Step-wise and pre-organization induced synthesis of a crossed alkene-bridged nisin Z DE-ring mimic by ring-closing metathesis

Article abstract of DOI:10.1039/b618085j

This paper describes two approaches for the synthesis of a crossed alkene-bridged mimic of the thioether ring system of the nisin Z DE-fragment. The first approach comprised the stepwise total synthesis featuring a cross metathesis and a macrolactamizatio

Full text of DOI:10.1039/b618085j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Step-wise and pre-organization induced synthesis of a crossed alkene-bridged
nisin Z DE-ring mimic by ring-closing metathesis†‡
Nourdin Ghalit,a Johan Kemmink,a Hans W. Hilbers,a Cees Versluis,b Dirk T. S. Rijkersa and
Rob M. J. Liskamp*a
Received 12th December 2006, Accepted 16th January 2007
First published as an Advance Article on the web 7th February 2007
DOI: 10.1039/b618085j
This paper describes two approaches for the synthesis of a crossed alkene-bridged mimic of the
thioether ring system of the nisin Z DE-fragment. The first approach comprised the stepwise total
synthesis featuring a cross metathesis and a macrolactamization on a solid support followed by a
ring-closing metathesis in solution. Via this route the title compound was obtained in an overall yield of
7% (85% on average for 16 reaction steps). In the second approach, the linear precursor peptide was
subjected to ring-closing metathesis and the bicyclic peptide with the correct side chain connectivity
pattern was obtained in yields up to 95%. The preferred formation of the bicyclic crossed alkene-bridged
mimic of the DE-ring suggests a favorable pre-organization of the linear precursor peptide.
mixture of three bicyclic products, in addition to monocyclic in-
Introduction
termediates, starting material and alkene-isomerization products.
The antimicrobial peptide nisin Z belongs to the lantibiotics
From such a mixture the desired product would have be isolated
which form an important natural class of antibiotics.1 A general
and its structure proven, a non-trivial task.
feature of the lantibiotics is the presence of lanthionine residues
Hence, a step-wise approach was developed; it was consid-
as the cyclic constraint to give the peptide its specific bioactive
ered that this would more likely lead to the desired crossed
conformation (Fig. 1).2 The lanthionine moiety is introduced
alkene-bridged DE-ring mimic. The planned route featured cross
via an enzymatically-assisted,3 posttranslational modification via
a chemoselective Michael-addition of a cysteine residue toward
a dehydrated serine (dehydroalanine, Dha) or a dehydrated
threonine (dehydrobutyrine, Dhb) residue to give the lanthionine
(Lan) or the 3-methyllanthionine (MeLan) moiety, respectively
(Fig. 2).4
metathesis8 and macrolactamization reactions on a solid support,
followed by a RCM reaction in solution. As such this represents the
first example of RCM being applied for the synthesis of a crossed
alkene-bridge9,10 to obtain mimics of thioether-bridges containing
lantibiotics.
The lanthionine moiety or thioether (sulfide) bridge as a natural
constraint in bioactive peptides can be replaced by an alkene-
or alkyne-bridge in order to increase the metabolic stability of
the newly designed peptide-derived antibiotics. Recently, it was
shown by us that such thioether bridges could successfully be
replaced by either alkene/alkyne-bridges or by a combination of
both alternative conformational constraints.5,6
In the DE-ring system of nisin the amino acid side chains cross
each other (connectivity pattern: [1→4], [3→6]) implying that an
alkene mimic of this ring system may be difficult to synthesize.7 The
most straightforward route towards the crossed alkene-bridged
DE-ring mimic is a direct synthesis from the linear peptide RCM-
precursor containing the required allylglycine residues. However,
it was assumed that this approach may result in a complex reaction
Results and discussion
The envisaged route (Scheme 1) started by attachment11 of Fmoc-
R
Alg-OH to plain Argogelꢀ (resin 1), enabling the determination of
the loading.12 The Fmoc group was replaced by a Boc functionality
(resin 2) to introduce orthogonality of the protecting groups (vide
infra). Then, the putative alkene-bridge of ring E was synthesized
by a cross metathesis in the presence of 2nd generation Grubbs’
catalyst13 (Fig. 3) in 1,1,2-trichloroethane at 60 ◦C with Fmoc-
Alg-OH and resin 2.
Fortunately, it was found that protection of the carboxyl moiety
of the Fmoc-protected allylglycine residue was not required, since
this would complicate the synthesis substantially. At this stage of
the synthesis (resin 3), a third orthogonal protecting group was
necessary. Therefore, after removal of the Fmoc group, azidoala-
nine hydroxysuccinimide ester (N3-Ala-ONSu) was coupled, in
which the azide was a masked amino group, orthogonal to the
Fmoc and Boc-group.14 This enabled us to complete the peptide
sequence of ring E (resin 4→6). The advantage of succinimide
esters in this and the next steps is that they can be used in the
presence of a free carboxylic acid moiety. Next, acidolysis of the
Boc-group by TFA was followed by coupling of Fmoc-Asn(Trt)-
ONSu. After removal of the Fmoc-group (resin 5), Fmoc-Alg-
ONSu was coupled to obtain resin 6.
aDepartment of Medicinal Chemistry and Chemical Biology, Utrecht In-
stitute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082,
3508, TB Utrecht, The Netherlands. E-mail: R.M.J.Liskamp@pharm.uu.nl;
Fax: +31 30 253 6655; Tel: +31 30 253 7396/7307
bDepartment of Biomolecular Mass Spectrometry, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, The Netherlands
† Part of this research was published as a communication: see ref. 5a.
‡ Electronic supplementary information (ESI) available: General methods
and procedures and details of the computational modeling and TOCSY
spectra of compounds 9 and 17. See DOI: 10.1039/b618085j
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Fig. 1 Amino acid sequence of nisin Z and its alkene-bridged mimic, the crossed DE-ring system is shown in more detail.
Fig. 2 Structures of the (2S,6R)-lanthionine and (2S,3S,6R)-3-methyllanthionine moieties and their corresponding alkene-bridged mimics formed by
(S)-2-amino-4-pentenoic acid, (R)-2-amino-4-pentenoic acid and (2R,3R)-2-amino-3-methyl-4-pentenoic acid. (Note: The stereochemistry of the chiral
centers is the same, their R/S configuration designations are opposite due to the CIP rules.)
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Scheme 1 Step-wise solid phase synthesis of the alkene-bridged DE-ring mimic.
500 MHz, COSY, TOCSY and ROESY) in combination with MS-
MS experiments proved that the correct ring structure was formed
and thus that the previously introduced alkene bridge of the E-ring
was not converted into different metathesis products. Based on
their distinct spin system, Ala2 and Asn5 could be assigned in first
instance (Fig. 5A, upper and middle panel). Furthermore, three
ROEs (Fig. 5A, lower panel) were visible which provided sequen-
tial assignments. ROE-1 is a cross peak between the NH of Ala2
and the aCH of Alg1, ROE-2 is a cross peak between the NH of
Alg4 and the aCH of Alg3, and ROE-3 is a cross peak between the
NH of Alg6 and the aCH of Asn5 (Fig. 4). The TOCSY data (the
whole spectrum is given in Fig. 1 of the Supporting Information‡)
proved the correct side chain connectivity pattern (Fig. 5B).
