59 (OCCH2- NaHCO3 and brine were added, the aqueous layer was washed with
OTrt), 84.62 (OCCH2N3), 81.06 (CHOH), 76.37 (OCH2Ph), 66.84 diethyl ether (5ϫ) and the organic layer was dried with MgSO4,
(CH2OTrt), 57.52 (CH2N3) ppm.
filtered and the solvents evaporated. Purification by silica gel col-
umn (eluents: 2:5 Ǟ 3:5 v/v EtOAc in PE) yielded 14 (4 g,
11.9 mmol, 70%) as a colorless oil. H NMR (200 MHz, CDCl3):
tert-Butyl [(2R,3S,4R,5S)-3-(Benzyloxy)-4-hydroxy-5-(trityloxy-
methyl)tetrahydrofuran-2-yl]methylcarbamate (12): Tritylated 11
(5.54 g, 10.64 mmol) was co-evaporated with toluene (2ϫ), re-dis-
solved in THF (55 mL), cooled to 0 °C under argon and PMe3
[21.3 mL (1  solution in toluene), 2 equiv.] was added. The reac-
tion mixture was stirred for 1 h and then H2O (0.38 mL,
21.3 mmol, 2 equiv.) was added, after which stirring was continued
for another 6 h. After that, the reaction mixture was concentrated,
co-evaporated with toluene (2ϫ), re-dissolved in toluene (50 mL)
and cooled to 0 °C and DiPEA (2.75 g, 3.5 mL, 21.3 mmol,
2 equiv.) and Boc2O (3.5 g, 15.96 mmol, 1.5 equiv.) were added.
The reaction was stirring for 12 h at room temp., EtOAc was added
and the organic layer was washed with satd. aq. NaHCO3, NH4Cl
and brine, dried with Na2SO4, filtered and concentrated. Protected
amine 12 was isolated by column chromatography (eluents: 1:20 Ǟ
3:20 v/v EtOAc in PE) (5.89 g, 9.9 mmol, 93% yield) as a white
foam. 1H NMR (200 MHz, CDCl3): δ = 7.68–7.01 (m, 20 H, HAr),
4.98 (br. s, 1 H, NHBoc), 4.64 (d, J = 2.0 Hz, 2 H, CH2Ph), 4.32
(dd, J = 0.7, 4.6 Hz, 1 H, CHOH), 4.21–4.05 (m, 1 H, CH2OTrt),
3.97 (m, 1 H, CH2OTrt), 3.77 (dd, J = 1.6, 3.7 Hz, 1 H, CHOPh),
3 . 6 1– 3 . 25 (m , 4 H , O CHCH2 OTr t , OCHCH2 NHBoc,
CH2NHBoc), 1.42 (s, 9 H, 3ϫ CH3) ppm. 13C NMR (50 MHz,
CDCl3): δ = 155.96 (C=O), 143.31 (CqAr), 137.49 (CqAr), 128.35
(CHAr), 128.30 (CHAr), 127.83 (CHAr), 127.65 (CHAr), 127.51
(CHAr), 127.05 (CHAr), 87.17 (CqTrt), 86.60 (CHOBn), 81.81
(OCHCH2NHBoc), 79.60 (OCHCH2OTrt), 79.10 (CqBoc), 76.51
(CHOH), 71.60 (CH2Ph), 62.32 (CH2OTrt), 42.84 (CH2NHBoc),
28.23 (CH3Boc) ppm.
1
δ = 7.39–7.28 (m, 5 H, HAr), 4.84 (br. s, 1 H, NHBoc), 4.52 (s, 2
H, CH2Ph), 4.29 (m, 1 H, CH2OH), 4.21–4.08 (m, 1 H, CH2OH),
4.08–3.86 (m, 3 H, CHOBn, OCHCH2NHBoc, CH2NHBoc), 2.03–
1.97 (m, 2 H, CCH2CHOBn), 1.44 (s, 9 H, CH3Boc) ppm. 13C
NMR (50 MHz, CDCl3): δ = 156.43 (C=O), 137.66 (CqPh),
128.17–127.35 (CHAr), 83.53 (CHOBn), 81.13 (OCHCH2NHBoc),
79.37 (OCHCH2OH), 79.20 (CqBoc) 70.94 (CH2OBn), 63.03
(CH2OH), 43.26 (CH2NHBoc), 32.37 (CCH2COBn), 28.09
(CH3Boc) ppm. MS: m/z = 338.2 [M + H]+, 282.1 [M – tBu + H]
+, 238.1 [M – Boc + H]+.
(2S,4S,5R)-4-(Benzyloxy)-5-[(tert-butoxycarbonylamino)methyl]te-
trahydrofuran-2-carboxylic Acid (15): Alcohol (14) (2.4 g, 7 mmol)
was diluted in a solution of TEMPO in CH3CN (18.2 mg in 14 mL)
and KBr in satd. NaHCO3 (88 mg, 0.7 mmol in 18.2 mL) and co-
oled to 0 °C. 87 mL of a solution of NaOCl (31 mL), NaHCO3
(19.6 mL) and NaCl (36.4 mL) was slowly added over a 1 h period,
after which there was no more change of color with extra addition
of the solution. TLC analysis (4:5 v/v EtOAc in PE) indicated com-
pletion of the reaction and the reaction mixture was quenched with
MeOH, after which it was washed with DCM and the aqueous
layer was acidified with 1  HCl solution to pH 6, washed with
DCM (4ϫ), dried with MgSO4, filtered and the solvents evapo-
rated. Compound 15 was retrieved as a yellow solid (1.96 g,
1
5.6 mmol, 80%). H NMR (CDCl3): δ = 7.27–7.09 (m, 5 H, HAr),
5.56 (br. s, 1 H, NHBoc), 4.69 (m, 1 H, CHCO2H), 4.61, (s, 2 H,
CH2Ph), 4.14 (m, 1 H, CHOBn), 3.91 (m, 1 H, OCHCH2NHBoc),
3.22 (CH2NHBoc), 2.44 (CCH2CHOBn), 2.07 (CCH2CHOBn),
1.41 (s, 9 H, CH3Boc) ppm. 13C NMR (CDCl3): δ = 175.22
(CO2H), 156.84 (C=OBoc), 137.25 (CqPh), 128.70–127.30 (CHAr),
84.61, (OCHCH2NHBoc), 79.60 (CHCO2H), 76.05 (CHOBn),
70.96 (CH2Ph), 42.61 (CH2NHBoc), 35.81 (CCH2COBn), 27.96
(CH3Boc) ppm. MS: m/z = 352.1 [M + H]+, 252.1 [M – Boc +
H]+.
