Cyclization of all-L-pentapeptides by means of 1-hydroxy-7-azabenzotriazole-derived uronium and phosphonium reagents

Article abstract of DOI:10.1021/jo951108d

Due to their restricted conformational flexibility, cyclic peptides are of great interest in connection with structure-activity relationships, especially the elucidation of bioactive conformations. For linear peptides that do not contain turn structure-inducing amino acid residues, the cyclization reaction may be an inherently improbable or slow process, and side reactions, such as cyclodimerization and epimerization at the C-terminal residue, may dominate. A number of 1-hydroxy-7-azabenzotriazole-based onium salts were examined for cyclization of thymopentin-derived pentapeptides and the results compared with data from more conventional coupling reagents. The azabenzotriazol-derived coupling reagents stood out as being the most effective by far. The cyclizations proceed extremely rapidly, and in contrast to other coupling reagents, C-terminal epimerization was generally less than 10%. C-terminal D-amino acid residues favor the formation of monocyclic pentapeptide rings. A similar effect was observed for cyclization of linear N-methylamino acid-containing peptides, suggesting that reversible amide bond alkylation such as Hmb-modification should be useful in promoting the cyclization of pepitdes devoid of turn-inducing amino acid residues.

Full text of DOI:10.1021/jo951108d

J . Org. Chem. 1996, 61, 8831-8838
8831
Cycliza tion of a ll-L-P en ta p ep tid es by Mea n s of
-Hyd r oxy-7-a za ben zotr ia zole-Der ived Ur on iu m a n d
P h osp h on iu m Rea gen ts
1
Angelika Ehrlich,* Hans-Ulrich Heyne, R u¨ diger Winter, Michael Beyermann, Hanka Haber,
†
Louis A. Carpino, and Michael Bienert
Research Institute of Molecular Pharmacology, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany, and
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
Received J une 20, 1995 (Revised Manuscript Received September 18, 1996X)
Due to their restricted conformational flexibility, cyclic peptides are of great interest in connection
with structure-activity relationships, especially the elucidation of bioactive conformations. For
linear peptides that do not contain turn structure-inducing amino acid residues, the cyclization
reaction may be an inherently improbable or slow process, and side reactions, such as cyclodimer-
ization and epimerization at the C-terminal residue, may dominate. A number of 1-hydroxy-7-
azabenzotriazole-based onium salts were examined for cyclization of thymopentin-derived pen-
tapeptides and the results compared with data from more conventional coupling reagents. The
azabenzotriazol-derived coupling reagents stood out as being the most effective by far. The
cyclizations proceed extremely rapidly, and in contrast to other coupling reagents, C-terminal
epimerization was generally less than 10%. C-terminal D-amino acid residues favor the formation
of monocyclic pentapeptide rings. A similar effect was observed for cyclization of linear N-
methylamino acid-containing peptides, suggesting that reversible amide bond alkylation such as
Hmb-modification should be useful in promoting the cyclization of pepitdes devoid of turn-inducing
amino acid residues.
In the synthesis of homodetic cyclic peptides, the
readiness of an open-chain precursor to cyclize depends
on the size of the ring to be closed, and usually no
difficulties arise for the cyclization of peptides containing
seven or more amino acid residues. Although ring
closure with hexa- and pentapeptides is more hampered,
the ease of cyclization is often enhanced by the presence
of turn structure-inducing amino acids such as glycine,
proline, or a D-amino acid.1
-3
For linear peptides that
do not contain amino acid residues that stabilize turn
structures, the cyclization reaction may be an inherently
improbable or slow process, and side reactions, such as
cyclodimerization, may dominate even at high dilutions
10 -10 M).4 For such slow cyclizations the increased
-
3
-4
(
lifetime of the intermediate activated linear peptide
provides an opportunity for increased epimerization at
the C-terminal residue. The extent of epimerization may
5
be diminished by application of the azide method or its
modification using DPPA.6 However, these methods are
,7
extremely slow, usually requiring many hours or even
5
,8,9
10
several days.
In comparison with DPPA, TBTU and
BOP11 provide for fast cyclization but may also lead to
C-terminal epimerization levels that are comparable to
those observed with DCC/HOBt.13
12
F igu r e 1. HOAt-derived coupling reagents used in this study.
Recently, a number of new peptide coupling reagents
1
4,16
(
Figure 1) derived from HOAt
have shown advantages
of these onium reagents to the cyclization of a linear
GnRH-derived decapeptide showed that they were also
highly effective for head-to-tail and side chain cycliza-
tion.18 These reagents led to complete cyclization within
less than 30 min, whereas standard reagents such as
over the corresponding HOBt-based reagents for solution-
and solid-phase peptide synthesis with regard to both
speed and maintenance of chiral integrity.17 Application
†
University of Massachusetts.
Abstract published in Advance ACS Abstracts, November 1, 1996.
19
TBTU, TOPPipU, and DPPA caused cyclization under
X
(
(
(
(
(
(
1) Dale, J . Angew. Chem. 1966, 78, 1070.
the same conditions to the extent of only 60, 10, and 12%,
2) Kessler, H.; Kutscher, B. Liebigs Ann. Chem. 1986, 869.
3) Kessler, H.; Kutscher, B. Liebigs Ann. Chem. 1986, 914.
4) Kopple, K. D. J . Pharm. Sci. 1972, 61, 1345.
5) Izumiya, N.; Kato, T.; Waki, M. Biopolymers 1981, 20,1785.
6) Brady, S. F.; Paleveda, W. J .; Arison, B. H.; Freidinger, R. M.;
18
respectively.
Although azabenzotriazole-based coupling reagents are
not expected to overcome difficulties arising from unfa-
vored conformations of short, linear peptides, the ob-
served high coupling efficiency prompted a reexamination
of the cyclization of all-L-penta- and -hexapeptides.
Nutt, R. F.; Veber, D. F. In Peptides. Structure and Function.
Proceedings of the 8th American Peptide Symposium; Hruby, V. J .,
Rich, D. H., Eds.; Pierce Chemical Company: Rockford, 1983; p 127.
S0022-3263(95)01108-X CCC: $12.00 © 1996 American Chemical Society

8
3
832 J . Org. Chem., Vol. 61, No. 25, 1996
Ehrlich et al.
As models the thymopentin-derived peptides 1, 2, and
were chosen since their cyclization is not easily
achieved.
