Combinatorial solid-phase synthesis of multivalent cyclic neoglycopeptides

Article abstract of DOI:10.1002/1521-3773(20001201)39:23<4348::AID-ANIE4348>3.0.CO;2-X

Conformationally restricted, multivalent cyclic neoglycopeptides, such as 1, are accessible by a convergent solid-phase synthesis in which an active carbonate is treated with immobilized cyclopeptides bearing free amino side chains. The libraries of glycoclusters obtainable by this procedure are suited for screening for lectin-binding properties.

Full text of DOI:10.1002/1521-3773(20001201)39:23<4348::AID-ANIE4348>3.0.CO;2-X

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[9] a) J. Bao, W. D. Wulff, V. Dragisich, S. Wenglowsky, R. G. Ball, J. Am.
Chem. Soc. 1994, 116, 7616; b) M. Mori, K. Kuriyama, N. Ochifugi, S.
Watanuki, Chem. Lett. 1995, 615; c) Y. Zhang, J. W. Herndon,
Tetrahedron 2000, 56, 2175.
[10] For a recent review of the reactivity of dienylcarbenes, see R.
Aumann, Eur. J. Org. Chem. 2000, 17 .
[11] J. Barluenga, F. Aznar, M. A. Palomero, S. Barluenga, Org. Lett. 1999,
1, 541.
[12] For a similar tandem 4cycloadition ± alkyne insertion ± cyclization)
process of Fischer carbenes, see: S. Chamberlain, W. D. Wulff, B. Bax,
Tetrahedron 1993, 49, 5531.
[13] As an example, compound 5b was prepared in 58% yield by the one-
pot process, while the overall yield of the two-step reaction was 70%
493% for the [2‡2] cycloaddition and 75% for the metallahexa-
triene ± alkyne coupling).
[14] For a review of the chemistry of Cr4CO)3-complexed trienyl com-
pounds, see J. H. Rigby, Tetrahedron 1999, 55, 4521.
pound was purified by flash chromatography, and its structure
was assigned by COSY and HMBC experiments. This com-
pound could be quantitatively converted into the metal-free
carbocycle 5e by exposure to air and sunlight.
Most of the compounds 5 are unstable in solution leading in
a few hours to complex and unidentifiable mixtures of
products. However, compound 5e gave a single diaster-
eoisomer of polycyclic system 9, in 90% yield, upon standing
in CHCl3 for ten days 4Scheme 4). Longer reaction times led
to the release of the trimethylsilyl 4TMS) group, and thus to
the formation of 10 492% yield). These products are evidently
derived from the six-electron electrocyclization of the starting
trienyl system 5e and the hydrolysis of the resulting enol
ether. The single diastereoisomer of 9 can best be explained as
arising from epimerization of the a-carbonyl stereocenter in
5e and the stereoselective cyclization of one of the epimeric
ketones.
In conclusion, we have reported the first Dötz-like reaction
between conjugated dienyl carbene chromium complexes and
terminal alkynes to afford eight-membered carbocycles. We
have also studied the ring closure of one of the mentioned
systems to yield new polycyclic compounds.
Combinatorial Solid-Phase Synthesis of
Multivalent Cyclic Neoglycopeptides**
Valentin Wittmann* and Sonja Seeberger
Experimental Section
Dedicated to Professor Horst Kessler
A solution of dienyl carbene 4 41 mmol) and of the alkyne 43 mmol) in THF
415 mL) was refluxed, under a nitrogen atmosphere, until analysis by thin-
layer chromatography revealed total consumption of the starting complex.
The reaction mixture was diluted with hexane 420 mL) and exposed to
sunlight and air in order to oxidize the metallic species to the corresponding
organic compounds. Filtration through a pad of Celite and flash chroma-
tography provided carbocycles 5.