tert-Butyl [(2R,3S,5R)-3-(Benzyloxy)-5-(trityloxymethyl)tetra-
hydrofuran-2-yl]methylcarbamate (13): Protected amine (12) (0.18 g,
0.3 mmol) was co-evaporated with toluene (1ϫ), 1,1-thiocarbonyl-
diimidazole (0.23 g, 1.2 mmol, 4 equiv.) was added, the reaction
mixture was heated to 90 °C for 5 h using an oil bath. EtOAc was
added and the organic layer was washed with demi water (3ϫ),
dried with Na2SO4, filtered and the solvents evaporated. The resi-
due was co-evaporated with toluene (2ϫ), re-dissolved in toluene
and AIBN (9.8 mg, 0.06 mmol, 0.2 equiv.) was added. Argon was
bubbled through the flask for 15 min at 80 °C and then Bu3SnH
(0.27 g, 0.244 mL, 0.92 mmol, 4 equiv.) was added. TLC analysis
indicated completion of the reaction after 4 h and the mixture was
concentrated. The residue was purified by column chromatography
(eluents: 1:20 Ǟ 2:10 v/v EtOAc in PE) yielding compound 13
(0.13 g, 0.22 mmol, 74%) as a colorless oil. 1H NMR (200 MHz,
CDCl3): δ = 7.58–7.11 (m, 20 H, HAr), 4.92–4.75 (br. s, 1 H,
NHBoc), 4.52 (s, 2 H, CH2Ph), 4.34 (m, 1 H, CH2OTrt), 4.23–3.87
(m, 2 H, CH2OTrt, CHOBn), 3.37 (m, 1 H, OCHCH2NHBoc), 3.17
(m, 3 H, OCHCH2OTrt, CH2NHBoc), 1.95–1.77 (m, 2 H,
CCH2CHOBn) 1.42 (s, 9 H, CH3Boc) ppm. 13C NMR (CDCl3,
50 MHz): δ = 154.64 (C=O), 143.83 (CqAr), 137.81 (CqAr), 128.56
(CHAr), 128.30 (CHAr), 127.64 (CHAr), 127.56 (CHAr), 127.49
(CHAr), 126.84 (CHAr), 86.39 (CqTrt), 82.94 (CHOBn), 80.66
(OCHCH2HBoc), 79.12 (CqBoc), 77.77 (OCHCH2OTrt), 71.18
(CH2 OTrt), 65.78 (CH2 Ph), 43.23 (CH2 NHBoc), 34.67
(CH2CHOBn), 28.23 (CH3Boc) ppm.
(2R,4S,5R)-5-(Azidomethyl)-4-(benzyloxy)tetrahydrofuran-2-carb-
oxylic Acid (4): Boc-protected amine 15 (175 mg, 0.5 mmol) was
treated with 50% TFA/DCM (2 mL) for 30 min before it was con-
centrated. Residual traces of TFA were removed by repeated coev-
apporation with toluene (5ϫ2 mL) to yield the crude TFA-salt
which was dissolved in H2O (1.6 mL). To this solution K2CO3
(207 mg, 1.5 mmol, 3 equiv.) was added, followed by a solution of
CuSO4 (8 mg, 0.05 mmol, 0.1 equiv.) in H2O (0.5 mL) and MeOH
(3.2 mL). To this homogeneous solution, freshly prepared TfN3
(5 mmol) in DCM (7 mL) was added and the reaction was allowed
to stir for 16 h before the reaction was diluted with DCM (10 mL)
and H2O (10 mL) and the aqueous phase washed with DCM
(2ϫ5 mL). The aqueous phase was acidified to pH = 2 by addition
of satd. aq. citric acid and extracted with DCM (3ϫ5 mL) to yield
1
the title compound (107 mg, 0.39 mmol, 77%) as colorless oil. H
NMR (200 MHz, MeOD): δ = 10.09 (s, 1 H, CO2H), 7.44–7.17 (m,
5 H), 4.71 (t, J = 7.9 Hz, 1 H, CHCO2H), 4.54 (d, J = 5.4 Hz,
2 H, CH2Ph), 4.26–4.16 (m, 1 H, CHOBn), 4.10–4.00 (m, 1 H,
OCHCH2N3), 3.46 (dd, J = 1.4, 4.7 Hz, 2 H, CH2N3), 2.46 (ddd,
J = 3.4, 7.4, 13.4 Hz, 1 H, CCH2CHOBn), 2.23 (ddd, J = 5.1, 7.2,
8.4 Hz, 1 H, CCH2CHOBn) ppm. 13C NMR (50 MHz, MeOD): δ
tert-Butyl [(2R,3S,5R)-3-(Benzyloxy)-5-(hydroxymethyl)tetra-
hydrofuran-2-yl]methylcarbamate (14): De-oxygenated (13) (9.9 g,
Eur. J. Org. Chem. 2009, 4231–4241
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4237