H-Arg(NO )-Lys(Z)-Asp(OBn)-Val-Tyr-OH (1)
2
H-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-OH
(2)
(3)
+
H-Val-Arg(H )-Lys(Ac)-Ala-Val-Tyr-OH
Peptides 1 and 3 gave by the DCC/DMAP technique2,3
the corresponding cyclic peptides, although in both cases
the C-terminal tyrosine unit had been completely epimer-
ized to the D-configuration. In the case of peptide 2 the
F igu r e 2. Cyclization of thymopentin analogs via HAPyU:
[
, H-Arg(NO
(Ac)-Asp(OBn)-Val-Tyr-OH; b, H-Arg-Lys(Z)-Ala-Val-Tyr-OH;
, H-Arg-Lys(Ac)-Asp(OBn)-Val-Tyr-OH; #, H-Arg-Lys(Ac)-
2
)-Lys(Z)-Asp(OBn)-Val-Tyr-OH; +, H-Arg-Lys-
desired cyclic peptide was obtained but the azide coupling
process was very slow (1 week) and the yield was low.9
0
Ala-Val-Tyr-OH; 9, H-Arg-Lys(Ac)-Asn-Val-Tyr-OH; O, H-Arg-
Lys(Z)-Ala-Val-Tyr-OH.
(7) Abbreviations: AcOH, acetic acid; AcHmb, 2-acetoxy-4-methoxy-
benzyl; BOP, (1-benzotriazolyloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; DCC, dicyclohexylcarbodiimide; DCM, dichlo-
romethane; DIEA, diisopropylethylamine; DMF, dimethylformamide;
DPPA, diphenylphosphoryl azide; Hmb, 2-hydroxy-4-methoxybenzyl;
HOBt, 1-hydroxybenzotriazole; HOAt, 1-hydroxy-7-azabenzotriazole;
HAPyU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium
hexafluorophosphate; HATU, N-[(dimethylamino)-H-1,2,3-triazolo[4,5-
b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophos-
phate; PyAOP, (7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium
hexafluorophosphate; PyBOP, (benzotriazol-1-yloxy)trispyrrolidino-
phosphonium hexafluorophosphate; TAPipU, O-(7-azabenzotriazol-1-
yl)-1,1,3,3-pentamethylenuronium tetrafluoroborate; TBPipU, O-(ben-
zotriazol-1-yl)-1,1,3,3-pentamethylenuronium tetrafluoroborate; TBTU,
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA,
trifluoracetic acid; TFE, trifluoroethanol; TOPPipU, 2-(2-oxopyrid-1-
yl)-1,1,3,3-pentamethylenuronium tetrafluoroborate.
Resu lts a n d Discu ssion
Initially, a number of HOAt-based onium salts were
examined for cyclization of pentapeptides 1 and 2, and
the results compared with data from more conventional
coupling reagents. The HOAt-derived coupling reagents
stood out as being the most effective by far, causing
conversion of 1 or 2 to the corresponding cyclopeptides
within 10 min in yields of 53-55% (Figure 2, Tables 1
and 2). The monocyclic structures of the isolated peptides
were confirmed by ES-MS and NMR 2D sequential cross
peaks.
(
8) Schmidt, R.; Neubert, K. Int. J . Peptide Protein Res. 1991, 37,
02.
9) Heavner, G. A.; Audhya, T.; Doyle, D.; Tjoeng, F. S.; Goldstein,
G. Int. J . Peptide Protein Res. 1991, 37,198.
10) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahe-
dron Lett. 1989, 30, 1927.
11) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, C. Tetrahedron Lett.
975, 14, 1219.
12) (a) Felix, A. M.; Wang, Ch.-T.; Heimer E. P.; Fournier, A. Int.
5
Investigation of the chiral integrity of the cyclic
products by means of GC-MS using a Chirasil-L-Val
column revealed that cyclization was not accompanied
by significant inversion of configuration of the activated
(
(
(
2
,3
1
tyrosine residue. In contrast to the DCC/DMAP case,
(
generally less than 10% of the D-Tyr isomer was detected
in the crude products (Tables 1 and 2). In the case of
all-L-pentapeptides 1 and 6 the less active reagents
TBTU and BOP induce significantly higher degrees of
epimerization (Tables 1 and 2; Figures 3 and 4). The
HAPyU-mediated cyclization proceeds extremely rapidly,
with about 90% of the cyclization products being formed
within 1 min after addition of the coupling reagent.
Similarly rapid cyclizations were observed with PyAOP
and HATU. Since the onium moieties in TBTU and
PyBOP on the one hand, and those of HATU and PyAOP
on the other, are identical, the differences in reactivity
of the two series points again18 to the favorable influence
J . Peptide Protein Res. 1988, 31, 231-238. (b) Zimmer, S.; Hoffmann,
E.; J ung, G.; Kessler, H. In Peptides 1992. Proceedings of the 22nd
European Peptide Symposium; Schneider, C. H., Eberle A. N., Eds.;
ESCOM: Leiden, 1993; p 393.
(13) Benoiton, N. L.; Lee, Y. C.; Steinauer, R.; Chen, F. M. F. Int. J .
2
0
Peptide Protein Res. 1992, 40, 559.
1
5
(
14) Recently, X-ray analysis has shown that HATU crystallizes
as the guanidinium salt shown in Figure 1 rather than as of the
isomeric O-substituted form i previously assigned to this compound.
HBTU ii was also shown to crystallize in guanidinium rather than
the uronium form. For analogous coupling reagents (HAPyU, TAPipU,
TBPipU, TBTU) for which X-ray data have not yet been obtained the
traditional structural representations and nomenclature have arbi-
trarily been retained in the present communication. To date there is
no evidence concerning the possibility of equilibration in solution
between the O- and N-forms of these compounds.
2
2
of the OAt residue on the cyclization process. On the
(20) The cyclization of various penta- and hexapeptides via HBTU
was recently described (ref 21) as yielding 2-21% of the cyclopeptides
after reaction times of 2-16 h. The stereochemical integrity of the
activated amino acid residues was not discussed. Considering our
results with TBTU (Figures 5 and 6; Table 1), it would be expected
that considerable C-terminal epimerization would also occur in the case
of HBTU.
(
15) Abdelmoty, I.; Albericio, F.; Carpino, L. A.; Foxman, B. M.;
Kates, S. A. Lett. Pept. Sci. 1994, 1, 57.
16) (a) Azev, Y.; Mokrushina, G. A.; Postovoskii, I. Ya.; Sheinker,
(21) Sowemimo, V.; Scanlon, D.; J ones, P.; Craik, D. J . J . Protein
Chem. 1994, 13, 339.
(
Yu. N.; Anisimova, O. S. Chem. Heterocycl. Compd. 1976, 1172. (b)
Mokrushina, G. A.; Azev, Y.; Postovoskii, I. Ya. Chem. Heterocycl.
Compd. 1975, 880. (c) Azev, Y.; Mokrushina, G. A.; Postovoskii, I. Ya.