The molecular recognition of carbohydrates by carbohy-
drate-binding proteins 4lectins) is the basis of numerous
intercellular recognition processes.[1] High-affinity lectin li-
gands are of considerable medicinal interest in the diagnosis
and manipulation of such processes.[2] Individual carbohy-
drate epitopes 4normally mono- to pentasaccharides) are,
however, mostly bound by lectins with only low affinity
4dissociation constants in the milli- to micromolar range) and
in part broad specificity.[3] Since many membrane-associated
lectins have several binding sites or occur in oligomeric or
clustered form,[4] the creation of multivalent carbohydrate
derivatives is a promising approach to arrive at effective lectin
ligands.[5, 6]
Several strategies have been described to achieve the
formation of a sufficient number of individual interactions
necessary for high avidity of a multivalent ligand. Glycopol-
ymers,[7] for example, are able to cover large areas of cell
surfaces and bridge several membrane-located lectins 4ªsta-
tisticalº multivalency). Small oligovalent carbohydrate deriv-
atives 4miniclusters)[6] on the other hand bind preferentially to
several binding sites of a single 4oligomeric) lectin proximate
Received: July 5, 2000 [Z15393]
[1] For general reviews of the thermal cyclization reactions of Fischer
carbene complexes see: a) W. D. Wulff in Comprehensive Organo-
metallic Chemistry II, Vol. 12 4Eds.: E. W. Abel, F. G. A. Stone, G.
Wilkinson), Pergamon Press, New York, 1995, p. 469; b) D. F. Harvey,
D. M. Sigano, Chem. Rev. 1996, 96, 271; c) F. Zaragoza Dörwald in
Metal Carbenes in Organic Synthesis, Wiley-VCH, 1999; d) K. H.
Dötz, P. Tomuschat, Chem. Soc. Rev. 1999, 28, 187.
[2] K. H. Dötz, Angew. Chem. 1975, 87, 67 2;Angew. Chem. Int. Ed. Engl.
1975, 14, 644.
[3] Iron, cobalt, or molybdenum complexes give rise to cyclopentadienes
or furans: a) W. D. Wulff, S. R. Gilbertson, K. A. Abboud, W. M.
Jones, J. Am. Chem. Soc. 1986, 108, 520; b) M. F. Semmelhack, J. Park,
Organometallics 1986, 5, 2550; c) D. F. Harvey, E. M. Grenzer, J. Org.
Chem. 1996, 61, 159.
[4] a) R. Aumann, H. Heinen, M. Dartmann, B. Krebs, Chem. Ber. 1991,
124, 2343; b) W. D. Wulff, A. M. Gilbert, R. P. Hsung, A. Rahm, J.
Â
Org. Chem. 1995, 60, 4566; c) J. Barluenga, F. Aznar, I. Gutierrez, A.
Ä
Martín, S. García-Granda, M. A. Llorca-Baragano, J. Am. Chem. Soc.
2000, 122, 1314.
[5] M. Duetsch, S. Vidoni, F. Stein, F. Funke, M. Noltemeyer, A.
de Meijere, J. Chem. Soc. Chem. Commun. 1994, 1679.
[6] a) F. Stein, M. Duetsch, R. Lackmann, M. Noltemeyer, A. de Meijere,
Angew. Chem. 1991, 103, 1669; Angew. Chem. Int. Ed. Engl. 1991, 30,
1658; b) F. Stein, M. Duetsch, M. Noltemeyer, A. de Meijere, Synlett
1993, 483; c) H. Schirmer, T. Labahn, B. Flynn, Y. T. Wu, A.
de Meijere, Synlett 1999, 2004.
[7] H. Schirmer, M. Duetsch, F. Stein, T. Labahn, B. Knieriem, A
de Meijere, Angew. Chem. 1999, 111, 1369; Angew. Chem. Int. Ed.
1999, 38, 1285.
[*] Dr. V. Wittmann, Dipl.-Chem. S. Seeberger
Institut für Organische Chemie
Johann Wolfgang Goethe-Universität
Marie-Curie-Strasse 11, 60439 Frankfurt 4Germany)
Fax : 4‡49)69-798-29148
E-mail: wittmann@chemie.uni-frankfurt.de
[**] This work was supported by the Deutsche Forschungsgemeinschaft.
We thank Prof. Joachim W. Engels for his support and the Degussa-
Hüls AG for amino acids. The abbreviations used are given in
reference [25].
[8] For
a review of the use of Fischer carbene complexes in the
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/angewandte/ or from the author.
preparation of five-membered carbocycles, see J. W. Herndon, Tetra-
hedron 2000, 56, 1257.
