Synthesis of orthogonally protected lanthionines: A reassessment of the use of alanyl β-cation equivalents

Article abstract of DOI:10.1016/S0040-4039(02)01981-0

Whilst developing a strategy for the solid-phase synthesis of lanthionine-containing peptides, we became aware of some problems with a previously published route for the synthesis of orthogonally-protected lanthionine. We report a structural reassignment

Full text of DOI:10.1016/S0040-4039(02)01981-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8359–8362
Synthesis of orthogonally protected lanthionines:
a reassessment of the use of alanyl b-cation equivalents
a
b,c
d
d
M. Firouz Mohd Mustapa, Richard Harris, Jessica Mould, Nathan A. L. Chubb,
d
b,c,e
a,
Darren Schultz, Paul C. Driscoll
and Alethea B. Tabor *
a
Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street,
London WC1H OAJ, UK
b
Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street,
London WC1E 6BT, UK
c
Bloomsbury Centre for Structural Biology, Department of Biochemistry and Molecular Biology, University College London,
Gower Street, London WC1E 6BT, UK
d
Pfizer Limited, Sandwich, Kent CT13 9NJ, UK
e
Ludwig Institute for Cancer Research, 91, Riding House Street, London W1W 7BS, UK
Received 27 June 2002; revised 13 August 2002; accepted 13 September 2002
Abstract—Whilst developing a strategy for the solid-phase synthesis of lanthionine-containing peptides, we became aware of some
problems with a previously published route for the synthesis of orthogonally-protected lanthionine. We report a structural
reassignment of the key iodoalanine intermediate and resulting lanthionine derivatives. © 2002 Elsevier Science Ltd. All rights
reserved.
6
The unusual amino acid lanthionine 1, a monosulfide
analogue of cystine, is the key component of lantibiotic
peptides with important antibiotic and other biologica1l
properties, such as nisin, duramycin and subtilin.
Incorporation of lanthionine into a peptide results in a
cyclic structure, bridged by a thioether, which cannot
be reductively cleaved, and additionally imparts stabil-
extrusion of sulfur from cystine, ring-opening of serine
7
8
b-lactone, Michael addition to dehydroalanine, and
approaches based on alanyl b-cation equivalents. We
chose to adapt the alanyl b-cation equivalent approach
9
developed by Dugave and M e´ nez as it was direct,
high-yielding and amenable to large-scale synthesis.
Moreover, it had none of the drawbacks of regio- or
2
6–8
ity to proteolytic cleavage. It has therefore also been
stereoselectivity
reported with the other strategies.
used in the design and synthesis of cyclic analogues of
bioactive peptides, for instance analogues of
However, during the course of the synthesis, some
problems with the original published report became
apparent, and we therefore carried out a full investiga-
tion of the regio- and stereochemical outcome of the
key reaction between the alanyl b-cation equivalent and
cysteine. In this paper, we report the results of this
investigation, which have led us to a reassessment of
the course of this reaction.
3
4
enkephalin and somatostatin. As part of an ongoing
programme to develop a new approach for the solid-
phase synthesis of cyclic peptides with unnatural side-
5
chain linkages, we are currently investigating the
synthesis of mono- and polycyclic lanthionine-bridged
peptides.
Our approach required the synthesis of the orthogo-
nally protected lanthionine 2. A few approaches to the
synthesis of lanthionine have been published, involving
Nucleophilic reaction at the b-position of a-amino acids
is known to be most successful when the amino group
is trityl-protected; this suppresses competing reactions,
1
0
particularly elimination, forming dehydroalanine.
D)-Serine was therefore converted to the N-trityl
(
1
1
*
Corresponding author. Fax: +44 20 7679 7463; e-mail: a.b.tabor@
derivative using a one-pot procedure, and esterified to
1
2
9
ucl.ac.uk
give 3 (Scheme 1). Following the reported method,
0
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01981-0