Since we had now the desired bicyclic 9 as a reference available,
it was possible to evaluate the feasibility of the ‘straightforward’
approach using linear precursor peptide 10 directly in RCM
(Scheme 2).
Protected peptide 10 was obtained after solid phase peptide syn-
thesis using Fmoc–tBu protocols followed by purification in 69%
yield. This peptide was now treated with 2nd generation Grubbs’
catalyst. After 2 h a sample was taken from the reaction mixture
and the catalyst was immediately removed by filtration over a small
silica plug. The remaining reaction mixture was refluxed overnight
after addition of more catalyst. First, the reaction intermediates in
the sample were analyzed and purified by HPLC and characterized
by LCES-TOF MS-MS.16 The observed mass in combination with
the obtained fragmentation pattern enabled the elucidation of the
structure of the formed monocylic intermediates.
Fig. 3 Structures of the first (I) and second (II) generation Grubbs’
ring-closing metathesis catalyst.
Now, the sequence of the E-ring (resin 6) was completed and
at this stage, the Fmoc group was removed followed by extensive
washing with DIPEA to remove any residual piperidine in order to
avoid truncation resulting from formation of a piperidinyl amide.
Lactamization between residues Alg3 and Alg4 to complete ring E
was performed on the resin with HATU–HOAt–DIPEA in DMF15
which resulted in resin 7. Reduction of the azide functionality
under Staudinger conditions gave the free amine.14 Finally, Boc-
Alg-OH was coupled with BOP–DIPEA and then the resulting
resin was treated with a catalytic amount of KCN in methanol to
give the monocyclic fully protected peptide ester 8 in 11% overall
yield after purification (86% on average per reaction step).
The correct side chain to side chain connectivity of ring E was
confirmed by NMR analysis and the correct fragmentation pattern
was found by mass analysis (LCES-TOF MS-MS) (Fig. 4).16
Peptide 8 was treated with 2nd generation Grubbs’ catalyst to
give the desired bicyclic peptide 9 in 50% yield. NMR analysis (1H-
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Fig. 4 MS-MS fragmentation pattern of peptide 816 and the observed c-proton and ROESY sequential connectivities evidencing the correct bicyclic
structure of 9.
Scheme 2 Intermediates in the one step double ring-closing metathesis pathway of the alkene bridged DE-ring mimic. (Note: Before the peptide could
be characterized by MS-MS, both protecting groups (Boc and Trt) were removed by treatment with TFA. This treatment is represented by (2. TFA) and
is carried out for analysis purposes only (also in Schemes 3 and 4).)
Theoretically, six monocyclic intermediates could have been
formed, however, only four (8, 11–13) corresponding to the [3,6],
[1,4], [4,6] and [1,6] RCM products were found (Scheme 2).
Remarkably, the [1,3] RCM-product was not observed, whereas
the [4,6] RCM product was. Absence of the [3,4] RCM product
might be explained by reluctance of the trans-amide bond to
assume a cisoid geometry necessary for the eight membered ring
of this product.17 The unique fragmentation pattern of each RCM-
product enabled unambiguous determination of the position of the
cyclic constraint.16 The ratio of product formation 8 : 11 : 12 : 13
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Next, the products obtained after refluxing overnight were
isolated and purified. Only two of the three possible bicyclic
compounds—based on the formed monocyclic compounds in
the reaction mixture sample—were observed. Both monocyclic
products 8 and 11 cyclized to the desired bicyclic product 9.
Intermediate 12 cyclized to product 14 i.e. the [1,3]–[4,6] product.
Not unexpectedly, (vide supra) [1,6] product 13 was not converted
to a bicycle, since this would require formation of a [3,4] cycle,
which is probably difficult. Thus, the desired bicyclic product was
obtained in 72% yield as compared to only 19% of one other
bicyclic product (14). The preferred formation of monocyclic
products 8 and 11 and the ensuing bicyclic product hints at a
favorable pre-organization of the linear peptide for formation the
DE-ring alkene mimic, which in view of their ring size (two 14-
membered rings) might be close to an a-helical structure.19,20 It is
tempting to speculate that this pre-organization might also play a
role in construction of the natural DE-ring system in nisin itself.
The natural DE-ring is a pair of (2S,3S,6R)-3-methyllanthio-
nine with a crossed side chain to side chain connectivity pattern
(Fig. 1 and 2). This implies that the stereochemistry of each
amino acid forming the 3-methyllanthionine moiety corresponds
to that of (2S,3S)-threonine (≡ D-Thr) and (R)-cysteine (≡ L-Cys).
Along these lines, two additional mimics of the DE-ring have been
designed, the first one, bicyclic peptide 17 (Scheme 3), has the same
stereochemistry as nisin at the a-position and is therefore derived
from D-allylglycine at residues 1 and 3. The second one, bicyclic
peptide 21 (Scheme 4), has in addition the same stereochemistry
at the b-position ((2R,3R)-2-amino-3-methyl-4-pentenoic acid ≡
D-Alg(R-bMe)(D-Amp) as nisin at residue 1 and 3).21
Precursor peptide 15 with D-allylglycine residues at position 1
and 3 respectively, was synthesized on the solid phase in 90%
yield (Scheme 3). This peptide was treated with 2nd generation
Grubbs’ catalyst and after 2 h a sample was drawn, purified over
silica gel and analyzed by MS-MS. The remaining reaction mixture
was refluxed for additional 16 h after addition of more catalyst.
Mass analysis of the sample showed a single peak apparently
corresponding to a bicyclic peptide with crossed side chains, since
MS-MS did not result in peptide fragment ions. On HPLC, three
major peaks could be identified which converged into one single
peak after hydrogenolysis of the sample. NMR analysis proved the
homogeneity of the hydrogenated sample. These results suggested
that ring-closure of 15 resulted in a mixture of, at least, three
cis/trans isomers of a single bicyclic peptide.
Fig. 5 (A) The upper panel (CbH–NH cross-peaks) and middle panel
(CaH–NH cross-peaks) are expansions of the TOCSY spectrum of bicyclic
peptide 9. The lower panel is a part of the ROESY spectrum of bicyclic
peptide 9. The observed sequential CaH–NH connectivities are indicated.
(B) Expansion of the TOCSY spectrum of bicyclic peptide 9 showing the
c-proton connectivities of Alg4–Alg1 and Alg3–Alg6 which proves the
bicyclic structure of 9.
1
Also with this bicyclic peptide 17, H TOCSY and ROESY
experiments were performed to determine the connectivity pattern
of the side chains (see Fig. 2 of the Supporting Information‡).
