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Solid Phase Synthesis of Peptides and Glycopeptides on Polymeric Supports with Allylic Anchor Groups.

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Solid Phase Synthesis of Peptides and Glycopeptides
on Polymeric Supports with Allylic Anchor Groups**
By Horsr Kunz* and Berthold Dombo
Merrifield's concept of carrying out the synthesis of peptides on polymeric supports constitutes one of the milestones in modern chemistry."] Both in the original['] as well
as in the present day form of this solid-phase peptide synthesis"] the C-terminal amino acid is anchored as substituted benzyl ester to the polymeric support (resin) either
via a coupling group ("handle", spacer) or directly. Strong
acids are used for detachment of the synthesized peptide
from the resin. The N-terminal protecting groups and most
of the side-chain protecting groups are removed at the
same time. Alkoxybenzyl ester^'^,^] as anchor groups enable removal of the peptide with trifluoroacetic acid. With
dialkoxybenzyl esters as anchor
even dilute trifluoroacetic acid can be used for removal of the peptide.
Then, the fluorenylmethoxycarbonyl (Fmoc) group[81has
to be used as N,-protecting group, since acid conditions
must be carefully avoided during synthesis of the chain
with such an acid-labile anchorage.
With the aim of constructing the acid-sensitive and in
part also base-sensitive glycopeptides['] in solid-phase syntheses, we have used the successful strategy of palladium(o)-catalyzed deblocking of peptide- and glycopeptide
allyl esters"'I as well as allyloxycarbonyl(Aloc)-peptides
and glycopeptides" 'I for the development of a polymeric
support with allylic anchor groups.I"I Thereby we were
able to achieve both a n acid- as well as base-stable anchorage of the peptide and glycopeptide chains to be synthesized on a resin, from which, however, they can be detached at room temperature under neutral, weakly acid o r
weakly basic
Suitable for the production
of the polymers containing allylic anchor groups such as 3
is the reaction of resins containing aminomethyl groups,
e.g. of aminomethylpolystyrene"31 2 with haloalkenoic
acids such as
which have an allyl halide partial
structure. 2 contains 0.8-3.3 mequiv. H,N-CH, per gram
resin. This reaction is particularly easy to carry out: 1 and
2 are condensed with dicyclohexylcarbodiimide (DCC)
and I-hydroxybenzotriazole (HOBt)L'51
in CH2C12to give
peptides and glycopeptides on the resin 3a are shown
here. The corresponding alcohol 3b can also be used, but
there is the danger of racemization during the esterification with the C-terminal amino acids.
Reaction of the allyl halide-resin 3a with the cesium
salts['61 of N-protected amino acids 4 (molar ratio 1 : 1.5)
furnishes the [4-(aminoacyloxy)crotonyl]aminomethyl resins 5 . The yields (determined by elemental analysis) of
the hitherto unoptimized reactions are 70-82% (Table I).
+ PG-Xaa-OQCse
Table I. Coupling of the amino acid salts 4 with the resin 3a to protected
aminoacyl-Hycram resins 5 and cleavage of the protecting group PG (Step
A) with 50% CF3COOH/CH2Cl2 (1 h) to give aminoacyl-hycram resins 6.
Owing to the high acid-stability of the allylic ester bond,
the N,-Boc group in the protected aminoacyl-Hycram resins 5a-c is selectively cleavable with trifluoroacetic acid/
dichloromethane (step A). The polymer-bound salts 6 thus
6 a >-
a, X = Br; b, X = OH
A cross-linking alkylation (formation of bromide ions) is
not observed in this reaction. Among the possible anchor
groups, the polar crotonyl group in 3 has the advantage of
being highly acid-stable; therefore the model syntheses of
[*] Prof. Dr. H. Kunz, DipLChem. B. Dombo
Institut fur Organische Chemie der Universitat
J.-J.-Becher-Weg 18-20, D-6500 Mainz (FRG)
This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
Angew Chem Int Ed Engl 27(1988)
No S
Table 2. Stepwise synthesis of the polymer-bound peptides and glycopeptides 7 to 10. Normal conditions for step B, see text.
