Synthetic polystyrene resin and its use in solid phase peptide synthesis

A synthetic resin based on a polystyrene that can be used as a support for solid phase peptide synthesis and that has been cross-linked with from 0 to 5 mol % of divinyl benzene, characterized in that it has been substituted at benzene rings of its skeletal structure by groups of the formula ##STR1## in which X represents --O-- or --NH-- and R represents C.sub.1 -C.sub.4 -alkyl, as a support renders possible the solid phase synthesis of peptides and peptide amides that are, if desired, protected at the N-terminal and/or at other functional groups. The resin is manufactured by reaction of a customary chloromethylated polystyrene resin with an alkali metal salt of the corresponding 4-hydroxybenzophenone and subsequent reduction of the carbonyl group and, optionally, amination.

The invention relates to a novel synthetic resin for use as a support for 
the synthesis of peptides and peptide amides in solid phase in accordance 
wth the known general principle of the Merrifield synthesis. The synthetic 
resin according to the invention consists of the skeletal structure of a 
polystyrene that can be used for such a Merrifield synthesis and that has 
optionally been cross-linked by copolymerisation with from 0 to 5 mol%, 
preferably from 1 to 2 mol%, of divinyl benzene and is characterised in 
that it has been substituted at benzene rings of its skeletal structure by 
groups of the formula 
##STR2## 
in which X represents --O-- or --NH-- and R represents C.sub.1 -C.sub.4 
-alkyl. X in a resin according to the invention is preferably --O--, and 
the symbol R is preferably a linear C.sub.1 -C.sub.4 -alkyl, especialy 
methyl. 
The invention also relates to a process for the manufacture of the 
above-characterised resin and to its use for the manufacture of peptides 
and peptide amides, especially those that are protected at the N-terminal 
amino group and/or at the remaining functional groups. The invention 
relates also to corresponding resins in which the radical --XH, when it 
represents --NH.sub.2, is in protected form, and to resins with attached, 
optionally protected, amino acid, peptide and peptide amide residues, and 
also to the free peptides and peptide amides manufactured by the 
above-mentioned process, especially those described in the Examples. 
The principle of the Merrifield synthesis of peptidic compounds, published 
in 1962, is generally known. In this process, an amino acid protected at 
one terminal (usually the N-terminal) is bonded (attached) to a suitably 
functionalised base polymer (in the most common form to a chloromethylated 
polystyrene). The bond should on the one hand be sufficiently strong to 
remain intact under the various reaction conditions of the synthetic 
construction of the peptide (especially the removal of the N-terminal 
amino-protecting group), but on the other hand should allow the finished 
peptide to be detached from the polymer support under conditions that do 
not impair the product. The actual synthesis is then carried out at the 
amino acid that is attached to the support; in this process usually the 
N-terminal protecting group is removed, the freed amino group is acylated 
by a suitable amino acid derivative protected at the N-terminal (that is 
to say a new peptidic bond is formed), and the removal of the terminal 
protecting group and acylation with a further amino acid residue is 
repeated until an amino acid sequence of the desired length is obtained. 
This sequence is then detached (removed) from the support as the finished 
peptide by means of a suitable reagent. This ideal reaction course suffers 
from a number of disadvantages and sources of errors when carried out in 
practice, and over the last 25 years peptide literature has been actively 
concerned with these problems. One of the most difficult problems is that 
the synthesised sequence on the support cannot be freed of secondary 
products, and a defective structure that has already been formed (for 
example a secondary product with a shortened sequence) co-reacts in every 
further stage of the synthesis and is carried over into subsequent stages 
so that, at the end, the desired product is often obtained only in a small 
quantity together with a predominant mass of secondary products of similar 
structure from which it has to be separated in a complicated manner. This 
situation is especially difficult if the amino acids used contain other 
side chain functional groups that must virtually always be protected 
(owing to the relatively energetic conditions in the acylation operation). 
These protecting groups, in turn, must be sufficiently resistant to remain 
intact when the N-terminal amino group is freed. Yet another problem is 
posed by peptide amides since their bond to the support (in contrast to 
the ester bond of conventional peptides), is also a peptidic bond like the 
bond between amino acids of the complete amino acid sequence. The 
difficulty therefore arises of selectively detaching a peptide amide from 
the support whilst retaining the other peptidic bonds. For these (and also 
other) reasons, solid phase synthesis (Merrifield synthesis) is currently 
regarded as a method having certain advantages only when synthesising 
peptides having a maximum of 30 amino acids. 
In order to broaden its field of application, it has been proposed, see, 
for example, E. Atherton et al., Proceedings of the 7th American Peptide 
Symposium, pages 163-195, Pierce Chemical Company, Rockford, IL, U.S.A. 
(1981), that entire amino acid partial sequences having protected 
functional groups (including the N-terminal amino group) be attached in 
the form of building blocks to a support and linked with other protected 
peptide fragments in the form of building blocks. It should, then, be 
possible also to produce these necessary building blocks (peptide 
fragments) by solid phase synthesis on a support. This, however, presents 
an entirely new problem. For this, a resin is required that permits the 
synthesised peptide fragment, which consists of several protected amino 
acid residues, to be released from attachment to the resin under such mild 
and/or selective conditions that neither the N-terminal protecting group 
nor the protecting groups of other functional groups (which of necessity 
must be different from the former) are affected. A detailed discussion of 
various proposals and partial solutions can be found in the 
above-mentioned publication of Atherton et al.; these authors have 
themselves proposed as their own best solution the use of a polyamide base 
resin that is bonded by way of norvaline to 2- or 
3-methoxy-4-hydroxymethylphenoxyacetic acid. The C-terminal of the first 
(protected) amino acid is then attached to the hydroxymethyl group by an 
ester bond; the cleaving of this bond (detachment from the resin) at the 
end of the synthesis is effected using 1% trifluoroacetic acid in 
methylene chloride. But even this method, which can probably be considered 
as one of the best in the State of the Art, is not universally applicable 
since, as stated by the authors themselves, under the removal conditions 
at the end of the synthesis at least two very important protecting groups 
of the side chains, that is the tert.-butoxycarbonyl protecting group of 
the .epsilon.-amino group of lysine and the tert.-butyl group protecting 
the hydroxy group of tyrosine, are so easily removed that the use of these 
protecting groups becomes questionable. Moreover, the resin is not 
suitable for a direct solid phase synthesis of peptide amides. All of this 
probably explains why this method has not been more widely used so far in 
spite of its clear advantages. 