Three ROEs (Fig. 4) (different from those found in 9) provided
sequential assignments. ROE-1 is a cross peak between the NH of
Ala2 and the aCH of D-Alg1, ROE-2 is a cross peak between the
NH of D-Alg3 and the aCH of Ala2, and ROE-3 is a cross peak
between the NH of Alg6 and the aCH of Asn5. The TOCSY data
proved the correct side chain connectivity pattern (see Fig. 3 of the
Supporting Information‡). Thus, from these experiments it can be
concluded that RCM of 15 resulted predominantly (>95%) in the
desired bicyclic peptide 17 in less than 2 h reaction time (Fig. 4).
As an additional proof, precursor peptide 15 was also treated
with the 1st generation Grubbs’ catalyst22 (Fig. 3). After 2 h, an
aliquot of the reaction mixture was analyzed (while the remaining
reaction mixture was refluxed overnight after the addition of
was found to be circa 1 : 4 : 1 : 2 and thus the reaction
mixture contained approximately 60% of the desired intermediates
8 and 11. A pure statistical distribution—assuming the formation
of six possible RCM-products—would only have resulted in
formation of approximately 33% of 8 and 11. Molecular mechanics
calculations were in agreement with the preferential formation of
8 and 11, since the energy of these intermediates appeared to be
significantly lower.18
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Scheme 3 RCM-intermediates starting from peptide 15.
more catalyst) and only two products were identified: monocyclic
intermediate 16 and bicyclic peptide 17 (Scheme 3). However,
after refluxing overnight, both peptides were still present in a
ratio 16 : 17 of 1 : 2. Apparently, ring-closing with the less
reactive 1st generation Grubbs’ catalyst did not result in complete
conversion. Moreover, monocyclic intermediate 16, corresponding
to RCM product [1,4], is probably preferred since this intermediate
product was also predominantly formed in the double ring-closing
metathesis reaction of peptide 10 (Scheme 2).
Finally, precursor peptide 18 was synthesized in which (2R,3R)-
2-amino-3-methyl-4-pentenoic acid23 (D-Alg(R-bMe); D-Amp)
was incorporated at positions 1 and 3 (Scheme 4) correspond-
ing to the stereochemistry of the a- and b-carbons in the 3-
methyllanthionine residue of native nisin Z.
Treatment of peptide 18 with 2nd generation Grubbs’ catalyst
resulted predominantly in the formation of monocyclic peptide
19 (>90%) with the incorrect [4,6] connectivity pattern and
the desired, correctly folded [3,6] monocyclic intermediate was
isolated in only 5%.
Preference for the formation of 19 can be explained by the
reduced steric hindrance in the RCM reaction, since in formation
of either the [1,4] or [3,6] RCM product a b-substituted allylglycine
derivative has to be incorporated. The decreased reactivity of b-
substituted allylglycine compared to allylglycine in RCM has been
recently described in the literature.24 Longer reaction times did not
change the outcome of this one step double ring-closing metathesis
pathway. Monocyclic intermediate 19 did not react further since
two b-substituted allylglycine residues were sterically too hindered
for any subsequent RCM reaction. However, correctly folded
intermediate product 20 was converted into bicyclic peptide 21,
unfortunately, this product was formed in very small amounts
(as judged by LC-MS-MS) which hampered isolation and further
analysis and characterization.
To address the issue whether an alkene-bridge is a good mimic
of the thioether moiety of the lanthionine functionality, modeling
studies using MacroModel18 were carried out. The conformational
search for obtaining the global minimum of compounds 9, 17
and 21 was carried out in chloroform since, the ring-closing
metathesis reactions as well as the NMR experiments were carried
out in comparable apolar solvents. From each structure the energy
content (kJ mol−1) of the global minimum of the E,E; E,Z; Z,E
and the Z,Z geometry of the double bond was calculated and are
shown in Table 1.
In compound 9, with all-L stereochemistry of the peptide
backbone, the order of energy content was: Z,Z ≈ E,Z < E,E
ꢀ Z,E. In the case of compound 17, with the same backbone
stereochemistry as nisin, this rank order was found to be exactly
reversed: Z,E < E,E ꢀ E,Z < Z,Z. Not unexpectedly, if 17 was
compared with compound 21, with the same backbone and side
chain stereochemistry as nisin, the order of energy content of the
four E/Z conformers was nearly identical: Z,E ꢀ E,E ≈ Z,Z ≈
E,Z.
In contrast to formation of 9, ring-closing metathesis leading to
17 was very fast and efficient since no side products with undesired
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Scheme 4 RCM-intermediates starting from peptide 18.
Table 1 Modeling studies using MacroModel18 of the bicyclic alkene-
bridged DE-ring mimics of nisin
was based on the superimposition of carbon atoms aC1, aC3,
aC4 and aC6 of each derivative. The following RMS-values were
˚
calculated: 9(Z,Z)–native nisin DE: 0.79 A, 17(Z,E)–native nisin
Compounda
Double bond geometry
Energy (kJ mol−1
)
˚
˚
DE: 1.27 A and 21(Z,E)–native nisin DE: 1.43 A. These results as
well as the overall view of the superimposition may imply that an
alkene-bridge is at least a reasonable mimic of a thioether moiety.
9
E,E
E,Z
Z,E
Z,Z
E,E
E,Z
Z,E
Z,Z
E,E
E,Z
Z,E
Z,Z
—
291.8
286.2
298.5
285.9
263.2
276.1
261.3
279.8
312.1
314.6
299.9
313.5
298.1
9
9
9
17
17
17
17
21
21
21
21
Native nisin DE
a The conformational search to obtain the global minimum was carried
out with Na-acetylated hexapeptide methyl ester derivatives with a free
asparagine side chain.
different side-chain to side-chain connectivities were found. In
general, the energy content of the isomers of 17 were significantly
lower than those of 9 (261.3 respectively 285.9 kJ mol−1) which
may partially explain the faster and more selective RCM reaction.
There is a good superimposition of the lowest energy conformers
of each RCM product (9(Z,Z), 17(Z,E) and 21(Z,E)) with the
DE-ring system of native nisin as is shown in Fig. 6. The overlay
Fig. 6 Superimposition of the lowest energy conformers of bicyclic
alkene-bridged mimics 9, 17 and 21 with the native DE-fragment. The
overlay was based on the superimposition of carbon atoms aC1, aC3, aC4
and aC6 of each derivative.
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In conclusion, we have prepared three alkene-bridged mimics
of the structurally most challenging part of the nisin Z sequence
comprising the DE-ring system. The stepwise total synthesis
featuring a cross metathesis and a macrolactamization on the solid
support followed by a ring-closing metathesis in solution resulted
in the first DE-ring mimic (all-L stereochemistry) with an overall
yield of 7% (85% on average for 16 reaction steps). In view of
the complexity of the products, this approach was an absolute
prerequisite for obtaining the necessary reference compound in
order to evaluate whether RCM of linear peptide RCM-precursors
would also lead to the desired mimics. Indeed, it was found that
there was a preferred formation of the intermediate monocyclic
mimic, resulting in a preferred formation of the bicyclic alkene
mimic (all-L stereochemistry), which might be explained by
invoking a certain degree of pre-organization of linear peptide
RCM-precursor. This was further evidenced by two additional
ring-closing metathesis reactions. A second DE-ring mimic was
synthesized, addressing the same backbone-stereochemistry as in
native nisin, and this peptide was found to undergo the double
RCM reaction leading to a single compound with the correct
[1→4], [3→6] side chain connectivity pattern with an increased
overall yield (95%) compared to the all-L derivative. However, the
precursor peptide corresponding to the stereochemistry of the a-
and b-carbons in the 3-methyllanthionine residues of the native
DE-ring system in nisin Z, cyclized only in trace amounts into a
third DE-ring mimic since the b-substituted allylglycine residues
were sterically too hindered for efficient RCM reaction.
