Synthesis of cyclic peptides by ring-closing metathesis

Article abstract of DOI:10.1021/jo000759t

The synthesis of a series of 'amide to amide' cyclized peptides by ring-closing metathesis (RCM) as well as a convenient synthesis for the linear precursors is described. In addition, the influence of the length of the alkene substituents and the influence of the peptide sequence is investigated, leading to a set of general rules to obtain 'amide to amide' cyclized peptides by RCM.

Full text of DOI:10.1021/jo000759t

J . Org. Chem. 2000, 65, 6187-6195
6187
Syn th esis of Cyclic P ep tid es by Rin g-Closin g Meta th esis
J ohn F. Reichwein,† Cornelis Versluis,‡ and Rob M. J . Liskamp*,†
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
P.O. Box 80082, 3508 TB Utrecht, The Netherlands, and Department of Biomolecular Mass Spectrometry,
Bijvoet Center for Biomolecular Research, Utrecht University
r.m.j.liskamp@pharm.uu.nl
Received May 18, 2000
The synthesis of a series of “amide to amide” cyclized peptides by ring-closing metathesis (RCM)
as well as a convenient synthesis for the linear precursors is described. In addition, the influence
of the length of the alkene substituents and the influence of the peptide sequence is investigated,
leading to a set of general rules to obtain “amide to amide” cyclized peptides by RCM.
Sch em e 2
In tr od u ction
Cyclization of peptides is an appropriate way to reduce
the flexibility of a peptide andsamong otherssto increase
the affinity toward a receptor.1 Methods for cyclization
can be divided into cyclizations involving C and N
termini,2 the so-called “backbone to backbone” cycliza-
tion,3 and methods involving the side chains of individual
amino acids.2 Examples of the latter method include the
formation of disulfide bridges between cysteine residues
and the formation of lactam bridges between glutamic/
aspartic acid and lysine residues.
Sch em e 1
In our approach denoted as “amide to amide” cycliza-
tion, we wanted to use N-substituted amides in such a
way that we could perform cyclization by ring-closing
metathesis (RCM).4 Several cyclic peptides by RCM were
reported5 but so far only one cyclic peptide 3 involving
N-substituted amino acids was prepared from 1 using
Grubbs ruthenium catalyst 2 (Scheme 1).6 Recently, we
synthesized a number of cyclic dipeptides derived from
the cyclization of amide substituents in bis-N-alkylated
dipeptides7 (Scheme 1) as well as a number of cyclic Leu-
Enkephalin derivatives originating from the cyclization
of bis amide substituted peptides in a rolling loop scan.8
*To whom correspondence should be addressed. Fax: (+31) 30-
2536655.
(5) Piscopio, A. D.; Miller, J . F.; Koch, K. Tetrahedron Lett. 1998,
39, 2667-2670. Piscopio, A. D.; Miller, J . F.; Koch, K. Tetrahedron Lett.
1997, 38, 7143-7146. Piscopio, A. D.; Miller, J . F.; Koch, K. Tetrahe-
dron 1999, 55, 8189-8198. J arvo, E. R.; Copeland, G. T.; Papaioannou,
N.; Bonitatebus, P. J ., J r.; Miller, S. J . J . Am. Chem. Soc. 1999, 121,
11638-11643. Pernerstorfer, J .; Schuster, M.; Blechert, S. Chem.
Commun. 1997, 1949-1950. Fink, B. E.; Kym, P. R.; Katzenellenbogen,
J . A. J . Am. Chem. Soc. 1998, 120, 4334-4344. Ripka, A. S.; Bohacek,
R. S.; Rich, D. H. Bioorg., & Med. Chem. Lett. 1998, 8, 357-360.
Grubbs, R. H.; Blackwell H. E. Angew. Chem., Int. Engl. 1998, 37,
3281-3284. Clark, T. D.; Ghadiri, M. R. J . Am. Chem. Soc. 1995, 117,
12364-12365.
† Utrecht Institute for Pharmaceutical Sciences.
‡ Bijvoet Center for Biomolecular Research.
(1) See e.g. Adang, A. E. P.; Hermkens, P. H. H.; Linders, J . T. M.;
Ottenheijm, H. C. J .; van Staveren, C. J . Recl. Trav. Chim. Pays-Bas
1994, 113, 63-78.
(2) See, for example, Hruby, V. J .; Al-Obeidi, F.; Kazmierski, W.
Biochem. J . 1990, 268, 249-262.
(3) See, for example, Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.;
Byk, G. Biopolymers 1991, 31, 745-750.
(4) For recent reviews, see Grubbs, R. H.; Miller, S. J .; Fu, G. C.
Acc. Chem. Res. 1995, 28, 446-452. Schmalz, H.-G. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1833-1836. Schuster, M.; Blechert, S. Angew.
Chem., Int. Ed. Engl. 1997, 36, 2036-2056. Grubbs, R. H.; Chang, S.
Tetrahedron 1998, 54, 4413-4450. Armstrong, S. K. J . Chem. Soc.,
Perkin Trans. 1 1998, 371-388.
(6) Miller, S. J .; Blackwell, H. E.; Grubbs, R. H. J . Am. Chem. Soc.
1996, 118, 9606-9614. Miller, S. J .; Grubbs, R. H. J . Am. Chem. Soc.
1995, 117, 5855-5856.
(7) Reichwein, J . F.; Liskamp, R. M. J . Eur. J . Org. Chem. 2000,
65, 2335-2344.
10.1021/jo000759t CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/25/2000

6188 J . Org. Chem., Vol. 65, No. 19, 2000
Reichwein et al.
Sch em e 3
Sch em e 4
Sch em e 5
We were interested to investigate the preparation of
larger cyclic peptides. One of the restrictions found so
far was the synthesis of the appropriate starting building
blocks (N-substituted amino acids) for synthesis of bis-
N-alkene peptides. The previously required relatively
simple bis-N-alkylated dipeptides were prepared by dif-
ferent methods. However, a few peptides were prepared
according to our earlier method for site-specific alkyla-
tion,9 which enabled the introduction of a substituent
onto every desired peptide bond nitrogen.
In this procedure the Fmoc group of the first amino
acid was removed and replaced by an o-nitrobenzene
sulfonyl (oNBS) group, which increases the acidity of the
N-H proton enough for a Mitsunobu reaction using an
(8) Reichwein, J . F.; Wels, B.; Kruijtzer, J . A. W.; Versluis, C.;
Liskamp, R. M. J . Angew. Chem., Int. Ed. 1999, 38, 3684-3687.
(9) Reichwein, J . F.; Liskamp, R. M. J . Tetrahedron Lett. 1998, 39,
1243-1246.

Synthesis of Cyclic Peptides
J . Org. Chem., Vol. 65, No. 19, 2000 6189
appropriate unsaturated alcohol for introduction of the
alkene substituent. After removal of the oNBS group by
application of a thiolate, peptide synthesis was continued
until the site for introduction of the required second
alkene side chain was reached, which was carried out as
just described. All cyclization precursors used for these
studies could be prepared according to this general and
versatile protocol (Scheme 2).9 RCM was carried out after
cleavage of the bis-N-alkylated peptides from the resin,
leading to the cyclized peptide.10
pected that this change in the backbone would have a
significant effect on the ring-closure results. The num-
ber of rotamers is reduced as is clear from the H and
1
13C NMR spectra which show sharper and more defined
signals. The absence of proline is reflected in the lower
yields of cyclized peptides 9a ,b obtained from the smaller
peptides 8a ,b. Not quite unexpectedly, the larger pep-
tides 8c,d gave comparable results (Scheme 5) as the
corresponding peptides 6c,d containing proline (Scheme
4), showing that in larger sequences proline is no longer
required for a “cis” like backbone conformation which is
favorable for cyclization.
Resu lts a n d Discu ssion
Tri- up to hexapeptides containing N-allyl substituents
4a -d both on the first and last amide bond were prepar-
ed. A proline residue was incorporated in order to favor
ring closure by facilitating rotation (of the peptide bond)
within the peptide backbone. Unfortunately, all attempts
to cyclize these compounds using the Grubbs ruthenium
catalyst 2 failed (Scheme 3). Despite the presence of a
proline residue, it is likely that these peptides cannot
adopt the proper conformation for ring-closure. The only
products obtained were either isomers, in which the
double bond had migrated one carbon atom,11 or dimers.
Increasing the distance between the N-allyl groups by
the introduction of even more amino acids between them
did not lead to any ring-closure product by RCM. The
only parameter which still could be varied was the length
of the alkenyl substituent. For this purpose one carbon
atom homologation to a homoallyl group was chosen and
synthesized. Remarkably, after subjection to RCM condi-
tions the corresponding peptides 6a -d all underwent
ring closure in moderate to very good yields affording
7a -d (Scheme 4). Apparently, the length of the alkene
chain has a tremendous effect on formation of the ring,
which cannot be fully explained by the increased ring size
(compare Schemes 3 and 4). However, the increase in
length will allow the existence of more conformations that
Although tripeptide 8a could not be cyclized, we were
interested in cyclic structures based on tripeptides with-
out a proline residue. We reasoned that the only way this
could be achieved is by further elongation of the N-alkene
substituents. Therefore an analogue of 8a was synthe-
sized, i.e., 10 containing 4-pentenyl substituents instead
of the homoallyl group. This tripeptide 10 was ring-closed
to 11 in a very good yield of 83% (Scheme 6). However,
1H and 13C NMR spectra revealed the presence of several
conformers.