Surprisingly, it has now been found that the synthetic resin according to 
the invention is free from the known disadvantages of the earlier 
proposals and thus permits a wide application of the solid phase synthesis 
of protected and unprotected peptides and peptide amides. Preferably, the 
most used support polymer, that is polystyrene, is used as the polymer 
skeleton for the synthesis of the synthetic resin. 
Practically any synthetic resin that contains phenyl groups in its skeleton 
can be used as the base polymer. Polymers of styrene, which have been in 
general use for 20 years as supports for the solid phase synthesis of 
peptides and which are available in the form of various commercial 
preparations for that purpose, are preferred. In order to increase the 
stability and insolubility in organic solvents, polystyrene resins that 
have been cross-linked by copolymerisation with at most 5 mol%, and 
preferably approximately from 1 to 2 mol%, divinyl benzene, are preferred. 
Such base polymers are substituted at their phenyl groups (benzene rings) 
by chloromethylation or bromomethylation and then the actual anchor groups 
are introduced at the methylene group by exchanging chlorine or bromine, 
respectively. Also, halomethylated, especially chloromethylated, 
polysytrene resins are common commercial products which, under the name 
"Merrifield resin", are widely used in the solid phase synthesis of 
peptides. 
The process according to the invention for the manufacture of the 
above-defined novel synthetic resins comprises reacting a suitable 
polystyrene, for example an above-described polystyrene cross-linked with 
from 0 to 5 mol% divinyl benzene and chloromethylated or bromomethylated 
at benzene rings of the skeletal structure, in succession, 
(a) with a compound of formula 
##STR3## 
in which M is an alkali metal and R has the meaning given above, 
(b) with a reducing agent and, if X represents --NH--, 
(c) with a reagent that introduces the amino group. 
The reaction according to process step (a) is carried out in the presence 
of a highly polar solvent that preferably has a good dissolving capacity 
for salts, for example a dipolar aprotic solvent, such as dimethyl 
sulphoxide, acetonitrile, hexamethylphosphorus triamide, N,N'-propylene 
urea or an aliphatic amide, such as, especially, dimethylformamide, or 
also mixtures of the mentioned solvents and, preferably, with the strict 
exclusion of moisture. Preferably a solution of the alkali metal salt of 
formula II is used; a caesium salt (M is caesium) is especially suitable 
for that purpose. The reaction can be carried out at temperatures of from 
0.degree. to 50.degree. C., preferably in the region of room temperature, 
the reaction times accordingly extending to several, for example from 8 to 
72, hours. During that time, the reaction mixture is preferably stirred or 
shaken mechanically. The 2,4-dialkoxy-4'-hydroxybenzophenones used as 
starting material can be obtained in a manner known per se, for example by 
C-acylation of a corresponding resorcinol diether with p-hydroxybenzoyl 
chloride catalysed by aluminium chloride, or by a modification of this 
acylation; the desired salt is obtained therefrom by conventional reaction 
with the corresponding alkali metal hydroxide, such as caesium hydroxide. 
Process step (b) is carried out in a manner known per se by reaction with a 
reducing agent, for example one that is customary for reducing oxo groups 
to hydroxy groups. Suitable reducing agents are, for example, diborane or 
complex hydrides, such as, especially, alkali metal borohydrides (for 
example sodium borohydride, lithium borohydride or potassium borohydride) 
as well as, also, alkali metal aluminium hydrides (for example sodium 
aluminium hydride or lithium aluminium hydride), which are used in 
expedient non-reactive organic solvents, especially open-chained or cyclic 
ethers (for example diisopropyl ether or 1,2-dimethoxy- or 
-diethoxy-ethane, or dioxan or tetrahydrofuran, respectively) at 
temperatures of from 0 .degree. to approximately 100.degree. C., depending 
on the reagent. The reaction times depend on the reagents and reaction 
conditions employed and as a rule range from 1 to 48 hours; shaking or 
stirring facilitates contact between the solid and liquid components. If 
necessary, the reduction process can be repeated with a fresh amount of 
reducing agent; excess reagent is advantageously destroyed at the end of 
the reaction, for example by a ketone, such as acetone. 
The optional process step (c), which is used to convert the "hydroxy resin" 
obtained in accordance with (b) into the "amino resin", is carried out, 
for example, with ammonia. For this purpose, for example ammonia gas is 
introduced into a stirred suspension of the "hydroxy resin" in a polar 
solvent, for example one of the solvents mentioned in process step (a) or 
(b), at temperatures of from 0.degree. C. to room temperature; this 
process can especially also be carried out under elevated pressure at 
temperatures of up to 50.degree. C. with shaking. 