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in space and may be tailored to lectins with known three-
dimensional 43D) structure 4ªdirectedº multivalency).[8] With
the carbohydrates connected by an inflexible scaffold, partic-
ularly affine ligands are obtainedÐprovided that the sugars
are oriented in a way required for multidentate binding.[5]
Moreover, conformationally restricted miniclusters are in
principle able to differentiate between various multivalent
lectins with the same carbohydrate specificity but varying
orientation of their binding sites. If the 3D structure of the
targeted lectin is unknown and especially if conformationally
restricted linkers are used, many potential ligands have to be
synthesized and screened in order to ªhitº the required
orientation of the sugar residues.
We now report a synthetic concept to generate libraries
of conformationally restricted miniclusters, comprising the
following steps: a) ªsplit ± mixº synthesis[9] of a library of
scaffold molecules with side chain amino groups in varying
amounts and spatial orientation, b) attachment of several
identical copies of a carbohydrate ligand to the amino
groups.
peptides. Reaction of literature-known glycosyl donor 2[15]
with mono TBDPS-protected cis-but-2-ene-1,4-diol under
methyl triflate activation gave b-configurated O-glycoside 3
in high yield. Substitution of the Troc protecting group with an
acetyl group followed by removal of the TBDPS group with
HF ´ pyridine complex led to N-acetylglucosamine derivative
4. Condensation with 4-nitrophenyl chloroformate finally
gave active carbonate 5 as a crystalline substance that could
be stored at room temperature 4Table 1).
Table 1. Selected physical data of compounds 5 and 12.
5: Rf ˆ 0.43 4silica, EtOAc); m.p. 1338C 4EtOAc/n-hexane); 1H NMR
4400 MHz, CDCl3, 300 K, TMS): d ˆ 8.29 ± 8.25 4m, 2H; arenes), 7.40 ± 7.36
4m, 2H; arenes), 5.85 ± 5.75 4m, 2H; vinyl-H), 5.64 4d, J ˆ 8.7Hz, 1H; NH),
5.28 4dd, J ˆ 9.3, 10.6 Hz, 1H; H-3), 5.05 4dd, J ˆ 9.3, 10.0 Hz, 1H; H-4),
4.90 ± 4.79 4m, 2H; allyl-H), 4.75 4d, J ˆ 8.3 Hz, 1H; H-1), 4.44 ± 4.29 4m,
2H; allyl-H), 4.22 4dd, J ˆ 4.7, 12.3 Hz, 1H; H-6a), 4.13 4dd, J ˆ 2.5,
12.3 Hz, 1H; H-6b), 4.83 4ddd, J ˆ 8.3, 8.7, 10.6 Hz, 1H; H-2), 3.69 4ddd,
J ˆ 2.5, 4.7, 10.0 Hz, 1H; H-5), 2.06 4s, 3H; C4O)CH3), 2.01 4s, 3H;
C4O)CH3), 2.00 4s, 3H; C4O)CH3), 1.93 4s, 3H; C4O)CH3); 13C NMR
4100 MHz, CDCl3, 300 K, TMS): d ˆ 170.8, 170.6, 170.2, 169.3, 155.4, 152.4,
145.4, 131.0 and 125.8 4olefins), 125.3 and 121.8 4arene CH), 99.5 4C-1), 72.2
4C-3), 71.8 4C-5), 68.6 4C-4), 64.6 and 64.2 4CH2allyl), 62.0 4C-6), 54.74C-2),
23.3 4CH3), 20.674CH 3), 20.61 4CH3), 20.55 4CH3); elemental analysis calcd
for C25H30N2O14 4%): C 51.55, H 5.19, N 4.81; found: C 51.52, H 5.28, N
4.79.