Chem. Heterocycl. Compd. 1974, 687. (d) Sacher, R. M.; Alt, G. H.;
Darlington, W. A. J . Agric. Food Chem. 1973, 21, 132.
(22) It is expected that the use of HOAt-derived coupling reagents
would also improve the cyclization of resin-bound peptides. In fact,
HATU was found to give better results than BOP in the final head-
to-tail cyclization of a resin-bound pentapeptide library constructed
in four variable positions from glycine, proline, arginine, and three
2
3
(17) (a) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397. (b)
nonspecified amino acids. Similarly, in side-chain to side-chain
cyclizations HAPyU and HATU gave far better yields of the desired
cyclic peptides than HBTU. However, in contrast to peptide cyclization
in solution as described in the present communication, HATU-,
HAPyU-, and HBTU-mediated cyclizations of resin-bound peptides are
reported to be accompanied by significant amounts of alkylguanidinium
peptides formed by reaction of the uronium reagent with the free amino
Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J . Chem. Soc.,
Chem. Commun. 1994, 201.
(
18) Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Car-
pino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781.
19) Henklein, P.; Beyermann, M.; Bienert, M.; Knorr, R. In Peptides
990, Proceedings of the 21st European Peptide Symposium; Giralt,
E., Andreu, D., Eds.; ESCOM: Leiden, 1991; p 67.
(
1
2
4
group.

Cyclization of all-L-Pentapeptides
J . Org. Chem., Vol. 61, No. 25, 1996 8833
Ta ble 1. Cycliza tion of H-Ar g(NO2)-Lys(Z)-Asp (OBn )-Va l-Tyr -OH, 1. In flu en ce of Cou p lin g Rea gen ts, Ba se, a n d P ep tid e
Con cen tr a tion on (a ) F or m a tion of Cyclom on om er vs Cyclod im er a n d (b) Ep im er iza tion of th e Cyclom on om er
C-Ter m in a l Tyr osin e Resid u e As Deter m in ed a fter a Rea ction Tim e of 1 h by GC-MS An a lysis
cyclomonomera
%)
D-Tyr-isomer
(%)
cyclodimera
(%)
concn of linear
peptide (mM)
(
reagent
base
2
5
8.0a
8.8
nd
nd
8.7
40
25
2
nd
16
HAPyU
HAPyU
HAPyU
HAPyU
HAPyU
3 equiv of DIEA
3 equiv of DIEA
3 equiv of DIEA
3 equiv of coll.
1 equiv of DIEA
10
1
0.1
1
5
8
1
6
5
2
3
0
b
1
2
equiv of coll.
5
5
5
3
5
6
3
0
8
2
4
10.9
8.3
15.1
20.2
13.0
nd
20
22
22
13
17
nd
PyAOP
HATU
TBTU
BOP
PyBOP
DPPA
3 equiv of DIEA
3 equiv of DIEA
3 equiv of DIEA
3 equiv of DIEA
3 equiv of DIEA
2 equiv of DIEA
1
1
1
1
1
1
.3c
a
Area percentages as determined by analytical HPLC analysis. b After 24 h 39% cyclomonomer, 22.5% D-Tyr-isomer, and 9.6% cyclodimer
c
were formed. After 24 h 23% cyclomonomer, 19.8% D-Tyr-isomer, and 7% cyclodimer were formed.
Ta ble 2. HAP yU-Med ia ted Cycliza tion of P en ta - a n d Hexa p ep tid es. In flu en ce of Sequ en ce, Sid e-Ch a in P r otection , a n d
Rever sible Ba ck bon e Alk yla tion on (a ) F or m a tion of Cyclom on om er vs Cyclod im er a n d (b) Ep im er iza tion of th e
C-Ter m in a l Tyr osin e Resid u ea
cyclomonomerb
(%)
cyclodimerb
(%)
D-Tyr-isomerb
(%)
concn of linear
peptide (mM)
peptides
2
3
4
5
6
H-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-OH
53
25
43
55
0
25
42
33
17
35
38
56
43
16
nd
11
13
nd
6.2c
7.0
7.0
1
1
1
1
10
1
1
1
0.1
1
1
+
H-Val-Arg(H )-Lys(Ac)-Ala-Val-Tyr-OH
+
H-Arg(H )-Lys(Z)-Asp(OBn)-Val-Tyr-OH
+
H-Arg(H )-Lys(Ac)-Asp(OBn)-Val-Tyr-OH
14.0
7.0
+
H-Arg(H )-Lys(Ac)-Ala-Val-Tyr-OH
3
3
8.4c
nd
+
7
8
H-Arg(H )-Lys(Z)-Ala-Val-Tyr-OH
32
33
+
H-Arg(H )-Lys(Ac)-Asn-Val-Tyr-OH
7.0
7
0
7.0
+
8.3c
nd
9
0
1
2
H-Arg(H )-Lys(Ac)-Ala-Val-D-Tyr-OH
80
76
60
64
+
1
1
1
H-Arg(H )-Lys(Ac)-MeGly-Val-Tyr-OH
+
5.2c
8.6c
H-Arg(H )-Lys(Ac)-MeAla-Val-Tyr-OH
1
1
+
H-Arg(H )-Lys(Ac)-(Hmb)Ala-Val-Tyr-OH
a
All cyclizations were carried out in the presence of 3 equiv of DIEA for 1 h. b Determined by HPLC analysis except where indicated
c
otherwise. Determined by GC-MS.
+
F igu r e 3. Time course for cyclization of H-Arg(NO
2
)-Lys(Z)-
F igu r e 4. Time course for cyclization of H-Arg(H )-Lys(Ac)-
Asp(OBn)-Val-Tyr-OH, 1. Comparison of different coupling
reagents. Data in parentheses refer to C-terminal epimeriza-
tion, as determined by GC-MS: [, HAPyU (8.7%); +, PyAOP
Ala-Val-Tyr-OH, 6. Comparison of different coupling reagents.
Data in parentheses refer to C-terminal epimerization, as
determined by GC-MS: [, HAPyU (8.4%); +, PyAOP (8.7%);
b, HATU (8.6%); 0, TBTU (16.1%); #, PyBOP (16.8%); 9, BOP
(20.7%).
