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Enzymatic Synthesis of Peptides and Ras Lipopeptides Employing Choline Ester as a Solubilizing Protecting and Activating Group.

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COMMUNICATIONS
Experimental Procedure
[6] H. Sasai, T. Arai, M. Shibasaki, J. A m . Chem. Soc. 1994. 116, 1571, and
references therein.
[7] Prepared from diisobutylaluminum hydride, two equivalents of (R)-BINOL,
and one equivalent of NaOtBu, KN(TMS),, and/or Ba(OrBu),.
[8] The crystalline I ( l 0 mol%) efficientlypromoted the Michael reaction o f 6 with
4 to give 7 (81 % ee, 75 % yield).
[9] The crystalline I1 (10 mol%) efficiently promoted Michael reaction of 6 with
4 to give 7 (89% ee. 67% yield).
[lo] Crystal structure data for I1 (C,,H,,O,AILi/C,H,O/(C,H,O),):
T = 223 K ;
space group P1. a = 9.510(1), h = 11.063(1), c = 13.052(3)A; a = 96.42(1),
fl = 95.71(1), y = 64.97(1)", 2 = 1. The structure was solved by direct methods
and refined to R(F) = 0.0520. Rw(F) = 0.0490; further details of the crystal
structure investigation are available on request from the Director of the Cambridge Crystallographic Data Centre, 12 Union Road, GB-Cambridge
CB2 1EZ (UK) on quoting the full journal citation.
[ l l ] C. Lambert, P. von R. Schleyer, Angew. Chem. 1994,106,1187; Angew. Chem.
Inr. Ed. Engl. 1994,33, 1129.
1121 The chemical shift of the a-proton of 1 in the 'H a n d I3C NMR spectra does
not change on mixing 1 and the AILi-BINOL complex.
[13] a) D. Canet, J. J. Delpuech, M. R. Khaddar, P. R. Rubini, J. Mug. Res. 1973,
9, 329; b) J. J. Delpuech, M. R. Khaddar, A. A. Peguy, P. R. Rubini, J. Chem.
SOC.Chem. Commun. 1974,154; c) J. Am. Chem. Sue. 1975,97,3373.
[14] The mixture rue3 does not react with 12 in the presence of 10mol% of the
AlLi - (R)-BINOL complex.
I: To a solution of LiAIH, (94.9 mg, 2.5 mmol) in THF (5.0 mL) was added a
solution of (R)-BINOL (20 mL. 5.0 mmol, 0.25 M in THF) at 0'C. After 30 min
stirring at O'C, this solution of I(O.1 M in THF) was directly used for Michael
reactions.
7: To a stirred solution of the catalyst I(0.05 mmol) in THF (0.5 mL) was successively added cyclohexenone ( 6 ) (48 pL, 0.5 mmol) and dibenzyl malonate (4)
(125 pL, 0.5 mmol) at 0°C. After 72 h stirring at room temperature, the reaction
mixture was treated with 1 N HCI (2.0 mL) and extracted with EtOAc ( 3 x 10 mL).
The combined organic extracts were washed with brine, dried (NaSO,). and concentrated to give an oily residue. Purification by flash chromatography (SO,. 25%
acetone/hexane) gave the Michael adduct 7 (172.0 mg, 88% yield) in 99% ee. The
spectral and analytical data of 7 were in agreement with those of Ref. [2].
13:To a solution of cyclopentenone (1) (84 pL, 1.O mmol), methyl diethylmalonate
(2) (138 pL, 1.0 mmol) and 3-phenylpropanal (12)(158 pL, 1.2 mmol) in THF
(1.0 mL) was added a solution of the AILi-(R)-BINOL complex I ( l . 0 mL, 0.1 M in
T H E 0.1 mmol) at 0°C. After 36 h stirring at room temperature, the reaction was
quenched with 1 N HCI at 0°C and worked up as described for 7.The oily residue
was purified by flash chromatography (SO,, acetone:hexane 1 :9) to give 13
(250 mg, 0.64 mmol. 64% yield). The optical purity of 13 was determined by HPLC
analysis on a chiral phase (DAICEL CHIRALPAK AD, iF'rOH:hexane 1:9).
