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Chain Elongation of Thiodipeptides with Proteases.

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pentane solutions were concentrated to 10 mL and cooled to -40°C. A colortivity and selectivity. Thiopeptides are attractive as peptiless solid crystallized (0.28 g, 42%). which sublimed at 2O0C/0.O01Tom.domimetics1'1 because of their enhanced stability towards
NMR spectra measured in C,D, at 25 "C; 'H NMR (499.843 MHz, TMS):
proteases['I
and the conformational changes they induce.l3]
6 = 1.83 (s, Me), 2.16 (H2-H6), 2.47 (H7-Hll), 3.03 (H12), measurement and
Their general application has been, however, prevented by
assignment of the three B-H signals as cross peaks in the 2D IIB-'H NMR
spectrum; "B NMR (160.364 MHz, Et,O-BF,): 6 = -11.2 (d, J = 1 4 7 Hz,
insufficient synthetic availability. The OjS exchange in pep87-Bl1,crosspeaksin the2D "B-"BNMRspectrum with B2-B6and with
tides by Lawesson's reagentL41or its variantsI51may be selec812). -5.1 (d, /=183Hz, B2-B6, no cross peak with B12), -0.1 (d,
tive in cyclic p e p t i d e ~ [6bl~ ,but its effectiveness is difficult to
J = 147 Hz, Bl2).
predict. Only protected dipeptides 1 allow the selective OjS
3: To a solution of 2 (39.0 mg, 0.245 mmol) in 2 mL of CH,CI, at room temperexchange at the amide position (+ 2) in high yields without
ature was added 0.10 mL of methanol. This mixture was stirred for 30min and
then treated with a solution of [N(PPh,),CI] (140 mg, 0.256 mmol) in 3 mL of
affecting urethane or ester groups. Deprotection of thiopepCH,CI,. Pentane was added to the reaction mixture; 3 precipitated and was
tides 2 generally causes no difficulties.
filtered, washed with pentane three times, and dried. Analytically pure bis(triphenylphosphorany1idene)ammonium [2-methoxy-l-methyl-8,9-p-hydrodecahydro-1-aza-nido-dodecaborate](3) was obtained (175 mg, 98 "A).Single
R O
R' 0
crystals of 3 were obtained from hexane/dichloromethane at -40 "C. - NMR
I II
I II
spectra were measured at 25 "C in CDCI, (same frequencies and standard as for
Boc-NH- CH - C-NH-CHC-OMe
2); 'H{"B} NMR: 6 = - 3.81 (broad, p-H8/9), 0.32 (H13). 1.41 (HlO/ll),
1.52(H4/5),1.85(H6/7),2.15(s,3H,NMe),2.44(H12),3.16(H8/9),3.80(s,
3H. OMe), 7.42-7.68 (30H, Ph), assignment of the exo B-H signals as cross
peaks in the 2D "B-IH NMR spectrum; I'B NMR spectrum: 6 = - 34.7 (d,
J = 1 3 4 H z , B13), -18,6(d,J=134Hz,B6/7), -13,4(d,J=134Hz, B4/5),
- 11.8 (d, J =134 Hz, BlOjll), 8.2 (d, J = 134 Hz, B12), 1.9 (d, J = 1 4 7 Hz,
B8/9), 9.3 (s, B2), assignment with help of the cross peaks in the 2D I l E
IiB NMR spectrum; "C NMR (125.697 Hz, TMS): 6 = 37.7 (NMe), 53.4
(OMe), 126.9 (d, Jpc=lo8 Hz, @so-C of Ph), 129.6, 132.0, 133.8 (Ph).
-
Received: April 8, 1992 [ZS288IE]
German version: Angew. Chem. 1992, 104, 1221
CAS Registry numbers:
1, 142581-18-8; 2, 142581-19-9; 3.PPN, 142581-21-3; 3 - H 1 , 142611-00-5;
Me-, 15194-58-8; rBuO-, 16331-65-0; F-, 16984-48-8.
[l] J. Miiller, J. Runsink, P. Paetzold, Angew. Chem. 1991, 103, 201; Angew.
Chem. lnt. Ed. Engl. 1991, 30, 115.
[2] R. A. Wiesboeck, M. F. Hawthorne, .IAm. Chem. Soc. 1964, 86, 16421643.
131 L. D Brown, W. N. Lipscomb, Inorg. Chem. 1977, 16, 2989-2996.
[4] J. Muller, P. Paetzold, R. Boese, Hereroutom Chem. 1990, I, 461-465.
