HYCRON, an Allylic Anchor for High-Efficiency Solid Phase Synthesis of Protected Peptides and Glycopeptides

Article abstract of DOI:10.1021/jo960743w

The recently developed allylic HYCRON anchor¹ exhibits excellent properties for the solid phase synthesis of protected peptides and glycopeptides. Model reactions with analogous low molecular weight compounds assessed the acid- and base-stability of the polar and flexible HYCRON linkage. The new anchor is available in a two-step synthesis and allows the use of both the Boc- and the Fmoc-strategy, which can even be combined within one synthesis. Protected glycopeptides are released under almost neutral conditions, taking advantage of the Pd(0)-catalyzed allyl transfer to a weakly basic nucleophile such as N-methylaniline. The highly efficient synthesis of O-αGalNAc-(TN)-peptides of the MUC-1 repeating unit is described. Acid- and base-stability of the allyl ester linkage enabled the synthesis of an O-glucosylated peptide by first removing a threonine tert-butyl group on the solid phase and subsequently glycosylating the liberated resin-bound hydroxyl component.

Full text of DOI:10.1021/jo960743w

J . Org. Chem. 1997, 62, 813-826
813
HYCRON, a n Allylic An ch or for High -Efficien cy Solid P h a se
Syn th esis of P r otected P ep tid es a n d Glycop ep tid es
Oliver Seitz and Horst Kunz*
Institut fu¨r Organische Chemie der Universita¨t Mainz, J .-J . Becher-Weg 18-20, D-55099 Mainz, Germany
Received April 23, 1996X
The recently developed allylic HYCRON anchor1 exhibits excellent properties for the solid phase
synthesis of protected peptides and glycopeptides. Model reactions with analogous low molecular
weight compounds assessed the acid- and base-stability of the polar and flexible HYCRON linkage.
The new anchor is available in a two-step synthesis and allows the use of both the Boc- and the
Fmoc-strategy, which can even be combined within one synthesis. Protected glycopeptides are
released under almost neutral conditions, taking advantage of the Pd(0)-catalyzed allyl transfer to
a weakly basic nucleophile such as N-methylaniline. The highly efficient synthesis of O-RGalNAc-
(TN)-peptides of the MUC-1 repeating unit is described. Acid- and base-stability of the allyl ester
linkage enabled the synthesis of an O-glucosylated peptide by first removing a threonine tert-butyl
group on the solid phase and subsequently glycosylating the liberated resin-bound hydroxyl
component.
In tr od u ction
and T-antigen structures (Ser/Thr(R-3-(âGal)-GalNAc))
even tumor-specific in breast tissue.8
Proteins and peptides exist in a variety of conjugated
forms, the most important being glycoconjugates.2 N-
and O-Glycosidically bound carbohydrates affect the
conformation of proteins and regulate their activity and
biological half-lives.3 Glycoproteins participate in bio-
logical recognition processes such as cell adhesion, regu-
lation of cell growth, and cell differentiation. During
tumor-transformation, the glycosylation pattern of gly-
coproteins, such as mucins, changes markedly.4 Mucins
are excessively O-glycosylated proteins with a carbohy-
drate portion reaching 50-80%.5 Mucins expressed from
various epithelial cell types in soluble and membrane-
associated forms typically consist of several serine- and
threonine-rich repeating units which serve as carbohy-
drate scaffolds. During tumor progression the balance
between the mucin-mediated adhesion6 and antiadhesion
processes is no longer under control. Simultaneously,
alterations of the glycosylation pattern occur, because
certain glycosyltransferases are expressed in lower con-
centrations.7 These two phenomena seem to be causally
related. For instance, TN-antigen structures (Ser/Thr-
(RGalNAc)) were demonstrated to be tumor-associated
Oligonucleotides9 and oligopeptides10 are readily avail-
able via solid phase synthesis. The properties of the
anchor group positioned between the oligomer to be
synthesized and the polymeric support are crucial for the
success of a solid phase synthesis. There is an increasing
demand for linkage groups which provide an additional
orthogonal stability and can, thus, accommodate a wider
range of reaction conditions. This particularly holds true
for the synthesis of glycopeptides and for the growing
field of combinatorial chemistry.11 The synthesis of
glycopeptides and phosphopeptides as well as peptides
with acid- and base-labile protecting groups, which are
to be employed in fragment condensations,12 requires
anchor groups which allow the release of the target
molecule under conditions that leave the labile structures
unaffected.
Most of the commonly used anchors for the solid phase
synthesis of protected peptides and glycopeptides are
either acid-labile13 or base-labile. Nearly all of the acid-
labile anchor groups belong to the benzyl ester type14 and
produce long-lived, stable carbocations during their
cleavage. These may cause undesired alkylations of
nucleophilic moieties.15 Furthermore, acid-labile protect-
ing groups such as the tert-butyloxycarbonyl(Boc)-group
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
(1) Abbreviations: AA, amino acid; Ac, acetyl; Acm, acetamido-
methyl; Bn, benzyl; Boc, tert-butyloxycarbonyl; DCC, N,N ′-dicyclo-
hexylcarbodiimide; DIC, N,N ′-diisopropylcarbodiimide; DIPEA, diiso-
propylethylamine; DKP, diketopiperazine; EtSMe, ethyl methyl sulfide;
Fmoc, 9-fluorenylmethoxycarbonyl; Gal, D-galactopyranose; GalNAc,
2-acetamido-2-deoxy-D-galactopyranose; Glc, D-Glucopyranose; GPC,
gel permeation chromatography; HOBt, 1-hydroxybenzotriazole; HON-
Su, N-hydroxysuccinimide; HYCRAM, hydroxycrotonoyl (aminometh-
yl)polystyrene; HYCRON, hydroxycrotyl-oligoethylene glycol-n-al-
kanoyl; MPLC, medium performance liquid chromatography; Mtr,
4-methoxy-2,3,6-trimethylbenzenesulfonyl; Muc, mucine; NMM, N-
methylmorpholine; Pac, phenacyl; PS, polystyrene; tBu, tert-butyl; Trt,
trityl; TBTU, 2-O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; Z, Benzyloxycarbonyl.
(7) Vavasseur, F.; Dole, K.; Yang, J .; Matta, K. L.; Myerscough, N.;
Corfield, A.; Paraskeva, C.; Brockhausen, I. Eur. J . Biochem. 1994,
222, 415.
(8) Karsten, U.; Papsdorf, G.; Pauly, A.; Vojtesek, B.; Moll, R.; Lane,
E. B.; Clausen, H.; Stosiek, P.; Kasper, M. Differentiation 1993, 54,
55.
(9) (a) Narang, S. A. Tetrahedron 1983, 39, 3. (b) Bannwarth, W.
Chimia 1987, 41, 302.
(10) (a) Merrifield, R. B. J . Am. Chem. Soc. 1963, 85, 2149. (b) Fields,
G. B.; Noble, R. L. Int. J . Pept. Protein Res. 1990, 35, 161. (c) Williams,
P. L.; Albericio, F.; Giralt, E. Tetrahedron 1993, 347, 11065.
(11) a) J ung, G.; Beck-Sickinger, A. G. Angew. Chem., Int. Ed. Engl.
1992, 31, 375. (b) Gallop, M. A.; Barrett, R. W.; Dower, W. J .; Fodor,
S. P. A.; Gordon, E. M. J . Med. Chem. 1994, 37, 1233. (c) Gallop, M.
A.; Barrett, R. W.; Dower, W. J .; Fodor, S. P. A.; Gordon, E. M. J . Med.
Chem. 1994, 37, 1385.
(2) (a) Montreuil, J . Adv. Carbohydr. Chem. Biochem. 1980, 37, 157
(b) Lis, H.; Sharon, N. Eur. J . Biochem. 1993, 218, 1.
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Downing, A. K.; Campbell, I. D.; Derek, R. A. Biochim. Biophys. Acta.
1995, 1248, 1.
(12) Benz, H. Synthesis 1993, 337.
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Taylor-Papadimitriou, J . Eur. J . Biochem. 1995, 233, 607.
(5) Hilkens, J .; Ligtenberg, M. J . L.;Vos, H. L.; Litvinov, S. L. Trends
Biochem. Sci. 1992, 17, 359.
(6) Berg, E. L.; McEvoy, L. M.; Berlin, C.; Bargatze, R. F.; Butcher,
E. C. Nature 1993, 366, 695.
(13) (a) Paulsen, H.; Merz, G.; Weichert, U. Angew. Chem., Int. Ed.
Engl. 1988, 27, 1365. (b) Lu¨ning, B.; Norberg, T.; Tejbrant, J .
Glycoconjugate J . 1989, 6, 5. (c) Laczko, I.; Hollosi, M.; Urge, L. E.;
Ugen, K.; Weiner, D. B.; Mautsch, H. H.; Thurin, J .; Otvos, L.
Biochemistry 1990, 4282. (d) Mucin-type glycopeptides: Meldal, M.;
Mouritsen, S.; Bock, K. Am Chem. Soc. Symp. Ser. 1993, 519, 19.
S0022-3263(96)00743-8 CCC: $14.00 © 1997 American Chemical Society

814 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
Sch em e 1
Sch em e 2
cannot be removed on solid phase without detaching the
peptide. Base-labile linkages such as in Kaiser’s oxime
resin are not compatible with the widely used fluorenyl-
methoxycarbonyl(Fmoc)-strategy. Most of the existing
photolabile linkers can only be cleaved in low yields if
longer peptide sequences are synthesized. Photolabile
anchors with high cleavability are usually base-sensi-
tive16 and not fully compatible with Fmoc chemistry.
Linkers of the general allyl-type17 are of particular
value, because they are removable under almost neutral
conditions and are orthogonally stable to the commonly
used acid- and base-labile protecting groups.18 Allylic
anchoring offers the possibility of applying both the Boc-
and the Fmoc-group for temporary N-terminal protection
in solid phase peptide synthesis. The strategies may
even be combined in one synthesis. The cleavage of the
allylic linkage is achieved by a palladium(0)-catalyzed
transfer reaction19 of the allyl group to a nucleophile,
which acts as an allyl scavenger. Nucleophiles such as
morpholine,20 the less basic N-methylaniline,21 or dime-
done22 irreversibly trap the allylic moiety.
Solid phase peptide synthesis with allylic anchoring
was introduced using hydroxycrotonic acid linkers (â-
HYCRAM 1, Scheme 1). A range of complex glycopep-
tides have been successfully synthesized on the â-HY-
CRAM-support.23 While Boc-strategy provided glyco-
peptides in high yield, the application of the Fmoc-
strategy showed losses of some peptide during the
synthesis.24 Moreover, complete release of the peptide
was sometimes difficult to achieve.
In the design of a new allylic anchoring group, a more
flexible spacer was inserted between the anchor and the
polymer in order to facilitate an efficient access of the
Pd(0) complex during the detachment reaction. The
success achieved with craft copolymers25 of polyethylene
glycol and polystyrene inspired the incorporation of a
polar spacer of the ethylene glycol type. In addition, in
this HYCRON anchor26 2 (Scheme 1), the R,â-unsatur-
ated carbonyl structure was avoided, since it could be
responsible for the occasionally low yields in Fmoc
chemistry with â-HYCRAM 1. As in â-HYCRAM, â-ala-
nine is used as a standard amino acid, since it simplifies
the determination of the peptide loading.27
Resu lts a n d Discu ssion
Syn th esis of a Low Molecu la r Weigh t Mod el. The
first step in synthesis of an anchor derivative 4, which
allows the coupling of the starting amino acid via
nucleophilic esterification consists in the sodium glycolate
catalyzed 1,4-addition of triethylene glycol to tert-butyl
acrylate (Scheme 2). The subsequent reaction of the
adduct 3 with 1,4-dibromo-2-butene is carried out under
phase transfer conditions, which avoids the retro-Michael
reaction leading to unseparable mixtures. This elimina-
tion is initiated by abstraction of an R-proton, which is
unlikely to occur in the resin-bound molecule, because
proton abstraction would take place at the amide function
disabling further deprotonation. Treatment of 4a with
TFA removes the tert-butyl group.
The carboxylic acid 5 is activated with DCC28 and
N-hydroxysuccinimide29 (HONSu) and reacted with â-Ala-
NH-Bn to give 6 (Scheme 3). A slight excess of the acid
compound must be used in order to prevent the concomi-
tant allylation of HONSu. Compound 6 can be consid-
ered a low molecular weight model of an (aminomethyl)-
polystyrene (H2N-PS) resin functionalized with the
standard amino acid â-alanine and the anchor-bromide.
The reaction of the cesium salt of Z-protected alanine
(14) (a) Wang, S. S. J . Am. Chem. Soc. 1973, 95, 1328. (b) Mitchell,
A. R.; Erickson, B. W.; Ryabster, M. N.; Hodges, R. S.; Merrifield, R.
B. J . Am. Chem. Soc. 1976, 98, 7357. (c) Flo¨rsheimer, A.; Riniker, B.
Peptides 1990. Proceedings of the 21st European Peptides Symposium;
Giralt, E., Andreu, D., Eds.; ESCOM: Leiden, 1991; p 131. (d) Mergler,
M.; Tanner, R.; Gosteli, J .; Grogg, P. Tetrahedron Lett. 1988, 29, 4005.
(e) Mergler, M.; Nyfeller, R.; Tanner, R.; Gosteli, J .; Grogg, P.
Tetrahedron Lett. 1988, 29, 4009. (f) Albericio, F.; Barany, G. Tetra-
hedron Lett. 1991, 32, 1015.
(15) (a) Atherton, E.; Cameron, L. R.; Sheppard, R. C. Tetrahedron
1988, 44, 843. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.;
Geva, L.; Masada, R. I.; Hudsen, D.; Barany, G. J . Org. Chem. 1990,
55, 3730.
(16) Tjoeng, F. S.; Tam, J . P.; Merrifield, R. B. Int. J . Pept. Protein
Res. 1979, 14, 262.
(17) Kunz, H.; Dombo, B. (ORPEGEN GmbH), DE-A P 3720269.3,
1987; USA. 4 929 671, 1990.
(18) (a) Kunz, H.; Dombo, B. Angew. Chem., Int. Ed. Engl. 1988,
27, 711. (b) Kunz, H.; Dombo, B.; Kosch, W. In Peptides 1988; (J ung,
G., Bayer, E., Eds.; W. de Gruyter: Berlin, 1989, 154. (c) Blankemeyer-
Menge, B.; Frank, R. Tetrahedron Lett. 1988, 29, 5871. (d) Guibe´, F.;
Dangles, O.; Balavoine, G.; Loffet, A. Tetrahedron Lett. 1989, 30, 2641.
(e) Zhang, X.; J ones, R. A. Tetrahedron Lett. 1996, 22, 3789.?
(19) (a) Trost, B. M. Acc. Chem. Res. 1980, 13, 385. (b) Trost, B. M.
Pure Appl. Chem. 1980, 53, 2357.
(25) (a) Rapp, W.; Zhang, L.; Ha¨bich, R.; Bayer, E. In Peptides 1988,
J ung, G.; Bayer, E., Eds.; de Gruyter: Berlin 1989, 199. (b) Bayer, E.
Angew. Chem., Int. Ed. Engl. 1991, 30, 113.
(26) Seitz, O.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 807.
(27) (a) Atherton, E.; Clive, D. I.; Sheppard, R. C. J . Am. Chem. Soc.
1975, 97, 6584. (b) Albericio, F.; Barany, G. Int. J . Pept. Protein Res.
1985, 26, 92.
(20) (a) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl., 1984,
23, 71. (b) Friedrich-Bochnitschek, S.; Waldmann, H.; Kunz, H. J . Org.
Chem. 1989, 54, 751.
(21) Ciommer, M.; Kunz, H. Synlett 1991, 593.
(22) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1984,
23, 436.
(23) Kosch, W.; Ma¨rz, J .; Kunz, H. React. Polym. 1994, 22, 181 and
references therein.
(24) Kosch, W. Dissertation, Universita¨t Mainz, 1992.
(28) Sheehan, J . C.; Hess, G. P. J . Am. Chem. Soc. 1955, 77, 1067.
(29) Weygand, F.; Hoffmann, D.; Wu¨nsch, E. Z. Naturforsch. 1966,
21b, 426.