Sch em e 6
1
have the proper geometry for ring closure. The H and
13C NMR spectra showed the presence of many (seven
for 7d ) conformers which originated from mixtures of E
and Z isomers and from rotations around the tertiary
amide bonds.
As was clearly demonstrated above, the length of the
two alkene substituents was important for the success
of the ring closure. So far only peptides were used having
two N-alkene substituents of equal length, and it was in-
teresting to investigate if also alkene substituents of un-
equal length could be employed. In addition, peptides con-
Thus, it seemed that the length of the alkene substit-
uents was crucial for ring closure. Therefore, at this point
it was important to investigate if the originally intro-
duced proline was still required for cyclization to occur.
To study this, proline was replaced by valine. It was sus-
Sch em e 7

6190 J . Org. Chem., Vol. 65, No. 19, 2000
Reichwein et al.
Sch em e 8
taining, for example, a homoallyl and an allyl substitu-
-ent as in 12, will lead to “identical size mimetics” of head
to tail cyclic peptides. Fortunately, peptides 12 could be
ring-closed, albeit in low yields, to obtain 13 (Scheme 7).
The role of proline with respect to successfully obtain-
ing cyclic peptides has been discussed above. Another
way to preorganize the backbone toward a cyclic struc-
ture is the introduction of a D-amino acid which will
supposedly induce a â-turn like structure.12 The presence
of D-amino acids, is also an essential requirement for the
cyclization of small peptides.13 The effect of the presence
of a D-amino acid was investigated in peptides 14. These
were subjected to RCM using 2 and the all-L peptide
barely gave ring closure except for the tetra- and pen-
tapeptide. In contradistinction the peptides containing
a D-amino acid all gave the cyclic peptides in moderate
to high yields (Scheme 8). It is noteworthy to mention
that the cyclic tetrapeptide 15d consisted of only one
conformation according to 1H, 13C, and NOESY NMR
spectra. It is possible that the formation of 15c was
accomplished by racemization because this peptide is
identical to 15d as judged by its NMR spectra and
retention time on HPLC.
hexapeptidessinvolving four, five, or six amide bonds,
respectivelyscan be obtained starting from bis N-ho-
moallyl (or longer) alkene substituents. Introduction of
either a proline residue or one D-amino acid residue as
part of the ring increases the yield of the cyclization.
F igu r e 1.
Con clu sion
Based on the above-described results, “rules” for cycli-
zation of bis N-alkylated peptides by RCM can be inferred
(Figure 1). Cyclic dipeptidessinvolving two amide bondss
can be obtained starting from bis N-allyl (or longer) al-
kene substituents. Cyclic tripeptidessinvolving three am-
ide bondsscan be obtained starting from bis N-4-pentenyl
(or longer) alkene substituents. Cyclic tetra-, penta-, and
In conclusion, we have prepared a variety of cyclic
peptides employing ring-closing metathesis. The results
(10) Although RCM can be carried out on the solid phase, so far
higher yields were obtained if RCM was carried out in solution: see
ref 7.
(11) Based on the presence of the ω-CH3 signals at 1.8 ppm in 1H
NMR spectra; see also ref 7.

Synthesis of Cyclic Peptides
J . Org. Chem., Vol. 65, No. 19, 2000 6191
and 80.0 (C Boc), 116.3, 116.4, 117.1, 117.9, and 118.3 (dCH2),
132.1, 132.3, 132.6, 133.6, and 134.0 (dCH), 155.4 and 155.9
(CO Boc), 167.5, 167.6, 169.4, 169.9, 170.6, 172.6, 172.7, and
173.4 (CO). MS-MS (FAB) m/z: 869 [2M + Na]+, 847 [2M +
H]+, 446 [M + Na]+, 424 [M + H]+, 368 [M - tBu + H]+, 324
[M - Boc + H]+, 227 [(H-Pro-(Nall)Gly-OMe) + H]+.
were used to define a set of rules with respect to the
length of the alkene substituents which have to be obeyed
for ring closure to take place. We have demonstrated that
the length of the alkene substituent has a tremendous
influence on the cyclization results. Incorporation of
proline-facilitated ring closure probably due to the pres-
ence of a “cis” rotamer. A â-turn structure favored by
introduction of a D-amino acid residue also led to a large
increase of the ring closure yield.
Boc-(Na ll)Gly-P r o-P h e-(Na ll)Gly-OMe 4b was prepared
as described for 4d (vide infra). Crude 4b was purified by
chromatography (2.5% MeOH/DCM) and obtained as a color-
less oil (0.198 mmol, 113 mg) in 88% yield. Rf 0.53 (10% MeOH/
DCM). The purity according to HPLC was 95%. 1H NMR
(CDCl3): δ 1.42 (s, 9H, CH3 Boc), 1.78-2.16 (m, 4H, âCH2 and
γCH2 Pro), 2.86-3.13 (m, 2H, âCH2 Phe), 3.33 (br m, 1H, δCH2
Pro) 3.52-4.15 (m, 9H, CH2 (Nall)Gly 8H and δCH2 Pro 1H),
3.68 (s, 3H, OCH3), 4.48 (br m, 1H, RCH Pro), 4.80-5.12 (m,
Exp er im en ta l Section
Gen er a l. Unless otherwise stated, chemicals were obtained
from commercial sources and used without further purification.
TEA14 and DiPEA were distilled subsequently from ninhydrin
and KOH. 2,4,6-Collidine was distilled from CaH2. “Dry
solvents” were obtained as peptide grade solvents from Bio-
solve and stored on molecular sieves (4 Å). THF was distilled
from LiAlH4. Reactions were run at ambient temperature
R
5H, dCH2 and CH Phe), 5.56-5.82 (m, 2H, dCH), 7.16-7.22
(m, 5H, ArH), 7.35-7.39 (m, 1H, NH). 13C NMR (CDCl3): δ
γ
24.8 and 27.3 (âCH2 and CH2 Pro), 38.9 (âCH2 Phe), 46.3, 47.1,
48.0, 50.6 and 51.1 (CH2), 28.3 (CH3 Boc), 50.1, 50.4, 52.0, 52.4,
and 60.0 (RCH and OCH3), 80.2 (C Boc), 116.4, 118.2, and 118.5
(dCH2), 126.7, 128.0.2, 128.5, 129.5, and 129.7 (CH Ar), 132.3
and 134.0 (dCH), 136.6 (C Ar), 155.9 (CO Boc), 168.9, 169.4,
170.5, 170.7, 171.5, and 171.8 (CO). MS-MS (FAB) m/z: 1163
[2M + Na]+, 1141 [2M + H]+, 593 [M + Na]+, 571 [M + H]+,
471 [M - Boc + H]+, 442 [(Boc-(Nall)Gly-Pro-Phe) + H]+, 374
[(H-Pro-Phe-(Nall)Gly-OMe) + H]+, 295 [(Boc-(Nall)Gly-Pro)
+ H]+, 277 [(H-Phe-(Nall)Gly-OMe) + H]+, 130 [(H-(Nall)Gly-
OMe) + H]+.
1
unless stated otherwise. H NMR spectra were also recorded
using the COSY sequence. 13C spectra were recorded using the
attached proton test (APT) sequence. Rf values were deter-
mined by thin-layer chromatography (TLC) on Merck pre-
coated silica gel 60F-254 (0.25 mm) plates. Spots were visu-
alized with I2, UV light, or Cl2-TDM (N,N,N′,N′-tetramethyl-
4,4′-diaminodiphenylmethane).15 Solvents were evaporated
under reduced pressure at 40 °C. Column chromatography was
performed on silica 60. Analytical and preparative HPLC was
performed on a automated HPLC system with an auto sampler
and a UV-vis detector with either an analytical or preparative
reverse-phase column (Alltech Adsorbosphere C8, 5 µm, 250
× 4.6 mm; Alltech Adsorbosphere C8, 10 µm, 250 × 22 mm,
resectively), or a UV detector operating at 254 and 220 nm,
at a flow of 1 mL/min (11.5 mL/min for preparative HPLC).
Elution was effected using a gradient from 0.1% TFA in water
to 0.085% TFA in acetonitrile/water (95/5, v/v) in 20 min. All
solid-phase reactions were run under a nitrogen atmosphere
with dry peptide-grade solvents.