M. Overhand et al.
FULL PAPER
= 176.10 (CO2H), 137.16 (CqPh), 128.54 (CHAr), 128.05 (CHAr), (CH2OH), 54.09 (CH2N3) ppm. [α]2D0 = –17.4 (c = 1.00, in MeOH)
127.70 (CHAr), 83.70 (OCHCH2NHBoc), 79.60 (CHCO2H), 76.41
(R)-1-Azido-3-(benzyloxy)propan-2-ol (20): Diol 17 (1.15 g,
9.8 mmol) was co-evaporated with toluene (3ϫ25 mL) and dis-
solved in toluene (25 mL) before addition of Bu2SnO (3.6 g,
15 mmol, 1.5 equiv.). The reaction mixture was stirred at 100 °C
for 3 h, before it was concentrated. The residue was coevapporatd
with toluene (3ϫ25 mL) and redissolved in toluene (25 mL). To
this solution, benzyl bromide (2.4 mL, 20 mmol 2 equiv.) was
added and reaction stirred at 100 °C until TLC analysis (25%
EtOAc/PE) indicated the formation of a higher running product.
The reaction mixture was concentrated, the residue partitioned be-
tween EtOAc/H2O and the aqueous phase extracted with EtOAc
(3ϫ25 mL). The combined organic phase was washed with satd.
aq. sodium hydrogen carbonate (25 mL) and brine (25 mL), dried
with Na2SO4, filtered and concentrated. The residue was purified
by silica column chromatography (0–8% EtOAc/PE) to yield the
(CHOBn), 71.67 (CH2Ph), 52.38 (CH2N3), 35.91 (CH3Boc) ppm.
MS: m/z = 555.2 [2M + H]+, 277.1 [M + H]+, 250.1 [M – N2
+
H]+.
(R)-3-Azidopropane-1,2-diol (18): A solution of (S)(–)-glycidol 16
(1.26 mL, 20 mmol) in tBuOH (100 mL) was charged with a solu-
tion of LiBF4 (375 mg, 4 mmol) in H2O (4 mL) and NaN3 (1.6 g,
25 mmol) and stirred for 5 h at 70 °C before the reaction mixture
was concentrated. The residue was directly applied to a silica col-
umn and eluted with 5% MeOH/EtOAc to afford the title com-
pound (1.15 g, 9.8 mmol, 49%) as colorless oil. 1H NMR
(200 MHz, MeOD): δ = 3.85–3.68 (m, 1 H, CCHOH), 3.53 (s, 1
H, OH), 3.34–3.29 (m, 4 H, CH2N3, CH2OH) ppm. 13C NMR
(50 MHz, MeOD): δ = 71.98 (CCHOH), 64.20 (CH2OH), 54.20
(CH2N3) ppm. [α]2D0 = +12.2 (c = 1.00, in MeOH)
1
(S)-3-Azidopropane-1,2-diol (19): Diol 19 was prepared similarly to
18 and obtained as colorless oil (1.06 g, 9.1 mmol, 44%). 1H NMR
title compound (1.25 g, 6.0 mmol, 62%) as colorless oil. H NMR
(200 MHz, CDCl3): δ = 7.59–7.13 (m, 5 H, HAr), 4.87 (br. s, 1 H,
(200 MHz, MeOD): δ = 3.91–3.70 (m, 1 H, CCHOH), 3.52 (s, 1 OH), 4.53 (s, 2 H, CH2Ph), 3.99–3.78 (m, 1 H, CHOH), 3.55–3.44
H, OH), 3.37 (m, 2 H, CH2OH), 3.34–3.28 (m, 2 H, CH2N3) ppm.
(m, 2 H, CH2OBn), 3.36–3.24 (m, 2 H, CH2N3) ppm. 13C NMR
(50 MHz, CDCl3): δ = 139.27 (CqPh), 129.46 (CHAr), 128.86
13C NMR (50 MHz, MeOD):
δ = 72.05 (CCHOH), 64.29
1
Table 2. H NMR + NOE (500 MHz, CD3OH) of compound 7.[a]
Atom 1
Chemical shift (m, J [Hz])
Atom 2
Atom 1
Chemical shift (m, J) Atom 2
Val2 NH
7.75 (d, 7.4)
SAA βH (w)
Leu9 NH (w)
Val7 NH
7.63 (d, 9.2)
Pro6 δH (m)
Val2 αH
Val2 βH
Val2 γH
Val2 γHЈ
Orn3 NH
4.01 (t, 7.6)
2.20 (m)
1.03 (d, 6.7)
0.97 (d, 6.7)
8.68 (d, 7.4)
Val7 αH
Val7 βH
Val7 γH
Val7 γHЈ
Orn8 NH
4.28 (m)
2.26 (m)
0.93 (d, 6.7)
0.92 (d, 6.7)
8.49 (d, 9.4)
Val2 αH (s)
Val2 βH (m)
Val2 γH (w)
Val7 αH (s)
Val7 βH (m)
Orn3 αH
Orn3 βH
Orn3 βHЈ
Orn3 γH
Orn3 γHЈ
Orn3 δH
Orn3 δHЈ
Orn3 NH2
Leu4 NH
4.80 (m)
2.02 (m)
2.02 (m)
1.75 (m)
1.75 (m)
3.04 (m)
2.93 (m)
Orn8 αH
Orn8 βH
Orn8 βHЈ
Orn8 γH
Orn8 γHЈ
