Orthogonally protected lanthionines: Synthesis and use for the solid-phase synthesis of an analogue of nisin ring C

Article abstract of DOI:10.1021/jo048222t

(Chemical Equation Presented) Lanthionine, a thioether analogue of cystine, is a key component of the lantibiotics, a family of modified peptides bearing multiple thioether bridges resulting from posttranslational modifications between side chains. It is also used as a conformational constraint in medicinally active peptides. We have explored two synthetic routes to give lanthionine, orthogonally protected with Alloc/allyl and Fmoc groups. One route utilized a carbamate-protected iodoalanine that yielded a mixture of diastereoisomers, and one utilized a trityl-protected iodoalanine, formed via a Mitsunobu reaction, that gave the single desired lanthionine, in complete regio-and diastereoselectivity. We then used this orthogonally protected lanthionine in the solid-phase synthesis of an analogue of a fragment of nisin containing its ring C. The chemoselective deprotection of the allyl and Alloc groups of the incorporated lanthionine unit was followed by regio- and stereoselective cyclization on resin to give the desired lanthionine-bridged peptide.

Full text of DOI:10.1021/jo048222t

Orthogonally Protected Lanthionines: Synthesis and Use for the
Solid-Phase Synthesis of an Analogue of Nisin Ring C
Sarah Bregant and Alethea B. Tabor*
Department of Chemistry, UCL, Christopher Ingold Laboratories, 20, Gordon Street,
London WC1H 0AJ, UK
a.b.tabor@ucl.ac.uk
Received October 8, 2004
Lanthionine, a thioether analogue of cystine, is a key component of the lantibiotics, a family of
modified peptides bearing multiple thioether bridges resulting from posttranslational modifications
between side chains. It is also used as a conformational constraint in medicinally active peptides.
We have explored two synthetic routes to give lanthionine, orthogonally protected with Alloc/allyl
and Fmoc groups. One route utilized a carbamate-protected iodoalanine that yielded a mixture of
diastereoisomers, and one utilized a trityl-protected iodoalanine, formed via a Mitsunobu reaction,
that gave the single desired lanthionine, in complete regio-and diastereoselectivity. We then used
this orthogonally protected lanthionine in the solid-phase synthesis of an analogue of a fragment
of nisin containing its ring C. The chemoselective deprotection of the allyl and Alloc groups of the
incorporated lanthionine unit was followed by regio- and stereoselective cyclization on resin to
give the desired lanthionine-bridged peptide.
Introduction
tibiotics typically include between two and five lanthion-
ine, or â-methyllanthionine, residues, and therefore
have two to five thioether bridges. These bridges can be
mostly sequential, as in Type A lantibiotics such as nisin
and subtilisin, or highly overlapping, as in Type B
lantibiotics such as cinnamycin. The thioether linkage
acts as a conformational constraint, which mimics the
disulfide bridge of cystine but is stable to reducing agents.
For this reason, lanthionine and analogues have also
been incorporated into medicinally relevant peptides
to afford biologically active, thioether-bridged peptido-
mimetics.3
The development of methodology for the synthesis of
peptides containing lanthionine and indeed for the
synthesis of lanthionine and methyllanthionine them-
selves are therefore of great interest. To date, however,
the formidable challenge presented by the lantibiotics has
The unusual bisamino acid lanthionine, a thioether
analogue of cystine, is a key component of the lantibiot-
1
ics, a group of multiply bridged peptides with a range
of biological properties, and it is also found as a cross-
linking unit in bacterial cell walls. Incorporation of
both amino acid moieties of lanthionine into a linear
peptide sequence necessarily results in a cyclic peptide
with a thioether linkage between two side chains. Lan-
2
(
1) For reviews, see: (a) Jung, G. Angew. Chem., Intl. Ed. Engl.
1
991, 30, 1051-1192. (b) Sahl, H.-G.; Jack, R. W.; Bierbaum, G. Eur.
J. Biochem. 1995, 230, 827-853. (c) Sahl, H.-G.; Bierbaum, G. Annu.
Rev. Microbiol. 1998, 52, 41-79. (d) van Kraaij, C.; de Vos, W. M.;
Siezen, R. J.; Kuipers, O. P. Nat. Prod. Rep. 1999, 16, 575-587. (e)
Breukink, E.; de Kruijff, B. Biochem. Biophys. Acta 1999, 1462, 223-
2
6
34. (f) Guder, A.; Wiedemann, I.; Sahl, H.-G. Biopolymers 2000, 55,
2-73. (g) Pag, U.; Sahl, H.-G. Curr. Pharm. Des. 2002, 8, 815-833.
(
2) Richaud, C.; Mengin-Lecreulx, D.; Pochet, S.; Johnson, E. J.;
Cohen, G. N.; Marli e` re, P. J. Biol. Chem., 1993, 268, 26827-26835.
10.1021/jo048222t CCC: $30.25 © 2005 American Chemical Society
2430
J. Org. Chem. 2005, 70, 2430-2438
Published on Web 03/02/2005

Solid-Phase Synthesis of an Analogue of Nisin Ring C
-11
meant that only one total synthesis has been disclosed.
Shiba et al. have reported the total synthesis of nisin,
by solution-phase synthesis of individual cystine-bridged
cyclic peptides, followed by desulfurization to give lanthion-
ine-bridged fragments, and finally by segment coupling
residues are within a linear peptide.7 The ring opening
of serine â-lactones13 with protected cysteines gives
mixtures of lanthionines and thioesters, although steri-
cally hindered analogues may be successfully pre-
pared.1
3,14
The ring opening of aziridines with cysteine
4
15
of these fragments. This desulfurization approach for the
has also been studied; however, mixtures of regio-
isomers arising from competing attack at the R- and
â-positions have been observed. Recent reports have
suggested that lanthionine can be expediently synthe-
sized from the attack of suitably protected cysteine on
in situ generation of lanthionine bridges (and related
structures) from disulfide-bridged cyclic peptides has
been explored in detail by Spatola et al., who have
demonstrated that the mechanism of this transformation
involves generation of a dehydroalanine residue, followed
by Michael addition to form the thioether. Under these
â-iodo-1
6,17
or â-bromoalanine.
18
We have developed a flexible, general method for the
solid-phase synthesis of other side chain bridged peptides
using orthogonally protected bisamino acids as building
(
base-catalyzed) conditions, however, the reaction does
5
not appear to be diastereoselective. The methodology has
also recently been adapted for solid-phase methods.6
Several other groups have approached the synthesis of
lanthionine-bridged cyclic peptides by explicitly generat-
ing dehydroalanine residues within a preassembled pep-
tide and subsequently carrying out a Michael addition
of cysteine sulfhydryl to form the thioether bridge.7-11
This biomimetic approach has been most extensively
explored by Bradley et al.11 using mildly basic conditions
19,20
blocks.
In this general approach, the required bisami-
no acid is first synthesized with the side chain linkage
(Z: e.g., Z ) aliphatic) in place. One amino acid moiety
is protected for conventional Fmoc-based synthesis, and
the other amino acid moiety is protected with allyl and
Alloc groups, orthogonal to both the transient (Fmoc) and
t
permanent ( Bu/Boc) protecting groups used in solid-
phase peptide synthesis. This bisamino acid is then
incorporated into a linear peptide using standard solid-
phase peptide synthesis conditions (Scheme 1). The ring
closure is formed by sequential selective deprotection of
the allyl/Alloc, and Fmoc groups followed by on-resin
cyclization, affording the desired side chain bridged cyclic
peptide, which can then be further chain-extended.
In this paper, we report two new approaches to the
synthesis of orthogonally protected lanthionine (Z ) S),
and in an extension of our solid-phase approach, we
demonstrate the use of this residue in the solid-phase
synthesis of an analogue of ring C of the lantibiotic nisin.
(
buffer, pH 8) for the cyclization step. Under these
conditions, the majority of the precursor peptides appear
to be preorganized such that the Michael addition
proceeds in a diastereoselective fashion to give a single
diastereoisomer. This approach has also recently been
adapted for solid-phase methods.1 Finally, the PCOR
1b
(
peptide cyclization on an oxime resin) method has also
3
been employed. In this method, a linear peptide contain-
ing an orthogonally protected lanthionine is first syn-
thesized, and head-to-tail cyclization, with simultaneous
cleavage of the cyclic peptide from the resin, then gives
the desired cyclic peptide. All of these approaches have
shown potential for the synthesis of singly bridged
lanthionine-containing peptides. However, the expedient
and regioselective formation of multiple and overlapping
thioether bridges, as seen in the families of lantibiotics,
still presents a considerable challenge.
