Pre-organization induced synthesis of a crossed alkene-bridged nisin Z DE-ring mimic by ring-closing metathesis

Article abstract of DOI:10.1039/b415555f

An alkene-bridged mimic of the complex DE-bisthioether-ring system of the antibiotic nisin was prepared in one step from the linear precursor.

Full text of DOI:10.1039/b415555f

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Pre-organization induced synthesis of a crossed alkene-bridged nisin Z
DE-ring mimic by ring-closing metathesis{
Nourdin Ghalit,a Dirk T. S. Rijkers,a Johan Kemmink,a Cees Versluisb and Rob M. J. Liskamp*a
Received (in Cambridge, UK) 7th October 2004, Accepted 10th November 2004
First published as an Advance Article on the web 2nd December 2004
DOI: 10.1039/b415555f
is particularly difficult to synthesize.4,5 Here we report the first time
An alkene-bridged mimic of the complex DE-bisthioether-ring
that RCM has been applied for the synthesis of a crossed alkene
bridge for obtaining mimics of the thioether bridges6 containing
lantibiotics.
system of the antibiotic nisin was prepared in one step from the
linear precursor.
Stable, naturally occurring, conformational constraints, such as
thioether (sulfide) or disulfide bridges are very important for the
three-dimensional shape and thereby biological activity of
peptides. In order to investigate whether the repertoire of nature
involving these conformational constraints can be extended, we are
interested in using among others an alkene bridge as an alternative
constraint for bioactive molecules.1
The most straightforward route towards the crossed alkene-
bridged DE mimic is a direct synthesis from the linear peptide
RCM-precursor containing the required allylglycine residues.
Thus, protected peptide 2 was obtained after solid phase peptide
synthesis using Fmoc/tBu protocols followed by purification in an
overall yield of 69% (Scheme 1). 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 by purification on HPLC and by characterization using
LCES-TOF MS/MS. The observed mass in combination with the
obtained fragmentation pattern enabled the elucidation of the
structure of the formed monocylic intermediates. Theoretically, six
monocyclic intermediates could have been formed, however, only
four (3–6) corresponding to the [3,6], [1,4], [1,6] and [4,6] RCM
products were found (Scheme 1). 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. The unique
fragmentation pattern of each RCM-product enabled unequivocal
The lantibiotic nisin Z produced by a broad range of bacteria
e.g., Bacillus, Lactococcus, Streptomyces and Staphylococcus
species,2 poses a particular challenge as a target for this aim, since
it contains five thioether bridges, two of which form a ‘‘knot’’
comprising the DE-ring system (Fig. 1). It was planned to
synthesize ultimately the complete alkene-bridged nisin Z mimic
(Fig. 1) starting from the linear fragments constituting the
individual ring systems followed by cyclization using ring-closing
metathesis (RCM).3
In the DE-ring system of nisin the amino acid side chains cross
each other. As a consequence, an alkene mimic of this ring system
{ Electronic supplementary information (ESI) available: Experimental
procedures and characterization details for 2, 7 and 14. See http://
www.rsc.org/suppdata/cc/b4/b415555f/
*R.M.J.Liskamp@pharm.uu.nl
Fig. 1 Nisin Z and its alkene-bridged mimic 7.
192 | Chem. Commun., 2005, 192–194
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Scheme 1 Possible and observed intermediates in the one step double RCM leading to alkene-bridged mimic 7.
determination of the position of the cyclic constraint.1a The ratio
of product formation 3 : 4 : 5 : 6 was found to be ca. 1 : 4 : 1 : 2
and thus the reaction mixture contained 63% of the desired
intermediates 3 and 4. A purely statistical distribution—assuming
the formation of six possible RCM-products—would only have
led to formation of ca. 33% of 3 and 4. Molecular mechanics
calculations were in agreement with this preferential formation of 3
and 4, since the energy of these intermediates was significantly
lower.7 Next, the products obtained after refluxing overnight were
isolated and purified. Only two of the three possible bicylic
compounds—based on the formed monocyclic compounds in the
reaction mixture sample—were observed. Both monocyclic
products 3 and 4 cyclized to the desired bicyclic product 7.
Intermediate 6 cyclized to product 8 i.e. the [1,3]–[4,6] product. Not
unexpectedly (vide supra), [1,6] product 5 was not converted to a
bicyclic product, since this would require formation of a [3,4] cycle,
which is probably difficult (vide infra). Thus, the desired bicyclic
product was obtained in 72% yield as compared to only 19% of
one other bicyclic product (8). The preferred formation of
monocyclic products 3 and 4 and the ensuing bicyclic product
hints at a favorable pre-organization of the linear peptide for
formation of the DE-ring alkene mimic, which in view of their ring
Scheme 2 Step-wise synthesis featuring subsequent cross metathesis and ring-closing metathesis to afford alkene-bridged mimic 7.