Orn8 δH
Orn8 δHЈ
Orn8 NH2
Leu9 NH
4.91 (m)
1.62 (m)
1.62 (m)
1.50 (m)
1.50 (m)
2.90 (m)
2.90 (m)
8.70 (d, 9.0)
Orn3 αH (s)
Val7 NH (w)
8.79 (d, 9.1)
Orn8 αH (s)
Leu4 αH
Leu4 βH
Leu4 βHЈ
Leu4 γH
Leu4 δH
Leu4 δHЈ
dPhe5 NH
dPhe5 αH
4.64 (dd, 7.5, 15.8)
1.55 (m)
1.55 (m)
1.42 (m)
0.91 (m)
0.91 (m)
8.91 (d, 3.1)
4.55 (m)
Leu9 αH
Leu9 βH
Leu9 βHЈ
Leu9 γH
Leu9 δH
Leu9 δHЈ
4.52 (m)
1.62 (m)
1.62 (m)
1.47 (m)
0.87 (m)
0.86 (m)
SAA αHЈ (m)
SAA δH (w)
Leu4 αH (s)
Pro6 δH (s)
Pro6 δHЈ(m)
SAA NH
8.27 (t, 6.2)
Leu9 αH (s)
Val2 αH (s)
dPhe5 βH
dPhe5 βHЈ
dPhe5 HAr
Pro6 αH
Pro6 βH
Pro6 βHЈ
Pro6 γH
Pro6 γHЈ
Pro6 δH
3.08 (m)
2.96 (m)
7.24–7.34 (m)
4.36 (d, 7.5)
2.00 (m)
2.00 (m)
1.66 (m)
1.66 (m)
3.73 (m)
SAA αH
SAA αHЈ
SAA βH
SAA γH
SAA δH
SAA δHЈ
SAA εH
SAA CH2Ph
SAA HAr
4.29 (m)
4.09 (d, 5.2)
3.79 (dd, 6.3, 15.9)
3.07 (m)
2.38 (dd, 5.2, 13.1)
1.54 (m)
4.44 (dd, 5.4, 11.2)
4.54 (d, 4.8)
7.24–7.34 (m)
Pro6 δHЈ
2.49 (m)
1
[a] Columns 1 and 2 contain the interpretation of the H NMR spectroscopy. Column 1: proton notation. Column 2: chemical shift in
ppm. Between brackets are the multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet) and the coupling
constant in Hertz. Column 3: NOE cross peaks. Intensities are given in terms of weak (w), medium (m) or strong (s).
4238
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 4231–4241

Novel Gramicidin S Analogues
(CHAr), 74.30 (CH2OBn), 72.64 (CH2Ph), 70.67 (CHOH), 54.82 phase was washed with Et2O (2ϫ25 mL) and the combined organic
(CH2N3) ppm. [α]2D0 = +17.0 (c = 1.00, in MeOH)
phase with satd. aq. sodium hydrogen carbonate (25 mL) and brine
(25 mL), dried (Na2SO4), filtered and concentrated. The residue was
applied to a silica column and eluted with 0–3% EtOAc/PE to af-
ford the title compound (1.14 g, 3.56 mmol, 64%) as colorless oil.
1H NMR (200 MHz, CDCl3): δ = 7.29 (m, 5 H, HAr), 4.54 (s, 2 H,
CH2Ph), 4.15 (s, 2 H, OCH2C=O), 3.80–3.66 (m, 1 H, CCHO),
3.66–3.57 (m, 2 H, CH2OBn), 3.52–3.44 (m, 2 H, CH2Ph), 1.47 (s,
9 H, CH3tBu) ppm. 13C NMR (50 MHz, CDCl3): δ = 169.50 (C=O),
137.78 (CqPh), 128.41 (CHAr), 127.76 (CHAr), 127.68 (CHAr), 81.71
(CCHO), 78.25 (CH2CHOCH2), 73.53 (CH2Ph), 69.87 (CH2OBn),
(S)-1-Azido-3-(benzyloxy)propan-2-ol (21): Benzyl ether 21 was pre-
pared according to the procedure described for 20 to yield the prod-
uct (1.87 g, 9 mmol, 28%) as colorless oil. 1H NMR (400 MHz,
MeOD): δ = 7.38–7.32 (m, 5 H, HAr), 5.13 (br. s, 1 H, OH), 4.53
(s, 2 H, CH2Ph), 3.83–3.70 (m, 1 H, CHOH), 3.61 (dd, J = 5.2,
10.2 Hz, 2 H, CH2OBn), 3.53–3.35 (m, 2 H, CH2N3) ppm. 13C
NMR (101 MHz, CDCl3): δ = 137.38 (CqPh), 128.27 (CHAr),
127.70 (CHAr), 127.61 (CHAr), 127.29 (CHAr), 126.75 (CHAr),
73.26 (CH2OBn), 71.18 (CH2Ph), 69.39 (CHOH), 53.25 (CH2N3)
ppm. [α]2D0 = –12.6 (c = 1.00, in MeOH)
68.24 (OCH2C=O), 52.15 (CH2N3), 28.04 (CH3tBu) ppm. [α]2D0
=
+7.2 (MeOH). MS: m/z = 344.0 [M + Na]+, 339.0 [M + NH4]+,
322.0 [M + H]+, 288.1 [M – tBu + Na]+, 296.1 [M – N2 + H]+,
266.0 [M – tBu + H]+, 238 [M – tBu – N2 + H]+.
(R)-tert-Butyl 2-[1-Azido-3-(benzyloxy)propan-2-yloxy]acetate (22):
Alcohol 20 (1.15 g, 5.55 mol) was dissolved in DMF (25 mL) at 0 °C
under argon before addition of NaH (266 mg, 6.67 mmol, 1.2 equiv.