[z]g4 = + 18.3 (c = 0.66 in chloroform) (91 % ee); 'H NMR (270 MHz, CDCI,):
6 = 7.40-7.13(m, 5 H),4.13(q, J = 7.0 Hz,2 H),4.11 (4, J = 7.0 Hz, 1 H).4.06(q,
J = 7.0 Hz, 1 H). 3.75 (dt, J = 3.8, 9.6 Hz, 1 H), 2.97 (dt, J = 6.3, 8.0 Hz, 1 H),
2.89-2.76 (m, 1 H), 2.70-2.57 (m, 2 H), 2.35 (dd. J = 3.8. 6.3 Hz, 1 H), 2.24 (t.
J = 8.4 Hz, 2 H), 2.19-2.01 (m. 2 H), 1.87-1.68 (m. 2 H), 1.40 (s, 3 H). 1.22 (t.
J = 7.0 Hz. 3 H), 1.19 (t. J = 7.0 Hz, 3 H); I3C NMR (CDCI,): 6 = 218.9, 171.8,
171.6, 141.8, 128.4, 125.9. 72.0, 61.6. 56.9, 55.9. 43.3, 38.5, 36.1, 32.5, 22.9, 18.6,
,
14.0.13.9; IR(neat): G = 3518,3085.1728.1253 cm-';MS:m/;(%)391 [ M i ]373
[M' - H,O], 175 (100); anal. calcd for C,,H,,O,: C, 67.67; H, 7.74; found: C ,
67.41; H, 7.70.
16: To a solution of cyclopentenone (1) (84 pL, 1.0 mmol), methyl diethylmalonate
(2) (138 pL, 1.0 mmol) and henzaldehyde (14)(122 pL, 1.2 mmol) in THF (1.0 mL)
was added a solution of the AILi-(R)-BINOL complex (1.0mL. 0.1 M in THF,
Michael Schelhaas, Simone Glomsda, Marion Hansler,
0.1 mmol) at 0°C. After 72 h stirring at room temperature, the reaction was
quenched with 1 N HCI at 0 "C and worked up as described for 7.The crude product
Hans-Dieter Jakubke,* and Herbert Waldmann*
was purified by flash chromatography (SiO,, acetone:hexane 1.9) to give the
diastereomixture of 15 (297 mg, 82%). To a solution of 15 (100 mg, 0.277 mmol)
For the synthesis of peptides and sensitive complex peptide
was added molecular sieves (4.k 200 mg) and pyridinium chlorochromate (PCC,
conjugates, enzymatic transformations offer advantageous al65 mg, 0.3 mmol) at 0'C. After l h of stirring at room temperature, the reaction
mixture was diluted by Et,O (20 mL). The insoluble materials were filtered off
ternatives to classical chemical methods.", *I Thus, for instance,
through a Celite pad. The filtrate was evaporated to dryness in vacuo. The residue
biocatalyzed deblocking reactions['] have proven their efficienwas purified by silica gel chromatography (SiO,, acetone:hexane 1.9) to give 16
cy in the construction of glyc0-[~1and lipopeptide~,[~]
and
(100 mg, 100% yield). The optical purity of 16 was determined by HPLC analysis
protease-mediated peptide synthesis in ice[51can be used to couon a chiral phase (DAICEL CHIRALCEL OJ. iPrOH: hexane 1:4). [a]i4 = - 22.0
( c = 1.02 in chloroform) (89% ee); 'HNMR (270 MHz. CDCI,): 6 = 8.00-8.05
ple amino acids and peptides without racemization.