Results and Discussion
Synthesis of Lanthionines via Carbamate-Pro-
20
tected Iodoalanine. We have previously shown that
16
the synthesis of protected lanthionines from the reac-
The synthesis of lanthionine itself also presents several
challenges. Michael addition of cysteine to separate
dehydroalanine residues has been shown to give a 1:1
mixture of lanthionine diastereoisomers,12 in the absence
of any conformational preference that arises when these
t
tion of N-trityl-â-iodoalanine esters with Fmoc-Cys-O -
Bu and Cs CO is prone to rearrangement during the
2 3
synthesis of the N-trityl-â-iodoalanine esters. This gives
R-iodo-â-alanine derivatives and hence nor-lanthionines
as the major product and not a mixture of lanthionine
rotamers, as previously reported. This rearrangement is
assumed to take place via an aziridine intermediate,
which is generated both during the synthesis of N-trityl-
â-iodoalanine from N-trityl-O-methanesulfonyl serine
esters and during its reaction with thiol nucleophiles.
(3) Recent examples include: (a) O¨ sapay, G.; Prokai, L.; Kim, H.-
S.; Medzihradszky, K. F.; Coy, D. H.; Liapakis, G.; Reisine, T.; Melacini,
G.; Zhu, Q.; Wang, S. H.-H.; Mattern, R.-H.; Goodman, M. J. Med.
Chem. 1997, 40, 2241-2251. (b) Melacini, G.; Zhu, Q.; O¨ sapay, G.;
Goodman, M. J. Med. Chem. 1997, 40, 2252-2258. (c) Rew, Y.;
Malkmus, S.; Svensson, C.; Yaksh, T. L.; Chung, N. N.; Schiller, P.
W.; Cassel, J. A.; DeHaven, R. N.; Goodman, M. J. Med. Chem. 2002,
4
5, 3746-3754.
4) Fukase, K.; Kitazawa, M.; Sano, A.; Shimbo, K.; Horimoto, S.;
Fujita, H.; Kubo, A.; Wakamiya, T.; Shiba, T. Bull. Soc. Chem. Jpn.
992, 65, 2227-2240.
5) Galande, A. K.; Trent, J. O.; Spatola, A. F. Biopolymers (Pept.
(
(12) Probert, J. M.; Rennex, D.; Bradley, M. Tetrahedron Lett. 1996,
37, 1101-1104.
(13) Shao, H.; Wang, S. H. H.; Lee, C. W.; O¨ sapay, G.; Goodman,
M. J. Org. Chem. 1995, 60, 2956.
(14) Smith, N. D.; Goodman, M. Org. Lett. 2003, 5, 1035-1037.
(15) (a) Nakajima, K.; Oda, H.; Okawa, K. Bull. Chem. Soc. Jpn.
1983, 56, 520-522. (b) Hata, Y.; Watanabe, M. Tetrahedron 1987, 43,
3881-3888.
(16) Dugave, C.; M e´ nez, A. Tetrahedron: Asymmetry 1997, 8, 1453-
1465.
(17) Swali, V.; Mateucci, M.; Elliot, R.; Bradley, M. Tetrahedron
2002, 58, 9101-9109.
(18) Zhu, X.; Schmidt, R. R. Eur. J. Org. Chem. 2003, 4069-4072.
(19) Alexander McNamara, L. M.; Andrews, M. J. I.; Mitzel, F.;
Siligardi, G.; Tabor, A. B. J. Org. Chem. 2001, 66, 4585-4594.
(20) Mohd Mustapa, M. F.; Harris, R.; Esposito, D.; Chubb, N. A.
L.; Mould, J.; Schultz, D.; Driscoll, P. C.; Tabor, A. B. J. Org. Chem.
2003, 68, 8193-8198.
1
(
Sci.) 2003, 71, 543-551.
(
6) Galande, A. K.; Spatola, A. F. Lett. Pept. Sci. 2001, 8, 247-251.
7) Polinsky, A.; Cooney, M. G.; Toy-Palmer, A.; O¨ sapay, G.; Good-
(
man, M. J. Med. Chem. 1992, 35, 4185-4194.
(
8) Toogood, P. L. Tetrahedron Lett. 1993, 34, 7833-7836.
(
9) Mayer, J. P.; Zhang, J.; Groeger, S.; Liu, C.-F.; Jarosinski, M. J.
Pept. Res. 1998, 51, 432-436.
(
10) (a) Okeley, N. M.; Zhu, Y.; van der Donk, W. A. Org. Lett. 2000,
2
1
, 3603-3606. (b) Zhou, H.; van der Donk, W. A. Org. Lett. 2002, 4,
335-1338.
(11) (a) Burrage, S.; Raynham, T.; Williams, G.; Essex, J. W.; Allen,
C.; Cardno, M.; Swali, V.; Bradley, M. Chem. Eur. J. 2000, 6, 1455-
1466. (b) Matteucci, M.; Bhalay, G.; Bradley, M. Tetrahedron Lett.
2004, 45, 1399-1401.
J. Org. Chem, Vol. 70, No. 7, 2005 2431

Bregant and Tabor
SCHEME 1. General Scheme for the Solid-Phase
Synthesis of Side Chain Bridged Peptides Using
Orthogonally Protected Bisamino Acids as
Building Blocks
SCHEME 2. Synthesis of Lanthionines via
Carbamate-Protected Iodoalaninea
a
(
i) Alloc-Cl, Na2CO3; (ii) allyl bromide, NaHCO3, DMF (62%
over 2 steps); (iii) Ms-Cl, Et3N, THF (92%); (iv) NaI, acetone (78%);
v) Cs2CO3, Fmoc-Cys-O Bu, DMF (88% overall).
t
(
To circumvent this problem, as the electron-donating
trityl group is believed to promote the formation of
aziridines, we decided to explore routes using carbamate-
protected â-iodoalanine. Since our approach to the solid-
phase synthesis of side chain bridged peptides required
Alloc/allyl protection on one amino acid moiety, we
envisaged that this combination of protecting groups on
the precursor â-iodoalanine should enable a very concise
synthesis of the desired protected lanthionine.
FIGURE 1. Excerpt of the 13C DEPT (100.61 MHz) VT
experiments on the mixture A-1: 4 + 5 (85:15).
D-Serine was sequentially protected to give alcohol 1.
This was used to access mesylate 2, which was subse-
quently converted to â-iodoalanine 3 using NaI (Scheme
SCHEME 3. Different Routes to Mixtures of 4 and
and to the Mixture 11a
5
2). Under these conditions, only the â-iodo derivative was
obtained, as expected, and neither â-alanine nor the
corresponding aziridine was formed. The only side prod-
uct formed during the latter reaction corresponded to the
dehydroalanine 6 resulting from a â-elimination process
on 3. This side product was easily removed by flash
chromatography, and the unstable iodo derivative 3 was
isolated. The freshly obtained iodo derivative 3 was
t
reacted with Fmoc-Cys-O Bu in the presence of cesium
carbonate as a base to afford the thioether in 82% yield.
However, careful inspection of the 13C NMR revealed the
presence of two isomeric lanthionines, 4 and 5, in an
approximately 85:15 ratio, as determined by integration
1
3
of the C signals (Figure 1).
a
t
(
i) Cs2CO3, Fmoc-Cys-O Bu, DMF.
Variable temperature NMR experiments (Figure 1)
showed no coalescence of the 13C signals at temperatures
up to 333 K, indicating that the additional peaks arise
from the presence of two diastereomers rather than
conformers. To establish that the two inseparable com-
pounds obtained were diastereoisomeric at the CR-
position of the Alloc/allyl-protected amino acid moiety,
t
dehydroalanine 6 was reacted with Fmoc-Cys-O Bu
(Scheme 3). The 13C peaks corresponding to the Câ of the
mixture obtained indicated that 4 and 5 were produced
in a 1:1 ratio, indicating that the formation of 5 under
2432 J. Org. Chem., Vol. 70, No. 7, 2005

Solid-Phase Synthesis of an Analogue of Nisin Ring C
our original conditions must arise from Michael addition
to the dehydroalanine 6 formed under the coupling
reaction conditions. As a further test, the enantiomeric
SCHEME 4. Synthesis of Lanthionine via the
Mitsunobu Reactiona
t
iodoalanine 7 was reacted with Fmoc-Cys-O Bu and Cs
2
-
CO
0:90 ratio. Boc-protected iodoalanine 10 was also syn-
thesized from D-serine (via Boc-Ser-Oallyl 8 and Boc-Ser-
Ms)-Oallyl 9) and subjected to the same coupling reac-
3
under the same conditions, affording 4 and 5 in a
1
(
tion. This gave 11, also as two diastereoisomers, which
in this case could be differentiated in both the C and
13
1
H spectra.