Carbamates from which the alcohol moiety can be removed by treatment with a 
base but which are stable under acidic reaction conditions are especially 
suitable as the reagents for introducing the amino group used in process 
step (c). Examples of such carbamates are substituted ethylcarbamates that 
carry activating, especially electron-attracting, substituents in the 
.beta.-position. The following are suitable: .beta.-(lower alkane- or 
arenesulphonyl)-ethylcarbamates, for example 62 
-(methanesulphonyl)-ethylcarbamate or 
.beta.-(benzenesulphonyl)-ethylcarbamate, .beta.-(nitro-, cyano- or 
halo-phenyl)-ethylcarbamates, for example 62 
-(p-nitrophenyl)-ethylcarbamate, .beta.-di-(p-nitrophenyl)-ethylcarbamate 
or .beta.-(pentafluorophenyl)-ethylcarbamate, or especially 
9-fluorenylmethylcarbamate. In the reaction with the above-mentioned 
carbamates a suspension of the "hydroxy resin" in an inert organic 
solvent, for example a halohydrocarbon, such as methylene chloride, 
chloroform or 1,2-dichloroethane, or an ether, such as diisopropyl ether, 
diisobutyl ether, dimethoxyethane, diethoxyethane, tetrahydrofuran or 
dioxan, is stirred at temperatures of from 20.degree. to 80.degree. C., 
preferably of approximately 50.degree. C., with the carbamate and a strong 
acid, for example an organic sulphonic acid, preferably a lower 
alkanesulphonic or arenesulphonic acid, for example methanesulphonic acid, 
benzenesulphonic acid or p-toluenesulphonic acid, for a few hours, for 
example from 2 to 48 hours. In this manner a synthetic resin of the 
above-defined structure is obtained that carries instead of the --X--H 
group a --NH--W group in which W represents an amino-protecting group that 
can be removed by treatment with a base, especially a substituted 
ethoxycarbonyl group. To free the "amino resin" the protecting group is 
then removed with a base, for example with a solution of a tertiary or, 
preferably, secondary, open-chained or cyclic amine, for example 
triethylamine, tributylamine, diethylamine, piperidine, pyrrolidine or 
morpholine, in an inert organic solvent, preferably in one of the 
above-mentioned ethers or in a di-lower alkylamide, for example 
dimethylformamide or dimethylacetamide, at temperatures of from 0.degree. 
to 50.degree. C., preferably at approximately room temperature, with 
reaction times of from a few minutes, for example 1 minute, to a few 
hours, for example 6 hours. It is also possible to use an alkali metal 
hydroxide, for example sodium hydroxide, or an ammonium hydroxide, for 
example benzyltrimethylammonium hydroxide, instead of an amine, in which 
case, even at lower temperatures, the cleaving is already concluded after 
shorter reaction times, for example after less than 1 minute. 
As already mentioned hereinbefore, the invention also relates to the use of 
the synthetic resin according to the invention as a support for the solid 
phase syhthesis of peptides and peptide amides, especially those that are 
protected at the N-terminal amino group and/or at functional groups of the 
side chains. 
In accordance with the invention, the peptidic compounds are manufactured 
using the above-defined synthetic resin as follows: 
(a) the synthetic resin is reacted with a compound of formula W.sup.1 
--AM.sup.1 --Y--H in which Y represents --O-- or --NH--, W.sup.1 
represents an N-terminal amino-protecting group and AM.sup.1 represents 
the acyl radical of an amino acid sequence consisting of from 1 to 25 
amino acid residues which is, if desired, protected at functional groups, 
or with a reactive functional derivative thereof, for the purpose of 
attaching the residue W.sup.1 --AM.sup.1 -- to the --X-- group of the 
resin, 
(b) the N-terminal protecting group W.sup.1 is removed, 
(c) the freed N-terminal amino group is acylated by reaction with an acid 
of formula W.sup.2 --AM.sup.2 --OH in which W.sup.2 and AM.sup.2 have 
meanings analogous to those of the above-defined radicals W.sup.1 and 
AM.sup.1, or with a reactive functional derivative thereof, 
(d) the operation of alternate removal according to (b) and acylation 
according to (c) is repeated as required until the desired amino acid 
sequence is obtained and 
(e) the resulting peptide or peptide amide, if desired after the removal or 
simultaneously with the removal of protecting groups, is removed from the 
resin by acidolysis. 
When attaching the C-terminal protected amino acid or amino acid sequence 
to the resin of the invention, the conditions are always chosen taking 
into account the reacting functional groups concerned. The support resin 
preferably used is the "hydroxy resin" of formula I in which X represents 
--O--; if the end product is desired in the amide form, that resin can be 
reacted with a protected amino acid amide or with a protected amino acid 
sequence that is in the form of an amide, for example with a compound of 
formula W.sup.1 --AM.sup.1 --NH.sub.2 in which W.sup.1 and AM.sup.1 are as 
defined above, in which reaction the hydroxy groups --XH of the resin are 
exchanged for the amidic C-terminal amino group. The attchment is usually 
acid-catalysed, for example by means of an organic sulphonic acid, 
preferably a lower alkanesulphonic or an arenesulphonic acid, for example 
methanesulphonic acid, benzenesulphonic acid or p-toluenesulphonic acid, 
and carried out in the presence of inert organic solvents, such as 
chlorinated alkanes (for example methylene chloride, chloroform or 
1,2-dichloroethane) and/or open-chained or cyclic ethers (for example 
diethyl, diisopropyl or dibutyl ether, or 1,2-dimethoxy- or 
1,2-diethoxyethane, or dioxan or tetrahydrofuran, respectively) for 
several, for example from 2 to 48 hours at temperatures of from 20.degree. 
to 80.degree. C., preferably at approximately 50.degree. C. If necessary 
the attachment operation can be repeated. The resin with attached 
C-terminal amide, that is to say one in which the benzene rings have been 
substituted by radicals of the formula 
##STR4## 
in which R, W.sup.1 and AM.sup.1 have the meanings given above, is then 
subjected to the synthesis of the desired amino acid sequence in 
accordance with process steps (b) to (d). The detachment of the finished 
peptide amide (process step e) can be carried out with hydrogen fluoride, 
but is preferably carried out with milder acidic agents, such as 
trifluoroacetic acid, preferably in an inert organic diluent, such as a 
haloalkane, or in water. For example, the detachment is carried out using 
a mixture of trifluoroacetic acid and methylene chloride or 
1,2-dichloroethane, for example in a ratio by volume of 1:1. However, not 
all of the acidolytically removable protecting groups customarily used are 
resistant under these conditions. Depending on the reaction procedure, 
therefore, at the same time as the detachment from the resin the 
corresponding protecting groups are also removed, resulting in a peptide 
amide having free functional groups. 