(m, 2 H), 7.62-7.55 (m, 1 H). 7.52-7.45 (m, 2 H), 4.77 (d, J = 8.6 Hz, 1 H), 4.13
However, enzymatic reactions often are hampered or even
(4, J = 7.3 Hz, 2 H), 4.03 (dq, J = 7.3, 10.9 Hz, 1 H), 3.87 (dq, J = 7.3, 10.9 Hz,
prevented
by the low solubility of the protected peptides in
1 H), 3.68-3.55 (m. 1 H), 2.48-2.26 (m, 3 H), 2.03- 1.03 (m, 1 H), 1.49 (s, 3 H),
aqueous reaction media, resulting in limited accessibility of the
1.21 (t,J=7.3Hz,3H),1.04(t,J=7.3Hz,3H);'3CNMR(CDCI,):6=211.0,
195.1, 171.1, 136.5, 133.3, 129.4, 128.5, 61.4, 59.4, 55.6, 44.4, 38.9. 22.7, 19.7, 13.9,
substrates to the biocatalysts. This difficulty can be overcome by
13.6;1R(neat):C= 3463, 1728,1677, 1260cm-';MS:m/i(%)361 [Mt+1 ] ) , 3 6 0
employing solubilizing blocking groups.l6I Their removal, how[M '1, 105 (100); anal. calcd for C,,H,,O,: C , 66.65; H, 6.71; found: C. 66.38; H,
ever, often requires reaction conditions that cannot be tolerated
6.76.
in the construction of sensitive peptide conjugates. We now reReceived: August 18, 1995 [Z8324IE]
port that choline esters are advantageous solubility-enhancing
German version: Angew. Chem. 1996.108. 103-105
Enzymatic Synthesis of Peptides and Ras
Lipopeptides Employing Choline Ester as a
Solubilizing, Protecting, and Activating Group**
Keywords: aldol reactions . aluminum compounds
reactions . catalysis . Michael additions
.
cascade
[l] a) H. Sasai, T. Suzuki, S. Arai, T. Arai, M. Shibasaki, J. Am. Chewi. Sue. 1992.
114, 4418; b) H. Sasai, T. Suzuki, N. Itoh, M. Shihasaki, Tetrahedron Len.
1993,34.851; c) H.Sasai, N. Itoh, T. Suzuki, M. Shibasaki, ibid. 1993,34,855;
d) H.Sasai, T. Suzuki, N. Itoh, S. Arai, M. Shibasaki. ibid. 1993,34,2657; e)
H. Sasai, W-S. Kim, T. Suzuki, M. Shibasaki, M. Mitsuda. J. Hasegawa, T.
Ohashi, ibid. 1994,35,6123; f) H. Sasai, T. Suzuki, N. Itoh, K. Tanaka. T. Date,
K. Okamura, M. Shibasaki, J. Am. Chem. Sue. 1993,IlS, 10372; g) H. Sasai,
T. Tokunaga, S. Watanabe, T. Suzuki, N. Itoh, M. Shibasaki, J. Org. Chem.
1995,60, 7388: h) H. Sasai, S. Arai, Y. Tahara, M. Shibasaki, ibid. 1995,60,
6656.
[2] H. Sasai, T. Arai, Y. Satow, K. N. Houk, M. Shibasaki, J. Am. Chem. Suc. 1995,
117, 6194, and references therein.
131 For the previously known asymmetric AI-BINOL reagents and the related
compounds, see: a) R. Noyori. Tetrahedron. 1994,50, 4259, and references
therein; b) K. Maruoka, H. Yamamoto in Cutdytic Asymmetric ,yyntheSis
(Ed.: 1. O J ha) , VCH, New York, 1993,p. 413, and references therein,
[41 Calculated on the assumption of quantitative formation of the catalyst.
[51 For information on the absolute configurations and enantiomeric exceSxS of
all Michael adducts, see Refs. [2, 61.
106
'C
VCH Vrrlugsgesellschaft mhH, 049451 Weinheim.1996
protecting groups for the synthesis of peptides and lipopeptides
under extremely mild conditions.