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 815
Ta ble 1. Nu cleop h ilic Ester ifica tion of F m oc-Am in o Acid s w ith An ch or -Br om id es 4a ,b a n d Rem ova l of th e C-Ter m in a l
P r otectin g Gr ou p (see a lso Sch em e 4)
Ala
(R ) tBu),
% yield
Gly
(R ) tBu),
% yield
Pro
(R ) tBu),
% yield
Val
(R ) tBu),
% yield
Thr(tBu)
(R ) Pac),
% yield
ester formation
R removal
8a , 72
8b, 84
9a , 65
9b, 88
10a , 81
10b, 85
11a , 67
11b, 57
12a , 83
12b, 97
Sch em e 3
Sch em e 4
with bromide 6 provides the model compound 7. This
nucleophilic esterification is known to proceed without
any detectable racemization.30 Unfortunately, the chro-
matographic behavior of 6 and 7 is nearly identical
resulting in a small contamination with 6 even after two-
fold MPLC and preparative HPLC. However, the mate-
rial is useful for stability tests, since the reactions of both
6 and 7 can be monitored by analytical HPLC. In order
to assess the stability of the HYCRON system toward
the conditions of N-Boc- and N-Fmoc removal, compound
7 is treated with dichloromethane/TFA (1:1) and mor-
pholine, respectively. Several reaction cycles are simu-
lated by an exposure of two days. HPLC analysis shows
that formation of new substances occurs in an amount
of less then 4%. This is a promising result for the
application of HYCRON-anchoring in solid phase peptide
synthesis.
Sch em e 5
Syn th esis of An ch or Con ju ga tes a n d Resin Loa d -
in g. The starting amino acids should be loaded onto the
polymer via their anchor conjugates, since attempts to
couple amino acid cesium salts to a PS-resin functional-
ized with â-alanine and the bromo-anchor 5 resulted in
low loading yields. Using the linkage agent 4a , anchor
conjugates of Fmoc-amino acids can be synthesized. For
conjugate synthesis of amino acids with acid-labile side
chain protecting groups, the tert-butyl group of 4a is
exchanged (Scheme 2). The phenacyl (Pac) ester 4b
provides orthogonally stable protection to most side chain
protecting groups in SPPS.
Usually Fmoc-amino acids are alkylated via the cesium
salt method. This reaction is less favorable for the
HYCRON-system, because the rate of the alkylation
reaction is slow and Fmoc-cleavage competes in the basic
environment. In contrast, this nucleophilic esterification
with the anchor bromides 4a and 4b proceeds mildly
under phase transfer conditions using the base NaHCO3
(Scheme 4). Reactants need not to be applied in excess.
Conjugates 8a -12a are easily accessible (Table 1) using
this method. TFA liberates the carboxylic function in the
case of tert-butyl protection to give 8b-11b, and zinc
powder in acetic acid reductively cleaves the Pac-group
from 12a to form 12b.
tions. For synthesis of conjugate 16b containing Boc-
Ala, the anchor-bromide 5 is condensed with â-Ala-OPac.
Amide 14 is employed in an alkylation of the Boc-Ala
cesium salt followed by reductive removal of the Pac-
group (Scheme 5). Conjugate 16a is synthesized by
coupling 8b to â-Ala-OPac and subsequent reductive
cleavage of the Pac-ester 13.
In order to attach the amino acid-anchor-conjugates
to the polymer, Boc-â-Ala loaded (aminomethyl)polysty-
rene (H2N-PS) is treated with TFA and neutralized with
diisopropylethylamine (DIEA). The conjugates 8b-12b
are coupled to this resin by the DIC/HOBt31 method
(Scheme 6). The same reaction conditions are applied
for binding the conjugates 16a and 16b to unmodified
H2N-PS resin. Any amino groups that have not been
acylated are capped with acetic anhydride/pyridine.
Table 2 shows the loadings and yields achieved by these
reactions.
In the previously described synthesis of anchor conju-
gates the allylic ester is formed by allylation of the
carboxylic acid. However, nucleophilic esterifications can
be accompanied by alkylations of other nucleophilic
structures. For instance, the esterification of Fmoc-Cys-
(Acm) under the above mentioned conditions proceeds
Conjugates consisting of starting amino acid, anchoring
group, and standard amino acid are synthesized via two
possible routes, which differ in the sequence of condensa-
(31) (a) Sarantakis, D.; Teichmann, J .; Lien, E. L.; Fenichel, R. L.
Biochem. Biophys. Res.Commun. 1976, 73, 336. (b) Ko¨nig, W.; Geiger,
R. Chem. Ber. 1970, 103, 788.
(30) Wang, S.-S.; Gisin, B. F.; Winter, D. P.; Makofske, R.; Kulesha,
I. D.; Tsougraki, C.; Meienhofer, J . J . Org. Chem. 1977, 42, 1286.

816 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
Ta ble 2. Atta ch m en t of Am in o Acid -An ch or -Con ju ga tes
to th e P olystyr en e Resin (see a lso Sch em e 6)
Sch em e 7
loading/
AA
Ala
PG
anchor-conjugate
(mmol of AA/g), yielda
Fmoc
Fmoc
Boc
Fmoc
Fmoc
Fmoc
Fmoc
8b
16a
16b
9b
10b
11b
12b
17a , 0.52, 89%
17b, 0.71, 89%
17c, 0.67, 75%
18, 0.50b, 80%
19, 0.52, 61%
20, 0.41, 75%
21, 0.40c, 66%
Gly
Pro
Val
Thr(tBu)
a
Yield ) lAA/lâ-Ala × 100 for resins 17a and 18-21, lAA/lNH
×
2
100 17b,c (l ) loading, determined by amino acid analysis).
Content of Fmoc group (photometrically). c Original loadings and
b
yields are higher, and determination by amino acid analysis gives
values that are too low.
Sch em e 6
Sch em e 8
with a concomitant allylation of the thiol function. The
side reactions are avoided if the carboxylic group is
reacted with the anchor-alcohol. In contrast to the amino
acid loadings with HYCRON-bromides as substrates, this
procedure can also be performed on the solid support.
Acetoxylation of the anchor-bromide 4a and removal of
the tBu-group produces the protected form 23 of the
allylic anchor-alcohol (Scheme 7). Using DIC/HOBt,
compound 23 is coupled to â-Ala-NH-PS, and 24 is
deacetylated by means of aqueous NaOH/dioxane. The
attachment of the starting amino acid Fmoc-Cys(Acm)
is achieved by activating the C-terminus with 2,6-
dichlorobenzoyl chloride32 in situ and then coupling to
the resin-bound allylic alcohol in the presence of pyridine.
This reaction takes place without affecting the thiol
function as was demonstrated by the esterification of
compound 26 in solution. It should be taken into account
that the application of this more convenient method
results in a higher propensity of racemization.
Syn th esis of Tr i- a n d Tetr a p ep tid es. In order to
investigate the properties of the HYCRON-anchor re-
garding stability and cleavability, protected tri- and
tetrapeptides 28-31 have been synthesized (Scheme 8).
The coupling reactions were accomplished with a three-
to four-fold excess of the N-protected amino acids acti-
vated by DIC and HOBt. Temporary N-Boc-groups
(Scheme 8, PG ) Boc) were removed by treating the
peptide-resin with dichloromethane/TFA (1:1). Reactions
with DMF/morpholine (4:3) for 2 h deprotected the amino
groups if the Fmoc-strategy was applied (Scheme 8, PG
) Fmoc). Removal of the N-Fmoc-groups is usually
complete in less than 45 min. However, in these syn-
theses, the cleavage times were extended in order to
facilitate the detection of undesired anchor cleavage
(Table 3). The final release of the peptides was achieved
by treating the peptide-resin with a catalytic amount of
the palladium(0)-catalyst and a ten- to fifteen-fold excess
of the scavenger nucleophile under exclusion of oxygen.
Morpholine was used for the release of N-Boc-protected
peptides and N-methylaniline for the liberation of N-
Fmoc protected peptides, since this base does not to
cleave the Fmoc-group. Detachment yields were dem-
onstrated to be in the range of 90% on average. Table 3
shows peptide losses, which occur at the stage of the
dipeptide-resin due to the formation of diketopiperazines
(DKP) and the total peptide losses caused by any other
undesired cleavage of the HYCRON-linkage. It is clear
that the application of the Boc-strategy (peptide 28) does
not result in any premature losses at all. Fmoc-cleavage
with DMF/morpholine (4:3) for 2 h leads to a considerable
amount of DKP formation (see peptides 29-31). The
(32) Sieber, P. Tetrahedron Lett. 1987, 28, 6147.

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 817
Ta ble 3. Syn th esis of P r otected Tr i- a n d Tetr a p ep tid es (see a lso Sch em e 9)
peptide-DKP,a
% yield
losses,
scavenger
R2NH
detachment,
% yieldc
peptide
PG removal
% totalb
28
29
30
31
CH2Cl2/TFA, 1:1, 45 min
DMF/morpholine, 4:3, 2 h
DMF/morpholine, 4:3, 2 h
DMF/morpholine, 4:3, 2 h
0
20
38
33
0
22
33
34
morpholine
89
81
93
96
N-methylaniline
N-methylaniline
N-methylaniline
a
b
Diketopiperazine formation ) [(lAla/lâ-Ala
)tripep-resin/(lAla/lâ-Ala
)
- 1] × 100. ((lAla/lâ-Ala
)pep-resin/(lAla/lâ-Ala
)
- 1) × 100.
dipep-resin
AA-resin
c (1 - [(lstart-AA/lâ-Ala)a/(lstart-AA/lâ-Ala)b] × 100, (l ) loading, determined by amino acid analysis, a ) after cleavage, AA ) amino acid, b
) before cleavage).
Ta ble 4. Solid P h a se Syn th esis of P ep tid e 33 a n d O-Glycop ep tid es 32, 34, a n d 35
peptide (n-mer)
PG removal (PG, conditions)
couplings
detachment, % yielda
total, % yieldb
32 (4-mer)
33 (9-mer)
1.-3.: Fmoc, DMF/morpholine 1:1, 50 min
1.: Fmoc, DMF/morpholine 1:1, 5 min
2.: Boc, CH2Cl2/TFA 1:1, 50 min
1.-3.: DIC/HOBt
1.-8.: DIC/HOBt
96
87
77c
83
3.-9.: Fmoc, DMF/morpholine 1:1, 50 min
1.: Fmoc, DMF/morpholine 1:1, 50 min
2.: Boc, CH2Cl2/TFA 1:1, 50 min
3.-9.: Fmoc, DMF/morpholine 1:1, 50min
1.: Fmoc, DMF/morpholine 1:1, 50 min
2.: Boc, CH2Cl2/TFA 1:1, 50 min
34 (9-mer)
1.-4.: DIC/HOBt
93
95
5.-6.: TBTU/HOBt/NMM
7.-8.: DIC/HOBt
35 (20-mer)
1.-19.: TBTU/HOBt/NMM
95d
45d
3.-19.: Fmoc, DMF/morpholine 1:1, 50min
a
b
(1 - [(lstart-AA/lâ-Ala)a/(lstart-AA/lâ-Ala)b] × 100. npeptide/(mstart-AA-resin/lstart-AA) × 100. c Based on loading with â-Alafminimum yield.
d
Loadings determined photometrically by Fmoc-cleavage (l ) load, a ) after cleavage, b) before cleavage, n ) amount, m ) mass).
Sch em e 9
total losses are solely caused by DKP formation since they
(1:1) for 50 min removed the Fmoc-group (Table 4). The
carbohydrate portion was introduced by coupling the
preformed glycosyl amino acid (Fmoc-Thr(RAc3GalNAc)-
OH.34 This, as well as the other coupling reactions, was
performed using DIC/HOBt. In the presence of O-acetyl
groups long coupling times, which may be necessary due
to the bulkiness of the glycoside, may result in an acetyl
shift to the amino group that is to be acylated. In order
to accelerate the elongation reaction of the resin-bound
glycodipeptide, the reagents were employed in a larger
excess. Release of the O-glycosylated tetrapeptide 32 was
accomplished in a yield of 96% by palladium(0)-catalyzed
transfer of the allyl moiety to N-methylaniline. An exact
overall yield can not be given. Calculations based on the
initial amino acid load is not reasonable because the
amino acid analysis of hydroxyamino acids generally
gives low values resulting in overestimated yields. As-
are identical the DKP losses within the experimental
error. No additional losses occur, and it can be concluded
that the HYCRON-anchor is orthogonally stable to both
the Boc- and the Fmoc-group. The amount of intramo-
lecular aminolysis of the resin-bound dipeptides can be
decreased by reducing the Fmoc-cleavage times.
Syn th esis of O-Glycop ep tid es. The efficiency of
HYCRON-anchoring in solid phase glycopeptide synthe-
sis was demonstrated by the synthesis of the protected
O-glycotetrapeptide 32 (Scheme 9). Peptide 32 is a
glycosylated variant of the N-terminal part of peptide T,
a segment of the HIV-envelope glycoprotein gp120.33
Starting from resin 21, treatment with DMF/morpholine
(33) (a) Kowalski, M.; Potz, J .; Basiripour, L.; Dorfman, T.; Goh,
W. C.; Terwilliger, E.; Dayton, A.; Rosen, G.; Haseltine, W.; Sodroski,
J . Science 1987, 237, 1351. (b) Pert, C. B.; Hill, J . M.; Ruff, M. R.;
Berman, R. M.; Robey, W. G.; Arthur, L. O.; Ruscetti, F. W.; Farrar,
W. L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9254.
(34) Kunz, H.; Birnbach, S. Angew. Chem., Int. Ed. Engl. 1986, 25,
360.

818 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
Sch em e 10
suming a threonine/â-alanine-ratio of 1, which requires
a quantitative coupling of 12b to â-Ala-NH-PS, a mini-
mum overall yield of 77% can be calculated.
Taking advantage of the HYCRON-system, glycopep-
tide structures of the MUC-1 mucine were synthesized.
Synthesis of the protected forms of the nonapeptide 33,
its TN-variant 34 and of the TN-eicosapeptide 3535 (Scheme
9), which spans the entire 20-mer MUC-1 repeating unit,
starts from resins 18 and 19, respectively. The 20-mer
is continuously repeated up to 90 times in the MUC-1
mucin. It is obvious that it would be convenient to couple
two 20-mers in order to obtain a dimer of the repeating
unit. If possible, a Pro-Gly bond should be formed in
fragment condensations, since proline is the amino acid
least prone to racemization, and the fastest couplings are
achieved when the C-terminal fragment contains N-
terminal glycine. For that reason the frame of the
repeating unit in peptide 35 is slightly moved to C-
terminal proline. Removal of the Fmoc-groups was again
accomplished by treatment with DMF/morpholine (1:1)
for 50 min. N-Boc-amino acids were used for elongation
of the amino acid-resins (Table 4). The subsequent
acidolysis of the Boc-group reduces the formation of
diketopiperazines during cleavage. The strategy was
then returned to Fmoc-protection. All acylations were
carried out in DMF. For the synthesis of 33 and 34 DIC/
HOBt-activation was used except for the couplings of the
O-glycosyl-threonine and the following aspartic acid
building block. These acylations were anticipated to be
slow, and therefore the amino acids were activated with
TBTU,36 HOBt, and N-methylmorpholine (NMM). Cou-
plings of the N-terminal amino acids proline and alanine
were each repeated once. The N-terminal alanine was
acetylated after Fmoc-removal. TBTU-activation was
applied for all coupling reactions in the synthesis of TN-
eicosapeptide 35. Treating the peptide-resins with cata-
lytic amounts of the palladium(0)-complex in the presence
of morpholine or N-methylaniline in DMSO/DMF de-
tached the peptides in yields between 87 and 95%.
Compounds 33, 34, and 35 were obtained in overall yields
of 83%, 95% and 45% respectively. The overall yields are
based on the initial amino acid load determined by amino
acid analysis or photometrically after Fmoc-cleavage. In
the case of 34 the overall yield is higher than the
detachment yield. Of course, this is impossible, but the
deviation is within the experimental error of the amino
acid analysis.
cosyl amino acids or by glycosylation of an unprotected
hydroxyl group on the solid phase. Due to the difficulties
involved in the latter method, only very little data have
been published so far. Solid phase glycosylation using
sugar oxazolines was reported, but unfortunately no
yields were given.39 A yield of 5% was achieved in a silver
triflate promoted mannosylation of a serine employing
the corresponding glycosyl bromide.40
We had developed a method in which soft electrophiles
transform carbohydrates carrying an allylic carbamate
at the anomeric position into potent glycosyl donors.41 In
order to examine the efficiency of this glycosylation, a
resin-linked peptide with an deblocked hydroxyl group
was prepared starting from 20 (Scheme 11). Fmoc-
removal was achieved with DMF/piperidine (4:3) in 10
min, and couplings were performed using DIC/HOBt.
TFA cleaved the tert-butyl ether of the fully protected
resin-bound tripeptide. The tripeptide-resin was divided
and one part subjected to the palladium(0)-catalyzed allyl
transfer to N-methylaniline. Tripeptide 38, a partial
structure of MUC-1, was obtained in a total yield of 96%.
Before glycosylation, the remaining resin-linked peptide
was dried for several days under high vacuum and
repeatedly washed with dry dichloromethane. The resin-
bound tripeptide with an unprotected hydroxyl group was
reacted with 7 equiv of the glucose donor 1-O-(N-
allylcarbamoyl)-2,3,4,6-tetra-O-acetylglucose and 2.5 equiv
of the promotor S-methylbis(methylthio)sulfonium hexa-
chloroantimonate.41 The large excess of the donor rela-
tive to the promotor was used in order to prevent an
electrophilic attack of the promotor at the double bond
of the anchor system. The glycosylation reaction was
repeated twice. After palladium(0)-catalyzed cleavage,
the glycosylated Fmoc-tripeptide 39 was isolated in an
overall yield of 4%, the unchanged Fmoc-tripeptide 38
in 64%. In the solid phase O-glucosylation of a sterically
hindered threonine-peptide a yield comparable to the
O-mannosidation of the serine-peptide described previ-
ously was achieved.
The O-glycononapeptide 34 was deprotected with TFA
in the presence of ethyl methyl sulfide and anisole as
scavengers37 (Scheme 10). A sodium methylate-catalyzed
transesterification38 removed the O-acetyl groups of the
sugar moiety yielding 37, which was purified by gel
permeation chromatography and isolated in an overall
yield of 74% relative to the loaded starting amino acid
(22 steps).
Rem ova l of P er m a n en t P r otectin g Gr ou p s a n d
O-Glycosyla tion on Solid P h a se. Solid phase synthe-
sis of O-glycopeptides is possible following two routes.
The carbohydrate can be introduced by preformed gly-
Con clu sion
The allylic HYCRON-anchoring allows the efficient
solid phase synthesis of peptides and glycopeptides in
high yields and high purity. The acid- and base-stability
(35) Dupradeau, F.-Y.; Stroud, M. R.; Boivin, D.; Li, L.; Hakomori,
S.-E.; Singhal, A. K.; Toyokoni, T. Bioorg. Med. Chem. Lett. 1994, 4,
1813.
(36) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahe-
dron Lett. 1989, 30, 1927.
(37) Atherton, E.; Sheppard, R. C.; Wade, J . D. J . Chem. Soc., Chem.
Commun. 1983, 1060.
(38) Peters, S.; Bielefeldt, T.; Meldal, M.; Bock, K.; Paulsen, H. J .
Chem. Soc., Perkin Trans. 1 1992, 1163.
(39) Hollo´si, M.; Kolla´t, E.; Laczko´, I.; Medzihradszky, K. F.; Thurin,
J .; Otvo¨s, L. Tetrahedron Lett. 1991, 32, 1531.
(40) Andrews, D. M.; Seale, P. W. Int. J . Pept. Protein Res. 1993,
42, 165.
(41) Kunz, H.; Zimmer; J . Tetrahedron Lett. 1993, 34, 2907.