If the peptide at the end of the synthesis is desired in the form of a free 
acid (if desired with an N-terminal protecting group and/or other 
protecting groups), then the support resin used is again the "hydroxy 
resin" of formula I in which X represents --O--. This is reacted with a 
protected amino acid or with a protected amino acid sequence in the form 
of the free acid or in the form of a reactive functional derivative of the 
acid, for example with a compound of formula W.sup.1 --AM.sup.1 --OH in 
which W.sup.1 and AM.sup.1 are as defined hereinbefore, in which reaction 
the hydroxyl group of the resin is esterified. The attachment reaction is 
carried out under acylation conditions customary in peptide synthesis. The 
reactive functional derivative used is, for example, an anhydride of the 
acid to be attached, especially a symmetrical anhydride, preferably in the 
presence of organic tertiary bases, suchas those mentioned hereinafter. 
Another suitable functional derivative is an active ester of the acid to 
be attached, which is also reacted in the presence of the organic tertiary 
bases mentioned hereinafter. Suitable active esters are especially esters 
of phenols that carry electron-attracting substituents, and esters of 
N-hydroxyimides. Preferably, however, the compound to be attached is used 
in the form of the free acid and is acylated with the aid of 
carbodiimides, for example diisopropylcarbodiimide or especially 
dicyclohexylcarbodiimide, as condensation agent. The reaction is carried 
out in the presence of organic bases, especially tertiary bases (such as 
pyridine, quinoline, 4-dimethylaminopyridine, N-methylpiperidine, 
N-methylmorpholine, N,N'-dimethylpiperazine, or triethylamine or 
diisopropylethylamine) and in inert organic solvents, such as chlorinated 
alkanes (for example methylene chloride or chloroform) and/or open-chained 
or cyclic ethers (for example diethyl, diisopropyl or dibutyl ether, or 
1,2-dimethoxy- or 1,2-diethoxy-ethane, or dioxan or tetrahydrofuran, 
respectively). An activated N-hydroxy compound, for example 
1-hydroxybenzotriazole, is preferably added. The reaction time is as a 
rule several, such as from 2 to 48, hours at temperatures of from 
0.degree. to 40.degree. C.; if desired the acylation process can be 
repeated. As a means of avoiding undesired secondary products it is 
advantageous to treat the resulting resin with benzoyl chloride (or a 
similar simple acid derivative) in the presence of a base, such as one of 
those mentioned hereinbefore, and thus block the hydroxy groups of the 
resin which may still be free. The resin with attached C-terminal ester, 
that is to say one in which the benzene rings have been substituted by 
radicals of formula 
##STR5## 
in which R, W.sup.1 and AM.sup.1 have the meanings given hereinbefore, is 
then subjected to the synthesis of the desired amino acid sequence in 
accordance with process steps (b) to (d). If an end product without 
protecting groups, especially without acid-labile protecting groups, is 
desired, the detachment of the finished peptide acid (process step e) can 
be carried out with hydrogen fluoride, but is preferably carried out with 
milder acidic agents, such as trifluoroacetic acid, which is preferably 
diluted with an inert organic solvent, such as a haloalkane. If a peptide 
(amino acid sequence) is desired in which the N-terminal amino group 
and/or the other functional groups are retained in protected form, as is 
usually desirable in the case of peptide fragments that are to be used for 
further synthesis, the detachment of the end product from the resin is 
carried out under especially mild acidolytic conditions, for example with 
an organic acid, especially a lower alkanecarboxylic acid, preferably 
formic or propionic acid or, more especially, acetic acid, which may be 
diluted with neutral organic solvents, for example chlorinated alkanes or 
aliphatic or cyclic ethers; there may be mentioned as being especially 
advantageous, for example, a mixture of acetic acid and methylene chloride 
or chloroform in a ratio by volume of from approximately 1:1 to 
approximately 1:19, especially of 1:9. Also suitable are highly diluted 
trifluoroacetic acid, for example in the form of a 0.1 to 2% solution in 
methylene chloride or chloroform, or a pyridinium salt, for example 
pyridinium hydrochloride, in a polar, salt- and peptide-dissolving 
solvent, for example in dimethylacetamide or dimethylformamide. The 
detachment is usually carried out in a temperature range of from 0.degree. 
to 50.degree. C., preferably in the region of room temperature, the 
reaction time extending from a few minutes to several (for example from 2 
to 8) hours. 
It is also possible to obtain peptide amides using as the support resin the 
"amino resin" of formula I in which X represents --NH-- and reacting it 
with a protected amino acid or protected amino acid sequence in the form 
of the free acid or in the form of a reactive functional derivative of the 
acid, for example with a compound of formula W.sup.1 --AM.sup.1 --OH in 
which W.sup.1 and AM.sup.1 are as defined hereinbefore. This results in a 
resin with an attached C-terminal amide, that is to say a resin in which 
the benzene rings have been substituted by radicals of formula III. The 
reaction is preferably carried out under the reaction conditions mentioned 
above for the esterification of the "hydroxy resin" with free acid or a 
reactive functional derivative thereof. The synthesis of the desired amino 
acid sequence according to the process steps (b) to (d) and the detachment 
of the finished peptide amide according to process step (e) are then 
carried out exactly as described hereinbefore. 
The relative ease with which the amino acid sequence can be detached from 
the resin is determined by the special choice of the N-terminal 
amino-protecting group, the removal of which must be strictly selective, 
with the sequence remaining attached to the resin (and also with the 
retention of other protecting groups), but nevertheless quantitative. This 
N-terminal amino-protecting group W.sup.1 or W.sup.2 is preferably a group 
that can be removed under basic conditions, especially a group of the 
oxycarbonyl type. Preferred N-terminal amino-protecting groups W.sup.1 and 
W.sup.2 are substituted ethoxycarbonyl groups that carry in the 
.beta.-position activating, especially electron-attracting, substituents, 
such as those mentioned hereinbefore in the case of the corresponding 
carbamates. Especially suitable are the 
.beta.-(methanesulphonyl)-ethoxycarbonyl group, the 
.beta.-(p-nitrophenyl)-ethoxycarbonyl group and the 
.beta.-di-(p-nitrophenyl)-ethoxycarbonyl group, and more especially the 
9-fluorenylmethoxycarbonyl group (Fmoc). These protecting groups can be 
removed with inorganic or organic bases, especially with tertiary or 
secondary amines, such as those mentioned hereinbefore for the manufacture 
of the "amino resin". The conditions for the removal of the N-terminal 
amino-protecting group in accordance with process step (b), especially of 
the Fmoc group, are described hereinbefore in connection with the 
manufacture of the "amino resin" and are generally known. 