[5] Crystal data: a =18.181[5], b = 9.015(3), c = 24.401(8), a = p = y = 90",
V = 4000(2) A', Z = 4, pca,c= 1.413 gcm-', Pcu2, (No. 29), Nicolet
R3m/V: recording temperature 125K; p(MoK.) =1.6 cm-'; 4465 independent reflections with 3<28<45", 3919 of these observed (F0<4o(F));
structure solution and refinement with 398 parameters with SHELXTLPLUS (version 4.2); R = 0.058, R, = 0.066, ~ r - ' = oz(Fo) + 0.00l5Fo2.
All H atoms bound to B atoms were located by difference Fourier synthesis
and refined with a common isotropic dislocation factor. The H atoms on
C2 are disordered; the H atoms on the cation were treated as riding groups.
Further details of the crystal structure investigation may be obtained from
the Fachinformationszentrum Karlsruhe, Gesellschaft fur wissenschaftlich-technische Information mbH, D-W-7514 Eggenstein-Leopoldshafen 2 (FRG) on quoting the depository number CSD-320454, the
names of the authors, and the journal citation.
[6] X. L. R. Fontaine, H. Fowkes, N. N. Greenwood, J. D. Kennedy, M.
Thornton-Pett, .IChem. SOC.Dalton Trans. 1987, 2417-2429.
171 G. D. Friesen, A. Barriola, P. Daluga, P. Ragatz, J. C. Huffmann, L. J.
Todd, Inorg. Chem. 1980, 19, 458-462.
[8] T. L. Venable, R. B. Maynard, R. N. Grimes, J. Am. Chem. SOC.1984,106,
6187-6193.
[9] R. E. Williams in Eleclron Defcienl Boron und Carbon Cluslers, (Eds.:
G. A. Olah, K. Wade, R. E. Williams), Wiley, New York, 1991, pp. 11 -93.
[lo] F. Meyer, Dissertation, Technische Hochschule Aachen, in preparation.
Chain Elongation of Thiodipeptides with
Proteases"*
By Carlo Unverzagt,* Armin Geyer, and Howl Kessler*
The thioamide bond is a useful element for the structural
variation of biologically active peptides to increase their ac[*I Dr. C. Unverzagt, Prof. Dr. H. Kessler, Dipl.-Chem. A. Geyer
Organisch-Chemisches Institut
der Technischen Universitat Miinchen,
Lichtenbergstrasse 4, D-W-8046 Garcbing (FRG)
["I This work was supported by the Fonds der Chemischen Industrie, the
Deutschen Forschungsgemeinschaft, and the Leonhard-Lorenz-Stiftung.
Angew. Chem. Int. Ed. Engl. 1992, 31, No. 9
0 VCH
R
I
S
II
R'
I
Boc-o-Ala-Y[CS-NHIPhe-OMe 2a
0
II
Boc-NH-CH-C-NH-CH-C-OMe
Boc-LeuY[CS-NHILeu-OMe 2b
2
= Boc-ASY[CS-NHIAS-OMe
Boc-PheYtCS-NHIGiy-OMe 2c
Peptide elongation at the C terminus of thiopeptides by
means of classical peptide chemistry fails;[61instead the activated thiopeptide 3 forms a sulfur-containing heterocycle 4.
These thiazolones 4 are poor acylating agents and are prone
to racemization of the two chiral centers. Hence, only few
elongations of thiopeptides have been performed with achiral C termini, such as glycine or a-aminobutyric acid.[61
3
4
We report here on the enzymatic C-terminal elongation of
thiopeptides 2a, b and the N-terminal elongation of the
derivative of 2c deblocked at the N terminus (= 5e).Subtilisin Carlsberg and subtilisin BPN' accept 2a-c as substrates:
in 50 % dimethylformamide (DMF) the corresponding free
carboxylic acids are obtained in almost quantitative yield.
Under these conditions proteases do not cleave amide
bonds.[7981
Proteases can also be used to catalyze the elongation of amino acid esters and peptide esters at their C terminus.[81 We have successfully applied this mild synthetic
method to sensitive thiopeptides. The subtilisin BPN'
catalyzed reaction of thiopeptide methyl ester 2a with
leucine amide H-Leu-NH, (5c) yields mainly the hydrolyzed
dipeptide acid (60%) but also the desired tripeptide 6c
(24%) (see Table 1). Obviously the enzyme activates 2a mildly, thereby allowing the elongation to thiopeptide 6c,whereas the undesired side product (thiazolone) is not formed. The
250 MHz 'H NMR spectrum of 6c exhibits a characteristic
signal for the thioamide proton at 6 =10.2 and the resonances of all three amino acids. It also proves the sterochemical uniformity of the peptide bond formation.