60% susp. mineral oil). After the gas evolution ceased, tert-butyl
bromoacetate (1.6 mL, 11.1 mmol, 2 equiv.) was added and the reac-
tion mixture was allowed to stir at room temp. until TLC analysis
(10% EtOAc/PE) indicated the formation of a higher running spot.
The reaction was quenched by the addition of MeOH (5 mL) and
partitioned between Et2O (50 mL) and H2O (100 mL). The aqueous
(S)-tert-Butyl 2-[1-Azido-3-(benzyloxy)propan-2-yloxy]acetate (23):
tert-Butyl ester 23 was prepared in a similar fashion as described
for 22 resulting in the isolation of the target compound (153 mg,
0.48 mmol, 40%) as colorless oil. 1HNMR (200 MHz, CDCl3): δ =
7.32 (m, 5 H, HAr), 4.53 (s, 2 H, CH2Ph), 4.14 (s, 2 H, OCH2C=O),
3.78–3.65 (m, 1 H, CCHO), 3.65–3.55 (m, 2 H, CH2OBn), 3.50–
1
Table 3. H NMR + NOE (500 MHz, CD3OH) of compound 8.
Atom 1
Chemical shift (m, J [Hz])
Atom 2
Atom 1
Chemical shift (m, J [Hz])
Atom 2
Val2 NH
7.54 (d, 8.1)
OCH2C=O (w)
SAA αH (w)
Leu9 βH (m)
Val7 NH
7.64 (9.2)
dPhe5 αH (m)
Pro6 αH (m)
3.72 Pro6 (m)
Val2 αH
Val2 βH
Val2 γH
Val2 γHЈ
Orn3 NH
4.15 (t, 7.7)
2.14 (m)
0.98 (d, 6.7)
0.98 (d, 6.7)
8.70 (d, 9.5)
Val7 αH
Val7 βH
Val7 γH
Val7 γHЈ
Orn8 NH
4.25 (t, 8.4)
2.25 (m)
0.93 (m)
0.88 (m)
8.54 (d, 9.3)
Val2 NH (w)
Val2 αH (s)
Val7 βH (m)
Val7 NH (s)
Val7 βH (m)
Orn3 αH
Orn3 βH
Orn3 βHЈ
Orn3 γH
Orn3 γHЈ
Orn3 δH
Orn3 δHЈ
Orn3 NH2
Leu4 NH
4.84 (m)
2.02 (m)
2.02 (m)
1.78 (m)
1.68 (m)
3.02 (m)
2.91 (m)
Orn8 αH
Orn8 βH
Orn8 βHЈ
Orn8 γH
Orn8 γHЈ
Orn8 δH
Orn8 δHЈ
Orn8 NH2
Leu9 NH
4.83 (m)
2.90 (m)
1.70 (m)
1.59 (m)
1.49 (m)
8.65 (d, 9.1)
SAA NH (w)
Val7 NH (w)
Orn8 αH (s)
8.72 (d, 9.5)
Val2 NH (w)
Val2 αH (s)
Val2 βH (s)
Leu4 αH
Leu4 βH
Leu4 βHЈ
Leu4 γH
Leu4 δH
Leu4 δHЈ
dPhe5 NH
4.55 (m)
1.58 (m)
1.58 (m)
Leu9 αH
Leu9 βH
Leu9 βHЈ
Leu9 γH
Leu9 δH
Leu9 δHЈ
SAA NH
4.64 (m)
1.54 (m)
1.54 (m)
1.42 (m)
0.89 (m)
0.88 (m)
8.03 (t, 6.1)
0.88 (m)
0.87 (m)
8.95 (d, 3.2)
Leu9 NH (w)
Leu9 αH (s)
3.72 Pro6 (m)
2.46 Pro6 (m)
Leu9 αH (s)
Val2 βH (w)
dPhe5 αH
4.49 (m)
SAA αH
SAA αHЈ
SAA βH
OCH2C=O
SAA CH2Ph
SAA HAr
3.63 (m)
3.55 (m)
3.29 (m)
3.91 (d, 14.7)
4.54 (d, 2.8)
7.32–7.23 (m)
dPhe5 βH
dPhe5 βHЈ
dPhe5 HAr
Pro6 αH
Pro6 βH
Pro6 βHЈ
Pro6 γH
Pro6 γHЈ
Pro6 δH
3.07 (dd, 4.8, 12.6)
2.94 (m)
7.31–7.23 (m)
4.36 (d, 7.9)
2.00 (m)
2.00 (m)
1.67 (m)
1.56 (m)
3.72 (t, 8.2)
2.46 (m)
Pro6 δHЈ
Eur. J. Org. Chem. 2009, 4231–4241
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4239

M. Overhand et al.
FULL PAPER
3.41 (m, 2 H, CH2Ph), 1.46 (s, 9 H, CH3tBu) ppm. 13CNMR
ppm. [α]D20 = +13.6 (c = 1.00, in MeOH). MS: m/z = 283.3 [M +
(50 MHz, CDCl3): δ = 169.30 (C=O), 137.65 (CqPh), 128.22 Na]+, 266.1 [M + H]+, 238.1 [M – N2 + H]+.
(CHAr), 127.55 (CHAr), 127.47 (CHAr), 81.43 (CCHO), 78.07
(S)-2-[1-Azido-3-(benzyloxy)propan-2-yloxy]acetic Acid (6): Follow-
ing the same procedure as described for 5. (S)-tert-Butyl ester 23
(CH2CHOCH2), 73.30 (CH2Ph), 69.69 (CH2OBn), 68.03
(OCH2C=O), 51.94 (CH2N3), 27.86 (CH3tBu) ppm. [α]2D0 = –9.0 (c
= 1.00, in MeOH). MS: m/z = 344.1 [M + Na]+, 321.9 [M + H]+,
296.1 [M – N2 + H]+, 265.9 [M – tBu + H]+.