As scaffolds for the multivalent presentation of carbohy-
drate ligands, we have chosen cyclic peptides of general type 1
4Bal ˆ b-alanine) in which the residues Xaa are varied
12: 1H NMR 4600 MHz, H2O/D2O 9:1, 298 K): Boc-Lys1: d ˆ 6.767 4aNH),
3.9174 aH), 1.565 4bH2), 1.27± 1.17 4 gH2), 1.379 4dH2), 3.083 and 3.003
4eH2), 7.688 4eNH), 1.304 4Boc); Orn: d ˆ 8.262 4aNH), 4.254 4aH), 1.722
and 1.633 4bH2), 1.47± 1.38 4 gH2), 3.023 4dH2), 6.791 4dNH); Gly3: 8.201
4NH), 3.855 and 3.788 4aH2); Ala4: 7.999 4NH), 4.203 4aH), 1.277 4bH3); d-
Lys5: 8.148 4aNH), 4.185 4aH), 1.690 and 1.615 4bH2), 1.248 and 1.215
4gH2), 1.382 4dH2), 2.998 4eH2), 6.733 4eNH); Orn: d ˆ 8.316 4aNH), 4.254
4aH), 1.731 and 1.633 4bH2), 1.478 and 1.420 4gH2), 3.032 4dH2), 6.767
4dNH); d-Val7: d ˆ 8.136 4NH), 3.982 4aH), 2.041 4bH), 0.820 and 0.786 42
gH3); Glu8: d ˆ 8.201 4NH), 4.1074 aH), 1.831 and 1.999 4bH2), 2.201 and
2.136 4gH2); Bal9: d ˆ 8.049 4NH), 2.41 ± 2.33 4aH2), 3.40 ± 3.30 4bH2);
3 equivalent GlcNAc: d ˆ 8.103 4NH), 4.4274H-1), 3.588 4H-2), 3.429 4H-
3), 3.38 ± 3.31 4H-4, H-5), 3.810 and 3.651 42 H-6), 1.9274Ac); 5.72 ± 5.60 46
Boc-Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Glu-Bal-NH
1
combinatorially.[10] Using commercially available d- and l-
configurated amino acids, a high degree of conformationally
diversity may be easily generated.[11] Diamino acids with Ddv-
protected[12] side chains of varying length serve as sugar
attachment points. To this end, a new urethane-type linker
based on the Aloc protecting group[13] has been developed. In
contrast to glycosylation reactions employing solid-phase-
bound peptides,[14] the formation of an urethane bond
proceeds in virtually quantitative yield. Furthermore, after
the binding assay the carbohydrates may be cleaved off again
under Pd catalysis regenerating the unmodified side chain
cyclized peptides. Thus, the analysis of compounds bound to
single beads which has to be carried out when using the split ±
mix method is confined to an automated microsequencing
4Edman degradation) under standard conditions.
‡
vinyl-H), 4.52 ± 4.48 and 4.30 ± 4.18 4each 6 allyl-H); ESI-MS [M‡H ]:
calcd. 1963.0, found 1963.7.
The assembly of the cyclic neoglycopeptides was optimized
by using model compound 12 as an example 4Schemes 2 and 3,
Table 1). For attachment of the first amino acid to the solid
support 4TentaGel) the Sieber linker was applied.[16] Thus it
was possible to release the product of each reaction step by
treatment with a 1% solution of TFA in dichloromethane
4!7 ± 9, 11, 12) and monitor the course of the reaction by
analytical HPLC 4Figure 1) and subsequent ESI-MS. Syn-
thesis of the linear peptide fol-
Scheme 1 illustrates the synthesis of carbohydrate-linker
derivative 5 activated as p-nitrophenyl carbonate which was
applied to attach N-acetylglucosamine residues to the cyclo-
lowed the Fmoc strategy;[17] sole-
ly in the last coupling step an Na-
OAc
O
OAc
O
1. Zn, HOAc
2. Ac2O, Pyr (98 %, 2 steps)
HO
OTBDPS
AcO
AcO
AcO
AcO
SEt
O
O
OTBDPS
Boc-protected amino acid was
applied. For protection of the w-
NH2 groups of lysine and orni-
thine we preferred the Ddv
group[12] which distinguishes it-
self from the frequently used
Dde group[18] by a higher stabil-
ity under the conditions of Fmoc
cleavage. Preparatory for the
HN
NHTroc
3. HF • pyr (94 %)
MeOTf, CH2Cl2 (86 %)
O
2
3
Troc
CCl3
Cl
O
OAc
O
OAc
O
O
AcO
AcO
AcO
AcO
NO2
O
OH
O
O
O
O
NHAc
NHAc
THF, pyr (94 %)
NO2
4
5
Scheme 1. Synthesis of carbohydrate derivative 5 activated as its p-nitrophenyl carbonate.