Synthesis of the monocyclic peptide 8
(Boc-Alg1-Ala2-cyclo(3→6)[Alg3-Alg4-Asn(Trt)5-Alg6]-OMe)
The resin loaded with the cross-metathesis product (0.7 g,
0.14 mmol) was washed with DCM (3 × 10 mL, 2 min) and DMF
(3 × 10 mL, 2 min), subsequently, the Fmoc group was removed by
treatment with 20% piperidine in DMF (3 × 10 mL, 8 min) and the
resin was washed with DMF (3 × 10 mL, 2 min), DCM (3 × 10 mL,
2 min) and DMF (3 × 10 mL, 2 min). The resin was suspended
in DMF (15 mL) and N3-Ala-OSu14,26 (80 mg, 0.68 mmol) was
coupled to the free amine in the presence of DIPEA (215 lL,
1.2 mmol). After 2 h the resin was washed with DMF (3 × 10 mL,
2 min) and DCM (3 × 10 mL, 2 min), and the resin was suspended
in TFA–DCM (10 ml, 1 : 1 v/v) for 20 min to remove the Boc group.
Subsequently, the resin was washed with DCM (6 × 10 mL, 2 min),
DIPEA–DCM (1 : 9 v/v; 3 × 10 mL, 2 min) and DCM (3 × 10 mL,
2 min). Then, Fmoc-Asn(Trt)-OSu (695 mg, 1.0 mmol) in DMF
(10 mL) followed by DIPEA (215 lL, 1.2 mmol) were added. After
1 h the coupling was complete according to the Kaiser test. The
resin was washed with DMF (3 × 10 mL, 2 min), DCM (3 ×
10 mL, 2 min) and DMF (3 × 10 mL, 2 min) and the Fmoc group
was removed with piperidine in DMF (1 : 4 v/v; 3 × 10 mL, 8 min).
After washing with DMF (3 × 10 mL, 2 min), DCM (3 × 10 mL,
2 min) and DMF (3 × 10 mL, 2 min), Fmoc-Alg-ONSu (438 mg,
1.0 mmol) was coupled for 1 h. After Fmoc removal and washing of
the resin, an extra washing step with DIPEA–DMF (1 : 9 v/v; 3 ×
10 mL, 2 min) was carried out to remove any residual piperidine
salt. The macrocyclization was carried out with HATU27 (176 mg,
0.46 mmol), HOAt27 (63 mg, 0.46 mmol) in the presence of DIPEA
(241 lL, 1.4 mmol) in DMF (10 mL) in 16 h at room temperature
to obtain the ring E. The resin was washed with DMF (5 × 10 mL,
2 min) and all remaining free amines were acetylated with acetic
anhydride (47 lL, 0.5 mmol) with pyridine (81 lL, 1.0 mmol)
as base in DMF (5 mL) for 30 min subsequently followed by
extensive washing of the resin with DMF (3 × 10 mL, 2 min),
DCM (3 × 10 mL, 2 min), DMF (3 × 10 mL, 2 min) and dioxane
(3 × 10 mL, 2 min). The N-terminal azide was converted into the
corresponding amine by treatment with trimethylphosphine (1 M
in toluene; 1.5 mL, 1.5 mmol) in dioxane–H2O (4 : 1 v/v) for 1 h.
Then, the resin was washed with dioxane (6 × 10 mL, 2 min), DCM
(3 × 10 mL, 2 min) and DMF (3 × 10 mL, 2 min) followed by
the addition of Boc-Alg-OH (151 mg, 0.7 mmol), BOP (310 mg,
0.7 mmol) and DIPEA (244 lL, 1.4 mmol) in DMF (10 mL).
After 1 h, the resin was washed with DMF (3 × 10 mL, 2 min),
DCM (3 × 10 mL, 2 min), DMF (3 × 10 mL, 2 min) and MeOH
(3 × 10 mL, 2 min). The peptide was cleaved from the resin by
a catalytic amount KCN in methanol (15 mL) during 16 h. The
resin was filtered and washed with methanol (3 × 10 mL). The
filtrate was concentrated in vacuo and the residue was purified by
column chromatography with DCM–MeOH as eluent (97 : 3 →
9 : 1 v/v) followed by preparative HPLC to yield 16.1 mg (overall
yield 11%, average yield per step: 86%) of pure monocyclic peptide
Experimental
For general methods and procedures and details of the computa-
tional modeling, see the Supporting Information‡.
Cross metathesis reaction
ArgoGel-OH resin was loaded with Fmoc-Alg-OH using the
method of Sieber.11 The Fmoc group enabled determination of the
loading and also the efficiency of the coupling reaction.12 Fmoc-
R
Alg-O-ArgoGelꢀ (0.36 mmol g−1, 0.7 g, 0.25 mmol) was washed
with DCM (3 × 10 mL, 2 min) and DMF (3 × 10 mL, 2 min).
The Fmoc group was removed by treatment with 20% piperidine
in DMF (3 × 10 mL, 8 min) and the resin was subsequently
washed with DMF (3 × 10 mL, 2 min), DCM (3 × 10 mL, 2 min)
and DMF (3 × 10 mL, 2 min). Reprotection of the free amine
was performed with Boc2O (764 mg, 3.5 mmol) in DMF (10 mL)
in the presence of DIPEA (1.2 mL, 4.7 mmol) for 2 h at room
temperature. The deprotection–reprotection steps were monitored
with the Kaiser test.25 The resin containing Boc-Alg-O-ArgoGel
was washed with DMF (3 × 10 mL, 2 min), and DCM (3 × 10 mL,
2 min). The obtained resin was swelled in 1,1,2-trichloroethane
(20 mL), Fmoc-Alg-OH (768 mg, 2.28 mmol) was added and the
◦
reaction mixture was purged with N2 at 60 C for 20 min. Then,
8. Rf
: 0.51 (DCM–MeOH 9 : 1 v/v)28; R : 18.1 min; EI-MS: m/z
2nd generation Grubbs’ catalyst (129 mg, 0.16 mmol) was added
and the obtained reaction mixture was allowed to react overnight
at 60 ◦C under a nitrogen atmosphere. Subsequently, the resin was
washed with DCM (6 × 10 mL, 2 min) and diethyl ether (3 ×
10 mL, 2 min) and dried under vacuum. The yield of the cross-
metathesis reaction was calculated from an Fmoc determination12
and was found to be 56% (0.20 mmol g−1).