(
10.9%); b, HATU (8.3%); 0, TBTU (15.1%), #, PyBOP (13.0%),
9, BOP (20.2%); O, DPPA (19.8% after 24 h).
intermediate formed prior to the OAt ester is responsible
for the exceptional speed of the reaction and the reduced
loss of chirality remains to be elucidated. Since TBTU
leads to the formation of only 10-20% of the cyclopeptide
within 1 min (Figure 3), the intermediacy of less reactive
species (OBt ester, oxazolone) may account for the
enhanced stereomutation at the C-terminal amino acid
residue.
other hand, for some peptides, e.g., (Figure 4), the
differences among HAPyU, HATU, and PyAOP, with
HAPyU providing a significantly faster cyclization, sug-
gest that other factors are also involved. Whether some
(
23) Darlak, K.; Romanovskis, P.; Spatola, A. F. In Peptides,
Chemistry, Structure and Biology, Proceedings of the 13th American
Peptide Symposium 1993 Hodges, R. S., Smith, J . A., Eds.; ESCOM:
Leiden, 1994; p 981.
The superiority of coupling reagents derived from
HOAt for promoting peptide cyclization quickly and with
a minimum of stereomutation was also demonstrated in
(
24) Story, S. C.; Aldrich, J . V. Int. J . Peptide Protein Res. 1994, 43,
92. Arttamanykul, S.; Leelasvatanakij, L.; Aldrich, J . V. 23rd Eur.
Pept. Symp., Braga, Portugal, 1994, poster P76.
2

8
834 J . Org. Chem., Vol. 61, No. 25, 1996
Ehrlich et al.
the cyclization of an all-L-hexapeptide. Cyclization of 3
with the two HOBt-based reagents BOP and TBPipU25
gave only 5-10% of the desired cyclopeptide, whereas
with the HOAt-based reagents HAPyU and TAPipU a
1
8
moderate yield (25%) was obtained. In addition, the
extensive inversion at the C-terminal tyrosine residue
observed in the case of BOP (24%)18 is significantly
diminished with HAPyU (7%) (Table 2) as demonstrated
by HPLC analysis of the reaction mixture.
In an attempt to suppress further the racemization
observed in the HAPyU-mediated formation of cyclic
pentapeptides, DIEA was replaced by the less basic
tertiary amine collidine, which has been shown to dimin-
ish racemization in both solution- and solid-phase syn-
thesis of peptide segments.26 Unexpectedly, in the
presence of collidine, the HAPyU-mediated cyclization of
peptide 1 was very slow, allowing the activated linear
intermediate to undergo C-terminal inversion thereby
leading to the formation of up to 25% of the D-Tyr-isomer.
On the other hand, use of 1 equiv of DIEA and 2 equiv of
collidine permit efficient cyclization of 1 within 1 h (Table
F igu r e 5. Influence of N-methylamino acid substitution and
reversible backbone alkylation (Hmb) in position 3 on the
+
HAPyU-assisted cyclization of H-Arg(H )-Lys(Ac)-Ala-Val-Tyr-
3
3
3
OH, 6, 1 mM in DMF: [, MeGly ; +, HmbAla ; b, MeAla ; 0,
3
3
5
Ala ; #, Ala , D-Tyr .
Sch em e 1
1
). Although this technique reduced the amount of
cyclodimer formed relative to the case involving the use
of 3 equiv of DIEA, the amount of C-terminal epimer-
ization (about 8%) was not significantly affected.
Cyclization of 1 is accompanied by formation of 20-
2
5% of the cyclodimer when the reaction is performed at
a peptide concentration of 1 mM, whereas dimer forma-
tion is reduced to only 2% (80% cyclomonomer) at 0.1
mM. These results contrast with those obtained in the
HAPyU-assisted head-to-tail cyclizations of GnRH-de-
capeptides (80-90 % cyclomonomer), which proceeded
smoothly without cyclodimer formation even at high
peptide concentration18 (100 and 10 mM).
Although the use of HAPyU allows for the first time
efficient ring closure of thymopentin sequences 1 and 2,
the results presented are not inconsistent with the view
that peptide conformation strongly influences the prob-
ability of cyclization. Initial attempts to cyclize the
human splenin-derived pentapeptide 618 using HAPyU
at a peptide concentration of 10 mM resulted predomi-
nantly in the formation of the corresponding cyclodimer
with only 7% of the monocyclic D-Tyr-isomer being
formed. Even at peptide concentrations of 1 mM the
formation of the monocyclic ring is less effective (33%)
than for the pentapeptides 1 and 2 (>50%). These
differences might be caused at least in part by variations
in the side-chain protecting groups. Structural differ-
ences in the side chains of the arginine, lysine, and
tyrosine residues of peptides 1, 2, 4, and 5 do not
significantly influence either the cyclization rate or the
cyclomonomer/cyclodimer ratio, whereas in the case of
peptides 6-8, which lack a side chain benzyl group at
position 3, the yields of the corresponding cyclomonomers
are strikingly diminished. However, on the basis of the
present results it is not possible to decide whether this
effect is caused by the lack of space-filling protecting
groups or by changes in the amino acid sequence (Asp f
Ala or Asn) (see Figure 2 and Tables 1 and 2). As noted
earlier,2 the incorporation of turn-inducing amino acids
favors the formation of monocyclic pentapeptide rings.
Thus, changing the C-terminal configuration of peptide
(to peptide 9) speeds up the cyclization significantly
Table 2, Figure 5), thus supporting the data of Kessler
6
(
3
,27
et al.
A similar effect was observed for cyclization of
the linear N-methylglycine- or N-methylalanine-contain-
ing peptides 10 and 11 (Table 2), suggesting that revers-
ible amide bond alkylation2
9-31
should be useful in pro-
moting the cyclization of peptides devoid of turn-inducing
amino acid residues. Indeed, for (Hmb)Ala-substituted-
(27) Naturally, the effect of changing the C-terminal amino acid from
the L- to the D-configuration is negligible or at least less pronounced
when, for whatever reasons, the L-isomer itself undergoes rapid
2
8
cyclization. Thus, Feiertag et al. found no influence of the C-terminal
amino acid configuration on the cyclization of hexapeptides RGDSPX,
which gave the cyclized peptides in high yield (80%) even for the all-
L-peptides.
-4
(28) Feiertag, S.; Wiesm u¨ ller, K.-H.; Nicholson, G. J .; J ung, G. In
Peptides: Chemistry, Structure and Biology, Proceedings of the 14th
American Peptide Symposium 1995 Kaumaya, P. T. P., Hodges, R. S.,
Eds.; ESCOM: Leiden, 1996, in press.
(29) Blaakmeer, J .; Tijsse-Klasen, T.; Tesser, G. I. Int. J . Peptide
Protein Res. 1991, 37, 556.
(30) J ohnson, T.; Quibell, M. Tetrahedron Lett. 1994, 35, 463.
(31) Quibell, M.; Turnell, W. G.; J ohnson, T. Tetrahedron Lett. 1994,
35, 2237.
(
25) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Peptides
990, Proceedings of the 21st European Peptide Symposium; Girald,
E., Andrew, D., Eds.; ESCOM: Leiden, 1991; p 62.