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O
O
Fmoc-HN
N
Fmoc-Bal-NH
Sieber
H
O
6
Coupling cycle:
1. 20% piperidine
2. Fmoc-Xaa-OH, HBTU, HOBt, DIEA
Last Cycle:
1. 20% piperidine
2. Boc-Lys(Aloc)-OH, HBTU, HOBt, DIEA
O
O
Ddv =
iBu
Aloc Ddv
Ddv Ddv
OAll
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH
Sieber
Aloc Ddv
1% TFA
Ddv Ddv
OAll
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH2
7
[Pd(PPh3)4], morpholine
Figure 1. RP-HPLC analysis of the crude peptides 7 ± 9, 11, and 12
obtained according to Scheme 2 4a ± d), Scheme 3, route A 4e), and
Scheme 3, route B 4f).
Ddv Ddv Ddv
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH
Sieber
Ddv
Ddv Ddv
1% TFA
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH2
cyclization step the allyl ester and the Aloc group were
simultaneously removed by palladium40)-catalyzed allyl trans-
fer to morpholine.[13]
8
HBTU, HOBt, DIEA
Ddv Ddv Ddv
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH
Sieber
Addition of a mixture of HBTU, HOBt, and Hünigꢁs base
4DIEA) led to the desired cyclopeptide which after cleavage
from the resin was obtained in excellent purity 4Figure 1c).
Side products due to reaction of coupling reagent with the free
amino group[19] 4guanylation) was not observed. Other
cyclization methods 4N,N'-diisopropyl carbodiimide/HOBt
or activation of the carboxyl group as a Pfp ester[20]) were
clearly less effective. Subsequently, the Ddv groups were
removed by batchwise treatment with a 4% solution of
hydrazine hydrate which turned out to be advantageous over
the literature-recommended[12] 2% solution.[21]
Ddv
Ddv Ddv
1% TFA
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH2
9
1. 4% H2N-NH2 • H2O
2. 5, DIEA
R
R
R
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH
Sieber
10
R
R
R
1% TFA
Boc-Lys-Orn-Gly-Ala-D-Lys-Orn-D-Val-Glu-Bal-NH2
Attachment of the sugars was effected by addition of excess
45 equiv per free NH2 group) of 5 in the presence of DIEA.
After complete reaction, the absence of free amino groups
was verified by Kaiser test[22] and by addition of bromophenol
blue.[23] In order to deacetylate the sugars, 10 was treated with
a solution of sodium methoxide in methanol 4Scheme 3,
route A). After cleavage from the resin, the HPL-chromato-
gram shown in Figure 1e was obtained in which beside the
desired glycopeptide 12 noticeable amounts of side products
are visible. Since their appearance is possibly due to the acid
lability of 12[24] and therefore the chromatogram might not
reflect the realities on the solid support, we carried out the
deacetylation after cleavage from the resin for comparison
4Scheme 3, route B). The obtained chromatogram of the
crude product 4Figure 1 f) now indicates only slight amounts
of side products demonstrating the high efficiency of the
whole synthesis.
In order to verify that the described reaction conditions,
especially those of the critical cyclization step, are trans-
ferable to other peptide sequences, we synthesized the
neoglycopeptide library 15 comprising 18 compounds by
using the split ± mix method 4Scheme 4). Again, the course of
the synthesis was monitored by withdrawal of small resin
samples and analysis of the cleavage products by HPLC in
combination with ESI-MS 4Figure 2, Table 2). The expected
11
OAc
O
AcO
O
O
R =
AcO
NHAc
O
Scheme 2. Solid-phase synthesis of cyclic neoglycopeptide 11.
10
11 (crude product)
A)
B)
NaOMe,
1. NaOMe, MeOH
2. 1% TFA
MeOH, CHCl3
R–HN
NH–R
H
N
HN
H3C
O
O
O
O
O
O
N
O
O
H
NH
NH
HN
N
NH2
H
H
N
O
O
HN
HN
Boc
R–HN
OH
O
HO
HO
O
O
R =
NHAc
O
12
Scheme 3. Synthesis of deprotected neoglycopeptide 12.