Reagents etc.
B2,A, B3
B4, A, B5, A, BS,
A, 8 6
B1: Boc-Leu-OH, 2 h
8 2 : Boc-Asn(Ac3GlcNAc)-OH in
CHZC12/DMF, 12 h
8 3 : Boc-Leu-OH, CH2C12,8 h
8 4 : Boc-Phe-OH, 2 h
8 5 : Boc-Gly-OH, 2 h
B6: 2-Tyr(Bz1)-OH, 5 h
B7: Fmoc-Asn-OH, DMF, 3 h
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(1 :lo v/v)
< 30 rnin
12, 2 1 0 0 %
obtained are subjected to the synthesis cycle typical for
solid-phase peptide syntheses."] In the coupling step B
ethyldiisopropylamine, N-protected amino acid (PG-XaaOH), DCC and HOBt ( 5 :5 : 6 : 6 )are allowed to react with
1 mequiv. of aminoacyl-Hycram resin 6 with shaking in ca.
50 mL of dichloromethane or dichloromethane/DMF. The
same solvent is used for the rewashing. The polymerbound peptides and glycopeptides 7 to 10 are thus synthesized by stepwise chain lengthening (Table 2).
The coupling with Fmoc-Asn-OH (step B7) is carried
out with a small excess of reagent (2 :2 :2 :2) so as to avoid
nitrile formation. It is obviously incomplete, since free alanine is found during later cleavage of the resin.
For a better assessment of the cleavage reaction we have
synthesized the low molecular counterpart 11 of 7 from
the N-benzylamide of 4-bromocrotonic acid.f141
The Pdo-catalyzed cleavage of 11 proceeds selectively
and q ~ a n t i t a t i v e l y ~in' ~less
~ ' ~ than
30 min in the way previously described by US.^'^.^'^
The polymer-bound peptide 7 reacts in a completely
analogous way. Almost quantitative cleavage from the resin is achieved within 2 h (step C). Even the glycotripeptide
14 and the Leu-enkephalin 15 protected on the tyrosine
are smoothly removed from the Hycram polymer in this
way (Table 3).
Because of the incomplete coupling to 10, the yield of
Fmoc-dipeptide 16 is low. But no dipeptide with complete
loss of Fmoc is found. Any alanine (formed from un7
d Boc-Leu-ALa-OH
9 d Z-Tyr(Bzl)-GLy-Gly-Phe-Leu-OH
10 d Frnoc-Asn-Ala-OH
( + 30% H-Ala-OH)
Table 3. Detachment of the peptides and glycopeptides 12 and 14 to 16
from the resin. Normal conditions for step C : 90 rng (Ph,P),Pd per mequiv.
peptide or glycopeptide in 50 rnL T H F and 5 mL morpholine.
Reagents etc.
Yield [%]
7 - 1 2 1191
see above, 2 h
8- 14 1201
see above, 10 h
see above, 12 h
see above, but in 30 mL T H F
and 1 g dimedone, 2 h: from
washing 16 with methanol
98 (based
80 (based
76 (based
63 (based
9-15 [21]
10- 16 [22]
7 12
48 (based on 5a)
0 VCH Verlugsgesellschajt mbH, 0-6940 Weinheim, 1988
changed 6a) and dimedone are separated off chromatographically on a short silica gel column (methanol/dichloromethane). The total yields of glycopeptide 14 and pentapeptide 15 are substantial, considering that no repeated
coupling was applied in any of the reactions. It can be concluded therefore that the Pdo-catalyzed cleavage reaction
proceeds very effectively even in the case of the more complex resin-bound products 8 and 9 . The products 15 purified chromatographically with chloroform/methanol or reprecipitated from methanol/chloroform entrain some methanol. Their identity is confirmed by monitoring the 400MHz 'H-NMR spectra.120.''1
In summary, the polymeric supports such as 3a with
allylic anchor groups have the following advantages:
1. The solid-phase synthesis of peptides and glycopeptides on these supports can be carried out both with acidas well as base-labile protecting groups.