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 819
DMF/morpholine (1:1), from which 50 µL was taken after 1 h.
This solution was diluted and subjected to UV-analysis (300.5
nm). Chromatography was performed on open columns if no
explanations are given in the text. MPLC was performed with
silica gel (20-45 µm) and a 430 × 80 mm column. GPC was
performed on 950 × 38 mm columns. Some compounds were
obtained in form of highly viscous oils, and complete removal
of solvents often was impossible. In these cases, yields were
calculated including the content of solvent which reaches
values up to 5 mass % as determined by NMR-spectroscopy.
These corrections are indicated by “cor”.
Sch em e 11
Analytical HPLC was performed on a LKB-system using
columns as follows: 1. Eurospher-100, C4/7 µm, 125 × 4 mm
(Knauer); 2. Eurospher-100, C4/7 µm, 250 × 8 mm (Knauer);
3. Vydac C4, 250 × 4 mm (Separations Group); 4. Spherisorb
ODSII, C18/5 µm, 250 × 4 mm (Bischoff). Gradients used for
elution were as follows: 1. 0-42 min: 20% B-100% B in A,
42-48 min: 100% B; 2. 0-28 min: 10% B-50% B in A, 28-
48 min: 50% B-100% B in A; 3. 0-2 min: 20% B in A, 2-42
min: 20% B-70% B in A, 42-43 min: 70% B-100% B in A,
43-48 min: 100% B; 4. 0-2 min: 50% B in A, 2-42 min:
50% B-80% B in A; 5. 0-2 min: 10% B in A, 2-42 min: 10%
B-40% B in A; 6. 0-2 min: 1% B in A, 2-24 min: 1% B-30%
B in A, 24-44 min: 30% B-100% B in A, 44-48 min: 100%
B; 7. 0-2 min: 30% B in A, 2-40 min: 30% B-70% B in A,
40-42 min: 70% B-100% B in A, 42-48 min: 100% B.
Eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in
acetonitrile.
12-Hyd r oxy-4,7,10-tr ioxa d od eca n oic Acid ter t-Bu tyl
Ester (3). To a solution of anhydrous triethyleneglycol (128.0
mL, 0.94 mol) in 500 mL of THF were added 0.2 g (8.7 mmol)
of sodium. When the sodium was dissolved, tert-butyl acrylate
(48.0 mL, 0.33 mol) was added. The solution was stirred for
20 h and neutralized with 8 mL of 1 M HCl. After removal of
the solvent, the residue was suspended in brine and extracted
three times with ethyl acetate. The combined organic layers
were washed with brine and dried over Na2SO4 before the
solvent was removed. The resulting colorless oil was dried in
1
vacuo to give 78.7 g (86%) of 3. 90 MHz H-NMR (CDCl3) δ )
3.75-3.50 (m, 14H, (CH2CH2O)3, H-3), 2.74 (s, 1H, OH), 2.48
(t, 2H, H-2, J ) 6.5), 1.42 (s, 9H).
(E)-17-Br om o-4,7,10,13-tetr aoxa-15-h eptadecen oic Acid
ter t-Bu tyl Ester (4a ). A solution of tetrabutylammonium
hydrogensulfate (40.74 g, 0.12 mol) and NaOH (9.75 g, 0.24
mol) in 195 mL of water was added to a mixture of 3 (33.42 g,
0.12 mol) and (E)-1,4-dibromo-2-butene (51.6 g, 0.24 mol) in
375 mL of dichloromethane. The two-phase system was
vigorously stirred for 45 min. The aqueous layer was sepa-
rated and extracted twice with 150 mL of dichloromethane.
The combined organic layers were concentrated. Addition of
400 mL of diethyl ether led to precipitation of tetrabutylam-
monium bromide which was separated by filtration. The
filtrate was washed twice with brine. The organic layer was
dried over MgSO4. After removal of the solvent, chromatog-
raphy (petroleum ether/EtOAc, 1:1) furnished 22.3 g (45%) of
a yellowish oil. 200 MHz 1H-NMR (CDCl3) δ ) 5.95-5.69 (m,
2H, H-15, H-16), 3.96 (d, 2H, H-14, J ) 4.5), 3.88 (d, 2H, H-17,
J ) 6.3), 3.65-3.54 (m, 14 H, (CH2CH2O)3, H-3), 2.42 (t, 2H,
H-2, J ) 6.3), 1.36 (s, 9H, tBu). 50.3 MHz 13C-NMR (CDCl3):
170.71 (CO), 131.55 (C-15), 128.45 (C-16), 80.29 (CMe3), 70.43,
70.33, 70.19 ((CH2CH2O)3), 69.53 (C-14), 66.71 (C-3), 36.10 (C-
2), 31.82 (C-17), 27.93 (tBu).
(E)-17-Br om o-4,7,10,13-tetr aoxa-15-h eptadecen oic Acid
P h en a cyl Ester (4b). A solution of 4a (0.80 g, 1.94 mmol)
in 13 mL of TFA was stirred for 1 h. TFA was evaporated.
The residue was dissolved in 40 mL of chloroform and washed
with 20 mL of 1 M HCl. The aqueous layer was reextracted
twice with chloroform. The combined organic layers were
dried over MgSO4 and concentrated in vacuo to yield crude 5.
Then potassium fluoride (0.32 g, 5.5 mmol) and bromoac-
etophenone (0.45 g, 2.25 mmol) were suspended in DMF (5
mL). A solution of carboxylic acid 5 in DMF (2 mL) was added.
The mixture was stirred for 45 min before the precipitate was
separated by filtration over Hyflo. The filtrate was diluted
with 50 mL of ethyl acetate. The solution was washed twice
with saturated NaHCO3-solution. The combined aqueous
of the HYCRON-anchor makes the application of both
the Fmoc- and the Boc-strategy possible. They may even
be combined in one synthesis. Peptides and glycopep-
tides are liberated under almost neutral conditions via
the palladium(0)-catalyzed allyl transfer to scavenger
nucleophiles such as morpholine or the less basic N-
methylaniline. The Fmoc-group, the Boc-group, tert-
butyl-esters, tert-butyl ethers, the O-acetyl groups in
carbohydrates, and particularly O-glycosidic bonds are
left intact. Therefore, the HYCRON-anchor will be a
versatile tool in solid phase synthesis of compounds which
demand high standards of orthogonality and also shows
properties promising for combinatorial chemistry.
Exp er im en ta l Section
Gen er a l.42 Fmoc-amino acids, (aminomethyl)polystyrene,
and Boc-â-Ala-loaded polystyrene were kindly donated by
ORPEGEN GmbH, Heidelberg, Germany. THF was freshly
distilled from potassium/benzophenone before use. Dichlo-
romethane used in the glycosylation reaction was dried over
P4O10 and freshly distilled. Reactions were carried out at room
temperature if no specifications are given. Solid phase syn-
thesis was performed manually using a reaction vessel similiar
to the Merrifield-reactor.
Amino acid-analysis (AAA) was carried out by ORPEGEN
GmbH. Photometric determination of the Fmoc-loadings was
performed by treating 1 aliquot of the resin with 2.00 mL of
(42) See also Eberling, J .; Braun, P.; Kowalczyk, D.; Schultz, M.;
Kunz, H. J . Org. Chem. 1996, 61, 2638.

820 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
layers were extracted twice with ethyl acetate. The combined
organic layers were washed with brine and dried over MgSO4.
After removal of the solvent, the residue was purified by
chromatography (petroleum ether/EtOAc, 1:2), yielding 0.58
g (63% based on 4a ) of a clear, yellowish, and viscous liquid.
200 MHz 1H-NMR (CDCl3) δ ) 7.89 (dd, 2H, o-Pac, J o,m ) 8.3,
J o,p ) 1.2), 7.59-7.43 (m, 3H, m-Pac, p-Pac), 5.89-5.85 (m,
2H, H-15, H-16), 5.34 (s, 2H, CH2-Pac), 4.01 (d, 2H, H-14, J )
4.6), 3.93 (d, 2H, H-17, J ) 6.3), 3.81 (t, 2H, H-3, J ) 6.6),
3.64-3.55 (m, 12 H, (CH2CH2O)3), 2.78 (t, 2H, H-2, J ) 6.6).
50.3 MHz 13C-NMR (CDCl3) δ ) 191.80 (CO-Pac), 170.73 (CO),
133.69 (p-Pac), 133.60 (i-Pac), 131.50 (C-15), 128.65 (o-Pac),
128.37 (C-16), 127.53 (m-Pac), 70.36, 70.25 ((CH2CH2O)3),
69.45 (C-14), 66.20, 66.11 (CH2-Pac, C-3), 34.52 (C-2), 31.86
(C-17). Anal. Calcd for C21H29BrO7: C, 53.29; H, 6.17; Br,
16.88. Found: C, 53.20; H, 6.14; Br, 16.67.
pure for stability tests. 200 MHz 1H-NMR (CDCl3) δ ) 7.28-
7.18 (m, 11H, Har, NH-Bn), 6.99 (m, 1H, NH-â-Ala), 5.77-5.68
(m, 3H, H-15, H-16, NH-Ala), 5.03 (s, 2H, CH2-Z), 4.55 (d, 2H,
H-17, J ) 3.5), 4.35-4.28 (m, 3H, AR, CH2-Bn), 3.93 (d, 2H,
H-14, J ) 3.3), 3.64-3.40 (m, 16H, (CH2CH2O)3, â-Alaâ, H-3),
2.37, 2.35 (2 × t, 4H, â-AlaR, H-2, J 1 ) 6.2, J 2 ) 5.8), 1.35 (d,
3H, Aâ, J ) 7.2). 50.3 MHz 13C-NMR (CDCl3) δ ) 172.51,
171.56, 171.16, 155.52 (CO-Z), 138.39, 136.24 (i-Bn, i-Z),
131.25, 128.39, 128.31, 127.91, 127.81, 127.59, 127.13 (C-15,
o-,m-,p-Bn, o-,m-,p-Z), 125.73 (C-16), 70.51, 70.43, 70.34, 70.16,
70.05, 69.50 (C-14), 66.97 (C-3), 64.84 (C-17), 49.63 (AR), 43.26
(CH2-Bzl), [36.75, 35.79, 35.55 (C-2, â-AlaR, â-Alaâ)], 18.29 (Aâ).
Anal. Calcd for C34H47O10N3: C, 58.08; H, 6.53; N,6.17.
Found: C, 57.84; H, 5.86; N, 6.17.
Gen er a l P r oced u r e for Syn th esis of An ch or Con ju -
ga tes 8a -12a . A 0.25 molar solution of 1 equiv of the Fmoc-
amino acid and 1 equiv of the “anchor-bromide” 4a or 4b in
dichloromethane was added to a solution consisting of 1 equiv
of tetrabutylammonium bromide in a volume of aqueous
saturated NaHCO3 which equals the volume of the organic
solvent. The two-phase system was stirred until TLC moni-
toring showed the completion of the reaction (2-18 h). The
organic layer is separated and the aqueous phase extracted
twice with dichloromethane. The combined organic layers
were dried over MgSO4, and solvents were evaporated in
vacuo. Further purification is accomplished by chromatogra-
phy.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid ter t-Bu tyl Es-
ter (8a ). Yield: 72% (cor). Rf : 0.30 (petroleum ether/EtOAc,
1:2). [R]22D ) -11.6 (c ) 1, MeOH). 200 MHz 1H-NMR (CDCl3)
δ ) 7.72 (d, 2H), 7.56 (d, 2H), 7.39-7.22 (m, 4H), 5.82-5.77
(m, 2H, H-15, H-16), 5.55 (d, 1H, ANH, J ) 7.7), 4.60 (d, 2H,
H-17, J ) 3.8), 4.40-4.34 (m, 3H, AR, CH2-Fmoc), 4.17 (t, 1H,
H-9-Fmoc, J ) 6.9), 3.97 (d, 2H, H-14, J ) 3.4), 3.67 (t, 2H,
H-3, J ) 6.6), 3.60-3.56 (m, 12H), 2.46 (t, 2H, H-2, J ) 6.6),
1.41-1.38 (m, 12H, tBu, Aâ). 50.3 MHz 13C-NMR (CDCl3) δ
) 172.51, 170.68, [155.51, 143.77, 143.64, 141.1 (Fmoc)], 131.46
(C-15), [127.51, 126.88 (Fmoc)], 125.59 (C-16), [124.88, 119.77
(Fmoc)], 80.26 (CMe3), 70.51, 70.43, 70.40, 70.31, 70.17 ((CH2-
CH2O)3), 69.54 (C-14), 66.78, 66.71 (CH2-Fmoc, C-3), 64.96 (C-
17), 49.54 (AR), 47.01 (C-9-Fmoc), 36.12 (C-2), 27.92 (tBu), 18.39
(Aâ).
(E)-17-(N-9-(F lu or en ylm et h oxyca r b on yl)glycyloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid ter t-Bu tyl Es-
ter (9a ). Yield: 65%. Rf : 0.33 (petroleum ether/EtOAc, 1:2).
200 MHz 1H-NMR (CDCl3) δ ) 7.74 (d, 2H), 7.58 (d, 2H), 7.41-
7.25 (m, 4H), 5.91-5.79 (m, 2H), 5.37 (d, 1H, GNH, J ) 5.4),
4.63 (d, 2H, H-17, J ) 4.3), 4.38 (d, 2H, CH2-Fmoc, J ) 7.0),
4.21 (t, 1H, J ) 7.0), 4.00-3.87 (m, 4H, H-14, GR), 3.71-3.54
(m, 14H), 2.47 (t, 2H, J ) 6.5), 1.42 (s, 9H). 50.3 MHz 13C-
NMR (CDCl3) δ ) 170.72, 169.58, 156.21, 143.62, 141.07,
131.60, 127.51, 126.88, 125.50, 124.89, 119.77, 80.30, 70.47,
70.37, 70.28, 70.14, 69.50, 66.92, 66.68, 64.95, 46.90, 42.54 (GR),
36.06, 27.90.
(E)-17-Br om o-4,7,10,13-tetr aoxa-15-h eptadecen oic Acid
(5). A solution of 4a (0.22 g, 3.38 mmol) in TFA (10 mL) was
stirred for 45 min. TFA was evaporated in vacuo, and the
residue was dissolved in chloroform and washed with 1 M HCl.
The aqueous phase was reextracted with chloroform. The
combined organic layers were dried over Na2SO4 and concen-
trated in vacuo. Chromatography (EtOAc/AcOH, 100:1) yielded
0.16 g (82%) of a clear, yellowish, and highly viscous oil. 200
1
MHz H-NMR (CDCl3) δ ) 5.93-5.68 (m, 2H), 3.95 (d, 2H, J
) 4.6), 3.87 (d, 2H), 3.70-3.48 (m, 14H, J ) 6.4), 2.53 (t, 2H,
J ) 6.3). 50.3 MHz 13C-NMR (CDCl3) δ ) 175.72, 131.39,
128.51, 70.27, 70.20, 70.18, 69.33, 66.11, 34.57, 31.77. Anal.
Calcd for C13H23BrO6: C, 43.96; H, 6.53. Found: C, 43.91; H,
6.51.
(E)-17-Br om o-4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-
a la n in e Ben zyla m id e (6). To a solution of the carboxylic
acid 5 (0.40 g, 1.13 mmol) in 10 mL of dry ethyl acetate were
added HONSu (0.12 g, 1.08 mmol) and DCC (0.22 g, 1.08
mmol) at 0 °C. After stirring for 30 min, the mixture was
allowed to warm up to room temperature and was stirred for
additional 16 h. The urea which precipitated upon cooling to
0 °C was filtered off. The solvent was removed, and the
residue was treated with 20 mL of acetone to complete
precipitation of urea. The filtrate was concentrated in vacuo
before 5 mL of ethanol was added. A solution of â-alanine
benzylamide (0.20 g, 1.13 mmol) in 6 mL of ethanol/water (2:
1) was added in portions. The solvents were removed after 3
h of stirring. The residue was purified by chromatography
(petroleum ether/EtOAc, 1:1, elution of 0.13 g starting material
with EtOAc, elution of product with EtOAc/EtOH, 1:1). A 0.28
g (48%) amount of a yellowish, highly viscous oil was obtained.
200 MHz 1H-NMR (DMSO-d6) δ ) 8.41 (bs, 1H), 8.38 (bs, 1H),
7.34-7.21 (m, 5H), 5.96-5.68 (m, 2H), 4.27-3.82 (m, 6H),
3.57-3.47 (m, 14H), 3.27 (dt, 2H), 2.35-2.27 (m, 4H). 50.3
MHz 13C-NMR (DMSO-d6) δ ) 170.26, 169.99, 139.43, 131.56,
128.16, 127.54, 127.11, 126.62, 70.25, 69.71, 69.61, 69.45,
69.06, 68.74, 66.73, 41.93 39.83, 35.98, 35.24.
(E)-17-(N-(Ben zyloxyca r bon yl)-L-a la n yloxy)-4,7,10,13-
tetr a oxa -15-h ep ta d ecen oyl-â-a la n in e Ben zyla m id e (7).
To a stirred solution of Z-alanine (1.66 g, 7.46 mmol) in 50
mL of methanol was added Cs2CO3 (1.21 g, 3.73 mmol). The
solvent was removed after 45 min. The residue was coevapo-
rated three times with toluene. The salt was dried in vacuo
and dissolved in 30 mL of DMF. A solution of 6 (3.5 g, 6.79
mmol) in DMF (20 mL) was added. After 18 h of stirring, the
mixture was filtered over Hyflo. The filtrate was concentrated
in vacuo and the residue dissolved in 200 mL of chloroform.
The solution was extracted twice with 80 mL of saturated
NaHCO3-solution. The aqueous layers were extracted with
50 mL of chloroform, and the combined organic layers were
dried over Na2SO4. After removal of the solvent, 5.1 g of raw
material were obtained. Purification by chromatography
(EtOAc/EtOH) yielded 3.99 g of a product which was further
purified by MPLC (EtOAc/EtOH 20:1). A second MPLC-
purification (EtOAc/EtOH 40:1) furnished 2.80 g (63%) of a
colorless, highly viscous oil with a purity of 80% as determined
by HPLC.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-p r olyloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid ter t-Bu tyl Es-
ter (10a ). Yield: 81% (cor). Rf : 0.35 (petroleum ether/EtOAc,
1:1). [R]22 ) -20.0 (c ) 0.88, CHCl3). 400 MHz 1H-NMR
D
(CDCl3, signal doubling due to Pro-(E,Z)-rotamers) δ ) 7.73,
7.72 (2x d, 2H), 7.61-7.51 (m, 2H), 7.39-7.40 (m, 2H), 7.30-
7.25 (m, 2H), 5.84-5.71 (m, 2H), 4.62-4.52 (m, 2H), 4.44-
4.22 (m, 4H, PR, CH2-Fmoc, H-9-Fmoc), 3.97 (d, 1.1H, H-14, J
) 4.6), 3.88 (d, 0.9H, H-14, J ) 5.0), 3.68-3.43 (m, 16H), 2.47
(t, 2H, J ) 6.5), 2.27-2.19 (m, 1H, Pâ1), 2.04-1.85 (m, 3H,
Pâ2, Pγ), 1.41 (s, 9H). 100.6 MHz 13C-NMR (CDCl3, signal
doubling due to Pro-(E,Z)-rotamers) δ ) 172.22, 172.15, 170.77,
154.76, 144.16, 144.12, 143.86, 143.72, 141.23, 131.49, 131.14,
127.56, 126.97, 126.16, 125.77, 125.13, 125.03, 124.92, 119.86,
80.35, 70.72, 70.52, 70.44, 70.30, 69.60, 67.40, 66.84, 64.77,
[59.23, 58.80 (2 × PR)], 47.29, 47.20, [46.92, 46.40 (2 × Pδ)],
36.25, [31.00, 29.82 (2 × Pâ)], 28.04, [24.29, 23.31 (2 × Pγ)].
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-va lyloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid ter t-Bu tyl Es-
A small amount was further purified by preparative HPLC
(Spherisorb ODSII, C18/5 µm, 250 × 40 mm, 45% H2O in
acetonitrile, 20 mL/min) in order to obtain material sufficiently
ter (11a ). Yield: 67%. Rf : 0.42 (petroleum ether/EtOAc, 1:1).
1
[R]22 ) -2.3 (c ) 1.10, CHCl3). 200 MHz H-NMR (CDCl3) δ
D