t
920.75 [M + H]+, 942.90 [M + Na]+, 820.65 [(M − C5H8O2) + H]+;
1H NMR (CDCl3–CD3OH 14.5 : 1 v/v at 283 K, 500 MHz):
d Alg1: 5.62 (m, 1H, cCH), 5.42 (d (J 6.99 Hz), 1H, NH),
5.06 (m, 2H, dCH2), 4.09 (m, 1H, aCH), 2.45 (m, 2H, bCH2),
1.41 (s, 9H, (CH3)3-Boc); Ala2: 7.44 (d (J 6.99 Hz), 1H, NH),
4.34 (m, 1H, aCH), 1.28 (d (J 7.02 Hz), 3H, bCH3); Alg3: 7.62
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(d (J 6.99 Hz), 1H, NH), 5.18 (m, 1H, cCH), 4.36 (m, 1H, aCH),
2.50/2.36 (double m, 2H, bCH2); Alg4: 7.83 (d (J 8.0 Hz), 1H,
NH), 5.62 (m, 1H, cCH), 5.06 (m, 2H, dCH2), 4.26 (m, 1H, aCH),
2.46 (m, 2H, bCH2); Asn(Trt)5: 7.82 (d (J 6.99 Hz), 1H, CONH),
7.62 (s, 1H, CONHTrt), 7.30-7.15 (br m, 15H, arom H Trt), 4.70
(m, 1H, aCH), 3.11/2.46 (double m, 2H, bCH2); Alg6: 6.87 (d (J
7.5 Hz), 1H, NH), 5.18 (m, 1H, cCH), 4.83 (m, 1H, aCH), 3.72 (s,
3H, COOCH3), 2.60/2.09 (double m, 2H, bCH2).
(m, 1H, aCH), 2.53/2.31 (double m, 2H, bCH2); Asn(Trt)5: 7.90
(s, 1H, CONHTrt), 7.69 (d, 1H, CONH), 7.27–7.19 (br m, 15H,
arom H Trt), 4.74 (m, 1H, aCH), 2.88/2.78 (double m, 2H, bCH2);
Alg6: 7.55 (d (J 6.99 Hz), 1H, NH), 5.67 (m, 1H, cCH), 5.07 (d (J
11.5 Hz), 2H, dCH2), 4.47 (m, 1H, aCH), 3.70 (s, 3H, COOCH3),
2.46/2.42 (double m, 2H, bCH2).
Boc-D-Alg1-Ala2-D-Alg3-Alg4-Asn5(Trt)-Alg6-OMe (15)
Synthesis of the bicyclic peptide 9 (Boc-bicyclo(1→4,3→6)[Alg1-
Peptide 15 was synthesized on an Applied Biosystems 433A
Ala2-Alg3-Alg4-Asn(Trt)5-Alg6]-OMe)
peptide synthesizer using the FastMoc protocol29 on Fmoc-Alg-
R
O-ArgoGelꢀ on a 0.25 mmol scale as described for 10. Peptide
Peptide 8 (7 mg, 8 lmol) was dissolved in DCM (3 mL) and
refluxed in a nitrogen atmosphere during 30 min then followed by
the addition of 2nd generation Grubbs’ catalyst (1 mg, 1.1 lmol)
and the reaction mixture was allowed to react for 4 h. The
solvent was removed in vacuo and the residue was purified by
column chromatography with DCM–MeOH as eluent (97 : 3 →
9 : 1 v/v) to obtain bicyclic peptide 9 in 50% (4 mg) yield. Rf:
0.42 (DCM–MeOH 9 : 1 v/v); Rt: 17.1 min; EI-MS: m/z 892.80
15 was purified by column chromatography with DCM–MeOH as
eluent (95 : 5 v/v) to yield 224 mg (90%). Rf: 0.57 (DCM–MeOH
9 : 1 v/v); Rt: 35.9 min31; EI-MS m/z 948.55 [M + H]+, 970.70 [M +
Na]+; 1H NMR (CDCl3–CD3OH 14.5 : 1 v/v at 283 K, 500 MHz):
d D-Alg1: 5.93/5.73 (m, 1H, cCH), 5.75 (m, 1H, NH), 5.14/5.01
(double m, 2H, dCH2), 4.14 (m, 1H, aCH), 2.52/2.17 (double m,
2H, bCH2), 1.44 (s, 9H, (CH3)3-Boc); Ala2: 7.54 (m, 1H, NH), 4.14
(m, 1H, aCH), 1.23 (d (J 7.02 Hz), 3H, bCH3); D-Alg3: 7.54 (m, 1H,
NH), 5.93/5.73 (double m, 1H, cCH), 5.14/5.01 (double m, 2H,
dCH2), 4.14 (m, 1H, aCH), 2.52/2.17 (double m, 2H, bCH2); Alg4:
7.54 (m, 1H, NH), 5.93/5.73 (double m, 1H, cCH), 5.14/5.01
(double m, 2H, dCH2), 4.14 (m, 1H, aCH), 2.52/2.17 (double m,
2H, bCH2); Asn(Trt)5: 7.96 (d, 1H, NH), 7.88 (s, 1H, CONHTrt),
7.27–7.17 (br m, 15H, arom H Trt), 4.72 (m, 1H, aCH), 2.71 (m,
2H, bCH2); Alg6: 7.62 (d, 1H, NH), 5.93/5.73 (double m, 1H,
cCH), 5.14/5.01 (double m, 2H, dCH2), 4.44 (m, 1H, aCH), 3.70
(s, 3H, COOCH3), 2.52/2.17 (double m, 2H, bCH2).
1
[M + H]+, 915.60 [M + Na]+; H NMR (CDCl3–CD3OH 14.5 :
1 v/v at 283 K, 500 MHz): d Alg1: 5.77 (d, 1H, NH), 5.40 (m, 1H,
cCH), 4.18 (m, 1H, aCH), 2.53/2.37 (double m, 2H, bCH2), 1.45
(s, 9H, (CH3)3-Boc); Ala2: 7.86 (d, 1H, NH), 4.21 (m, 1H, aCH),
1.35 (double d (J 7.02 Hz), 3H, bCH3); Alg3: 7.32 (d, 1H, NH),
5.32 (m, 1H, cCH), 4.15 (m, 1H, aCH), 2.53/2.21 (double m, 2H,
bCH2); Alg4: 8.10 (d, 1H, NH), 5.48 (m, 1H, cCH), 4.30 (m, 1H,
aCH), 2.68/2.06 (double m, 2H, bCH2); Asn(Trt)5: 7.94 (d, 1H,
CONH), 7.57 (s, 1H, CONHTrt), 7.31–7.18 (br m, 15H, arom H
Trt), 4.82 (m, 1H, aCH), 3.23/2.51 (double m, 2H, bCH2); Alg6:
6.73 (d, 1H, NH), 5.12 (m, 1H, cCH), 4.84 (m, 1H, aCH), 3.75 (s,
3H, COOCH3), 2.67/1.96 (double m, 2H, bCH2).
