Pseudo-prolines as a solubilizing, structure-disrupting protection technique in peptide synthesis

Article abstract of DOI:10.1021/ja961509q

Serine-, threonine-, and cysteine-derived cyclic building blocks (pseudo-prolines, ΨPro) serve as reversible protecting groups for Ser, Thr, and Cys and prove to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry.

Full text of DOI:10.1021/ja961509q

9218
J. Am. Chem. Soc. 1996, 118, 9218-9227
Pseudo-Prolines as a Solubilizing, Structure-Disrupting
Protection Technique in Peptide Synthesis
Torsten Wo1hr, Franck Wahl, Adel Nefzi, Barbara Rohwedder, Tatsunori Sato,
Xicheng Sun, and Manfred Mutter*
Contribution from the Institute of Organic Chemistry, UniVersity of Lausanne,
BCH-Dorigny, CH-1015 Lausanne, Switzerland
ReceiVed May 6, 1996X
Abstract: Serine-, threonine-, and cysteine-derived cyclic building blocks (pseudo-prolines, ΨPro) serve as reversible
protecting groups for Ser, Thr, and Cys and prove to be versatile tools for overcoming some intrinsic problems in
the field of peptide chemistry. The presence of ΨPro within a peptide sequence results in the disruption of â-sheet
structures considered as a source of intermolecular aggregation during chain elongation, thus increasing solvation
and coupling kinetics in peptide assembly. Due to their easy synthetic access and variability in the chemical stability
by modifications introduced in the C-2 position of the oxazolidine/thiazolidine ring system, this protection technique
is adaptable to all common strategies in peptide synthesis. We describe new types of ΨPro building blocks suitable
for standard Fmoc/tBu-based solid phase peptide synthesis, convergent strategies, and chemoselective ligation
techniques as well as their use as a structure-disrupting, solubilizing protection technique for the example of peptides
generally considered as “difficult sequences”.
Introduction
chaotropic salts6 or solubilizing protecting groups7 which have
been shown to have variable efficiencies. Hydrogen-bonded
association has also been prevented by the introduction of an
amide protecting group within the peptide chain.8
Since the advent of solid phase peptide synthesis (SPPS),1 a
multitude of synthetic peptides have been produced using this
approach. Subsequently, many efforts have been devoted to
improving specific protecting groups, support systems, activation
methods, and the automation of protocols. However, successful
peptide assembly is still hampered by inherent problems such
as poor solvation of the growing peptide chain during solid phase
synthesis as well as limited solubility of fully protected peptide
fragments in the solution approach, often leading to incomplete
coupling steps.2 These undesirable physicochemical problems
originate from intermolecular hydrophobic aggregation of the
protected peptide chains and/or the formation of secondary
structures, most notably of â-sheets.3 Reported attempts to
suppress the degenerative effect of such associations during
aminoacylation reactions involve essentially “external factors”
like solvent composition,4 elevated temperature,5 and use of
Recently, we reported that Ser/Thr-derived oxazolidine and
Cys-derived thiazolidine derivatives (pseudo-prolines, ΨPro)
exert a pronounced effect upon backbone conformation due to
their structural similarity with proline itself (Scheme 1).9 Due
to the induction of a “kink” conformation in the peptide
backbone, originating in the preference for cis amide bond
formation, ΨPro prevent peptide aggregation, self-association,
and â-structure formation, thus improving the solvation and
coupling kinetics of the growing peptide chain considerably.
These new building blocks are readily accessible by cyclization
of Ser, Thr, or Cys with aldehydes or ketones (Scheme 1) and
serve as a reversible protecting group in peptide synthesis. As
a particular feature, variation of the C-2 substituents directly
affects the ring stability, thus allowing for differential chemical
stabilities in a variety of synthetic strategies.
Consequently, pseudo-prolines represent a valuable tool for
combining the protection of Ser, Thr, and Cys residues with
the simultaneous solubilization of the peptide chain. After
having established some basic structural features of ΨPro and
their potential in accessing difficult sequences, we elaborate here
on the synthesis and chemical stability of ΨPro derivatives
specifically designed for standard SPPS and for convergent
strategies. In particular, their incorporation as dipeptide building
X Abstract published in AdVance ACS Abstracts, September 15, 1996.
(1) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-54. (b)
Merrifield, R. B. Science 1986, 232, 341-7.
(2) (a) Mutter, M. Angew. Chem., Int. Ed. Engl. 1985, 24, 639-53. (b)
Kent, S. B. H. Annu. ReV. Biochem. 1988, 57, 959-89. (c) Kent, S. B. H.
In Peptides: Structure and Function, Proc. Am. Pept. Symp. 9th; Deber,
C. M., Hruby, V. J., Kopple, K. D., Eds.; Pierce Chemicals: Rockford,
1985; pp 407-414.
(3) (a) Mutter, M.; Altmann, K. H.; Bellof, D.; Floersheimer, A.; Herbert,
J.; Huber, B.; Klein, B.; Strauch, L.; Vorherr, T.; Gremlich, H. U. In
Peptides: Structure and Function, Proc. Am. Pept. Symp. 9th; Deber, C.
M., Hruby, V. J., Kopple, K. D., Eds.; Pierce Chemicals: Rockford, 1985;
pp 397-405. (b) Mutter, M.; Pillai, V. N. R.; Anzinger, H.; Bayer, E.;
Toniolo, C. In Peptides. Proc. Eur. Pept. Symp. 16th; Brunfeldt, K., Ed.;
Scriptor: Copenhagen, 1981; pp 660-665. (c) Pillai, V.; Mutter, M. Acc.
Chem. Res. 1981, 14, 122-30. (d) Narita, M.; Chen, J. Y.; Sato, H.; Lim,
Y., Bull. Chem. Soc. Jpn. 1985, 58, 2494-2501.
(4) (a) Zhang, L.; Goldhammer, C.; Henkel, B.; Zu¨hl, F.; Panhaus, G.;
Jung, G.; Bayer, E. In InnoVation and PerspectiVes in Solid Phase Synthesis,
Proc. Int. Conf. 3rd; Epton, R., Ed.; Mayflower Worldwide Limited:
Birmingham, England, 1994; pp 711-716. (b) Milton, S. C. F.; De L.
Milton, R. C. Int. J. Peptide Protein Res. 1990, 36, 193-96. (c) Yamashiro,
D.; Blake, J.; Li, C. H. Tetrahedron Lett. 1976, 18, 1469-72. (d) Ogunjobi,
O.; Ramage, R. Biochem. Soc. Trans. 1990, 18, 1322-1333.
(5) Lloyd, D. H.; Petrie, G. M.; Noble, R. L.; Tam, J. P. In Peptides:
Proc. Am. Pept. Symp. 11th; Rivier, J. E., Marshall, G. R., Eds.; Escom:
Leiden, Netherlands, 1990; pp 909-910.
(6) (a) Seebach, D.; Thaler, A.; Back, A. K. HelV. Chim. Acta 1989, 72,
857-867. (b) Thaler, A.; Seebach, D.; Cardinaux, F. HelV. Chim. Acta 1991,
74, 628-43. (c) Pugh, K. C.; York, E. H.; Steward, J. M. Int. J. Peptide
Protein Res. 1992, 40, 208-213.
(7) Mutter, M.; Oppliger, H.; Zier, A. Makromol. Chem., Rapid Commun.
1992, 13, 151-157.
(8) (a) Johnson, T.; Quibell, M.; Owen, D.; Sheppard, R. C. J. Chem.
Soc., Chem. Commun. 1993, 11, 369-372. (b) Narita, M.; Fukunaga, T.;
Wakabayashi, A; Ishikawa, K. Int. J. Peptide Protein Res. 1984, 23, 306-
14. (c) Blaakmeer, J.; Tijsse-Klasen, T.; Tesser, G. I. Int. J. Peptide Protein
Res. 1991, 37, 556-564.
(9) (a) Haack, T.; Mutter, M. Tetrahedron Lett. 1992, 33, 1589-1592.
(b) Wo¨hr, T.; Mutter, M. Tetrahedron Lett. 1995, 36, 3847-3848. (c)
Mutter, M.; Nefzi, A.; Sato, T.; Sun, X.; Wahl, F.; Wo¨hr, T. Peptide Res.
1995, 8, 145-153.
S0002-7863(96)01509-0 CCC: $12.00 © 1996 American Chemical Society

ΨPro as a Protection Technique in Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9219
Scheme 1. Pseudo-Proline Unit and Overview of the ΨPro
using the C-2-unsubstituted ΨPro as a temporary protection
technique, these building blocks may serve as proline mimics.
In contrast, by introducing C-2 substituents as in compound 1
or 2, ring opening can be achieved under moderately strong
acidic conditions. The kinetics of the ring opening turns out to
be strongly dependent on the acid/solvent system.
Building Block Synthesis According to Routes A and Ba
For example, the acidolysis of Fmoc-Ala-Thr(ΨMe,Mepro)-
NHBzl in methanol at room temperature was complete within
minutes in a 70% TFA/methanol solution, whereas a 10%
decrease in acid concentration resulted in an increase of the
deprotection time by a factor >10 (Figure 1a). The same
observations were made for the Ser and Cys analogues. The
solvent dependency of the ring-opening reaction was investi-
gated in methanol, THF, and DCM with the model dipeptide
Fmoc-Ala-Ser(ΨMe,Mepro)-NHBzl (Figure 1c). DCM was found
to be the appropriate solvent for rapid deprotection, since a 5%
TFA/DCM solution led to quantitative ring opening within
minutes. In 50% TFA/THF and 60% TFA/methanol the
cleavage time increased to 90 min and 24 h, respectively.