The yields in the enzymatic peptide elongation of 2a with
H-Leu-NH, (5c) could be increased with chymotrypsin as a
biocatalyst (Table 1). Under the reaction conditions the
thiodipeptides are completely converted into 6 by hydrolysis
Verlugsgesellschaft mbH, W-6940 Weinheim, 1992
0570-0833/92j0909-1229 $3.50+ ,2510
1229
Table 1. Thiopeptides 6a-g synthesized with chymotrypsin.
Starting
Thiopeptides
materials [a]
Za
Za
2a
2a
+ 5a
+ 5b
+ 5c
+ 5e
2b
2b
+ 5d
+ 5e
Yield [ %]
Boc-D-AlaY[CS-NHIPhe-Val-OAlI 6a
B O C - D - A I ~ Y ~ C S - N H ] P ~ ~ - V6b
~I-OM~
Boc-D-AlaY[CS-NHIPhe-Leu-NH, 6c
70
49
48
Boc-o-AlaY[CS-NHIPhe-PheY[CS-NHIGly-OMe
6d
63
Boc-D-AI~Y[CS-NH]P~~-(P~~Y[CS-NH]G~~),-OM~
6e 8 [b]
Boc-LeuY[CS-NHILeu-Phe-NH, 6f
66
Boc-LeuY[CS-NHILeu-PheY[CS-NHIGly-OMe 6g
26
+
[a] 5a = H-Val-OAI1, 5b = H-Val-OMe, 5c = H-Leu-NH,, 5d = H-Phe-NH,,
Phe'f"CS-NHIGly-OMe. [b] Byproduct in the synthesis of 6d.
=
H-
+ H-Val-OAII
2a
chymotrypsin
DMF/H,O
-
5a
Boc- o-AlaY[CS-NH]Phe-Val-OAll
6a
Chymotrypsin cleaves peptides after aromatic amino acids
and leucine with high specifity,[*]but does not attack valine
esters.[g1Hence, the reaction of thiopeptide 2a with valine
allyl and methyl esters 5a, b proceeds to thiotripeptides esters. It is remarkable that the slightly larger and more
lipophilic allyl ester group in the nucleophile increases the
yield by more than 20 %. The resulting tripeptides 6a (70 YO
yield) and 6b (49 % yield) can be deprotected and incorporated into larger fragments. Removal of the allyl ester protecting group in thiotripeptide 6a under Pdo catalysis was
not affected by the nucleophilic thioamide sulfur atom
(Scheme l).[lo* Subsequent coupling with 7 in the pres-
N-/ert-Butyloxycarbonyl-o-thioalanyl-L-phenylalanyl-~-valine
allyl ester (6a):
A 500 mg portion (1.37 mmol) of 2a and 860 mg (5.48 mmol) of 5a were dissolved in 21.2mL of DMF/H,O ( l / l ) in a plastic vessel. 250mg of chymotrypsin was added, and the solution was stirred for 48 h. The mixture was
concentrated in high vacuum and chromatographed on 30 g silica gel eluting
with CH,Cl,/MeOH (lOO/l). Yield 472 mg (70%) 6a. [a];' = - 4.0 (c =1,
MeOH).
CAS Registry numbers:
Za, 142765-11-5;2b, 128421-81-8;5a, 88224-01-5;5b,4070-48-8;5~,687-51-4;
5d, 5241-58-7; 5e, 142765-12-6; 6a, 142765-13-7; 6b, 142765-14-8; 6c, 14276515-9; 6d, 142765-16-0; 6e, 142765-17-1; 61, 142765-18-2; 6g, 142765-19-3; 7,
142765-20-6; 8, 142765-21-7; 9, 142765-22-8; chymotrypsin, 9004-07-3;
protease, 9001-92-7.
[l] A. E Spatola in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, Vol. 7 (Ed.: B. Weinstein), Marcel Dekker, New York, 1983, pp.
267-357.
[2] R. E. Beattie, D. T. Elmore, C. H. Williams, D. J. S. Guthrie, Biochem. J.
1987, 24S, 285-288, and references therein.
[ 3 ] D. Seebach, S. Y. KO, H. Kessler, M. Kock, M. Reggelin, P. Schrnieder,
M. D. Walkinshaw. J. J. Bolsterli, D. Bevec, Helv. Chim. Acta 1991, 74,
1953-1990; H. Kessler, H. Matter, A. Geyer, H.-J. Diehl, M. Kock, G.
Kurz, F. R. Opperdoes, M. Callens, R. K. Wierenga, Angew. Chem. 1992,
104, 343-345; Angew. Chem. Int. Ed. Engl. 1992, 31, 328-330.
[4] K. Clausen, M. Thorsen, S O . Lawesson, Terrahedron 1981, 37, 36353639.
