Transesterification and other conversion reactions of acid derivatives, using an amidine base

A process employs amidine or amidine base and a metal compound in the presence of an alcohol or water to transesterify, or saponify esters or amides. Since the process employs relatively mild conditions, it is especially suitable for the production of optically active substances and biomolecules, e.g. peptides, amino acids and nucleic acids which are sensitive to elevated temperatures, extreme pH values and/or long reaction times since these compounds are easily racemised or denatured. The conditions additionally find use in solid phase systems. When amino acid or peptide esters are saponified, the splitting is brought about with lithium hydroxide alone under mild conditions. The use of an amidine base, more particularly DBU or DBN, in combination with the metal salt additionally accelerates the reaction so strongly that even sensitive acid derivatives can be reacted under mild conditions. The amidine based system lends itself to the manufacture of complex esters, gentle splitting of peptides from the carrier, gentle saponification of amides or esters.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to a method of reacting a phosphoric acid, phosphonic 
acid or carboxylic acid derivative with an alcohol, water or NH.sub.3 in 
the presence of an amidine base, more particularly a method of 
transesterification, ammonolysis or saponification of carboxylic acid, 
phosphonic acid or phosphoric acid derivatives and splitting of amino 
acid, peptide or nucleic acid derivatives from a polymer carrier. The 
invention also relates to a method of saponifying a peptide ester in an 
aqueous medium in the presence of a base in the form of a metal compound. 
2. Background Information 
Various methods of transesterifyinq or saponifying the aforementioned 
compounds are already described in the literature. Operation is e.g. under 
strongly acid or basic conditions, with enzymes (D. Seebach, Angew. Chem. 
1990, 102, 1363), with titanates (D. Seebach, B. Weidmann, L. Widler in 
"Modern Synthetic Methods", 1983), or KF/cyclic ethers (B. Lejczak, P. 
Kafarski, J. Szewczyk, Synthesis 1982, 412) or with ion exchange resins 
(W. Pereira, V. Close, W. Patton, B. Halpfern, J. Org. Chem. 1969, 34, 
2032) or distannic oxanes (J. Otera, S. Ioka, H. Nozaki, J. Org. Chem. 
1989, 5*, 4013). There is also a description of a method of 
transesterifying or saponifying ester derivatives using the amidine base 
DBU in solution (EP 0 110 629 and 0 150 962) or polymer-bonded (T. 
Ishikawa, Y. Ohsumi, T. Kawai, Bull. Chem. Soc. Jpn. 1990, 63, 819). 
EP 0 110 629 Al discloses use of amidine bases for trans- esterification. 
The amidine is usually supported by epoxides. The citation does not 
mention particularly mild conditions, as necessary particularly when 
reacting optically active biomolecules. All the examples describe simple 
stable compounds without additional functional groups or optical activity. 
Transesterification using calcium acetate in methanol is known from Int. J. 
Peptide Protein Res. 37, 1991, 451-456. In the great majority of cases, 
the substrates are only peptides with a C-terminal glycine radical. These 
peptides are insensitive to racemisation. When a C-terminal alanyl radical 
is used, the reaction is already inhibited. Also, this reaction requires 
specific protective groups. A universally applicable mild method, more 
particularly for reacting biomolecules, cannot be obtained by using 
calcium acetate. 
The splitting of carboxylic acid methyl esters with DBU at 165.degree. C. 
in 48 hours is known from J. Org. Chem. 38, (1973), 1223-1225. These 
conditions are too drastic for most ester splitting, particularly for 
optically active esters having a center of chirality in the .alpha. 
position relative to the ester group. 
.beta.-elimination with DBU or DBN is known from CA 114, 186011 (1991) and 
Tetrahedron Letters 21 (1980), 1181-1184. As is universally the case in 
.beta.-elimination and also mentioned in this citation, theoretically any 
base, even potassium hydroxide, can be used. The citation does not 
describe universally applicable mild reaction conditions. 
All the previously-mentioned methods, except for the enzymatic method, 
normally require elevated temperatures, extreme pH values and/or long 
reaction times. Owing to the reaction conditions, most of the 
aforementioned methods are unsuitable for sensitive ester derivatives, 
e.g. containing additional functional groups or one or more chiral C 
atoms, more particularly compounds containing a chiral C atom in the 
.alpha. position relative to the ester function. 