26) (a) Carpino, L. A.; El-Faham, A. J . Org. Chem. 1994, 59, 695.
b) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett. 1994,
5, 2279.
1
(
(
3

Cyclization of all-L-Pentapeptides
J . Org. Chem., Vol. 61, No. 25, 1996 8835
Ta ble 3. HP LC-Reten tion Tim es a n d ES-MS Da ta of Lin ea r a n d Cyclized P ep tid es
linear peptide
ES-MS
cyclomonomer
ES-MS
cyclodimer
ES-MS
HPLC tR
HPLC tR
HPLC tR
peptides
calcd
found
(min)
calcd
found
(min)
calcd
found
(min)
1
2
3
4
5
6
7
8
9
949.04
1148.40
776.94
904.00
811.90
677.80
769.90
720.80
677.80
677.80
691.83
813.96
949.5
1148.6
778.0
904.8
812.0
678.7
770.6
721.0
678.7
678.7
692.7
814.8
15.90
25.62
14.12
14.02
25.87
11.27
6.27
931.02
1130.30
758.93
886.00
793.90
659.80
751.90
702.80
659.80
659.80
673.82
795.94
931.5
1130.3
759.8
886.8
794.3
660.6
752.7
703.3
660.6
660.2
674.6
796.8
20.90
30.03
20.98
18.70
32.13
19.78
11.55
16.17
15.18
16.63
19.50
29.45
1862.04
2260.60
1517.86
1772.00
1587.80
1319.60
1503.80
1405.60
1319.60
1319.60
1347.64
1591.88
1863.0
2261.0
1519.5
1772.5
1689.0
1320.4
1505.0
1406.0
nd
27.05
36.70
25.73
24.45
35.30
24.77
18.62
22.68
nd
9.88
13.78
11.42
13.67
23.75
1
1
1
0
1
2
1320.0
1348.5
nd
25.80
23.40
nd
Ta ble 4. Am in o Acid An a lysis of Cyclized P en ta p ep tid es
amino acid analysis
Arg
Lys
Xxx
Val
Tyr
cyclopeptides
found
1
2
3
4
5
6
7
8
9
0
1
2
cyclo[-Arg(NO2)-Lys(Z)-Asp(OBn)-Val-Tyr-]
cyclo[-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-]
0.82
0.94
0.83
0.99
0.98
0.97
0.97
0.96
0.94
0.96
1.02
0.99
0.98
0.98
1.0
0.99
1.0
1.06
1.01
1.04
1.0
1.02
1.04
1.09
1.01
0.99
1.08
1.18
1.01
0.98
0.93
1.21
0.9
1.0
1.0
1.78
1.0
1.0
1.0
1.0
1.0
0.95
1.0
1.0
1.0
0.56
0.67
0.9
+
cyclo[-Val-Arg(H )-Lys(Ac)-Ala-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Z)-Asp(OBn)-Val-Tyr-]
0.42
0.78
0.53
0.34
0.39
0.98
0.45
0.53
0.28
+
cyclo[-Arg(H )-Lys(Ac)-Asp(OBn)-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Ac)-Ala-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Z)-Ala-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Ac)-Asn-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Ac)-Ala-Val-D-Tyr-]
+
1
1
1
cyclo[-Arg(H )-Lys(Ac)-MeGly-Val-Tyr-]
1.01
nd
1.0
+
cyclo[-Arg(H )-Lys(Ac)-MeAla-Val-Tyr-]
+
cyclo[-Arg(H )-Lys(Ac)-(Hmb)Ala-Val-Tyr-]
peptide 12, the synthesis of which is outlined in Scheme
, the yield of cyclomonomer is double that obtained from
the unmodified sequence 6 (Table 1, Figure 5).
Standard methods were used to assemble the tripep-
tide sequence 15 on a chlorotrityl resin using the 2-hy-
droxy-4-methoxybenzyl group 13 attached to the R-amino
residue of alanine. Because of hindrance toward coupling
to a N-substituted amino acid, the next amino acid (Lys)
was added by overnight coupling. The final amino acid
cap A 300 column, 250 × 4 mm. Preparative isolation of
peptides was carried out by HPLC on a 10-µm Polyencap A
1
3
00 250 × 20 mm column. Peptides were eluted using linear
gradients formed as follows: (a) system 1, eluant A, 0.1% TFA
in water, and eluant B , 0.1% TFA in 50% ACN/50% water
v/v), or (b) system 2, eluant A and eluant B , 0.1% TFA in
1
(
2
8
0% ACN/20% water (v/v).
Amino acid analyses were performed following gas-phase
hydrolysis for 20 h at 110 °C. ES-MS was performed by
sample flow 1 µL/min methanol/water (1:1), 3-5 kV, 80 °C.
1
H-NMR spectra (1 D, 2 D Cosy,TOCSY with spin-lock time
(Arg) was added normally. Acetylation to stabilize the
7
5-80 ms and ROESY with mixing time 150 ms) were
Hmb residue during removal of the peptide from the resin
followed by TFA treatment gave protected pentapeptide
recorded at 300 K.
Racemization tests by the GC-MS technique33 were per-
formed on a Chirasil-Val column, 50 m, 0.25 µm. Hydrolysis
1
8 that was then deblocked by piperidine at both Fmoc
and AcHmb groups to give the desired backbone-
protected pentapeptide 12 (Scheme 1). An alternate
method of inducing cyclization-prone conformations by
2
was carried out in 6 N DCl/D O for 24 h (110 °C), and the
hydrolysate was converted to the N-(trifluoracetyl)amino acid
isopropyl ester.
reversible N-acylation of the peptide bond via (Boc)
2
O was
Rea gen ts. The Fmoc amino acid derivatives, 2-chlorotrityl
resin, p-alkoxybenzyl resin, and HOBt-derived coupling re-
agents were purchased from NovaBiochem, Bad Soden/Ts,
Germany. The synthesis of HOAt was carried out by methy-
lation of commercially available 2-nitro-3-pyridinol followed
by treatment of the resulting methyl ether with excess
recently reported.32
In conclusion, it has been demonstrated that the
recently developed HOAt-derived coupling reagents sig-
nificantly improve the head-to-tail cyclization of all-L-
penta- and -hexapeptides devoid of turn-inducing amino
acids. Although highly effective, these reagents could not
fully prevent the formation of cyclodimers or the occur-
rence of some C-terminal epimerization. The probability
of obtaining the cyclomonomer cleanly can be enhanced
by application of reversible backbone alkylation. Similar
methodology may also be helpful in the synthesis of cyclic
peptide libraries.
17a
hydrazine.
HOAt-derived coupling reagents were prepared
1
0
according to the procedure described by Knorr.