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 821
) 7.71 (d, 2H), 7.57 (d, 2H), 7.39-7.30 (m, 4H), 5.84-5.77 (m,
2H), 5.55 (d, 1H, VNH, J ) 9.1), 4.60 (d, 2H, J ) 4.1), 4.38-
4.19 (m, 4H, VR, CH2-, H-9-Fmoc), 3.97 (d, 2H, J ) 3.6), 3.67
(t, 2H, J ) 6.6), 3.60-3.57 (m, 12H), 2.46 (t, 2H, J ) 6.6), 2.16
CDCl3) δ ) 7.74 (d, 2H), 7.58 (d, 2H), 7.41-7.13 (m, 5H), 5.86-
5.80 (m, 2H), 5.40 (d, 1H, VNH, J ) 9.2), 4.62 (d, 2H, J ) 4.2),
4.40-4.17 (m, 4H, VR, CH2-, H-9-Fmoc), 4.01 (d, 2H, J ) 3.8),
3.74 (t, 2H, H-3, J ) 6.2), 3.62-3.57 (m, 12H), 2.60 (t, 2H,
H-2, J ) 6.2), 2.33-2.06 (m, 1H, Vâ), 0.95, 0.88 (2 × d, 6H,
Vγa, Vγb, J a ) J b ) 6.8).
(mc, 1H, Vâ), 1.41 (s, 9H), 0.94, 0.87 (2 × d, 6H, Vγa, Vγb, J a
)
J b ) 6.8). 50.3 MHz 13C-NMR (CDCl3) δ ) 171.50, 170.57,
156.00, 143.64, 143.45, 140.97, 131.49, 127.39, 126.77, 125.50,
124.80, 119.66, 80.10, 70.37, 70.27, 70.18, 70.05, 69.37, 66.67,
66.57, 64.69, 58.80 (VR), 46.89, 35.96, 30.94 (Vâ), 27.79, 18.75
(Vγa), 17.33 (Vγb).
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-O-ter t-bu tyl-
L-th r eon yloxy)-4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid
P h en a cyl Ester (12a ). Yield: 83% (cor). Rf : 0.32 (petroleum
ether/EtOAc, 1:2). 200 MHz 1H-NMR (CDCl3) δ ) 7.88 (dd,
2H, o-Pac), 7.74 (d, 2H, J ) 7.3), 7.65-7.24 (m, 9H, H-1-, H-8-,
H-2-, H-7-, H-3-, H-6-Fmoc, m-, p-Pac), 5.86-5.81 (m, 2H), 5.61
(d, 1H, TNH), 5.33 (s, 2H), 4.64-4.22 (m, 7H, Tâ, H-17, TR, CH2-,
H-9-Fmoc), 3.99 (d, 2H), 3.80 (t, 2H, J ) 6.6), 3.63-3.55 (m,
12H), 2.78 (t, 2H, J ) 6.6), 1.22 (d, 3H, Tγ, J ) 6.6), 1.11 (s,
9H).
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-O-ter t-bu tyl-
L-th r eon yloxy)-4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid
(12b). To a stirred solution of 12a (2.62 g, 3.13 mmol)cor in 40
mL of glacial acetic acid was added 3.5 g of freshly activated
zinc. After 2 h zinc was removed by filtration over Hyflo
followed by distillation of the filtrate. The residue was
dissolved in dichloromethane and washed with 0.5 M HCl. The
aqueous layer was reextracted with dichloromethane. The
combined organic layers were dried over MgSO4, and the
solvent was removed by distillation. The residue was purified
by chromatography (EtOAc/AcOH, 100:1). Coevaporation with
toluene yielded 2.11 g (97%, cor) of a clear and colorless, highly
viscous oil. Rf: 0.11 (EtOAc/AcOH, 100:1). [R]22 ) 1.54 (c )
D
1
1.0, toluene). 200 MHz H-NMR (CDCl3) δ ) 7.74 (d, 2H, J )
Gen er a l P r oced u r e for Syn th esis of An ch or -Con ju -
ga tes 8b-11b. The tert-butyl esters 8a -11a were treated
with TFA for 45 min. After evaporation of TFA in vacuo, the
residue was dissolved in chloroform. The solution was washed
with 1 M HCl. The aqueous phase was extracted twice with
chloroform. The combined organic layers were washed with
brine and dried over Na2SO4 prior to removal of the solvent.
The residue is purified by chromatography. The eluents
usually contain small amounts of acetic acid, which were
removed by coevaporation with toluene.
7.3), 7.65-7.59 (m, 2H), 7.41-7.25 (m, 4H), 5.86-5.80 (m, 2H),
5.68 (d, 1H, TNH, J ) 9.6), 4.65-4.22 (m, 7H, Tâ, H-17, TR,
CH2-, H-9-Fmoc), 4.00 (d, 2H, J ) 3.6), 3.73 (t, 2H, J ) 6.3),
3.67-3.59 (m, 12H), 2.60 (t, 2H, H-2, J ) 6.3), 1.22 (d, 3H, Tγ,
J ) 6.2), 1.12 (s, 9H). 50.3 MHz 13C-NMR (CDCl3) δ ) 175.03,
170.64 (TCO), 156.63, 143.74, 143.49, 140.98, 131.37, 127.43,
126.83, 125.64, 124.98, 124.92, 119.68, 73.89, 70.42, 70.26,
70.18, 70.06, 69.34, 67.14 (Tâ), 67.00, 66.26, 64.94, 59.66 (TR),
46.88, 34.53 (C-2), 28.12, 20.63 (Tγ).
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oyl-â-a la n in e P h en a cyl
Ester (13). A solution of DCC (1.06 g, 5.14 mmol) in 10 mL
of dichloromethane was added to a solution of 8b (3.53 g, 4.67
mmol)korr and HONSu (0.54 g, 4.70 mmol) in 25 mL of
dichloromethane and was stirred for 25 min. The resulting
solution was combined with a mixture of â-alanine phenacyl
ester hydrochloride (1.13 g, 4.67 mmol) and diisopropylethyl-
amine (DIEA, 0.80 mL, 4.67 mmol) in 20 mL of dry ethyl
acetate, which had been stirred for 15 min. The urea precipi-
tated after 17 h and was filtered off. The filtrate was
concentrated in vacuo. The residue was dissolved in 100 mL
of acetone and cooled to 0 °C for 16 h. The mixture was filtered
again prior to removal of the solvent in vacuo. The residue
was dissolved in chloroform and washed with saturated
NaHCO3 and 1 M HCl solution and brine. After each extrac-
tion the aqueous layers were reextracted two to three times.
The combined organic layers were dried over Na2SO4, and the
solvent was evaporated in vacuo. The residue was purified
by chromatography (EtOAc/EtOH, 9:1), which yielded 3.17 g
(82%, cor) of a clear, yellowish and viscous oil. Rf: 0.29
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-tetr aoxa-15-h eptadecen oic Acid (8b). Yield: 84%
(cor). Rf : 0.27 (EtOAc/AcOH, 100:1). [R]22 ) 6.0 (c ) 1.00,
D
toluene). 200 MHz 1H-NMR (CDCl3) δ ) 7.75 (d, 2H), 7.59 (d,
2H), 7.43-7.14 (m, 4H), 5.94-5.80 (m, 2H), 5.47 (d, 1H, J )
7.7), 4.64 (d, 2H, J ) 3.7), 4.43-4.30 (m, 3H, AR, CH2-Fmoc),
4.21 (t, 1H, J ) 6.8), 4.02 (d, 2H, J ) 3.3), 3.75 (t, 2H, H-3, J
) 6.2), 3.65-3.52 (m, 12H), 2.60 (t, 2H, H-2, J ) 6.2), 1.43 (d,
3H, Aâ, J ) 7.2). 50.3 MHz 13C-NMR (CDCl3) δ ) 175.25
(COOH), 172.69 (ACO), 155.62, 143.73, 143.58, 141.10, 131.26,
127.54, 126.91, 125.79, 124.91, 119.81, 70.52, 70.37, 70.28,
70.20, 70.13, 69.45, 66.82, 66.27, 65.02, 49.52 (AR), 46.93, 34.70
(C-2), 18.38 (Aâ).
(E)-17-(N-(9-F lu or en ylm et h oxyca r b on yl)glycyloxy)-
4,7,10,13-tetr aoxa-15-h eptadecen oic Acid (9b). Yield: 88%.
1
Rf : 0.37 (EtOAc/AcOH, 100:1). 200 MHz H-NMR (CDCl3) δ
) 7.73 (d, 2H), 7.58 (d, 2H), 7.41-7.24 (m, 4H), 5.85-5.78 (m,
2H), 5.50 (d, 1H, GNH, J ) 5.5), 4.63 (d, 2H, J ) 4.1), 4.38 (d,
2H, J ) 7.0), 4.20 (t, 1H, J ) 7.0), 4.00-3.90 (m, 4H, H-14,
GR), 3.75 (t, 2H, H-3, J ) 6.2), 3.61-3.54 (m, 12H), 2.58 (t,
2H, H-2, J ) 6.2). 100.6 MHz 13C-NMR (CDCl3) δ ) 175.03
(COOH), 169.74 (GCO), 156.34, 143.68, 141.12, 131.45, 127.56,
126.93, 125.72, 124.94, 119.81, 70.49, 70.37, 70.29, 70.19,
70.14, 69.48, 67.02, 66.32, 64.97, 46.96, 42.59 (GR), 34.72 (C-
2).
1
(EtOAc/EtOH, 9:1). 200 MHz H-NMR (CDCl3) δ ) 7.88 (dd,
2H), 7.74 (d, 2H, J ) 7.7), 7.60-7.23 (m, 9H), 7.11 (m, 1H,
â-AlaNH), 5.84-5.79 (m, 2H), 5.41 (d, 1H, ANH), 5.37 (s, 2H),
4.62 (d, 2H), 4.41-4.34 (m, 3H, AR, CH2-Fmoc), 4.21 (t, 1H, J
) 6.9), 3.98 (d, 2H, J ) 3.5), 3.74 (t, 2H, J ) 6.0), 3.65-3.56
(m, 14H, (CH2CH2O)3, â-Alaâ), 2.68 (t, 2H, â-AlaR, J ) 6.0),
2.50 (t, 2H, H-2, J ) 6.0), 1.42 (d, 3H, Aâ, J ) 7.2). 50.3 MHz
13C-NMR (CDCl3) δ ) 192.10, 172.50, 171.32, 155.50, 143.68,
143.53, 141.00, 133.87, 133.62, 131.23, 128.68, 127.52, 127.46,
126.83, 125.59, 124.83, 119.72, 70.44, 70.30, 70.25, 70.07,
69.99, 69.85, 69.40 (C-14), 66.90, 66.67, 65.86, 64.88, 49.45,
46.85, [36.68, 34.79, 34.04 (C-2, â-AlaR, â-Alaâ)], 18.24.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-p r olyloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid (10b). Yield:
85% (cor). Rf: 0.25 (EtOAc/AcOH, 100:1). [R]22 ) -15.83 (c
D
1
) 1.05, toluene). 200 MHz H-NMR (CDCl3, signal doubling
due to Pro-(E,Z)-isomers) δ ) 7.74 (d, 2H), 7.60-7.56 (m, 2H),
7.42-7.03 (m, 4H), 5.83-5.75 (m, 2H), 4.63, 4.54 (2 × d, 2H,
H-17, J 1 ) 3.4, J 2 ) 4.4), 4.44-4.28 (m, 4H, PR, CH2-, H-9-
Fmoc), 4.01 (d, 1.1H, H-14, J ) 3.9), 3.91 (d, 0.9H, H-14, J )
4.3), 3.74 (t, 2H, H-3, J ) 6.2), 3.62-3.51 (m, 14H, Pδ, (CH2-
CH2O)3), 2.60 (t, 2H, H-2, J ) 6.2), 2.32-2.15 (m, 1H, Pâ1),
2.08-1.96 (m, 3H, Pâ2, Pγ). 100.6 MHz 13C-NMR (CDCl3, signal
doubling due to Pro-(E,Z)-isomers) δ ) 175.30 (COOH), 172.12
(PCO), 154.82, 154.39, 143.94, 143.67, 143.54, 141.10, 131.15,
130.80, 128.85, 128.05, 127.52, 126.88, 126,29, 125.90, 125.12,
125.02, 119.79, 70.61, 70.48, 70.33, 70.19, 70.11, 69.33, 67.41,
66.26, 64.74, [59.10, 58.70 (2 × PR)], 47.09, 47.01, [46.84, 46.34
(2 × Pδ)], 34.68 (C-2), [30.88, 29.74 (2 × Pâ)], [24.18, 23.21 (2
× Pγ)].
(E)-17-Br om o-4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-
a la n in e P h en a cyl E st er (14). To a solution of 5 (9.74 g,
27.42 mmol) in 150 mL of dichloromethane HONSu (3.15 g,
27.37 mmol) was added. At 0 °C DCC (5.94 g, 28.79 mmol)
was added. After stirring for 30 min, the mixture was allowed
to warm up to room temperature. Urea was removed by
filtration. For completion of precipitation the filtrate was
concentrated in vacuo and triturated with acetone. After 14
h at 0 °C the solution was filtered and concentrated to dryness.
The residue was dissolved in a mixture of ethyl acetate (190
mL) and ethanol (50 mL). The resulting solution was com-
bined with a mixture of â-alanine phenacyl ester hydrochloride
(6.67 g, 27.4 mmol) and DIEA (4.7 mL, 27.4 mmol) in 160 mL
of ethyl acetate, which already had been stirred for 10 min.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-va lyloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oic Acid (11b). Yield:
57%. Rf : 0.39 (EtOAc/AcOH, 100:1). 1H-NMR (200 MHz,