8
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M. F. Mohd Mustapa et al. / Tetrahedron Letters 43 (2002) 8359–8362
this was then converted to mesylate 4 and reacted with
sodium iodide to give iodoalanine 5. At this point, the
presence of two distinct isomeric forms was observed in
this chiral centre does not occur during the coupling of
iodoalanines to cysteine. We independently corrobo-
rated these observations by the synthesis of the
9
1
the H NMR spectrum, in a ratio of between 2:1 and
diastereomeric 9 from L-serine, via 10 and 11 (Scheme
13
13
1
3
:2 (dependent on the reaction temperature). Dugave
2). As before, two isomers were observed in the H
NMR spectra of 10 and 11, and the two isomers
observed for 11 were different from those observed for
and M e´ nez had also observed the presence of two
1
isomers in the H NMR spectrum when synthesising
15
iodoalanines such as 6. In the original report, this was
attributed to the presence of at least two rotameric
forms of the iodoalanines in which the conformations
were locked due to the steric bulk of the trityl and iodo
moieties.
7
.
1
13
A H– C HSQC spectrum was acquired on iodoala-
nine 5, and this revealed that the chemical shift of the
a-carbon in the major component was ꢀ30 ppm
upfield of that in the minor component (20.1 ppm
versus 55.8 ppm), and that of the b-carbon was ꢀ40
ppm downfield (48.2 ppm versus 9.55 ppm). These
chemical shifts are only compatible with attachment of
iodine at the a-carbon in the major component and at
the b-carbon in the minor component (Fig. 1a). This
implies that the major component resulting from the
reaction of mesylate 4 with NaI is the regioisomer 12,
and the minor component is the desired 5, not rotamers
as previously reported. In order to confirm these obser-
vations, we carried out an HMBC experiment on the
Again following the published procedure, 5 was reacted
t
with Fmoc-Cys-O Bu in the presence of Cs CO to give
2
3
lanthionine 7 in excellent yield. Removal of the trityl
group and replacement with Aloc to give 8, followed by
removal of the tert-butyl ester, appeared to afford 2,
with the desired orthogonal protecting groups for
SPPS. Dugave and M e´ nez had also reported the pres-
ence of two rotameric forms for lanthionines formed
from iodoalanines such as 6, and indeed we also
1
observed two clearly distinct sets of H NMR signals
for 7, in ratios as high as 4:1. However, we were
disturbed to find that, even on removal of the bulky
trityl group and its replacement by Aloc, multiple
isomers were still observed in the NMR spectrum.
Moreover, although these compounds appeared homo-
geneous by tlc, careful HPLC of 5 and 8 revealed two
peaks. Compound 5 was partially resolved by prepara-
tive HPLC, and 8 was completely separated to give two
3
mixture of lanthionine isomers 7 (Fig. 1b). J Correla-
tions between the b-CH on the Fmoc-protected side of
2
the lanthionine and the a-C on the trityl-protected side
and also between the a-CH on the Fmoc-protected side
of the lanthionine and the b-C on the trityl-protected
side were observed for the major isomer. This is only
compatible with the formation of the regioisomer 13
from the major component 12. Finally, the complexity
of the spectra suggested that a mixture of two
diastereoisomers of 13 is formed, resulting from racemi-
sation of the a-iodo-b-alanine 12.
1
4
distinct isomers (Scheme 1).
Clearly these results threw doubt on the original assign-
ment of these isomers as the result of restricted rota-
tion. Two other possibilities remained; diastereoisomers
a
resulting from racemisation at the C -position derived
In summary, we have demonstrated that the synthesis
of lanthionines using N-trityl iodoalanine as an alanyl
b-cation equivalent is problematic. The major product
of this synthetic strategy is the regioisomeric nor-lan-
thionine 12, formed from the regioisomeric a-iodo-b-
from the serine precursor; or regioisomer formation.
Dugave and M e´ nez had previously demonstrated, via
desulfurisation, derivatisation and chiral HPLC mea-
surements of the resulting alanines, that racemisation of
Scheme 1. Reagents and conditions: (i) Me SiCl, Et N, DCM, Trt–Cl, then MeOH (88%); (ii) Cs CO , allyl bromide (87%); (iii)
3
3
2
3
t
MsCl, Et N (91%); (iv) NaI, acetone (87%); (v) Fmoc-CysO Bu, Cs CO (90%); (vi) TFA/CH Cl ; (vii) Alloc–Cl, NaHCO ,
3
2
3
2
2
3
dioxane/H O (87%); (viii) TFA/CH Cl (95%).
2
2
2
Scheme 2.