Synthesis of the bicyclic peptide 17 (Boc-bicyclo(1→4,3→6)[D-
Alg1-Ala2-D-Alg3-Alg4-Asn(Trt)5-Alg6]-OMe)
Boc-Alg1-Ala2-Alg3-Alg4-Asn5(Trt)-Alg6-OMe (10)
Peptide 15 (67 mg, 70 lmol) was dissolved in 1,2-dichloroethane
(25 mL) and heated to 60 ◦C in a nitrogen atmosphere during
30 min then followed by the addition of 2nd generation Grubbs’
catalyst (11 mg, 13 lmol) and the reaction mixture was allowed
to react for 2 h. A sample was taken from the reaction mixture
and the catalyst was immediately removed by filtration over a
small silica plug. The remaining reaction mixture was allowed to
react overnight after addition of more catalyst (7 mg, 8 lmol).
The solvent was removed in vacuo and the residue was purified by
column chromatography with DCM–MeOH as eluent (97 : 3 →
9 : 1 v/v) and isolated in 80% yield (50 mg). Rf: 0.53 (DCM–
MeOH 9 : 1 v/v); Rt: 27.3 min31; EI-MS m/z 892.8 [M + H]+, 915.5
Peptide 10 was synthesized on an Applied Biosystems 433A
peptide synthesizer using the FastMoc protocol29 on Fmoc-Alg-
R
O-ArgoGelꢀ on a 0.25 mmol scale. Each synthetic cycle consisted
of Na-Fmoc removal by a 10 min treatment with 20% piperidine in
NMP, a 6 min NMP wash, a 45 min coupling step with 1.0 mmol
of preactivated Fmoc amino acid in the presence of 2 equivalents
DIPEA, and a 6 min NMP wash. Na-Fmoc amino acids were
activated in situ with 1.0 mmol HBTU–HOBt30 (0.36 M in NMP)
in the presence of DIPEA (2.0 mmol). The peptide was detached
from the resin by treatment with a catalytic amount of KCN in
MeOH. After washing the resin with MeOH (3 × 10 mL) the
filtrate was concentrated in vacuo and the residue was purified by
column chromatography with DCM–MeOH as eluent (97 : 3 →
9 : 1 v/v) to yield 187 mg (69%) of the linear peptide 10. Rf:
0.57 (DCM–MeOH 9 : 1 v/v); Rt: 18.9 min; EI-MS m/z 948.65
1
[M + Na]+; H NMR (CDCl3–CD3OH 14.5 : m1 v/v at 283 K,
500 MHz): d D-Alg1: 6.52 (br s, 1H, NH), 5.45/5.32 (double m,
1H, cCH), 4.44 (m, 1H, aCH), 2.73/2.27 (double m, 2H, bCH2),
1.51 (s, 9H, (CH3)3-Boc); Ala2: 7.01 (br s, 1H, NH), 4.01 (m, 1H,
aCH), 1.05 (d (J 7.02 Hz), 3H, bCH3); D-Alg3: 8.00 (m, 1H, NH),
5.22/4.69 (double m, 1H, cCH), 4.57 (m, 1H, aCH), 2.73/2.02
(double m, 2H, bCH2); Alg4: 6.66 (d (J 6.99 Hz), 1H, NH),
5.45/5.32 (double m, 1H, cCH), 4.57 (m, 1H, aCH), 2.80/2.26
(double m, 2H, bCH2); Asn(Trt)5: 8.00 (m, 1H, NH), 7.58 (s, 1H,
CONHTrt), 7.25–7.18 (br m, 15H, arom H Trt), 4.78 (m, 1H,
aCH), 3.24/2.87 (br m, 2H, bCH2); Alg6: 6.72 (d (J 6.99 Hz), 1H,
NH), 5.22/4.69 (double m, 1H, cCH), 4.68 (m, 1H, aCH), 3.74 (s,
3H, COOCH3), 2.54/2.01 (double m, 2H, bCH2).
1
[M + H]+, 970.70 [M + Na]+; H NMR (CDCl3–CD3OH 14.5 :
1 v/v at 283 K, 500 MHz): d Alg1: 5.67 (m, 1H, cCH), 5.50 (d (J
6.99 Hz), 1H, NH), 5.07 (d (J 11.5 Hz), 2H, dCH2), 4.06 (m, 1H,
aCH), 2.48/2.31 (double m, 2H, bCH2), 1.44 (s, 9H, (CH3)3-Boc);
Ala2: 7.55 (d (J 6.99 Hz), 1H, NH), 4.22 (m, 1H, aCH), 1.28 (d
(J 7.02 Hz), 3H, bCH3); Alg3: 7.43 (d (J 6.99 Hz), 1H, NH), 5.67
(m, 1H, cCH), 5.07 (d (J 11.5 Hz), 2H, dCH2), 4.21 (m, 1H, aCH),
2.56/2.43 (double m, 2H, bCH2); Alg4: 7.34 (d (J 6.99 Hz), 1H,
NH), 5.67 (m, 1H, cCH), 5.07 (d (J 11.5 Hz), 2H, dCH2), 4.35
932 | Org. Biomol. Chem., 2007, 5, 924–934
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Boc-D-Alg(R-bMe)1-Ala2-D-Alg(R-bMe)3-Alg4-Asn5(Trt)-Alg6-
OMe (18)
7 One report describes the chemical synthesis of the natural DE-ring:
K. Fukase, Y. Oda, A. Kubo, T. Wakamiya and T. Shiba, Bull. Chem.
Soc. Jpn., 1990, 63, 1758(a) Two reports deal with the total synthesis
of nisin: K. Fukase, M. Kitazawa, A. Sano, K. Shimbo, H. Fujita, S.
Horimoto, T. Wakaiya and T. Shiba, Tetrahedron Lett., 1988, 29, 795;
(b) K. Fukase, M. Kitazawa, A. Gano, K. Shimbo, S. Horimoto, H.
Fujita, A. Kubo, T. Wakamiya and T. Shiba, Bull. Chem. Soc. Jpn.,
1992, 65, 2227–2240.
Peptide 18 was obtained with a yield of 82% (210 mg). Rf: 0.64
(DCM–MeOH 9 : 1 v/v); Rt: 18.9 min; EI-MS m/z 976.8 [M +
1
H]+, 999.55 [M + Na]+; H NMR (CDCl3–CD3OH 14.5 : 1 v/v
at 283 K, 500 MHz):d D-Alg(R-bMe)1: 5.70/5.56 (double m, 1H,
cCH), 5.46 (d (J 6.99 Hz), 1H, NH), 5.11/5.03 (double m, 2H,
dCH2), 4.04 (m, 1H, aCH), 2.81/2.36 (double m, 1H, bCH), 1.44
8 For a review on cross metathesis: S. J. Connon and S. Blechert, Angew.
Chem., Int. Ed., 2003, 42, 1900.
9 This is an example of a single reaction step involving a double ring-
closing metathesis reaction, see reference 5a. Other examples of tandem
ring-closing metathesis: (a) T.-L. Choi and R. H. Grubbs, Chem.