822 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
After 3 h the solvents were evaporated. The residue was
dissolved in chloroform and washed twice with 0.5 M HCl. The
aqueous layers were reextracted with chloroform. The com-
bined organic layers were washed with saturated NaHCO3
solution and brine and dried over Na2SO4 before the solvent
was removed in vacuo. Chromatography (EtOAc/EtOH, 8:1)
yielded 9.5 g (64%) of a clear and colorless oil. Rf : 0.25 (EE:
14H, (CH2CH2O)3, H-3), 3.48 (q, 2H, â-Alaâ, J â,R ) J â,NH ) 6.1),
2.50 (t, 2H, â-AlaR, J ) 6.2), 2.43 (t, 2H, J ) 5.8), 1.39 (s, 9H),
1.34 (d, 3H, J ) 7.1). 50.3 MHz 13C-NMR (CDCl3) δ ) 174.33
(COOH), 172.89, 172.24, 130.92, 125.95, 79.37, 70.49, 70.12,
69.31, 66.96, 64.72, 49.04, 36.46, 34.80, 33.74, 28.09, 18.25.
Gen er a l P r oced u r e for Atta ch in g Am in o Acid -An ch or -
Con ju ga tes 8b-12b to th e Resin . tert-Butyloxycarbonyl-â-
alanyl-(aminomethyl)polystyrene (Boc-â-Ala-PS, 1 g, 1.0-1.2
mmol of â-Ala/g) was shaken with 20 mL of dichloromethane/
TFA (1:1) for 45 min. The resin was washed with dichlo-
romethane (6 × 10 mL). After 10 min of shaking with 20 mL
of 10% DIEA in dichloromethane, the resin was washed with
dichloromethane (6 × 10 mL). Conjugates 8b-12b were
coupled using 3.0 equiv of the carboxylic acid, 3.3 equiv of DIC,
and 4.5 equiv of HOBt in the lowest amount of dichlo-
romethane needed for suspending the resin. After 10-15 h
of shaking the resin was washed (6 × 10 mL dichloromethane)
and treated with 20 mL of pyridine/acetic anhydride (3:1) for
10 min in order to accomplish capping. The resin was washed
(6 × 10 mL of dichloromethane) and dried in vacuo.
1
EtOH, 8:1). 200 MHz H-NMR (CDCl3) δ ) 7.81 (dd, 2H, J o,m
) 8.3, J o,p ) 1.2), 7.57-7.36 (m, 3H), 7.11 (t, 1H, NH-â-Ala, J
) 5.5), 5.80-5.73 (m, 2H), 5.30 (s, 2H), 3.95-3.82 (m, 4H,
H-14, H-17), 3.64 (t, 2H, H-3, J ) 6.1), 3.55-3.32 (m, 14 H,
(CH2CH2O)3, â-Alaâ), 2.60 (t, 2H, â-AlaR, J ) 6.2), 2.41 (t, 2H,
H-2, J ) 6.1). 50.3 MHz 13C-NMR (CDCl3) δ ) 192.10, 171.30,
133.88, 133.60, 130.88 (C-15), 128.67, 127.99 (C-16), 127.51,
70.21, 70.04, 69.95, 69.34 (C-14), [66.90, 65.85 (Pac-CH2, C-3)],
[36.64, 34.74, 33.99 (C-2, â-AlaR, â-Alaâ, C-17)].
(E)-17-(N-(ter t-Bu tyloxyca r bon yl)-L-a la n yloxy)-4,7,10,-
13-tetr a oxa -15-h ep ta d ecen oyl-â-a la n in e P h en a cyl Ester
(15). A solution of Boc-Ala-OH (3.62 g, 19.74 mmol) and Cs2-
CO3 (3.12 g, 9.57 mmol) in 100 mL of methanol was stirred
for 45 min. After removal of methanol in vacuo, the residue
was coevaporated with toluene. The dry salt was dissolved
in 120 mL of dry DMF, and 3.47 g (6.37 mmol) of bromide 14
was added. After 18 h of stirring, cesium bromide was
removed by filtration over Hyflo. The filtrate was concentrated
in vacuo. The residue was dissolved in chloroform and washed
twice with saturated NaHCO3 solution. The combined aque-
ous phases were reextracted with chloroform. After drying
the combined organic layers over Na2SO4, the solvents were
evaporated. Chromatography (EtOAc/EtOH, 8:1) furnished
3.84 g (92%) of a clear, yellowish, and highly viscous oil. Rf :
0.34 (EE:EtOH, 6:1). 200 MHz 1H-NMR (CDCl3) δ ) 7.80 (dd,
2H), 7.56-7.35 (m, 3H), 7.11 (t, 1H, J ) 5.8), 5.74-5.67 (m,
2H), 5.30 (s, 2H), 5.20 (d, 1H, ANH, J ) 8.5), 4.50 (d, 2H, H-17,
J ) 4.2), 4.18-3.97 (m, 1H, AR), 3.89 (d, 2H, H-14, J ) 3.7),
3.64 (t, 2H, J ) 6.1), 3.54-3.43 (m, 14H), 2.59 (t, 2H, J ) 6.2),
2.40 (t, 2H, J ) 6.1), 1.32 (s, 9H), 1.26 (d, 3H, Aâ, J ) 7.2).
50.3 MHz 13C-NMR (CDCl3) δ ) 192.06, 172.72, 171.23, 133.83,
133.60, 130.94, 128.63, 127.48, 125.69 (C-16), 79.37, 70.40,
70.18, 70.01, 69.92, 69.31, 66.87, 65.82, 64.59 (C-17), 48.95 (AR),
36.60, 34.71, 33.95, 28.00 (Boc), 18.14 (Aâ).
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-a la n yl (Am in o-
m eth yl)p olystyr en e (17a ). Starting from 1.62 g (1.57 mmol
of â-Ala) of Boc-â-Ala-NH-PS (0.97 mmol of â-Ala/g) and 3.25
g (4.72 mmol)cor of Fmoc-Ala-HYCRON-OH (8b), 1.66 g of
amino acid-resin was obtained. Loading (AAA): 0.52 mmol
of Ala/g, 0.58 mmol of â-Ala/g. Yield: 89%.
(E)-17-(N-(9-F lu or en ylm et h oxyca r b on yl)glycyloxy)-
4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-a la n yl (Am in o-
m eth yl)p olystyr en e (18). Starting from 1.61 g (1.93 mmol
of â-Ala) of Boc-â-Ala-NH-PS (1.20 mmol of â-Ala/g) and 3.60
g (6.2 mmol)cor of Fmoc-Gly-HYCRON-OH (9b), 2.24 g of amino
acid-resin was obtained. Loading (AAA): 0.50 mmol of Gly/g,
0.63 mmol of â-Ala/g. Yield: 80%.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-p r olyloxy)-
4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-a la n yl (Am in o-
m eth yl)p olystyr en e (19). Starting from 2.20 g (2.64 mmol
of â-Ala) of Boc-â-Ala-NH-PS (1.20 mmol â-Ala/g) and 5.53 g
(8.5 mmol)cor of Fmoc-Pro-HYCRON-OH (10b), 3.11 g of amino
acid-resin was obtained. Loading (photometrically by Fmoc-
cleavage): 0.52 mmol of Fmoc/g. Yield: 61%.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-va lyloxy)-
4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-a la n yl (Am in o-
m eth yl)p olystyr en e (20). Starting from 1.10 g (1.32 mmol
â-Ala) of Boc-â-Ala-NH-PS (1.20 mmol â-Ala/g) and 2.47 g (3.9
mmol)cor of Fmoc-Val-HYCRON-OH (11b), 1.60 g of amino
acid-resin was obtained. Loading (AAA): 0.42 mmol Val/g,
0.56 mmol â-Ala/g. Yield: 75%.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-O-ter t-bu tyl-
L-t h r eon yloxy)-4,7,10,13-t et r a oxa -15-h ep t a d ecen oyl-â-
a la n yl (Am in om eth yl)p olystyr en e (21). Starting from 0.83
g (1.00 mmol â-Ala) of Boc-â-Ala-NH-PS (1.20 mmol â-Ala/g)
and 2.04 g (2.9 mmol)cor of Fmoc-Thr(tBu)-HYCRON-OH (12b),
1.33 g of amino acid-resin was obtained. Loading (AAA):
>0.40 mmol of Thr/g, 0.61 mmol of â-Ala/g. Yield: >66%.
Gen er a l P r oced u r e for Atta ch in g Am in o Acid -An ch or -
â-Ala n in e Con ju ga tes 16a a n d 16b to th e Resin . (Ami-
nomethyl)polystyrene (1 g, 1.55 mmol NH2/g) was suspended
in a solution of 3 equiv of the amino acid-anchor-â-alanine
conjugate, 3.3 equiv of DIC, and 4.5 equiv of HOBt in 40 mL
of dichloromethane and shaken for 15 h. After washing
(dichloromethane, 6 × 15 mL), the resin was shaken in 30 mL
of pyridine/acetic anhydride (3:1) for 10 min in order to
accomplish capping. The resin was washed (dichloromethane,
7 × 15 mL) and dried in vacuo.
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-tetr a oxa -15-h ep ta d ecen oyl-â-a la n in e (16a ). The
Pac-ester 13 (2.97 g, 3.6 mmolcor) was dissolved in 45 mL of
glacial acetic acid. After addition of 3.76 g (57.5 mmol) of
activated zinc the suspension was stirred for 2.5 h. Workup
was performed as described in synthesis of 12b. The chro-
matographic purification (EtOAc/EtOH/AcOH, 4:1:0.1) yielded
2.27 g (88%, cor) of a clear, yellowish, and highly viscous oil.
Rf : 0.18 (EtOAc/EtOH/AcOH, 4:1:0.05). [R]22 ) -0.93 (c )
D
1.25, EE). 200 MHz 1H-NMR (CDCl3) δ ) 7.75 (d, 2H, J )
7.1), 7.59 (d, 2H, J ) 8.5), 7.42-7.26 (m, 4H), 7.08 (t, 1H,
â-AlaNH, J ) 8.4), 5.84-5.78 (m, 2H), 5.55 (d, 1H, J ) 7.8),
4.63 (d, 2H, J ) 3.3), 4.43-4.30 (m, 3H), 4.21 (t, 1H, J ) 6.9),
4.00 (d, 2H, J ) 3.0), 3.70-3.46 (m, 16H, (CH2CH2O)3, H-3,
â-Alaâ), 2.53 (t, 2H, â-AlaR, J ) 6.0), 2.45 (t, 2H, J ) 5.7), 1.43
(d, 3H, J ) 7.1). 50.3 MHz 13C-NMR (CDCl3) δ ) 174.26
(COOH), 172.69, 172.05, 155.66, 143.74, 143.59, 141.11, 131.12,
127.57, 126.93, 125.89, 124.94, 119.83, 70.51, 70.40, 70.18,
69.35, 67.08, 66.83, 64.98, 49.54, 46.94, [36.63, 34.81, 33.91
(C-2, â-AlaR, â-Alaâ)], 18.36.
(E)-17-(N-(ter t-Bu tyloxyca r bon yl)-L-a la n yloxy)-4,7,10,-
13-tetr a oxa -15-h ep ta -d ecen oyl-â-a la n in e (16b). To a solu-
tion of 15 (5.9 g, 9.0 mmol) in 90 mL of glacial acetic acid
freshly activated zinc (9.4 g, 0.144 mol) was added. The
suspension was stirred for 3 h. Work-up was performed as
described in synthesis of 12b. In addition, the combined
organic layers were washed with brine, which after extraction
was reextracted four times with small portions of chloroform.
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. Purification by chromatography (EtOAc/
EtOH/AcOH, 4:1:0.05) yielded 4.09 g (82%, cor) of a clear,
colorless, and highly viscous oil. Rf: 0.19 (EtOAc/EtOH/AcOH,
4:1:0.05). 200 MHz 1H-NMR (CDCl3) δ ) 7.02 (m, 1H,
â-AlaNH), 5.82-5.76 (m, 2H), 5.20 (d, 1H), 4.59 (d, 2H, J ) 3.9),
4.27-4.23 (m, 1H, AR), 3.98 (d, 2H, J ) 3.5), 3.69-3.58 (m,
(E)-17-(N-(9-F lu or en ylm eth oxyca r bon yl)-L-a la n yloxy)-
4,7,10,13-tetr aoxa-15-h eptadecen oyl-â-alan yl(Am in om eth -
yl)p olystyr en e (17b). Starting from 0.65 g (1.0 mmol NH2)
of (aminomethyl)polystyrene and 2.27 g (3.15 mmol)cor of Fmoc-
Ala-HYCRON-â-Ala-OH (16a ), 1.24 g of amino acid-resin was
obtained. Loading (AAA): 0.71 mmol of Ala/g, 0.73 mmol of
â-Ala/g. Yield: 89%.
(E)-17-(ter t-Bu t yloxyca r b on yl-L-a la n yloxy)-4,7,10,13-
tetr a oxa -15-h ep ta d ecen oyl-â-a la n yl (Am in om eth yl)p oly-
styr en e (17c). Starting from (aminomethyl)polystyrene (0.73
g, 1.13 mmol NH2) and 1.82 g (3.28 mmol)cor of Boc-Ala-