SUMMARY OF THE INVENTION 
The invention therefore is concerned with a system for reaction, more 
particularly saponification, ammonolysis or transesterification, of acid 
derivatives and splitting of polymer-bonded molecules, under conditions 
which are so mild that, more particularly, optically active substances and 
biomolecules such as peptides, amino acids or nucleic acids can be used. 
Dipeptides having C-terminal glycines are not envisioned as desired 
substrates. 
This problem is solved by combined use of an amidine base and a metal 
compound, more particularly during the transesterification, saponification 
or splitting of polymer-bonded molecules, e.g. in Merrifield synthesis. In 
the case where peptide esters were saponified, it was found that splitting 
could be brought about under mild conditions with lithium hydroxide alone, 
without the amidine base. We have unexpectedly found that use of an 
amidine base in combination with a metal salt accelerates 
transesterification, ammonolysis or saponification of the aforementioned 
acid derivatives so strongly that even sensitive acid derivatives can be 
successfully reacted under mild conditions, i.e. low temperatures and 
short reaction times. 
For example, sensitive peptide esters can be transesterified or saponified 
without racemisation of the C-terminal amino acid on the .alpha.-C atom. 
There is no effect on any protected side-chain functional groups not 
containing an ester group. For example, a heptapeptide ester was 
quantitatively saponified with DBU/LiBr in THF/H.sub.2 O in a short 
reaction time and without racemisation. On the other hand, saponification 
with aqueous NaOH was accompanied by some decomposition and resulted in 
racemisation. 
It is also unexpectedly possible, by the method according to the invention, 
to convert simple esters such as methyl ester into complex esters such as 
menthyl ester. 
DETAILED DESCRIPTION OF THE INVENTION 
Conventionally peptides and polynucleotides are synthesized on polymer 
carriers by novel techniques (Merrifield synthesis). The link with the 
polymer carrier is usually an ester or amide bond. The carrier material 
often has to be split off under drastic reaction conditions, usually 
resulting in loss of all the protective groups of any protected functional 
groups in the peptide or polynucleotide. Trifluoroacetic acid/HBr/TMS 
(trifluoromethane sulphonate) HF/anisole, NaOH/dioxane/H.sub.2 O.sub.2 or 
dimethyl aminoethanol/thallium ethanolate are examples of conventional 
splitting reagents. In contrast to these drastic conditions, the 
combination of an amidine base and a metal salt, particularly a lithium 
salt, constitutes an excellent reagent for splitting the bond between a 
peptide or polynucleotide and the polymer carrier. As a result of 
splitting, esters, amides or the corresponding free acids of the peptide 
or polynucleotide can be obtained as required, without decomposition or 
racemisation. 
There is no effect on the non base labile protective groups of any other 
functional groups in the molecule. This method is particularly suitable 
for liberating sensitive molecules and for producing protected peptide 
segments on the polymer carrier as required for subsequent segment 
coupling to longer peptide chains. One particular advantage is that there 
is no need for sensitive and sometimes dangerous reagents such as HF or 
TlOEt. 
Usually, the acid derivatives are transesterified by the method according 
to the invention as follows: 
The ester is dissolved or suspended in an alcohol, optionally with addition 
of another solvent such as THF or CH.sub.2 Cl.sub.2. After adding the 
amidine base, e.g. DBU or DBN in a 0.01-10 molar proportion, preferably in 
a 0.2-4 molar proportion, and after adding the metal compound, preferably 
salts of magnesium or caesium and particularly preferably lithium in a 
0.1-20 molar proportion, more particularly in a 2-10 molar proportion, the 
reaction is brought about at temperatures between -30.degree. C. and 
120.degree. C., preferably temperatures between -20.degree. C. and 
65.degree. C. In the case of esters sensitive to racemisation, the 
preferred temperatures are between -20.degree. C. and 30.degree. C. for 
short reaction times (the minimum necessary). 
When esters of low alcohols are transesterified with more complex alcohols 
it may be advantageous to distill off the lower alcohol evolved during the 
reaction. 