Alternatively, TFA can be replaced by Lewis acids such as boron
trifluoride which was shown to catalyze the deprotection reaction
of Fmoc-Ala-Thr(ΨMe,Mepro)-NHBzl even more efficiently
(Figure 1b). Whereas the various ΨPro derivatives are ef-
fectively cleaved by acids, they proved to be stable toward the
conditions normally used for NR-Fmoc deprotection (e.g., 20%
piperidine in DMF).
The convenient preparation of oxazolidine- or thiazolidine-
containing dipeptides allows for tailoring the chemical stability
according to the strategy used for peptide synthesis. Hence,
the ΨPro building blocks 2, 4, and 5 (Table 1) are compatible
with standard Fmoc/tBu protection (cleavable with 90% TFA
within minutes) whereas the ΨPro ring systems 1, 6, and 7 are
stable under tBu/Boc cleavage conditions; here ring opening
needs strong acidic conditions and proceeds within hours or
days, depending on the sequence and chain length of the peptide
(see below).
The incorporation of ΨPro residues into a growing peptide
chain in SPPS proceeds preferentially Via their preformed
dipeptide derivatives of the type Fmoc-Xxx-Xaa(ΨR′,R′′pro)-OH
(see Table 1). These commercially available12 ΨPro-containing
building blocks can be coupled without racemization (in analogy
to Pro) according to standard procedures (see below). The
synthesis of C-2-unsubstituted Ser- and Thr-derived oxazolidine
dipeptides is readily accomplished by in situ N-acylation with
NR-Fmoc-protected amino acid fluorides or N-carboxyanhy-
drides (route B, Scheme 1), whereas the 2,2-dimethyloxazolidine
building blocks 4 and 5 from Table 1 were obtained by direct
insertion of oxazolidine in the suitably protected preformed
dipeptides9b (route B, Scheme 1). In contrast, thiazolidine
building blocks 1, 2, and 3 could readily be obtained by direct
N-acylation of the stable 1,3-thiazolidine-4-carboxylic acids with
NR-Fmoc-protected amino acid fluorides or N-carboxyanhy-
drides (route A, Scheme 1) with yields ranging from 60 to 95%.
It is noteworthy that the condensation of L-cysteine with alde-
a
L-serine (X ) O, R ) H) and L-threonine (X ) O, R ) methyl)
derived 4-oxazolidinecarboxylic acid (Ser(ΨR′,R′′pro); Thr(ΨR′,R′′pro));
L-cysteine (X ) S, R ) H) derived 4-thiazolidinecarboxylic acid
(Cys(ΨR′,R′′pro)); R′ and/or R′′ ) alkyl, aryl.
Table 1. Overall Yields of Synthesis Calculated from Ser, Thr, or
Cys and Cleavage Conditions of Prototype ΨPro Building Blocks
Compatible with Fmoc-Peptide Synthesis Strategiesa
overall
yield (%)
prototype ΨPro building blocks
cleavage conditions
1, Fmoc-Xxx-Cys(ΨMe,Mepro)-OHb
2, Fmoc-Xxx-Cys(ΨH,Rpro)-OHb,c
3, Fmoc-Xxx-Cys(ΨH,Hpro)-OH
4, Fmoc-Xxx-Ser(ΨMe,Mepro)-OH
5, Fmoc-Xxx-Thr(ΨMe,Mepro)-OH
6, Fmoc-Xxx-Ser(ΨH,Hpro)-OH
7, Fmoc-Xxx-Thr(ΨH,Hpro)-OH
60-80
85-95
85-95
70-90
70-90
60-80
60-80
TFA within hours
TFA within minutes
stable to strong acids
TFA within minutes
TFA within minutes
TFMSA within hours
TFMSA within hours
a ΨPro building blocks 1, 2, 3, 6, and 7 synthesized according to
route A, 4 and 5 according to route B (Scheme 1). b Structural formula;
see Scheme 1. c R ) 2,4-dimethoxyphenyl.
blocks into peptide backbones and their differential cleavage
in connection with commonly applied protecting groups in
peptide synthesis are exemplified for various structurally and
functionally important peptides.
On the basis of the results obtained in these studies, ΨPro
are established as a convenient side-chain protection technique
for Ser, Thr, and Cys adaptable to most common strategies for
peptide synthesis, opening new prospects for the chemical
preparation of large peptides and proteins.
Results
Pseudo-prolines. The pseudo-proline (ΨPro)-containing
dipeptides presented in Table 1 emerged as the most suitable
building blocks compatible with the Fmoc stepwise synthetic
approach as well as for the segment assembly approach. Their
chemical stability toward acid, which predetermines the depro-
tection (ring-opening) conditions, largely depends on the nature
of the C-2 substituents, due to the strong relationship between
electronic effects of the substituents and ring stability: Whereas
dilute TFA10 was sufficient to open the ring in the 2,2-
dimethyloxazolidine-dipeptide 4, strong acids like TFMSA10
were required to deprotect the exceptionally stable unsubstituted
oxazolidine as in dipeptide 6. Furthermore, it has been estimated
that the relative stability of the thiazolidine ring is more than
104 times higher than that of the corresponding oxazolidine.11
This was confirmed by attempts to cleave the thiazolidine ring
system, as in 3, with strong acids. Consequently, rather than
(10) The following abbreviations were used: ATR, attenuated total
reflection; Bum, tert-butoxymethyl; CD, circular dichroism; DIC, N,N′-
diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; EDTA, eth-
ylenediaminetetraacetic acid; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt,
1-hydroxybenzotriazole; MALDI, matrix-assisted laser desorption time of
flight; NCA, N-carboxyanhydride; Pfp, pentafluorophenyl; ΨPro, pseudo-
proline; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluo-
rophosphate; RP-HPLC, reversed phase high-performance liquid chroma-
tography; Rt, retention time; TASP, template-assembled synthetic protein;
tBu, tert-butyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TFMSA,
trifluoromethanesulfonic acid; Trt, trityl; Xaa, Xxx, amino acid.
(11) (a) Fu¨lo¨p, F.; Pihalaja, K. Tetrahedron Lett. 1993, 49, 6701-6706.
(b) Fu¨lo¨p, F.; Mattinen, J.; Pihlaja, K. Tetrahedron 1990, 46. 6545-6552.
(12) Fmoc-ΨPro building blocks are now readily available from a
commercial source (Calbiochem-Novabiochem) for routine use in peptide
synthesis.

9220 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Wo¨hr et al.
Figure 1. Kinetics of the deprotection of Fmoc-Ala-Thr(ΨMe,Mepro)-NHBzl in solution (9.2 µmol/5 mL) at room temperature as monitored by RP-
HPLC: (a) effect of increasing TFA concentration on ΨPro cleavage (deprotection); O in 50% TFA/MeOH, shaded circle, in 60% TFA/MeOH,
b, in 70% TFA/MeOH; (b) comparison of TFA- and BF3-mediated deprotection; O, Fmoc-Ala-Thr(ΨMe,Mepro)-NHBzl in 5% BF3‚C4H10O/MeOH;
4, Fmoc-Ala-Thr(ΨMe,Mepro)-NHBzl in 50% TFA/MeOH; (c) influence of solvent choice on deprotection; 0, in 5% TFA/DCM, shaded box, in
50% TFA/THF, 9, in 60% TFA/MeOH.
hydes or unsymmetrical ketones generates a mixture of C-2 epi-
mers (2S,4R) and (2R,4R) as in ΨPro building blocks 2. Al-
though the diastereoisomeric mixture could be separated by
RP-HPLC or crystallization, this was generally not undertaken
since acidic deprotection results in the loss of the C-2 chiral
center.
Oxazolidine- or thiazolidine-containing dipeptides present a
much greater polar character than conventionally protected Ser,
Thr, or Cys derivatives, eluting at retention times similar to those
of the parent free Fmoc amino acids on RP-HPLC columns but
earlier, for example, than tBu- or trityl-protected Fmoc-amino
acids. This finding can be rationalized by the formation of
hydrogen bonds to the solvent-exposed ring heteroatom O or S
similar to cyclic ethers. Consequently, ΨPro act as a polar
protecting technique contributing to higher solvation of protected
peptides.
of time at room temperature. In addition, ΨPro dipeptides are
generally isolated as crystalline compounds and prove to be
readily soluble in solvents used in peptide synthesis.
As well as the influence upon ring stability, the ΨPro C-2
substituents exert strong influence upon the conformation of
the Xxx-Xaa(ΨPro) peptide bond. NMR studies followed by
NOE experiments revealed that ΨPro dipeptides existed in two
distinctive rotameric forms around the tertiary amide bond
commonly assigned as cis and trans isomers.13 However,
differences were observed for C-2-disubstituted ΨPro as for
example Ac-Ala-Ser(ΨMe,Mepro)-NH-Me (data not shown). The
absence of two sets of signals at room temperature indicated a
stable conformer with a high activation barrier for the isomer-
ization about the ΨPro peptide bond. By irradiating the CRi-
proton and the NHi proton specifically, a NOE upon the CR(i+1)
-
proton was observed, thus indicating a preference for the cis
All the ΨPro derivatives investigated showed remarkable
thermodynamic stability. They were readily characterized using
CI-MS and proved to be stable when stored over a long period
(13) (a) Haack, T. Ph.D. Thesis, University of Lausanne, 1994. (b) Dumy,
P.; Keller, M.; Ryan, D; Rohwedder, B.; Wo¨hr, T.; Mutter, M. J. Am. Chem.