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 823
HYCRON-â-Ala-OH (16b), 1.22 g of amino acid-resin was
obtained. Loading (AAA): 0.67 mmol of Ala/g, 0.74 mmol of
â-Ala/g. Yield: 75%.
3.62-3.55 (m, 12H), 2.47 (t, 2H, J ) 6.6), 1.42 (s, 9H). 50.3
MHz 13C-NMR (CDCl3) δ ) 170.91, 132.50 (C-15), 127.34 (C-
16), 80.49, 70.53, 70.41, 70.28, 69.35, 66.81, 62.59 (C-17), 36.20,
28.03. Anal. Calcd for C17H32O7: C, 58.60; H, 9.26. Found:
C, 59.05; H, 9.32.
(E)-17-Acetoxy-4,7,10,13-tetr aoxa-15-h eptadecen oic Acid
ter t-Bu tyl Ester (22). To a solution of cesium acetate (4.50
g, 23.4 mmol) in 250 mL of DMF was added 6.24 g (15.2 mmol)
of 4a . After 3 h of stirring, 1.50 g (7.8 mmol) of cesium acetate
was added. The mixture was stirred for additional 2 h.
Cesium bromide was separated by filtration over Hyflo, the
filtrate was concentrated in vacuo, and the residue was
dissolved in diethyl ether. The solution was washed four times
with brine and dried over MgSO4. The solvents were removed
in vacuo. After careful drying, 5.06 g (85%) of a clear oil was
obtained. Rf : 0.36 (petroleum ether/EtOAc, 1:1). 200 MHz
1H-NMR (CDCl3) δ ) 5.70-5.60 (m, 2H), 4.45 (s, 2H, H-17),
3.92 (s, 2H, H-14), 3.62-3.50 (m, 14H), 2.38 (t, 2H, J ) 7.0),
1.95 (s, 3H, Ac), 1.33 (s, 9H). 50.3 MHz 13C-NMR (CDCl3) δ )
170.58, 170.30, 130.78 (C-15), 126.33 (C-16), 80.17, 70.61,
70.44, 70.41, 70.32, 70.17, 69.49, 66.70, 63.96 (C-17), 36.12,
27.89, 20.60 (Ac). Anal. Calcd for C19H34O8: C, 58.44; H, 8.78.
Found: C, 57.76; H, 8.95.
(E)-17-(N-(9-F lu or en ylm et h oxyca r b on yl)-S-(a cet a m i-
d om et h yl)-L-cyst ein yloxy)-4,7,10,13-t et r a oxa -15-h ep t a -
d ecen oic Acid ter t-Bu tyl Ester (27). Pyridine was distilled
from 26 (174 mg, 0.50 mmol) for drying. The residue was
dissolved in a solution of Fmoc-Cys(Acm)-OH (415 mg, 1.0
mmol) in DMF (4 mL). After addition of pyridine (1.28 mL,
1.50 mmol), 2,6-dichlorobenzoyl chloride (0.14 mL, 1.00 mmol)
was added. The solution was stirred for 24 h followed by
removal of the solvent in vacuo. The residue was dissolved in
dichloromethane and washed with saturated NaHCO3 solu-
tion, 1 M HCl, and brine. The aqueous layers were reextracted
with dichloromethane. The combined organic layers were
dried over MgSO4 and concentrated in vacuo. The residue was
purified by chromatography (EtOAc), which furnished 251 mg
(68%) of a highly viscous oil. Rf : 0.24 (EtOAc). [R]22D ) -2.7
1
(c ) 1.00, CHCl3). 200 MHz H-NMR (DMSO-d6) δ ) 8.52 (t,
1H, NH-Acm, J ) 6.7), 7.94-7.87 (m, 3H, H-4-, H-5-Fmoc,
CNH), 7.71 (d, 2H, J ) 7.2), 7.45-7.26 (m, 4H), 5.82-5.73 (m,
2H), 4.60 (d, 2H, H-17, J ) 5.2), 4.31-4.19 (m, 6H, CR, CH2-,
H-9-Fmoc, CH2-Acm), 3.90 (d, 2H, H-14, J ) 4.2), 3.56 (t, 2H,
J ) 6.3), 3.50-3.31 (m, 12H), 3.00 (dd, 1H, Câa, J â,â ) 13.7,
J â,R ) 5.0), 2.85 (dd, 1H, Câb, J â,â ) 13.7), 2.39 (t, 2H, J ) 6.2),
1.83 (s, 3H, CH3-Acm), 1.38 (m, 9H). 50.3 MHz 13C-NMR
(DMSO-d6) δ ) 170.54, 170.34, 169.47, 155.92, 143.66, 140.66,
130.67 (C-15), 127.58, 127.00, 125.40 (C-16), 125.16, 120.02,
79.63, 69.69, 68.99, 66.14, 65.78, 64.35 (C-17), 54.04 (CR), 46.53,
39.80 (CH2-Acm), 35.76, 31.41 (Câ), 27.63, 22.45 (CH3-Acm).
Gen er a l P r oced u r e for Solid P h a se Syn th esis of 28-
35 a n d 38. The following amounts of chemicals are based on
a synthesis starting from 1 g of the amino acid loaded resin.
Boc-removal: 20 mL of dichloromethane/TFA (1:1), 45 min,
washing (dichloromethane, 6 × 15 mL). Neutralization:
dichloromethane/DIEA (10:1, 15 mL), 10 min, washing (dichlo-
romethane, 6 × 15 mL). Fmoc-removal: Method A: DMF/
morpholine (4:3, 25 mL), 2 h 30 min. Method B: DMF/
morpholine (1:1, 20 mL), 50 min. Method C: DMF/piperidine
(4:3, 20 mL), 10 min; washing (DMF, 6 × 15 mL). Couplings:
3.0-5.0 equiv of Boc- or Fmoc-amino acid, 4.5-7.5 equiv of
HOBt in dichloromethane/DMF (10:1) or DMF (resulting
concentration 0.12-0.17 M), 3.3-5.5 equiv of DIC (method A)
or 3.0-5.0 equiv of TBTU, 6.0-10.0 equiv of NMM (method
B), 3-16 h. Washing: dichloromethane or DMF (6 × 15 mL).
Incorporation of amino acids with side chain functions was
achieved by using amino acid derivatives as follows: Fmoc-
Arg(Mtr)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH. The
GalNAc-structure was introduced by coupling the preformed
glycosyl-amino acid Fmoc-Thr(RAc3GalNAc)-OH.34 Capping:
pyridine/acetic anhydride (3:1, 15 mL), 10 min, washing
(dichloromethane or DMF, 6 × 15 mL).
(E)-17-Acetoxy-4,7,10,13-tetr aoxa-15-h eptadecen oic Acid
(23). A solution of 3.00 g (7.68 mmol) of 22 in 40 mL of TFA
was stirred for 45 min. TFA was removed in vacuo and the
residue coevaporated with toluene. Chromatography (petro-
leum ether/EtOAc/AcOH) yielded 2.06 g (90%) of a yellowish
oil. Rf : 0.11 (petroleum ether/EtOAc, 1:1). 200 MHz 1H-NMR
(CDCl3) δ ) 9.70 (s, 1H, COOH), 5.77-5.72 (m, 2H), 4.47 (d,
2H, J ) 4.1)), 3.95 (d, 2H, J ) 3.8), 3.67 (t, 2H, H-3, J ) 6.3),
3.56-3.49 (m, 12 H), 2.53 (t, 2H, H-2, J ) 6.3), 1.98 (s, 3H).
50.3 MHz 13C-NMR (CDCl3) δ ) 175.33 (COOH), 170.63,
130.58, 126.47, 70.58, 70.29, 70.24, 70.10, 69.30, 66.24 (C-3),
64.03, 34.67 (C-2), 20.67. Anal. Calcd for C15H26O8: C, 53.88;
H, 7.84. Found: C, 54.01; H, 7.66.
(E)-17-Acetoxy-4,7,10,13-tetr a oxa -15-h ep ta d ecen oyl-â-
a la n yl (Am in om eth yl)p olystyr en e (24). Boc-removal from
1.11 g (1.67 mmol of â-Ala) of Boc-â-Ala-NH-PS (1.5 mmol of
â-Ala/g) and subsequent neutralization was performed as
described (see 17a -21). The â-alanyl-resin was suspended in
a solution of 23 (1.67 g, 5.00 mmol), HOBt (1.22 g, 7.50 mmol),
and DIC (0.85 mL, 5.50 mmol) in 20 mL of dichloromethane,
shaken for 16 h, and washed (dichloromethane, 6 × 20 mL).
Unreacted amino groups were capped by shaking in pyridine/
acetic anhydride (3:1, 20 mL). After washing (dichloromethane,
6 × 20 mL) and drying in vacuo, 1.30 g of 24 was obtained.
(E)-17-(N-(9-F lu or en ylm et h oxyca r b on yl)-S-(a cet a m i-
d om et h yl)-L-cyst ein yloxy)-4,7,10,13-t et r a oxa -15-h ep t a -
d ecen oyl-â-a la n yl (Am in om eth yl)p olystyr en e (25).
A
solution of 1 N NaOH (34 mL) in 1,4-dioxane (100 mL) was
added to 1.30 g of resin 24. The suspension was shaken for
20 min. After washing, this procedure was repeated once. The
resin was washed [2 × 20 mL of dioxane, 2 × 20 mL of dioxane/
water (1:1), 2 × 20 mL of water, 2 × 20 mL of DMF, 2 × 20
mL of dichloromethane] and dried, yielding 1.26 g of the dry
hydroxyallyl-polymer. A solution of 1.24 g (3.00 mmol) of
Fmoc-Cys(Acm)-OH, 0.36 mL (4.50 mmol) of pyridine, and 0.42
mL (3.00 mmol) of 2,6-dichlorobenzoyl chloride in DMF (10
mL) was added to 1.0 g of this resin. After 24 h the resin was
washed (6 × 20 mL of DMF) followed by addition of 15 mL of
pyridine and 4 mL of benzoyl chloride. The suspension was
shaken for 10 min, washed (4 × 20 mL of DMF, 4 × 20 mL of
dichloromethane), and dried in vacuo yielding 1.38 g of amino
acid-resin. Loading (photometrically by Fmoc-cleavage): 0.59
mmol Fmoc/g.
Boc-Leu -P h e-Ala -OH (28). The synthesis was carried out
via Boc-strategy starting from 1.18 g (0.79 mmol Ala) of resin
17c (0.67 mmol Ala/g, 0.74 mmol â-Ala/g). Boc-amino acids
were coupled using DIC. The peptide-resin was dried in vacuo,
yielding 1.40 g of the polymer (0.43 mmol Phe/g, 0.45 mmol
Ala/g, 0.45 mmol â-Ala/g). The peptide was released by
suspending the peptide-resin in a solution of dichloromethane/
THF (3:1, 20 mL) and of morpholine (0.7 mL, 8.0 mmol)
followed by multiple evacuations and subsequent argon-
streamings. After adding tetrakis(triphenylphosphine)pal-
ladium (50 mg, 0.04 mmol) the oxygen-free suspension was
shaken for 17 h under exclusion of light in an argon atmo-
sphere. Washing (dichloromethane, 6 × 20 mL) and drying
in vacuo yielded a polymer with loadings as follows: 0.08 mmol
of Leu/g, 0.08 mmol of Phe/g, 0.08 mmol of Ala/g, 0.73 mmol
of â-Ala/g (89% detachment yield).
(E)-17-H yd r oxy-4,7,10,13-t et r a oxa -15-h ep t a d ecen oic
Acid ter t-Bu tyl Ester (26). To a stirred solution of 22 (1.01
g, 2.58 mmol) in 1,4-dioxane (150 mL) was added 0.5 M
aqueous NaOH (50 mL). After 20 min the mixture was
neutralized with 1 M HCl, and 50 mL of dichloromethane were
added. The aqueous layer was extracted three times with
dichloromethane. The combined organic layers were washed
twice with brine and dried over MgSO4. The solvents were
removed in vacuo. After drying, 0.82 g (91%) of a clear and
colorless oil were obtained. Rf : 0.16 (petroleum ether/EtOAc,
The combined filtrates were washed twice with 30 mL of
0.5 M HCl. The aqueous solutions were extracted with 20 mL
of dichloromethane. The combined organic layers were dried
over Na2SO4 and concentrated in vacuo. Purification was
performed by dissolving the material obtained by chromatog-
1
1:3). 200 MHz H-NMR (CDCl3) δ ) 5.87-5.79 (m, 2H), 4.13
(s, 2H, H-17), 4.00 (d, 2H, H-14, J ) 4.5), 3.68 (t, 2H, J ) 6.7),

824 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
raphy (petroleum ether/EtOAc) in ethyl acetate followed by
addition of petroleum ether. The colorless solid was collected
and dried in vacuo to give 220 mg (63%) of 28. tR: 11 min 40
NMR (CDCl3) δ ) 7.73 (d, 2H), 7.50 (d, 2H), 7.41-7.22 (m,
5H), 7.13 (d, 1H, J ) 7.3), 7.00 (d, 2H, Yar-2,6, J ) 8.3), 6.83
(d, 2H, Yar-3,5, J ) 8.4), 5.60 (d, 1H, NH), 4.58-4.12 (m, 6H,
YR, AR, CH2-, H-9-Fmoc), 3.02-2.96 (m, 2H, Yâ), 1.58-1.40 (m,
6H, Lγ, Lâ, Aâ), 1.25 (s, 9H), 0.83 (d, 6H, Lδ, J ) 5.3). 50.3
MHz 13C-NMR (Acetone-d6) δ ) 174.21, 172.80, 172.51, 156.90,
154.99, 144.89, 144.82, 141.92, 133.11, 130.73, 128.41, 127.87,
126.16, 126.09, 124.45, 120.65, 78.21, 67.31, 57.20, 52.19,
48.60, 47.80, 42.18, 38.38, 29.02, 25.13, 23.36, 22.25, 17.83.
Anal. Calcd for C37H45N3O7: C, 69.03; H, 7.05; N, 6.53.
Found: C, 69.07; H, 6.97; N, 6.55.
s (column 1, grad 1, 1 mL/min). [R]22 ) -41.32 (c ) 1.0,
D
1
MeOH). Mp: 118 °C. 00 MHz H-NMR (DMSO-d6) δ ) 8.36
(d, 1H, ANH, J ) 7.2), 7.73 (d, 1H, FNH, J ) 8.4), 7.22-7.10 (m,
5H, Far), 6.90 (d, 1H, LNH, J ) 8.5), 4.61-4.53 (m,1H, FR), 4.20
(quint, 1H, AR, J R,NH ) J R,â ) 7.2), 3.94-3.78 (m, 1H, LR), 3.02
(dd, 1H, Fâa, J â,â ) 13.8, J â,R ) 3.9), 2.78 (dd, 1H, Fâb, J â,â
)
13.8, J â,R ) 9.5), 1.44-1.18 (m, 15H), 0.80 (2 × d, 6H, Lδ, J )
6.7). 100.6 MHz 13C-NMR (CDCl3) δ ) 174.90, 173.06, 170.80,
155.79, 136.15, 129.42, 128.37, 126.80, 80.22, 53.94, 53.30,
48.30, 41.21, 38.20, 28.22, 24.61, 22.74, 21.85, 17.85. Anal.
Calcd for C23H35N3O6: C, 61.45; H, 7.85; N, 9.35. Found: C,
61.34; H, 7.84; N, 9.27.
F m oc-Gly-Leu -Tyr (tBu )-Ala -OH (31). The synthesis was
performed applying Fmoc-strategy (method A) and started
from 2.31 g (1.44 mmol Ala) of 8b (0.63 mmol Ala/g, 0.71 mmol
â-Ala/g). Fmoc-amino acids were activated by DIC. Drying
yielded 2.43 g of peptide-resin (0.35 mmol of Gly/g, 0.32 mmol
of Leu/g, 0.28 mmol of Tyr/g, 0.35 mmol of Ala/g, 0.60 mmol
of â-Ala/g). Peptide-release was accomplished as described
above (see 29) using 2.39 g of peptide-resin, DMF/DMSO (1:1,
50 mL), N-methylaniline (2.0 mL, 18.7 mmol), and 130 mg (0.1
mmol) of palladium(0)-catalyst. After cleavage, 1.85 g of dry
polymer (0.02 mmol of Gly/g, 0.02 mmol of Leu/g, 0.00 mmol
of Tyr/g, 0.01 mmol of Ala/g, 0.64 mmol of â-Ala/g, 96%
detachment yield) was obtained. Aqueous workup (see 29) and
separation of the catalyst by crystallization (see 30) furnished
a brown oil, which was purified by chromatography (EtOAc).
The yellowish solid was dissolved in dichloromethane and the
colorless solid, which had precipitated upon addition of n-
heptane, was collected to give 478 mg (47%) of Fmoc-tetrapep-
F m oc-Leu -P h e-Ala -OH (29). The synthesis was carried
out via Fmoc-strategy (method A) starting from 1.64 g (0.81
mmol Ala) of resin 17a (0.50 mmol Ala/g, 0.69 mmol â-Ala/g).
Fmoc-amino acids were activated by DIC. After drying in
vacuo 1.40 g of peptide-resin were obtained (0.40 mmol of Leu/
g, 0.33 mmol of Phe/g, 0.40 mmol of Ala/g, 0.78 mmol of â-Ala/
g). The resin was suspended in dichloromethane/DMSO (1:1,
30 mL) prior to addition of 1.1 mL (10.1 mmol) of N-
methylaniline. The reaction vessel was evacuated until bub-
bling of the suspension occurred and then streamed by argon.
After five repeats, tetrakis(triphenylphosphine)palladium (100
mg) was added. The mixture was shaken in argon under
exclusion of light for 15 h. The resin was washed (DMF, 3 ×
20 mL, dichloromethane, 4 × 20 mL) and dried in vacuo,
yielding 1.42 g of polymer (0.08 mmol of Leu/g, 0.07 mmol of
Phe/g, 0.09 mmol of Ala/g, 0.85 mmol of â-Ala/g, 81% detach-
ment yield). The combined filtrates were concentrated in
vacuo. The residue was dissolved in chloroform (50 mL) and
washed with 30 mL of 1 M HCl. The aqueous solution was
reextracted four times with dichloromethane. The combined
organic layers were dried over Na2SO4 and evaporated in
vacuo. Two-fold chromatography (1. EtOAc/AcOH, 100:1; 2.
chloroform/MeOH) gave 343 mg of peptide, which was dis-
solved in dichloromethane and precipitated by addition of
n-heptane. The colorless solid was collected and dried in vacuo
to give 290 mg (62%) of tripeptide. Rf: 0.15 (EtOAc/AcOH,
tide. tR: 17 min 30 s (column 1, grad 1, 1 mL/min). [R]22
)
D
-24.50 (c ) 1.00, MeOH). Mp: 120-140 °C. 400 MHz 1H-
NMR (DMSO-d6) δ ) 8.04 (m, 3H), 7.93 (d, 1H, J ) 8.3), 7.88
(d, 2H, J ) 7.5), 7.69 (d, 1H, J ) 7.4), 7.59 (t, 1H, J ) 5.6),
7.40 (t, 2H, J ) 7.4), 7.31 (t, 2H, J ) 7.4), 7.11 (d, 2H, Yar-2,6
,
J ) 8.2), 6.82 (d, 2H, Yar-3,5, J ) 8.2), 4.50-4.46 (m, 1H, YR),
4.28-4.12 (m, 5H, AR, CH2-, H-9-Fmoc, LR), 3.64-3.60 (m, 2H,
GR), 3.04 (dd, 1H, Yâa, J â,â ) 13.9, J â,R ) 3.4), 2.75 (dd, 1H,
Yâb, J â,â ) 13.7, J â,R ) 10.3), 1.54-1.49 (m, 1H, Lγ), 1.32-1.22
(m, 14H, Lâ, Aâ, tBu), 0.80, 0.78 (2 × d, 6H, Lδ, J ) 6.5). 100.6
MHz 13C-NMR (DMSO-d6) δ ) 173.84, 171.59, 170.65, 169.31,
156.57, 153.39, 143.80, 140.71, 132.39, 129.62, 127.60, 127.04,
125.16, 123.26, 120.05, 77.49, 65.84, 53.41, 51.44, 47.62, 46.63,
43.47, 40.71, 36.48, 28.53, 24.04, 22.94, 21.53, 17.08. Anal.
Calcd for C39H48N4O8: C, 66.84; H, 6.90; N, 7.99. Found: C,
66.21; H, 6.99; N, 7.85.
100:1). [R]22 ) -39.44 (c ) 1.00, MeOH). Mp: 182 °C. 400
D
1
MHz H-NMR (DMSO-d6) δ ) 8.27 (d, 1H, NH, J ) 7.2), 7.87
(d, 3H), 7.69, 7.68 (2 × d, 2H), 7.46-7.05 (m, 11H), 4.54 (ddd,
1H, FR, J R,âa ) 4.1, J R,âb ∼ J R,NH ) 8.7), 4.33-4.07 (m, 4H),
3.98-3.92 (m, 1H, LR), 3.02 (dd, 1H, Fâa, J â,â ) 13.9, J â,R
)
Fm oc-Ala-Ser (tBu )-Th r (rAc3GalNAc)-Th r (tBu )-OH (32).
The synthesis was performed via Fmoc-strategy (method B)
starting from 1.31 g (0.52 mmol Thr) of 21 (0.40 mmol of Thr/
g, 0.61 mmol of â-Ala/g). All amino acids were activated by
DIC. After drying, 1.79 g of peptide-resin (0.42 mmol of Ala/
g, 0.08 mmol of Ser/g, 0.23 mmol of Thr/g, 0.48 mmol of â-Ala/
g) was obtained. Peptide-release was accomplished by treating
1.76 g of peptide-resin with DMF/DMSO (1:1, 30 mL), N-
methylaniline (1.2 mL, 11.2 mmol), and tetrakis(triphenylphos-
phine)palladium (100 mg, 0.09 mmol) (see 29) to give 1.02 g
of dried polymer (0.02 mmol of Ala/g, 0.01 mmol of Ser/g, 0.02
mmol of Thr/g, 0.62 mmol of â-Ala/g, 96% detachment yield).
Aqueous workup (see 29) followed by removal of palladium
by crystallization (see 30) yielded 1.1 g of raw material which
was purified by two-fold chromatography (EtOAc/AcOH, 50:
1). Lyophilization using benzene furnished 630 mg (77%) of
a colorless solid. tR: 8 min 10 s (column 1, grad 1, 1 mL/min).
[R]22D ) +33.53 (c ) 1.0, MeOH). Mp: 120-125 °C. 400 MHz
1H-NMR (1H,1H-COSY, DMSO-d6) δ ) 8.08 (d, 1H, TNH, J )
9.1), 7.90-7.85 (m, 3H, H-4,5-Fmoc, SNH, J 4,3 ) J 5,6 ) 7.6, J NH,R
) 9.9), 7.79 (d, 1H, TNH, J ) 9.1), 7.72 (d, 2H, J ) 7.2), 7.75
(d, 1H, NH-Ac, J ) 7.8), 7.42 (t, 2H, J ) 7.4), 7.33 (t, 2H, J )
7.4), 6.97 (d, 1H, ANH, J ) 8.4), 5.29 (d, 1H, H-4′, J 4′,3′ ) 3.2),
4.98 (dd, 1H, H-3′, J 3′,2′ ) 11.4, J 3′,4′ ) 3.2), 4.80 (d, 1H, H-1′,
J 1′,2′ ) 3.4), 4.76 (dd, 1H, TR, J R,NH ) 9.2, J R,â ) 1.3), 4.57-
4.53 (m, 1H, SR), 4.41-4.15 (m, 9H, AR, CH2-, H-9-Fmoc, TR,
Tâ, H-3′, H-5′), 4.03-3.95 (m, 2H, H-6′), 3.53-3.45 (m, 2H, Sâ),
2.10, 1.96, 1.87, 1.84 (4 × s, 4 × 3H, 4 × Ac), 1.24 (d, 3H, Aâ,
J ) 7.1), 1.16-1.02 (m, 24H, Tγ, tBu, J 1 ) 6.1, J 2 ) 6.2). 100.6
MHz 13C-NMR (DMSO-d6) δ ) 172.45, 171.63, 170.13, 169.89,
169.84, 169.74, 169.65, 169.18, 155.60, 143.77, 143.69, 140.61,
4.0), 2.77 (dd, 1H, Fâb, J â,â ) 13.9, J â,R )9.7), 1.50-1.43 (m,
1H, Lγ), 1.36-1.29 (m, 2H, Lâ), 1.27 (d, 3H, Aâ, J ) 7.3), 0.82
(d, 3H, Lδa, J ) 6.5), 0.77 (d, 3H, Lδb, J ) 6.5). 50.3 MHz 13C-
NMR (DMSO-d6): 173.93, 171.96, 170.67, 155.75, 143.85,
143.62, 140.66, 137.53, 129.25, 127.83, 127.56, 127.00, 125.19,
120.02, 65.48, 53.25, 53.18, 47.54, 46.67, 38.14, 37.47, 24.05,
22.89, 21.42, 17.14. Anal. Calcd for C33H37N3O6: C, 69.33;
H, 6.52; N, 7.35. Found: C, 68.92; H, 6.50; N, 7.17.
F m oc-Tyr (tBu )-Leu -Ala -OH (30). The synthesis started
from 1.21 g (0.84 mmol of Ala) of 17b (0.71 mmol of Ala/g,
0.73 mmol of â-Ala/g). The Fmoc-strategy was applied (method
A). Fmoc-amino acids were coupled using DIC. After drying
in vacuo 1.29 g of peptide-resin (0.37 mmol of Tyr/g, 0.42 mmol
of Leu/g, 0.46 mmol of Ala/g, 0.70 mmol of â-Ala/g) were
obtained. Peptide-release was accomplished as described for
the synthesis of 29 using 1.25 g of the peptide-resin, dichlo-
romethane/DMSO (1:1, 30 mL), N-methylaniline (1.1 mL, 10.1
mmol), and tetrakis(triphenylphosphine)palladium (200 mg).
Drying furnished 0.94 g of polymer (0.03 mmol of Tyr/g, 0.03
mmol of Leu/g, 0.04 mmol of Ala/g, 0.71 mmol of â-Ala/g, 93%
detachment yield). After aqueous workup (see synthesis of 29),
the raw material was dissolved in wet MeOH (25 mL) and
allowed to stand for 2 h. The palladium was removed by
filtration and the filtrate concentrated in vacuo. The residue
was purified by chromatography (CHCl3/MeOH), precipitation
(MeOH), and then chromatography again (petroleum ether/
EtOAc), yielding 330 mg of a yellow foam which was crystal-
lized from EtOAc/n-heptane to furnish 270 mg (50%) of a
colorless solid. tR: 15 min 20 s (column 1, grad 1, 1 mL/min.
1
[R]22 ) -30.5 (c ) 1.0, MeOH). Mp: 163 °C. 200 MHz H-
D