Boc-(Na ll)Gly-Leu -P r o-P h e-(Na ll)Gly-OMe 4c was pre-
pared as described for 4d (vide infra). Crude 4c was purified
by chromatography (2.5% MeOH/DCM) and obtained as a
colorless oil (0.204 mmol, 140 mg) in 91% yield. Rf 0.60 (10%
MeOH/DCM). The purity according to HPLC was 93%. 1H
NMR (CDCl3): δ 0.90-0.96 (m, 6H, CH3 Leu), 1.31-1.49 (m,
γ
2H âCH2 Leu), 1.43 (s, 9H, CH3 Boc), 1.63-1.66 (m, 1H, CH
Leu), 1.91-2.16 (m, 4H, âCH2 and γCH2 Pro), 2.86-3.13 (m,
2H, âCH2 Phe), 3.46-3.47 (m, H, δCH2 Pro), 3.60-4.11 (m, 9H,
CH2 (Nall)Gly 8H and δCH2 Pro 1H), 3.66 and 3.68 (s, 3H,
OCH3), 4.46-4.48 (m, 1H, RCH Pro), 4.78-4.83 (m, 1.25H, RCH
For the synthesis of Fmoc-(Nall)Gly-OH, see ref 7.
R
Typ ica l P r oced u r e for RCM. A 10 mM solution of the
linear peptide in TCE was purged with nitrogen for 15 min,
followed by the addition of 10 mol % of Cl2(PCy3)2RudCHPh
2 and refluxed for 16 h under a nitrogen atmosphere. After
concentrating in vacuo, the crude cyclic peptide was obtained
and was purified by column chromatography.
Leu and Phe 0.25H), 4.99-5.14 (m, 4.75H, dCH2 and CH Phe
0.75 H), 5.46-5.78 (m, 2H, dCH), 6.60 (m, 1H, NH Leu), 7.17-
7.27 (m, 6H, ArH and NH Phe). 13C NMR (CDCl3): δ 21.7,
23.4, and 24.7 (γCH and CH3 Leu), 24.9, 27.7, 27.8, 39.1, 41.9,
47.0, 47.1, 47.9, 49.6, 50.2, and 51.1 CH2), 28.2 (CH3 Boc), 48.7,
50.2, 50.6, 52.0, 52.4, 59.8, and 59.9 (RCH and OCH3), 80.8 (C
Boc), 118.3 and 118.5 (dCH2), 126.9, 126.9, 128.4, 129.4, and
129.6 (CH Ar), 132.1, 132.2 and 133.0 (dCH), 136.2 and 136.4
(C Ar), 155 (CO Boc), 169.2, 169.3, 169.5, 170.4, 170.5, 171.6,
171.8, and 172.1 (CO). MS-MS (FAB) m/z: 706 [M + Na]+,
684 [M + H]+, 584 [M - Boc + H]+, 374 [(H-Pro-Phe-(Nall)-
Gly-OMe) + H]+, 130 [(H-(Nall)Gly-OMe) + H]+, 555.3 [(Boc-
(Nall)Gly-Leu-Pro-Phe) + H]+.
Boc-(Na ll)Gly-P r o-(Na ll)Gly-OMe 4a was prepared as
described for 4d (vide infra). Crude 4a was purified by
chromatography (2.5% MeOH/DCM) and obtained as a color-
less oil (0.187 mmol 79 mg) in 75% yield. Rf 0.43 (10% MeOH/
DCM). The purity according to HPLC was 98%. 1H NMR
(CDCl3): δ 1.40 (s, 9H, CH3 Boc), 1.94-2.13 (m, 4H, âCH2 and
γCH2 Pro), 3.42-4.28 (m, 9H, CH2 Nall, Gly and δCH2 Pro),
3.67 and 3.71 (two s, 3H, OCH3), 4.43, 4.49, 4.86, and 4.92
(four lines, 1H, CH2 Gly), 4.51-4.55 and 4.81-4.84 (m, 1H,
RCH Pro), 5.05-5.31 (m, 4H, dCH2), 5.67-5.91 (m, 2H, dCH).
13C NMR (CDCl3): δ 21.9, 24.6, 25.0, 28.7, 29.1, and 31.5 (â-
Boc-(Nall)Gly-Leu -P r o-Ala-P h e-(Nall)Gly-OMe 4d. Fmoc-
(Nall)Gly-OH was coupled to TentaGel-OH according to the
method of Sieber.16 The Fmoc group was removed from Fmoc-
(Nall)Gly-O-TentaGel (0.224 mmol, 995 mg) by treatment with
20% piperidine in NMP for 30 min, followed by washings with
NMP (3×) and DCM (3×). The resulting amine was treated
with Boc-Phe-OH (0.896 mmol, 237 mg), PyBroP (0.896 mmol,
418 mg), and DiPEA (1.79 mmol, 312 µL) in NMP for 1 h,
followed by washings with NMP (3×) and DCM (3×). Removal
of the Boc-group was achieved by treatment (twice) with 4 N
HCl in Dioxane for 15 min, followed by washings with DCM
(6×). The hydrochloride salt was treated with Fmoc-Ala-OH
(0.896 mmol, 295 mg), HATU (0.896 mmol, 340 mg), and
DiPEA (1.79 mmol, 312 µL) in NMP for 1 h, followed by
washings with NMP (3×) and DCM (3×). The Fmoc group was
removed by treatment with 20% piperidine in NMP for 30 min,
followed by washings with NMP (3×) and DCM (3×). This
procedure was repeated for the introduction of the proline,
leucine and N-allyl-glycine residues. Treatment of the resin
with a catalytic amount of NaCN in MeOH for 16 h afforded
γ
CH2 and CH2 Pro), 28.1 (CH3 Boc), 46.3, 46.4, 46.9, 47.2, 47.5,
47.7, 47.9, 48.8, 49.8, 49.9, 50.1, 50.9, and 51.4 (CH2), 51.9,
52.1, and 52.2 (OCH3), 56.2, 56.5, and 56.8 (RCH Pro), 79.8
(12) See, for example, Kessler, H.; Gratias, R.; Gurrath, M.; Muller.
Pure Appl. Chem. 1996, 68, 1201-1205.
(13) See, for example, Schmidt, U.; Langner, J . J . Pept. Res. 1997,
49, 67-73.
(14) Abbreviations used: (Nall)Aaa: N-allyl amino acid; all: allyl;
(Nhal)Aaa: N-homoallyl amino acid; hal: homo allyl; (Npen)Aaa: N-4-
pentenyl amino acid; pen: 4-pentenyl; DCE: 1,2-dichloroethane; TCE:
1,1,2-trichloroethane; DCM: dichloromethane; DMF: N,N′-dimethylfor-
mamide; PyBroP, bromotripyrrolidinophosphonium hexafluorophos-
phate; BOP, (1H-benzotriazol-1-yloxy)tris(dimethyl)phosphonium hex-
afluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HATU, O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
TEA: triethylamine; DiPEA: N,N-diisopropylethylamine: DEAD: dieth-
yl azodicarboxylate; DIAD: diisopropyl azodicarboxylate; NMP: 1-meth-
yl-2-pyrrolidinone.
(15) von Arx, E.; Faupel, M.; Bruggen, M. J . Chromatogr. 1976, 120,
224
(16) Sieber, P. Tetrahedron Lett. 1987, 28, 6147-6150.

6192 J . Org. Chem., Vol. 65, No. 19, 2000
Reichwein et al.