Soc., submitted for publication.

ΨPro as a Protection Technique in Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9221
Figure 3. Peptides containing ΨPro building blocks for studying the
solubilizing and conformational effects during SPPS and in subsequent
segment assembly steps.
ΨPro residues in the fully deprotected peptide. Finally, by
concomitant cleavage of the acid labile protecting groups Y (e.g.,
tBu, Trt), the resin anchor (e.g., Wang, Sasrin, Rink), and the
ΨPro ring system, a fully deprotected peptide can be obtained
in one single step according to route V, Figure 2.
The strategies outlined in Figure 2 offer a large choice for
accessing the beneficial properties of ΨPro building blocks in
peptide synthesis. First, when used in conventional SPPS as a
temporary protection technique for Ser, Thr, and Cys, the
stepwise elongation of the peptide chain is strongly facilitated
by the pronounced solubilizing effect of ΨPro building blocks.
Cleavage of the ΨPro-containing, fully protected peptides
(strategy I) allows the further condensation of hydrophobic
peptides to larger polypeptides, thus increasing the potential of
convergent strategies for the synthesis of proteins. Finally,
differential cleavage of the side chain protecting groups (in
preserving the ΨPro system) gives access to water soluble
peptide fragments as required in chemoselective ligation tech-
niques, preventing the problem of self-association and aggrega-
tion due to the secondary structure-disrupting effect of ΨPro.
These various applications of ΨPro are exemplified by SPPS
of some representative peptides, as described below.
ΨPro as a Solubilizing Protection Technique in Conver-
gent Syntheses. (1) Bis-Amphiphilic Secondary Structure
Forming Peptides (Switch Peptides). The potentially second-
ary structure forming peptide 8c was designed according to the
general principles of switch peptides22 and included a Cys,
protected as a ΨPro derivative at position 7 (Figure 3) for
probing the effects of ΨPro.
The synthesis of peptide 8a (Figure 3) was performed on a
Fmoc-Ala-Sasrin resin using Fmoc chemistry and allyl protect-
ing groups for Lys and Glu. A 4-fold orthogonality was used
for preserving the potential of differential cleavage in 8a. The
incorporation of the ΨPro residue proceeded smoothly Via the
Fmoc-Ala-Cys(ΨMe,Mepro)-OH dipeptide and DIC/HOBt activa-
tion; the fully protected peptide 8a was obtained in high yield
after RP-HPLC purification. In contrast to the corresponding
switch peptide devoid of a ΨPro residue (Cys(ΨMe,Mepro)
replaced by Ala22), peptide 8a proved to be soluble in polar
organic solvents like methanol, DMF, or water/acetonitrile
mixtures reflecting the ΨPro-induced conformational effects.
Peptide 8b was obtained after selective cleavage of the allyl
protecting groups with a Pd0/tributyltin hydride treatment20 and
subsequent RP-HPLC purification. Finally, the fully deprotected
peptide 8c was obtained by cleavage of the 2,2-dimethylthi-
Figure 2. Strategic use of ΨPro building blocks in peptide chemistry
according to standard Fmoc SPPS and convergent approaches using
fully protected peptides or with ΨPro-containing deprotected peptides.
Synthetic pathways I-V are described in the text. Abbreviations: Nt,
Ct, N-terminal and C-terminal functional groups; Y, conventional
protecting group; Xaa(Ψpro), Cys, Ser, or Thr protected as pseudo-
proline; Xxx, any amino acid.
conformation. Subsequent molecular dynamics calculations as
well as X-ray diffraction analysis supported these findings.14
As a major goal of this study, general strategies for using
ΨPro building blocks (Table 1) in peptide synthesis have been
elaborated. When applied to segment condensation (synthetic
route I, Figure 2), ΨPro have the potential to simplify the
purification of peptide fragment intermediates and to enhance
the segment coupling kinetics.15 Here, the synthesis of the fully
protected peptide is achieved by selective cleavage of the
protected peptide fragment from super acid labile resins, for
example, the 2-Cl-Trt16 resin. Indeed, under these mild cleavage
conditions (20% acetic acid), the C-2-substituted ΨPro blocks
(e.g., 2,2-dimethyloxazolidine-4-carboxylic acid or 2-(2,4-
dimethoxyphenyl)thiazolidine-4-carboxylic acid residues) remain
stable. Similarly, ΨPro building blocks 2, 4, or 5 (Table 1)
can be used with the Sasrin17 resin if the cleavage conditions
(1% TFA) are carefully controlled.
In some cases (e.g., in chemoselective ligation techniques)18
it is desirable to preserve the ΨPro ring system intact while
the other amino acid side chains are deprotected (strategy IV
in Figure 2). In these circumstances, orthogonal side chain
protecting groups Y to ΨPro, for instance, the benzylic,19
allylic,20 or Dde21 protecting groups are applied. Alternatively,
the use of the TFA stable proline isosteric oxazolidine or
thiazolidine building blocks allows the preservation of intact
(14) Nefzi, A.; Schenk, K.; Mutter, M. Protein Peptide Lett. 1994, 1,
66-69.
(15) Haack, T.; Nefzi, A.; Zier, A.; Mutter, M. In Peptides, Proc. Eur.
Pept. Symp. 22th; Schneider, C. H., Eberle, A. N., Eds.; ESCOM: Leiden,
1992; pp 595-596.
(16) Barlos, K.; Gatos, D.; Kallitsis, J.; Pappaphotiou, G.; Wenquing,
Y.; Schafer, W. Tetrahedron Lett. 1989, 30, 3943.
(17) Mergler, M.; Nyfeler, R.; Gosteli, J.; Grogg, P. In Peptides:
Chemistry and Biology, Proc. Am. Pept. Symp. 10th; Marschall, G. R., Eds.;
1988; pp 259-60.
(18) (a) Dawson, P. E.; Kent, S. B. H. J. Am. Chem. Soc. 1993, 115,
7263. (b) Vilaseca, L. A.; Rose, K.; Werlen, R.; Meunier, A.; Offord, C.
L.; Nichols, C. L.; Scott, W. L. Bioconjugate Chem. 1993, 4, 515. (c) Shao,
J.; Tam, J. P. J. Am. Chem. Soc. 1995, 117, 3893.
(20) (a) Loffet, A.; Zhang, H. X. Int. J. Peptide Protein Res. 1993, 42,
346-351. (b) Waldmann, H.; Kunz, H. Liebigs Ann. Chem. 1983, 1712-
1725.
(21) Bycroft, B. W.; Chan, W. C.; Chhabr, S. R.; Hone, N. D. J. Chem.
Soc., Chem. Commun. 1993, 778.
(22) Mutter, M.; Gassmann, R.; Buttkus, U.; Altmann, K. H. Angew.
Chem., Int. Ed. Engl. 1991, 30, 1514-1516.
(19) Hartung, W. H.; Simonoff, R. Org. React. 1953, VII, 263-326.

9222 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Wo¨hr et al.
Figure 4. CD spectra of peptide 8b (pH 4, 11) and peptide 8c (pH 4,
11) (c ) 0.25 mM).
azolidine ring in TFA for 4 h. The intrinsic capability of peptide
8c to undergo medium dependent conformational transitions22
was demonstrated by CD spectroscopy, as shown in Figure 4.
Peptide 8c adopts a â-structure conformation at pH 4, and shows
a transition to a partially helical conformation at pH 11. It is
significant to note that the replacement of Ala-7 with a Cys
residue had no marked influence on the conformational proper-
ties of the peptide. However, the insertion of a single ΨPro
residue within the peptide sequence as in 8b results in a
complete disruption of secondary structures (Figure 4) paralleled
by increased solvation of the ΨPro-containing peptide.
The solubilizing power of ΨPro in peptide 8a was further
exemplified in the convergent synthesis of a TASP (template-
assembled synthetic protein)23 molecule. In this case, the fully
protected peptide fragments 8a were ligated to a topological
template including the â-turn mimetics AMTA (8-(amino-
methyl)-5,6,7,8-tetrahydro-2-naphthoic acid)24 Via amide bond
formation to the lysine side chains. Due to the solubilizing
effect of the ΨPro, peptide 8a was readily soluble in a minimum
volume of DMF, thus allowing the condensation reaction
(PyBOP-mediated activation) to proceed to completion after 3
h as confirmed by analytical RP-HPLC and ES-MS.