Usually the acid derivatives, preferably an ester, are saponified by the 
method according to the invention as follows: 
The acid derivative is dissolved or suspended in a solvent, preferably 
ethers such as THF or dioxane. After adding water (1+, preferably 
10-100-fold molar proportion), the amidine base, e.g. DBU or DBN, used in 
a 1-10 molar proportion, preferably a 1-4 molar proportion, and adding the 
metal compound, preferably salts of lithium, magnesium or caesium in 
0.1-20 molar proportion, particularly in 2-10 molar proportion, the 
reaction is brought about at temperatures between -20.degree. and 
65.degree. C. The sequence of additions is arbitrary. Derivatives 
sensitive to racemisation are preferably reacted at between -20.degree. C. 
and 30.degree. C. for short reaction times. 
The metal compounds in the aforementioned method are preferably halides, 
particularly a bromide or chloride, or a hydroxide (specially for 
saponification), perchlorate, acetate, sulphate or carbonate. The 
alcoholates of metal compounds are also suitable for trans-esterification 
or alcoholysis reactions. 
For the purpose of ammonolysis of a carboxylic acid ester, preferably an 
amino acid or peptide ester, the ester is dissolved or suspended in a 
polar solvent, more particularly THF or dioxane, to which some DMF (up to 
30 vol. %) can be added. The amidine base and the metal compound, 
preferably a lithium salt, a palladium salt, a copper (I) compound as the 
anion halides and perchlorate are particularly suitable) are added and NH3 
is introduced with cooling. LiBr, LiClO.sub.4, KF, CuCl or PdCl.sub.2 are 
particularly suitable metal compounds, and these salts, more particularly 
KF, can be introduced on Al.sub.2 O.sub.3 into the reaction mixture. 
Amidine bases are organic compounds containing the structural element 
##STR1## 
the free valencies of the nitrogen atoms being bonded to hydrogen and 
preferably (more particularly all) being bonded to carbon atoms. The free 
valency on the carbon atom is preferably bonded to an additional carbon 
atom, or alternatively to an additional nitrogen atom. 
The amidine bases are preferably non-nucleophilic tertiary bases. The 
following bicyclic compounds are particularly suitable: 
1,8-diazabicyclo[5,4,0)undec-7-ene (DBU) or 
1,5-diazabicyclo[4.3.0]non-5-ene (DBN). The amidine base is normally used 
in a 0.01-10 molar proportion relative to the acid derivative. The best 
results are obtained with 0.2-4 molar proportions. During saponification 
of esters a free acid group is produced, and consequently the amidine base 
must be used in at least a molar proportion, unless the acid group is 
trapped by an additional auxiliary base, preferably a tertiary amine such 
as triethyl amine. The auxiliary base can also be present in a buffer 
system. 
The metal compound, particularly advantageously lithium or magnesium or 
caesium salts, is usually used in a 0.1 20 molar proportion. 2-10 molar 
proportions of the metal compound are particularly advantageous, in each 
case relative to the acid derivative. 
The research leading to the present invention also showed that in some 
cases lithium hydroxide alone, or another lithium salt and a base (i.e. so 
that lithium and hydroxide ions are present in the reaction solution) can 
be used to saponify amino acid or peptide esters. A 1.0-20 molar 
proportion of lithium hydroxide is suitable, or preferably a 2-20 molar 
proportion of lithium hydroxide, relative to the compound to be 
saponified. If the lithium hydroxide is in a buffer system or if an 
auxiliary base is added, the lithium compound can also be added in a 0.1 
molar proportion. The resulting free acid is then neutralized by the 
auxiliary base and the buffer system, so that the reaction solution for 
saponification retains its alkaline character.

The invention will be explained in detail with reference to the following 
examples. 
General Instructions for Transesterifying Carboxylic Acid Esters 
A: LiBr (5 eq.) and the corresponding carboxylic acid ester (1 eq.) are 
dissolved or suspended under dry argon in a suitable quantity of the 
desired absolute alcohol, giving a concentration of 0.2 to 0.3 M. Freshly 
distilled DBU (0.5 eq.) is added and the solution is agitated at room 
temperature. The course of the reaction is followed by thin-layer 
chromatography or gas chromatography. As soon as the reaction ceases, the 
reaction mixture is concentrated in a rotary evaporator in vacuo and 
hydrolysed with saturated aqueous NH.sub.4 Cl or a 1 N HCl solution. The 
product is shaken out twice with diethyl ether, the combined organic 
fractions are washed with brine until the reaction is neutral, and are 
then dried over Na.sub.2 SO.sub.4. After removal of the solvent in vacuo, 
the raw product is purified by distillation or flash chromatography. 