crude 4d . Column chromatography (gradient: 2.5% MeOH/
DCM to 5% MeOH/DCM) afforded 4d (0.218 mmol, 165 mg)
as a colorless oil in 97% yield. Rf 0.61 (10% MeOH/DCM). The
purity according to HPLC was 96%. 1H NMR (CDCl3): δ 0.92-
0.98 (m, 6H, CH3 Leu), 1.25-1.28 (m, 3H, CH3 Ala), 1.46 (s,
9H, CH3 Boc), 1.46-1.51 (m, 1H, γCH Leu), 1.53-1.65 (m, 2H
and DCM (3×). The amine group was treated with oNBS-Cl
(1.25 mmol, 278 mg) and DiPEA (1.29 mmol, 224 µL) in DMF
for 30 min, followed by washings with DCM (6×). Mitsunobu
reaction of the sulfonamide was carried out using PPh3 (1.25
mmol, 328 mg), 3-butene-1-ol (2.35 mmol, 202 µL), and DIAD
(1.18 mmol, 232 µL) in DCM for 30 min, followed by washings
with DCM (3×) and DMF (6×). The oNBS group was removed
by DBU (1.21 mmol, 180 µL) in 0.50 M 2-mercaptoethanol/
DMF for 30 min, followed by washings with 25% HOAc/DMF
(1×), DMF (3×), 2.5% DiPEA/DMF (3×), and DMF (3×). Now
the secondary amine was treated with Boc-Phe-OH (0.706
mmol, 187 mg), PyBroP (0.706 mmol, 329 mg), and DiPEA
(1.45 mmol, 252 µL) in DMF for 1 h, followed by washings with
DMF (3×) and DCM (3×). Removal of the Boc-group was
achieved with 4 N HCl in dioxane for 15 min, this step was
repeated once, followed by washings with DCM (6×). The
amine group was treated with Fmoc-Ala-OH (0.706 mmol, 232
mg), HATU (0.706 mmol, 268 mg), and DiPEA (1.45 mmol,
252 µL) in DMF for 1 h, followed by washing with DMF (3×)
and DCM (3×). The Fmoc group was removed by treatment
with 20% piperidine in DMF for 30 min, followed by washings
with DMF (3×) and DCM (3×). The amine group was treated
with Fmoc-Pro-OH (0.706 mmol, 302 mg), BOP (0.706 mmol,
396 mg), and DiPEA (1.45 mmol, 252 µL) in DMF for 1 h,
followed by washings with DMF (3×) and DCM (3×). The
leucine and glycine residues were introduced as described for
the proline residue. Introduction of the oNBS-group, the
Mitsunobu reaction, and removal of the oNBS-group were
carried out as described above. Now the secondary amine was
treated with Ac-Cl (1.25 mmol, 89 µL) and DiPEA (1.25 mmol,
217 µL) in DCM for 30 min, followed by washings with DCM
(6×). Treatment of the resin with a catalytic amount of NaCN
in MeOH for 16 h afforded crude 6d . Column chromatography
(gradient: 2.5% MeOH/DCM to 10% MeOH/DCM) afforded 6d
(76 µmol, 54 mg) as a colorless oil in 32% yield. Rf 0.36 (10%
MeOH/DCM). 1H NMR (CDCl3): δ 0.88-0.94 (m, 6H, CH3
Leu), 1.16-1.23 (m, 3H, CH3 Ala), 1.46-1.63 (m, 3H, âCH2 and
γCH Leu), 1.88-2.11 (m, 4H, âCH2 and γCH2 Pro), 2.02 and
â
γ
âCH2 Leu), 1.87-2.31 (m, 4H, CH2 and CH2 Pro), 2.92-3.15
(m, 2H, âCH2 Phe), 3.51-3.58 (m, 1H, δCH2 Pro), 3.65-4.16
δ
(m, 9H, CH2 (Nall)Gly 8H and CH2 Pro 1H), 3.71 and 3.72 (s,
3H, OCH3), 4.30-4.35 (m, 1H, RCH Ala), 4.46-4.50 (m, 1H,
RCH Pro), 4.81-4.86 (m, 1.20H, RCH Leu and Phe 0.20H),
R
5.04-5.18 (m, 4.8H, dCH2 and CH Phe 0.8 H), 5.53-5.79 (m,
2H, dCH), 6.74-6.77 (m, 2H, NH Leu and Phe), 7.08 (m, 1H,
NH Phe), 7.16-7.30 (m, 5H, ArH). 13C NMR (CDCl3): δ 17.9,
21.8, 23.3, and 24.7 (γCH and CH3), 28.3 (CH3 Boc), 25.1, 27.6,
39.1, 41.9, 47.1, 47.3, 48.1, 49.6, 50.1, and 51.2 (CH2), 48.9,
50.1, 50.5, 52.1, 52.5, and 59.8 (RCH and OCH3), 80.7 (C Boc),
118.4 and 118.5 (dCH2), 126.9, 128.4, 129.4, and 129.6 (CH
Ar), 132.1 and 133.1 (dCH), 136.1 and 136.4 (C Ar), 169.2,
169.5, 170.9, 171.4, 171.6, 171.9, and 172.3 (CO). MS-MS (FAB)
m/z: 1532 [2M + Na]+, 777 [M + Na]+, 755 [M + H]+, 655 [M
- Boc + H]+, 558 [(H-Leu-Pro-Ala-Phe-(Nall)Gly-OMe) + H]+,
445 [(H-Pro-Ala-Phe-(Nall)Gly-OMe) + H]+, 277 [(H-Phe-
(Nall)Gly-OMe) + H]+, 130 [(H-(Nall)Gly-OMe) + H]+, 626
[(Boc-(Nall)Gly-Leu-Pro-Ala-Phe-H) + H]+, 379 [(Boc-(Nall)-
Gly-Leu-Pro-Ala-H) + H]+, 408 [(Boc-(Nall)Gly-Leu-Pro-H) +
H]+.
Ac-(Nh a l)Gly-P r o-(Nh a l)Gly-OMe 6a was prepared as
described for 6d (vide infra). Crude 6a was purified by
chromatography (gradient: 2.5% MeOH/DCM to 10% MeOH/
DCM) and obtained as a colorless oil (108 µmol, 41 mg) in 46%
1
yield. Rf 0.60 (10% MeOH/DCM). H NMR (CDCl3): δ 1.95-
2.07 (m, 4H, âCH2 and γCH2 Pro), 2.14 and 2.16 (s, 3H, CH3
Ac), 2.18-2.47 (m, 4H, âCH2 hal), 3.36-3.75 (m, 7H, RCH2 hal,
δCH2 Pro and RCH2 Gly 1H), 3.70 (s, 3H, OCH3), 3.71-4.05
(m, 2H, CH2 Gly), 4.50-4.58 (m, 1.5H, CH2 Gly 1H and RCH
Pro 0.8H), 4.89-5.14 (m, 4.5H, dCH2 and RCH Pro 0.2H),
5.70-5.82 (m, 1H, dCH). 13C NMR (CDCl3): δ 21.0 (CH3 Ac),
24.7, 25.1, 28.8, 29.3, 31.8, 32.7, 33.0, 46.4, 46.6, 46.8, 47.3,
48.1, 48.2, 48.3, 48.7, 49.1, 49.5, 50.3, and 51.1 (CH2), 52.0,
52.3, 56.3, and 56.6 (RCH Pro and OCH3), 116.8, 117.6, and
117.7 (dCH2), 134.2, 134.5, and 135.2 (dCH), 167.0, 171.1, and
172.7 (CO). MS (ESI) m/z: 416 [M + Na]+.
â
2.13 (s, 3H, CH3 Ac), 2.18-2.34 (m, 4H, CH2 hal), 2.87-3.12
(m, 2H, âCH2 Phe), 3.21-3.48 (m, 4H RCH2 hal), 3.51-4.13 (m,
6H, δCH2 Pro and CH2 Gly), 3.68 and 3.69 (s, 3H, OCH3), 4.34-
4.48 (m, 2H, RCH Pro and RCH Ala), 4.73-4.83 (m, 1H, RCH
Leu), 4.95-5.14 (m, 5H, dCH2 and RCH Phe), 5.57-5.77 (m,
1H, dCH), 7.04-7.29 (m, 8H, ArH and NH). MS (ESI) m/z:
747 [M + Na]+.
Cyclo [Ac-(Nh a l)Gly-P r o-(Nh a l)Gly-OMe] 7a was pre-
pared as described in the typical procedure for RCM. Column
chromatography (gradient 2.5% MeOH/DCM to 10% MeOH/
DCM) afforded 7a (137 µmol, 48 mg) as a brownish oil in 46%
yield. Rf 0.32 (10% MeOH/DCM). The purity according to
HPLC was 95%. MS (FAB) m/z: 388 [M + Na]+, 366 [M +
H]+.
Cyclo [Ac-(Nh a l)Gly-P r o-P h e-(Nh a l)Gly-OMe] 7b was
prepared as described in the typical procedure for RCM.
Column chromatography (gradient 2.5% MeOH/DCM to 5%
MeOH/DCM) afforded 7b (72.3 µmol, 36 mg) as a brownish
oil in 92% yield. Rf 0.51 (10% MeOH/DCM). The purity
according to HPLC was >99%. MS (FAB) m/z: 1074 [2M +
Na]+, 1025 [2M + H]+, 535 [M + Na]+, 513 [M + H]+.
Cyclo [Ac-(Nh a l)Gly-Leu -P r o-P h e-(Nh a l)Gly-OMe] 7c
was prepared as described in the typical procedure for RCM.
Column chromatography (gradient 2.5% MeOH/DCM to 10%
MeOH/DCM) afforded crude 7c. After a second purification
by column chromatography (gradient 2.5% MeOH/DCM to 5%
MeOH/DCM) 7c (64.0 µmol, 40 mg was obtained) as a white
solid in 39% yield. Rf 0.38 (10% MeOH/DCM). Preparative
HPLC afforded pure (99+%) 7c in a recovery of 41%. MS (FAB)
m/z: 648 [M + Na]+, 626 [M + H]+.
Cyclo [Ac-(Nh a l)Gly-Leu -P r o-Ala -P h e-(Nh a l)Gly-OMe]
7d was prepared as described in the typical procedure for
RCM. Column chromatography (gradient 2.5% MeOH/DCM
to 10% MeOH/DCM) afforded 7d (50.0 µmol, 34 mg) as a
brownish solid in 65% yield. Rf 0.48 (10% MeOH/DCM). The
purity according to HPLC was 98%. MS (FAB) m/z: 719 [M +
Na]+, 697 [M + H]+.