(2) Transmembrane Peptides. The synthesis of a strongly
hydrophobic sequence such as the transmembrane peptide 9c
(Figure 3) is generally classified as a “difficult sequence”,
essentially due to aggregation and â-sheet formation of the
growing peptide chain resulting in low solvation and poor
coupling efficiency. In order to circumvent this problem, we
introduced a ΨPro moiety at position 14, as a structure-
disrupting, solubilizing protection technique for Thr. The
transmembrane peptide was synthesized on a Rink amide resin
following standard Fmoc chemistry.25 The incorporation of the
pseudo-proline residue was effected Via the dipeptide Fmoc-
Ala-Thr(ΨH,Hpro)-OH (2.7 equiv) mediated by HOBt/DIC
activation. Aib units were introduced as dipeptides Fmoc-Leu-
Aib-OH in a similar fashion. Most significantly, the coupling
of the ΨPro-containing dipeptide derivative as well as the
following coupling steps proceeded smoothly to quantitative
yields according to UV measurement of Fmoc release. After
resin cleavage by TFA/H2O (95/5) the ΨPro-containing peptide
9a was obtained in high yield (86%) as shown by analytical
HPLC of the crude product (Figure 5a). It is noteworthy that
the HPLC conditions had to be adapted to the hydrophobic
character of the peptide. The peptide products were best eluted
with a linear gradient of 90% 2-propanol/10% water (0.09%
TFA) (10-60%, 30 min) in 90% CH3CN/10% water (0.09%
Figure 5. (a) RP-HPLC spectrum of the crude transmembrane peptide
9a after resin cleavage. Column nucleosil 300-5C4 (solvent A, 2-pro-
panol, 90%/water, 10% (0.09% TFA); solvent B, acetonitrile, 90%/
water, 10% (0.09% TFA)); 1 mL/min. (b) ATR-FT-IR of 9a attached
to the Rink amide resin. (c) CD spectra of peptides 9a and 9b in TFE
(c ) 1 mg/mL).
TFA) with a C4 reversed phase column. The secondary
structure-disrupting effect of the ΨPro residue was confirmed
by the absence of â-sheet formation as seen from the ATR-IR
spectrum of the resin bound sequence (Figure 5b). Furthermore,
the ΨPro-containing peptide 9a was readily soluble in a large
number of organic solvents such as methanol, 2-propanol, DMF,
DCM, and chloroform, thereby facilitating the purification
protocol.
Selective cleavage of the oxazolidine ring system at position
14 was effected by treatment with strong acid (10% TFMSA
in TFA) at room temperature for 48 h, producing unprotected
peptide 9c. Peptide 9c showed a significantly lower solubility
in organic solvents and in water/TFE mixtures compared to the
precursor peptide 9a. The CD spectra of transmembrane
peptides 9c and its ΨPro-containing precursor 9a clearly reveal
the helix-disrupting effect of pseudo-prolines: The observed
helicity of the [ΨPro-14]-peptide 9a increased significantly by
transforming the Thr(ΨPro) building block to a regular Thr by
ring opening (Figure 5c).
The solubilizing properties of ΨPro were further demon-
strated for the convergent synthesis of an integral membrane
TASP molecule23,26 by chemoselective ligation. Peptide 9b
was found to be readily soluble in DMF, thus allowing the
condensation reaction with template 1024 to proceed homoge-
neously. Thioether formation proceeded with 5 equiv of peptide
9b at high dilution in DMF in the presence of DIEA (Figure
6). The condensation reaction was complete after 10 h as
followed by analytical RP-HPLC. The transmembrane TASP
protein 11 was finally obtained in 68% yield after RP-HPLC
purification and proved to be soluble in most organic solvents.
ΨPro as Temporary Protection for Cys-Containing Pep-
tides. The synthesis of [ΨPro-7]Sarafotoxin-S6b, a bicyclic
21 amino acid peptide isolated from the venom glands of the
Israeli burrowing asp Atractaspis engaddensis27 was used for
(23) (a) Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989,
28, 535-554. (b) Tuchscherer, G.; Mutter, M. J. Peptide Sci. 1994, 1, 3-10.
(c) Eggleston, I.; Mutter, M. Macromol. Symp. 1996, 101, 397-404.
(24) Ernest, I.; Kalvoda, J.; Sigel, C.; Rihs, G.; Fritz, H.; Blommers, M.
J. J.; Raschdorf, F.; Francotte, E.; Mutter, M. HelV. Chim. Acta 1993, 76,
1539-1563.
(25) (a) Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990,
35, 161-214. (b) Sheppard, R. C. Chem. Br. 1983, 402. (c) Chang, C. D.;
Meienhofer, J. Int. J. Peptide Protein Res. 1978, 11, 246-249.
(26) Sato, T.; Wo¨hr, T.; Wahl, F.; Rohwedder, B.; Mutter, M. In
Peptides: Chemistry and Biology, Proc. Am. Pept. Symp. 14th, in press.
(27) Takasaki, C.; Tamiya, N.; Bdolah, A.; Wolberg, Z.; Kochva, E.
Toxicon 1988, 26, 543-548.

ΨPro as a Protection Technique in Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9223
(Figure 8c). The folded peptides were isolated by RP-HPLC,
lyophilized, and analyzed by analytical HPLC. ESI-MS analysis
of the bicyclic peptides confirmed the integrity of the target
molecule.
Discussion
In this paper we have presented ΨPro building blocks as a
tool for modulation of the conformational and physicochemical
properties of peptides. ΨPro primarily offer a convenient
temporary protection technique for Ser, Thr, or Cys combined
with several attractive inherent features useful for the design
and synthesis of peptides. Most notably, ΨPro act as strongly
solubilizing building blocks during peptide synthesis and in
convergent strategies for the synthesis of large peptides.
ΨPro consist of oxazolidine- and thiazolidine-4-carboxylic
acid ring systems which can readily be prepared from Thr, Ser,
or Cys by cyclocondensation with aldehydes or ketones.29 The
cyclic structure is in equilibrium with the open chain imine form
governed by the ring-chain tautomeric equilibrium constant of
oxazolidines and thiazolidines. Furthermore, the presence of a
heteroatom in position 1 of the ring system reduces the
nucleophilic character of the nitrogen atom in position 3,
resulting in poor acylation yields of a N-terminal ΨPro peptide.
Consequently, rather than applying ΨPro in their monomeric
form, they are introduced in a peptide preferably as preformed
dipeptides of the type shown in Table 1. ΨPro building blocks
have been developed for Fmoc synthetic strategies; however,
they offer a degree of adaptability to other strategies as well,
since varying the C-2 substituents directly affects the acid lability
of the ring system. Besides their easy access, ΨPro building
blocks are efficiently incorporated into solid phase peptide
assembly according to conventional procedures such as HOBt
activation, thus making their use as a routine technique (e.g.,
for automatic synthesis) very attractive. When applied in
convergent strategies, ΨPro offer the additional advantage of
increasing the choice of segmentation sites of the target
sequence. Indeed, the coupling of C-terminal ΨPro-protected
peptide fragments proceeds without racemization as for C-
terminal proline or glycine fragments. Furthermore, it was
demonstrated that ΨPro greatly expand our ability to synthesize
so far inaccessible polypeptides in solubilizing hydrophobic
protected segments and in preventing self-association of peptide
fragments in convergent strategies or in chemoselective ligation
approaches. The synthesis of a bis-amphiphilic peptide (switch
peptide), designed for the investigation of the structural and
dynamic factors involved in secondary and tertiary structure
formation of peptides and proteins,22 provided an ideal model
for studying the influence of ΨPro upon secondary structure
formation and self-association. Here, the solid phase assembly
of the medium-sized fully protected peptide 8a proceeded more
efficiently and to higher purity compared to its analogue devoid
of a ΨPro building block.22 Owing to the solubilizing effect
of the incorporated ΨPro residue, the fully protected fragment
8b could be efficiently coupled in solution to a topological
template, resulting in a four-bundle TASP molecule of M >
8000.30 This representative example shows that the general
problem of low solvation in segment assembly strategies can
be overcome with the use of the ΨPro protection technique.
Due to the structural and functional importance of membrane-
spanning peptides, much attention has been devoted to the
Figure 6. Synthesis of a four R-helix transmembrane TASP molecule
(TASP T4-(4R20)) by chemoselective ligation via thioether formation.
Template 10 was prepared as described previously.24
probing ΨPro as a temporary Cys protection technique in SPPS.
In the strategy employed, Cys-3, -11, and -15 were protected
as TFA labile 2,2-dimethylthiazolidines during the course of
chain assembly, thereby preventing peptide aggregation on the
solid support and allowing simultaneous deprotection of the thiol
side chains during resin cleavage. Additionally, a TFA stable
ΨPro residue (2,2-dihydrooxazolidine-4-carboxylic acid) was
placed at the strategic position 7 for inducing backbone
conformations (e.g., turns, kinks) favorable for the correct
disulfide formation (isomer A, Figure 7).
The synthesis of Sarafotoxin-S6b (SRTX-b) was carried out
according to Fmoc-PyBOP stepwise solid phase chemistry
starting with a Fmoc-Trp(Boc)-functionalized Wang type resin.
The amino acid side chain protections were chosen as follows:
tBu (Asp, Tyr, Ser, Glu), Boc (Lys), Bum (His), Trt (Gln, Cys-
1). Thr-7, Cys-3, -11, and -15 were introduced as dipeptides
in their pseudo-proline form. The progress of the assembly was
monitored by UV detection of the dibenzofulvene adduct from
the Fmoc deprotection step. The step-by-step synthesis pro-
ceeded smoothly since no coupling reaction needed to be
repeated following the ninhydrin test. ATR-IR of resin samples
revealed an absorption band at 1641 cm-1 (amide I band)
throughout the build-up, consistent with the absence of â-sheet
formation and pointing to the benefit of ΨPro in preventing
peptide aggregation. Cleavage and deprotection of the peptide
resin was effected by treatment with TFA/ethanedithiol/thio-
anisole/water/phenol (82.5/2.5/5/5/5) for 2 h under argon at room
temperature. After filtration and washing with TFA and DCM,
the crude product was precipitated with cold tert-butyl methyl
ether. A further treatment for 32 h in TFA/water (95/5) was
required to cleave the 2,2-dimethylthiazolidine ring completely.
