Introduction of the Aib-Pro unit into peptides by means of the 'azirine/oxazolone method' on solid phase

Article abstract of DOI:10.1016/j.tet.2006.07.082

A method for the direct introduction of Aib-Pro into peptides on solid phase was developed. The Aib-Pro unit was introduced by means of the 'azirine/oxazolone method' using allyl N-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate as the synthon. After the reacti

Full text of DOI:10.1016/j.tet.2006.07.082

Tetrahedron 62 (2006) 9671–9680
Introduction of the Aib-Pro unit into peptides by means of the
‘azirine/oxazolone method’ on solid phase
y
*
Simon Stamm and Heinz Heimgartner
Institute of Organic Chemistry, University of Zu€rich, Winterthurerstrasse 190, CH-8057 Zu€rich, Switzerland
Received 19 May 2006; revised 13 July 2006; accepted 25 July 2006
Available online 21 August 2006
Abstract—A method for the direct introduction of Aib-Pro into peptides on solid phase was developed. The Aib-Pro unit was introduced by
means of the ‘azirine/oxazolone method’ using allyl N-(2,2-dimethyl-2H-azirin-3-yl)-L-prolinate as the synthon. After the reaction of the
resin-bound amino or peptide acid with allyl N-(2,2-dimethyl-2H-azirin-3-yl)-L-prolinate, the allyl protecting group of the resulting extended
peptide could be removed by a mild Pd0-promoted procedure. Cleavage of the peptide from the resin was performed with UV light at 352 nm
and yielded C-terminal protected peptides. The method found a successful application in the syntheses of different Aib-Pro containing pep-
taibol segments. Furthermore, a protected derivative of the peptide antibiotic Trichovirin I 1B was prepared by segment condensation.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
stituted a-amino acids into oligopeptides,4–13 endothiopep-
tides,14–16 cyclic peptides,17–18 and cyclic depsipeptides.19–23
2H-Azirin-3-amines are highly strained systems with versa-
tile reactivity.1 Oneveryinterestingandusefulreactionistheir
applicationinpeptidesynthesis. Inthe‘azirine/oxazoloneme-
thod’, 2H-azirin-3-amines such as 1 or 2 are used as synthons
for the introduction of sterically demanding a,a-disubstituted
a-aminoacidsintopeptides.1–3 Thus,thereactionof2H-azirin-
3-amines, e.g., the a-aminoisobutyric acid (Aib) synthon 1a,
with amino or peptide acids leads to peptide amides, the termi-
nal amide bonds of which can be hydrolyzed selectively to
give extended peptide acids. In solution-phase chemistry, the
‘azirine/oxazolone method’ has proven to be successful for
the introduction of a variety of sterically demanding a,a-disub-
Recently, we adapted the ‘azirine/oxazolone method’ to
solid-phase conditions, in order to additionally benefit
from their advantages,24 e.g., the rapid access to peptides
without the need for the isolation of the sometimes cumber-
some peptide acid intermediates. In this method, the growing
peptide was attached through a carbamate linker to a [4-(hy-
droxymethyl)phenyl]acetamidomethyl (PAM) polystyrene
t
resin (3) (Scheme 1). After deprotection of Bu ester 4a,
resin-bound amino acid 4b was treated with a solution of
1a. It is worth mentioning that unconsumed 1a could easily
be recovered and re-used. The terminal amide 5a was selec-
tively hydrolyzed with 3 M HCl in THF/H2O to provide the
resin-bound peptide acid 5b. Further extension of the peptide
R2
t
chain could be achieved either with a Bu-protected amino
N
N
Ph
R1
acid and a coupling reagent or with 1a. Cleavage of the pep-
tide from the resin was achieved with HBr (33%) in acetic
acid, and yielded the tripeptide 6. In a recent paper, we
showed that the method is not restricted to the Aib synthon
1a, and that it was successfully extended to the 1-amino-
cyclopentane-1-carboxylic acid synthon 1b, the 4-amino-
3,4,5,6-tetrahydro-2H-pyran-4-carboxylic acid synthon 1c,
and the a-methylphenylalanine synthon 1d.25
N
N
OR
O
1a R1 = R2 = Me
1b
1 – R2 = –(CH2)4–
1c R1 – R2 = –(CH2)2O(CH2)2–
1d
1 = Me, R2 = PhCH2
2a R = Me
2b R = CH2CHCH2
2c R = PhCOCH2
R
R
Keywords: a-Aminoisobutyric acid; Azirine/oxazolone method; Peptaibols;
Peptide synthesis; Photocleavable linker.
Peptaibols are linear, amphiphilic oligopeptides from fungal
sources with a high proportion of a,a-disubstituted a-amino
acids, primarily, Aib.26–27 Peptaibols show antibiotic pro-
perties due to self-association in lipid membranes forming
ion channels.28 Several peptaibols, or segments thereof,
were synthesized by means of the ‘azirine/oxazolone
method’.4,8–13 The 2H-azirin-3-amine 2a played an impor-
tant role in these syntheses, since 2a allowed the direct
Abbreviations: Aib, a-aminoisobutyric acid; DIPEA, N,N-diisopropylethyl-
amine; HOBt, 1-hydroxybenzotriazole; PyBOP, (1H-benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate; TBTU, O-(1H-benzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic
acid; TIPS, triisopropylsilane; Z, benzyloxycarbonyl; Z-ONSu, N-[(benzyl-
oxycarbonyl)oxy]succinimide.
* Corresponding author. Tel.: +41 44 6354282; fax: +41 44 6356836;
e-mail: heimgart@oci.unizh.ch
y
€
Part of the projected Ph.D. thesis of S. Stamm, Universitat Zurich.
€
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.07.082

9672
S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
O
R
1. COCl2 (1.9M in PhMe), THF
N
H
O
OH
O
O
t
O
2. H-Ala-O Bu · HCl, DIPEA, CH2Cl2
N
H
N
H
t
3
4a R = O Bu
TFA, CH2Cl2, TIPS
1
4b R = OH
N
Ph
O
O
N
H
N
N
H
1a
CH2Cl2
O
O
R
O
N
H
Ph
O
H
5a R = N(Me)Ph
1
t
.
1. H-Phe-O Bu HCl, HOBt,
HCl (3M),
THF/ H2O
N
OH
N
H
1. PyBOP, DIPEA, DMF
H2N
5b R = OH
O
O
2. 33% HBr/AcOH
6
Scheme 1.
introduction of the frequently present, but relatively acid
labile, Aib-Pro unit.9 Unexpectedly, the use of 2a on solid
phase was not successful (vide infra).
HBr (33%) in acetic acid to give H-Ala-Aib-Pro-Val-OH
(9) in 20% yield. In order to guarantee proper swelling of the
resin during the saponification, the experiment was repeated
with a Tentagel resin. However, the yield of 21% was still
conspicuously lower in comparison with the introduction
of the synthons 1a–d (37–50%). The problem was recog-
nized, when the synthesis of the model peptide H-Ala-Aib-
Pro-Leu-Aib-Val-OH (on Tentagel resin) and the peptaibol
segment (A8–A14 of Trichovirin Ia) H-Val-Aib-Gly-Aib-
Aib-Pro-Leu-OH (on polystyrene resin) failed. Only peptide
fragments, which are caused by Aib-Pro fissions, were
detected. The analysis of the fragments revealed that the
cleavage of the Aib-Pro amide bond occurred during the
HBr-promoted cleavage of the peptide from the resin, and
not during the hydrolysis of the terminal amide, which was
necessary after the incorporation of 1a.
Herein we report a method for the introduction of the Aib-
Pro unit into peptides on solid phase, using a photolabile
linker and a new Aib-Pro synthon (2b), which was especially
developed for this purpose.29
2. Results and discussion
The most obvious approach to the introduction of the Aib-
Pro unit was the use of synthon 2a in analogy to the method
outlined in Scheme 1. In doing so, the resin-bound amino
acid 4b was treated with a solution of 2a, and the resulting
resin-bound tripeptide methyl ester 7 was saponified with
LiOH in a mixture of THF, MeOH, and H2O (Scheme 2).
After coupling with H-Val-OtBu by using PyBOP as the cou-
pling reagent, the peptide was cleaved from the resin with
Thus, a linker was required, which can be cleaved under
milder conditions, but is still stable in TFA (50%) and HCl
(3 M). Some years ago, Kunz introduced a Pd0-labile allyl
O
O
O
H
N
OH
N
H
2a
N
H
O
O
O
O
N
R
O
O
CH2Cl2
N
H
N
H
O
7 R = OMe
1
4b
LiOH, THF/MeOH/H2O
8 R = OH
t
.