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6
Combinatorial
solid-phase synthesis
Aloc
OAll
-Leu-Glu-Bal-NH
Ala
Ile
Pro
Val
Gly
D-Lys(Ddv)
Leu
Boc-Lys-
-Lys(Ddv)-
-Pro-
Sieber
D-Lys(Ddv)
13
1. [Pd(PPh3)4], morpholine
2. HBTU, HOBt, DIEA
Ala
Ile
Gly
D-Lys(R)
Leu
Boc-Lys- Pro -Lys(R)-
-Pro-
-Leu-Glu-Bal-NH
Sieber
D-Lys(R)
Val
14 R = Ddv
1. 4% H2N-NH2 • H2O
2. 5, DIEA
OAc
O
AcO
AcO
O
O
15 R =
NHAc
O
Scheme 4. Split ± mix synthesis of neoglycopeptide library 15. At the
positions denoted by square brackets the resin was distributed at two and
three reaction vessels, respectively, and each coupled with one of the given
amino acids.
Figure 2. RP-HPLC analysis of the compound libraries obtained from
treatment of a) 13, b) 14, and c) 15 with a 1% solution of TFA. The
assignment of peaks resulted from mass spectrometric analysis and is given
in Table 2. Peaks of corresponding peptide derivatives are marked with
identical numbers. Those marked with asterisks stem from non-peptide
impurities.
18 products were identified in all chromatograms. It turned
out that all peptides underwent cyclization without forming
noteworthy amounts of side products. Particularly, after mass
spectrometric analysis of 14 we were not able to detect any
starting materials or linear or cyclic peptide dimers.
The introduced synthetic strategy is suitable for efficient
construction of large libraries of neoglycopeptides presenting
any desired carbohydrate ligands in variable quantity and
varying distances to each other. With the convergent ap-
proach it is easily possible to apply a once prepared cyclo-
peptide library in studying different lectins by simply attach-
ing appropriate carbohydrate ligands to it.
Experimental Section
The solid-phase peptide synthesis was carried out on NovaSyn TG Sieber
resin 4Novabiochem) 4abbreviation: Sieber-TG) following standard proto-
cols[17] 4loading after immobilization of the first amino acid: 0.17mmolg À1).
Couplings were performed in NMP. For cleavage solid-phase aliquots 4ca.
5 mg) were repeatedly treated with TFA/iPr3SiH/CH2Cl2 41:1:98). Products
Table 2. Calculated and experimentally found masses of the library of cyclic neoglycopeptides obtained by treatment of 15 with a 1% solution of TFA 4peak
numbers correspond to the assignment given in Figure 2c).
‡
Peak
Compound[a]
[M‡H]
calcd
found
1
2
8
3
7
4
10
9
cyclo[Boc-Lys-Pro-Lys4R)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Pro-Lys4R)-d-Lys4R)-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-Ala-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Pro-Lys4R)-Ala-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Pro-Lys4R)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-d-Lys4R)-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-Ala-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2 or
cyclo[Boc-Lys-Ile-Lys4R)-d-Lys4R)-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-d-Lys4R)-Pro-Gly-Leu-Glu]-Bal-NH2 or
cyclo[Boc-Lys-Val-Lys4R)-Ala-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Pro-Lys4R)-d-Lys4R)-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-Ala-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-d-Lys4R)-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-Leu-Pro-Gly-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Pro-Lys4R)-Leu-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Val-Lys4R)-Leu-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-d-Lys4R)-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
cyclo[Boc-Lys-Ile-Lys4R)-Leu-Pro-d-Lys4R)-Leu-Glu]-Bal-NH2
1434.71435.4
1436.71437
1934.9
1450.8
1949.0
1476.8
1937.0
1951.0
.3
1935.7
1451.2
1950.0
1477.4
1938.0
1952.0
5
12
1478.8
1951.0
1479.4
1952.0
16
11
17
6
13
14
18
15
2449.2
1965.0
2451.2
1492.8
1991.0
1993.0
2465.2
2007.0
2450.1
1966.2
2452.8
1493.3
1991.8
1993.6
2465.9
2008.0
OAc
O
AcO
AcO
[a]
O
O
O
R =
NHAc
Angew. Chem. Int. Ed. 2000, 39, No. 23
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1433-7851/00/3923-4351 $ 17.50+.50/0
4351

COMMUNICATIONS
that were hardly soluble in the cleavage cocktail were washed off the solid
support with a suitable solvent. Before concentration cleavage solutions
were neutralized with pyridine. Analytical HPLC was carried out on C18
reversed-phase columns 4250 Â 4 mm) with linear gradients of acetonitrile
in water/0.1% TFA and a flow of 1 mLminÀ1. Product peaks were
characterized by ESI-MS. Experimental details for the synthesis of 8, 9, 11,
and 12 are found in the Supporting Information.