Surprisingly, the oxazolidine ring in position 7 was found to
be stable when treated with the standard method for ring opening
(10% TFMSA/90% TFA) compared to a corresponding dipep-
tide model. The preservation of a ΨPro system at position 7
turned out to be advantageous since the insolubility problems
often encountered in SRTX or Endothelin28 intermediates were
eliminated, providing simple and efficient purification steps. The
crude product was purified by preparative RP-HPLC to give
the pure [ΨPro-7]-tetrathiol peptide in a very satisfactory overall
yield of 18%, based on the resin loading (Figure 8b). The
reduced peptide was subjected to air oxidation for 3 h under
high dilution. As expected, the peptide with the correct disulfide
bridge (isomer A) eluted earlier by RP-HPLC. Whereas an
isomer ratio of 3/1 of the correct form A over B was obtained,
a small peak characterized as Met(O)-peptide was also observed
(29) (a) Kallen, R. G. J. Am. Chem. Soc. 1971, 17, 6236-6246. (b)
Schmolka, I. R.; Spoerri, P. E. J. Org. Chem. 1957, 22, 943-946. (c)
Rasshofer, W. In Houben-Weyls Methoden der Organischen Chemie;
Hagemann, H., Klamann, D., Eds.; Georg Thieme Verlag: Stuttgart, New
York, 1991; Band E 14a/2, pp 595-679. (d) Wolfe, S.; Militello, G.; Ferrari,
C.; Hasan, S. K.; Lee, S. L. Tetrahedron Lett. 1979, 41, 3913-3916.
(30) The conformational properties of this switch TASP molecule will
be the subject of another publication.
(28) (a) Akaji, K.; Nishiuchi, H.; Kiso, Y. Tetrahedron Lett. 1995, 36,
1875-1878. (b) Wade, J. D.; McDonald, M. R.; Treggear, G. W. In
InnoVation and PerspectiVes in Solid Phase Synthesis, Proc. Int. Conf. 1st;
Epton, R., Ed.; SPCC UK Limited: Kingswinford, England, 1990; pp 585-
591.

9224 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Wo¨hr et al.
Figure 7. Solid phase synthesis of [Ψpro-7]Sarafotoxin-S6b with Cys-3, -11, and -15 protected as ΨPro followed by resin cleavage and random
air oxidation of the Cys side chains, giving the three possible disulfide isomers: s, 1-15, 3-11, A, - - -, 1-11, 3-15, B; - - -, 1-3, 11-15,
C.
studies directed toward the construction of supramolecular
assembly systems using TASP molecules,23c the ΨPro concept
appeared to be an ideal tool for overcoming these intrinsic
problems in the synthesis of fragments derived from integral
membrane proteins.32 For this, we modified a hydrophobic
transmembrane segment reported recently32 by incorporating a
ΨPro residue in a central position of the peptide 9a. As well
as the expected solubilizing effect, the incorporation of ΨPro
as a proline mimetic provided an elegant tool for probing the
structural and functional role of a Pro residue in a transmem-
brane helical peptide33 (induction of a “kink”, Figure 6).
R-Aminoisobutyric acid (Aib) residues at positions 6, 9, and
16 were incorporated in order to enhance the helicity of this
segment. As before, the solid phase assembly of this strongly
hydrophobic sequence proceeded in single coupling steps to
completion. In particular, the introduction of the ΨPro dipeptide
was achieved according to standard protocols of automated
SPPS; the target peptide 9c containing the ΨPro building block
and a N-terminal Cys was readily purified and coupled to a
topological template Via thioether formation. The presence of
a single ΨPro residue prevented this hydrophobic peptide from
intermolecular aggregation, thus providing excellent solubility
Figure 8. Analytical RP-HPLC of [ΨPro-7]SRTX-b: (a) crude, (b)
purified tetrathiol, (c) after air oxidation, (d) after HPLC purification
(conditions: Nucleosil C18 (250 × 4 mm) column; gradient of 10-
60% B in 30 min (solven A, 0.09% TFA in water; solvent B,
acetonitrile/water/TFA (90/10/0.09)); λ ) 214 nm).
in organic solvents for chemoselective thioether formation. The
resulting TASP molecule will be the subject of detailed
biofunctional investigations. The structure and biological
function of Sarafotoxin-S6b (SRTX-b), Figure 7, are closely
related to those of the mammalian family of vasoconstrictor
design, synthesis, and biostructural characterization of this
particular class of peptides. Due to their hydrophobic nature,
the chemical synthesis and purification protocols prove to be
extremely difficult. For example, transmembrane peptides of
the type (Val)n-(Ser)m could not be prepared according to
standard protocols of SPPS.31 As part of our on-going structural
(31) (a) Altmann, K. H.; Floersheimer, A.; Mutter, M. Int. J. Peptide
Protein Res. 1986, 27, 314-319. (b) Mutter, M. Angew. Chem., Int. Ed.
Engl. 1985, 24, 639-653.
(32) Whitley, P.; Nilsson, I.; Heijne, G. V. Struct. Biol. 1994, 1, 858-
862.
(33) Hall, J. E.; Vodyanov, I.; Balasubramanian, T. M.; Marshall, G. R.
Biophys. J. 1984, 45, 233-247.

ΨPro as a Protection Technique in Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9225
peptides endothelins (ET-1, ET-2, ET-3),28 and SRTX-b was
chosen as a model for the use of Cys-derived ΨPro residues
for improving the SPPS of bioactive peptides of larger size. So
far, there has been no consensus as to the three-dimensional
structure of SRTX-b, but it is suggested to be potentially
identical to ET-1.28 The secondary structure would therefore
be characterized by an R-helix spanning from Lys-9 to Cys-15
and an extended strand of Cys-1 to Cys-3. In the natural form
both structures are stabilized by disulfide bonds pairing Cys-
1-15 and Cys-3-11, joining the two segments in antiparallel
alignments. These two segments are linked by the â-turn-
forming peptide Lys-4-Asp-Met-Thr-7 moiety. Although three
disulfide isomers are possible (isomer C, 1-3, 11-15; isomer
B, 1-11, 3-15; isomer A, 1-15, 3-11) as shown in Figure 7,
only the A and B types are formed during random oxidation of
the tetrathiol product. It has been shown for ET-1 that the
exchange of amino acids may result in analogues, producing
an altered isomer distribution during thiol oxidation. We
therefore introduced a Thr-derived ΨPro of orthogonal chemical
stability at position 7 for inducing backbone conformations
promoting the correct disulfide formation. However, the
observed isomer ratio (A/B ) 3/1) was identical to the reported
ratio for the oxidation of reduced sarafotoxin,34 indicating that
the conformational restriction induced by the ΨPro residue at
position 7 did not significantly affect disulfide bridge formation.
The stepwise synthesis of SRTX-b including ΨPro dipeptide
derivatives as temporary protection for Cys-3, -11, and -15
proceeded smoothly and in high yields due to the solubilizing,
â-sheet-disrupting effect of ΨPro. Most notably, the strategy
applied here adapts perfectly to standard Fmoc/tBu protocols
and provides a general scheme for the SPPS of Cys-containing
peptides. Obviously, by incorporating Cys-derived ΨPro build-
ing blocks of differential chemical stability, the ΨPro concept
enlarges the potential for selective disulfide formation in
bioactive peptides.
The above examples clearly demonstrate the crucial role that
ΨPro may play in the stepwise synthesis of Thr-, Ser-, or Cys-
containing peptides. Moreover, ΨPro have been essential in
solubilizing protected segments in convergent strategies and
in increasing the solvation of unprotected fragments for
chemoselective ligation techniques. From the results obtained
in this study, the following general features of the ΨPro concept
in peptide synthesis can be derived: (1) ΨPro represent a
convenient, cheap, and easily accessible temporary protection
technique for Ser, Thr, and Cys of comparable chemical stability
as tBu or Trt side chain protecting groups.12 (2) ΨPro building
blocks are compatible with common strategies for peptide
synthesis. (3) ΨPro are introduced in peptides as N-protected
dipeptides following the same protocols as conventional Fmoc-
amino acids (e.g., HOBt/DIC) without racemization, essentially
because of their C-terminal proline-like structure. (4) The
coupling of dipeptides or fragments containing C-terminal ΨPro
residues proceeds without racemization, offering a new tool
in the convergent synthesis of peptide fragments exhibiting
C-terminal Ser, Thr, or Cys. (5) ΨPro disrupt secondary
structures (most notably â-sheets) and increase the solvation
of the growing peptide chain during solid phase assembly. This
solubilizing effect may be preserved in fragment condensation
or chemoselective ligation procedures by selective cleavage
protocols.
(e.g., by using multifunctional aldehydes or ketones) opens new
prospects in peptide modification and targeting and in the de
noVo design of proteins.