Ac-(Nh a l)Gly-P r o-P h e-(Nh a l)Gly-OMe 6b was prepared
as described for 6d (vide infra). Crude 6b was purified by
chromatography (gradient: 2.5% MeOH/DCM to 5% MeOH/
DCM) and obtained as a colorless oil (78 µmol, 41 mg) in 33%
1
yield. Rf 0.55 (10% MeOH/DCM). H NMR (CDCl3): δ 1.68-
2.12 (m, 4H, âCH2 and γCH2 Pro), 2.14 and 2.17 (s, 3H, CH3
Ac), 2.20-2.35 (m, 4H, âCH2 hal), 2.89-3.16 (m, 2H, âCH2 Phe),
3.20-3.36 (m, 2H, δCH2 Pro), 3.39-3.54 (m, 4H, RCH2 hal),
3.70 (s, 3H, OCH3), 3.70-4.41 (m, 4H CH2 Gly), 4.44-4.49 (m,
1H, RCH Pro), 4.78-4.83 (0.2H, RCH Phe), 4.98-5.13 (m, 4.8H,
dCH2 and RCH Phe 0.8H), 5.58-5.84 (m, 1H, dCH), 6.91-
6.94 (m, 1H, NH), 7.16-7.34 (m, 5H, ArH). MS (ESI) m/z: 563
[M + Na]+.
Ac-(Nh a l)Gly-Leu -P r o-P h e-(Nh a l)Gly-OMe 6c was pre-
pared as described for 6d (vide infra). Crude 6c was purified
by chromatography (gradient: 2.5% MeOH/DCM to 5% MeOH/
DCM) and obtained as a colorless oil (0.165 mmol, 108 mg) in
66% yield. Rf 0.54 (10% MeOH/DCM). 1H NMR (CDCl3): δ
γ
0.91-0.99 (m, 6H, CH3 Leu), 1.35-1.66 (m, 3H, âCH2 and CH
γ
Leu), 1.87-2.12 (m, 4H, âCH2 and CH2 Pro), 2.16 (s, 3H, CH3
Ac), 2.26-2.39 (m, 4H, âCH2 hal), 2.87-3.18 (m, 2H, âCH2 Phe),
3.23 (t, 2H, J ) 7.3 Hz, δCH2 Pro), 3.36-3.56 (m, 4H, RCH2
hal), 3.69 and 3.71 (s, 3H, OCH3), 3.76-4.17 (m, 4H CH2 Gly),
R
R
4.49 (br m, 1H, CH Pro), 4.69-4.82 (m, 1H, CH Leu), 4.95-
5.13 (m, 5H, dCH2 and RCH Phe), 5.54-5.80 (m, 1H, dCH),
6.62-6.77 (m, 1H, NH), 7.08-7.26 (m, 6H, ArH and NH). MS
(ESI) m/z: 676 [M + Na]+.
Ac-(Nh al)Gly-Leu -P r o-Ala-P h e-(Nh al)Gly-OMe 6d. Fmoc-
Gly-OH was coupled to TentaGel-OH according to the method
of Sieber.16 The Fmoc group was removed from Fmoc-Gly-O-
TentaGel (0.236 mmol, 1.00 g) by treatment with 20% piperi-
dine in DMF for 30 min, followed by washings with DMF (3×)

Synthesis of Cyclic Peptides
J . Org. Chem., Vol. 65, No. 19, 2000 6193
Ac-(Nh a l)Gly-Va l-(Nh a l)Gly-OMe 8a was prepared as
described for 6d . Crude 8a was purified by chromatography
(gradient: 2.5% MeOH/DCM to 5% MeOH/DCM) and obtained
as a colorless oil (116 µmol, 46 mg) in 46% yield. The purity
according to HPLC was 93%. Rf 0.53 (10% MeOH/DCM). 1H
NMR (CDCl3): δ 0.85-0.91 and 0.97-1.01 (m, 6H, CH3 Val),
2.04 and 2.15 (s, 3H, CH3 Ac), 1.93-2.15 (m, 1H, âCH Val),
2.22-2.39 (m, 4H, âCH2 hal), 3.38-3.48 (m, 4H, RCH2 hal),
3.20-4.40 (m, 4H, CH2 Gly), 3.70 and 3.74 (s, 3H, OCH3),
4.45-4.50 and 4.75-4.87 (m, 1H RCH Val), 5.00-5.14 (m, 4H,
dCH2), 5.66-5.82 (m, 1H, dCH), 6.70-6.80 (m, 1H, NH). MS-
MS (FAB) m/z: 813 [2M + Na]+, 791 [2M + H]+, 418 [M +
Na]+, 396 [M + H]+, 253 [(Ac-(Nhal)Gly-Val) + H]+, 154 [(Ac-
(Nhal)Gly) + H]+, 144 [(H-(Nhal)Gly-OMe) + H]+.
MeOH/DCM) afforded 9b (42.8 µmol, 22 mg) as an off white
solid in 27% yield. Rf 0.52 (10% MeOH/DCM). The purity
according to HPLC was 99+%. MS (FAB) m/z: 1051 [2M +
Na]+, 1027 [2M + H]+, 537 [M + Na]+, 515 [M + H]+.
Cyclo [Ac-(Nh a l)Gly-Leu -Va l-P h e-(Nh a l)Gly-OMe] 9c
was prepared as described in the typical procedure for RCM.
Column chromatography (gradient 2.5% MeOH/DCM to 10%
MeOH/DCM) afforded 9c (60.4 µmol, 37 mg) as a brownish
solid in 42% yield. Rf 0.45 (10% MeOH/DCM). Preparative
HPLC afforded pure (99+%) 9c in a recovery of 15%. MS (FAB)
m/z: 1277 [2M + Na]+, 1255 [M + H]+, 650 [M + Na]+, 628
[M + H]+.
Cyclo [Ac-(Nh a l)Gly-Leu -Va l-Ala -P h e-(Nh a l)Gly-OMe]
9d was prepared as described in the typical procedure for
RCM. Column chromatography (gradient 2.5% MeOH/DCM
to 10% MeOH/DCM) afforded 9d (48.2 µmol, 33 mg) as a
brownish oil in 54% yield. Rf 0.37 (10% MeOH/DCM). Prepara-
tive HPLC afforded pure (99+%) 9d in a recovery of 30%. MS
(FAB) m/z: 1420 [2M + Na]+, 1398 [M + H]+, 721 [M + Na]+,
699 [M + H]+.
Ac-(Nh a l)Gly-Va l-P h e-(Nh a l)Gly-OMe 8b was prepared
as described for 6d . Crude 8b was purified by chromatography
(gradient: 2.5% MeOH/DCM to 5% MeOH/DCM) and obtained
as a white solid (161 µmol, 87 mg) in 64% yield. Rf 0.57 (10%
MeOH/DCM). 1H NMR (CDCl3): δ 0.71-0.88 (m, 6H, CH3 Val),
1.98-2.18 (m, 3H âCH Val and âCH2 hal), 2.12 and 2.18 (s,
â
3H, CH3 Ac), 2.21-2.37 (m, 2H, CH2 hal), 2.81-3.11 (m, 4H,
Ac-(Np en )Gly-Va l-(Np en )Gly-OMe 10 was prepared as
described for 6d . Crude 10 was purified by chromatography
(2.5% MeOH/DCM) and obtained as a colorless oil (260 µmol,
107 mg) in 52% yield. Rf 0.58 (10% MeOH/DCM). 1H NMR
(CDCl3): δ 0.76-0.97 (m, 6H, CH3 Val), 1.52-1.72 (m, 4H,
â
R
RCH2 hal and CH2 Phe) 3.17-3.51 (m, 2H, CH2 hal), 3.68 (s,
3H, OCH3), 3.83-4.10 (m, 4H, CH2 Gly), 4.22-4.37 (m, 1H,
RCH Val), 4.82-5.16 (m, 5H, CH Phe and dCH2), 5.54-5.80
R
(m, 1H, dCH), 6.63-6.66 and 6.85-6.98 (m, 2H, NH), 7.16-
7.27 (m, 5H, ArH). MS (ESI) m/z: 565 [M + Na]+.
âCH2 pen), 1.92-2.17 (m, 8H, CH3 Ac and γCH2 pen and CH
â
Ac-(Nh a l)Gly-Leu -Va l-P h e-(Nh a l)Gly-OMe 8c was pre-
pared as described for 6d . Crude 8c was purified by chroma-
tography (gradient: 2.5% MeOH/DCM to 5% MeOH/DCM) and
obtained as a colorless oil (145 µmol, 93 mg) in 61% yield. Rf
0.26 (10% MeOH/DCM). 1H NMR (CDCl3): δ 0.84-0.93 (m,
Val), 3.21-3.49 (m, 4H, RCH2 pen), 3.60-4.43 (m, 4H, CH2
Gly), 3.66 and 3.70 (s, 3H, OCH3), 4.70-4.83 (m, 1H, RCH Val),
4.89-5.04 (m, 4H, dCH2), 5.65-5.81 (m, 1H, dCH), 6.71-6.82
(m, 1H, NH). 13C NMR (CDCl3): δ 17.2, 17.5, 19.4 (γCH3 Val),
21.0 and 21.5 (CH3 Ac), 26.2, 26.5, 27.4, 27.6, 30.4, 30.5, 30.8,
46.9, 47.2, 47.5, 48.3, 49.4, 49.6, 49.9, 50.0, and 52.1 (CH2),
31.3, 31.5, and 31.6 (âCH Val), 52.0, 52.4, 53.3, 53.4, and 53.8
(RCH Val and OCH3), 114.9, 115.1, 115.7, 115.8, and 115.9 (d
CH2), 136.7, 136.9. 137.5, and 137.6 (dCH), 168.0, 168.7, 169.3,
169.5, 171.2, and 171.9 (CO). MS (ESI) m/z: 446 [M + Na]+.