Syn th eses of Lin ea r P ep tid e Sequ en ces. (a ) Syn th eses
of Un p r otected Lin ea r P ep tid es. The syntheses of linear
unprotected peptides were carried out manually using a
p-alkoxybenzyl resin (0.6 mmol/g) by means of Fmoc amino
acids and TBPipU18 as activating agent. All amino acids were
coupled using 2 equiv of Fmoc amino acid (0.3 M in DMF), 2.2
equiv of TBPipU, and 4 equiv of DIEA. Coupling times were
3
0 min. Couplings were monitored by the Kaiser ninhydrin
3
4
Exp er im en ta l Section
test. The Fmoc group was cleaved by 20% piperidine/DMF
for 15 min. After completion of the syntheses the resins were
Gen er a l P r oced u r es. Analytical HPLC characterization
of all synthesized peptides was carried out on a 5-µm Polyen-
(
33) Kusomoto,S.; Matsukura, M.; Shiba, T. Biopolymers 1981, 20,
869.
(34) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970 34, 595.
1
(32) Cavelier-Frontin, F.; Achmad, S.; Verducci, J .; J acquier, R. J .
Mol. Struct. 1993, 286, 125.

8
836 J . Org. Chem., Vol. 61, No. 25, 1996
Ehrlich et al.
Ta ble 5. H NMR Sign a l Assign m en ts a n d H -Hr Cou p lin g Con sta n ts (DMSO-d 6, 300 K) of Cyclom on om er s,
1
N
Cyclod im er s, a n d Th eir D-Tyr -Isom er s
HN
Ha
Hb
others
3J HN-Ha
(a) Cyclo[-Arg(NO2)-Lys(Z)-Asp(OBn)-Val-Tyr-], 1a , Cyclo[-Arg(NO2)-Lys(Z)-Asp(OBn)-Val-D-Tyr-], 1b, and
Cyclo[-Arg(NO2)-Lys(Z)-Asp(OBn)-Val-Tyr-Arg(NO2)-Lys(Z)-Asp(OBn)-Val-Tyr-], 1c
1
2
3
ꢀ
1
1
1
a
b
c
Arg
Lys
Asp
Val
Tyr
Z,
8.26
7.95
8.10
8.05
8.04
3.87
4.01
4.41
3.52
4.21
1.76
1.65
2.91
2.08
Hγ 1.44; Hδ 3.13; HN 8.49
8.0
8.3
7.9
7.9
6.0
Hγ 1.20; Hδ 1.38; Hꢀ 2.95; HNú 7.21
4
5
Hγ 0.74/0.61
Hδ 6.97; Hꢀ 6.64; HO 9.17
2.91/2.87
H2C 4.99
H2C 5.07
aromatic 7.25-7.39
}
OBn
1
ꢀ
Arg
8.46
7.17
8.50
7.54
8.85
3.97
4.15
4.11
4.06
4.29
1.77
Hγ 1.44/1.30; Hδ 3.05; HN 8.36
Hγ 1.13; Hδ 1.37; Hꢀ 2.94; HN 7.20
8.1
8.7
7.1
8.9
7.0
2
ú
Lys
3.08/2.94
3.08/2.94
1.70
3
Asp
4
Val
Hγ 0.70/0.65
Hδ 7.00; Hꢀ 6.62; HO 9.12
5
D-Tyr
Z,
OBn
2.73
H2C 4.98
H2C 5.08
aromatic 7.25-7.38
}
1
,6
ꢀ
Arg
Lys
Asp
7.56
7.93
8.49
7.22
8.27
4.23
4.06
4.36
4.14
4.36
1.69
Hγ 1.43; Hδ 3.09; HN 8.50
Hγ 1.24/1.20; Hδ 1.34; Hꢀ 2.94; HN 7.18
2
,7
,8
,9
ú
1.54/1.47
3.01/2.79
1.89
3
4
Val
Tyr
Z,
Hγ 0.72/0.66
Hδ 6.96; Hꢀ 6.59; HO 9.11
5
,10
3.01/2.79
H2C 4.98
aromatic 7.25-7.38
}
OBn
H2C 5.06
(
b) Cyclo[-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-], 2a , Cyclo[-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-D-Tyr(Bn)-], 2b, and
Cyclo[-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-Arg(Tos)-Lys(Z)-Asp(OBn)-Val-Tyr(Bn)-], 2c
1
2
3
ꢀ
2
2
2
a
b
c
Arg
Lys
Asp
Val
Tyr
Tos
Z,
8.25
7.92
8.10
8.03
8.06
3.86
4.02
4.42
3.55
4.26
1.72
1.64
2.91
2.04
Hγ 1.34/1.31; Hδ 3.03; HN 6.80
7.7
8.4
8.0
8.3
8.6
ú
Hγ 1.20; Hδ 1.38; Hꢀ 2.95; HN 7.21
4
5
Hγ 0.73/0.57
Hδ 7.10; Hꢀ 6.90
aromatic 7.62/7.24; H3C 2.29
H2C 4.99
2.97/2.92
OBn,
Bn
H2C 5.07 aromatic 7.26-7.44
}
H2C 5.04
1
ꢀ
Arg
8.43
7.16
8.51
7.55
8.87
3.93
4.15
4.12
4.05
4.32
1.72
Hγ 1.37/1.15; Hδ 2.93; HN 6.73
Hγ 1.13; Hδ 1.37; Hꢀ 2.93; HN 7.20
8.2
8.8
7.1
8.8
7.0
2
ú
Lys
1.59/1.54
3.09/2.94
1.70
3
Asp
4
Val
Hγ 0.70/0.64
Hδ 7.11; Hꢀ 6.86
aromatic 7.62/7.24; H3C 2.30
H2C 4.98
5
D-Tyr
Tos
Z,
OBn,
Bn
2.78
H2C 5.08 aromatic 7.26-7.41
}
H2C 5.01
1
,6
ꢀ
Arg
Lys
Asp
7.46
7.94
8.52
7.15
8.36
4.23
4.00
4.32
4.16
4.50
1.64
Hγ 1.36; Hδ 3.00; HN 6.69
Hγ 1.25/1.19; Hδ 1.34; Hꢀ 2.93; HN 7.18
2
,7
,8
,9
ú
1.53/1.47
3.03/2.79
1.88
3
4
Val
Tyr
Tos
Z,
Hγ 0.72/0.66
Hδ 7.08; Hꢀ 6.82