2. The detachment of the synthesized product from the
support can be carried out very effectively under almost
neutral conditions.
3. Acid- (Boc) and base-labile (Fmoc or OAc in 14) protective groups as well as the sensitive glycoside bonds remain intact during the cleavage.
Received: December 18, 1987;
revised: February 9, 1988 [Z 2548 IE]
German version: Angew. Chem. 100 (1988) 732
Review: G. Barany, N. Kneib-Cordonier, D. G. Mullen, Int. J. P e p . Protein Res. 30 (1987) 705.
R. B. Merrifield, J. A m . Chem. SOC.85 (1963) 2149.
A. R. Mitchell, S. B. H. Kent, M. Engelhard, R. B. Merrifield, J. Org.
Chem. 43 (1978) 2845.
S. S. Wang, J . Am. Chem. SOC.9s (1973) 1328.
I. P. Tam, R. DiMarchi, R. B. Merrifield, Int. J. P e p . Protein Res. 16
(1980) 412.
R. C. Sheppard, B. J. Williams, Int. J. Pept. Protein Res. 20 (1982) 451.
M. Mergler, Poster P60, Absrr. 10th A m . Pept. Svmp.. St. Louis 1987.
L. A. Carpino, G. Y . Han, J. Org. Chem. 37 (1972) 3404.
H. Kunz, Angcw. Chem. 99 (1987) 297: Angew. Chem. l n t . Ed. Engl. 26
(1987) 294.
a) H. Kunz, H. Waldmann, Angew. Chem 96 (1984) 49; Angew. Chem.
Int. Ed. Engl. 23 (1984) 71; b) ibid. 97 (1985) 885 and 24 (1985) 883; c)
Helu. Chim. Acta 68 (1985) 618; d) H. Kunz, H. Waldmann, C. Unverzagt, Int. J. Pept. Protein Res. 26 (1985) 493.
a) H. Kunz, C. Unverzagt, Angew. Chem. 96 (1984) 426: Angew. Chem.
Int. Ed. Engl. 23 (1984) 436: b) H. Kunz, H. Waldmann, C . Unverzagt in
D. Theodoropoulos (Ed.): Peptides 1986, de Gruyter, Berlin 1987, p.
H. Kunz, B. Dombo, Dtsch. Pat.-Anm. 3720269.3 (June 19, 1987).
A. R. Mitchell, S. B. H. Kent, B. W. Erickson, R. B. Merrifield, Tetruhedron Lett. 1976. 3795.
K. Ziegler, W. Schumann, E. Winkelmann, Justus Liebigs Ann. Chem.
551 (1942) 117.
W. Konig, R. Geiger, Chem Ber. 103 (1970) 788.
B. F. Gisin, Helu. Chim. Actu 56 (1973) 1476.
For reviews on Pdo-catalyzed ally1 transfer for C C bond formation see:
a) B. M. Trost, Acc. Chem. Res. 13 (1980) 385: b) J. Tsuji, Pure Appl.
Chem. 51 (1979) 1235.
For the first observation of reductive allyl ester cleavage by Pdo-catalyzed hydride transfer see: H. Hey, H.-J. Arpe, Angew. Chem. 8S (1973)
986: Angew. Chem. I n t . Ed. Engl. I2 (1973) 928; see also b) J. Tsuji, T.
Yamakawa, Tetrahedron Lett. 1979. 613: c) Pdo-catalyzed hydrostannylation of allyl carbonates: F. Guibe, Y. Saint M'Leux, ibid. 22 (1981)
3591; d) allyl carbonate, carboxylate, and carbamate cleavage by Pd"catalyzed transesterification with 2-ethyl hexanoate: P. D. Jeffrey, S. W.