Peptide choline esters (peptide-Cho) 1 (Scheme 1) can be constructed by treating the corresponding 2-bromoethyl esters with
trimethylamine.171Alternatively, they are accessible by condensation of amino acid choline ester hydrobromides with C-terminally deblocked peptides (vide infra; Scheme 2).
[*] Prof. Dr. H. Waldmann, Dipl.-Chem. M. Schelhaas, Dipl.-Chem. S. Glomsda
lnstitut fur Organische Chemie der Universitat
Richard-Willstatter-Allee2, D-76128 Karlsruhe (Germany)
Fax: Int. code +(721)608-4825
Prof. Dr. H.-D. Jakubke, Dr. M. Hansler
Institut fur Biochemie der Universitat
Talstrasse 33, D-04103 Leipzig (Germany)
Fax: Int. code +(341)295-939
[**I This work was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium fur Forschung und Technologie. and the Fonds der Chemischen Industrie. We are grateful to Degussa AG for the donation of chemicals.
We thank Mrs. R. Schauf and Mrs. H. Spith for assistance with the experiments.
0570-0833/96/3501-0106S 10.00t ,2510
Angew. Chem. Int. Ed. En$. 1996, 3.5, N o . 1
COMMUNICATIONS
The high solubility of these amorphous hygroscopic solids in
water, water/dioxane, and water/methanol mixtures is advantageous for the selective removal of the C-terminal protecting
group by means of the enzymes butyrylcholine esterase
(EC 3.1.I .8) from horse serum or acetylcholine esterase
(EC 3.1.I .7) from electric eel. Butyrylcholine esterase accepts a
variety of dipeptides as substrates and removes the C-terminal
choline ester in high yield under very mild conditions (pH 6.5,
room temperature) and with complete selectivity (Scheme 1,
Table 1). In the majority of the cases the protected dipeptide
P G - - A A ' - - A A ~ - BOro ~ ~ ~ ~ ~
la-i
L
J
-
"Cho"
I
butyrylcholine
eSteraSe
PG = 2, BOC,PhAc, AIOC
sE:!
Na3P04
buffer, room temp.
size sensitive S-palmitoylated and S-farnesylated cysteinyl
lipopeptides, which, for instance, represent the characteristic
partial structures of the Ras lipoproteins.
The Ras proteins are a class of membrane-bound lipoproteins
found in organisms as diverse as mammals, flies, worms, and
yeast, and serve as central molecular switches.[101They translate
the signals given by growth factors through a well-balanced
series of noncovalent protein/protein interactions into a cascade
of highly specific protein phosphorylations terminating in the
activation of transcription factors, which then may influence the
transcription of the genetic code. Cell growth and proliferation
are regulated by this pathway, and if it is disturbed or interrupted, uncontrolled proliferation and finally cancer may occur.
Thus, in roughly 40 % of all human cancers a point mutation in
the ras oncogenes coding for the Ras proteins is found, a figure
that increases to 85 % for some of the major malignancies like
lung, colon, and pancreas cancer.["] The human H- and N-Ras
proteins terminate in an S-farnesylated cysteine methyl ester,
and towards the N-terminus one or more S-palmitoylated cysteines are found[lZ1(see for instance the C-terminal lipohexapeptide of the human N-Ras protein 9 (Scheme 2)). The
0
AIOC=
AIOC-CYS-OH
I
2a-i
Aloc-Cys-OH
Scheme 1. Enzymatic removal of the choline ester (Cho) from peptides by butyrylcholine esterase. AA = amino acid, PG = protecting group.
3
1
1) NaBH,,
2) palmitoyl chloride,
NaOH, O°C
DMAP, THF
26% overall yield
Table 1. Results of the enzymatic removal of the choline ester protecting group
from the dipeptide choline esters 1 by means of butyryl- and acetylcholine esterase.
No.