The acylation of the freed N-terminal amino group in accordance with 
process step (c) is carried out under generally customary conditions that 
are common for solid phase synthesis, especially on a Merrifield support. 
Of the numerous known variants, attention is drawn in particular to that 
in which the acylation is carried out with an amino acid (or amino acid 
sequence) protected at the N-terminal by Fmoc or by an equivalent 
base-labile protecting group, in which all the functional groups of the 
side chain are in protected form, or with a reactive functional derivative 
of that acid, for example the symmetrical anhydride or an active ester. If 
the free acid is used, then the condensation agent employed is 
dicyclohexylcarbodiimide (or an analogous reagent), preferably in 
combination with 1-hydroxybenzotriazole, and in the presence of tertiary 
organic bases, for exammple tertiary aliphatic or cyclic amines or 
heteroaromatic bases, such as triethylamine, diisopropylethylamine, 
N,N-dimethylaniline, N-methylpiperidine, N-ethylpiperidine, 
N-methylmorpholine, N,N'-dimethylpiperazine, or pyridine and homologues 
thereof, quinoline or 4-dimethylaminopyridine. The acylation with the 
symmetrical anhydride or the active ester is also carried out in the 
presence of the mentioned tertiary bases and preferably also in the 
presence of 1-hydroxybenzotriazole. Suitable active esters are esters with 
phenols that carry electron-attracting substituents, for example 
p-nitrophenol, pentafluorophenol or 2,4,5-trichlorophenol, or with 
N-hydroxyimides, for example with N-hydroxysuccinimide or 
N-hydroxy-norbornane- or 5-norbornene-2,3-dicarboxylic acid imide. The 
acylation conditions of the mentioned variants are generally known; if 
desired, the acylation process can be repeated and/or, in order to prevent 
the formation of incorrecnt sequences, the residual non-acylated starting 
material can be acylated in a conventional manner with a simple carboxylic 
acid derivative, such as acetic anhydride or acetyl chloride, and thus 
blocked from further undesired acylations by amino acid residues in later 
stages of the synthesis. 
Suitable amino acid residues for the synthesis according to the invention 
are those derived from naturally occurring .alpha.-amino acids of the 
L-series especially in the form of peptide building blocks, and closely 
related analogues thereof, such as, especially, the enantiomers of the 
"unnatural" D-series. Preferred .alpha.-amino acids are, for example, 
glycine, alanine, valine, leucine, isoleucine, phenylalanine, aspartic 
acid, glutamic acid, arginine, histidine and lysine, and also 
.alpha.-aminobutyric acid, norvaline, isovaline, norleucine, ornithine and 
citrulline, as well as asparagine, glutamine, tyrosine, tryptophan, 
methionine, threonine, serine, but also proline and hydroxyproline (in 
which the .alpha.-amino group is combined with the alkyl radical to form a 
ring), and also cysteine and cystine (the latter occurring as a pair of 2 
cysteine residues bonded together, which may also be located at positions 
of the sequence separated from one another). Also suitable are residues of 
other amino acids that are derived from C.sub.1 -C.sub.7 -alkanecarboxylic 
acids, especially linear ones, and that carry the amino group in any 
position of the chain, for example at the terminal C-atom (such as in 
.beta.-alanine, .gamma.-aminobutyric acid or .delta.-aminovaleric acid); 
in addition they may also carry other primary amino groups (as in 
.alpha.,.gamma.-diaminobutyric acid) or be substituted by other functional 
groups, such as hydroxy, mercapto, disulphido, guanidino, carboxy or 
carboxamide groups, or by cyclic hydrocarbyl or heterocyclyl radicals, 
such as phenyl, p-hydroxphenyl, indolyl or imidazolyl. If they contain 
asymmetric C-atoms these amino acids may be used in racemic form or, 
preferably, in optically active form. 
The specific character of the solid phase synthesis demands that functional 
groups in the amino acid residues used that do not participate in the 
reaction (and also those that can remain free in liquid phase syntheses) 
are as a rule in protected form. 
The choice of protecting groups depends on the conditions of the synthesis 
and on the use of the end product to be produced. If several functional 
groups are to be protected then advantageous combinations must be 
selected. In particular, care should be taken that protecting groups of 
functional groups of the amino acid side chains are resistant during the 
removal of the N-terminal protecting group, such as the Fmoc group, and, 
if a protected peptide building block for a further synthesis is desired 
as end product, that they are stable under the mild acidic conditions 
employed for the detachment from the resin at the end of the synthesis. 
To protect other amino groups present, such as the .epsilon.-amino group in 
the lysine residue, it is possible to use any amino-protecting group 
customary in peptide chemistry that is stable under weakly basic 
conditions. Suitable groups are described collectively in known reference 
works, for example in Houben-Weyl; Methoden der organischen Chemie, 4th 
edition, vol 15/I, E. Wunsch (editor), Synthese von Peptiden (Georg Thieme 
Verlag, Stuttgart, 1974). 