Clearly, the abstraction of the R-hydrogen from 3 to
give dehydroalanine 6 cannot be completely avoided
under these coupling reaction conditions and, as has been
previously observed, Michael additions between separate
dehydroalanines and cysteine nucleophiles show no di-
astereoselectivity.12 Although a range of conditions were
investigated (solvent, base, temperature, excess of cys-
teine nucleophile), we could not avoid the competing
elimination reaction leading to formation of an ap-
preciable amount of the undesired diastereomer 5. In
contrast, Schmidt has reported18 that similar reactions
between less reactive electrophilic bromoalanine deriva-
tives and cysteine nucleophiles are highly diastereose-
lective, implying that in this case elimination to the
dehydroalanine does not occur. However, we concluded
that partial elimination from the more reactive carbam-
ate-protected iodoalanines could not be avoided, making
these unsuitable for direct nucleophilic reaction with
cysteine nucleophiles. We therefore revisited the use of
trityl-protected iodoalanines, for which this problem does
not arise.
a
(i) Me SiCl, Et N, then MeOH, then Trt-Cl; (ii) Cs CO , allyl
3
3
2
3
bromide (72% over 2 steps); (iii) MeI, Ph3P, DEAD, CH2Cl2 (72%);
t
(
(
iv) FmocCysO Bu, Cs2CO3, DMF (90%); (v) TFA/CH2Cl2 (1:10 v/v);
vi) Alloc-Cl, NaHCO3, H2O/dioxane (85% over 2 steps); (vii) TFA/
CH2Cl2 (1:1 v/v) (41% after HPLC).
Synthesis of Lanthionines via the Mitsunobu
Reaction. We have previously reported21 that trityl
FIGURE 2. Excerpt of the 13C (100.61 MHz) spectrum (Câ
domain) of 4, prepared via the route shown in Scheme 4.
t
serine allyl ester can be reacted with Fmoc-Cys-O Bu,
using modified Mitsunobu conditions with zinc tartrate
as a cocatalyst, to give the desired lanthionine regioiso-
mer directly. This reaction proceeded with complete
selectivity to give a unique coupling compound. Unfor-
tunately, although this route would undoubtably be the
most direct method to give protected lanthionines, we
could not further optimize the moderate (50%) yield of
this key step as the reaction was too slow; competing
Fmoc deprotection and cysteine dimerization led instead
to the consumption of the starting materials.
ally observed during the reaction if the temperature or
the duration of the reaction was not carefully controlled.
Once 13 had been successfully produced as a single
t
regioisomer, reaction with Fmoc-Cys-O Bu/Cs
2
CO
3
then
gave the desired lanthionine 15 as a single regio- and
diastereoisomer.22 Subsequent manipulation of the pro-
tecting groups allowed us to access 4 as a single isomer,
confirmed by its 13C NMR spectrum showing only one
t
set of peaks (A-5, Figure 2). Finally, removal of the Bu
ester allowed lanthionine 16 to be obtained via a direct,
regio- and stereocontrolled route.
Synthesis of an Analogue of Ring C of Nisin. To
illustrate the utility of such orthogonally protected
lanthionines for the solid-phase synthesis of lantibiotics,
we undertook the synthesis of a cyclic fragment of nisin
We therefore returned to the synthesis of the key
intermediate, N-trityl-â-iodo serine 13, and found that
it can be directly synthesized via a Mitsunobu reaction
from the alcohol 12 (Scheme 4). (This avoids the synthesis
21
of an intermediate mesylate; we previously showed that
-
displacement of the mesylate by I is sufficiently slow
that the resulting iodo alanine can rearrange.) Provided
the reaction temperature was carefully controlled and did
not exceed -2 °C during the entire reaction, only the
â-iodo-alanine 13 was produced and it was possible to
avoid the production of the undesired regioisomeric
R-iodo-â-alanine. Only trace amounts of aziridine 14 were
observed as a byproduct. Formation of the aziridine 14
was observed during purification and was also occasion-
(
H-Ala-Lys-Lan-Gly-Ala-Leu-Met-Gly-Lan-Asn-Met-Leu-
NH ). Linear peptide C was first assembled manually on
2
-1
Sieber resin with a loading of 0.16 mmol g . Standard
coupling techniques (preactivation of the Fmoc-protected
amino acid with HBTU using DIEA, followed by coupling
for 25 min), followed by capping with acetylimidazole and
subsequent deprotection with 20% piperidine in DMF,
were used. Lanthionine 16 was incorporated at the fourth
(
21) Mohd Mustapa, M. F.; Harris, R.; Bulic-Subanovic, N.; Elliott,
(22) We have previously shown21 that the undesired aziridine 14
t
S. L.; Groussier, M. F. A.; Bregant, S.; Mould, J.; Chubb, N. A. L.;
cannot subsequently react with Fmoc-Cys-O Bu/Cs
2
CO
3
. It is also clear
Schultz, D.; Gaffney, P. R. J.; Driscoll, P. C.; Tabor, A. B. J. Org. Chem.
003, 68, 8185-8192.
that under these conditions the â-iodo-R-alanine 13 cannot rearrange
(via the aziridine) to give the undesired nor-lanthionine regioisomer.
2
J. Org. Chem, Vol. 70, No. 7, 2005 2433

Bregant and Tabor
TABLE 1. 1H NMR Parameters of Peptide 17 Deduced from Experiments in D2O (277 K) and H2O/D2O (9:1) (275 K)
1H NMR chemical shifts, δ (ppm)
JH,H (Hz)
amino acid residue
NHa
HR
Hâ
Hø
Hδ
Hꢀ
other H
3JR,â
3JR,ΝΗa
Lys1
Met2
Asn3
Lan4
Gly5
8.49
8.54
8.75
8.30
8.58
8.15
8.39
8.45
8.90
8.81
8.80
8.19
4.26
1.84; 1.78
2.12; 1.99
2.85; 2.76
3.07; 2.95
1.46; 1.41
2.60; 2.51
1.68
2.98
8.8; 5.4
9.6; 4.7
7; 6.9
6.7
6.8
6.6
7.5
6.1
7
4.48
2.09 SCH3
4.67
4.55
9.0; 4.6
2
4.05, 3.85
4.54
17.5 ( J)
Met6
Leu7
Ala8
2.08; 2.02
1.77; 1.74
1.43 CH3
2.61; 2.49
1.63
2.09 SCH3
9.3; 5.3
10; 5.4
7.1
4.22
4.41
0.88; 0.93 CH3
1.68
5.5
6
2
Gly9
4.09, 3.82
4.60
16.8 ( J)
6.1
6.3
5.6
nd
Lan10
Lys11
Ala12
2.99; 2.94
1.81; 1.77
1.51 CH3
7.6; 6.3
8.0; 6.4
7.2
4.31
4.07
1.45; 1.41
2.97
a
From H2O/D2O (9:1) experiments
SCHEME 5. Synthesis of an Analogue of Nisin
Removal of the Fmoc group was then followed by in-
tramolecular cyclization using PyAOP/HOAt to give the
a
24
Ring C
cyclic intermediate D. The remaining amino group was
further extended first with Fmoc-Lys(Boc)OH and then
with Boc-Ala-OH, using the same standard coupling
techniques as previously described.
The peptide was then cleaved from the resin, purified
by reverse-phase HPLC (C18), and freeze-dried. This
afforded the desired cyclic peptide 17 as a single isomer,
as a light powder with a yield of 19% (relative to the
mmol of resin functionality used). The peptide was
characterized by mass spectrometry using electrospray
positive mode (ESI+), and a single mass, corresponding
to the desired peptide, was observed. It is theoretically
possible for interstrand cross-linking to take place be-
tween two separate strands of the partially deprotected,
2
resin-bound peptide (with two free NH and one free
COOH group) during the cyclization with PyAOP to give
peptide D. We therefore also analyzed both the crude
peptide (prior to reverse-phase HPLC) and a sample of
peptide cleaved from the resin after the cyclization step
by ESI+ mass spectrometry. In all cases, no evidence of
peptides with molecular weights higher than that ex-
pected was seen, indicating that only intrastrand bond
formation occurs during the cyclization step. This selec-
tive bond formation may be favored by the low substitu-
tion of the resin.