2
PG
AA'
AA2
Vol%
CH,OH
Enzyme [a]
Yield
[%I [bl
l
2
3
a
a
b
6
l
8
Y
c
d
e
f
Phe
Phe
Phe
P he
Ala
Ala
Thr
Ala
Phe
Phe
Phe
Pro
BE
AE
BE
AE
BE
AE
BE
BE
BE
BE
BE
BE
65
46
b
c
Phe
Phe
Ala
Akd
Ile
Ile
G~Y
G~Y
Ser
Phe
Ile
Leu
-
4
5
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Z
Z
PhAc
PhAc
Aloc
10
11
12
g
h
i
~
~
~
-
10
10
~
~
-
AloC-CyS-OH
I
Pal
4
HBr H-Met-Gly-OCho
5
50
44
76
60
Aloc-Cys-Met-Gly-OCho
I
Pal
6
66
89
90
I
butyrylcholine
esterase,
59%
pH 6.5
70
91
95
[a] BE = butyrylcholine esterase from horse serum; AE = acetylcholine esterase
from electric eel. [b] Yields refer to pure deprotected dipeptide 2. All dipeptide
carboxylic acids 2 were characterized by means of their 200 MHz 'H NMR spectra
(in CDCI,) and their FAB mass spectra. The analytical purity of the carboxylic acids
2 was confirmed by HPLC.
Aloc-Cys-Met-Gly-OH
I
bai
I
7
H-Leu-Pro-Cys-OMe
I
Far
esters 1 are freely soluble in the aqueous buffer, and a solubilizing cosolvent is not needed (Table 1). In the enzymatic removal
of the N-terminal phenylacetamido (PhAc) groupL8]and the
C-terminal heptyl (Hep) esters, in particular, the combination of
two sterically demanding hydrophobic amino acids posed serious problems due to the low solubility of the respective peptide
derivatives in the aqueous media. However, butyrylcholine esterase accepts these combinations, too, and deprotects substrates l a , b, g, h, and l i in high yield. Since the acetylcholine
esterase catalyzed deprotection of peptide choline esters under
these mild conditions is slow (Table 1, nos. 2,4, and 6), butyrylcholine esterase is preferred.
The conditions for the enzymatic deprotection of the choline
esters are so mild that this method can be employed to syntheAngt'w C h m It11 Ed Engl. 1996, 35,No. I
0 VCH
88%
0
EDC. HoBt, CH2C12
64%
Aloc-Cys-Met-Gly-Leu-Pro-Cys-OMe
I
I
S.).yv.rof
s7
0
9
I
OH
Scheme 2. Enzymatic synthesis of the S-palmitoylated and S-farnesylated Ras
lipopeptide 9.
Verlugsgesellsehufi mbH. 0-49451 Wemherm. I996
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$ IO.OO+ .25/0
107
COMMUNICATIONS
synthesis of characteristic Rus peptides is severely complicated
by the acid sensitivity of the farnesyl residue and the pronounced base lability of the palmitic acid thioester (vide infra)
and excludes the use of acid- or base-labile blocking
For this purpose protecting groups have to be employed that
can be removed selectively under extremely mild, preferably
neutral conditions.
In a retrosynthetic sense the Ras peptide 9 was divided into
the masked S-palmitoylated tripeptide 7 and the N-terminally
deprotected S-farnesylated tripeptide ester 8 to ensure that the
final coupling would proceed efficiently (Scheme 2). For the
synthesis of 7, bis[allyloxycarbonyl(Aloc)]-protected cystine 3
was reduced to Aloc-cysteine, which was immediately acylated
with palmitoyl chloride to give the correctly functionalized cysteine derivative 4. Compound 4 was then condensed in high
yield with the dipeptide choline ester 5, which was readily obtained from the corresponding Boc-protected dipeptide choline
ester by treatment with HBr/AcOH. The selective removal of
the choline ester group from the fully protected tripeptide 6 in
the presence of the base-labile thio ester posed a serious challenge. Preliminary experiments revealed that palmitic acid is
cleaved from 6 in aqueous solution by nonenzymatic hydrolysis
already at pH 6.5, in other words under virtually neutral conditions, without saponification of the choline ester. However,
when 6 was treated with the enzyme butyrylcholine esterase at
pH 6.5, the chemoselectivity of the hydrolysis was completely
reversed, and the C-terminally deprotected lipotripeptide 7 was
obtained in 59% yield. We would like to stress that under these
extremely mild reaction conditions absolutely no p-elimination
of the S-acyl group is observed, a disturbing side reaction that
occurs readily for acylated serine and cysteine derivatives at
weakly basic pH.[131The synthesis of the completely functionalized lipopeptide 9[141was finally completed by carbodiimidemediated condensation of the S-palmitoylated peptide carboxylic acid 7 with the S-farnesylated N-terminally deblocked
peptide ester tir4](Scheme 2).