(150 mg, 0.47 mmol) yielded the title compound (100 mg,
1
0.40 mmol, 86%) as colorless oil. H NMR (200 MHz, MeOD): δ
= 7.24 (m 5 H, HAr), 4.43 (s, 2 H, CH2Ph), 4.16 (s, 2 H,
OCH2C=O), 3.75–3.59 (m, 1 H, CCHO), 3.53 (d, J = 2.9 Hz, 1 H,
CH2OBn), 3.50 (d, J = 3.1 Hz, 1 H, CH2OBn), 3.34 (t, J = 4.9 Hz,
2 H, CH2N3) ppm. 13C NMR (50 MHz, MeOD): δ = 173.81
(CO2H), 139.27 (CqPh), 129.36 (CHAr), 128.82 (CHAr), 79.68
(CCHO), 74.34 (CH2CHOCH2), 70.93 (CH2Ph), 68.22 (CH2OBn),
52.74 (CH2N3) ppm. [α]2D0 = –16.8 (c = 1.00, in MeOH). MS: m/z
= 266.1 [M + H]+, 238.1 [M – N2 + H]+.
(R)-2-[1-Azido-3-(benzyloxy)propan-2-yloxy]acetic Acid (5): tert-Bu-
tyl ester 22 (1.04 g, 3.2 mmol) was dissolved a mixture of DCM/
TFA/TIS (10 mL, 1:1:0.05) and stirred for 1 h before TLC analysis
(EtOAc/AcOH, 99:1) indicated complete conversion of the starting
material into a lower running spot. The reaction mixture was con-
centrated and repeatedly evaporated with toluene (3ϫ10 mL) and
partitioned between DCM (10 mL) and satd. aq. sodium hydrogen
carbonate (10 mL). The aqueous phase was washed with DCM
(2ϫ5 mL), acidified to pH = 2 with 1  HCl and washed with Compound 7 was prepared according to the general procedure on
1
DCM (3ϫ10 mL). The combined organic phase was dried
a 200-µmol scale; yield 54.5 mg, 40 µmol, 20%. For the H NMR
(Na2SO4), filtered and concentrated to afford the title compound and NOE data see Table 2. 13C NMR (151 MHz, CDCl3): δ =
(611 mg, 2.3 mmol, 72%) as colorless oil. 1H NMR (200 MHz, 174.50, 174.13, 173.68, 173.60, 173.41, 173.32, 173.12, 172.69,
MeOD): δ = 7.32 (m, 5 H, HAr), 4.50 (s, 2 H, CH2Ph), 4.23 (s, 2 H, 172.64, 139.14, 136.86, 130.36, 129.66, 129.39, 128.77, 128.72,
OCH2C=O), 3.79–3.65 (m, 1 H, CCHO), 3.59 (d, J = 2.9 Hz, 1 H, 128.48, 86.43, 81.72, 79.26, 71.98, 61.94, 60.24, 59.83, 55.86, 53.33,
CH2OBn), 3.57 (d, J = 3.1 Hz, 1 H, CH2OBn), 3.41 (t, J = 5.0 Hz, 2 53.04, 51.74, 47.83, 42.57, 42.06, 40.66, 40.61, 38.45, 37.30, 32.25,
H, CH2N3) ppm. 13C NMR (50 MHz, MeOD): δ = 173.83 (CO2H), 31.91, 30.62, 30.44, 25.82, 25.61, 24.38, 23.25, 23.04, 22.80, 19.87,
139.29 (CqPh), 129.38 (CHAr), 128.83 (CHAr), 79.71 (CCHO), 74.36 19.58, 19.31, 18.78 ppm. HRMS calcd. for [C59H91N11O11 + H]+
(CH2CHOCH2), 70.95 (CH2Ph), 68.24 (CH2OBn), 52.76 (CH2N3) 1130.68713; found 1130.69908.
1
Table 4. H NMR + NOE (500 MHz, CD3OH) of compound 9.