It is thus possible to use, for example, amino-protecting groups that can 
be removed by reduction or under energetic basic conditions, for example 
especially the benzyloxycarbonyl group and benzyloxycarbonyl groups that 
have been substituted in the aromatic moiety by halogen atoms, nitro 
groups, lower alkoxy groups and/or lower alkyl radicals, such as the 
p-chloro- or p-bromo-benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 
p-methoxybenzyloxycarbonyl or p-tolylmethoxycarbonyl group, and also the 
isonicotinyloxycarbonyl group, as well as also sulphonyl groups such as 
p-toluenesulphonyl, benzenesulphonyl, o-nitrobenzenesulphonyl and other 
substituted benzenesulphonyl groups or also acyl groups, such as formyl, 
trifluoroacetyl or phthaloyl. An advantageous amino-protecting group is an 
ethoxycarbonyl group that carries in the .beta.-position a silyl group 
substituted by three hydrocarbon radicals, such as triphenylsilyl, 
dimethyl-tert.-butyl-silyl or, especially, trimethylsilyl. Such a 
.beta.-(trihydrocarbylsilyl)-ethoxycarbonyl group, such as a 
.beta.-(tri-lower alkylsilyl)-ethoxycarbonyl group, for example especially 
the .beta.-(trimethylsilyl)-ethoxycarbonyl group, is resistant under the 
conditions of acidic hydrolysis and of hydrogenolysis, but can be removed 
under quite specific, very mild conditions by the action of fluoride ions. 
In this respect it behaves analogously to the .beta.-silylethyl ester 
group described hereinafter as a carboxy-protecting group. 
More especially preferred are groups that can be removed by acidolysis, 
such as, especially, tert.-butoxycarbonyl and analogous groups, for 
example the tert.-amyloxycarbonyl, isopropoxycarbonyl, 
diisopropylmethoxycarbonyl, allyloxycarbonyl, cyclopentyloxycarbonyl, 
cyclohexyloxycarbonyl, d-isobornyloxycarbonyl and adamantyloxycarbonyl 
groups, and also groups of the aralkyl type, such as benzhydryl and 
triphenylmethyl (trityl), and also aralkoxycarbonyl groups of the 
2-aryl-2-propoxycarbonyl type, for example the 2-phenyl- or 
2-p-biphenylyl-2-propoxycarbonyl groups. 
It is possible for one of the above-mentioned amino-protecting groups to be 
used as a protecting group of the guanidino function, as occurs, for 
example, in the natural amino acid arginine. Especially suitable are 
sulphonyl groups, especially lower alkanesulphonyl, for example methane-, 
ethane- or isopropane-sulphonyl groups, or arenesulphonyl, for example 
substituted benzenesulphonyl, groups, suitable substituents being lower 
alkyl, for example methyl, lower alkoxy, fpr example methoxy, fused lower 
alkoxy, for example 1-oxa-1,4-butylene, nitro or halogen, for example 
chlorine or bromine. The 2,3,5-trimethyl-4-methoxybenzenesulphonyl group 
and the 2,2,5,7,8-pentamethyl-chroman-6-sulphonyl group are especially 
preferred. 
It is possible to use a hydroxy-protecting group any group customary in 
peptide chemistry for that purpose, cf. the above-cited review in 
Houben-Weyl. Groups that can be removed by acidolysis, such as 
2-tetrahydropyranyl and more especially tert.-butyl, as well as, also, 
tert.-butoxycarbonyl, are preferred. It is also possible, however, to use 
hydroxy-protecting groups that can be removed by reduction or under basic 
conditions, for example benzyl or benzyloxycarbonyl groups which may be 
substituted in the aromatic moiety by halogen, nitro and/or by lower 
alkoxy, lower alkanoyl radicals, such as acetyl, or aroyl radicals, such 
as benzoyl. 
It is possible to use as a carboxy-protecting group any group customary for 
that purpose, cf. the above-cited review in Houben-Weyl. Thus, carboxy 
groups are protected, for example, by hydrazide formation or by 
esterification. The following, for example, are suitable for the 
esterification: lower, optionally substituted alkanols, such as methanol, 
ethanol, cyanomethyl alcohol, 2,2,2-trichloroethanol, benzoylmethyl 
alcohol or, especially, tert.-butyl alcohol, but also an optionally 
substituted benzyl alcohol. An especially advantageous category of 
substituted alkanols are ethanols carrying a trisubstituted silyl group, 
such as triphenylsilyl, dimethyl-tert.-butyl-silyl or, especially, 
trimethylsilyl, in the .beta.-position. These alcohols are especially 
suitable for the protection of carboxy groups because the corresponding 
.beta.-silylethyl esters, for example .beta.-(trimethylsilyl)ethyl ester, 
do indeed have the stability of customary alkyl esters, but can be removed 
selectively with fluoride ions whilst all other protecting groups are 
retained. 
There may be used as a mercapto-protecting group any group customary in 
peptide chemistry for that purpose. The mercapto groups are protected 
especially by suitable acylation or alkylation. The following, for 
example, are suitable for the acylation: the acetyl or benzoyl radical, a 
lower alkyl- (for example ethyl-) carbamoyl group, or a benzyloxycarbonyl 
group optionally substituted as indicated hereinbefore. The following, for 
example, are suitable for the alkylation: the tert.-butyl, 
isobutoxymethyl, benzylthiomethyl or tetrahydropyranyl radical, or 
arylmethyl radicals optionally substituted by halogen, lower alkoxy or by 
nitro, such as benzyl, p-methoxybenzyl, diphenylmethyl, 
dimethoxybenzhydryl or, more especially, trityl, as well as, also, 
phenylcyclohexyl, p-methoxyphenylcyclohexyl, or 2-thienylcyclohexyl. An 
acylaminomethyl radical in which acyl is, for example, acetyl or 
alternatively benzoyl, is also very advantageous. The acetylaminomethyl 
group is especially preferred. 
Preferably, the protecting groups of the side chains are so selected that 
they can all be removed under similar conditions; especially preferred are 
the groups to which attention has already been drawn that can be removed 
by acidolysis, especially those derived from tert.-butyl. The removal of 
all of these protecting groups is then advantageously carried out in a 
single operation. 