Cyclo [Ac-(Np en )Gly-Va l-(Np en )Gly-OMe] 11 was pre-
pared as described in the typical procedure for RCM. Column
chromatography (gradient 2.5% MeOH/DCM to 5% MeOH/
DCM) afforded 11 (152 µmol, 58 mg) as a brownish oil in 76%
yield. Rf 0.49 (10% MeOH/DCM). Preparative HPLC afforded
pure (99+%) 12 in a recovery of 25%. MS (ESI) m/z: 418 [M
+ Na]+, 396 [M + H]+.
â
12H, CH3 Val and Leu), 1.50-1.62 (m, 3H, γCH and CH2 Leu),
1.93-2.15 (m, 3H, âCH Val and âCH2 hal), 2.15 (s, 3H, CH3
Ac), 2.24-2.36 (m, 2H, âCH2 hal), 2.89-3.13 (m, 2H, âCH2 Phe),
3.17-3.50 (m, 4H, RCH2 hal), 3.60-3.76 (m, 1H, CH2 Gly), 3.67
(s, 3H, OCH3), 3.83-4.06 (m, 3H, CH2 Gly), 4.41-4.44 and
R
R
4.46-4.73 (m, 2H, CH Leu and Val), 4.82-5.26 (m, 5H, CH
Phe and dCH2), 5.54-5.80 (m, 2H, dCH), 6.67-6.83 (m, 1H,
NH), 7.08-7.27 (m, 6.1H, ArH and NH), 7.37-7.43 and 7.59-
7.62 (m, 0.8H, NH), 8.15-8.18 (m, 1H, 0.1H). 13C NMR
(CDCl3): δ 18.0, 18.3, 18.7, 18.8, 18.9, 21.1, 21.5, 21.8, 22.9,
24.7, 24.8, 31.2, 31.7 (CH3 and CH), 31.4, 31.8, 32.7, 32.8, 32.9,
39.3, 39.6, 40.8, 41.6, 46.9, 47.4, 47.9, 48.0, 48.2, 49.4, 49.7,
and 50.0 (CH2), 49.9, 50.1, 51.7, 52.0, 52.5, 58.0, and 58.1 (RCH
and OCH3), 116.7, 117.0, 117.9, 118.0, and 118.1 (dCH2), 127.0,
128.6, 129.3, 129.4, 129.5, and 129.6 (CH Ar), 133.8, 134.1,
134.8, and 135.4 (dCH), 136.3 and 136.4 (C Ar), 168.8, 169.2,
169.5, 170.4, 170.5, 171.4, 171.7, 172.0, 172.2, and 172.3 (CO).
MS (ESI) m/z: 678 [M + Na]+.
Ac-(Nh a l)Gly-Va l-(Na l)Gly-OMe 12a was prepared as
described for 6d . Crude 12a was purified by chromatography
(2.5% MeOH/DCM) and obtained as a colorless oil (320 µmol,
117 mg) in 64% yield. Rf 0.67 (10% MeOH/DCM). 1H NMR
(CDCl3): δ 0.84-0.95 (m, 6H, CH3 Val), 1.94-2.13 (m, 4H, CH3
â
â
Ac and CH Val), 2.17-2.38 (m, 2H, CH2 hal), 3.32-3.44 (m,
2H, RCH2 hal), 3.58-4.37 (m, 6H, CH2 Gly and RCH2 all), 3.65
and 3.69 (s, 3H, OCH3), 4.70-4.83 (m, 1H RCH Val), 4.95-
5.22 (m, 4H, dCH2), 5.62-5.81 (m, 2H, dCH), 6.85-6.88 (m,
1H, NH). 13C NMR (CDCl3): δ 17.2, 17.6, 19.5 (CH3 Val), 21.1
and 21.5 (CH3 Ac), 31.2, 31.3, and 31.4 (âCH Val), 31.8, 32.7,
46.6, 46.8, 48.1, 49.2, 49.6, 49.8, 49.9, 51.3 and 52.3 (CH2), 51.9,
53.4, 53.5, and 53.9 (RCH Val and OCH3), 116.8, 117.7, 118.1,
118.5, and 118.7 (dCH2), 132.1, 132.2, 133.8, and 135.0 (d
CH), 168.1, 168.7, 169.3, 169.4, 171.2, 171.8, 172.0, and 172.1
(CO). MS (ESI) m/z: 404 [M + Na]+.
Ac-(Nh a l)Gly-Leu -Va l-Ala -P h e-(Nh a l)Gly-OMe 8d was
prepared as described for 6d . Crude 8d was purified by
chromatography (gradient: 2.5% MeOH/DCM to 5% MeOH/
DCM) and obtained as a colorless oil (86 µmol, 61 mg) in 65%
yield. Rf 0.23 (10% MeOH/DCM). 1H NMR (DMSO-d6): δ 0.80-
0.90 (m, 12H, CH3 Val and Leu), 1.11-1.17 (m, 3H, CH3 Ala),
â
1.48-1.63 (m, 3H, γCH and CH2 Leu), 1.92-2.32 (m, 5H, âCH
Val and âCH2 hal), 1.92 and 2.05 (s, 3H, CH3 Ac), 2.76-3.04
â
(m, 2H, CH2 Phe), 3.24-3.58 (m, 6H, RCH2 hal and CH2 Gly
2H), 3.62 and 3.66 (s, 3H, OCH3), 3.86-4.07 (m, 2H, CH2 Gly),
R
R
4.13-4.18 (m, 2H, CH Val), 4.26-4.48 (m, 2H, CH Leu and
Ala), 4.60 (br m, 0.1H, RCH Phe), 4.91-5.14 (m, 4.9H, RCH
Phe 0.9 H and dCH2), 5.64-5.83 (m, 2H, dCH), 7.18-7.30
(m, 5H, ArH), 7.72-7.75, 7.86-7.92, 8.03-8.06 and 8.23-8.32
(m, 4H, NH). 13C NMR (DMSO, d6): δ 18.7, 18.8, 19.1, 19.2,
20.0, 21.9, 22.2, 22.3, 23.8, 24.9, 25.1, 31.1, and 31.3 (CH3 and
CH), 32.0, 32.3, 33.2, 33.4, 38.7, 41.3, 41.4, 46.6, 48.5, 48.8,
49.4, and 51.8 (CH2), 48.6, 50.4, 50.6, 52.0, 52.1, 52.5, 52.9,
58.2, and 58.4 (RCH and OCH3), 117.1, 117.4, 118.0, and 118.1
(dCH2), 127.3, 127.4, 129.1, 129.2, 130.0, and 130.1 (CH Ar),
135.8, 136.4, 136.5, and 136.9 (dCH), 138.2 and 138.4 (C Ar),
169.4, 169.5, 170.5, 171.0, 171.2, 171.4, 172.2, 172.6, 172.7,
and 172.9 (CO). MS (ESI) m/z: 749 [M + Na]+.
Ac-(Nh a l)Gly-Va l-P h e-(Na ll)Gly-OMe 12b was prepared
as described for 6d . Crude 12b was purified by chromatogra-
phy (gradient 2.5% MeOH/DCM to 5% MeOH/DCM) and
obtained as a colorless oil (154 µmol, 83 mg) in 61% yield. Rf
0.58 (10% MeOH/DCM). 1H NMR (CDCl3): δ 0.71-0.88 (m,
â
6H, CH3 Val), 2.00-2.17 (m, 1H, CH Val), 2.15 and 2.17 (s,
â
3H, CH3 Ac), 2.26-2.36 (m, 2H, CH2 hal), 2.88-3.09 (m, 2H,
âCH2 Phe), 3.36-3.48 (m, 2H, RCH2 hal), 3.60-4.06 (m, 6H,
CH2 Gly and RCH2 all), 3.66 and 3.67 (s, 3H, OCH3), 4.11-
R
4.29 (m, 1H RCH Val), 4.84-5.13 (m, 5H, dCH2 and CH Phe),
5.46-5.79 (m, 2H, dCH), 6.69-6.72 and 6.93-7.08 (m, 2H,
NH), 7.16-7.26 (m, 5H, ArH). MS (ESI) m/z: 551 [M + Na]+,
529 [M + H]+, 400 [M - ((Nall)Gly-OMe) + H]+.
Cyclo [Ac-(Nh a l)Gly-Va l-P h e-(Nh a l)Gly-OMe] 9b was
prepared as described in the typical procedure for RCM.
Column chromatography (gradient 2.5% MeOH/DCM to 10%
Ac-(Nh a l)Gly-Leu -Va l-P h e-(Na ll)Gly-OMe 12c was pre-
pared as described for 6d . Crude 12c was purified by chro-
matography (gradient: 2.5% MeOH/DCM to 5% MeOH/DCM)

6194 J . Org. Chem., Vol. 65, No. 19, 2000
Reichwein et al.
and obtained as a white solid (77 µmol, 50 mg) in 31% yield.