Experimental Section
Materials and Methods. All protected amino acids were purchased
from Calbiochem-Novabiochem AG (La¨ufelfingen, CH) except for NR-
Fmoc-Xaa-NCA (Propeptide, Vert-le-Petit, F) and NR-Fmoc-Xaa-Pfp
(Bachem, Bu¨bendorf, CH). Reagents and solvents were purchased from
Fluka (Buchs, CH) and used without further purification. HPLC was
performed on Waters equipment using columns packed with Vydac
Nucleosil 300 Å 5 µm C18 particles unless otherwise stated. The
analytical column (250 × 4.6 mm) was operated at 1 mL/min and the
preparative column (250 × 21 mm) at 18 mL/min, monitoring at 214
nm. Solvent A consisted of 0.09% TFA and solvent B of 0.09% TFA
in 90% acetonitrile unless otherwise stated. TLC was performed on
silica gel plates, Merck 60 F254, detection by UV and 5% vanillin in
concentrated sulfuric acid. Flash chromatography was performed with
Merck silica gel 60 (40-63 mesh). Amino acid analyses were
performed after 24 h of hydrolysis in 4 N methanesulfonic acid on a
Perkin-Elmer HPLC. Mass spectra were obtained by laser desorption
ionization (LDI-MS) with an LDI-1700 mass monitor, by electron spray
ionization (ESI-MS) on a Finnigan MAT SSQ 710C, or by chemical
ionization (CI-MS) with a Nermag R10-10C. Circular dichroism (CD)
spectra were recorded at room temperature on a Jobin Yvon CD Mark
V spectropolarimeter using quartz cells of 0.1 mm path length. The
instrument was calibrated with D-10 camphorsulfonic acid. 1H-NMR
spectra were obtained on a Bruker-WH250 FT or Bruker DPX-400
with trimethylsilane as the internal standard. Infrared spectra were
recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer using
KBr pills. Melting points are uncorrected values measured with a Bu¨chi
510 melting point apparatus.
Peptide Synthesis. All the peptides described hereby were syn-
thesized on a semiautomatic Advanced ChemTech ACT 200 peptide
synthesizer using standard Fmoc solid-phase peptide synthetic proto-
cols.25 The resins were purchased from Calbiochem-Novabiochem AG
(La¨ufelfingen, CH) with the C-terminal NR-Fmoc-amino acid already
attached to the anchor group. Before starting a peptide synthesis, the
resins were swollen in dichloromethane (DCM) for about 30 min.
Commercial N,N-dimethylformamide (DMF) was degassed for several
hours with nitrogen. The peptide chains were then assembled by
sequential couplings of preactivated NR-Fmoc-amino acid (3 equiv)
for 45-60 min at room temperature. The preactivation was carried
out manually in DMF in the presence of N,N′-diisopropylcarbodiimide
(DIC) (3 equiv), N-hydroxybenzotriazole (HOBt) (3 equiv), and N,N-
diisopropylethylamine (3 equiv) (DIEA) for 15 min at room temper-
ature. The completeness of each coupling was verified by the Kaiser
test. A positive Kaiser test was usually followed by an additional
coupling cycle and if unsatisfactory the remaining free amino groups
were blocked (capping step) by acylation with acetic anhydride (15
equiv) and pyridine (15 equiv) in DMF for 15 min. The NR-Fmoc
deprotection was carried out by treatment with piperidine (20% v/v in
DMF) (1 × 3 min and 1 × 15 min) and assessed by UV analysis of
the effluents. All the wash steps were performed with the automated
protocols of the peptide synthesizer. The protected peptides were
cleaved from the resin with trifluoroacetic acid (TFA) and appropriate
scavengers as indicated, concentrated, and precipitated with diethyl
ether. The crude products were purified by preparative reversed phase
HPLC to purities greater than 95% homogeneity.
General Procedure for the Preparation of ΨPro Building Blocks.
Fmoc-Xxx-Cys(ΨMe,Mepro)-OH (1), Fmoc-Xxx-Cys(ΨH,Rpro)-OH
(2), and Fmoc-Xxx-Cys(ΨH,Hpro)-OH (3). 1,3-Thiazolidine-4-car-
boxylic acid, 2,2-Dimethyl-1,3-thiazolidine-4-carboxylic acid, and
2-(2,4-Dimethoxyphenyl)-1,3-thiazolidine-4-carboxylic acid were readily
prepared according to published procedures.35 The appropriate thia-
zolidine derivative (10 mmol) was dissolved in dry DMF (100 mL) in
the presence of DIEA (19 mmol). A solution of Fmoc-Xxx-NCA or
Besides these practical applications, ΨPro as reversible
mimetics of Pro provide an interesting source for structure-
function studies in peptide and protein chemistry. Moreover,
the introduction of reactive sites into the ring system of ΨPro
(34) Kumagaye, S. I.; Kuroda, H.; Nakajima, K.; Watanabe, T. X.;
Kimura, T.; Masaki, T.; Sakakibara, S. Int. J. Peptide Protein Res. 1988,
32, 519-526.
(35) (a) Lewis, N. J.; Inloes, R. L.; Hes, J. J. Med. Chem. 1978, 21,
1070-1073. (b) Confalone, P. N.; Pizzolato, G.; Baggiolini, E. G.; Lollar,
D.; Uskokovic, M. R. J. Am. Chem. Soc. 1977, 99, 7020-7026.

9226 J. Am. Chem. Soc., Vol. 118, No. 39, 1996
Wo¨hr et al.
Fmoc-Xxx-F36 (9 mmol) in dry DMF (100 mL) was added dropwise
to the reaction mixture, and the mixture was stirred for 2 h at room
temperature. The solution was then evaporated and the residue taken
up in ethyl acetate, washed with aqueous 5% citric acid and brine, and
dried over sodium sulfate. The solvents were evaporated to dryness,
and the residue was purified by flash chromatography over silica gel,
eluting with petroleum ether/ethyl acetate/acetic acid to give a white
powder in 60-95% yield.
by successive treatment (5 min) with a TFA solution (1% v/v in DCM),
and the filtrates were neutralized with one equimolar amount of
pyridine. The combined filtrates were precipitated with diethyl ether,
centrifuged, and washed three times with diethyl ether. The crude
product (990 mg, 0.51 mmol, 72%) was purified by preparative RP-
HPLC with a gradient of 10-40% solvent B over 30 min. After
lyophilization the purity of the peptide (95%) was confirmed by
analytical HPLC with the same gradient at a retention time (Rt) of 23.2
min. Using a RP-C4 column with an identical gradient, the Rt was
33.5 min. The desired pure peptide was obtained as a white powder
(728 mg, 65%). IR (cm-1, KBr): 3293, 1652 (amide I), 1547 (amide
II). LDI-MS: m/z ) 1916 (M+), 1939 (M + Na), 1955 (M + K).
ASA: Glu, 1.76 (2), Ala, 6.43 (7), Leu, 4.27 (4), Lys, 2.25 (2).
Peptide 8b was obtained from 8a after deprotection of the allyl side
chain protecting groups according to the literature.20 Protected peptide
8a (20 mg, 0.01 mmol) was dissolved in dimethyl sulfoxide (DMSO)
(20 mL) and the solution treated with bis(triphenylphosphine)palladium-
(II) dichloride (0.58 mg, 0.08 equiv), tributyltin hydride (2.7 mL of a
1% v/v solution in DCM, 10 equiv), and acetic acid (0.68 mL of a 1%
v/v solution, 12 equiv). Allyl deprotection was complete after 30 min
of reaction time. Partially deprotected peptide 8b was isolated by
preparative HPLC to give a white powder (14 mg, 8.4 µmol, 81%).
The purity was confirmed by analytical HPLC, with a gradient 20-
80% solvent B over 40 min; the Rt was 21 min. LDI-MS: m/z )
1664.4 (M+).
Fmoc-Xxx-Ser(ΨMe,Mepro)-OH (4) and Fmoc-Xxx-Thr(ΨMe,Mepro)-
OH (5). L-Serine and L-threonine (30 mmol), respectively, were
dissolved in a minimal volume of aqueous sodium carbonate (10% w/v)
at pH 9, and the solution was added to a suspension of Fmoc-Xxx-
OPfp (10 mmol) in acetone (80 mL). After completion of the reaction
according to HPLC, the reaction mixture was acidified with aqueous
HCl (5% w/v) to pH ∼1 in an ice bath. The solution was concentrated
in vacuo to half the initial volume and the product extracted with ethyl
acetate (2 × 150 mL). The organic solution was washed with water
(100 mL) and brine (2 × 100 mL), dried over MgSO4, and finally
evaporated to dryness. The residue was recrystallized from ethyl
acetate/hexane to give pure Fmoc-Xxx-Ser-OH or Fmoc-Xxx-Thr-OH
as a white powder.
The dipeptide (5.0 mmol) was then suspended in dry THF (100 mL).
Pyridyl toluene-4-sulfonate (250 mg, 1.0 mmol) and 2,2-dimethoxy-
propane (3.0 mL, 25.0 mmol) were added. The suspension was
subsequently heated to reflux for several hours under an argon
atmosphere, the condensate being bypassed over molecular sieves (4
Å). After cooling, the yellow solution was added with triethylamine
(0.21 mL, 1.5 mmol) and evaporated to dryness. The residue was taken
up in ethyl acetate (150 mL), washed with water (3 × 70 mL), dried
over MgSO4, and evaporated to dryness. The foamy residue was
purified by flash chromatography over silica gel or crystallized from
ethyl acetate/hexane to give 4 or 5 as a white powder in 70-90% yield.