A Novel Allylic Anchor in Solid Phase Synthesis
J . Org. Chem., Vol. 62, No. 4, 1997 825
127.52, 126.99, 125.16, 119.98, 98.82 (C-1′), 76.90 (T(Ac3-
GalNAc)â), 73.16, 72.69, 68.54, 67.27, 66.98, 66.35, 65.63, 61.84,
57.33, 55.37 (T(Ac3GalNAc)R), 52.90, 49.90, 46.59, 46.50, 28.19,
26.95, 22.54, 20.39, 20.35, 20.30, 18.07, 17.89. Anal. Calcd
for C51H71N5O18: C, 58.74; H, 6.87; N, 6.72. Found: C, 58.65;
H, 6.91; N, 6.71.
layers were washed with brine, which again was reextracted.
The combined organic layers were evaporated in vacuo and
the residue dissolved in wet methanol. The solution was kept
at 0 °C overnight and the yellow solid, which had precipitated,
was removed by filtration. The filtrate was concentrated in
vacuo and dissolved in dichloromethane. The precipitate was
separated by filtration. After evaporation of the filtrate the
residue was purified by three-fold chromatography (CHCl3/
MeOH/AcOH). The material was dissolved in 2-propanol.
Benzene was added, and the solution was subjected to lyo-
philization to furnish 605 mg (95%) of a colorless solid. tR:
Ac-Ala-P r o-Asp(OtBu )-Th r (tBu )-Ar g(Mtr )-P r o-Ala-P r o-
Gly-OH (33). The synthesis started from 1.28 g (0.49 mmol
Gly) of 18 (0.38 mmol of Gly/g, 0.47 mmolof â-Ala/g). After
Fmoc-removal (method B), Boc-proline was coupled. Subse-
quent couplings were performed with Fmoc-amino acids
activated by DIC. Couplings were repeated for the N-terminal
threonine, aspartic acid, proline and alanine. The terminal
alanine was acetylated after Fmoc-cleavage. After drying, 1.85
g of peptide-resin (0.22 mmol of Asp/g, 0.18 mmol of Thr/g,
0.26 mmol of Arg/g, 0.46 mmol of Ala/g, 0.90 mmol of Pro/g,
0.30 mmol of Gly/g, 0.39 mmol of â-Ala/g) were obtained. For
peptide-release 1.83 g of peptide-resin were treated with
DMSO/DMF (1:1, 20 mL), dichloromethane (4 mL), N-meth-
ylaniline (1.4 mL, 12.9 mmol), and a small amount of the
palladium-catalyst (see 29). Washing (6 × 15 mL of DMF and
dichloromethane) and drying yielded 1.10 g of polymer (0.03
mmol of Asp/g, 0.03 mmol of Thr/g, 0.04 mmol of Arg/g, 0.09
mmol of Ala/g, 0.12 mmol of Pro/g, 0.05 mmol of Gly/g, 0.54
mmol of â-Ala/g, 87% detachment yield). The filtrates were
worked up as described (32). The raw material was purified
by chromatography (CHCl3/MeOH/AcOH, 8:2:0.01). The prod-
uct fractions were evaporated in vacuo and the residue
dissolved in 2-propanol followed by addition of n-heptane. The
colorless precipitate was collected and dried to give 509 mg
(83%) of nonapeptide. tR: 21 min 30 s (column 1, grad 2, 1
24 min 10 s (column 2, grad 3, 2 mL/min). [R]22 ) -20.50 (c
D
) 1.02, DMSO). Mp: 146-152 °C. 400 MHz 1H-NMR (1H,
1
1H-COSY, H, 13C-COSY, DMSO-d6) δ ) 8.27 (d, 1H, RR-NH, J
) 6.6), 8.12, 8.10 (2 × d, 2H, DNH, ANH or TNH, J 1 ) 7.0, J 2
)
6.1), 8.03 (t, 1H, GNH, J ) 5.8), 7.97 (d, 1H, ANH or TNH, J )
7.1), 7.69 (d, 1H, ANH, J ) 9.0), 7.05 (d, 1H, GalNAcNH, J )
9.5), 6.67 (mc, 2H, H-5-Mtr, RNH(ꢀ,ú,η)), 6.42 (s, 2H, RNH(ꢀ,ú,η)),
5.27 (s, 1H, H-4′), 4.91 (dd, 1H, H-3′, J 3′,2′ ) 11.5, J 3′,4′ ) 3.1),
4.77 (d, 1H, H-1′, J 1′,2′ ) 3.4), 4.64 (m, 1H, DR), 4.50-4.41 (m,
3H, AR, TR), 4.37-4.28 (m, 4H, RR (4.37), 3 × PR), 4.21-4.14
(m, 3H, Tâ, H-2′, H-5′), 4.01 (mc, 2H, H-6′), 3.77 (s, 3H, OCH3-
Mtr), 3.71-3.63 (m, 2H, GR), 3.61-3.35 (m, Pδ, H2O), 3.01 (mc,
2H, Rδ), 2.70 (dd, 1H, Dâa, J â,â ) 16.0, J â,R ) 6.1), 2.58 (s, 3H,
CH3-Mtr), 2.50-2.46 (m, CH3-Mtr, DMSO, Dâb), 2.10-1.64 (m,
30H, Pâ, Pγ, Ac, CH3-Mtr), 1.43-1.22 (m, 13H, Râ, Rγ, tBu),
1.18-1.15 (2 × d, 6H, 2 × Aâ, J 1 ) 6.2, J 2 ) 6.1), 1.09 (d, 3H,
Tγ, J ) 6.2). 100.6 MHz 13C-NMR (1H,13C-COSY, DMSO-d6)
δ ) 171.73, 171.58, 171.00, 170.96, 170.80, 170,45, 170.40,
169.90, 169.81, 169.70, 169.26, 169.26, 169.19, 169.12, 168.83,
157.41, 156.12, 137.50, 135.52, 134.61, 123.44, 111.67, 98.15
(C-1′), 80.17 (tBu), 75.42 (Tâ), 68.50 (C-3′), 67.17 (C-4′), 66.23
(C-5′), 61.90 (C-6′), [59.30, 59.07 (PR)], 55.53 (TR), 55.39, 50.12
(RR), 49.42 (DR), [46.55, 46.43 (Pδ, AR, C-2′)], 40.56 (GR), 39.70
(Rδ), 36.98 (Dâ), [28.91, 28.46, 28.11 (Râ, Pâ)], 27.52 (tBu),
[24.92, 24.22 (Rγ, Pγ)], 23.39, 22.64, 22.12, 20.88, 20.32, 18.14
(Tγ), 17.83, 16.76 (2 × Aâ), 11.59. HRMS (FAB, glyc, pos):
1521.6 (M(1 × 13C) + H+, 0.88%, 1521.7 calcd), 1520.9 (M +
H+, 1.25%, 1520.7 calcd), 1309.8 (M(1 × 13C) - Mtr + H +
H+, 1.41%, 1309.6 calcd), 1308.7 (M - Mtr + H + H+, 1.94%,
1308.6 calcd), 213.0 (Mtr+, 2.12%, 213.1).
mL/min). [R]22 ) -92.60 (c ) 1.00, MeOH). Mp: 150-160
D
1
°C. 400 MHz H-NMR (1H,1H-COSY, ROESY, DMSO-d6) δ )
8.27 (d, 1H, DNH, J ) 7.5), 8.13 (d, 1H, A1NH), 8.10-8.06 (m,
2H, GNH, A7NH), 7.98 (d, 1H, RR-NH, J ) 6.0), 7.19 (d, 1H, TNH
,
J ) 6.9), 6.66 (mc, 2H, H-5-Mtr, RNH(ꢀ,ú,η)), 6.27 (s, 2H, RNH(ꢀ,ú,η)),
R
R
4.61-4.50 (m, 4H, DR (4.56), A1 (4.52), RR (4.51), A7 (4.50)),
4.40-4.33 (m, 3H, PR), 4.29-4.22 (m, 1H, TR), 4.01 (mc, 1H,
Tâ), 3.80 (s, 3H, OCH3-Mtr), 3.72 (mc, 2H, GR), 3.60-3.30 (m,
Pδ, H2O), 3.03 (mc, 2H, Rδ), 2.70 (mc, 1H, Dâa), 2.59-2.51 (2 ×
s, 2 × CH3-Mtr, DMSO), 2.46 (mc, 1H, Dâ2), 2.18-1.75 (m, 18H,
Pâ, Pγ, Ac, CH3-Mtr), 1.45-1.39 (m, 13H, Râ, Rγ, tBu), 1.22-
Fm oc-Gly-Ser (tBu )-Th r (tBu )-Ala-P r o-P r o-Ala-His(Tr t)-
Gly-Va l-Th r (t Bu )-Ser (t Bu )-Ala -P r o-Asp (t Bu )-Th r (rAc3-
Ga lNAc)-Ar g(Mtr )-P r o-Ala -P r o-OH (35). The synthesis
started from 1.07 g (0.55 mmol of Fmoc) of 19 (0.52 mmol of
Fmoc/g). After Fmoc-removal (method B), Boc-alanine was
coupled. The subsequent couplings were performed with
Fmoc-amino acids, which were activated by TBTU. After
drying, 2.29 g of peptide resin (0.17 mmol of Fmoc/g) was
obtained. The peptide was released by adding DMSO/DMF
(1:1, 12 mL), N-methylaniline (1.0 mL, 9.2 mmol), and a small
amount of palladium-catalyst to 1.81 g of the peptide-resin (see
29). Washing and drying yielded 0.73 g of polymer (0.02 mmol
of Fmoc/g, 95% detachment yield). Workup was performed as
described above. The HCl-washing was substituted by wash-
ing with 0.5 M citric acid. Palladium was removed by
crystallization from methanol (see 32) to give a yellow amor-
phous solid. Purification was achieved by chromatography
(CHCl3/MeOH/AcOH) followed by GPC (Sephadex LH20,
CHCl3/MeOH, 1:1). Lyophilization from benzene/2-propanol
(10:1) yielded 611 mg (45%) of a colorless solid. tR: 21 min 10
â
â
1.16 (m, 6H, A1 (1.19) A7 (1.16)), 1.08 (s, 9H, tBu), 0.98 (m,
3H, Tγ, J ) 6.3). 100.6 MHz 13C-NMR (MeCN-d3) δ ) 173.37,
173.29, 173.08, 172.84, 172.61, 172.35, 172.02, 171.73, 171.37,
170.87, 170.06, 159.25, 157.76, 139.27, 137.38, 135.38, 125.39,
112.92, 82.02, 75.53, 67.59, 61.41, 61.30, 58.89, 56.32, 51.67,
50.98, 49.16, 48.28, 22.28, 47.54, 48.01, 41.82, 37.71, 32.20,
29.89, 29.67, 29.43, 28.67, 28.34, 25.77, 25.69, 25.23, 24.26,
22.87, 18.76, 18.50, 18.01, 17.74, 12.28. HRMS (FAB, nba,
neg): 1248.1 (M(2 × 13C)-H+, 6.81%, 1247.7 calcd), 1247.1 (M(1
×
13C) - H+, 21.40%, 1246.7 calcd), 1246.0 (M - H+, 32.25%,
1245.7 calcd).
Ac-Ala-P r o-Asp(OtBu )-Th r (rAc3GalNAc)-Ar g(Mtr )-P r o-
Ala -P r o-Gly-OH (34). The synthesis started from 0.85 g (0.42
mmol of Gly) of 18 (0.50 mmol of Gly/g, 0.63 mmol of â-Ala/g).
After Fmoc-removal (method B) and coupling of Boc-proline
the following couplings were performed with Fmoc-amino
acids. Fmoc-Thr(RAc3GalNAc)-OH and Fmoc-Asp(OtBu)-OH
were activated by TBTU. All other amino acids were activated
by DIC. Couplings were repeated for the N-terminal proline
and alanine. The terminal alanine was acetylated after Fmoc-
cleavage. Drying yielded 1.49 g of peptide-resin (0.30 mmol
of Asp/g, 0.24 mmol of Thr/g, 0.32 mmol of Arg/g, 0.58 mmol
of Ala/g, 0.90 mmol of Pro/g, 0.35 mmol of Gly/g, 0.40 mmol of
â-Ala/g). The peptide-release was accomplished using 1.47 g
of resin, DMSO/DMF (1:1, 16 mL), dichloromethane (2 mL),
morpholine (1.2 mL, 13.7 mmol), and a small amount pal-
ladium-catalyst (see 29). After washing and drying, 0.74 g of
polymer (0.03 mmol of Asp/g, 0.02 mmol of Thr/g, 0.03 mmol
of Arg/g, 0.06 mmol of Ala/g, 0.09 mmol of Pro/g, 0.04 mmol of
Gly/g, 0.66 mmol of â-Ala/g, 93% detachment yield) was
obtained. The combined filtrates were concentrated in vacuo
and coevaporated with DMF. The residue was dissolved in
dichloromethane followed by two-fold washing with 0.5 M HCl.
After reextraction of the aqueous layers the combined organic
s (column 3, grad 4, 1 mL/min). [R]22 -43.07 (c ) 0.76,
D
MeOH). HRMS (FAB, nba, pos): 3212.0 (Maverage + K+, 3212.7
calcd), 3176.9 (M(4 × 13C) + H+, 0.03%, 3176.6 calcd), 3175.7
(M(3 × 13C) + H+, 0.05%, 3175.6 calcd), 3174.6 (M(2 × 13C) +
H+, 0.06%, 3174.6 calcd), 3173.8 (M(1 × 13C) + H+, 0.04%,
3173.6 calcd), 3173.1 (M + H+, 0.04%, 3172.6 calcd).
Ac-Ala-P r o-Asp-Th r (rAc3GalNAc)-Ar g-P r o-Ala-P r o-Gly-
OH‚AcOH (36). To 51 mg (33.5 µmol) of glycononapeptide
35 were added anisole (0.06 mL) and ethyl methyl sulfide (0.06
mL). After 6 h of stirring the solution was cooled to 0 °C for
17 h. The solution was concentrated in vacuo, and the residue
was dissolved in methanol. The hydrotrifluoroacetate pre-
cipitated upon addition of dry diethyl ether. The colorless solid
was collected, washed with diethyl ether, and dried in vacuo.
The fitrate was concentrated in vacuo and the residue dis-