Rf 0.56 (10% MeOH/DCM). 1H NMR (CDCl3): δ 0.83-0.92 (m,
CH2), 5.60-5.78 (m, 2H, dCH), 6.68-6.71 and 6.92-6.95 (d,
2H, NH), 7.16-7.27 (m, 5H, ArH). 13C NMR (CDCl3): δ 14.8,
21.6, 21.8, 22.1, 22.2, 22.5, 22.6, 24.2, 24.5, and 24.6 (CH3 and
CH), 24.6, 27.1, 27.5, 37.7, 38.9, 46.6, 46.7, 46.8, and 48.4
(CH2), 49.7, 50.4, 51.9, 52.2, 55.6, 57.9, 59.6, and 59.8 (RCH
and OCH3), 116.1, 116.5, 117.6, and 117.7 (dCH2), 126.8, 128.3,
129.2, and 129.5 (CH Ar), 133.9 and 134.8 (dCH), 136.3 and
136.5 (C Ar), 170.2, 170.6, 171.6, 171.9, and 172.4 (CO). MS
(ESI) m/z: 607 [M + Na]+, 585 [M + H]+, 400 [M - ((Nall)-
Leu-OMe) + H]+.
â
12H, CH3 Val and Leu), 1.49-1.70 (m, 3H, γCH and CH2 Leu),
1.93-2.18 (m, 1H, âCH Val), 1.98 and 2.14 (s, 3H, CH3 Ac),
2.23-2.36 (m, 2H, âCH2 hal), 2.89-3.12 (m, 2H, âCH2 Phe),
R
3.37-3.48 (m, 2H, CH2 hal), 3.59-4.08 (m, 6H, CH2 Gly and
RCH2 all), 3.64, 3.66 and 3.67 (s, 3H, OCH3), 4.43-4.51 and
4.62-4.74 (m, 2H RCH Val and Leu), 4.91-5.25 (m, 5H, dCH2
R
and CH Phe), 5.44-5.80 (m, 2H, dCH), 6.75-6.86, 7.53-7.70
and 8.28-8.31 (m, 3H, NH), 7.15-7.30 (m, 5H, ArH). MS (ESI)
m/z: 664 [M + Na]+.
Ac-(Nh a l)Gly-Va l-D-P h e-(Na ll)Leu -OMe 14d was pre-
pared as described for 6d . Crude 14d was purified by chro-
matography (2.5% MeOH/DCM) and obtained as a colorless
oil (101 µmol, 59 mg) in 46% yield. Rf 0.36 (10% MeOH/DCM).
1H NMR (CDCl3): δ 0.69-0.94 (m, 12H, CH3 Val and Leu),
1.27-1.36 (m, 1H, γCH Leu), 1.42-1.80 (m, 2H, âCH2 Leu),
Cyclo [Ac-(Nh a l)Gly-Va l-(Na l)Gly-OMe] 13a was pre-
pared as described in the typical procedure for RCM. Column
chromatography (gradient: 2.5% MeOH/DCM to 5% MeOH/
DCM) afforded 13a (2 µmol, 7 mg) as a brownish oil in 35%
yield. Rf 0.41 (10% MeOH/DCM). Preparative HPLC afforded
pure (99+% according to HPLC) 13a in a recovery of 42%. MS
(ESI) m/z: 375 [M + Na]+.
â
1.97-2.22 (m, 1H, CH Val), 2.04, 2.14, 2.16 and 2.22 (s, 3H,
CH3 Ac), 2.28-2.39 (m, 2H, âCH2 hal), 2.88-3.11 (m, 2H, âCH2
Phe), 3.36-3.50 (m, 2H, RCH2 hal), 3.52-4.11 (m, 4H, RCH2
all and CH2 Gly), 3.62, 3.63 and 3.65 (s, 3H, OCH3), 4.26-
4.32 (m, 1H, RCH Val), 4.81-4.87 (m, 1H, RCH Leu), 5.00-
5.20 (m, 5H, RCH Phe and dCH2), 5.44-5.81 (m, 2H, dCH),
6.57-6.60, 6.66-6.69, 6.81-6.85 and 6.88-6.99 (m, 2H, NH),
7.13-7.27 (m, 5H, ArH). 13C NMR (CDCl3): δ 16.9, 19.1, 21.0,
21.8, 22.6, 24.5, and 30.2 (CH3 and CH), 31.8, 32.8, 37.6, 39.1
48.4, 50.0, and 51.0 (CH2), 50.8, 52.0, 55.6, and 58.0 (RCH and
OCH3), 117.8 and 118.0 (dCH2), 127.0, 128.5, and 129.7 (CH
Ar), 134.0 (dCH), 136.4 (C Ar), 169.7, 170.3, 171.8, 172.0, and
172.7 (CO). MS (ESI) m/z: 607 [M + Na]+.
Cyclo [Ac-(Nh a l)Gly-Leu -Va l-P h e-(Na ll)Gly-OMe] 13c
was prepared as described in the typical procedure for RCM.
Column chromatography (gradient 2.5% MeOH/DCM to 5%
MeOH/DCM) afforded 13c (1 µmol, 6 mg) as a white solidl in
13% yield. Rf 0.41 (10% MeOH/DCM). Preparative HPLC
afforded pure (99+%) 13c in a quant. recovery. MS (FAB)
m/z: 632 [M + Na]+, 614 [M + H]+.
Ac-(Nh a l)Va l-P h e-(Na ll)Leu -OMe 14a was prepared as
described for 6d . Crude 14a was purified by chromatography
(gradient: 2.5% MeOH/DCM to 5% MeOH/DCM) and obtained
as a colorless oil (63 µmol, 33 mg) in 24% yield. Rf 0.54 (10%
MeOH/DCM). 1H NMR (CDCl3): δ 0.67-0.98 (m, 12H, CH3
Val and Leu), 1.33-1.47 (m, 1H, γCH Leu), 1.50-2.12 (m, 4H,
Ac-(Nh a l)Gly-Leu -Va l-D-P h e-(Na ll)Gly-OMe 14e was
prepared as described for 6d . Crude 14f was purified by
chromatography (2.5% MeOH/DCM) and obtained as a white
solid (134 µmol, 86 mg) in 54% yield. Rf 0.54 (10% MeOH/
DCM). 1H NMR (CDCl3): δ 0.58-0.84 (m, 6H, CH3 Val), 0.88-
â
âCH2 hal and CH2 Leu), 1.98, 2.00, 2.03 and 2.06 (s, 3H, CH3
Ac), 2.20-2.32 (m, 1H, âCH Val), 2.82-3.19 (m, 4H, âCH2 Phe
and RCH2 hal), 3.62, 3.64, 3.65 and 3.67 (s, 3H, OCH3), 3.71-
3.95 (m, 1H, RCH2 all), 4.10-4.38 (m, 2H, RCH2 all and RCH
Val), 4.77-5.26 (m, 6H, RCH Leu, RCH Phe and dCH2), 5.49-
5.96 (m, 2H, dCH), 6.06-6.09, 6.65-6.57 and 7.03-7.12 (m,
1H, NH), 7.17-7.26 (m, 5H, ArH). 13C NMR (CDCl3): δ 18.0,
18.7, 18.8, 19.5, 21.7, 21.8, 21.9, 22.8, 23.0, 23.2, 24.6, 24.7,
26.1, 26.2, and 31.4 (CH3 and CH), 33.2, 37.8, 38.4, 38.7, 39.3,
45.5, 48.9, 49.7 (CH2), 50.5, 51.6, 51.7, 53.0, 55.1, 55.9, and
58.1 (RCH and OCH3), 116.9, 117.0, 117.2, 117.8 (dCH2), 126.7,
127.0, 128.4, 128.5, 128.6, 129.3, 129.5, and 129.5 (CH Ar),
133.8, 134.3, 134.4, 134.5 (dCH), 135.9 and 136.7 (C Ar), 169.8,
180.4, 170.5, 171.6, 171.9, 172.0, 172.1, and 172.7 (CO). MS
(ESI) m/z: 550 [M + Na]+.