Fmoc-Xxx-Ser(ΨH,Hpro)-OH (6) and Fmoc-Xxx-Thr(ΨH,Hpro)-
OH (7). L-Serine and L-threonine (10.3 mmol), respectively, were
dissolved in aqueous Na2CO3 (32 mL, 2.5 M). Then an aqueous
solution of formaldehyde (19.3 mL, 260 mmol, 37%) was added
dropwise under vigorous stirring and the resulting solution stored
overnight at 4 °C, after which time the pH was 8. In order to perform
the acylation, a solution of NR-Fmoc-Xxx-F36 or NR-Fmoc-Xxx-NCA
(12.8 mmol, 1 M) in acetone was added dropwise over a period of 70
min while the pH was adjusted between 8 and 9 with sodium carbonate
(∼1 g). The reaction mixture was stirred for another 30 min at room
temperature and then cooled to 0 °C. The solution was acidified with
concentrated hydrochloric acid (16 mL). The white suspension at pH
3-4 was extracted with ethyl acetate (4 × 50 mL). The organic phases
were combined and washed with brine (3 × 80 mL) and dried over
magnesium sulfate. After evaporation the crude product was purified
by flash chromatography and lyophilized from acetonitrile/water to give
a white powder in 60-80% yield.
Peptide 8b (5 mg, 3 µmol) was subsequently treated with TFA/water
(98/2) under nitrogen for 10 h. Peptide 8c was finally obtained by
diethyl ether precipitation and subsequently used as such (4 mg, 2.24
µmol, 82%). ESI-MS: m/z ) 813 ((M + 2H+)/2).
Synthesis of Transmembrane Peptides 9a-c. Peptide 9a was
synthesized on Rink Amide MBHA resin (1.0 g, 0.30 mmol) according
to the general procedure described above. Fmoc-Ala-Thr(ΨH,Hpro)-
OH was prepared according to the general procedure described above,
starting from Fmoc-Ala-NCA. The target compound was obtained in
60% yield. Mp: 103-108 °C (lyophilized). Rf (chloroform/methanol/
acetic acid, 85/15/5) ) 0.32. HPLC (C18, 214 nm, gradient of 40-
100% solvent B over 30 min): Rt ) 13.4 min. 1H-NMR (250 MHz,
CDCl3, two conformeres: (major (80%), minor (20%)): δ 7.69-7.08
(m, 8 arom H), 6.06 (d, J ) 9.0, HN), 5.85 (d, J ) 7.8, HN), 5.31 (d,
J ) 3.9, HRe-C2), 5.22 (d, J ) 5.0, HRe-C2), 4.85 (d, J ) 4.0, HSi-C2),
4.83 (HSi-C2), 4.35-3.91 (m, 6 H, H-CFmoc, H2-CFmoc, H-CRAla, H-C4,
H-C5), 1.39 (d, J ) 5.9, H3-C5), 1.38 (H3C-C5), 1.29 (d, J ) 7.0,
H3-CâAla), 1.26 (H3-CâAla). C23H24N2O6 (424.45). CI-MS (NH3): m/z
) 425 (0.5, [M + 1]+), 178 (100).
Double coupling was performed for every step except for the
dipeptide Fmoc-Ala-Thr(ΨH,Hpro)-OH, where a single coupling was
made with a prolonged reaction time (20 h). The N-terminal position
was acetylated according to the capping procedure described above.
The cleavage of the peptide from the resin (200 mg) was achieved by
successive treatment (3 × 10 min) with a mixture of TFA/water (95/
5), and the combined filtrates were concentrated in Vacuo. The crude
product was dissolved in a mixture of acetonitrile/methanol (2/1),
precipitated with water, centrifuged, and washed two times with water.
The crude peptide 9a was dried in Vacuo to give a white powder (58
mg, 86%) which was found to be 96% pure by analytical HPLC at Rt
) 9.6 min using a C4 column with a gradient 10-60% solvent B over
30 min (solvent A, 90% 2-propanol/10% water (0.09% TFA); solvent
B, 90% CH3CN/10% water (0.09% TFA). ESI-MS: m/z calculated
for C97H173N21O22 1983, found 993.9 ([M + 2H+]/2). MALDI-MS:
2025.1 (corresponding to [M + 2Na - 4]). ASA: Thr, 0.73 (1); Ala,
5.88 (6); Leu, 10.71 (10); Aib, 3.57 (3).
Synthesis of Switch Peptides 8a-c. Peptide 8a was synthesized
on NR-Fmoc-Ala-Sasrin resin (1.0 g, 0.68 mmol) according to the
general procedure described above. The following protected side chain
amino acids were used: NR-Fmoc-Glu(OAll)-OH, NR-Fmoc-Lys-
(Aloc)-OH, and NR-Fmoc-Ala-Cys(ΨMe,Mepro)-OH.
NR-Fmoc-Ala-Cys(ΨMe,Mepro)-OH was prepared according to the
general procedure described above, starting from Fmoc-Ala-F to give
the target compound in 62% yield. Mp: 107-112 °C. Rf (petroleum
ether/ethyl acetate/acetic acid, 8/8/1) ) 0.53. HPLC (C18, 214 nm,
gradient of 40-100% solvent B over 30 min): Rt ) 17.5 min (100%).
3
1H NMR (400 MHz, CDCl3): δ 1.52 (d, J ) 6.5 Hz, 3H, Ala-CH3),
2
1.99 (s, 3H, C2-CH3), 2.10 (s, 3H, C2-CH3), 3.36 (dd, J ) 11.5 Hz,
3J ) 5.6 Hz, 1H, C5-H), 3.54 (d, 2J ) 11.5 Hz, 1H, C5-H), 4.22-4.34
Peptide 9c was prepared from peptide 9a (3.0 mg, 1.5 µmol) which
was treated with 10% TFMSA in TFA (700 µl) at rt for 48 h.
Completion of the reaction was monitored by HPLC. After evaporation
of TFA, pyridine (56 µL) was added at 0 °C to neutralize TFMSA,
and the whole was dissolved in water (1 mL). The precipitate thus
formed was centrifuged, washed three times with water, and dried in
Vacuo overnight to give the deprotected peptide 9c (1.0 mg, 33%) as
a pale yellow solid. Analytical HPLC: Rt ) 23.2 min using a C4
column with a linear gradient of 10-60% solvent B over 30 min
(solvent A, 90% 2-propanol/10% water (0.09% TFA); solvent B, 90%
CH3CN/10% water (0.09% TFA). ESI-MS: m/z calculated for
C96H173N21O22 1971, found 987.6 ([M + 2H+]/2).
3
(m, 2H, Fmoc-CH2), 4.43 (m, 1H, Fmoc-CH), 4.65 (t, J ) 7.0 Hz,
1H, Ala-CR-H), 4.92 (d, 3J ) 5.0 Hz, 1H, C4-H), 6.14 (d br, 3J ) 6.8
Hz, 1H, NH), 7.27-7.72 (m, 8H, Fmoc-Ar-H). C24H26N2O5S (454.55).
ESI-MS: m/z ) 455.5 [M + H+].
The ΨPro dipeptide was introduced by single coupling according
to the standard procedure described above. Double coupling was
necessary for Ala in position 5 and Leu in position 6. The N-terminus
position was acetylated according to the capping procedure described
above. The protected peptide 8a was finally cleaved from the resin
(36) Carpino, L. A.; Aalaee, D. S.; Chao, H. G.; De Selms, R. H. J. Am.
Chem. Soc. 1990, 112, 9751-9752.

ΨPro as a Protection Technique in Peptide Synthesis
J. Am. Chem. Soc., Vol. 118, No. 39, 1996 9227
Peptide 9b was prepared from the nonacetylated precursor resin-
bound peptide of 9a. Prior to N-terminal acetylation of 9a the peptidyl
resin, an S-trityl-protected cysteine residue, was further coupled and
acetylated according to standard protocols described above. The resin-
bound peptide 9b thus obtained (355 mg, substitution of 0.24 mmol/g
after the last UV absorption of N-fluorenylmethylpiperidine) was
subjected to successive treatment (3 × 10 min) with a mixture of TFA/
triisopropylsilane/water (95/2.5/2.5) (3 × 3 mL) and washing with TFA
(3 × 1 mL). The combined filtrates were concentrated in Vacuo, and
the resulting crude material was purified by semipreparative HPLC (C4
column eluted with a linear gradient of 10-70% solvent B over 30
min (solvent A, 90% 2-propanol/10% water (0.09% TFA); solvent B,
90% CH3CN/10% water (0.09% TFA) to give the peptide 9b (76 mg,
71% based on the resin, substitution of 0.24 mmol/g). Analytical
HPLC: Rt ) 8.9 min using a C4 column with a linear gradient of 10-
60% solvent B over 30 min (solvent A, 90% 2-propanol/10% water
(0.09% TFA); solvent B, 90% CH3CN/10% water (0.09% TFA). ESI-
MS: m/z calculated for C100H178N22O23S 2086, found 1045.0 ([M +
2H+]/2).
Synthesis of Template 10. The bromoacetylated template 10 was
prepared from the precursor Z-protected template previously described
in the literature.24 The Z protection was removed by treatment with
hydrogen/10% Pd-carbon/concentrated HCl (6 equiv) in MeOH,
affording the deprotected template (4 × HCl salt) quantitatively. The
template thus obtained (30 mg, 27.7 µmol) was treated with bromoacetic
anhydride (333 µmol, prepared from bromoacetic acid (92.4 mg, 665
µmol) and N,N'-dicyclohexylcarbodiimide (69.0 mg, 333 µmol) in
dichloromethane (2 mL)) in the presence of DIEA (14.3 mg, 111 µmol)
in DMF (5 mL) at rt for 20 h. The volatiles were evaporated, and the
residue was again dissolved, filtered to remove a small amount of
dicyclohexylurea, and concentrated. The crude product was purified
by semipreparative HPLC using a C4 column eluted with a linear
gradient of 0-100% solvent B over 30 min to give the bromoacetyl
template 10 (22 mg, 56%). Analytical HPLC: Rt ) 19 min using a
C18 column with a linear gradient 10-100% solvent B over 30 min.