The protecting groups are removed in generally known manner. The acidolysis 
or acidic hydrolysis is carried out, for example, by means of 
trifluoroacetic acid, hydrochloric acid or hydrogen fluoride, and in the 
case of readily removable protecting groups also by means of a lower 
aliphatic carboxylic acid, such as formic acid and/or acetic acid, in the 
presence of a halogenated hydrocarbon, for example methylene chloride, or 
of water and optionally a polyhalogenated lower alkanol or lower alkanone, 
such as 1,1,1,3,3,3-hexafluoropropan-2-ol or hexafluoroacetone. The groups 
that can be removed by reduction, especially those that contain benzyl 
radicals, are removed preferably by hydrogenolysis, for example by 
hydrogenation catalysed by palladium. The isonicotinyloxycarbonyl group is 
preferably removed by zinc reduction. The .beta.-silylethoxycarbonyl group 
is cleaved by fluoride ions. 
The invention relates also to the intermediates and end products of the 
synthesis process using the resin according to the invention. The 
invention relates especially to a synthetic resin of the structure defined 
at the beginning that instead of the --X--H group carries an --NH--W, 
--X--AM--W or --X--AM--H group in which --X-- represents --O-- or --NH--, 
W represents an N-terminal amino-protecting group and AM represents the 
acyl radical of an amino acid sequence consisting of from 1 to 180 amino 
acid residues which, if desired, is protected at functional groups, 
especially one of the compounds described in the following Examples. The 
invention relates also to peptides and peptide amides of formula 
W--AM.sup.o --XH in which --X-- represents --O-- or --NH--, W represents a 
terminal amino-protecting group and AM.sup.o represents the acyl radical 
of an amino acid sequence consisting of from 2 to 180 amino acid residues 
which, if desired, is protected at functional groups, and to analogous 
compounds having a free N-terminal amino group of formula H--AM.sup.o 
--XH, insofar as they can be obtained by the synthesis process according 
to the invention, especially to the peptides and peptide amides described 
in the following Examples. 
The abbreviations used, for example for the designation of amino acids, 
peptides and protecting groups etc., are customary abbreviations, for 
example the abbreviations compiled in the above-cited review in 
Houben-Weyl. Unless stated otherwise, the names and abbreviations of amino 
acid residues refer to residues of .alpha.-amino acids of the naturally 
occurring L-series. Unless indicated to the contrary, the term "lower" 
when used in connection with an organic radical or compound indicates such 
a radical or compound having a maximum of 7, but preferably a maximum of 
4, carbon atoms.

In the following Examples the invention is illustrated further without the 
scope thereof being limited in any way. The abbreviations used have the 
following meanings: 
Boc--tert.-butoxycarbonyl 
But--tert.-butyl (in ether) 
TLC--thin layer chromatography 
DCCI--dicyclohexylcarbodiimide 
DMA--dimethylacetamide 
DMF--dimethylformamide 
DMSO--dimethyl sulphoxide 
EDC--1,2-ethane dichloride 
FAB-MS--Fast Atom Bombardment Mass Spectrum 
Fmoc--9-fluorenylmethoxycarbonyl 
HOBt--1-hydroxybenzotriazole 
HPLC--high pressure liquid chromatography 
IGF-2--Insulin-like Growth Factor-2 
MIF--Macrophage migration Inhibition Factor 
Mtr--2,3,5-trimethyl-4-methoxybenzenesulphonyl 
OBut--tert.-butyl (in ether) 
TFA--trifluoroacetic acid 
THF--tetrahydrofuran 
Trt--trityl 
EXAMPLE 1 
2,4-dimethoxy-4'-hydroxy-benzophenone (1) 
The title compound is synthesised analogously to the preparation of 
2,4'-dihydroxy-4-methoxy-benzophenone, see Ray, S., Grover, P. K. and 
Anand Nitya: Indian Journal of Chemistry 9, 619-623 (1971), from 
resorcinol dimethyl ether and 4-hydroxybenzoic acid. Elemental analysis, 
.sup.1 H-NMR spectrum and IR spectrum confirm the structure of (1). 
EXAMPLE 2 
Caesium salt of 2,4-dimethoxy-4'-hydroxybenzophenone (1A) 
5.00 g of (1) (19.3 mmol) are suspended in 40 ml of ethanol and 16 ml of 
water and a solution of 3.25 g (19 mmol) of caesium hydroxide monohydrate 
in 4.5 ml of water is added. The solution is concentrated in vacuo, taken 
up in 40 ml of water/tert.-butyl alcohol (1:1) and lyophilised. The 
slightly yellowish lyophilisate, when crystallised from 20 ml of 
tert.-butyl alcohol, yields the salt (1A) in the form of a white powder. 
EXAMPLE 3 
4-(2,4-dimethoxybenzoyl)-phenoxymethyl-polystyrene (1% DVB cross-linked) 
(2) 
20.0 g of chloromethyl-polysytrene-1% divinylbenzene (DVB) (=Merrifield 
polymer Fluka, Switzerland; 0.67 mmol Cl/g) (13.4 mmol) are dried under a 
high vacuum for 1 hour at 50.degree. C. 26.1 g of (1A) (67 mmol) are 
suspended three times, with approximately 500 ml of pyridine each time, 
followed by concentration by evaporation under a high vacuum, and the 
residue is dissolved in approximately 700 ml of dry DMF and concentrated 
to approximately 400 ml under a high vacuum. The resin is added and the 
reaction mixture is shaken at room temperature for 20 hours with the 
exclusion of moisture. The resin is filtered and washed with 100 ml 
portions of the following solvents: 3.times.isopropyl alcohol, 
3.times.DMF, 3.times.water, 3.times.DMF, 5.times.water, 4.times.isopropyl 
alcohol. The resin is dried under a high vacuum at from 40.degree. to 
45.degree. C. until constant mass is reached. The IR spectrum exhibits the 
expected C.dbd. O band. 