1. H-Val-O Bu HCl, HOBt,
1. PyBOP, DIPEA, DMF
2. 33% HBr/AcOH
O
H
N
H2N
N
O
OH
N
H
O
O
9
Scheme 2.

S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
9673
linker for the synthesis of peptide acids.30 Therefore, we syn-
thesized 2-{[(Z)-4-(triisopropylsilyloxy)but-2-enyl]oxy}-
ethanoic acid31 and attached its carboxy group via an alanine
spacer to an aminomethyl polystyrene resin.32 Additionally,
(E)-7-hydroxyhept-5-enoic acid was prepared,33 and its car-
boxy group was attached to an aminomethyl polystyrene
resin. In both cases, the N-terminus of the first amino acid
was immobilized through a carbamate group to the resin
(in the first case after removing the TIPS-protecting group
with TFA), and the resin-bound peptides 6 and 9 were synthe-
sized analogously to the ‘PAM/HBr-strategy’. Cleavage from
the resin was performed with Pd(Ph3P)4 and PhSiH3 in
DMSO/CH2Cl2, but the desired peptides could only be
obtained in low yield and after a painstaking purification of
the Pd-containing crude product.
and subsequently with H-Ala-OtBu$HCl under Schotten–
Baumann conditions to give NVOC-Ala-OtBu, which was
then subjected to acidic conditions. Apart from Bu hydro-
t
lysis, neither in TFA (50%) nor in HCl (3 M) cleavage or
decomposition of the linker was observed. Hence, the
chloroformate of the photocleavable resin 10 was reacted
with H-Ala-OtBu (Scheme 3). Deprotection of the tBu ester
11 with TFA afforded the resin-bound amino acid 12, which
was treated with a solution of 1a. Selective hydrolysis of the
terminal amide of the resin-bound dipeptide 13 with 3 M HCl
afforded the resin-bound peptide acid 14, which was coupled
with H-Phe-OtBu. Cleavage of the peptide from the resin was
performed with UV light (16ꢀ8 W, lmax¼352 nm) in MeCN/
H2O, but the initial yields were disappointing. Most proba-
bly, the remaining nitrosoaldehyde functionalized resin
captured the peptide 15 via imine formation with its amino
group.37 This assumption was supported by 1H NMR experi-
ments: the formyl signal of methyl 4-[(4-formyl-2-methoxy-
5-nitrophenyl)oxy]butanoate disappeared within 20 min on
reaction with isopropylamine, and a new signal at 8.65 ppm,
typical for imines, appeared. To overcome the interception
of the peptide, the cleavage from the resin was performed
in a solution of semicarbazide hydrochloride in THF/
MeOH.37 Although 15 was now obtained in much better yield
(ca. 30–40%), its purity was unsatisfactory. Therefore, the
general procedures had to be slightly modified again. The
In 1995, Holmes introduced a photolabile nitroveratryl linker
and immobilized peptidesby coupling their C-terminus to the
resin.34–36 This photolinker is among the most effective ever
described—it is stable to various chemical reactions, but can
easily be cleaved with UV light with l¼365 nm, a wave-
length that does not affect aromatic amino acids such as
Trp or Tyr. The corresponding photocleavable resin 10
(Scheme 3) is commercially available from Novabiochem.
With the aim of testing the stability under acidic conditions,
N-(4,5-dimethoxy-2-nitrobenzyloxy)carbonyl-L-alanine tert-
butyl ester (NVOC-Ala-OtBu) was prepared by reaction of
4,5-dimethoxy-2-nitrobenzyl alcohol and COCl2 in THF,
t
reaction time for the Bu ester hydrolysis was extended to
60 min to avoid the previously detected H-Ala-Phe-OtBu
side product. Moreover, the synthesis was carried out on
O
MeO
R
MeO
1) COCl2 (1.9M in PhMe), THF
N
H
O
OH
NO2
H
N
H
N
t
O
2) H-Ala-O Bu · HCl, DIPEA, CH2Cl2
O
3
NO2
O
3
O
O
t
10
11 R = O Bu
TFA, CH2Cl2, TIPS
12 R = OH
N
N
Ph
N
N
O
OMe
1a, CH2Cl2
2a, CH2Cl2
O
O
H
O
O
H
MeO
N
MeO
N
N
H
N
H
O
N
O
R
H
H
N
N
O
O
O
O
NO2
NO2
O
3
3
O
O
R
13 R = N(Me)Ph
14 R = OH
16 R = OMe
17 R = OH
HCl (3M), THF/ H2O
LiOH, THF/MeOH/H2O
t
.
t
.
1. H-Phe-O Bu HCl, HOBt,
1. H-Phe-O Bu HCl, HOBt,
1. PyBOP, DIPEA, DMF
1. PyBOP, DIPEA, DMF
2. h (352 nm), MeOH, THF,
2. h (352 nm), MeOH, THF,
.
.
2. H2NCONHNH2 HCl
2. H2NCONHNH2 HCl
Ph
O
O
H
H
N
t
N
O Bu
N
H
H2N
N
H2N
Ph
O
O
O
t
O Bu
N
H
O
15
18
O
Scheme 3.

9674
S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
Table 1. Peptides synthesized by means of the ‘azirine/oxazolone method’
on solid phase
a polystyrene instead of a Tentagel resin. The use of Tentagel
resin suffered from (poly)ethylene glycol loss. Finally, model
peptide 15 was prepared in high purity and in 33% yield (after
prep. HPLC, based on resin loading).
Sequence
Description
Yield
[%]a
H-Ala-Aib-Phe-OtBu (15)
Model peptide
33
35
42
H-Ala-Aib-Pro-Phe-OtBu (18) Model peptide
After having established the protocol for the use of Aib
synthon 1a, the preparation of Aib-Pro containing peptides
was attempted. But when dipeptide synthon 2a was used
to incorporate the Aib-Pro unit, the experiment failed again
(Scheme 3). During the saponification of resin-bound methyl
ester 16 to give 17, we observed a considerable darkening of
the resin, which prevented the subsequent photoinduced
cleavage of the peptide from the resin.
H-Val-Aib-Gly-Aib-Aib-
A8–A14 of Trichovirin Ia
A1–A6 of Trichovirin I 1B
Pro-Leu-OtBu (20)
H-Aib-Asn-Leu-Aib-Pro-
Ser(OBn)-OtBu (21)
H-Val-Aib-Pro-Aib-Leu-Aib-
34
A7–A14 of Trichovirin I 1B 34
Pro-Leu-OtBu (22)
a
Based on resin loading, after prep. HPLC.
the photocleavable resin 10, the Aib synthon 1a, and the
allyl-protected Aib-Pro synthon 2b. Gly and Leu were intro-
duced with PyBOP as the coupling reagent. Analogously,
the syntheses of the hexa- and octapeptides H-Aib-Asn-
Leu-Aib-Pro-Ser(OBn)-OtBu (21, A1–A6 of Trichovirin I
1B) and H-Val-Aib-Pro-Aib-Leu-Aib-Pro-Leu-OtBu (22,
A7–A14 of Trichovirin I 1B) were carried out. Sequence
21 was chosen to evaluate the introduction of non-aliphatic
amino acids, while in the synthesis of 22, an Aib-Pro unit
had to be installed prior to an Aib residue and another Aib-Pro
unit. Both peptides were prepared on the photocleavable
resin 10 using the Aib synthon 1a and the allyl-protected
Aib-Pro synthon 2b. All other amino acids were introduced
with PyBOP as the coupling reagent. The side chain of Asn
was not protected, while the hydroxy group of the serine
side chain was masked as benzyl ether, a protecting group
which is orthogonal to the tBu protecting group. The peptides
were obtained in 34% yield each (after prep. HPLC, based
on resin loading), which is comparable to that of tetrapeptide
18 and therefore indicating a good linker stability.
This issue was addressed by the synthesis of the new Aib-Pro
synthons allyl N-(2,2-dimethyl-2H-azirin-3-yl)-L-prolinate
(2b) and phenacyl N-(2,2-dimethyl-2H-azirin-3-yl)-L-proli-
nate (2c), which contain easily removable carboxy-protect-
ing groups.29 The resin-bound amino acid 12 was reacted
with a solution of 2c, and the resulting resin-bound phenacyl
ester was treated with tetrabutylammonium fluoride. Again,
considerable darkening of the resin was observed. On the
other hand, the allyl protecting group of resin-bound peptide
19, obtained from the reaction of 12 and 2b, was smoothly
38
removed with Pd(Ph3P)4 and PhSiH3 in CH2Cl2 to give
the resin-bound peptide acid 17 (Scheme 4). The latter was
then coupled with H-Phe-OtBu and PyBOP as the coupling
reagent affording the corresponding resin-bound tetrapep-
tide. Cleavage from the resin was achieved with UV light
(16ꢀ8 W, lmax¼352 nm) in a solution of semicarbazide
hydrochloride in THF/MeOH and gave H-Ala-Aib-Pro-
Phe-OtBu (18) in high purity and in 35% yield (after prep.