B: In the case of expensive alcohols, LiBr, the corresponding methyl ester 
and a stoichiometric or slightly super- stoichiometric quantity of the 
alcohol (1-2 eq.) is dissolved in a mixture of tetrahydro-furan/methylene 
chloride (3:1 v/v) as per method A. 
The reaction mixture is then reflux-heated under dry argon, and the 
released methanol is trapped in a 5 A molecular sieve disposed in a 
dropping funnel or an extractor between the reaction flask and the reflux 
condenser. The course of the reaction is followed as in method A, and the 
processing is similar. 
General Method of Processing Peptide Esters 
The reaction mixture is added to 200 ml ethyl acetate (150 ml ethyl acetate 
in a second separating funnel), and the extract is washed successively 
with 100 ml of 1 N HCl, 50 ml 1 N HCl, 100 ml 1 M KHCO.sub.3, 50 ml 1 M 
KHCO.sub.3 and twice with 50 ml H.sub.2 O, and is then dried over 
MgSO.sub.4 and concentrated in vacuo. The residue is dried at reduced 
pressure for a number of hours. 
EXAMPLE 1 
Transesterification of Phenylacetic Acid Methyl Ester to Phenylacetic Acid 
Ethyl Ester 
Following method A, phenylacetic acid methyl ester (4.51 g, 30 mmol) and 
LiBr (13.03 g, 150 mmol) were dissolved in ethanol (150 ml). DBU (2.28 g, 
15 mmol) was added and the reaction mixture was agitated at room 
temperature for an hour. It was then hydrolysed and processed as 
described. Vacuum distillation yielded 4.40 g (90% of the theoretical 
yield) of pure phenylacetic acid ethyl ester, b.p. 65.5-66.degree. C./l 
Torr. 
EXAMPLE 2 
Transesterification of phenylacetic acid methyl ester to phenylacetic 
acid-(R) menthyl ester 
Following method B, phenylacetic acid methyl ester (751 mg, 5 mmol), LiBr 
(2.17 g, 25 mmol) and (R)-(-)-menthol (751 mg, 5 mmol) were dissolved in 
THF/CH.sub.2 Cl.sub.2 (3:1 v/v, 20 ml). DBU (0.37 ml, 2.5 mmol) was added 
and the reaction mixture was heated at reflux for several hours. The 
thin-layer chromatogram (SiO.sub.2 :pentane/diethyl ether 4:1 v/v) showed 
that transesterification was not complete after boiling at reflux for 24 
hours. Even so, the reaction mixture was hydrolysed and processed. Flash 
chromatography (SiO.sub.2 pentane diethyl ether 4:1 v/v) showed 691 mg (a 
50% yield) of phenylacetic acid-(R)-menthyl ester in the form of an oil 
substantially pure in 1H-NMR. 
EXAMPLE 3 
Transesterification of phenylacetic acid methyl ester to phenylacetic 
Acid-2-trimethyl silyl thyl Ester 
Following method B, phenylacetic acid methyl ester (751 mg, 5 mmol) was 
transesterified with reflux with 2-trimethyl silyl ethanol (1.18 g, 1.43 
ml, 10 mmol) in THF/CH.sub.2 Cl.sub.2 (3:1 v/v, 20 ml). After refluxing 
for 8 hours, the reaction mixture was hydrolysed and processed. In the gas 
chromatogram and 1H-NMR the raw product, obtained in a quantitative yield, 
was shown to be substantially pure (&lt;99% in the GC). 