Commun., 2001, 2648; (b) F.-D. Boyer and I. Hanna, Tetrahedron Lett.,
2002, 43, 7469; (c) P. Børsting and P. Nielsen, Chem. Commun., 2002,
2140; (d) M. Rosillo, L. Casarrubios, G. Dominguez and J. Perez-
Castelles, Org. Biomol. Chem., 2003, 1, 1450; (e) A. E. Sutton, B. A.
Seigal, D. F. Finnegan and M. L. Snapper, J. Am. Chem. Soc., 2002,
124, 13390; (f) T. Honda, H. Namiki, K. Kaneda and H. Mizutani,
Org. Lett., 2004, 6, 87; (g) R. Garcia-Fandin˜o, E. M. Codesido, E.
Sobarzo-Sa´nchez, L. Castedo and J. R. Granja, Org. Lett., 2004, 6,
193; (h) F.-D. Boyer, I. Hanna and L. Ricard, Org. Lett., 2004, 6,
1817; (i) R. A. J. Wybrow, N. G. Stevenson and J. P. A. Harrity,
Synlett, 2004, 140; (j) H. T. ten Brink, D. T. S. Rijkers, J. Kemmink,
H. W. Hilbers and R. M. J. Liskamp, Org. Biomol. Chem., 2004,
2, 2658; (k) F.-D. Boyer and I. Hanna, Eur. J. Org. Chem., 2006,
471.
ꢁ
(s, 9H, (CH3)3-Boc), 1.06 (d (J 6.87 Hz), 3H, cCH3); Ala2: 7.87 (d
(J 6.99 Hz), 1H, NH), 4.32 (m, 1H, aCH), 1.26 (d (J 7.02 Hz), 3H,
bCH3); D-Alg(R-bMe)3: 7.32 (d (J 6.99 Hz), 1H, NH), 5.70/5.56
(double m, 1H, cCH), 5.11/5.03 (double m, 2H, dCH2), 3.89 (m
1H, aCH), 2.81/2.36 (double m, 1H, bCH), 0.99 (d (J 6.87 Hz),
3H, c’CH3); Alg4: 7.45 (d (J 6.99 Hz), 1H, NH), 5.70/5.56 (double
m, 1H, cCH), 5.11/5.03 (double m, 2H, dCH2), 4.42 (m, 1H, aCH),
2.81/2.36 (double m, 2H, bCH2); Asn(Trt)5: 7.90 (d (J 6.99 Hz),
1H, NH), 7.87 (s, 1H, CONHTrt), 7.28–7.18 (br m, 15H, arom
H Trt), 4.71 (m, 1H, aCH), 2.93 (m, 2H, bCH2); Alg6: 7.60 (d
(J 6.99 Hz), 1H, NH), 5.70/5.56 (double m, 1H, cCH), 5.11/5.03
(double m, 2H, dCH2), 4.42 (m, 1H, aCH), 3.70 (s, 3H, COOCH3),
2.81/2.36 (double m, 2H, bCH2).
10 For another elegant approach for the selective formation of carbon–
carbon bonds via ring-closing metathesis, see: (a) A. J. Robinson, J.
Elaridi, J. Patel and W. R. Jackson, Chem. Commun., 2005, 5544; (b) J.
Elaridi, J. Patel, W. R. Jackson and A. J. Robinson, J. Org. Chem., 2006,
71, 7538.
References and notes
11 P. Sieber, Tetrahedron Lett., 1987, 28, 647.
1 (a) G. Jung, Angew. Chem., Int. Ed. Engl., 1991, 30, 1051; (b) C.
Chatterjee, M. Paul, L. Xie and W. A. van der Donk, Chem. Rev.,
2005, 105, 633.
12 J. Meienhofer, M. Waki, E. P. Heimer, T. J. Lambros, R. C. Makofske
and C.-D. Chang, Int. J. Pept. Protein Res., 1979, 13, 35.
13 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Org. Lett., 1999, 1,
2 E. Gross and J. L. Morell, J. Am. Chem. Soc., 1971, 93, 4634.
3 (a) J. R. van der Meer, J. Polman, M. M. Beerthuyzen, R. J. Siezen,
O. P. Kuipers and W. M. de Vos, J. Bacteriol., 1993, 175, 2578;
(b) O. P. Kuipers, M. M. Beerthuyzen, R. J. Siezen and W. M. de Vos,
Eur. J. Biochem., 1993, 216, 281; (c) L. Xie, L. M. Miller, C. Chatterjee,
O. Averin, N. L. Kelleher and W. A. van der Donk, Science, 2004, 303,
679; (d) L. D. Kluskens, A. Kuipers, R. Rink, E. de Boef, S. Fekken,
A. J. M. Driessen, O. P. Kuipers and G. N. Moll, Biochemistry, 2005,
44, 12827.
4 Chemical approaches for the synthesis of lanthionines: (a) P. L.
Toogood, Tetrahedron Lett., 1993, 34, 7833; (b) S. Burrage, T. Rayham,
G. Williams, J. W. Essex, C. Allen, M. Cardino, V. Swali and M. Bradley,
Chem.–Eur. J., 2000, 6, 1455; (c) N. M. Okeley, Y. Zhu and W. A. van
der Donk, Org. Lett., 2000, 2, 3603; (d) H. Zhou and W. A. van der
Donk, Org. Lett., 2002, 4, 1335; (e) Y. Zhu, M. D. Gieselman, H. Zhou,
O. Averin and W. A. van der Donk, Org. Biomol. Chem., 2003, 1, 3304;
(f) M. Matteucci, G. Bhalay and M. Bradley, Tetrahedron Lett., 2004,
45, 1399; (g) S. Bregant and A. B. Tabor, J. Org. Chem., 2005, 70, 2430;
(h) R. S. Narayan and M. S. VanNieuwenhze, Org. Lett., 2005, 7, 2655;
(i) X. Zhang, W. Ni and W. A. van der Donk, J. Org. Chem., 2005, 70,
6685; (j) A. Avenoza, J. H. Busto, G. Jime´nez-Ose´s and J. M. Peregrina,
Org. Lett., 2006, 8, 2855.
953.
14 J. T. Lundquist, IV and J. C. Pelletier, Org. Lett., 2001, 3, 781.
15 Lactamization was found to be complete after 16 h according to
the Malachite Green test: M. E. Attardi, G. Porcu and M. Taddei,
Tetrahedron Lett., 2000, 41, 7391.
16 Before the peptides could be characterized by MS-MS, both protecting
groups were removed by treatment with TFA. Bicyclic, peptide 9 did
not result in a unique peptide fragmentation pattern due to the crossed
peptide-ring system, see also: J. F. Reichwein, B. Wels, J. A. W. Kruijtzer,
C. Versluis and R. M. J. Liskamp, Angew. Chem., Int. Ed., 1999, 38,
3684.
17 Eight-membered rings were conveniently prepared when the alkene
chain is present on the amide nitrogen, facilitating rotation about
the amide bond: (a) J. F. Reichwein and R. M. J. Liskamp,
Eur. J. Org. Chem., 2000, 2335; (b) J. F. Reichwein, C. Versluis
and R. M. J. Liskamp, J. Org. Chem., 2000, 65, 6187; (c) see also
reference 16.