McCornbie, J. Org. Chem. 47 (1982) 587.
12: [a]::= -28.4 ( c = 1, MeOH) (W. Danho, C. H. Li, Inr J. P e p . Protein Res. 3 (1971) 81: [a]8=-29.0 (c= I, MeOH)), R,=0.21 (CH2C1:/
MeOH 5 : I).-400 MHz-'H-NMR (CDCI,): 6=7.13 (m, I H, NH, Ala),
5.33 (rn. 1 H, NH, urethane), 4.53 (m, 1 H, a-CH, Ala), 4.22 (m, 1 H, aCH, Leu).
14: [a];;=-0.7 (c=0.65, MeOH), Rt=0.29 (CH:CI:/MeOH
5 : I).400MHz-'H-NMR ([DJlMSO): 6=8.72 (d, J = 10 Hz, 1 H, @-NH,Asn),
8.32 (m, 1 H, a - N H , Asn), 7.99 (d, br., J = 10 Hz, 1 H, NHAc), 7.08 (m.
0570-0833/88/050S-0712 $ 02.50/0
Angew. Chem. Int. Ed. Engl. 27 (1988) No. 5
I H, NH, He), 6.9 (d, J = 10 Hz, 1 H, NH, Leu), 5.25-5.1 1 (m. 2 H , H-I,
H-3, GlcNAc), 2.0, 1.95, 1.92 (3 x s, each 3 H, OAc), 1.34 (5. br. over multiplet, 9H, CH3, Boc), 0.88 (m. 12H, CHi, Leu, He).
[?I] 15:
+0.07, [ale6=+0.05, [a]%,= -0.07 (c=0.7, MeOH),
R1=0.48 (CH?CI,/MeOH 2 : 1).-400MHz-'H-NMR
6= 8.17, 8.08. 7.54 (each 1 H, NH), 7.46-7.05 (m, 17H, aromat.
H of Phe, Z, Bzl, 2 H, Tyr), 6.88 (d, J = 10 Hz, 2 H, Tyr), 5.03 (s,2 H, CHI,
Z): 4 8 9 (m, 2H, CH2, Bzl); 0.88 and 0.82 (each d, J = 7 Hz, each 3H,
CH,, Leu).
I221 16: [n]?= - 13.4 (c=O.8, DMF), R,=0.60 (CH2Clz/MeOH 1 :I).200MHz-'H-NMR ([D,IDMSO): 6=8.0-7.25 (m, 11 H, Fmoc, amide
NH), 6 97 (5, br., 1 H, NH, urethane), 3.92-3.70 (m, 4 H , a-CH, Asn,
CH-CH2, Fmoc), 1.2 (d, J = 7 Hz, 3 H , CHI, Ala).