The structure and connectivity of the peptide 17 was
established by NMR. NMR experiments were performed
2 2 2
in a H O/D O (9:1) mixture and in D O on a static sample
at 275 and 277 K, respectively. The complete assignment
of all proton resonances of 17, summarized in Table 1,
was based on the combined use of TOCSY and NOESY
experiments performed in H3
2 2
O/D O (9:1). Coupling con-
stants between NH and HR ( JR,NH) were estimated from
1
the 1D H spectrum recorded in the H
2
O/D
2
O mixture,
whereas coupling constants between HR and Hâ were
1
2
found in the 1D H spectrum recorded in D O (Table 1).
NOESY experiments allowed us to prove the cyclic
character of the target peptide by clearly showing a cross-
peak (marked with a square in Figure 3) between the
NH of the Gly9 and the HR of the Lan10 units.
The assignment of the R and side chain carbon reso-
nances (Table 2) was deduced using gradient hetero-
a
(
i) Pd(PPh3)4, CHCl3/AcOH/NMM; (ii) 20% piperidine in DMF;
(
(
iii) PyAOP, HOAt, DIEA; (iv) Fmoc-Lys(Boc)OH, HBTU/DIEA;
v) acetylimidazole, DMF; (vi) Boc-Ala-OH, HBTU/DIEA; (vii) 5%
TFA/5%TES/CH2Cl2, then TFA (90%) (19% overall).
residue (Scheme 5) using the same coupling protocol.
After linear extension of the peptide sequence to give
resin-bound intermediate C, the allyl and Alloc protecting
groups were then selectively removed with Pd(0).23
(
23) Kates, S. A.; Sol e´ , N. A.; Johnson, C. R.; Hudson, D.; Barany,
G.; Albericio, F. Tetrahedron Lett. 1993, 34, 1549-1552.
24) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.;
Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856.
(
2434 J. Org. Chem., Vol. 70, No. 7, 2005

Solid-Phase Synthesis of an Analogue of Nisin Ring C
TABLE 2. 13C NMR Parameters of Peptide 17 in D2O at 277 K
13C NMR chemical shifts, δ (ppm)
amino acid residue
CO
179.29
CR
Câ
Cø
Cδ
28.98
Cꢀ
other C
Lys1
Met2
Asn3
Lan4
Gly5
56.20
55.46
53.31
56.38
45.75
55.28
56.20
52.34
45.60
55.99
56.59
51.50
32.95
32.60
38.38
35.85
24.95
32.12
41.87
176.47
175.24/177.31
174.35
174.90
176.79
177.74
178.16
173.63
175.16
176.30
173.57
16.90 or 16.79 SCH3
a
Met6
Leu7
Ala8
32.46
41.87
19.8 CH3
31.96
27.17
16.90 or 16.79 SCH3
23.30, 24.86
29.16
Gly9
Lan10
Lys11
Ala12
35.57
33.29
19.3CH3
24.95
41.87
a
Side chain.
characterized by mass spectroscopy (ESI+). The major
1
isomer has been characterized by 1D and 2D H NMR
(
NOESY, TOCSY). As the synthesis was carried out using
the inseparable mixture of lanthionine diastereoisomers,
the diastereomeric character of the two resulting peptides
can be assumed to come from the two isomers at the CR
position of the original Alloc/allyl-protected amino acid
moiety.
Conclusion
In summary, a very concise route to protected lanthion-
ines as a C-2 diastereomeric mixture has been reported,
using a carbamate-protected iodo-alanine. An alternative
route, using a trityl-protected iodo-alanine formed by a
Mitsunobu reaction, gave the single desired lanthionine,
in complete regio- and diastereoselectivity. We have used
this orthogonally protected amino acid in an efficient
strategy for the synthesis of side chain bridged peptides.
In this approach, a linear peptide, incorporating the
orthogonally protected lanthionine, is first synthesized,
and at an appropriate point, chemoselective deprotection
of the allyl and Alloc groups is followed by regio- and
stereoselective cyclization on resin to give the desired
lanthionine-bridged peptide. In this way, the position of
on-resin cyclization may be completely controlled, and
furthermore, competing interchain coupling is not ob-
served. This approach was successfully used to synthesize
an analogue of nisin ring C.
FIGURE 3. NH-HR domain of NOESY spectrum of 17 (H
O, 9:1, 275 K) Inter-residue NOE cross-peaks (NH
are labeled with a f.
2
O/
D
2
i
-HRi+1)
1
13
nuclear single quantum coherence experiments ( H- C
gHSQC) performed in D O. The assignment of each
2
carbonyl resonance was established by performing gradi-
ent heteronuclear multiple bond coherence experiments
1
13
Experimental Section
For ease of assignment, the two amino acid moieties of all
lanthionine derivatives have been indicated thus:
(
H- C gHMBC). The latter confirmed the desired
connectivity of the core backbone.
Finally, we have also utilized the mixture of insepa-
rable diastereomeric lanthionines obtained via the car-
bamate-protected iodoalanine (Scheme 2) in a further
synthesis of an analogue of ring C of nisin. The mixture
of 4 and 5 was deprotected with TFA, as described above,
to give the protected lanthionine monomer as an insepa-
rable mixture of (2S,6R) 16 and its (2R,6R) diastereoi-
somer (85:15 ratio). This mixture was then used to
synthesize the sequence Ac-Lys-Lan8-Gly-Ala-Leu-Met-
N-Allyloxycarbonyl-â-iodo-(S)-alanine Allyl Ester (3).
N-Allyloxycarbonyl-(R)-serine(O-methanesulfonyl) allyl ester
Gly-Lan2-Asn-H, starting from Novasyn TGT resin,
preloaded with Fmoc-Asn(Trt), in a manner analogous
to that described above for the synthesis of 17. Cleavage
from the resin and purification by reverse-phase HPLC
2
(381 mg, 1.24 mmol) and NaI (931 mg, 6.2 mmol, 5 equiv)
were dissolved in dry acetone (10 mL). The reaction mixture
2
was stirred for 29 h at room temperature. Et O (40 mL) was
(
C18) afforded two peptide diastereoisomers in an ap-
proximately 80:20 ratio. These were separated and
added, and the resulting organic layer was washed with 10%
thiosulfate aqueous solution (1 × 20 mL) and with brine (2 ×
J. Org. Chem, Vol. 70, No. 7, 2005 2435

Bregant and Tabor
allyl M), 65.9 (CH
2
1
5 mL). It was then dried over MgSO
4
, filtered though Celite,
Fmoc m), 66.4 (CH
2
allyl m), 66.3 (CH
2
and concentrated to afford a crude mixture as pale yellow oil.
Purification by flash chromatography on silica gel (hexane/
EtOAc 10:1 to 8:1) afforded the desired compound 3 as a white
sticky oil (329 mg, 0.97 mmol, 78% isolated) which turned to
Alloc M + m), 54.3 (CR M, B side), 54.2 (CR m, B side) 53.8
(CR M, A side), 53.7 (CR m, A side), 47.0 (CH Fmoc M + m),
35.8 (Câ M, B side), 35.7 (Câ M, A side), 35.4 (Câ m, B side),
t
35.3 (Câ m, A side), 27.9 (CH
3
38 2 8
Bu M + m). C32H N O S (ES+)
+
+
+
a white solid at low temperature. R
f
) 0.4 (hexane/EtOAc 8:1);
) δ 5.88 (m, 1H, CHdCH ), 5.59 (bd,
H, J ) 6.2 Hz, NHAlloc), 5.33 and 5.25 (2dq, 2H, Jtrans
m/z: [MH] ) 611, [MNa] ) 633, [MK] ) 649. HPLC (reverse
1
-1
H NMR (400 MHz, CDCl
3
2
phase, flow rate of 1.5 mL min , λ ) 214 nm, t (mixture) )
R
3
3
1
1
5
1
)
2
42.8 min using a linear gradient buffer B (CH CN) in A (H O)
3
2
2
3
4
7.2 Hz, J ) 1.4 Hz, Jcis ) 10.4 Hz, J ) 1.2 Hz, CHdCH
),
.29 and 5.19 (2dq, 2H, Jtrans ) 17.2 Hz, Jcis ) 10.5 Hz, J )
.3 Hz, CHdCH ), 5.22 (4dd, 4H, CHdCH ), 4.65 (m, 2H, CH
), 4.55 (m, 3H, J ) 5.2 Hz, CH -CHdCH + HR),
â); C NMR (100.61 MHz, CDCl ) δ 168.8
CO allyl), 154.2 (CO Boc), 132.3 and 131.0 (CH -CHdCH ),
19.3 and 117.9 (CH -CHdCH ), 66.7 and 65.9 (CH -CHd
[90:10 to 10:90 within 60 min], t (mixture) ) 14.6 min using
R
3
3
4
a isocratic buffer B (CH CN) in A (H O) [40:60].