aromatic 7.62/7.24; H3C 2.29
H2C 4.98
5
,10
2.99/2.70
OBn,
Bn
H2C 5.04 aromatic 7.25-7.41
}
H2C 4.98
(
c) Cyclo[-Val-Arg-Lys(Ac)-Ala-Val-Tyr-], 3a , and Cyclo[-Val-Arg-Lys(Ac)-Ala-Val-D-Tyr-], 3b
3
3
a
Val1
7.21
8.26
8.31
8.05
7.61
7.99
4.24
3.80
3.62
4.29
3.78
4.33
2.16
1.66
1.95
1.26
1.82
3.14/2.69
Hγ 0.89/0.80
6.3
3.7
7.0
8.1
8.9
8.7
2
ꢀ
Arg
Lys
Ala
Val
Hγ 1.52; Hδ 3.14; HN 7.47
3
ú
Hγ 1.22; Hδ 1.37; Hꢀ 3.00; HN 7.82
4
5
Hγ 0.77/0.46
Hδ 7.02; Hꢀ 6.60; HO 9.07
H3C 1.79
6
Tyr
Ac
b
Val1
8.05
7.92
7.83
8.06
7.04
8.33
4.02
4.11
3.81
3.93
4.07
4.46
2.09
1.70
1.65
1.30
1.88
2.83/2.68
Hγ 0.75/0.69
8.9
7.3
5.7
6.3
8.1
6.8
2
ꢀ
Arg
Hγ 1.50/1.43; Hδ 3.09; HN 7.46
3
ú
Lys
Hγ 1.24; Hδ 1.38; Hꢀ 3.01; HN 7.81
4
Ala
5
Val
Hγ 0.71/0.61
Hδ 7.02; Hꢀ 6.60; HO 9.07
H3C 1.78
6
D-Tyr
Ac
(d) Cyclo[-Arg-Lys(Ac)-Ala-Val-Tyr-], 6a , cyclo[-Arg-Lys(Ac)-Ala-Val-D-Tyr-], 6b, and
Cyclo[-Arg-Lys(Ac)-Ala-Val-Tyr-Arg-Lys(Ac)-Ala-Val-Tyr-], 6c
1
ꢀ
6
a
Arg
Lys
Ala
Val
Tyr
Ac
8.13
7.84
7.93
7.75
8.09
4.03
4.07
4.08
3.69
4.20
1.79/1.70
1.74/1.68
1.32
1.92
2.95/2.89
Hγ 1.45/1.38; Hδ 3.09; HN 7.50
8.3
7.5
7.7
9.0
8.6
2
ú
Hγ 1.23; Hδ 1.39; Hꢀ 2.30; HN 7.83
3
4
Hγ 0.76/0.59
Hδ 6.98; Hꢀ 6.65; HO 9.19
H3C 1.78
5

Cyclization of all-L-Pentapeptides
J . Org. Chem., Vol. 61, No. 25, 1996 8837
Ta ble 5 (Con tin u ed )
HN
Ha
Hb
others
3J HN-Ha
(d) Cyclo[-Arg-Lys(Ac)-Ala-Val-Tyr-], 6a , cyclo[-Arg-Lys(Ac)-Ala-Val-D-Tyr-], 6b, and
Cyclo[-Arg-Lys(Ac)-Ala-Val-Tyr-Arg-Lys(Ac)-Ala-Val-Tyr-], 6c *
1
2
ꢀ
6
6
b
c
Arg
Lys
Ala
Val
8.54
7.17
8.25
7.32
8.91
4.01
4.21
3.79
4.06
4.28
1.81/1.42
1.79/1.60
1.37
1.69
2.74
Hγ 1.29; Hδ 3.01; HN 7.43
8.3
9.0
6.5
8.8
6.8
ú
Hγ 1.19; Hδ 1.39; Hꢀ 2.99; HN 7.81
3
4
Hγ 0.72/0.66
5
D-Tyr
Ac
Hδ 7.00; Hꢀ 6.62; HO 9.15
H3C 1.77
1
,6
ꢀ
Arg
Lys
Ala
Val
Tyr
Ac
8.12
7.79
7.60
8.01
8.02
3.96
4.16
4.34
4.10
4.48
1.73/1.64
1.75/1.53
1.16
1.84
3.14/2.74
Hγ 1.47; Hδ 3.11; HN 7.50
2
,7
ú
Hγ 1.20; Hδ 1.33; Hꢀ 2.96; HN 7.81
3
4
5
,8
,9
Hγ 0.76/0.74
,10
Hδ 7.01; Hꢀ 6.58; HO 9.11
H3C 1.77
(
e) Cyclo[-Arg-Lys(Ac)-MeAla-Val-Tyr-], 11a , and Cyclo[-Arg-Lys(Ac)-MeAla-Val-D-Tyr-], 11b
1
2
1
1
1a
1b
Arg
Lys
7.68
8.07
4.06
4.38
3.58
3.85
4.32
1.70/1.38
1.80
1.55
1.83
2.91/2.72
Hγ 0.79; Hδ 3.10; HNꢀ 7.49
8.4
7.7
ú
Hγ 1.32/1.17; Hδ 1.53; Hꢀ 2.97; HN 7.80
3
MeAla
H3C 2.67
Hγ 0.82/0.59
Hδ 6.96; Hꢀ 6.65; HO 9.20
H3C 1.77
4
Val
Tyr
Ac
7.45
7.77
10.0
9.5
5
1
Arg
Lys
8.57
7.20
3.86
4.56
3.54
4.11
4.23
1.77
1.66
1.62
1.70
2.72
Hγ 1.42; Hδ 2.99; HNꢀ 7.39
7.5
9.1
2
ú
Hγ 1.13; Hδ 1.37; Hꢀ 2.96; HN 7.77
3
MeAla
H3C 2.78
Hγ 0.81/0.73
Hδ 6.99; Hꢀ 6.62; HO 9.14
H3C 1.76
4
Val
7.87
8.79
9.8
6.1
5
D-Tyr
Ac
(
f) Cyclo[-Arg-Lys(Ac)-(Hmb)Ala-Val-Tyr-], 12a , and Cyclo[-Arg-Lys(Ac)-(Hmb)Ala-Val-D-Tyr-], 12b
1
2
ꢀ
1
1
2a
2b
Arg
Lys
Ala
Val
Tyr
Ac
8.31
7.87
3.77
4.82
3.47
3.91
4.03
1.87/1.83
1.74/1.49
1.45
1.90
2.98
Hγ1.37; Hδ 3.06; HN 7.44
7.3
9.1
ú
Hγ 1.22; Hδ 1.38; Hꢀ 2.99; HN 7.80
3
4
5
7.70
8.30
Hγ 0.83/0.76
9.1
7.5
Hδ 6.99; Hꢀ 6.65; HO 9.17
H3C 1.78
Hmb
H2C 4.47; H3CO 3.95; HO 9.81; aromatic 6.40, 6.99
1
ꢀ
Arg
8.57
7.26
3.90
4.83
3.43
4.14
4.24
1.75/1.42
1.72
1.48
1.70
2.75
Hγ 1.24; Hδ 2.99; HN 7.39
7.5
9.1
2
ú
Lys
Ala
Val
Hγ 1.14; Hδ 1.41/1.37; Hꢀ 2.97; HN 7.79
3
4
8.12
8.88
Hγ 0.81/0.73
9.7
6.3
5
D-Tyr
Ac
Hδ 7.01; Hꢀ 6.63; HO 9.14
H3C 1.77
Hmb
H2C 4.36; H3CO 3.69; HO 9.82; aromatic 6.41, 6.90
treated with TFA/thioanisol/m-cresol (9:0.5:0.5; v/v/v) for 3 h
to give the crude peptide trifluoracetates that were character-
ized by HPLC (system 1) and ES-MS (Table 3).