HPLC, based on resin loading).
To evaluate the described protocol for the introduction of the
Aib-Pro motif into peptides, the preparation of some peptai-
bol segments was attempted. The heptapeptide H-Val-Aib-
Gly-Aib-Aib-Pro-Leu-OtBu (20, A8–A14 of Trichovirin
Ia; Table 1), whose synthesis failed with the ‘PAM/HBr-
strategy’ (see above), was prepared in 42% yield using
In contrast to the ‘PAM/HBr-strategy’ (Scheme 1), the use of
the photolinker allows the preparation of tBu-protected pep-
tides, which, in turn, offers the possibility of segmentconden-
sations. For that purpose, the C-terminal Bu-protected
peptides had to be transformed into N-terminal protected
t
O
MeO
R
MeO
1) COCl2 (1.9M in PhMe), THF
N
H
O
OH
NO2
H
N
H
N
t
O
2) H-Ala-O Bu · HCl, DIPEA, CH2Cl2
O
3
NO2
O
3
O
O
t
10
11 R = O Bu
TFA, CH2Cl2, TIPS
12 R = OH
N
N
O
O
H
N
O
O
MeO
N
H
2b
O
N
R
H
N
O
CH2Cl2
O
3
NO2
O
O
O
t
1) H-Phe-O Bu . HCl, HOBt,
19 R = OAllyl
1
H
N
Pd(Ph3P)4, PhSiH3,
CH2Cl2
1. PyBOP, DIPEA, DMF
N
H2N
Ph
17 R = OH
O
2) h (352 nm), MeOH, THF,
2. H2NCONHNH2 . HCl
t
O Bu
N
H
O
18
O
Scheme 4.

S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
9675
O
O
O
H
N
H
N
1. Z-ONSu, DIPEA,
N
H
dioxane, rt
O
N
N
H2N
Ph
O
Ph
O
O
O
2. TFA, CH2Cl2,
TIPS, rt
t
OH
O Bu
N
H
N
H
O
O
23
18
Scheme 5.
1. Z-ONSu, DIPEA,
1. dioxane, rt
t
Z-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-OH
H-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-O Bu
2. TFA, CH2Cl2,
2. TIPS, H2O, rt
21
24
22, TBTU, HOBt,
DIPEA, CH2Cl2,
DMF, rt
t
Z-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-Val-Aib-Pro-Aib-Leu-Aib-Pro-Leu-O Bu
25
Scheme 6.
peptides. Therefore, the tBu-protected peptide 18 was reacted
with N-[(benzyloxycarbonyl)oxy]succinimide (Z-ONSu),
and the corresponding tBu protecting group was hydrolyzed
to give the N-protected peptide 23 (Scheme 5).
4. Experimental
4.1. General
Unless otherwise noted, materials were obtained from com-
mercial suppliers and were used without further purification.
Aminomethyl polystyrene resin (1% divinylbenzene, 100–
200 mesh, loading 1.14 mmol/g) and aminomethyl Tentagel
resin (90 mm, loading 0.28 mmol/g) from Rapp Polymere
Analogously, the heptapeptide 21 (A1–A6 of Trichovirin I
1B) was transformed into the N-protected peptide 24
(Scheme 6). Due to steric hindrance of Aib, the reaction
time for the introduction of the Z-protecting group was ex-
tended to 8 h. However, the transformation was not complete,
and 21 (20%) was partially recovered. The hydrolysis of the
corresponding tBu ester with TFA was accompanied by par-
tial cleavage of the Aib-Pro amide bond. Most probably, the
extentof this side reaction could be reduced by shortening the
reaction time (HPLC/MS indicates complete deprotection
already after 20 min). Finally, 24 was coupled with 22 (A7–
A14 of Trichovirin I 1B) and TBTU as the coupling reagent,
and the protected Trichovirin I 1B derivative (Z-Aib-Asn-
Leu-Aib-Pro-Ser(OBn)-Val-Aib-Pro-Aib-Leu-Aib-Pro-Leu-
OtBu; 25) was obtained in 73% yield (Scheme 5).39
€
(Rapp Polymere, Tubingen, Germany). Hydroxymethyl-
photolinker AM resin (10) (polystyrene, 1% divinylbenzene,
100–200 mesh, loading 0.75 mmol/g) from Novabiochem
€
(Calbiochem-Novabiochem, Laufelfingen, Switzerland).
N,2,2-Trimethyl-N-phenyl-2H-azirin-3-amine (1a) was
synthesized according to the method of Villalgordo and
Heimgartner.40,41 Methyl N-(2,2-dimethyl-2H-azirin-3-yl)-
L-prolinate (2a), allyl N-(2,2-dimethyl-2H-azirin-3-yl)-L-
prolinate (2b), and phenacyl N-(2,2-dimethyl-2H-azirin-3-
yl)-L-prolinate (2c) were synthesized according to Ref. 9
(2a) and Ref. 29 (2b, 2c), respectively. H-Ser(OBn)-OtBu
was prepared by treatment of Fmoc-Ser(OBn)-OH with
tert-butyl trichloroacetamidate and removal of Fmoc with
Et2NH. The 1H NMR spectra of H-Ser(OBn)-OtBu were in
accordance with the data given in Ref. 42. Reaction vessels
for solid-phase synthesis: Single fritted (20 mm) PE reservoir
(15 ml) (Separtis, Grenzach-Wyhlen, Germany) was wrap-
ped with aluminum foil and used on an Advanced ChemTech
PLS 4ꢀ6 Shaker (Advanced ChemTech, Inc., Louisville, KY,
USA) with a self-made adapter. The original Advanced
ChemTech reaction vessels were used for reactions with
COCl2. Photolysis was performed in a quartz tube (13.5 cm,
13 mm i.d.) and irradiation with circularly arranged sterilAir
BLB8 lamps (16ꢀ8 W, lmax¼352 nm) (sterilAir AG, Wein-
felden, Switzerland). High-performance liquid chromato-
graphy (HPLC) instrument: Waters 600E multisolvent
delivery system equipped with a Waters 996 PDA (Waters,
Milford, CA, USA); column: Interchim Uptisphere WOD
3. Conclusions
A method for the direct introduction of Aib-Pro into peptides
via ‘azirine coupling’ on solid phase was developed. The
protocol is based on a photocleavable resin (10) and the
Aib-Pro synthon 2b. After the coupling of the resin-bound
peptide acid with 2b, the allyl protecting group of the
extended peptide was removed by a mild Pd0-promoted
procedure. In contrast to the previously described ‘PAM/
HBr-strategy’, this protocol allows the isolation of peptides
with a protected C-terminus. The method found a successful
application in the syntheses of different Aib-Pro containing
peptaibol segments. A subsequent segment condensation
led to the Trichovirin I 1B derivative 25, an oligopeptide
containing three Aib-Pro units and two additional Aib
residues.
˚
C18, 300 A, 10 mm, 250ꢀ21.2 mm (Interchim, Montluc¸on,

9676
S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
France); eluents: A¼H2O/TFA (0.1%), B¼MeCN/TFA
(0.1%); flow rate: 10 ml/min; various gradients. Column
chromatography (CC): Silica gel C-560 from Chemie Ueti-
kon (CU Chemie Uetikon GmbH, Uetikon, Switzerland).
IR spectra: Perkin–Elmer, Spectrum one FT-IR spectropho-
tometer (Perkin–Elmer, Wellesley, MA, USA), absorptions
in cmꢁ1. NMR spectra: Bruker AV-600 (Bruker Biospin,
Karlsruhe, Germany). Chemical shifts in parts per million
relative to tetramethylsilane as internal standard. 2D-NMR
experiments were performed for assignment of the signals.
The integer n in Xaan corresponds to the position of the amino
acid within the peptide, but is only given if the amino acid
was present more than once in the peptide and if the NMR
signal could be assigned unambiguously. HPLC/MS: The
system consists of a Rheos 2000 pump, a Rheos CPS-LC de-
gasser (Flux Instruments, Basel, Switzerland) and a Thermo
Finnigan Surveyor photodiode array detector (Thermo Finni-
gan, San Jose, CA, USA). The HPLC system is equipped with
a HTS PAL autosampler (CTC Analytics, Zwingen, Switzer-
land) and connected to a Thermo Finnigan MSQ linear quad-
4.2.5. GP 5: coupling with H-Xaa-OtBu$HCl. The resin
was swollen in DMF. HOBt (6 equiv) in DMF, PyBOP
(4 equiv) in DMF, H-Xaa-OtBu$HCl (4 equiv) in DMF,
and DIPEA (12 equiv) were added (concn of H-Xaa-
OtBu$HCl¼ca. 0.2 M), the resin was agitated at rt, and
then washed with DMF (3ꢀ) and CH2Cl2 (3ꢀ).