EXAMPLE 4 
Preparation of R-(4RS, 5SR)-5-isopropyl-2-oxazolidinone-4carboxylic acid 
ethyl ester (3b) 
R-(4SR, 5RS, 
8SR)-1-aza-3,7-dioxa-4-(2'-propyl)-8-(tert.-butyl)-bicyclo[3.3.0]-octane-2 
,6-dione (302 mg, 1.25 mmol) and LiBr (543 mg, 6.25 mmol) were dissolved in 
ethanol (30 ml). DBU (0.37 ml, 2.5 mmol) was added and the resulting 
solution was agitated at room temperature for 2 hours. After acid 
hydrolysis, usual processing and flash chromatography (SiO.sub.2 : 
CH.sub.2 Cl.sub.2 /ethyl acetate 4:1 v/v), 192 mg (a 76% yield) of 3b was 
obtained in the form of a colorless viscous oil. 
EXAMPLE 5 
Preparation of Boc-Phe-Ala-OEt 
After dissolving Boc-Phe-Ala-OMe (701 mg, 2 mmol) and LiBr (869 mg, 10 
mmol) in ethanol (10 ml), DBU (150 .mu.l, 1 mmol) was added at room 
temperature. After 6 minutes the reaction solution was treated with 1 N 
HCl (3 ml) and processed as previously described. 
Yield: 700 mg (96%) with 2% starting product (1H-NMR) and a D-Ala content 
of 4% (GC). 
EXAMPLE 6 
Preparation of Boc-Phe-Ala-OCHMe.sub.2 
After dissolving Boc-Phe-Ala-OMe (701 mg, 2 mmol) and LiBr (869 mg, 10 
mmol) in isopropanol (10 ml), DBU (150 .mu., 1 mmol) was added at 
-10.degree. C. After agitation for 44 hours at the same temperature, the 
reaction mixture was treated with dilute HCl/diethyl ether (3 ml) and 
processed as previously described. 
Yield: 664 mg (88%) with 4% starting product (1H-NMR) and a D-Ala content 
of 4% (GC). 
EXAMPLE 7 
Preparation of Boc-Phe-Ala-OCH.sub.2 CH.dbd.CH.sub.2 (7d) 
After dissolving Boc-Phe-Ala-OMe (701 mg, 2 mmol) and LiBr (869 mg, 10 
mmol) in allyl alcohol (10 ml), DBU (150 .mu.l, 1 mmol) was added at 
0.degree. C. After agitation at 0.degree. C. for 6 hours, dilute 
HCl/diethyl ether (3 ml) was added to the reaction mixture and processed 
as described hereinbefore. 
Yield: 686 mg (91%) of slightly brownish 7d with 3% of the starting product 
(1H-NMR) and a D-Ala content of 5% (GC). 
EXAMPLE 8 
Peptide-resin alcoholysis, Boc-Leu-Ala-Gly-Val-OMe (15b) (SEQ ID NO:1) 
After suspending Boc-Leu-Ala-Gly-Val-(PS-Pam resin) (15a) (300 mg, 0.168 
mmol peptide) in 3 ml of a 0.28 M LiBr/methanol solution (487 mg LiBr/20 
ml methanol) and agitation at room temperature for 15 minutes, DBU (50 
.mu.l, 0.34 mmol) was added. After agitation at room temperature for 4 
hours, the reaction mixture was filtered and the resin was washed with 
ethyl acetate (about 10 ml), treated with 1N HCl (about 10 ml) and twice 
extracted with ethyl acetate (about 10 ml). After drying the combined 
organic extracts with magnesium sulphate, filtering, evaporation of the 
solvent and drying in a high vacuum, 78 mg of 15b were obtained, slightly 
contaminated with a D-Val content of 1% (GC). Additional purification by 
flash chromatography (5% methanol in diethyl ether), after drying for 24 
hours in a high vacuum, yielded 66 mg (83%) of 15b in the form of a white 
powder melting at 71-72.degree. C. 