Annelation of Carbene Ligands by 13-Phosphaalkynes,
an Entry to Functionalized Phosphaarenes**
By Karl Heinz Dotz, * Athanassios Tiriliomis, Klaus Harms,
Manfred Regitz, and Ulrich A m e n
Following the synthesis of the first stable h3-phosphaalkynel*] under normal conditions, a systematic investigation
of this class of compounds became possible. [3 21 and
[4 + 21 c y c l ~ a d d i t i o n sas
[ ~well
~ as metal-induced oligomerizationsi4]reveal close parallels with the reaction behavior
of alkynes. We were interested in the question of whether
phosphaalkynes can also be used for the annelation of carbene ligands. This type of reaction has proven useful for a
direct entry to highly substituted h y d r o q ~ i n o n e s [and
~ ' for
syntheses in the natural products ~ e r i e s . ~ ' , ~ ]
The pentacarbonyl[al koxy( 1-naphthyl)carbene]chromium
complex 1 reacts with 2,Z-dimethylpropylidynephosphane-
5 bar A r
nated to the newly constructed phosphaaromatic ring,
whose 3'P-NMR signal is characteristically shifted upfield
with 6 = -26.50 (3a) and -24.77 (3b), as in the case of
the only previously known tricarbonyl(ph0sphaarene)chromium complex tricarbonyl(2,4,6-triphenylphosphinine)chromium 5.['l Within the limits of NMR detection the
incorporation of the P=C moiety is regiospecific; the coupling constants of 3Jp,c=34.3 (3a) and 35.3 Hz (3b), respectively, observed in the I3C-NMR spectrum for the
OCH3- and OCH,-groups suggest the 3-phosphaphenanthrene structure. As in the carbene annelation with alkynes,I8] the regioselectivity is apparently sterically controlled. By ligand-exchange under CO pressure,['] the
phosphaarene 4 can be liberated almost quantitatively
from 3b; the Cr(C0)6 formed in the reaction can be further used for the synthesis of the carbene complex l . As
expected, in the 13C-and 3'P-NMR spectra of 4 the signals
for the ring atoms of the heterocycle are shifted downfield
(A6(I3C) = 30-40 ppm; A6(3'P) = 150 ppm) compared to
those of the coordinated phosphaarene 3b.
In contrast to tricarbonyl(phenanthrene)chromium, in
which the metal is coordinated unsymmetrically to the terminal ring,"'] in the phosphaphenanthrene complex 3a the
tricarbonylchromium group is bound centrally to the phosphinine ring (Fig. 1).[ITJ The metal is in a pseudo-octahe-
[*] Prof. Dr. K. H. Dotz, DipLChem. A. Tiriliomis, Dr. K. Harms
2, and by coupling of the phosphaalkyne, the carbene and
a carbonyl ligand gives the phosphaphenanthrenehydroquinone complexes 3 . The Cr(C0)3 fragment is coordi-
Fachbereich Chemie der Universitat
Hans-Meerwein-Strasse, D-3550 Marburg (FRG)
Prof. Dr. M. Regitz, Dipl.-Chem. U. Annen
Fachbereich Chemie der Universitat
Erwin-Schrodinger-Strasse,D-6750 Kaiserslautern (FRG)
Reactions of Complex Ligands, Part 33. This work was supported by the
Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.-Part 32: [I].
Angen'. Chem. In,. Ed. Engl. 27(1988) No. 5
Fig. I . Structure of 3a in the crystal. Projection onto the plane of the phosphinine ring. Important bond lengths [A] and angles ["I: Cr-PI 2.4591(4), CrChe,eroclclc
2.290( 1) (mean value), Cr-Ccrmonyl1.836(2) (mean value), CI-PI
1.768(1), C13-PI 1.759(1), CI-C2 1.400(2), C2-C3 1.438(2), C3-CI2 1.428(2),
C12-CI3 1.437(2); C,,,hon,l-Cr-C,,,b,,,I 89.5(1) (mean value), Cr-C-OLrrhonyl
178.2(1) (mean value), CI-PI-C13 101.73(6), PI-CI-C2 121.4( I), CI-C2-C3
l26.4( I), C2-C3-C12 l24.0( I), C3-CI2-Cl3 1 l7.9( I), Pl-CI3-Cl2 l28.2( I):
torsional angle: CI-C2-C3-C12 0.3(4), C2-C3-C12-C13 4.8(4), C3-CI2-Cl3PI - 4.2(4), C12-C 13-PI-C 1 - 0.5(3), C 13-PI-CI-C2 5.2(2), PI -C I -C2-C3
- 5.8(4).
drat environment. On comparison with the only previously
X-ray crystallographically investigated phosphaarenechromium complex 5:Iz1 the following differences were
noted: the Cr-Cring distances (average: 2.290(1) A) are
somewhat longer and the Cr-P bond (2.4591(4)A) is
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synthesis, solis, anchor, group, support, glycopeptides, polymeric, phase, allylic, peptide
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