4.2.6. GP 6: removal of the allyl protecting group. The
resin was dried i.v. All manipulations were carried out under
Ar. The resin was swollen in CH2Cl2. A mixture of
Pd(Ph3P)4 (0.3 equiv) and PhSiH3 (20 equiv) in CH2Cl2
(ca. 2 ml/100 mg resin) was added, the resin was agitated
at rt for 1.5 h, and then washed with CH2Cl2 (3ꢀ), DMF,
CH2Cl2 (2ꢀ), THF (1% H2O) (2ꢀ), THF (1ꢀ), MeOH,
and CH2Cl2 (2ꢀ).
4.2.7. GP 7: cleavage. The resin (ca. 0.075 mmol) was swol-
len in a solution of semicarbazide hydrochloride (24 mg) in
MeOH/THF (2:1, 6 ml), and the mixture was degassed by
bubbling Ar into the mixture. Under vigorous stirring, the
resin was irradiated with 16ꢀ8 W (lmax¼352 nm) for 2 h,
then the supernatant solution was removed, and the irradia-
tion process was repeated two times. Afterwards, the resin
was washed with MeOH/THF (2:1, 3ꢀ). All solutions
were combined, concentrated under reduced pressure, and
the crude product was purified by means of HPLC. The
purified product was lyophilized.
˚
rupole instrument. Interchim Uptisphere C18-ODB, 120 A,
3 mm, 50ꢀ2.0 mm column; eluents: A¼H2O, B¼MeCN,
C¼HCOOH (1%) in H2O; flow rate: 0.2 ml/min, gradient
(A/B/C): 0.0–15.0 min: 87:3:10 to 40:50:10. MS: Bruker
ESQUIRE-LC quadrupole instrument (Bruker Daltonik
GmbH, Bremen, Germany)or Finnigan TSQ-700 triple quad-
rupole instrument (Finnigan MAT, San Jose, CA, USA).
Direct infusion ESI-MS were performed with a syringe infu-
sion pump at a flow rate of 5 ml/min.
4.3. Synthesis of peptides
4.2. General procedures 1–7 (GP 1–GP 7)
4.3.1. H-Ala-Aib-Pro-Val-OH (9). Aminomethyl Tentagel
resin (350 mg, 0.098 mmol) was swollen in DMF.
4-(Hydroxymethyl)phenylacetic acid (33 mg, 0.199 mmol),
DIPEA (103 ml, 0.602 mmol), and PyBOP (104 mg,
0.200 mmol) in DMF (1.5 ml) were added, and the resin
was agitated at rt for 30 min (negative Kaiser test), then
washed with DMF (3ꢀ), CH2Cl2 (3ꢀ), and THF (3ꢀ). The
resin was treated as described in GP 1–3. Then, the resin
was swollen in a mixture of THF (1 ml) and MeOH
(0.7 ml), and LiOH$H2O (33 mg, 0.787 mmol) in H2O
(0.3 ml) was added. The resin was agitated at rt overnight,
washed with THF/MeOH/H2O (4:3:1, 3ꢀ), and DMF (3ꢀ).
The resin was treated as described in GP 5 (2 h), then swollen
in CH2Cl2. HBr in AcOH (33%, 3 ml) and two drops of H2O
were added, and the resin was agitated at rt for 4 h. The resin
was separated by filtration and washed with AcOH/CH2Cl2
(1:1, 3ꢀ) and MeCN/CH2Cl2 (1:1, 3ꢀ). The solvents were
evaporated under reduced pressure and the crude product
was purified by means of HPLC. The purified product was
lyophilized and yielded 9 (10 mg, 21%) as a colorless
powder. IR (KBr): 3435s, 3271s, 3068s, 2969s, 2882s,
1674vs, 1630vs, 1548s, 1472m, 1422s, 1369m, 1338w,
1310w, 1269m, 1202vs, 1183vs, 1138vs, 1005w, 979w,
929w, 837w, 800w, 722m. 1H NMR (DMSO-d6, 600 MHz):
ca. 10.0–7.0 (br s, NH3(Ala)); 8.61 (s, NH(Aib)); 7.73
(d, J¼8.2 Hz, NH(Val)); 4.42–4.41 (m, CH(a)(Pro));
4.06–4.04 (m, CH(a)(Val)); 3.88–3.87 (m, CH(a)(Ala));
3.57–3.54, 3.38–3.34 (2m, CH2(d)(Pro)); 2.04 (dsept.,
J¼6.6, 6.6 Hz, CH(b)(Val)); 1.88–1.83, 1.81–1.76 (2m,
CH2(b)(Pro), CH2(g)(Pro)); 1.37, 1.34 (2s, 2 Me(Aib));
1.36 (d, J¼7.6 Hz, Me(Ala)); 0.90, 0.89 (2d, J¼6.8 Hz,
2 Me(Val)). 13C NMR (DMSO-d6, 150 MHz): 173.0 (s,
CO(Val)); 171.9 (s, CO(Pro)); 170.4 (s, CO(Aib)); 168.4
4.2.1. GP 1: attachment of the first amino acid. All mani-
pulations were carried out under N2. Hydroxymethyl-photo-
linker AM resin was swollen in THF. After filtration,
a solution of COCl2 (CAUTION) in toluene (ca. 20%,
10 equiv) and THF (ca. 0.7 ml/100 mg resin) were added to
the resin, which was agitated at rt for 2 h, then washed with
THF (2ꢀ) and CH2Cl2 (2ꢀ). In a separate vial, H-Xaa-
OtBu$HCl (4 equiv) was dissolved in DIPEA (8 equiv) and
CH2Cl2 (concn of H-Xaa-OtBu$HCl¼ca. 0.2 M). This mix-
ture was added to the resin and any ammonium salt that
occurred was removed by filtration. The resin was agitated at
rt overnight, then washed with DMF (3ꢀ) and CH2Cl2 (3ꢀ).
4.2.2. GP 2: removing the tBu protecting group. The resin
was swollen in CH2Cl2. TFA in CH2Cl2 (2 ml, 1ꢀ15 s, 25%;
2 ml, 1ꢀ60 min, 50%) and TIPS (5%, in each case) were
added, and the resin was agitated at rt. Then, the resin was
washed with CH2Cl2 (3ꢀ), DMF (2ꢀ), and CH2Cl2 (3ꢀ).
4.2.3. GP 3: coupling with 2H-azirin-3-amines 1a, 2a, and
2b. The corresponding resin was swollen in CH2Cl2. A solu-
tion of 2H-azirin-3-amine (4 equiv) in CH2Cl2 (concn of 2H-
azirin-3-amine¼ca. 0.2 M) was added, the resin was agitated
at rt overnight, and then washed with CH2Cl2 (3ꢀ). Uncon-
sumed 2H-azirin-3-amine could easily be recovered.
4.2.4. GP 4: hydrolysis of the terminal amide. The resin
was swollen in THF. Aq HCl (ca. 2 ml/100 mg resin, 3 M
in THF/H2O, prepared from concd HCl and THF) was
added, and the resin was agitated at rt overnight, then
washed with THF (3ꢀ), DMF (3ꢀ), and CH2Cl2 (3ꢀ).

S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
t
9677
(s, CO(Ala)); 60.6 (d, CH(a)(Pro)); 57.2 (d, CH(a)(Val));
56.0 (s, C(a)(Aib)); 47.9 (d, CH(a)(Ala)); 47.6 (t,
CH2(d)(Pro)); 29.9 (d, CH(b)(Val)); 27.9, 25.2 (2t,
CH2(b)(Pro), CH2(g)(Pro)); 24.9, 24.5 (2q, 2 Me(Aib));
19.1, 18.1 (2q, 2 Me(Val)); 17.3 (q, Me(Ala)). ESI-MS:
371 (100, [M+H]+), 215 (34, [Pro-Val]+).