[22] E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem.
1970, 34, 595 ± 598.
[23] Monitoring of single beads under a microscope.
[24] The stability of a glycosidic bond is enhanced upon acylation of the
carbohydrate: H. Kunz, C. Unverzagt, Angew. Chem. 1988, 100,
1763 ± 1765; Angew. Chem. Int. Ed. Engl. 1988, 27, 1697.
[25] Abbreviations used: Aloc ˆ allyloxycarbonyl, Bal ˆ b-alanine, Boc ˆ
tert-butoxycarbonyl, Dde ˆ 1-44,4-dimethyl-2,6-dioxocyclohexylide-
ne)ethyl), Ddv ˆ 1-44,4-dimethyl-2,6-dioxocyclohexylidene)isovaler-
yl, DIEA ˆ ethyldiisopropylamine, ESI-MS ˆ electrospray ionization
mass spectrometry, Fmoc ˆ 9-fluorenylmethoxycarbonyl, HBTU ˆ O-
Received: May 16, 2000
Revised: August 4, 2000 [Z15129]
benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophos-
phate, HOBt ˆ 1-hydroxybenzotriazole, Mtt ˆ 44-methylphenyl)di-
phenylmethyl, NMP ˆ 1-methyl-2-pyrrolidone, MeOTf ˆ methyl tri-
fluoromethanesulfonate, Pfp ˆ pentafluorophenyl, RP-HPLC ˆ re-
versed-phase HPLC, TBDPS ˆ tert-butyldiphenylsilyl, TFA ˆ tri-
fluoroacetic acid, Troc ˆ trichloroethoxycarbonyl.
[1] Essentials of Glycobiology 4Eds.: A. Varki, R. Cummings, J. Esko, H.
Freeze, G. Hart, J. Marth), Cold Spring Harbor Lab. Press, Cold
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[2] a) Y. C. Lee, R. T. Lee, J. Biomed. Sci. 1996, 3, 221 ± 237; b) J. M.
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gates. Preparation and Applications 4Eds.: Y. C. Lee, R. T. Lee),
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[7] a) G. B. Sigal, M. Mammen, G. Dahmann, G. M. Whitesides, J. Am.
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N. S. Pannu, R. J. Read, D. R. Bundle, Nature 2000, 403, 669 ± 672;
b) E. Fan, Z. Zhang, W. E. Minke, Z. Hou, C. L. M. J. Verlinde,
W. G. J. Hol, J. Am. Chem. Soc. 2000, 122, 2663 ± 2664.
Wound-Activated Chemical Defense in
Unicellular Planktonic Algae**
Georg Pohnert*
Diatoms are highly successful unicellular algae occurring in
ocean and fresh water phytoplankton, as well as in biofilms on
solid substrates. They are exceedingly abundant and are
among the most important primary sources sustaining the
marine food chain. Despite this, little is known about the
chemical defense of these micro algae. Two of the few
reported examples are the aldehydes decadienal 5 and
decatrienal 6 4see Scheme 1) from the diatom Thalassiosira
rotula, which reduce the hatching success from eggs of
copepods 4zooplankton grazers).[1] This observed activity
explains the paradox that herbivorous copepods are less
successful feeding on diatoms, although these algae are
considered as high-quality food.
Here I provide biosynthetic and kinetic data on the
formation of fatty acid derived metabolites in planktonic
diatoms, demonstrating that the release of a,b,g,d-unsaturat-
ed dienals is widespread among this class of algae. The
enzymatic mechanism to produce these metabolites is effi-
ciently activated seconds after cell disruption and leads to
high local concentrations of the defensive metabolites 5 and 6
or of structurally related potentially active aldehydes like 9.