[5] a) M. Yokoyama, Y Hasegawa, H. Hatanaka, Y Kawazoe, T. Imamoto,
Synthesis 1984, 827-829; b) G. Lajoie, F. Lepine, B. Belleau, Tetrahedron
Lett. 1983, 24, 3815-3818; c) H. Davy, .
I
Chem. Soc. Chem. Commun.
1982,457-458.
[6] a) 0. E. Jensen, A. Senning, Tetrahedron 1986, 42, 6555-6564; b) D. B.
Sherman, A. F. Spatola, J. Am. Chem. Soc. 1990, l f Z , 433-441; c) D. W.
Brown, M. M. Campbell, M. S. Chambers, C. V. Walker, Tetrahedron
Lett. 1987,28, 2171-2174.
I
Am. Chem. SOC.
[7] C. F. Barbas 111, J. R. Matos, J. B. West, C. H. Wong, .
1988,110, 5162-5166.
[8] H. D. Jakubke in The Peptides, Vol. 9 (Eds.: S. Udenfriend, J. Meienhofer),
Academic Press, London, 1987, pp. 103-165.
[9] G. Kloss, G. Schroeder, Hoppe-Seyler's Z. Ph.ysiol. Chein. 1964,336,248256.
[lo] H. Waldrnann, H. Kunz, Liebigs Ann. Chem. 1983, 1712-1725.
[ l l ] H. Kunz, C. Unverzagt, Angew,. Chem. 1984,96,426-427; Angew. Chem.
hi.Ed. Engl. 1984, 27, 1697-1699.
2,4-Didehydrophenol-First Proof of a
meta-Aryne by IR Spectroscopy**
Boc-o-AlaY[CS-NH]Phe-Val-OAl1 6 a
i
i
Experimental Precedure
Received: March 13,1992 [Z 5238181
German version: Angew. Chem. 1992, 104, 1231
or peptide elongation. Exceptions are the reactions of
2a + 5c and 2b + 5e in which 46 and 30% of the respective
starting peptides were recovered. The results of the enzymatic reactions (Table 1 ) are not optimized, and yields may be
increased by variation of cosolvent, substrate, and enzyme.
When thiopeptides such as 5e are used as the amino component in peptide couplings, tetrapeptides with alternating
thioamide bonds (6d, g) are formed. The hexapeptide 6e
containing three thioamide groups was isolated as a
byproduct (8 %) in the synthesis of 6d.Apparently some of
the amine 5e dimerized to the tetrapeptide H-PheY[CSNHIGly-Phe Y[CS-NHIGly-OMe, which was then preferentially elongated by chymotrypsin to 6e.
Boc-o-AlaY[CS-NH]Phe-OMe
choice of suitable enzymes and appropriate protecting
groups should allow the selective introduction of thioamide
bonds in a variety of peptide linkages.
1. morpholine, [Pd(PPh,),]
By Gotz Bucher, Wolfram Sander,* El$ Kraka,
and Dieter Cremer*
2. EDCI/HOBt, H-Lys(Z)-Trp-Phe-OMe 7
Boc-~-AlaY[CS-NH]Phe-Val-Lys(Z)-Trp-Phe-0Me
8
Dedicated to Professor Giinther Maier
on the occasion of his 60th birthday
1. NaOH, iPrOH
2. HCI, Et,O
H-D-AlaY[CS-NHIPhe-Val-Lys(Z)-Trp-Phe-OH
9
Since the pioneering work of Wittig et al."] arynes have
enjoyed a special status in mechanistic and preparative organic chemistry.[*' This area has received added impetus re-
Scheme 1. Reactions sequence starting from thiopeptide 6a
ence of N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide
hydrochloride/l-hydroxybenzotriazole(EDCI/HOBt) gave
thiohexapeptide 8 in 85% yield. Deprotection yielded 9
which is presently under investigation in cyclization reactions.
The method described here for the enzymatic elongation
of thiodipeptides can certainly be applied for the elongation
of a number of other amino acids at the C terminus. The
1230
0 VCH
VerlagsgesellschaJl mbH, W-6940 Weinheim, 1992
[*I Prof. W. Sander, Dip1.-Chem. G. Bucher
Institut fur Organische Chemie der Technischen Universitat
Hagenring 30, D-W-3300 Braunschweig (FRG)
Prof. D. Cremer, Prof. E. Kraka
Department of Theoretical Chemistry, University of Goteborg
KemigPrden 3, S-41296 Goteborg (Sweden)
[**I This work was supported by the Deutsche Forschungsgemeinschaft, the
Fonds der Chemischen Industrie, the Swedish Natural Science Research
Council, and the Nationellt Superdatorcentrum (Linkoping, Sweden).
OS70-0833~92/0909-1230$3.50+ .2SjO
Angew. Chem. In[. Ed. Engl. 1992, 31, N o . 9
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