[Mꢁ Bu]+), 319 (7, [Mꢁ(Ala-Aib)]+). HPLC/MS: tR
9.9 min, m/z 497 (10, [M+Na]+), 475 (30, [M+H]+), 419
t
(24, [Mꢁ Bu]+), 319 (32, [Mꢁ(Ala-Aib)]+), 263 (100,
[Mꢁ(Ala-Aib)ꢁ Bu]+).
t
4.3.4. H-Val-Aib-Gly-Aib-Aib-Pro-Leu-OtBu (20). Hy-
droxymethyl-photolinker AM resin (100 mg, 0.075 mmol)
was treated as described in GP 1–5 (overnight), 2–4, 3, 6, 5
(2 h), and 7 to yield 20 (25.7 mg, 42%) as a colorless powder
after prep. HPLC purification and lyophilization. IR (KBr):
3433vs, 3317vs, 3063m, 2978s, 2939s, 2877m, 1725m,
1671vs, 1543vs, 1537vs, 1469m, 1440m, 1416m, 1397m,
1384m, 1368m, 1333w, 1282m, 1248m, 1203vs, 1178vs,
4.3.2. H-Ala-Aib-Phe-OtBu (15). Aminomethyl polysty-
rene resin (62 mg, 0.071 mmol) was swollen in DMF. 4-
[(4-Hydroxymethyl-2-methoxy-5-nitrophenyl)oxy]butanoic
acid (40 mg, 0.140 mmol), PyBOP (72 mg, 0.138 mmol),
and DIPEA (72 ml, 0.421 mmol) in DMF (1.5 ml) were
added, and the resin was agitated at rt for 30 min, then
washed with DMF (3ꢀ), CH2Cl2 (2ꢀ), and THF (2ꢀ).
The resin was treated as described in GP 1–5 (overnight),
and 7 to yield 15 (11.4 mg, 33%) as a colorless powder after
prep. HPLC purification and lyophilization. IR (KBr):
3426s, 3399s, 3288s, 3065m, 3032m, 2983s, 2939s, 1724s,
1674vs, 1549s, 1516s, 1457m, 1440m, 1393m, 1368s,
1324w, 1259s, 1204vs, 1180vs, 1156vs, 1139vs, 1079w,
1030w, 1015w, 1003w, 948w, 929w, 881w, 839m, 800w,
754w, 739w, 722m, 700m. 1H NMR (DMSO-d6,
600 MHz): 8.31 (s, NH(Aib)); 8.01 (br s, NH3(Ala)); 7.67
(d, J¼7.8 Hz, NH(Phe)); 7.29–7.19 (m, 5 arom. H); 4.34
(ddd, J¼8.2, 7.8, 6.4 Hz, CH(a)(Phe)); 3.84 (q, J¼6.9 Hz,
CH(a)(Ala)); 3.02 (dd, J¼13.8, 6.4 Hz, 1 H of CH2(Phe));
2.95 (dd, J¼13.8, 8.2 Hz, 1 H of CH2(Phe)); 1.38, 1.37
(2s, 2 Me(Aib)); 1.33 (s, Me3C); 1.32 (d, J¼7.3 Hz,
Me(Ala)). 13C NMR (DMSO-d6, 150 MHz): 172.9 (s,
CO(Aib)); 170.4 (s, CO(Phe)); 168.8 (s, CO(Ala)); 137.4
(s, arom. C); 129.2, 128.1, 126.4 (3d, 5 arom. CH); 80.7
(s, Me3C); 56.3 (s, C(a)(Aib)); 54.2 (d, CH(a)(Phe)); 48.3
(d, CH(a)(Ala)); 36.8 (t, CH2(Phe)); 27.5 (q, Me3C); 24.7,
24.6 (2q, 2 Me(Aib)); 17.1 (q, Me(Ala)). ESI-MS: 378
1
1146s, 1018w, 947w, 878w, 837w, 801w, 722w. H NMR
(DMSO-d6, 600 MHz): 8.82 (s, NH(Aib2)); 8.20 (s,
NH(Gly)); 8.08 (br s, NH3(Val)); 7.81 (d, J¼8.1 Hz,
NH(Leu)); 7.72 (s, NH(Aib4)); 7.43 (s, NH(Aib5)); 4.37–
4.35 (m, CH(a)(Pro)); 4.11–4.07 (m, CH(a)(Leu)); 3.61–
3.59 (m, CH(a)(Val), 1 H of CH2(Gly), 1 H of CH2(d)(Pro));
3.55–3.51 (m, 1 H of CH2(Gly)); 3.43–3.39 (m, 1 H of
CH2(d)(Pro)); 2.12 (dsept., J¼6.7, 6.7 Hz, CH(b)(Val));
2.02–1.98 (m, 1 H of CH2(b)(Pro)); 1.74–1.69 (m, 1 H of
CH2(b)(Pro), CH2(g)(Pro)); 1.63–1.57 (m, CH(g)(Leu), 1
H of CH2(b)(Leu)); 1.51–1.45 (m, 1 H of CH2(b)(Leu));
1.403, 1.398 (2s, 2 Me(Aib2), 2 Me(Aib4)); 1.38 (s, Me3C);
1.35, 1.33 (2s, 2 Me(Aib5)); 0.96, 0.94 (2d, J¼7.0 Hz, 2
Me(Val)); 0.89, 0.82 (2d, J¼6.3 Hz, 2 Me(Leu)). 13C NMR
(DMSO-d6, 150 MHz): 174.6 (s, CO(Aib2)); 174.3 (s,
CO(Aib4)); 171.7 (s, CO(Pro)); 171.1 (s, CO(Leu)); 171.0
(s, CO(Aib5)); 168.4 (s, CO(Gly)); 168.0 (s, CO(Val)); 79.9
(s, Me3C); 60.8 (d, CH(a)(Pro)); 57.5 (d, CH(a)(Val));
56.13 (s, C(a)(Aib4)); 56.09 (s, C(a)(Aib2)); 56.0 (s,
C(a)(Aib5)); 50.9 (d, CH(a)(Leu)); 47.2 (t, CH2(d)(Pro));
43.8 (t, CH2(Gly)); 39.3 (t, CH2(b)(Leu)); 29.5 (d,
CH(b)(Val)); 28.4 (t, CH2(b)(Pro)); 27.5 (q, Me3C); 25.8
(q, 1 Me of 2 Me(Aib4)); 25.6 (q, 1 Me of 2 Me(Aib2));
25.2 (q, 1 Me of 2 Me(Aib5)); 25.0 (t, CH2(g)(Pro)); 24.5
(q, 1 Me of 2 Me(Aib4)); 24.2 (d, CH(g)(Leu)); 24.0 (q, 1
Me of 2 Me(Aib5)); 23.7 (q, 1 Me of 2 Me(Aib2)); 22.7,
21.2 (2q, 2 Me(Leu)); 18.4, 17.5 (2q, 2 Me(Val)). ESI-MS:
718 (17, [M+Na]+), 696 (100, [M+H]+). HPLC/MS: tR
(53, [M+H]+), 322 (100, [Mꢁ Bu]+). HPLC/MS: tR
t
t
10.3 min, m/z 378 (16, [M+H]+), 322 (100, [Mꢁ Bu]+),
157 (27, [Mꢁ(Phe-OtBu)]+).
4.3.3. H-Ala-Aib-Pro-Phe-OtBu (18). Hydroxymethyl-
photolinker AM resin (101 mg, 0.075 mmol) was treated
as described in GP 1–3, 6, 5 (2 h), and 7 to yield 18
(15.6 mg, 35%) as a colorless powder after prep. HPLC pu-
rification and lyophilization. IR (KBr): 3428s, 3293s, 3065s,
3032s, 2983s, 2940s, 1728s, 1674vs, 1548s, 1536s, 1499m,
1472m, 1456m, 1422m, 1415m, 1395m, 1369m, 1258m,
1203vs, 1177vs, 1156vs, 1135vs, 1052w, 1029w, 1004w,
11.5 min, m/z 696 (100, [M+H]+), 640 (27, [Mꢁ Bu]+),
t
412 (24, [Mꢁ(Pro-Leu-OtBu)]+), 339 (16).