3
2
2
2
2
-
N-Triphenylmethyl-(S)-alanine Allyl Ester (12).17 To a
3
CHdCH
.56 (m, 2H, CH
2
2
2
suspension of D-serine (3.85 g, 36.4 mmol, 1 equiv) in dry CH
Cl (150 mL) was added trimethylsilyl chloride (14.25 mL,
12.7 mmol, 3.1 equiv) at room temperature. The mixture was
heated under reflux for 40 min and allowed to cool to room
temperature. Et N (15.7 mL, 112.7 mmol, 3.1 equiv) diluted
in dry CH Cl (50 mL) was then added, and the reaction
2
-
1
3
3
2
3
2
(
2
2
1
1
2
2
2
CH ), 53.9 (CR), 7.21 (Câ); IR νmax 1750, 1720, 1500 +1200
2
3
-
1
+
cm . C10
+
H
14NO
4
I (ES+) m/z: [M - HI + H] ) 212, [M - HI
2
2
+
+
Na] ) 234. HRMS (FAB) C10
H
14NO
4
I calcd for [MH]
mixture was heated under reflux for a further 1 h. It was then
cooled to 0 °C using an ice-water bath before being subjected
to the dropwise addition of dry MeOH (1.5 mL, 36.4 mmol, 1
equiv). The mixture was allowed to warm to room temperature.
3
40.00458, found 340.00507.
The only impurity formed during this reaction and its
purification corresponds to the corresponding N-allyloxycar-
bonyl dehydroalanine allyl ester 6: R ) 0.7 (hexane/EtOAc
) δ 7.22 (s, 1H, NHAlloc), 6.18
f
3
Et N (5.07 mL, 36.4 mmol, 1 equiv) was then added followed
1
1
0:1); H NMR (400 MHz, CDCl
2
bs, 1H, Câ-H), 5.87 (m, 1H, CHdCH ), 5.75 (d, 1H, J ) 1.3
2
3
by a portionwise addition of triphenylmethyl chloride (10.13
g, 36.4 mmol, 1 equiv). After stirring at room temperature for
2
(
3
Hz, Câ-H), 5.29 and 5.21 (2dq, 2H, Jtrans ) 17.3 Hz, J ) 1.6
2
3
7 h, Et N (20 mL) and MeOH (200 mL) were added to the
3
4
Hz, Jcis ) 10.4 Hz, J ) 1.3 Hz, CHdCH
2
), 5.26 and 5.17 (2dq,
reaction mixture. Removal of the solvents in vacuo led to a
residue that was partitioned between EtOAc (500 mL) and 5%
aqueous citric acid, precooled to 4-5 °C using an water-ice
bath (300 mL). The organic layer was washed with further
3
2
3
4
2
H, Jtrans ) 17.3 Hz, J ) 1.6 Hz, Jcis ) 10.4 Hz, J ) 1.3 Hz,
3
4
CHdCH
2
), 4.65 (dt, 2H, J ) 5.7 Hz, J ) 1.3, CH
2
-CHdCH
2
2
),
3
4
13
4
.55 (dt, 2H, J ) 5.7 Hz, J ) 1.3, CH
2
-CHdCH
); C NMR
(
(
(
100.61 MHz, CDCl
CHdCH
CHdCH
3
) δ 163.2 (CO allyl), 152.8 (CO Alloc), 132.2
5
% precooled aqueous citric acid (2 × 300 mL), dried over
2
Alloc), 131.11 (CR), 130.9 (CHdCH
allyl), 118.0 (CHdCH Alloc), 105.8 (Câ), 66.3 (CH
Alloc); IR νmax 1735, 1716,
2
allyl), 118.7
MgSO and concentrated to afford a crude solid that was used
4
2
2
2
-
in the next step without further purification.
CHdCH
2
allyl), 65.6 (CH
2
-CHdCH
13NO (ES+) m/z: [MH] ) 212, [MNa]
5
2
-1
+
+
This solid was dissolved in dry MeOH (100 mL) in the
presence of cesium carbonate (5.92 g, 18.2 mmol, 0.5 equiv).
The reaction mixture was stirred at room temperature for 1 h
before being concentrated under vacuum. The resulting cesium
salt was dissolved in DMF (50 mL), and allylbromide (3.5 mL,
1
527 + 1310 cm . C10
H
+
)
5
234. HRMS (CI-methane) C10H13NO calcd for [MH]
2
12.09228, found 212.09265.
3
-[(R)-2-tert-Butoxycarbonyl-2-(fluoren-9-ylmethoxy-
carbonylamino)ethylsulfanyl]-(S)-(allyloxycarbonylamino)-
propionic Acid Allyl Ester (4) and 3-[(R)-2-tert-Butoxy-
carbonyl-2-(fluoren-9-ylmethoxycarbonylamino)ethyl-
sulfanyl]-(R)-(allyloxycarbonylamino)propionic Acid
Allyl Ester (5). (4:5, 85:15) A mixture of N-9-fluorenyl-
methoxycarbonyl-(R)-cysteine tert-butyl ester (45 mg, 1.13 ×
3
9.99 mmol, 1.1 equiv) was then added dropwise at room
temperature. The reaction mixture was stirred for 14 h, and
then EtOAc (250 mL) was added directly to the mixture. The
layer was washed with 5% aqueous citric acid, precooled to
4
-5 °C using a water-ice bath (5 × 200 mL), dried over
MgSO , and concentrated in vacuo. Purification by flash
4
-4
1
0
mol, 0.96 equiv) and N-allyloxycarbonyl-â-iodo-(S)-alanine
chromatography on silica gel afforded the compound 12 as a
-4
allyl ester 3 (40 mg, 1.18 × 10 mmol, 1 equiv) were dissolved
white sticky foam (10.85 g, 26.92 mmol, 74% over two steps):
-4
in DMF (0.5 mL). Cesium carbonate (36 mg, 1.11 × 10 mmol,
1
R
f
) 0.2 (hexane/EtOAc 4:1); H NMR (400 MHz, CDCl
3
) δ
0
.94 equiv) was added portionwise over 2 h. The reaction was
7
.61 (m, 6H, Trt), 7.33 (m, 6H, Trt), 7.26 (m, 3H, Trt), 5.78
monitored by TLC and was complete after 2.5 h. EtOAc (5 mL)
was then added to the reaction mixture, and the organic layer
was washed with 5% aqueous citric acid (4 × 10 mL) and brine
3
(
m, 1H, CHdCH
2
), 5.27 and 5.18 (2dq, 2H, Jtrans ) 17.2 Hz,
2
3
4
J ) 1.5 Hz, Jcis ) 10.5 Hz, J ) 1.5 Hz, CHdCH
2
), 4.28 and
-CHdCH ),
.72 (m, 1H, HR), 3.87 and 3.68 (2 dd, 2H, J ) 10.4 Hz, J )
2
3
4
3
4
.17 (2 dd, 2H, J ) 13.2 Hz, J ) 5.9, 5.7 Hz, CH
2
2
(
1 × 5 mL), dried over MgSO
4
, and concentrated in vacuo.
2
3
Purification by flash chromatography on silica gel (hexane/
EtOAc 4:1 to 2:1) afforded a mixture of two inseparable
diastereomers (C-2 epimers) 4 (M) and 5 (m) in a ratio [M]:[m]
1
3
. 3, 5.6 Hz, CH
) δ 173.1 (CO), 145.9 (Cq. arom. Trt), 131.9 (CHdCH
129.9, 128.1, 126.7 (C arom. Trt), 118.5 (CHdCH ), 71.5 (Cq.
aliph. Trt), 65.8 (CH -CHdCH ), 65.2 (Câ), 58.1 (CR).
S (ES+) m/z: [Trt] ) 243, [MH] ) 388, [MNa] )
2
â), 3.16 (s, 1H, OH); C NMR (100.61 MHz,
CDCl
3
2
),
-
4
2
)
85:15 (62 mg, 0.99 × 10 mol, 88% overall): R
f
) 0.3
1
2
2
(
hexane/EtOAc 2:1); H NMR (500.13 MHz, CDCl
3
) δ 7.78-
2
+
+
+
33 42 2 8
C H N O
410.