γ
â
0.93 (m, 6H, CH3 Leu), 1.47-1.77 (m, 3H, CH Leu and CH2
â
Leu), 1.86-2.06 (m, 1H, CH Val), 2.00, 2.12 and 2.13 (s, 3H,
CH3 Ac), 2.45-2.35 (m, 2H, âCH2 hal), 2.91-3.13 (m, 2H, âCH2
R
Phe), 3.39-3.44 (m, 2H, CH2 hal), 3.64, 3.65 and 3.72 (s, 3H,
OCH3), 3.68-5.21 (m, 13H, RCH2 all and CH2 Gly, RCH and
dCH2), 5.57-5.76 (m, 2H, dCH), 7.14-7.26 (m, 5H, ArH), 6.8-
8.3 (m, 3H, NH). 13C NMR (CDCl3): δ 17.6, 17.7, 18.8, 18.9,
19.1, 21.1, 21.6, 21.9, 22.3, 22.8, 24.8, 30.9, 31.2, and 31.7 (CH3
and CH), 31.9, 32.8, 38.7, 41.0, 47.4, 48.4, and 49.9 (CH2), 49.8,
50.2, 50.4, 51.4, 51.9, 52.0, 52.5, 57.5, and 58.0 (RCH and
OCH3), 116.5, 117.6, 117.7, 118.2, 118.4, and 118.5 (dCH2),
126.8, 128.4, 129.3, and 129.4 (CH Ar), 131.9, 132.2, 134.0,
and 135.3 (dCH), 136.2, 136.3, and 136.8 (C Ar), 168.6, 169.1,
169.2, 169.3, 169.9, 170.3, 170.5, 171.3, 171.5, 171.6, 172.2,
and 172.6 (CO). MS (ESI) m/z:664 [M + Na]+.
Ac-(Nh a l)Va l-D-P h e-(Na ll)Leu -OMe 14b was prepared as
described for 6d . Crude 14b was purified by chromatography
(gradient 2.5% MeOH/DCM to 5% MeOH/DCM) and obtained
as a colorless oil (25 µmol, 13 mg) in 10% yield. Rf 0.68 (10%
MeOH/DCM). 1H NMR (CDCl3): δ 0.72-0.98 (m, 12H, CH3
Val and Leu), 1.27-1.43 (m, 1H, γCH Leu), 1.46-1.80 (m, 2H,
Cyclo [Ac-(Nh a l)Va l-D-P h e-(Na ll)Leu -OMe] 15b was
prepared as described in the typical procedure for RCM.
Column chromatography (gradient: 2.5% MeOH/DCM to 5%
MeOH/DCM) afforded 15b (2 µmol, 8 mg) as a brownish oil in
36% yield. Rf 0.50 (10% MeOH/DCM). MS (ESI) m/z: 522 [M
+ Na]+, 500 [M + H]+.
Cyclo [Ac-(Nh a l)Gly-Va l-P h e-(Na ll)Leu -OMe] 15c was
prepared as described in the typical procedure for RCM.
Column chromatography (gradient: 1% MeOH/DCM to 10%
MeOH/DCM) afforded 15c (40 µmol, 22 mg) as a brownish oil
in low purity. Rf 0.30 (10% MeOH/DCM). Preparative HPLC
afforded pure (99+%) 16c in 5% recovery, overall 2% yield.
MS (FAB) m/z: 579 [M + Na]+, 557 [M + H]+.
â
âCH2 Leu), 1.99-2.21 (m, 2H, CH2 hal), 2.11 and 2.14 (s, 3H,
CH3 Ac), 2.43 (m, 1H, âCH Val), 2.84-3.09 (m, 2H, âCH2 Phe),
3.19-3.25 (m, 2H, RCH2 hal), 3.56-3.76 (m, 1H RCH2 all), 3.62
and 3.65 (s, 3H, OCH3), 4.19-4.37 (m, 1H, RCH2 all), 4.83-
R
4.88 (m, 1H, RCH Leu), 5.00-5.25 (m, 6H, RCH Val, CH Phe
and dCH2), 5.57-5.74 (m, 2H, dCH), 7.18-7.26 (m, 5H, ArH),
7.40-7.50 (m, 1H, NH). 13C NMR (CDCl3): δ 19.0, 19.5, 22.0,
22.7, 24.7, 26.3 (CH3 and CH), 33.4, 37.9, 39.0 and 48.6 (CH2),
50.6, 52.0, and 55.8 (RCH and OCH3), 117.2 and 117.5 (dCH2),
126.7, 128.3, and 129.5 (CH Ar), 134.3 (dCH), 136.6 (C Ar),
170.6, 171.7, 172.1, and 172.5 (CO). MS (ESI) m/z: 550 [M +
Na]+.
Cyclo [Ac-(Nh al)Gly-Val-D-P h e-(Nall)Leu -OMe] 15d was
prepared as described in the typical procedure for RCM.
Column chromatography (gradient: 2.5% MeOH/DCM to 5%
MeOH/DCM), followed by another purification by column
chromatography (gradient: EtOAc:hexanes, 1:1 to EtOAc)
afforded 15d (68 µmol, 38 mg) as a colorless oil in 90% yield.
Rf 0.30 (10% MeOH/DCM). Preparative HPLC afforded pure
Ac-(Nh a l)Gly-Va l-P h e-(Na ll)Leu -OMe 14c was prepared
as described for 6d . Crude 14c was purified by chromatogra-
phy (2.5% MeOH/DCM) and obtained as a colorless oil (101
µmol, 59 mg) in 46% yield. Rf 0.36 (10% MeOH/DCM). 1H NMR
(CDCl3): δ 0.75-0.87 (m, 12H, CH3 Val and Leu), 1.40-1.48
(m, 1H, γCH Leu), 1.51-1.77 (m, 2H, âCH2 Leu), 2.05-2.18
(m, 1H, âCH Val), 2.17 and 2.18 (s, 3H, CH3 Ac), 2.30-2.45
(m, 2H, âCH2 hal), 2.87-3.07 (m, 2H, âCH2 Phe), 3.38-3.50
(m, 2H, RCH2 hal), 3.61 and 3.62 (s, 3H, OCH3), 3.67-4.04 (m,
4H, RCH2 all and CH2 Gly), 4.19-4.24 (m, 1H, RCH Val), 4.74-
4.79 (m, 1H, RCH Leu), 5.06-5.15 (m, 5H, RCH Phe and d
1
(99+%) 15d in 39% recovery. H NMR (CDCl3): δ 0.69-0.74
(m, 6H, CH3 Val), 0.77-0.88 (m, 6H, CH3 Leu), 1.31-1.36 (m,
1H, γCH Leu), 1.51-1.59 (m, 1H, âCH2 Leu), 1.60-1.74 (m,
â
â
1H, CH Val), 1.77-1.86 (m, 1H, CH2 Leu), 2.04 (s, 3H, CH3
Ac), 2.06-2.19 (m, 1H, âCH2 hal), 2.44-2.49 (m, 1H, âCH2 hal),
2.88-2.96 (m, 1H, CH2 Phe), 3.00 and 3.05 (s, 1H, CH2 Gly),

Synthesis of Cyclic Peptides
J . Org. Chem., Vol. 65, No. 19, 2000 6195
R
3.20 and 3.26-3.25 (s, 1H, CH2 hal), 3.27-3.44 (m, 2H, CH2
in 61% yield. Rf 0.40 (10% MeOH/DCM). MS (ESI) m/z: 636
[M + Na]+, 614 [M + H]+.
Phe and CH2 all), 3.59 (s, 3H, OCH3), 3.55-3.69 (m, 1H, RCH2
R
hal), 4.05-4.09 (m, 1H, RCH Val), 4.35 and 4.41 (s, 1H, RCH2
all), 4.75-4.80 (m, 1H, RCH Leu), 4.85 and 4.90 (s, 1H, CH2
Gly), 5.06-5.14 (m, 1H, dCH (Nall)Gly), 5.32-5.42 (m, 2H,
dCH (Nhal)Gly and RCH Phe), 6.26 (d, 1H, J ) 10.2 Hz, NH
Phe), 7.16-7.26 (m, 5H, ArH), 8.04 (d, 1H, J ) 8.4 Hz, NH
Val). 13C NMR (CDCl3): δ 18.0, 18.8, 21.5, 21.6, 22.8, 24.6,
and 31.4 (CH3 and CH), 31.4, 36.3, 38.2, 47.2, 51.5, and 53.1
(CH2), 49.9, 51.9, 54.6 and 58.3 (RCH and OCH3), 126.7, 128.4,
128.8, 129.6, and 130.9 (CH Ar and dCH), 136.9 (C Ar), 169.7,
170.3, 170.5, 172.2, and 172.4 (CO). MS (FAB) m/z: 579 [M +
Na]+, 557 [M + H]+.
Ack n ow led gm en t. These investigations were sup-
ported by the Council for Chemical Sciences of The
Netherlands Organization for Scientific Research (CW-
NWO) with financial aid from The Netherlands Tech-
nology foundation.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra of compounds 4a -d , 6a -d , 7a -d , 8a -d , 9b-e, 10,
11, 12a -c, 13a , 13c, 14a -e, and 15b-e. Copies of 13C NMR
spectra of compounds 4a -d , 6a , 7b-d , 8c-d , 9b-d , 10-11,
12a , 14a -e, and 15b-e. HPLC traces of compounds 4a -d ,
7a -d , 8a , 9b-d , 11, 13a , 15b-e. This material is available
free of charge via the Internet at http://pubs.acs.org.
Cyclo [Ac-(Nh al)Gly-Leu -Val-D-P h e-(Nall)Gly-OMe] 15e
was prepared as described in the typical procedure for RCM.
Column chromatography (gradient: 2.5% MeOH/DCM to 5%
MeOH/DCM) afforded 15f (82 µmol, 50 mg) as a white solid
J O000759T