4.3.5. H-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-OtBu (21). Hy-
droxymethyl-photolinker AM resin (100 mg, 0.075 mmol)
was treated as described in GP 1, 2, 5 (overnight), 2, 5
(90 min), 2, 3, 6, 5 (90 min), and 7 to yield 21 (21.7 mg,
34%) as a colorless powder after prep. HPLC purification
and lyophilization. IR (KBr): 3427s, 3301s, 3066m,
2979m, 2961m, 2938m, 2875m, 1673vs, 1535s, 1470m,
1453m, 1424m, 1412m, 1369m, 1247m, 1203vs, 1182vs,
1138vs, 837w, 800w, 742w, 722m, 700w. 1H NMR
(DMSO-d6, 600 MHz): 8.39–8.37 (m, NH(Asn)); 8.17–
8.13 (m, NH(Leu), NH3(Aib1), NH(Aib4)); 7.99 (d,
J¼8.0 Hz, NH(Ser)); 7.42–7.41 (m, 1 H of CONH2(Asn));
7.36–7.32, 7.29–7.27 (2m, 5 arom. H); 6.98 (s, 1 H of CON-
H2(Asn)); 4.70 (ddd, J¼7.5, 7.5, 7.5 Hz, CH(a)(Asn)); 4.49,
4.53 (AB, J¼12.1 Hz, OCH2Ph(Ser)); 4.42–4.40 (m,
CH(a)(Pro)); 4.36–4.33 (m, CH(a)(Ser)); 4.29–4.25 (m,
CH(a)(Leu)); 3.72 (dd, J¼9.7, 5.8 Hz, 1 H of CH2(b)(Ser));
3.65 (dd, J¼9.7, 4.4 Hz, 1 H of CH2(b)(Ser)); 3.46–3.44,
3.42–3.38 (2m, CH2(d)(Pro)); 2.64–2.60, 2.43–2.39 (2m,
1
928w, 879w, 836w, 800w, 740w, 721m, 701m. H NMR
(DMSO-d6, 600 MHz): 8.65 (s, NH(Aib)); 8.08 (br s,
NH3(Ala)); 7.91 (d, J¼7.4 Hz, NH(Phe)); 7.29–7.20 (m, 5
arom. H); 4.33–4.31 (m, CH(a)(Pro)); 4.26 (ddd, J¼7.3,
7.3, 7.3 Hz, CH(a)(Phe)); 3.88–3.87 (m, CH(a)(Ala));
3.52–3.50, 3.38–3.35 (2m, CH2(d)(Pro)); 2.97–2.95 (m,
CH2(Phe)); 1.86–1.76, 1.69–1.67 (2m, CH2(b)(Pro),
CH2(g)(Pro)); 1.36 (d, J¼7.1 Hz, Me(Ala)); 1.35, 1.34 (2s,
2 Me(Aib)); 1.30 (s, Me3C). 13C NMR (DMSO-d6,
150 MHz): 171.7 (s, CO(Pro)); 170.4 (s, CO(Phe)); 170.3
(s, CO(Aib)); 168.3 (s, CO(Ala)); 137.3 (s, arom. C);
129.2, 128.0, 126.3 (3d, 5 arom. CH); 80.3 (s, Me3C); 60.7
(d, CH(a)(Pro)); 55.9 (s, C(a)(Aib)); 54.2 (d, CH(a)(Phe));
47.8 (d, CH(a)(Ala)); 47.5 (t, CH2(d)(Pro)); 36.6 (t,
CH2(Phe)); 27.9 (t, CH2(b)(Pro)); 27.4 (s, Me3C); 25.0 (t,
CH2(g)(Pro)); 24.7, 24.6 (2q, 2 Me(Aib)); 17.1 (q, Me(Ala)).
ESI-MS: 497 (10, [M+Na]+), 475 (100, [M+H]+), 419 (17,

9678
S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
CH2(b)(Asn)); 1.90–1.87, 1.77–1.72 (2m, CH2(b)(Pro),
CH2(g)(Pro)); 1.60–1.55 (m, CH(g)(Leu)); 1.49–1.43
(m, CH2(b)(Leu)); 1.45, 1.43 (2s, 2 Me(Aib1)); 1.37 (s,
Me3C); 1.34, 1.30 (2s, 2 Me(Aib4)); 0.86, 0.80 (2d,
J¼6.6 Hz, 2 Me(Leu)). 13C NMR (DMSO-d6, 150 MHz):
171.9 (s, CO(Pro)); 171.2 (s, CO(Leu), CONH2(Asn));
171.1 (s, CO(Aib1)); 170.8 (s, CO(Aib4)); 170.3 (s,
CO(Asn)); 169.0 (s, CO(Ser)); 137.9 (s, arom. C); 128.1,
127.5, 127.4 (3d, 5 arom. CH); 80.5 (s, Me3C); 72.1 (t,
OCH2Ph(Ser)); 69.3 (t, CH2(b)(Ser)); 60.5 (d, CH(a)(Pro));
56.2 (s, C(a)(Aib1)); 55.6 (s, C(a)(Aib4)); 52.8 (d,
CH(a)(Ser)); 50.8 (d, CH(a)(Leu)); 49.8 (d, CH(a)(Asn));
47.3 (t, CH2(d)(Pro)); 40.3 (t, CH2(b)(Leu)); 36.8 (t,
CH2(b)(Asn)); 28.0 (t, CH2(b)(Pro)); 27.5 (q, Me3C); 25.0
(t, CH2(g)(Pro)); 24.9, 24.7 (2q, 2 Me(Aib4)); 24.1 (d,
CH(g)(Leu)); 23.3, 23.1 (2q, 2 Me(Aib1)); 22.9, 21.3 (2q, 2
Me(Leu)). ESI-MS: 768 (100, [M+Na]+), 746 (48,
4.3.7. Z-Ala-Aib-Pro-Phe-OH (23). Z-ONSu (4.3 mg,
17.3 mmol) and DIPEA (8.1 ml, 47.3 mmol) were added to
a solution of 18 (9.3 mg, 15.8 mmol) in dioxane (2.5 ml) at
rt, and the solution was stirred at rt for 3 h. The mixture
was concentrated, and the crude product was purified by
prep. HPLC. After lyophilization, Z-Ala-Aib-Pro-Phe-
OtBu (HPLC/MS: tR 16.5 min, m/z 631 (100, [M+Na]+))
(8.5 mg, 89%) was obtained as a colorless powder, which
was dissolved in CH2Cl2/TFA (1:1, 2 ml) and TIPS
(100 ml). The solution was stirred at rt for 1 h, then it was con-
centrated under reduced pressure, and the crude product was
purified by prep. HPLC. The purified product was lyophi-
lized and yielded 23 (7.5 mg, 97%) as a colorless powder.
IR (KBr): 3412vs, 3292vs, 3063m, 3032m, 2984s, 2940m,
2876m, 1722vs, 1650vs, 1535vs, 1499s, 1469m, 1454s,
1411s, 1380m, 1366m, 1340m, 1328m, 1317m, 1282m,
1243vs, 1215s, 1203s, 1177s, 1116m, 1072m, 1040m,
t
1
[M+H]+), 712 (28, [Mꢁ Bu+Na]+), 622 (17). HPLC/MS: tR
1028m, 1002w, 977w, 911w, 824w, 743m, 700s. H NMR
12.4 min, m/z 746 (100, [M+H]+), 398 (34, [Mꢁ(Pro-
(DMSO-d6, 600 MHz): ca. 13.2–11.2 (br s, COOH); 8.20
(s, NH(Aib)); 7.77 (d, J¼8.0 Hz, NH(Phe)); 7.44 (d,
J¼7.6 Hz, NH(Ala)); 7.38–7.18 (m, 10 arom. H); 5.01,
5.06 (AB, J¼12.6 Hz, CH2(carbamate)); 4.37 (ddd, J¼8.4,
8.4, 5.3 Hz, CH(a)(Phe)); 4.29–4.27 (m, CH(a)(Pro)); 4.10
(dq, J¼7.2, 7.2 Hz, CH(a)(Ala)); 3.54–3.50, 3.35–3.31
(2m, CH2(d)(Pro)); 3.05 (dd, J¼13.9, 5.1 Hz, 1 H of
CH2(Phe)); 2.93 (dd, J¼13.9, 9.2 Hz, 1 H of CH2(Phe));
1.79–1.73 (m, 1 H of CH2(b)(Pro)); 1.61–1.58 (m,
CH2(g)(Pro)); 1.51–1.48 (m, 1 H of CH2(b)(Pro)); 1.30,
1.29 (2s, 2 Me(Aib)); 1.21 (d, J¼7.1 Hz, Me(Ala)). 13C
NMR (DMSO-d6, 150 MHz): 172.6 (s, CO(Phe)); 171.9 (s,
CO(Ala)); 171.7 (s, CO(Pro)); 170.9 (s, CO(Aib)); 155.6
(s, CO(carbamate)); 137.6, 137.0 (2s, 2 arom. C); 129.1,
128.2, 128.0, 127.7, 127.6, 126.2 (6d, 10 arom. CH); 65.2
(t, CH2(carbamate)); 60.9 (d, CH(a)(Pro)); 55.5 (s,
C(a)(Aib)); 53.2 (d, CH(a)(Phe)); 49.9 (d, CH(a)(Ala));
47.2 (t, CH2(d)(Pro)); 36.6 (t, CH2(Phe)); 27.8 (t,
CH2(b)(Pro)); 25.0 (q, 1 Me of 2 Me(Aib)); 24.9 (t,
CH2(g)(Pro)); 24.6 (q, 1 Me of 2 Me(Aib)); 17.9 (q,
Me(Ala)). ESI-MS: 591 (67, [M+K]+), 575 (100,
[M+Na]+), 553 (44, [M+H]+), 263 (96, [Mꢁ(Pro-
Phe)ꢁCO]+, [Pro-Phe]+). HPLC/MS: tR 14.0 min, m/z 591
(51, [M+K]+), 263 (100, [Mꢁ(Pro-Phe)ꢁCO]+, [Pro-Phe]+).