7
.25 (m, 8H, H arom. Fmoc), 5.86 (m, 2H, CHdCH
allyl and
3
3
Alloc), 5.77 (d, 1H, J ) 7.65 Hz, NHFmoc), 5.69 (d, 1H, J )
3
N-Triphenylmethyl-â-iodo-(S)-alanine Allyl Ester (13).
N-Triphenylmethyl-(S)-alanine allyl ester 12 (1.57 g, 4.06
mmol, 1 equiv) was dissolved in dry CH Cl (5 mL) at room
2 2
7
.10 Hz, NHAlloc), 5.30 and 5.22, (2m, 2H, Jtrans ) 17.1 Hz,
3
3
J
J
cis ) 10.4 Hz, CH
2
-CHdCH
2
allyl), 5.28 and 5.18 (2m, 2H,
trans ) 17.1 Hz, Jcis ) 10.4 Hz, CH -CHdCH Alloc), 4.61
-CHdCH Alloc + HR A side), 4.56 (m, 2H, CH
allyl), 4.47 (m, 1H, HR B side), 4.37 (m, 2H, CH
3
2
2
(
m, 3H, CH
CHdCH
Fmoc), 4.21 (t, 1H, J ) 7 Hz, CH Fmoc), 3.09-2.91 (m, 4H,
2
2
2
-
temperature in the presence of triphenylphosphine (1.60 g,
6.09 mmol, 1.5 equiv). After 10 min, the reaction mixture was
cooled to -10 °C using an ethanol bath refrigerated with a
cryostat probe. DEAD (937 µL, 6.04 mmol, 1.5 equiv) was
added dropwise to the reaction mixture over 1 min. After a
further 5 min this was followed by a careful addition of
iodomethane (375 µL, 6.04 mmol, 1.5 equiv). The temperature
of the reaction mixture was then maintained at -2 °C over 3
h. Formation of the undesired aziridine 14 can occur during
the reaction if the temperature of the reaction is not properly
controlled; this must be avoided, as if the aziridine forms
2
2
3
1
3
CH
2
â A and B sides), 1.47 (s, 9H, C(CH
3 3
) ); C NMR (100.6
MHz, CDCl
3
) δ 170.12 (CO allyl m.), 170.09 (CO allyl M),
t
t
1
69.33 (CO Bu m), 169.27 (CO Bu M), 155.7 (CO Fmoc M +
m), 155.6 (CO Alloc M + m), 143.8, 143.8, 141.21, 141.20 (Cq.
Fmoc M + m), 132.4 (CHdCH Alloc M + m), 131.2 (CHdCH
allyl M + m), 128.8, 127.6, 127.0, 125.1, 119.9 (C. arom. Fmoc
M + m), 119.2 (CHdCH allyl M + m), 117.9 (CHdCH Alloc
M + m), 83.0 (Cq. Bu M + m), 67.1 (CH Fmoc M), 67.1 (CH
2
2
2
2
t
2
2
2436 J. Org. Chem., Vol. 70, No. 7, 2005

Solid-Phase Synthesis of an Analogue of Nisin Ring C
t
during the reaction itself, this can lead to rearrangement to
allyl and Bu), 155.7 (CO Fmoc), 145.6, 145.5, 143.7, 141.2 (Cq
arom.), 131.6 (CHdCH ), 129.2, 129.0, 128.4, 128.2, 127.6,
127.0, 126.9, 125.7, 125.7, 120.4 (C arom. Fmoc and Trt.), 119.0
give the undesired and inseparable R-iodo-â-alanine.21
2
The whole reaction mixture was directly filtered by chro-
matography on silica gel using a nonresolving eluant (hexane/
EtOAc 4:1) to remove the phosphorus derivatives. The fractions
collected containing the target compound were concentrated,
and the residue obtained was purified by flash chromatography
on silica gel (hexane/EtOAc 20:1) to afford the desired iodo
derivative 13 as a white foam (1.45 g, 2.92 mmol, 72%). Iodo
derivative 13 has to be kept and stored at a low temperature
t
(CHdCH
2
), 83.2 (Cq. aliph. Bu), 71.0 (Cq aliph. Trt), 67.1 (CH
-CHdCH ), 56.1 (CR A side), 54.0 (CR B
2
Fmoc), 65.6 (CH
side), 47.0 (CH Fmoc), 37.8 (Câ A side), 35.2 (Câ B side), 28.9
Bu). C47
[MNa] ) 791.
3-[(R)-2-tert-Butoxycarbonyl-2-(fluoren-9-ylmethoxy-
carbonylamino)ethylsulfanyl]-(S)-(allyloxycarbonylami-
2
2
t
+
+
(CH
3
H
48
N
2
O
6
S (ES+) m/z: [Trt] ) 243, [MH] ) 769,
+
1
7
(
-4 °C). At this temperature, the derivative is a solid.
no)propionic Acid Allyl Ester (4). An excess of TFA (0.5
mL) was added at room temperature to a solution of 15 (300
The main side product of this reaction was the aziridine
-
4
derivative 14. As well as forming during the Mitsunobu
mg, 3.9 × 10 mol) dissolved in 5 mL of CH
2 2
Cl (5 mL). (The
reaction itself if the temperature was not carefully controlled,
rate of the reaction could be increased by the addition of TES
(triethylsilane) in the mixture 5% v/v.) After 3 h, the reaction
mixture was concentrated, helped by several additions of
MeOH. The residue obtained was then suspended in water (4
mL) in the presence of sodium hydrogen carbonate (100 mg,
1
4 was observed if the iodo derivative is allowed to stand for
prolonged periods in solvents. Collected fractions from the
column chromatography steps must therefore be concentrated
quite quickly. It is preferable to use moderate heating to
perform the concentration and avoid the addition of dichloro-
methane, as aziridine formation is favored by high tempera-
ture and (surprisingly) by acidic conditions, even slight.
-
4
15.6 × 10 mol, 4 equiv). Dioxane (4 mL) was then added to
the reaction mixture, which was cooled to 1 °C using an
ethanol bath refrigerated with a cryostat probe. At this
-
4
N-Triphenylmethyl-â-iodo-(S)-alanine Allyl Ester (13).
temperature, allylchloroformate (84 µL, 7.8 × 10 mol, 2
equiv) was added, and the reaction was stirred overnight at
+1 °C. The dioxane was then carefully removed under vacuum,
and the aqueous layer obtained was diluted in further water
(25 mL). The layer was extracted with EtOAc (2 × 50 mL).
The collected organic layers were then washed with water (5
1
f 3
R ) 0.6 (hexane/EtOAc 15:1); H NMR (400 MHz, CDCl ) δ
7
.59 (m, 6H, Trt), 7.31 (m, 6H, Trt), 7.23 (m, 3H, Trt), 5.74
3
(
m, 1H, CHdCH
2
2
), 5.18 and 5.11 (2dq, 2H, Jtrans ) 17.2 Hz,
), 4.31 and 4.17 (2 dd,
-CHdCH ), 3.51 (dd,
3
J
cis ) 10. 3 Hz, J ) 1.5 Hz, CHdCH
2
2
3
2
H, J ) 13.0 Hz, J ) 5.9, 4.5 Hz, CH
2
2
3
2
3
1
H, J ) 6.9, 3.4, HR), 3.33 and 3.22 (2 dd, 2H, J ) 9.8 Hz, J
× 35 mL), dried over MgSO
4
and concentrated. Purification
1
3
)
6.7, 3.4 Hz, CH
2
I); C NMR (100.61 MHz, CDCl
), 129.0, 127.9, 126.6 (C
), 71.0 (Cq. aliph. Trt), 65.9 (CH
3
) δ 171.8
of the resulting residue by flash chromatography on silica gel
(
CO), 145.5 (Cq. Trt), 131.6 (CHdCH
2
(hexane/EtOAc 4:1 to 2:1) afforded the desired compound 4 as
-
4
arom. Trt), 118.6 (CHdCH
CHdCH ), 56.1 (CR), 9.8 (Câ). C25
243, [MNa] ) 520. IR νmax 1730, 1510 cm . HRMS (FAB)
2
2
-
a single isomer (183 mg, 3.3 × 10 mol, 85%): R
f
) 0.3
+
1
2
H
24NO
2
I (ES+) m/z: [Trt]
(hexane/EtOAc 2:1); H NMR (500.13 MHz, CDCl
3
) δ 7.78-
2
+
-1
)
C
7.25 (m, 8H, H arom. Fmoc), 5.86 (m, 2H, CHdCH
allyl and
+
3
3
25
H
24NO
2
I calcd for [MNa] 520.0732, found 520.0750.