Atom 1
Chemical shift (m, J [Hz])
Atom 2
Atom 1
Chemical shift (m, J [Hz])
Atom 2
Val2 NH
7.95 (d, 8.7)
Val7 NH
7.63 (d, 9.1)
Pro6 αH (w)
Pro6 δH (m)
Val2 αH
Val2 βH
Val2 γH
Val2 γHЈ
Orn3 NH
4.23 (m)
2.14 (m)
0.99 (d, 6.7)
0.97 (d, 6.8)
8.73 (d, 7.7)
Val7 αH
Val7 βH
Val7 γH
Val7 γHЈ
Orn8 NH
4.28 (m)
2.26 (m)
0.90 (d, 6.7)
0.89 (d, 6.8)
8.58 (d, 9.2)
Val2 αH (s)
Val7 αH (s)
Val7 βH (w)
Orn3 αH
Orn3 βH
Orn3 βHЈ
Orn3 γH
Orn3 γHЈ
Orn3 δH
Orn3 δHЈ
Orn3 NH2
Leu4 NH
4.80 (m)
2.02 (m)
2.02 (m)
1.77 (m)
1.65 (m)
3.00 (m)
2.89 (m)
7.90 (br. s)
8.72 (d, 9.3)
Orn8 αH
Orn8 βH
Orn8 βHЈ
Orn8 γH
Orn8 γHЈ
Orn8 δH
Orn8 δHЈ
Orn8 NH2
Leu9 NH
4.85 (m)
1.76 (m)
1.76 (m)
1.55 (m)
1.44 (m)
2.81 (m)
2.73 (m)
7.77 (br. s)
8.66 (d, 9.0)
Val7 NH (w)
Val2 NH (m)
Orn8 αH (m)
Leu4 αH
Leu4 βH
Leu4 βHЈ
Leu4 γH
Leu4 δH
Leu4 δHЈ
dPhe5 NH
dPhe5 αH
dPhe5 βH
dPhe5 βHЈ
dPhe5 HAr
Pro6 αH
Pro6 βH
Pro6 βHЈ
Pro6 γH
Pro6 γHЈ
Pro6 δH
4.65 (m)
1.53 (m)
1.53 (m)
1.43 (m)
0.89 (m)
0.89 (m)
8.94 (d, 3.2)
4.51 (m)
3.08 (dd, 4.8, 12.5)
2.94 (m)
7.36–7.23 (m)
4.37 (d, 7.8)
2.00 (m)
2.00 (m)
1.65 (m)
1.57 (m)
3.71 (m)
2.46 (m)
Leu9 αH
Leu9 βH
Leu9 βHЈ
Leu9 γH
Leu9 δH
Leu9 δHЈ
SAA NH
SAA αH
SAA αHЈ
SAA βH
OCH2C=O
SAA CH2Ph
SAA HAr
4.95 (m)
1.64 (m)
1.64 (m)
1.53 (m)
0.87 (m)
0.87 (m)
7.93 (m)
3.93 (m)
3.77 (m)
3.02 (m)
3.56 (m)
4.54 (s)
Leu4 αH (s)
Pro6 δH (s)
Leu9 αH (s)
7.36–7.23 (m)
Pro6 δHЈ
4240
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 4231–4241

Novel Gramicidin S Analogues
haney, Biochemistry 2005, 44, 2103–2112; c) M. Jelokhani-Nia-
raki, L. H. Kondejewski, S. W. Farmer, R. E. W. Hancock,
C. M. Kay, R. S. Hodges, Biochem. J. 2000, 349, 747–755.
[9] J. H. Kondejewski, S. W. Farmer, D. S. Wishart, C. M. Kay,
R. E. W. Hancock, R. S. Hodges, J. Biol. Chem. 1996, 241,
25261–25268.
Compound 8 was prepared according to the general procedure on
a 200-µmol scale; yield 123 mg, 91 µmol, 46%. For the H NMR
1
and NOE data see Table 3. 13C NMR (151 MHz, CDCl3): δ =
174.34, 173.54, 173.51, 173.40, 173.00, 172.97, 172.73, 172.67,
172.43, 162.86, 162.63, 139.23, 136.86, 130.56, 130.36, 129.64,
129.36, 128.79, 128.75, 128.44, 117.05, 80.69, 74.35, 71.41, 70.32,
61.90, 59.86, 59.45, 55.86, 55.77, 53.27, 53.25, 52.74, 51.69, 47.80,
47.24, 42.27, 42.02, 40.73, 40.52, 40.44, 37.27, 32.27, 30.57, 30.40,
29.81, 27.27, 25.84, 25.65, 25.04, 24.63, 24.35, 23.44, 23.16, 23.08,
22.25, 19.81, 19.56, 19.02, 18.86 ppm. HRMS calcd. for
[C58H91N11O11 + H]+ 1118.69723; found 1118.69896.
[10]
Y. Shimmohigashi, H. Kodama, S. Imazu, H. Horimoto, K.
Sakaguchi, M. Waki, H. Uchida, M. Kondo, T. Kato, N.
Izumiya, FEBS Lett. 1987, 222, 251–255.
[11]
[12]
J. Xioa, B. Weisblum, P. Wipf, Org. Lett. 2006, 8, 4631–4734.
G. M. Grotenbreg, M. S. M. Timmer, A. L. Llamas-Saiz, M.
Verdoes, G. A. van der Marel, M. J. van Raaij, H. S. Overk-
leeft, M. Overhand, J. Am. Chem. Soc. 2004, 126, 3444–3446.
Reviewed in: a) S. A. W. Grunner, E. Locardi, E. Lohof, H.
Kessler, Chem. Rev. 2002, 102, 491–514; b) T. K. Chakraborty,
P. Srinivasu, S. Tapadar, B. K. Mohan, Glycoconjugate J. 2005,
22, 83–93; c) M. D. P. Risseeuw, M. Overhand, G. W. J. Fleet,
M. I. Simone, Tetrahedron: Asymmetry 2007, 18, 2001–2010.
a) T. K. Chakraborty, D. Koley, R. Ravi, V. Krishnakumari, R.
Nagaraj, A. C. Kunwar, J. Org. Chem. 2008, 73, 8731–8744;
b) G. M. Grotenbreg, A. E. M. Buizert, A. L. Llamas-Saiz, E.
Spalburg, P. A. van Hooft, A. J. de Neeling, D. Noort, M. J.
van Raaij, G. A. van der Marel, H. S. Overkleeft, M. Over-
hand, J. Am. Chem. Soc. 2006, 128, 7559–7565.
Compound 9 was prepared according to the general procedure on
a 200-µmol scale; yield 56.1 mg, 41.7 µmol, 21%. For the 1H NMR
and NOE data see Table 4. 13C NMR (151 MHz, CDCl3): δ =
174.32, 173.60, 173.54, 173.38, 173.05, 172.93, 172.68, 172.52,
172.11, 138.79, 136.85, 130.36, 129.66, 129.60, 129.08, 129.00,
128.48, 78.77, 74.17, 71.30, 69.89, 69.77, 61.92, 59.72, 59.67, 55.85,
53.52, 53.34, 52.83, 51.66, 49.85, 49.43, 49.28, 49.14, 49.00, 48.86,
48.72, 48.57, 47.83, 47.83, 42.03, 41.60, 40.68, 40.59, 32.51, 32.30,
30.61, 29.82, 25.85, 25.60, 24.36, 23.29, 23.23, 23.01, 22.35, 19.80,
19.50, 18.94, 18.79 ppm. HRMS calcd. for [C58H91N11O11 + H]+
1118.69723; found 1118.69870.