ESI-MS: m/z calculated for C50H78B14N14O14 1417, found 1419.1,
1420.4, 1421.4, 1422.6, 1423.4 in the ratio of 1/4/6/4/1 ([M + H]+),
711.3 ([M + 2H]+/2).
2.57 (m, 6 H, H-CFmoc, H2-CFmoc, H-CRMet, HâThr, HRThr), 2.08 (s, CH3-
Met), 2.1-1.89 (m, HâMet) 1.49 (d, J ) 6.1, CH3-Thr). C23H24N2O6
(424.45). EI-MS: 485 [M + H+].
Fmoc-Ser(tBu)-Cys(ΨMe,Mepro)-OH. Starting from Fmoc-Ser(tBu)-
F, the target compound was obtained in 64% yield. Mp: 110-112
°C. Rf (petroleum ether/ethyl acetate/acetic acid, 14/5.5/0.5) ) 0.32.
HPCL (C18, 214 nm, gradient of 40-100% solvent B over 30 min):
Rt ) 26.0 min. 1H NMR (400 MHz, CDCl3): δ 1.16 (s, 9H, tBu-
CH3), 1.84 (s, 3H, -CH3), 1.91 (s, 3H, -CH3), 3.18-3.61 (m, 4H,
Ser-CH2, C5-H), 4.16 (m, 1H, Fmoc-CH), 4.30 (m, 2H, Fmoc-CH2),
4.65 (m, 1H, Ser-CR-H), 5.52 (m, 1H, C4-H), 7.27-7.75 (m, 8H, Fmoc-
Ar-H). C28H34N2O6S (526.66). ESI-MS: m/z ) 527.5 [M + H+].
Fmoc-Glu(OtBu)-Cys(ΨMe,Mepro)-OH. Starting from Fmoc-Glu-
(OtBu)-F, the target compound was obtained in 64% yield. Mp: 94-
95 °C. Rf (petroleum ether/ethyl acetate/acetic acid, 14/5.5/0.5) ) 0.31.
HPCL (C18, 214 nm, gradient of 40-100% solvent B over 30 min):
Rt ) 24.8 min. 1H NMR (400 MHz, CDCl3): δ 1.44 (s, 9H, OtBu-
CH3), 1.81 (s, 3H, -CH3), 1.91 (s, 3H, -CH3), 2.23-2.44 (m, 4H,
Glu-CH2), 3.28 (m, 1H, C5-H), 3.42 (m, 1H, C5-H), 4.11 (m, 1H, Fmoc-
CH), 4.18-4.32 (m, 2H, Fmoc-CH2), 4.58 (m, 1H, Glu-CR-H), 5.93
(d, 3J ) 8.9 Hz, 1H, C4-H), 7.24-7.77 (m, 8H, Fmoc-Ar-H).
C30H36N2O7S (568.69). ESI-MS: m/z ) 569.6 [M + H+].
Fmoc-Phe-Cys(ΨMe,Mepro)-OH. Starting from Fmoc-Phe-F, the
target compound was obtained in 79% yield. Mp: 106-110 °C. Rf
(petroleum ether/ethyl acetate/acetic acid, 14/5.5/0.5) ) 0.30. HPCL
(C18, 214 nm, gradient of 40-100% solvent B over 30 min): Rt )
24.7 min. 1H NMR (400 MHz, CDCl3): δ 1.74 (s, 3H, -CH3), 1.86
(s, 3H, -CH3), 2.35 (m, 1H, Phe-CH2), 2.90 (m, 1H, C5-H), 3.06 (m,
2H, Phe-CH2, C5-H), 4.06-4.30 (m, 4H, Fmoc-CH, Fmoc-CH2, Phe-
CR-H), 4.79 (m, 1H, C4-H), 7.23-7.73 (m, 13H, Phe-Ar-H, Fmoc-
Ar-H). C30H30N2O5S (530.65). ESI-MS: m/z ) 531.1 [M + H+].
The step by step assembly proceeded smoothly with single couplings.
Cleavage and deprotection of the peptide resin (1.0 g) was carried out
by treatment with a freshly prepared solution of 82.5% TFA/2.5%
ethanedithiol/5% thioanisole/5% water/5% phenol (7 mL) for 2 h under
argon at room temperature. After filtration and washing with TFA (2
× 3 mL) and DCM (3 mL), the volatiles were evaporated and the crude
peptide material was precipitated with chilled tert-butyl methyl ether
and isolated by centrifugation. After HPLC analysis, a further treatment
of 32 h in 95% TFA/5% water (5 mL) was carried out followed by
precipitation in cold tert-butyl methyl ether and isolated by centrifuga-
tion as a white solid. The crude product was subsequently purified by
preparative RP-HPLC using a C18 column with a gradient of 10-
60% solvent B over 30 min to give the pure C1,3,11,15-thiol, T7-ΨPro
peptide in 18% yield (80 mg) after lyophilization. Analytical HPLC:
Rt ) 19.7 min using a C18 column with a linear gradient of 10-60%
solvent B over 30 min. ESI-MS: found ((M + 3)/3) 860.8, calculated
861.0. ASA: Asp + Asn, 3.06 (3); Glu + Gln, 1.98 (2); Ser, 0.64
(1); Thr, 1.00 (1); Val, 0.71 (1); Met, 1.07 (1); Ile, 0.68 (1); Leu, 0.98
(1); Phe, 1.09 (1); Lys, 2.19 (2); His, 0.95 (1); Tyr, 0.60 (1).
Synthesis of 4r-Helix Transmembrane TASP. To the bromoacetyl
template 10 (2.0 mg, 1.4 µmol) were added sequentially a solution of
cysteinyl peptide 9b (14.6 mg, 7.0 µmol) in DMF (2 mL) and DIEA
(905 µg, 7.0 µmol) at rt (the pH of the solution was 8.2-8.5). The
reaction mixture was further stirred for 16 h. The solvent was
evaporated, and the resulting crude mixture was purified by semi-
preparative HPLC using a C4 column eluting with a linear gradient of
10-80% solvent B over 30 min, to give the TASP molecule 11 (9.0
mg, 68%). Analytical HPLC: Rt ) 30.6 min using a C4 column with
a linear gradient 10-70% solvent B over 30 min followed by 70-
80% solvent B over 10 min. MALDI-MS: m/z calculated for
C450H786N102O106S4 9438, found 9561.0.
Synthesis of [ΨPro-7]Sarafotoxin-S6b. Peptide assembly was
The reduced peptide (50 mg) was dissolved at a concentration of
10-4 M in Tris‚HCl (10 mM) and EDTA (1 mM) buffer at pH 8.0 for
3 h. After lyophilization the two oxidized forms of [ΨPro-7]-
Sarafotoxin-S6b (isomers A and B) were isolated by semipreparative
RP-HPLC using a C18 column with a linear gradient of 10-60% solvent
B over 30 min, and lyophilized. Analytical HPLC of isomers A and
B: Rt (A) ) 18.1 min, Rt (B) ) 18.5 min, using a C18 column with a
linear gradient of 10-60% solvent B over 30 min. ESI-MS: found
((M + 3)/3) 859.1, calculated 859.6.
carried out on a Fmoc-Trp(Boc)-functionalized Wang type resin (1.88
g, 0.31 mmol/g resin loading) according to Fmoc-PyBOP chemistry.
The amino acid side chain protections were chosen as follows: tBu
(Asp, Tyr, Ser, Glu), Boc (Lys), Bum (His), Trt (Gln, Cys-1). Thr-7
and Cys-3, -11, and -15 were introduced as dipeptides Fmoc-Met-Thr-
(ΨH,Hpro)-OH, Fmoc-Ser(tBu)-Cys(ΨMe,Mepro)-OH, Fmoc-Glu(OtBu)-
Cys(ΨMe,Mepro)-OH, and Fmoc-Phe-Cys(ΨMe,Mepro)-OH, respectively.
These ΨPro building blocks were readily prepared according to the
procedures described above.
Fmoc-Met-Thr(ΨH,Hpro)-OH. Starting from Fmoc-Met-F, the
target compound was obtained in 80% yield. Mp: 105-115 °C. Rf
(ethyl acetate/hexane/acetic acid, 90/10/1) ) 0.27. HPLC (C18, 214
nm, gradient of 40-100% solvent B over 30 min): Rt ) 17 min. 1H-
NMR (400 MHz, CDCl3): δ 7.77-7.30 (m, 8 arom H), 6.05 (d, J )
8.8, HN), 5.9 (d, J ) 8.4, HN), 5.53 (d, J ) 4.1, HRe-C2), 5.3 (d, J )
5.4, HRe-C2), 5.05 (d, J ) 4.1, HSi-C2), 4.95 (d, J ) 5.4, HSi-C2), 4.57-
Acknowledgment. This work was supported by the Swiss
National Science Foundation. The authors gratefully acknowl-
edge Dr. P. Dumy and Dr. I. M. Eggleston for helpful
discussions.
JA961509Q