Besides the choline esterases, proteases like chymotrypsin,
papain, trypsin, subtilopeptidase A, nagarse, and proteinase K
also cleave N-protected amino acid and peptide choline esters
under mild conditions. For instance, in the chymotrypsin-catalyzed hydrolysis of Z-Phe-OCho 10 the specificity
has a value of 1.2 x lo6 mM-'S-', which is significantly higher
than the values determined for comparable water-soluble Nmaleinamido amino acid and peptide benzyl and p-nitrobenzyl
esters. This difference is caused in part by the PIspecificity of
chymotrypsin for positively charged residues, as we determined
previously,['61 and also by the coulombic effect of the positive
charge, which leads to enhanced reactivity of the choline ester
bond." 71 Due to these excellent substrate properties, we tested
choline esters as acyl donors in kinetically controlled peptide
syntheses.
In these reactions, too, the excellent solubility of the protected
amino acid choline esters in aqueous solvents is a great advantage. Thus, in the presence of chymotrypsin the Z-protected
phenylalanine choline ester 10 is coupled in only 30 min with the
amino acid amides 11 and 12, and also with the amino acid
benzyl esters 15 and 16, to give the fully protected peptides 13,
14, 17, and 18, respectively, in high yield (Scheme 3). The undesired competing hydrolysis of the choline ester can be suppressed simply by adding a precooled solution (0 "C) of the acyl
donor slowly to an ice-cold solution (pH 7.8) of the acyl acceptor and the biocatalyst.
The application of the amino acid choline esters for enzymatic
peptide synthesis is not restricted to reactions with chymotrypsin as the biocatalyst. For instance, the dipeptide 13 also
108
0 VCH
Verlugsgeseilschufl mbH, 0.69451 Weinheim,1996
Z -Phe-OCho
10
chymotrypsin
A.
2-Phe-AA-NH,
pH 7.8, 0%;
30 min
H-AA-NH,
13: AA = Leu 82%
14: AA = Arg 86%
11: AA = Leu
12: AA = Arg
+
10
trypsin
11
*
pH 7.8, O"C,
30 min
13
72%
Z -Phe-OCho
10
chvmotlvDsin
,.
+
*
~
pH 7.8, O°C,
30 min
H-AA-OBzl
15: AA = Ala
16: AA = Gly
Z-Phe-AA-OBzl
17: AA = Ala 60%
18: AA = Gly 61%
2-Leu-Gly-OCho
19
papain
+
DH 7.8, 1h,
room temp.
H-LeU-NH,
.- Z-Leu-Gly-Leu-NH,
20
90%
11
19
+
11
butyrylcholine
esterase
20
pH 7.8, -15"C,
37%
Scheme 3. Enzymatic peptide synthesis employing amino acid and peptide choline
esters as acyl donors.
is obtained in high yield by employing trypsin (Scheme 3). In
addition, the N-protected peptide choline ester 19 and leucine
amide 11 are condensed by papain to give the tripeptide 20 in
90 YOyield (Scheme 3). Particularly surprising is the finding that
butyrylcholine esterase is also able to transfer the acyl moiety
from amino acid choline esters to N-nucleophiles; in other
words, it catalyzes not only the hydrolysis of choline esters but
also the formation of peptide bonds. However, in initial experiments with this biocatalyst tripeptide 20 was obtained from 19
and 11 in 37% yield only at -15°C in a frozen aqueous systernr5]but not at room temperature.