[13]
[14]
[15]
[16]
F. Kazemi, A. R. Kiasat, S. Ebrahimi, Synth. Commun. 2003,
33, 999–1004.
In the case of AAAs 4 and 5 it is imperative to avoid prolonged
existence of the intermediate iminophosphorane by using anhy-
drous reaction conditions. In anhydrous THF, the intermediate
iminophosphorane suffered from nucleofilic attack of the imi-
nophosphorane nitrogen on the AAA carbonyl, resulting in
loss of the AAA moiety as depicted below. Premixing the THF
with H2O prevented this side reaction to occur as gauged by
LCMS analysis (See the proposed side reaction during Staud-
inger reduction.)
Acknowledgments
We are very grateful for generous financial support by TNO De-
fense, Security and Safety, Rijswijk, the Netherlands. We thank
Hans van der Elst for assistance with the LCMS analyses and
HPLC purifications and Kees Erkelens and Fons Lefeber for assist-
ance with the recording of NMR spectra.
[1] A number of reviews have been published in relation to CAPs:
a) R. M. Epand, H. J. Vogel, Biochim. Biophys. Acta 1999,
1462, 11–28; b) A. Giuliani, G. Pirri, S. F. Nicoletto, Cent. Eur.
J. Biol. 2007, 2, 1–33; c) R. E. W. Hancock, D. S. Chapple,
Antimicrob. Agents Chemother. 1999, 43, 1317–1323.
[2] G. F. Gause, M. G. Brazhnikove, Nature 1944, 154, 703–703.
[3] a) L. Zhang, J. Parente, S. M. Harris, D. E. Wods, R. E. W.
Hancock, T. J. Falla, Antimicrob. Agents Chemother. 2005, 49,
2921–2927; b) R. E. W. Hancock, Lancet infect. dis. 2001, 1,
156–164.
[4] M. M. Shemyakin, Yu. A. Ovchinnikov, V. T. Ivanov, I. D. Rya-
bova, Experientia 1967, 23, 326.
3
[17]
[18]
The JHNα in peptides is dependent on the dihedral angle φ
[C(O)-N(H)-Cα-C(O)] allowing the qualitative assignment of
secondary structureN. Ramachandran, R. Chandrasekaran,
K. D. Kopple, Biopolymers 1971, 10, 2113–2131.
The position of the Hα resonance signal of a residue in a par-
ticular peptide, relative to the value of that same residue in
a random coil configuration (the chemical shift perturbation,
∆δHα) can be used as an indication of the configuration of
that residue. A value of ∆δHα Ͼ 0.1 ppm indicates a β-strand,
–0.1 Ͻ ∆δHα Ͻ 0.1 indicates a rondam coil and ∆δHα Ͻ
–0.1 ppm indicates a helical configuration. D. S. Wishart, B. D.
Sykes, F. M. Richards, Biochemistry 1992, 31, 1647–1651.
K. Wüthrich, NMR of proteins and nucleic acids, John Wiley &
Sons, New York, 1986.
[5] a) E. J. Prenner, R. N. A. H. Lewis, R. N. McElhaney, Biochim.
Biophys. Acta 1999, 1462, 201–221; b) E. Staudegger, E. J.
Prenner, M. Kriechbaum, G. Degovics, R. N. A. H. Lewis,
R. N. McElhaney, K. Lohner, Biochim. Biophys. Acta 2000,
1468, 213–230; c) M. Kiricsi, L. I. Horváth, L. Dux, T. Páli, J.
Mol. Struct. 2001, 563–564, 469–475.
[6] a) M. Jelokhani-Niaraki, R. S. Hodges, J. E. Meissner, U. E.
Hassenstein, L. Weaton, Biophys. J. 2008, 95, 3306–3321; b) M.
Ashrafuzzaman, O. S. Anderson, R. N. McElhaney, Biochim.
Biophys. Acta 2008, 1778, 2814–2822.
[7] a) R. Pankiewicz, A. Gurzkowska, B. Brzezinski, G. Zundel,
F. Bartl, J. Mol. Struct. 2003, 646, 67–74; b) A. Stern, W. A.
Gibbons, L. C. Craig, Proc. Natl. Acad. Sci. USA 1968, 61,
734–741; c) S. E. Hull, R. Karlsson, P. Main, M. M. Woolfson,
E. J. Dodson, Nature 1978, 75, 206–207; d) A. L. Llamas-Saiz,
G. M. Grotenbreg, M. Overhand, M. J. van Raaij, Acta Crys-
tallogr., Sect. D 2007, 63, 401–407.
[8] See for examples a) S. L. Grage, A. V. Suleymanova, S. Afonin,
P. Wadhwani, A. S. Ulrich, J. Magn. Reson. 2006, 183, 77–86;
b) T. Abraham, R. N. A. H. Lewis, R. S. Hodges, R. N. McEl-
[19]
[20]
J. V. Olsen, L. M. F. de Godoy, G. Q. Li, B. Macek, P. Mort-
ensen, R. Pesch, A. Makarov, O. Lange, S. Horning, M. Mann,
Mol. Cell Proteomics 2005, 4, 2010–2021.
E. Kaiser, R. L. Colescott, C. D. Bossering, P. I. Cook, Anal.
Biochem. 1970, 34, 595.
[21]
Received: April 28, 2009
Published Online: July 21, 2009
Eur. J. Org. Chem. 2009, 4231–4241
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4241