Ser(OBn)-tBu)]+).
4.3.6. H-Val-Aib-Pro-Aib-Leu-Aib-Pro-Leu-OtBu (22).
Hydroxymethyl-photolinker AM resin (100 mg, 0.075
mmol) was treated as described in GP 1–3, 6, 3–5(overnight),
2, 3, 6, 5 (2 h), and 7 to yield 22 (24.8 mg, 34%) as a colorless
powder after prep. HPLC purification and lyophilization.
IR (KBr): 3440vs, 3330sh, 3058m, 2961s, 2874m, 1724sh,
1653vs, 1627vs, 1536vs, 1471s, 1451m, 1440m, 1416s,
1385m, 1368m, 1344w, 1249m, 1241m, 1203vs, 1178vs,
1146s, 839w, 833w, 800w, 722w. 1H NMR (DMSO-d6,
600 MHz): 9.00 (s, NH(Aib2)); 8.13 (br s, NH3(Val)); 7.77
(d, J¼7.8 Hz, NH(Leu8)); 7.72 (s, NH(Aib4)); 7.69 (s,
NH(Aib6)); 7.38 (d, J¼8.5 Hz, NH(Leu5)); 4.37–4.35 (m,
CH(a)(Pro7)); 4.17–4.12 (m, CH(a)(Leu5), CH(a)(Pro3));
4.04–4.00 (m, CH(a)(Leu8)); 3.76–3.73 (m, CH(a)(Val));
3.60–3.55 (m, 3 H of 2 CH2(d)(Pro)); 3.40–3.36 (m, 1 H
of 2 CH2(d)(Pro)); 2.21–2.18 (m, CH(b)(Val)); 2.12–2.08
(m, 1 H of 2 CH2(b)(Pro)); 2.00–1.95 (m, 1 H of 2
CH2(b)(Pro), 1 H of 2 CH2(g)(Pro)); 1.89–1.85 (m, 1 H of
2 CH2(g)(Pro)); 1.74–1.68 (m, 2 H of 2 CH2(b)(Pro), 2 H
of 2 CH2(g)(Pro)); 1.66–1.57 (m, 3 H of 2 CH2(b)(Leu), 2
CH(g)(Leu)); 1.48–1.44 (m, 1 H of 2 CH2(b)(Leu)); 1.42,
1.40 (2s, 2 Me of 6 Me(Aib)); 1.38 (s, Me3C, 1 Me of 6
Me(Aib)); 1.36, 1.35 (2s, 3 Me of 6 Me(Aib)); 1.00, 0.94
(2d, J¼7.0 Hz, 2 Me(Val)); 0.91, 0.87, 0.82, 0.78 (4d,
J¼6.2 Hz, 4 Me(Leu)). 13C NMR (DMSO-d6, 150 MHz):
173.9 (s, CO(Aib4)); 172.6 (s, CO(Pro3)); 172.2 (s,
CO(Aib2)); 171.7 (s, CO(Leu5), CO(Pro7)); 171.3 (s,
CO(Leu8)); 170.8 (s, CO(Aib6)); 167.5 (s, CO(Val)); 79.9
(s, Me3C); 63.2 (d, CH(a)(Pro3)); 60.8 (d, CH(a)(Pro7));
56.9 (d, CH(a)(Val)); 56.2 (s, C(a)(Aib4)); 56.1 (s,
C(a)(Aib2)); 55.6 (s, C(a)(Aib6)); 51.1 (s, 2 CH(a)(Leu));
4.3.8. Z-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-OH (24). Z-ONSu
(6.0 mg, 24.1 mmol) and DIPEA (11.6 ml, 67.8 mmol) were
added to a solution of 21 (19.4 mg, 22.6 mmol) in MeCN
(5 ml) at rt, and the solution was stirred at rt for 6 h. Addi-
tional Z-ONSu (0.6 mg, 2.3 mmol) and DIPEA (3.9 ml,
22.6 mmol) were added, and the solution was stirred at rt
for further 2 h. Then, the mixture was concentrated, and
the crude product was purified by prep. HPLC. After lyoph-
ilization, Z-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-OtBu (HPLC/
MS: tR 16.6 min, m/z 880 (62, [M+H]+)) (13.8 mg, 70%;
additionally, 21 (3.8 mg, 20%) was isolated) was obtained
as a colorless powder, which was dissolved in CH2Cl2/TFA
(1:1, 2 ml), H2O (1 drop), and TIPS (100 ml). The solution
was stirred at rt for 45 min, then it was concentrated under
reduced pressure, and the crude product was purified by
prep. HPLC. The purified product was lyophilized and
yielded 24 (8.9 mg, 67%) as a colorless powder. IR (KBr):
3418sh, 3308vs, 3063m, 3033m, 2957m, 2872m, 1660vs,
1532vs, 1469s, 1454s, 1412s, 1387m, 1366m, 1267s,
1203s, 1176s, 1094s, 1078m, 1028w, 740m, 698m.
48.0, 47.5 (2t,
2 CH2(d)(Pro)); 39.6, 39.2 (2t, 2
CH2(b)(Leu)); 29.6 (d, CH(b)(Val)); 28.3, 27.6 (2t, 2
CH2(b)Pro); 27.5 (q, Me3C); 26.4 (q, 1 Me of 6 Me(Aib));
25.5 (t, 1CH2 of 2CH2(g)(Pro)); 25.4 (q, 1Me of 6Me(Aib));
25.1 (t, 1 CH2 of 2 CH2(g)(Pro)); 24.6, 24.32, 24.29 (3q, 3
Me of 6 Me(Aib)); 24.27, 24.2 (2d, 2 CH(g)(Leu)); 23.9
(q, 1 Me of 6 Me(Aib)); 23.0, 22.9, 21.2, 20.6 (4q, 4
Me(Leu)); 18.6, 16.7 (2q, 2 Me(Val)). ESI-MS: 871
(19, [M+Na]+), 849 (100, [M+H]+). HPLC/MS: tR
13.9 min, m/z 849 (100, [M+H]+), 565 (93, [Mꢁ(Pro-Leu-
OtBu)]+).

S. Stamm, H. Heimgartner / Tetrahedron 62 (2006) 9671–9680
9679
1H NMR (DMSO-d6, 600 MHz): ca. 13.2–11.3 (br s,
COOH); 8.49 (d, J¼6.0 Hz, NH(Asn)); 8.06 (s, NH(Aib1));
7.96 (d, J¼7.9 Hz, NH(Ser)); 7.74 (d, J¼8.5 Hz, NH(Leu));
7.49 (s, 1 H of CONH2(Asn)); 7.48 (s, NH(Aib4)); 7.40–7.26
(m, 10 arom. H); 7.04 (s, 1 H of CONH2(Asn)); 5.08, 5.00
(AB, J¼12.4 Hz, CH2(carbamate)); 4.51 (s, OCH2Ph(Ser));
4.43–4.38 (m, CH(a)(Ser), CH(a)(Pro)); 4.32–4.29 (m,
CH(a)(Asn)); 4.25–4.21 (m, CH(a)(Leu)); 3.75 (dd,
J¼9.8, 5.9 Hz, 1 H of CH2(b)(Ser)); 3.68 (dd, J¼9.8,
4.2 Hz, 1 H of CH2(b)(Ser)); 3.45–3.42 (m, CH2(d)(Pro));
2.67–2.63, 2.57–2.54 (2m, CH2(b)(Asn)); 1.90–1.86, 1.75–
1.56 (2m, CH2(b)(Pro), CH2(g)(Pro), CH2(b)(Leu),
CH(g)(Leu)); 1.36 (s, 1 Me of 2 Me(Aib1)); 1.35, 1.332
(2s, 2 Me(Aib4)); 1.325 (s, 1 Me of 2 Me(Aib1)); 0.81,
0.74 (2d, J¼6.3 Hz, 2 Me(Leu)). 13C NMR (DMSO-d6,
150 MHz): 175.3 (s, CO(Aib1)); 172.1 (s, CONH2(Asn));
172.0 (s, CO(Pro)); 171.4 (s, CO(Ser)); 171.2 (s, CO(Leu));
170.9 (s, CO(Asn)); 170.8 (s, CO(Aib4)); 156.2 (s,
CO(carbamate)); 138.1 (s, arom. C(Bn)); 136.4 (s, arom.