Alloc), 5.77 (d, 1H, J ) 7.65 Hz, NHFmoc), 5.69 (d, 1H, J )
3
7
.1 Hz, NHAlloc), 5.30 and 5.22, (2m, 2H, Jtrans ) 17.1 Hz,
(
R)-1-Triphenylmethylaziridine-2-carboxylic Acid Al-
3
2
1
1
J
cis ) 10.4 Hz, CH
2
-CHdCH
2
allyl), 5.28 and 5.18 (2m, 2H,
-CHdCH Alloc), 4.61
lyl Ester (14).
R
f
) 0.5 (hexane/EtOAc 15:1); H NMR (500
3
3
J
trans ) 17.1 Hz, Jcis ) 10.4 Hz, CH
2
2
MHz, CDCl
H, Trt), 5.95 (m, 1H, CHdCH
3
) δ 7.35 (m, 6H, Trt), 7.29 (m, 6H, Trt), 7.23 (m,
3
2 2 2
(m, 3H, CH -CHdCH allyl + HR A side), 4.56 (m, 2H, CH -
3
)
2
), 5.36 and 5.27 (2dq, 2H, Jtrans
2
3
4
CHdCH Alloc), 4.47 (m, 1H, HR B side), 4.37 (m, 2H, CH
2
17.2 Hz, J ) 1.5 Hz, Jcis ) 10.5 Hz, J ) 1.2 Hz, CHd
2
3
3
2
Fmoc), 4.21 (t, 1H, J ) 7 Hz), 3.09-2.91 (m, 4H, CH â A and
13
CH
2
), 4.69 (m, 2H, CH
2
-CHdCH
2
), 2.29 (dd, 1H, J ) 2.7 Hz,
2
3
3 3
B sides), 1.47 (s, 9H, C(CH ) ); C NMR (125.75 MHz, CDCl3)
J ) 1.6 Hz, Hâu), 1.94 (dd, 1H, J ) 2.7, 6.2 Hz, HR), 1.44
t
3
2
13
δ 170.1 (CO allyl), 169.3 (CO Bu), 155.7 (CO Fmoc), 155.6 (CO
Alloc), 143.78, 143.78, 141.21, 141.20 (Cq. Fmoc), 132.4 (CHd
(
dd, 1H, J ) 6.2 Hz, J ) 1.6 Hz, Hâd); C NMR (125.75 MHz,
CDCl ) δ 171.1 (CO), 143.6 (Cq arom.), 131.9 (CHdCH ), 129.0,
29.8, 126.9 (C arom.), 118.5 (CHdCH ), 74.3 (Cq aliph.), 65.5
CH -CHdCH ), 31.7 (CR), 28.7 (Câ). C25 (ES+) m/z:
3
2
CH
2
Alloc), 131.2 (CHdCH
2
allyl), 128.8, 127.6, 127.0, 125.1,
allyl), 117.9 (CHdCH
Alloc), 83.0 (Cq. Bu), 67.1 (CH Fmoc), 66.3 (CH allyl), 65.9
1
(
[
2
1
19.9 (C. arom. Fmoc), 119.2 (CHdCH
2
2
2
+
2
H23NO
2
t
+
+
Trt] ) 243, [MH] ) 370, [MNa] ) 392.
2
2
(
CH
2
Alloc), 54.3 (CR, B side), 53.8 (CR, A side), 47.0 (CH
Fmoc), 35.8 (Câ, B side), 35.7 (Câ, A side), 27.9 (CH
HRMS (FAB) C32
633.2247.
3
-[(R)-2-tert-Butoxycarbonyl-2-(fluoren-9-ylmethoxy-
t
3
Bu).
38 2 8
H N O S calcd for [MNa] 633.2265, found
carbonylamino)ethylsulfanyl]-(S)-(triphenylmethylami-
no)propionic Acid Allyl Ester (15). N-Triphenylmethyl-â-
+
-
4
iodo-(S)-alanine allyl ester 13 (450 mg, 9.1 × 10 mol, 1.1
equiv) and N-9-fluorenylmethoxycarbonyl-(R)-cysteine tert-
3-[(R)-2-tert-Butoxycarbonyl-2-(fluoren-9-ylmethoxy-
carbonylamino)ethylsulfanyl]-(S)-(allyloxycarbonylami-
no)propionic Acid (16). 3-[(R)-2-tert-Butoxycarbonyl-2-
(fluoren-9-ylmethoxycarbonylamino)ethylsulfanyl]-(S)-
(allyloxycarbonylamino)propionic acid allyl ester 4 (200 mg,
-
4
butyl ester (328 mg, 8.23 × 10 mol, 1 equiv) were dissolved
-
4
in DMF (15 mL). Cesium carbonate (267 mg, 8.23 × 10 mol,
1
equiv) was added portionwise over 30 min at room temper-
ature, and the reaction was complete within 4 h. EtOAc (80
mL) was then added to the reaction mixture, and the resulting
organic layer was washed with distilled water (7 × 30 mL)
-
4
3.28 × 10 mol) was treated with a mixture of TFA/CH
2 2
Cl
(1:1, 10 mL), and the resulting mixture was stirred at room
temperature for 5 h. The solvents were then removed under
high vacuum. Removal of TFA was helped by several additions
of toluene followed by evaporation; MeOH has to be excluded
for coevaporation to help to avoid the formation of the methyl
ester derivative. This gave 16 in quantitative yield. The final
traces of TFA were removed by purifying the crude mixture
and brine (1 × 30 mL) before being dried over MgSO
4
and
concentrated under vacuum. The residue was purified by
chromatography on silica gel (hexane/EtOAc 10:1, then 4:1 to
2
:1) to afford the desired compound 15 as a light white solid
-
4
(
569 mg, 7.41 × 10 mol, 90%): R
f
) 0.15 (hexane/EtOAc 10:
) δ
), 5.62 (d,
1
1
7
1
1
); R
f
) 0.8 (hexane/EtOAc 2:1); H NMR (500 MHz, CDCl
3
.95-7.17 (m, 15H, H arom.), 5.68 (m, 1H, CHdCH
2
either by reverse phase chromatography (isocratic eluant CH
3
-
3
3
H, J ) 7.8 Hz, NHFmoc, 5.18 and 5.14 (2dq, 2H, Jtrans
)
CN/H O 60:40) or by HPLC using a C-18 column, monitoring
2
2
3
4
7.2 Hz, J ) 1.5 Hz, Jcis ) 10.5 Hz, J ) 1.5 Hz, CHdCH
2
),
at 214, 254, or 301 nm. Purification was performed at a flow
-1
4
.48 (m, 1H, HR B side), 4.38 (m, 2H, CH Fmoc), 4.23 (t, 1H,
2
rate of 15 mL min using a linear gradient buffer B in A (A:B
3
2
J ) 7.2 Hz, CH Fmoc), 4.15 and 3.98 (2dd, 2H, J ) 13.1 Hz,
from 50:50 to 30:70 within 20 min; A ) 0.05% aqueous TFA;
3
3
J ) 6, 5.9 Hz, CH
2
-CHdCH
2
), 3.54 (dd, 1H, J ) 7.4, 4.8,
3 3
B ) 0.05% TFA in CH CN). The CH CN was then removed
2
3
HR A side), 2.97 and 2.84 (m, 4H, J ) 13.1, 14 Hz, J ) 7.4,
under vacuum, and the aqueous layer either evaporated with
6.2, 4.8, 4.4 Hz, 4Hâ), 1.6 (bs, 1H, NHTrt), 1.30 (s, 9H,
moderate heating or freeze-dried. This procedure yielded 16
1
3
C(CH
3
)
3
); C NMR (125.75 MHz, CDCl
3
) δ 173.0, 169.5 (CO
f
(72 mg, 41%) suitable for solid-phase peptide synthesis: R )
J. Org. Chem, Vol. 70, No. 7, 2005 2437

Bregant and Tabor
0
.2 (CH
2
Cl
2
/MeOH/ꢀ AcOH 10:1); 1H NMR (500.13 MHz,
temperature in order to cleave the side chain protecting
groups. The presence of the remaining water allowed the trityl
carbocation, generated by the asparagine side chain deprotec-
tion, to be trapped. TFA was removed under high vacuum, and
the resulting aqueous layer was extracted twice with ether,
allowing a expedient removal of the trityl subproduct.