826 J . Org. Chem., Vol. 62, No. 4, 1997
Seitz and Kunz
solved in methanol followed by addition of diethyl ether. The
precipitate was collected and washed with diethyl ether. The
precipitates were combined and purified by GPC (Sephadex
G15, 0.1 M aqueous NH4OAc, 0.8 mL/min). Lyophilization
yielded 41 mg (93%) of glycopeptide 36. tR: 22 min 0 s (column
4, grad 5, 1 mL/min). 400 MHz 1H-NMR (D2O) δ ) 5.42 (d,
1H, J ) 2.6), 5.17 (dd, 1H, J 1 ) 11.2, J 2 ) 2.9), 5.02 (d, 1H, J
) 3.6), 4.88-4.70 (m, DR, HDO), 4.59-4.32 (m, 10H), 4.23-
4.4.20 (m, 2H), 3.82-3.61 (m, 8H), 3.22 (mc, 2H), 2.77 (dd, 1H,
J 1 ) 16.o, J 2 ) 7.0), 2.62 (dd, 1H, J 1 ) 16.0, J 2 ) 6.0), 2.30-
2.21 (m, 6H), 2.08-1.85 (m, 24H), 1.80-1.67 (m, 4H), 1.38,
1.35 (2 × d, 6H, J 1 ) J 2 ) 7.1), 1.26 (d, 3H, J ) 6.3). 100.6
MHz 13C-NMR (D2O) δ ) 173.85, 173.74, 173.68, 173.59,
173.44, 173.30, 173.06, 171.37, 171.14, 156.94, 98.67, 76.22,
69.25, 68.19, 67.05, 62.63, 60.73, 60.42, 60.18, 57.00, 51.44,
51.29, 47.74, 47.62, 40.61, 29.38, 29.28, 27.39, 24.68, 24.63,
24.57, 24.17, 22.15, 21,55, 20.16, 20.09, 17.99, 15.64, 15.52.
HRMS (FAB, glyc, pos): 1254.8 (M(2 × 13C) + H+, 2.46%,
1254.6 calcd), 1253.9 (M(1 × 13C) + H+, 5.72%, 1253.6 calcd),
1252.9 (M + H+, 10.90%, 1252.6 calcd).
4.00-3.96 (m, 1H), 3.92-3.86 (m, 1H, LR), 2.04-1.99 (m, 1H,
Vâ), 1.65-1.59 (m, 1H, Lγ), 1.47-1.44 (m, 2H, Lâ), 1.03 (d, 3H,
Tγ, J ) 6.2), 0.86-0.81 (m, 12H, Lδ, Vγ). 100.6 MHz 13C-NMR
(DMSO-d6) δ ) 172.57, 171.97, 169.78, 155.94, 143.80, 143.66,
140.62, 127.53, 126.97, 125.18, 119.97, 66.71 (Tâ), 65.71, 60.37
(TR), 57.21 (VR), 50.84 (LR), 46.62, 40.74 (Lâ), 29.70 (Vâ), 23.95
(Lγ), 23.00, 21.57, [9.49, 18.91, 17.97 (Lδ, Vγ, Tγ)]. Anal. Calcd
for C30H39N3O7: C, 65.08; H, 7.10; N, 7.59. Found: C, 64.43;
H, 6.94; N, 7.44.
F m oc-Th r (âAc4Glc)-Leu -Va l-OH (39). The Fmoc-TLV-
resin (0.61 g, ≈0.27 mmol of peptide), which had been dried
carefully in vacuo over P4O10, was washed intensively with dry
dichloromethane. The resin was suspended in 30 mL of dry
dichloromethane followed by addition of 800 mg (1.85 mmol)
of 1-O-(N-allylcarbamoyl)-2,3,4,6-tetra-O-acetyl-â-D-glucopyr-
anose.41 After addition of 290 mg (0.61 mmol) of S-methylbis-
(methylthio)sulfonium hexachloroantimonate, the suspension
was shaken for 30 min. The resin was washed (dry dichlo-
romethane, 3 × 10 mL) and the glycosylation reaction was
repeated twice: 1. with 800 mg (1.85 mmol) of donor and 290
mg (0.61 mmol) of promotor, 2. with 800 mg donor and 270
(0.57 mmol) of promotor. The resin was washed (dichlo-
romethane, 3 × 10 mL; DMF, 6 × 10 mL; pyridine, 4 × 10
mL; DMF, 7 × 10 mL; dichloromethane, 4 × 10 mL) and dried
to give 0.70 g of resin (0.35 mmol of Thr/g, 0.51 mmol of Leu/
g, 0.56 mmol of Val/g, 0.73 mmol of â-Ala/g). The peptide-
release was accomplished using 0.68 g of resin, DMSO/DMF
(1:1, 10 mL), dichloromethane (1 mL), N-methylaniline (0.9
mL, 8.3 mmol), and a small amount of palladium(0)-catalyst
(see 29). After washing (13 × 15 mL of DMF, 4 × 15 mL of
pyridine, 4 × 15 mL of dichloromethane) the cleavage-reaction
was repeated twice. Intensive washing and drying in vacuo
furnished 0.53 g of polymer (0.16 mmol of Thr/g, 0.21 mmol of
Leu/g, 0.22 mmol of Val/g, 0.71 mmol of â-Ala/g, 59% detach-
ment yield). The combined filtrates were worked up as
described above (see 32). Two-fold chromatography (EtOAc/
AcOH, 100:1) gave glycopeptide 39 and unglycosylated peptide
38. The product fractions were evaporated in vacuo and
lyophilized from benzene/2-propanol (10:1), yielding 96 mg
(64% based on peptide load) of unglycosylated peptide 38 and
10 mg (4.2% based on peptide load) of glycopeptide 39. tR: 31
Ac-Ala -P r o-Asp -Th r (rGa lNAc)-Ar g-P r o-Ala -P r o-Gly-
OH‚AcOH (37). A solution of 79 mg (60.2 µmol) of 36 in 10
mL of dry methanol was adjusted to pH 8.5 by addition of a
0.014 M solution of sodium methanolate in methanol. After
1.5 h of stirring, the solution was acidified to pH 6 by addition
of acetic acid. The solution was concentrated in vacuo. The
residue was dissolved in water and lyophilized. Purification
by GPC (Sephadex G15, 0.1 M NH4OAc, 0.8 mL/min) and
lyophilization yielded 59 mg (83%) of glycopeptide. tR: 17 min
0 s (column 4, grad 6, 1 mL/min). [R]22 ) -112.49 (c ) 1.00,
D
H2O). Mp: 205-210 °C. 400 MHz 1H-NMR (1H,1H-COSY,
1H,13C-COSY, H2O) δ ) 8.32 (d, 1H, J ) 6.4), 8.25-8.23 (m,
2H), 8.15 (d, 1H, J ) 7.2), 8.05 (d, 1H, J ) 5.6), 7.89 (s, 1H,
GNH), 7.60 (d, 1H, GalNAcNH, J ) 9.6), 7.14 (m, 1H, RꢀNH), 6.53
(s, 2H, RNH), 5.0-4.6 (m, H-1′, DR, H2O), 4.59-4.20 (m, 8H, 2
× AR, RR (4.50), PR, TR (4.33), Tâ (4.27)), 4.05 (mc, 1H, H-2′),
3.96-3.90 (m, 2H, H-5′ (3.96), H-4′ (3.92)), 3.85-3.45 (m, 11H,
H-3′ (3.83), H-6′ (3.59), GR (3.75), Pδ (3.79-3.54)), 3.17 (mc,
2H, Rδ), 2.82-2.61 (m, 2H, Dâ), 2.25 (mc, 3H, Pâa), 2.06-1.73
(m, 15H, Pâb, Pγ, Ac), 1.71-1.60 (m, 4H, Râ, Rγ), 1.22, 1.19 (2
× d, 6H, 2 × Aâ, J 1 ) 7.2, J 2 ) 6.8), 1.09 (d, 3H, Tγ, J ) 6.4).
100.6 MHz 13C-NMR (1H,13C-COSY, DEPT, D2O) δ ) 173.97,
173.88, 173.75, 173.45, 171.25, 171.07, 156.89, 98.60 (C-1′),
75.24 (Tâ), 71.45 (C-5′), 68.65 (C-4′), 68.12 (C-3′), 61.31 (C-6′),
[60.66, 60.46, 60.12 (PR)], 57.19 (TR), 51.29 (RR), 50.85 (DR),
49.77 (C-2′), 47.71 (Pδ, AR, GR), 40.60 (Rδ), 31.25 (Dâ), 29.36
(Pâ), 27.53 (Râ), [24.64, 24.56 (Pγ)], 24.16 (Rγ), 22.36, 21.50 (Ac),
18.33 (Tγ,), [15.56, 15.44 (Aâ)]. HRMS (FAB, glyc, pos): 1128.7
(M(2 × 13C) + H+, 2.44%, 1128.5 calcd), 1127.7 (M(1 × 13C) +
H+, 6.34%, 1127.5 calcd), 1126.8 (M + H+, 10.67%, 1126.5
calcd).
m 20 s (column 2, grad 7, 1 mL/min). [R]22 ) 0.33 (c ) 1.91,
D
MeOH). Mp: 84 °C. 400 MHz 1H-NMR (1H,1H-COSY, DMSO-
d6) δ ) 7.91-7.87 (m, 3H, H-4-, H-5-Fmoc, VNH), 7.81 (d, 1H,
LNH, J ) 7.8), 7.69 (mc, 2H), 7.40 (t, 2H, J ) 7.4), 7.31 (t, 2H,
J ) 7.5), 6.97 (d, 1H, TNH, J ) 7.9), 5.23 (dd, 1H, H-3′, J 3′,2′
)
J 3′,4′ ) 9.6), 4.90-4.84 (m, 2H, H-1′, H-4′), 4.70 (dd, 1H, H-2′,
J 2′,1′ ) J 2′,3′ ) 8.8), 4.38 (mc, 1H, LR), 4.24-4.14 (m, 4H, CH2-
Fmoc, H-6′), 4.11-4.05 (m, 2H, VR, TR), 3.99-3.91 (m, 3H, H-9-
Fmoc, Tâ, H-5′), 2.03-1.89 (m, 13H, Ac, Vâ), 1.63-1.59 (m, 1H,
Lγ), 1.43-1.37 (m, 2H, Lâ), 1.07 (d, 3H, Tγ, J ) 6.2), 0.86-
0.80 (m, 12H, Lδ, Vγ). 100.6 MHz 13C-NMR (DEPT, DMSO-d6
(signal doubling in DEPT-spectra, no doubling in 1D-spec-
trum)) δ ) 172.6, 171.60, 169.8, 169.1, 169.0, 168.8, 168.5,
143.7, 140.59, 128.89, 127.61, 127.25, 127.02, 125.19, 121.33
(?), 120.06, 119.98, 98.10, 97.84 (C-1′), [77.75, 75.66, 71.98,
71.91, 70.91, 70.48, 68.19 (C-2′, -3′, -4′, -5′, Tâ)], 65.79, 61.81,
61.70 (C-6), 58.69 (TR), 57.22, 50.95, 50.53, 46.53, 41.17, 40.78,
29.76, 23.87, 23.07, 22.99, 21.71, 21.61, 20.39, 20.28, 20.20,
18.96, 17.98, 16.41, 16.33. HRMS (FAB, nba, pos): 885.9 (M(1
F m oc-Th r -Leu -Va l-OH (38). The synthesis was per-
formed via Fmoc-strategy (method C) and started from 1.56 g
(0.65 mmol Val) of 20 (0.42 mmol of Val/g, 0.56 mmol of â-Ala/
g). The Fmoc-Thr(tBu)-Leu-Val-resin (0.30 mmol of Thr/g, 0.44
mmol of Leu/g, 0.48 mmol ofVal/g, 0.53 mmol of â-Ala/g) was
treated with 20 mL of TFA for 2 h. After washing with DMF
(5 × 15 mL) and dichloromethane (4 × 15 mL) and drying,
1.65 g of tripeptide-resin (0.30 mmol of Thr/g, 0.43 mmol of
Leu/g, 0.44 mmol of Val/g, 0.55 mmol of â-Ala/g) with unpro-
tected hydroxyl group was obtained. The peptide was released
by treating 0.73 g (≈0.32 mmol of peptide) of peptide-resin with
DMF/DMSO (1:1, 8 mL), N-methylaniline (0.9 mL, 7.1 mmol),
and a small amount of palladium(0)-catalyst (see 29). Washing
(6 × 15 mL of DMF, 5 × 15 mL of dichloromethane) and drying
yielded 0.60 g of polymer (0.09 mmol of Thr/g, 0.11 mmol of
Leu/g, 0.12 mmol of Val/g, 0.91 mmol of â-Ala/g, 84% detach-
ment yield). The filtrates were worked-up as described (see
32). Purification was achieved by two-fold chromatography
(1. CHCl3/MeOH/AcOH, 2. EtOAc/AcOH. 100:1) yielding 170
mg (96%) of a colorless solid. tR: 24 min 0 s (column 2, grad
×
13C) + H+, 1.42%, 885.4 calcd), 884.9 (M + H+, 3.26%, 884.4
calcd).
Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft and by the Fonds der
Chemischen Industrie. O.S. is grateful for an award of
the Boehringer Ingelheim Foundation.
Su p p or tin g In for m a tion Ava ila ble: 1H-NMR spectra of
3, 4a , 6, 8a -12a , 8b-12b, 13-15, 16a /b, 27, 33-37 and 39;
1H,1H-COSY of 33 and 34; 1H,13C-COSY of 37; RP-HPLC of
35 (29 pages). This material is contained in libraries on
microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
7, 1 mL/min). [R]22 ) -36.39 (c ) 1.00, MeOH). Mp: 92 °C.
D
400 MHz 1H-NMR (DMSO-d6) δ ) 7.94-7.87 (m, 4H, H-4-,
H-5-Fmoc, VNH, LNH), 7.73 (mc, 2H,), 7.40 (t, 2H, J ) 7.4), 7.31
(t, 2H, J ) 7.4), 7.17 (d, 1H, TNH, J ) 8.7), 4.83 (s, 1H, TOH),
4.45-4.39 (m, 1H), 4.29-4.19 (m, 3H), 4.09-4.06 (m, 1H),
J O960743W