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E-mail: R.M.J.Liskamp@pharm.uu.nl; Fax: 31 30 2536655;
size (two 14-membered rings) might be close to an a-helical
structure. 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.
Tel: 31 30 2537396
bDepartment of Biomolecular Mass Spectrometry, Bijvoet Center for
Biomolecular Research and Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, The Netherlands
The structure of the desired mimic was confirmed by
independent step-wise synthesis (Scheme 2). Starting from Boc-
Alg-Argogel2 (9) the alkene bridge of ring E was synthesized by a
cross metathesis with Fmoc-Alg-OH thus affording 10.8 It was
found that protection of the carboxyl moiety was not required. A
third orthogonal protecting group was now necessary and after
removal of the Fmoc group, N3-Ala-ONSu was coupled, in which
the azide is orthogonal to the Fmoc and Boc-group. Next,
acidolysis of the Boc-group in 11 by TFA was followed by
coupling of Fmoc-Asn(Trt)-ONSu. After removal of the Fmoc
group, Fmoc-Alg-ONSu was coupled to give 12. Removal of the
Fmoc group was followed by lactamization using HATU/HOAt/
DIPEA in DMF between residues Alg3 and Alg4 to afford ring E.
Reduction of the azide functionality in 13 under Staudinger
conditions gave the free amine.9 Finally, Boc-Alg-OH was coupled
and the resulting resin was treated with a catalytic amount of
KCN in methanol to give the monocyclic fully protected
peptide ester 14 (after purification: 11% overall yield, average of
82% per step). The correct side chain to side chain connectivity of
ring E was confirmed by NMR analysis (1H-500 MHz, TOCSY,
NOESY and ROESY) and the correct fragmentation pattern was
found by mass analysis (LCES-TOF MS/MS). 14 was treated with
2nd generation Grubbs catalyst to give the desired bicyclic peptide
7 in 50% yield. NMR analysis in combination with MS/MS
experiments proved that the ring structure was correct and
identical with the product obtained by a one step double ring-
closing metathesis.
Notes and references
1 Recent examples of our investigations include: (a) Leu-enkephalin:
J. F. Reichwein, B. Wels, J. A. W. Kruijtzer, C. Versluis and R. M.
J. Liskamp, Angew. Chem., Int. Ed., 1999, 38, 3684; (b) Grb2 SH2
ligands: F. J. Dekker, N. J. de Mol, M. J. E. Fischer, J. Kemmink and
R. M. J. Liskamp, Org. Biomol. Chem., 2003, 1, 3297; (c) MC4-receptor
ligands: B. Wels, PhD Thesis, University of Utrecht, 2004; (d) B. Wels,
J. A. W. Kruijtzer, K. M. Garner, W. A. J. Nijenhuis, W. H. Gispen,
R. A. H. Adan and R. M. J. Liskamp, manuscript in preparation; (e)
vancomycin: H. T. ten Brink, D. T. S. Rijkers, J. Kemmink, H. W.
Hilbers and R. M. J. Liskamp, Org. Biomol. Chem., 2004, 2, 2658.
2 (a) G. Jung, Angew. Chem., Int. Ed. Engl., 1991, 30, 1051; (b) H.-G. Sahl,
R. W. Jack and G. Bierbaum, Eur. J. Biochem., 1995, 230, 827.
3 For reviews on alkene metathesis: (a) T. M. Trnka and R. H. Grubbs,
Acc. Chem. Res., 2001, 34, 18; (b) A. Fu¨rstner, Angew. Chem., Int. Ed.,
2000, 39, 3012; (c) R. H. Grubbs and S. Chang, Tetrahedron, 1998, 54,
4413.
4 Synthesis of the natural DE-ring: (a) K. Fukase, Y. Oda, A. Kubo,
T. Wakamiya and T. Shiba, Bull. Chem. Soc. Jpn., 1990, 63, 1758; total
synthesis of nisin: (b) K. Fukase, M. Kitazawa, A. Sano, K. Shimbo,
H. Fujita, S. Horimoto, T. Wakaiya and T. Shiba, Tetrahedron Lett.,
1988, 29, 795; (c) 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.
5 (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.
6 For recent examples of an alkene-bridge as a mimic of a disulfide bond
see: S. J. Miller, H. E. Blackwell and R. H. Grubbs, J. Am. Chem. Soc.,
1996, 118, 9606; R. M. Williams and J. Liu, J. Org. Chem., 1998, 63,
2130; Y. Gao, P. Lane-Bell and J. C. Vederas, J. Org. Chem., 1998, 63,
2133; J. L. Stymiest, B. F. Mitchell, S. Wong and J. C. Vederas, Org.
Lett., 2003, 5, 47.
In conclusion, we have prepared an alkene mimic of the
challenging nisin DE-ring in a single reaction step involving a
double RCM. Preferred formation of the intermediate monocyclic
mimic, resulting in formation of the desired bicyclic mimic, may be
due to a considerable degree of pre-organization of the linear
peptide RCM-precursor.
7 Energy content in kJ mol21 of the E/Z conformers: 3: 263.3/249.3, 4:
238.6/243.0, 5: 295.6/278.8, 6: 286.9/264.8. These values were obtained
using MacroModel: F. Mohamadi, N. C. J. Richards, W. C. Guida,
R. Liskamp, C. Caufield, G. Chang, T. Hendrickson and W. C. Still, J.
Comput. Chem., 1990, 11, 440.
8 For a review on cross metathesis: S. J. Connon and S. Blechert, Angew.
Chem., Int. Ed., 2003, 42, 1900.
9 J. T. Lundquist, IV and J. C. Pelletier, Org. Lett., 2001, 3, 781.
Nourdin Ghalit,a Dirk T. S. Rijkers,a Johan Kemmink,a Cees Versluisb
and Rob M. J. Liskamp*a
aDepartment of Medicinal Chemistry, Utrecht Institute for
Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Utrecht
University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands.
194 | Chem. Commun., 2005, 192–194
This journal is ß The Royal Society of Chemistry 2005