EXAMPLE 9 
Splitting of Peptide and Resin. Production of Boc-Leu-Ala- Gly-Val-OH (15c) 
After suspension of Boc-Leu-Ala-Gly-Val-(PS-Pam resin) (15a) (150 mg, 0.093 
mmol peptide) in a solution of LiBr (40 mg, 0.46 mmol) in THF (1.8 ml) and 
water (0.2 ml) and agitation for 15 minutes at room temperature, DBU (7 
.mu.l, 0.047 mmol) was added. After agitation at room temperature for 4 
hours, the reaction mixture was filtered and the resin was washed with 
ethyl acetate (about 10 ml), treated with 1N HCl (about 10 ml) and 
extracted twice with ethyl acetate (about 10 ml). After drying the 
combined organic extracts with MgSO.sub.4, filtering and distilling of the 
solvent, the mixture was dried in a high vacuum. The resulting product (81 
mg) was slightly contaminated with a 1% content of D-Val (GC). The yield 
was determined by .sub.1 H-NMR on the crude product using acetonitrile as 
the internal standard and was 34 mg (81%). Esterification of the crude 
product with CH.sub.2 N.sub.2 resulted in a product with a 1H-NMR spectrum 
corresponding to the spectrum of 15b. 
EXAMPLE 10 
Peptide Resin Alcoholysis. Boc-Leu-Ala-Glv-Phe-OMe (16b) (SEQ ID NO:2) 
After suspension of Boc-Leu-Ala-Gly-Phe-(PS-Pam resin) (16a) (150 mg, 0.084 
mmol peptide) in a solution of LiBr (36 mg, 0.41 mmol) in MeOH (2 ml) and 
after agitation for 15 minutes at 0.degree. C., DBU (6.3 .mu.l, 0.042 
mmol) was added. After agitation at 0.degree. C. for 8 hours, the reaction 
mixture was filtered and the resin was washed with ethyl acetate (about 10 
ml), treated with 1N HCl (about 10 ml) and processed as before (half the 
quantity of solvent). The product was 64 mg of 16b, a colorless oil with a 
D-Phe content of 2% (GC). The content of 16b was 38 mg (86%) as determined 
by 1H-NMR on the crude product using acetonitrile as an internal standard. 
Further purification by flash chromatography (10% v/v MeOH/diethyl ether), 
after drying for 24 hours over a high vacuum, yielded 36 mg (82%) of 16b 
in the form of a white powder. 
EXAMPLE 11 
Peptide-resin Splitting. Production of Boc-Leu-Ala-Gly-Phe-OH (16c) 
After suspension of Boc-Leu-Ala-Gly-Phe-(PH-Pam resin) (16a) (150 mg, 0.084 
mmol peptide) in a solution of LiBr (36 mg, 0.41 mmol) in THF/10% v/v 
H.sub.2 O (2 ml) and after agitation at room temperature for 15 minutes, 
DBU (6.3 .mu.l, 0.042 mmol) was added. After agitation at room temperature 
for 4 hours, the reaction mixture was filtered and the resin was washed 
with ethyl acetate (about 10 ml), treated with 1N HCl (about 10 ml) and 
extracted twice with ethyl acetate (about 10 ml). After processing as 
before, 96 mg of impure 16c was isolated with a D-Phe content of 2% (GC). 
The content of 16c, measured over 1H-NMR on the crude product using 
acetonitrile as the internal standard, was 40 mg (93%). Esterification of 
the crude product with CH.sub.2 N.sub.2 yielded a 1H-NMR spectrum 
corresponding to 16b. 
EXAMPLE 12 
Ac-D-Nal-D-p-Cl-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-OH 
250 mg (0.223 mmol) of Ac-D-Nal-D-p-Cl-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-OMe was 
suspended in 15 ml of THF, the suspension was mixed with 1 ml water and a 
solution of 11.2 mg (0.468 mmol) LiOH in 1 ml water, and the reaction 
mixture was agitated at room temperature for 4 hours, after which the HPLC 
failed to show any more educt. The reaction solution was brought to pH 4 
with 1N hydrochloric acid, the THF was removed in vacuo, and the residue 
was diluted with 15 ml water and suction-filtered. The product was 
digested with 30 ml acetonitrile while hot at 80.degree. C. and again 
suction-filtered and dried. The final product was 220 mg (90%) of 
Ac-D-Nal-D-p-Cl-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-OH, with 98.5% purity as per 
HPLC. The 1H-NMR spectrum did not contain the signal of the methyl ester 
at 3.6 ppm, but in other respects the spectrum was similar to a spectrum 
of Ac-D-Nal-D-p-Cl-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-OH prepared independently 
for comparison. A GC racemate test showed no significant racemisation of 
leucine (D-Leu 0.5%). 