The resulting mixture was then purified via reverse-phase
HPLC using a C-18 column, monitoring at 214 nm. Purification
MeOD) (7.78-7.25 (m, 8H, H arom. Fmoc), 5.90 (m, 2H, CHd
3
CH
2
allyl and Alloc), 5.29 and 5.17, (2m, 2H, Jtrans ) 17.2 Hz,
3
J
cis ) 10.5 Hz, CH
2
-CHdCH
2
allyl), 5.28 and 5.18 (2m, 2H,
-CHdCH Alloc), 4.58
allyl), 4.52 (m, 2H, CH -CHdCH
Alloc), 4.44 (m, 1H, HR A side), 4.33 (m, 1H, HR B side), 4.37
3
3
J
trans ) 17.4 Hz, Jcis ) 10.7 Hz, CH
2
2
(
m, 2H, CH
2
-CHdCH
2
2
2
2
3
and 4.28 (2dd, 2H, J ) 10.4 Hz, J ) 7.1 Hz, CH
2
Fmoc), 4.22
3
2
-1
(
t, 1H, J ) 7 Hz, CH Fmoc), 3.09 and 2.93 (2dd, 2H, J )
was performed at a flow rate of 15 mL min using a linear
3
1
3.6 Hz, J ) 3.6 Hz, CH
2
â A side), 3.03 and 2.94 (2dd, 2H,
gradient buffer B in A (A:B from 90:10 to 0:100 within 40 min;
2
3
13
J ) 14.3 Hz, J ) 4.9 Hz, CH
2
â B side); C NMR (125.75
A ) 0.05% aqueous TFA in CH
3 3
CN; B ) 0.05% TFA in CH -
MHz, MeOD) δ 175.4 (COOH), 172.0 (CO allyl), 158.4 (CO
Fmoc), 158.3 (CO Alloc), 145.3, 142.6 (Cq. Fmoc), 134.2 (CHd
CH
CN). The CH CN was then removed under vacuum, and the
3
peptide solution was freeze-dried. The lyophilization yielded
2
Alloc), 133.1 (CHdCH
2
allyl), 128.8, 128.2, 126.4, 120.9
allyl), 117.7 (CHdCH Alloc),
allyl), 66.7 (CH Alloc), 56.0 (CR,
the peptide 17 as white light solid (11 mg purified, 19%
-
1
(
C. arom. Fmoc), 118.8 (CHdCH
2
2
isolated). HPLC (reverse phase, flow rate of 1.5 mL min , λ
6
8.2 (CH
2
Fmoc), 67.0 (CH
2
2
) 214 or 254 nm, t
B (CH CN) in A (H
(ES+) m/z: [MH] ) 1191, [MNa] ) 1213,
R
) 6.1 min using a linear gradient buffer
2
B side), 55.7 (CR, A side), 49.0 (CH Fmoc estimated from
HMQC), 36.0 (Câ, B side); IR νmax 3446, 2590, 1668, 1446,
1
3
O) [90:10 to 0:100 within 20 min].
+
+
C
48
H
86
N
16
O
13
S
3
-
1
+
110+973 cm ; HPLC (reverse phase, flow rate of 1.5 mL
[MK] ) 1229.
-1
min , λ ) 214 or 254 nm, t
buffer B (CH CN) in A (H
) 5.91 min using a linear gradient buffer B (CH
30
O) [50:50 to 30:70 within 10 min]: HRMS (ES+) C28H -
R
) 19.2 min using a linear gradient
O) [90:10 to 0:100 within 20 min],
CN) in A
All NMR experiments were performed on a NMR spectrom-
eter equipped with a pulse field gradient, operating at 500.13
3
2
1
13
t
R
3
and 125.75 MHz, respectively, for H and C observations.
Data acquisition and processing were realized using Xwin
(
H
2
+
N
O
2 8
S calcd for [MNa] 577.16151, found 577.16182.
Peptide Synthesis and Characterization. Unless men-
tioned otherwise, all numbers of equivalents of reagents are
NMR software. All experiments were performed in D
2
O or in
a mixture of H O/D O on a static sample at 277 and 275 K,
2
2
respectively. Temperature was kept constant using air flow.
External reference signals were used for calibration of both
-
1
given relative to the resin loading (mmol g ). Peptide 17 was
synthesized manually using solid-phase Fmoc strategy starting
from Fmoc-Sieber resin (0.3 g, 0.048 mmol, loading 0.16 mmol
1
13
1D and 2D spectra. H and C chemical shifts were measured
1
13
relative to the methyl resonance present in H and C spectra
-
1
g
). Fmoc was removed by a treatment with 20% piperidine
of 3-(trimethylsilyl)-1-propane sulfonic acid (TMS-PSA, Ald-
1
in dimethylformamide. Apart from the lanthionine unit (used
in a 1.5-fold excess), a 4-fold excess of the respective com-
mercially available Fmoc amino acids was preactivated for 3
min using HBTU (0.95 equiv relative to the amount of Fmoc
amino acid) and diisopropylethylamine (DIEA, 2 equiv relative
to the amount of Fmoc amino acid) in DMF. The whole mixture
was then added to the growing N-terminus of the unprotected
peptide, and each coupling was allowed to proceed for 25 min.
Capping of unreacted amino groups was performed by acety-
lation using N-acetyl imidazole in DMF (0.3 M) during 20 min.
Prior to the cyclization step, the fully protected, resin-bound
peptide C (Scheme 5) was treated with tetrakis(triphenyle-
rich) recorded in D
2
O at 277 and 275 K respectively ( H: sr )
13
-162.89 (277 K), sr ) -177.31 (275 K). C: sr ) -338.13 (277
K)). Mixing times were set to 80 ms for total correlation
spectroscopy (TOCSY) and 600 ms for nuclear Overhauser
enhancement spectroscopy (NOESY). For 1D and 2D experi-
2 2
ments in H O/D O, the water resonance was suppressed with
1
13
a presaturation pulse. Complete assignments of H and
C
are summarized in Tables 1 and 2 of Results and Discussion.
Acknowledgment. We thank Dr. Abil E. Aliev
(Chemistry Department, University College London) for
his valuable help and guidance with the NMR experi-
ments. We are grateful to the Leverhulme Trust for a
Research Project Grant (to S.B.).
0
phosphine)palladium (2 equiv) previously dissolved in a
mixture of DMF/CHCl
0
0
3
/NMM/AcOH (1.85/1.85/1/2, [Pd ] )
.050 M). After 2 h protected from light, the resin was then
washed several time successively with a solution of DIEA in
DMF (5% v/v) followed by DMF, diethyldithiocarbamate tri-
hydrate in DMF (0.5% w/v), and further DMF. Subsequent
Fmoc removal lead to the cyclization substrate. Intramolecular
cyclization was allowed to occur in the presence of DIEA (10
equiv) using a PyAOP/HOAt mixture (5 equiv) previously
dissolved in DMF and added to the resin. The resin was
subjected twice to this treatment to give the cyclized resin-
bound peptide D. The resin was then washed several times
with DMF, and the following Fmoc lysine activated unit was
added. For the final alanine residue incorporated, Boc-Ala-
OH was used, activated as described above. Cleavage of the
peptide from the solid support was carried out by treating the
Supporting Information Available: Descriptions of gen-
eral procedures and experimental for the preparation of 1, 2,
and 8-11; experimental procedures for the formation of 4 and
13
5
(1:1 from dehydroalanine 6, and 1:9 from 7); C NMR spectra
of all compounds described in the Experimental Section and
1
Supporting Information; H NMR of the mixture of 4 and 5
13
13
(
85:15 and 50:50 ratios); excerpts from C DEPT and C NMR
spectra (Câ region, around 36 ppm) of mixtures of 4 and 5
1
(
85:15, 50:50, 10:90 ratios); sample H NMR spectra at various
1
stages during the synthesis and purification of 13; H NMR
1
2
spectra of 13, 15, and 16. For peptide 17: H spectra in D O
1
(
H
500.13 MHz, 277 K), showing HR assignments, H spectra in
O:D O 9:1 (500.13 MHz, 275 K), showing NH assignments,
TOCSY spectrum (H
2
2
resin several times with a solution of TFA (5%) in CH
2
Cl
2
in
13
2
O:D
2
O 9:1, 275K), C spectrum (D
2
O,
the presence of 2.5% of water and 2.5% of triethylsilane. The
collected layers were then concentrated in vacuo to a small
volume mainly consisting of an aqueous layer. (Coevaporation
of TFA was facilitated by addition of MeOH.) The resulting
solution was then treated with pure TFA for 30 min at room
1
1
25.75 MHz, 277 K). H NMR of diastereomeric mixture of
peptides (80:20 ratio). This material is available free of charge
via the Internet at http://pubs.acs.org.
JO048222T
2438 J. Org. Chem., Vol. 70, No. 7, 2005