M. F. Mohd Mustapa et al. / Tetrahedron Letters 43 (2002) 8359–8362
8361
1
13
Figure 1. (a) Selected region of the assigned 2D H– C HSQC spectrum of the mixture of 5 and the regioisomer 12 in CDCl
3
1
13
recorded on a 600 MHz Varian UNITYplus spectrometer at 283 K. (b) Selected region of the 2D H– C HMBC spectrum of
the mixture of 7 and the regioisomer 13 in CD OD recorded on a 500 MHz Varian UNITYplus spectrometer at 283 K. The J
3
3
correlations across the thioether bridge of the regioisomer 13 are shown.
alanine 12, and not mixtures of rotameric forms of the
desired lanthionine 7 and iodoalanine 5, respectively, as
3. Polinsky, A.; Cooney, M. G.; Toy-Palmer, A.; O
Goodman, M. J. Med. Chem. 1992, 35, 4185–4194.
4. Melacini, G.; Zhu, Q.; Osapay, G.; Goodman, M. J. Med.
8
sapay, G.;
9
previously reported. Work is in progress to establish
8
the mechanistic pathway for this rearrangement, and
will be published in due course.
Chem. 1997, 40, 2252–2258.
5. Alexander McNamara, L. M.; Andrews, M. J. I.; Mitzel,
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. Shao, H.; Wang, S. H. H.; Lee, C.-W.; O
6
Acknowledgements
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8
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sapay, G.;
We thank the Overseas Research Students’ Award
Scheme for the award of a Scholarship (to MFMM),
UCL for the award of a Graduate School Research
Scholarship (to MFMM) and Pfizer Limited for a
CASE award (to MFMM). We also acknowledge the
considerable preliminary work of Dr. Marianne
Groussier (UCL). This is a publication from the
Bloomsbury Centre for Structural Biology, funded by
the BBSRC.
Goodman, M. J. Org. Chem. 1995, 60, 2956–2957.
. Probert, J. M.; Rennex, D.; Bradley, M. Tetrahedron Lett.
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chromatography and, in the case of 5 and its enantiomer,
also by recrystallisation. Found for 10: C, 60.15; H, 4.8;
N, 2.7; I, 25.6. C H NO I requires C, 60.4; H, 4.9; N, 2.8;
1
. van Kraaij, C.; de Vos, W. M.; Siezen, R. J.; Kuipers, O.
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2
2. O
8
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C H N O S requires C, 73.4; H, 6.3; N, 3.6; S, 4.2%.
47
48
2
6
14. Preparative normal phase HPLC separation of 5: Tech-
−
1
sil5SILICA column, 22 mm×25 cm, flow rate 15 ml min :

8
362
M. F. Mohd Mustapa et al. / Tetrahedron Letters 43 (2002) 8359–8362
gradient 2–5% EtOAc in hexane over 40 min: two par-
15. The chemical shifts of each isomer of the two indepen-
dently prepared diastereoisomers 11 and 7 are similar,
but not identical. Previous work in which the two lan-
thionine diastereoisomers were unambiguously prepared
have also shown the chemical shifts to be similar:
tially resolved peaks at 14.5 and 15.5 min corresponding
1
to the two isomers observed by H NMR. HPLC separa-
tion of 8 was carried out using the same conditions:
gradient 17–23% EtOAc in hexane over 45 min: two
peaks at 20.4 and 23.6 min were completely separated,
O
8
sapay, G.; Zhu, Q.; Shao, H.; Chadha, R. K.; Good-
1
corresponding to the two isomers observed by H NMR.
man, M. Int. J. Pept. Prot. Res. 1995, 46, 290–301.