Experimental Procedure
General procedure for the enzymatic cleavage of choline esters by means of butyrylcholine esterase: A solution of 1 mmol of the choline ester and 1000 n of butyrylcholine esterase in 50 mL of 0.05 M Na,PO, buffer (pH 6.5; when necessary 5 1 0 ~ 0 1 %of methanol was added as cosolvent) was shaken at 37°C. After the
conversion was complete (24&36h; reaction monitored by thin-layer chromdtography with n-hexaneiethyl acetate/methanol), the pH was adjusted to 2 - 3 with 1 N
HCI. Depending on the substrate the solution was extracted four to eight times with
40 mL of dichloromethane. The combined organic layers were dried over MgSO,,
the solvent was removed in vdcuo, and the residue was purified by chromatography
on silica gel eluting with n-hexanelethyl acetateimethanol.
7: Colorless oil; R, = 0.25 (n-hexanejethyl acetate/methanol 60/30/5 [vivjv]);
[u]:' = - 59 (c = 0.5 in CH,OH); ' H N M R (400 MHz. CDCI,. 2 5 ° C TMS):
b = 0.88 (t, J = 7 Hz, 3H, o-CH, Pal), 1.25 (m, 24H, CH, Pal), 1.60-1.68 (m, 2H,
P-CH, Pal). 1.95-2.09 (m, 2H. P-CH, Met), 2.10 (s, 3H. CH, Met), 2.53-2.57 (m,
4H, a-CH, Pal. ;-CH, Met). 3.15 -3.27 (m, 2H, fl-CH, Cys). 3.83-3.93 (m, IH,
CH,, Gly), 4.10-4.20 (m. 1 H, CH,, Gly), 4.39--4.78 (m. 4H, 1-CH Met, a-CH Cys,
OCH, Aloc), 5.18-5.38 (m, 3H, CH,=CH Aloc), FAB-MS (2-NBA/TFA 5jl): m / z
( Y o ) :654 (63) [ M +Na]+.
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Angew. Chem. Int. Ed. Engl. 1996. 35. N o . 1
COMMUNICATIONS
General procedure for chymotrypsin-catalyzed peptide synthesis employing N-protected amino acid choline esters: A precooled (0°C) solution of 1 mmol of the
choline ester in 10 mL of water was added with stirring at 0°C within 3 min to a
solution of 2 mmol of the amino acid ester or the amino acid amide and 7.5 mg of
chymotrypsin in 7.5 mL of water (pH 7.8), and the reaction mixture was stirred for
30 min. The precipitate was removed by filtration, washed three times with water,
dried in vacuo. and recrystallized from methanol/water.
13: M.p. 189-193 "C: FAB-MS (2-NBA): mjz: 654 [ M + H I + ; the purity of 13 was
confirmed by HPLC (eluent: CH,CN/H,O 2/3 [v/v] and 0.1 vol% trifluoroacetic
acid).
Received: August 17, 1995
Revised version: October 19, 1995 [Z8334/8335IE]
German version: Angew. Chem. 1996, 108, 82-85
Keywords: choline esterase . enzymatic catalysis . peptides . Ras
proteins
[l] Review on enzymatic protecting group techniques: H. Waldmann, D. Sebastian, Chem. Rev. 1994, 94, 911.
[2] Review on enzymatic peptide syntheses: V. Schellenberger, H.-D. Jakubke,
Angew. Chem. 1991,103, 1440; Angew. Chem. Inr. Ed. Engl. 1991, 30, 1437.
[3] P. Braun, H. Waldmann, H. Kunz, Bioorg. Med. Chem. 1993, 1, 197.
[4] E. Ndgele, H. Waldmann, Angew. Chem. 1995,107, 2425; Angew. Chem. Int.
Ed. Engl. 1995, 34, 2259.