EXAMPLE 13 
Ammonolysis, Preparation of Boc-Phe-Ala-NH.sub.2 
300 mg (0.86 mmol) of Boc-Phe-Ala-OMe and 274 mg (2.6 mmol, 3 eq) were 
dissolved in 30 ml dry THF and 400 mg KF were suspended on Al.sub.2 
O.sub.3 (approx. 2.2 mmol F.). A stream of dried NH.sub.3 gas was sent 
through at 0.degree. C. After 24 hours the educt had substantially been 
used up. 1H-NMR showed an ester content of about 10%. No side-products 
were shown in the DC. 
EXAMPLE 14 
(Comparative)--Preparation of Boc-Phe-Ala-OEt by Means of the 
Interesterification of Boc-Phe-Ala-OMe with Ca(OAc).sub.2 in Ethanol 
(method of Miranda et al. Int. J. Pep. Prot. Res. Vol. 37, (1991) pp. 
451-6) 
1.7 g calcium acetate were dissolved and suspended in 200 ml ethanol abs. 
After the addition of 0.7 g (2 mmoles) Boc-Phe-Ala-OMe the mixture was 
agitated 2 days at room temperature. After this time only educt was able 
to be demonstrated in the HPLC. Even after several hours of agitation at 
40.degree. C. no conversion (reaction) was able to be observed. The 
solvent was removed in a vacuum and the residue taken up in 100 ml ethyl 
acetate and 100 ml water. The ethyl acetate phase was washed successively 
with 50 ml of a saturated, aqueous NaHCO.sub.3 solution and a saturated, 
aqueous NaCl solution and dried with sodium sulfate. After the removal of 
the sodium sulfate by suction the solvent was removed in a vacuum and the 
residue dried in an oil pump vacuum. Finally, 0.7 g of a solid was 
obtained which was, according to .sup.1 H-NMR, the educt used. 
In contrast thereto, the interesterification of Boc-Phe-Ala-OMe to the 
corresponding diethyl ester according to the method of Seebach succeeds in 
a 96% yield (example 5 of the U.S. application). 
Comparative example 1 demonstrates that the method of Miranda doesn't work 
at all by a transesterification reaction of Boo-Phe-Ale-OMe with 
Ca(OAc).sub.2. In contrast, our claimed method yields 96% of the desired 
product (example 5 of the above-identified patent application). 
EXAMPLE 15 
(Comparative)--Preparation of Z-Asn-Leu-1 Ome by Means of the 
Interesterification of Z-1-ASM-Leu-OEt with DBU/LiBr in Methanol (Seebach 
method) Seebach et al. Helv. Chim. Actu. Vol. 74 (1991) pp 1102-1118 
0.81 g (2 mmoles) Z-Asn-Leu-OEt were dissolved in 25 ml anhydrous methanol. 
After the mixture cooled down to 0.degree. C. 0.87 g (10 mmoles) LiBr and 
0.15 ml DBU were added and the mixture agitated at this temperature 
overnight. According to HPLC no more educt was present after this time. 
After the addition of 1 ml in HCl the solvent was removed in a vacuum and 
the oily residue digested until complete crystallization with 100 ml 
water. The product was removed by suction and dried in an oil pump vacuum. 
Finally, 0.58 g (73.4%) of a white powder accumulated which was, according 
to .sup.1 H-NMR, the interesterification product Z-Asn-Leu-OMe. The 
diethyl ester was not able to be demonstrated any more in either the HPLC 
or in the .sup.1 H-NMR. According to a gas-chromatographic racemate test 
the portion of D-leucine in the product was 1% (educt 0.45%), that is, 
only a very slight racemization took place under the reaction conditions. 
In contrast thereto, only 13% of the methyl ester was produced with the 
method of Miranda (calcium acetate, methanol, 35.degree. C., 24 h reaction 
time) (no statement about the racemization). 
Comparative example 2 shows the superiority of our method with clearly 
higher yields (73.4% vs. 13%) than the method of Miranda. Additionally, we 
find nearly no racemization. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 2 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- Leu Ala Gly Val 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 4 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Leu Ala Gly Phe 
__________________________________________________________________________