18 F. Mohamadi, N. C. J. Richards, W. C. Guida, R. Liskamp, M.
Lipton, C. Caufield, G. Chang, T. Hendrickson and W. C. Still,
J. Comput. Chem., 1990, 11, 440. Energy content in kJ mol−1 of the
E/Z conformers: 8: 174.6/168.6, 11: 163.2/157.5, 12: 222.1/236.5, 13:
180.8/176.5.
19 For the stabilization of a-helices by RCM, see: (a) M. J. I. Andrews
and A. B. Tabor, Tetrahedron, 1999, 55, 11711; (b) H. E. Blackwell
and R. H. Grubbs, Angew. Chem., Int. Ed., 1998, 37, 3281; (c) C. E.
Schafmeister, J. Po and G. L. Verdine, J. Am. Chem. Soc., 2000, 122,
5891; (d) H. E. Blackwell, J. D. Sadowsky, R. J. Howard, J. N. Sampson,
J. A. Chao, W. E. Steinmetz, D. J. O’Leary and R. H. Grubbs, J. Org.
Chem., 2001, 66, 5291.
20 NMR studies of nisin showed that in aqueous solution the confor-
mation of the DE-ring system is ‘quite rigid’ and adopts an ‘overall
appearance of two consecutive b-turns or of a somewhat overwound
a-helix’, see: (a) F. J. M. van de Ven, H. W. van den Hooven, R. N. H.
Konings and C. W. Hilbers, Eur. J. Biochem., 1991, 202, 1181; (b) L.-Y.
Lian, W. C. Chan, S. D. Morley, G. C. K. Roberts, B. W. Bycroft and
D. Jackson, Biochem. J., 1992, 283, 413. However, in the presence
of membrane-mimicking micelles, the conformation of the intertwined
DE-ring system is determined as ‘two consecutive type II and II’ b-
turns’, see: H. W. van den Hooven, C. C. M. Doeland, M. van de
5 (a) N. Ghalit, D. T. S. Rijkers, J. Kemmink, C. Versluis and R. M. J.
Liskamp, Chem. Commun., 2005, 192; (b) N. Ghalit, A. J. Poot, A.
Fu¨rstner, D. T. S. Rijkers and R. M. J. Liskamp, Org. Lett., 2005, 7,
2961; (c) N. Ghalit, D. T. S. Rijkers and R. M. J. Liskamp, J. Mol.
Catal. A: Chem., 2006, 254, 68.
6 An alkene-bridge as a mimic of a disulfide bond has been described:
(a) S. J. Miller, H. E. Blackwell and R. H. Grubbs, J. Am. Chem. Soc.,
1996, 118, 9606; (b) R. M. Williams and J. Lui, J. Org. Chem., 1998,
63, 2130; (c) Y. Gao, P. Lane-Bell and J. C. Vederas, J. Org. Chem.,
1998, 63, 2133; (d) J. L. Stymiest, B. F. Mitchell, S. Wong and J. C.
Vederas, Org. Lett., 2003, 5, 47; (e) A. N. Whelan, J. Elaridi, M. Harte,
S. V. Smith, W. R. Jackson and A. J. Robinson, Tetrahedron Lett., 2004,
45, 9545; (f) J. L. Stymiest, B. F. Mitchell, S. Wong and J. C. Vederas,
J. Org. Chem., 2005, 70, 7799; (g) A. N. Whelan, J. Elaridi, R. J. Mulder,
A. J. Robinson and W. R. Jackson, Can. J. Chem., 2005, 83, 875; (h) I.
Berezowska, N. N. Chung, C. Lemieux, B. C. Wilkes and P. W. Schiller,
Acta Biochim. Pol., 2006, 53, 73; (i) D. J. Derksen, J. L. Stymiest and
J. C. Vederas, J. Am. Chem. Soc., 2006, 128, 14252.
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Kamp, R. N. H. Konings, C. W. Hilbers and F. J. M. van de Ven,
Eur. J. Biochem., 1996, 235, 382.
21 Due to the Cahn–Ingold–Prelog rules, the stereochemistry at the
corresponding chiral centers is the same, but the configurations are
opposite since sulfur has a higher priority than oxygen.
25 E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Anal.
Biochem., 1970, 34, 595.
26 G. W. Anderson, J. F. Zimmerman and F. M. Callahan, J. Am. Chem.
Soc., 1964, 86, 1839.
27 (a) L. A. Carpino, J. Am. Chem. Soc., 1993, 115, 4397; (b) L. A. Carpino,
A. El-Faham, C. A. Minor and F. Albericio, J. Chem. Soc., Chem.
Commun., 1994, 201.
22 P. Schwab, M. B. France, J. W. Ziller and R. H. Grubbs, Angew. Chem.,
Int. Ed. Engl., 1995, 34, 2039.
23 This amino acid was synthesized according to the procedures as
described by: (a) I. Izzo, E. Avallone, L. Della Corte, N. Maulucci and
F. De Riccardis, Tetrahedron: Asymmetry, 2004, 15, 1181; (b) based
on previous methods developed by: U. Kazmaier, Angew. Chem., Int.
Ed. Engl., 1994, 33, 998; (c) K. Sakaguchi, M. Fujita, H. Suzuki, M.
Higashino and Y. Ohfune, Tetrahedron Lett., 2000, 41, 6589; (d) K.
Sakaguchi, H. Suzuki and Y. Ohfune, Chirality, 2001, 13, 357; (e) Y.
Morimoto, M. Takaishi, T. Kinoshita, K. Sakaguchi and K. Shibata,
Chem. Commun., 2002, 42.
28 Rf values were determined by thin layer chromatography (TLC)
on Merck precoated silica gel 60F254 (0.25 mm) plates. Spots were
visualized with UV quenching, ninhydrin or TDM–Cl2: E. von Arx,
M. Faupel and M. J. Brugger, J. Chromatogr., 1976, 120, 224.
29 K. M. Otteson, R. L. Noble, P. D. Hoeprich, Jr., K. T. Shaw and R.
Ramage, Applied Biosystems Research News, June 1993, 1.
30 C. G. Fields, D. H. Lloyd, R. L. Macdonald, K. M. Otteson and R. L.
Noble, Peptide Res., 1991, 4, 95.
31 This analytical HPLC runs were performed at a flow of 1 mL min−1
using a linear gradient of buffer B (100% in 45 min) from 100% buffer
A (buffer A: 0.1% TFA in H2O; buffer B: 0.085% TFA in CH3CN–H2O
95 : 5 v/v) on a C8 column.
24 (a) U. Kazmaier, C. Hebach, A. Watzke, S. Maier, H. Mues and V.
Huch, Org. Biomol. Chem., 2005, 3, 136; (b) A. B. Smith, III, E. F.
Mesaros and E. A. Meyer, J. Am. Chem. Soc., 2005, 127, 6948.
934 | Org. Biomol. Chem., 2007, 5, 924–934
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