[5] a) M. Schuster, A. Aaviksaar, H.-D. Jakubke, Tetrahedron 1990, 46, 8093; b)
M. Schuster. G. Ullmann, U. Ullmann, H.-D. Jakubke, Tetrahedron L e f t .
1993,34, 5701.
[6] a) For an enzymatic peptide synthesis employing solubilizing protecting groups
see: A. Fischer. A. Schwarz, C. Wandrey, A. S . Bommarius. G. Knaup, K.
Drauz, Bionwd. Biochim. Acta 1991, 50, 169; b) for enzymatically removable.
solubility-enhancing, C-terminal protecting groups see: H. Kunz, D. Kowalczyk, P. Braun, G. Braum, Angew. Chem. 1994, 106, 353: Angen. Chem. Int.
Ed. Engl. 1994,33, 336; G. Braum, P. Braun, D. Kowalczyk, H. Kunz, Tetruhedron Lett. 1993, 34, 31 11.
(71 H. Kunz, M. Buchholz, Chem. Ber. 1979, 112, 2145.
[8] H. Waldmdnn, Liebigs Ann. Chem. 1988, 1175.
[9] P. Braun, H. Waldmann, W. Vogt, H. Kunz, Liebigs Ann. Chem. 1991, 165.
[lo] Reviews: a) S . E. Egan, R. A. Weinberg. Nature 1993, 365, 781; b) M. S. Boguski, F. McCormick, Nulure 1993, 366, 643; c) J. L. Bos. Mutation Res. 1988,
195, 255.
[ l l ] Review: A. Levitsky, Eur. J. Biochem. 1994, 226, 1.
I
1990, 4 , 3319, and references thcrein.
[12] W. A. Maltese, FASEB .
[13] K. Wakabayashi, W. Pigman, Curbohydr. Res. 1974, SS, 3.
[14] 9: Colorless oil; R, = 0.75 (n-hexane/ethyl acetate/methanol 50/50/10 [v/v/v]);
[a];' = - 34 (c = 0.7 in CHCI,); 'HNMR (400 MHz, CDCI,, 2 5 ° C TMS):
6 = 0.88 (t. J = 7 Hz, 3H, w-CH, Pal), 0.94 (d, J = 7 Hz. 3H, w-CH,. Leu),
0.98 (d, J = 7 Hz, 3H, w-CH,, Leu), 1.25 (m, 24H, CH, Pal), 1.55-1.75 (m.
14H, 8-CH, Pal, 4CH, Far), 1.95-2.2 (m, 19H, p-CH, Met, p-CH, Leu,
4CH, Far, 2CH, Pro, S-CH, Met), 2.35 (m. IH, y-CH Leu), 2.55 (m, 4H,
1-CH, Pal, yCH, Met), 2.65-2.68 (m, 2H, 8-CH, Cys-S-Far), 2.90-3.30 (m,
4H, 8-CH, Cys-S-Pal, a-CH, Far), 3.60-3.80 (m, 6H, CO,CH,, CH, Pro,
CH,, Gly). 4.24-4.28 (m, IH, CH,, Gly), 4.50-4.78 (m. 7H, r-CH Met,
2a-CH Cys, a-CH Leu, a-CH Pro, OCH, Aloc), 5.10-5.34(m, 5H, 3CH Far,
CH,=CH Aloc), 5.86-5.93 (m, l H , CH,=CH Aloc). 6.05 (d, br, NH urethane), 7.05 (d, br, IH, NH), 7.25 (d, br, l H , NH), 7.4 (br. l H , NH), 7.5X (d,
br, l H , NH); FAB-MS (2-NBA/TFA 5jl): m / z ( Y O )1163
:
(4) [ M +H I + , 1185
(20) [ M +Na]+.
[15] V. Schellenberger, U. Schellenberger,H.-D. Jakubke, CON. Czech. Chem. Commun. 1988, 53, 2884.
[I61 V. Schellenberger, H.-D. Jakubke, Biochim. Biophys. Acta 1986, 869, 54.
[I71 P. Sikk. A. Aaviksaar, Org. React. 1977. 14, 61.
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