The simultaneous production of C11 hydrocarbons and
9-oxonona-5Z,7E-dienoic acid from C20 fatty acids was
demonstrated with the benthic diatom Gomphonema parv-
ulum.[2, 3] The polar dienoic acid contains the same aldehydic
Ï Â
[9] K. S. Lam, M. Lebl, V. Krchnak, Chem. Rev. 1997, 97, 411 ± 448.
[10] On the use of cyclic peptides as scaffolds see: a) U. Sprengard, M.
Schudok, W. Schmidt, G. Kretzschmar, H. Kunz, Angew. Chem. 1996,
108, 359 ± 362; Angew. Chem. Int. Ed. Engl. 1996, 35, 321 ± 324; b) H.
Franzyk, M. K. Christensen, R. M. Jùrgensen, M. Meldal, H. Cordes,
S. Mouritsen, K. Bock, Bioorg. Med. Chem. 1997, 5, 21 ± 40; c) G.
Tuchscherer, M. Mutter, Pure Appl. Chem. 1996, 68, 2153 ± 2162; d) J.
Eichler, A. W. Lucka, R. A. Houghten, Peptide Res. 1994, 7, 300 ± 307.
[11] R. Haubner, D. Finsinger, H. Kessler, Angew. Chem. 1997, 109, 1440 ±
1456; Angew. Chem. Int. Ed. Engl. 1997, 36, 1374 ± 1389, and
references therein.
[12] S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft, W. C.
Chan, Tetrahedron Lett. 1998, 39, 1603 ± 1606.
[13] H. Kunz, C. Unverzagt, Angew. Chem. 1984, 96, 426 ± 427; Angew.
Chem. Int. Ed. Engl. 1984, 23, 436 ± 437.
[14] A. Schleyer, M. Meldal, M. Renil, H. Paulsen, K. Bock, Angew. Chem.
1997, 109, 2064 ± 2067; Angew. Chem. Int. Ed. Engl. 1997, 36, 1976 ±
1978.
[15] a) U. Ellervik, G. Magnusson, Carbohydr. Res. 1996, 280, 251 ± 260;
b) M. Schultz, H. Kunz, Tetrahedron: Asymmetry 1993, 4, 1205 ± 1220.
[16] P. Sieber, Tetrahedron Lett. 1987, 28, 2107± 2110.
[17] D. A. Wellings, E. Atherton, Methods Enzymol. 1997, 289, 44 ± 67.
[18] B. W. Bycroft, W. C. Chan, S. R. Chhabra, N. D. Hone, J. Chem. Soc.
Chem. Commun. 1993, 778 ± 779.
[19] S. C. Story, J. V. Aldrich, Int. J. Pept. Protein Res. 1994, 43, 292 ± 296.
[20] Using this cyclization method the Rink amide linker was applied for
attachment to the solid support and the e-NH2-group of the lysine
residue involved in cyclization was protected with the Mtt group and
liberated after formation of the Pfp ester: D. Tumelty, M. C. Needels,
V. V. Antonenko, P. R. Bovy in Peptides: Chemistry, Structure, and
Biology 4Eds.: P. T. P. Kaumaya, R. S. Hodges), Mayflower Scientific,
Kingswinford, 1996, pp. 121 ± 122.
[*] Dr. G. Pohnert
Max-Planck-Institut für Chemische Ökologie
Carl-Zeiss-Promenade 10, 07745 Jena 4Germany)
Fax : 4‡49)3641-643665
E-mail: pohnert@ice.mpg.de
[**] I gratefully acknowledge the gift of T. rotula by Prof. S. Poulet
[21] By using
a 2% solution of hydrazine hydrate, even after five
4Roscoff, France).
stimulating discussion during the preparation of the manuscript. I
thank J. Rechtenbach for technical assistance.
I am indebted to Prof. Dr. W. Boland for
treatments significant amounts of the cleavage product 3-iso-butyl-
6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole could be detected
by UV spectroscopy 4A290).
4352
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1433-7851/00/3923-4352 $ 17.50+.50/0
Angew. Chem. Int. Ed. 2000, 39, No. 23