residues via TFA/thioanisol/m-cresol (9:0.5:0.5; v/v/v) for 3 h
yielded the N-terminally Fmoc- and AcHmb-protected linear
peptide 18. Deprotection of the Fmoc group and the acetyl
residue was achieved by treatment with 20% piperidine/DMF
for 15 min. The crude linear Hmb-protected sequence was
(
b) Syn th eses of P r otected Lin ea r Sequ en ces. The
peptides were obtained via manual Fmoc solid phase synthesis
on a trityl resin using the side chain-protected amino acid
purified by preparative HPLC using mobile phase A and B
1
.
derivatives Tyr(Bn), Arg(Tos), Lys(Z), Lys(Ac), and Arg(NO
2
).
A linear gradient was formed from 1% B to 70% B over 70
1
1
Fmoc amino acid derivatives (2 equiv) were coupled in DMF
for 30 min following activation with 2 equiv of TBPipU and 4
equiv of DIEA. Couplings were monitored by the Kaiser
ninhydrin test. Deprotection of the Fmoc group was achieved
by treatment with 20% piperidine/DMF for 15 min. Cleavage
from the resin was carried out with AcOH/DCM/TFE (2:6:2;
v/v/v) for 1 h to give side chain-protected linear peptides, which
were used for cyclization studies without further purification.
The crude peptides were characterized by HPLC (system 2)
and ES-MS (Table 3). All peptides were found to be suf-
ficiently pure (>85% according to HPLC analysis) for use in
min with a flow rate of 7 mL/min. Pure fractions were pooled
and lyophilized, yielding 130 mg (26%) of pure backbone-
protected peptide 12.
Cycliza tion Exp er im en ts. (a ) Cycliza tion w ith Ur o-
n iu m a n d P h osp h on iu m Rea gen ts. The linear pentapep-
tides (0.1 mmol) were cyclized by treatment with the corre-
sponding coupling reagent (0.11 mmol) and 0.3 mmol of a
tertiary amine (see Tables 1 and 2) in DMF. The cyclization
reaction was monitored via analytical HPLC as described
below using system 1 for unprotected sequences and system 2
for side chain protected peptides. After 1 h the reaction
mixture was evaporated to dryness and the resulting residue
dissolved in methanol and precipitated with ethyl acetate/
ether. The crude peptides were examined for the desired
cyclopeptides and any byproducts by HPLC analysis. Ap-
propriate HPLC fractions were collected and examined by ES-
MS to distinguish and identify the cyclomonomers and cy-
clodimers (Table 3). The corresponding D-Tyr-containing
diastereoisomers were synthesized and used as standards in
the HPLC analysis of crude cyclization products.
cyclization studies without further purification.
+
(
c) Syn th esis of H-Ar g(H )-Lys(Ac)-(Hm b)Ala -Va l-Tyr -
OH, 12. This sequence was obtained via the manual Fmoc
strategy on the trityl resin using the following amino acid de-
rivatives: Arg(Pmc), Lys(Ac), Tyr(tBu), and (Hmb)Ala31 (see
Scheme 1). All Fmoc amino acids (2 equiv, 0.3 M in DMF)
were coupled using 2 equiv of TBPipU and 4 equiv of DIEA
for 30 min except in the case of Fmoc-Lys(Ac)-OH, which was
coupled via EDCI/HOBt in DCM overnight. The Fmoc-group
was cleaved by 20% piperidine/DMF for 15 min. After the last
coupling step the acid-labile Hmb group was temporarily
stabilized through acetylation of the 2-hydroxy moiety.30
Cleavage from the resin and deprotection of Pmc and tBu
(b) Cycliza tion w ith DP P A. The crude linear pentapep-
tide 1 (0.1mmol) was dissolved in DMF, the solution adjusted
to pH 8 with 0.2 mmol of DIEA, and 0.2 mmol DPPA added at
room temperature. The course of the reaction was monitored

8
838 J . Org. Chem., Vol. 61, No. 25, 1996
Ehrlich et al.
via analytical HPLC as described below using system 2. After
4 h solvent was evaporated and the residue dissolved in
methanol and precipitated with ethyl acetate/ether. The crude
peptides were examined by HPLC and ES-MS.
30% B
2
to 70% B
2
over 90 min with a flow rate of 10 mL/min
2
for side chain protected peptides. Fractions were collected at
0.5 min intervals (monitoring by analytical HPLC) pooled and
lyophilized, resulting in 15-30% yields of the purified peptides
which were analyzed by ES-MS, amino acid analysis (Tables
(
c) HP LC Mon itor in g. To examine the time course of the
1
cyclization reactions an aliquot (500 µL) of the reaction mixture
was withdrawn at definite time intervals. Reaction was
stopped by dilution with 500 µL of 0.1% TFA, and 50 µL of
the resulting solution was injected onto the reversed-phase
HPLC column with separation being performed by linear
gradient elution using system 1 or 2. The cyclization yields
were determined by integration of the appropriate peak areas.
3 and 4), and H-NMR analysis (Table 5a-f).
Ack n ow led gm en t. We are grateful for financial
support from the Deutsche Forschungsgemeinschaft. We
thank H. Hans for technical assistance, B. Pisarz for
performing amino acid analysis, and D. Runald for ES-
MS data. Access for one of us (R.W.) to high-field NMR
facilities in the laboratory of Prof. H. Kessler, Institute
of Organic Chemistry, Technical University Munich, is
gratefully acknowledged.
(
d ) Isola tion of Cyclom on om er s a n d Cyclod im er s by
P r ep a r a tive HP LC. The crude cyclopeptides (100 mg) were
purified by preparative HPLC using system 1 for unprotected
peptides and system 2 for protected peptides. A linear
gradient was formed from 1% B
to 80% B over 70 min with
1 1
a flow rate of 7 mL/min for unprotected sequences and from
J O951108D