C(Z)); 128.4, 128.2, 128.0, 127.7, 127.5, 127.4 (6d, 10
arom. CH); 72.2 (t, OCH2Ph(Ser)); 69.4 (t, CH2(b)(Ser));
65.7 (t, CH2(carbamate)); 60.7 (d, CH(a)(Pro)); 56.1 (s,
C(a)(Aib1)); 55.8 (s, C(a)(Aib4)); 52.4 (d, CH(a)(Ser));
51.6 (d, CH(a)(Asn)); 50.9 (d, CH(a)(Leu)); 47.5 (t,
CH2(d)(Pro)); 39.4 (t, CH2(b)(Leu)); 35.1 (t, CH2(b)(Asn));
27.9 (t, CH2(b)(Pro)); 26.1 (q, 1 Me of 2 Me(Aib1)); 25.1 (q,
1 Me of 2 Me(Aib4)); 25.0 (t, CH2(g)(Pro)); 24.8 (q, 1 Me
of 2 Me(Aib4)); 24.0 (d, CH(g)(Leu)); 23.9 (q, 1 Me of
2 Me(Aib1)); 23.1, 20.7 (2q, 2 Me(Leu)). ESI-MS: 868
(45, [MꢁH+2Na]+), 846 (100, [M+Na]+). HPLC/MS: tR
15.6 min, m/z 824 (11, [M+H]+), 532 (100, [Mꢁ(Pro-
Ser(OBn)-OtBu)]+), 447 (40, [Mꢁ(Aib-Pro-Ser(OBn)-
OtBu)]+).
2.68–2.64, 2.60–2.56 (2m, CH2(b)(Asn)); 2.24–2.19 (m,
2 H of 3 CH2(b)(Pro)); 2.09 (dsept., J¼6.8, 6.8 Hz,
CH(b)(Val)); 2.01–1.98 (m, 1 H of 3 CH2(b)(Pro)); 1.93–
1.83 (m, 4 H of 3 CH2(g)(Pro)); 1.74–1.57 (m, 3
CH(g)(Leu), 4 H of 3 CH2(b)(Leu), 3 H of 3 CH2(b)(Pro),
2 H of 3 CH2(g)(Pro)); 1.54–1.51 (m, 1 H of 3 CH2(b)(Leu));
1.48 (s, 1 Me of 10 Me(Aib)); ca. 1.44 (m, 1 H of 3
CH2(b)(Leu)); 1.43, 1.42, 1.38, 1.37, 1.364, 1.358, 1.34
(7s, 9 Me of 10 Me(Aib), Me3C); 0.91 (d, J¼6.4 Hz, 1 Me
of 6 Me(Leu)); 0.87, 0.83 (2d, J¼6.8 Hz, 2 Me(Val)); 0.82
(d, J¼6.4 Hz, 2 Me of 6 Me(Leu)); 0.749, 0.745 (2d,
J¼6.0 Hz, 2 Me of 6 Me(Leu)); 0.63 (d, J¼6.2 Hz, 1 Me
of 6 Me(Leu)). 13C NMR (DMSO-d6, 150 MHz): 175.7,
174.2, 173.0, 172.8, 172.74, 172.70, 172.6, 171.90,
171.86, 171.6, 171.3, 171.2, 171.1, 170.9, 169.6 (15s, 15
CO); 156.2 (s, CO(carbamate)); 137.9 (s, arom. C of
CH2Ph(Ser)); 136.2 (s, arom. C(Z)); 128.3, 128.1, 127.9,
127.6, 127.4, 127.2 (6d, 10 arom. CH); 79.8 (s, Me3C);
72.0 (t, OCH2Ph(Ser)); 68.6 (t, CH2(b)(Ser)); 65.8 (t,
CH2(carbamate)); 63.9, 62.9, 60.8 (3d, 3 CH(a)(Pro));
58.3 (d, CH(a)(Val)); 56.1, 56.0, 55.9, 55.8, 55.6 (5s, 5
C(a)(Aib)); 55.4, 51.9 (2d, CH(a)(Asn), CH(a)(Ser));
51.1, 51.0 (2d, 3 CH(a)(Leu)); 48.3, 48.2, 47.6 (3t, 3
CH2(d)(Pro)); 39.5, 39.1, 39.1 (3t, 3 CH2(b)(Leu)); 34.9
(t, CH2(b)(Asn)); 29.6 (d, CH(b)(Val)); 28.4, 28.3, 28.1
(3t, 3 CH2(b)(Pro)); 27.5 (q, Me3C); 26.5, 26.0, 25.7 (3q, 3
Me of 10 Me(Aib)); 25.6 (t, 1 CH2 of 3 CH2(g)(Pro));
25.5 (q, 1 Me of 10 Me(Aib)); 25.4 (t, 1 CH2 of
3 CH2(g)(Pro)); 25.3 (q, 1 Me of 10 Me(Aib)); 25.1 (t,
1 CH2 of 3 CH2(g)(Pro)); 24.2 (q, d, 1 Me of 10 Me(Aib),
2 CH of 3 CH(g)(Leu)); 23.81 (q, 1 Me of 10 Me(Aib));
23.76 (d, 1 CH of 3 CH(g)(Leu)); 23.3 (q, 2 Me of 10
Me(Aib)); 23.1 (q, 1 Me of 10 Me(Aib)); 23.0, 22.93,
22.89, 21.1, 20.3, 20.2 (6q, 6 Me(Leu)); 18.8, 17.9 (2q,
2 Me(Val)). ESI-MS: 1693 (17, [M+K]+), 1677 (85,
[M+Na]+). HPLC/MS: tR 18.1 min, m/z 1676 (2, [M+H]+),
846 (100), 819 (35).
4.3.9. Z-Aib-Asn-Leu-Aib-Pro-Ser(OBn)-Val-Aib-Pro-
Aib-Leu-Aib-Pro-Leu-OtBu (25). HOBt, TBTU, and DI-
PEA were taken from stock solutions. HOBt (1.7 mg,
11.1 mmol), TBTU (3.5 mg, 11.0 mmol), DIPEA (5.4 ml,
31.3 mmol), and 22 (10.6 mg, 11.0 mmol) were added to a
solution of 24 (8.6 mg, 10.4 mmol) in CH2Cl2/DMF (1:1,
3 ml). The mixture was stirred at rt for 4 h, the solvent was
evaporated under reduced pressure, and the crude product
was purified by prep. HPLC. After lyophilization, 25
(12.5 mg, 73%) was obtained as a colorless powder. IR
(KBr): 3435sh, 3306s, 2958m, 2872w, 1648vs, 1623vs,
1536vs, 1470s, 1454m, 1412s, 1385m, 1366m, 1269m,
1203m, 1172s, 1152m, 1094w, 1028w, 740w, 698w. 1H
NMR (DMSO-d6, 600 MHz): 8.57 (d, J¼5.5 Hz, NH(Asn));
8.11 (s, 1 NH of 5 NH(Aib)); 7.90 (d, J¼6.7 Hz, NH(Ser));
7.82 (d, J¼7.7 Hz, 1 NH of 3 NH(Leu)); 7.82, 7.79 (2s, 2 NH
of 5 NH(Aib)); 7.74 (d, J¼7.7 Hz, 1 NH of 3 NH(Leu));
7.55, 7.54 (2s, 2 NH of 5 NH(Aib)); 7.53 (s, 1 H of CON-
H2(Asn)); 7.39–7.30, 7.27–7.24 (2m, 10 arom. H, 1 NH of
3 NH(Leu), NH(Val)); 7.07 (s, 1 H of CONH2(Asn)); 5.07,
5.00 (AB, J¼12.3 Hz, CH2(carbamate)); 4.55, 4.51 (AB,
J¼11.9 Hz, OCH2Ph(Ser)); 4.38–4.36 (m, 1 H of 3
CH(a)(Pro)); 4.33–4.26 (m, CH(a)(Ser), CH(a)(Asn), 1 H
of 3 CH(a)(Leu), 1 H of 3 CH(a)(Pro)); 4.19–4.14 (m, 1 H
of 3 CH(a)(Leu), CH(a)(Val)); 4.08 (dd, J¼8.4, 8.4 Hz, 1
H of 3 CH(a)(Pro)); 4.04–4.00 (m, 1 H of 3 CH(a)(Leu));
3.86–3.83 (m, 1 H of CH2(b)(Ser)); 3.77–3.68 (m, 1 H
of CH2(b)(Ser), 2 H of 3 CH2(d)(Pro)); 3.65–3.61, 3.52–
3.48, 3.38–3.34, 3.30–3.25 (4m, 4 H of 3 CH2(d)(Pro));
Acknowledgements
We thank Dr. Daniel Obrecht, Polyphor Ltd, Allschwil
(Switzerland) for continuous advice and valuable discus-
sions, as well as the analytical sections of our institute for
2D-NMR measurements and mass spectra. Financial support
of the Swiss National Science Foundation and F. Hoffmann-
La Roche AG, Basel is gratefully acknowledged.
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