EXAMPLE 4 
4-(2,4-dimethoxyphenyl-hydroxy-methyl)-phenoxymethyl-polystyrene 
[dimethoxybenzhydryloxymethyl-polystyrene, "hydroxy resin"] (3) 
14.5 ml of 1M lithium borohydride in THF are added to a suspension of 7.07 
g of (2) (approximately 4.8 mmol ketone function) in 50 ml of dry 
tetrahydrofuran (THF) and the reaction mixture is refluxed for 1 hour with 
the exclusion of moisture. After the mixture has been cooled to 
approximately from 0.degree. to 5.degree. C., 24 ml of methanol and, 
dropwise while stirring well, approximately 6 ml of acetone, are added. 
The filtered resin is washed as follows using 30 ml portions of solvent: 
3.times.methanol, 1.times.water, 1.times.aqueous hydrochloric acid of pH 
approximately 3.0, 2.times.water, 4.times.methanol. The resin is dried 
under a high vacuum at from 40.degree. to 45.degree. C. until constant 
mass is reached. Elemental analysis: 3.67% O=2.31 mmol O/g, IR spectrum: 
no C.dbd.O band. 
EXAMPLE 5 
N-[Fmoc-Pro]-4-(2,4-dimethoxyphenyl-amino-methyl)-phenoxymethylpolystyrene 
[Fmoc-Pro-amino resin] (4) 
A mixture of 0.46 g of (3) (approximately 0.26 mmol), 0.31 g of 
Fmoc-Pro-NH.sub.2 (0.92 mmol), 76.5 .mu.l of 1M benzenesulphonic acid 
solution in dichloromethane (76.5 .mu.mol) and 10 ml of dioxan is stirred 
for 3 hours and, after the addition of a further 76.5 .mu.l of 1M 
benzenesulphonic acid solution, is stirred for a further 20 hours at 
50.degree. C. The resin is filtered and washed as follows, using 5 ml 
portions of solvent: 5.times.methanol, 12.times.dichloromethane, 
3.times.methanol, 3.times.dichloromethane. The resin is dried under a high 
vacuum at from 40.degree. to 45.degree. C. until constant mass is reached. 
Using a sample weighing approximately 20 mg, the Fmoc group is removed by 
4.times.2 minute treatments with 300 .mu.l of 20% piperidine each time and 
washing six times with dimethylacetamide (DMA). The spectrophotometric 
determination (300 nm) gives a specific loading of 0.23 mmol of Fmoc/g of 
resin. 
EXAMPLE 6 
N-[Boc-Pro-Glu(OBut)-Ile-Pro]-amino resin (5) 
0.42 g of (4) (97 .mu.mol) is reacted to form (5) in an automatic peptide 
synthesiser by means of the following process by successive alternate 
removal of the Fmoc group and condensing (coupling) with Fmoc-Ile, 
Fmoc-Glu(OBut) and Boc-Pro, in each case with unreacted amino groups being 
blocked by acetylation. 
Individual operations: 
Washing and removal (deblocking) of Fmoc at room temperature with in each 
case approximately 10 ml: 1.times.isopropyl alcohol (1 min.), 4.times.DMA 
degassed (0.5 min.), 1.times.isopropyl alcohol (1 min.), 3.times.DMA 
degassed (0.5 min.), 4.times.20% piperidine in DMA (2 mins.), 
2.times.water/dioxan (1:1) (1 min.), 5.times.DMA degassed (0.5 min.), 
3.times.DMA dist. (0.5 min.). 
Coupling: 0.39 mmol of N-protected amino acid, 0.68 ml of 0.57M 
1-hydroxybenzotriazole (HOBt) in DMA (0.39 mmol) and 0.18 ml of 2.4M 
dicyclohexylcarbodiimide (DCCI) in DMA with the addition of 0.20 ml of DMA 
(20 mins. at room temperature, 2 hours at 40.degree.). 
Washing and acetylation of the remaining free amino groups with: 
1.times.acetic anhydride/pyridine/DMA (1:1:8 vol.) (5 mins.), 3.times.DMA 
degassed, 1.times.isopropyl alcohol (1 min.), 3.times.DMA degassed (0.5 
min.). The resin is washed with isopropyl alcohol (3.times.5 ml) and dried 
at room temperature under a high vacuum. 
EXAMPLE 7 
H-Pro-Glu-Ile-Pro-NH.sub.2 
0.46 g of (5) (approximately 90 .mu.mol) are stirred at room temperature 
for 15 minutes in trifluoroacetic acid/dichloromethane (1:1 vol.) and the 
resin is filtered off and washed 3 times with approximately 10 ml of 
dichloromethane each time. The filtrate is concentrated in vacuo to 
approximately 2 ml and, while stirring, is introduced dropwise into 10 ml 
of ether. The precipitate is filtered off and dried in vacuo. According to 
thin layer chromatography and HPLC the resulting colourless powder is 
identical to authentic tetrapeptide amide produced in solution. 
EXAMPLE 8 
O-[Fmoc-Gly]-hydroxy resin (6) 
2.0 g of (3) (approximately 1 mmol), 1.19 g of Fmoc-Gly-OH (4 mmol) and 
0.87 g of DCCI (4.2 mmol) are stirred for 5 minutes at from 0.degree. to 
5.degree. C. in 20 ml of 1,2-ethane dichloride (EDC), 24 mg of 
4-dimethylaminopyridine (DMAP) (0.2 mmol) are added and, after a further 
20 minutes at approximately 5.degree. C., 110 .mu.l of N-methylmorpholine 
(1 mmol) are added. The mixture is stirred at room temperature for 4 
hours. The filtered resin is washed in a peptide synthesiser with 20 ml 
each time of the following solvents: 3.times.methanol, 3.times.EDC, 
3.times.DMA, in each case for 0.5 minutes. To block hydroxy groups still 
present in the resin, the latter is stirred at room temperature for 2 
hours with 1.23 g of benzoic acid anhydride (5.4 mmol) in 1.2 ml of 
pyridine and 6 ml of DMA and then washed as follows, each time with 20 ml: 
2.times.isopropyl alcohol, 3.times.DMA degassed, 2.times.isopropyl 
alcohol, 6.times.EDC, 2.times.isopropyl alcohol, 3.times.DMA degassed, 
3.times.isopropyl alcohol, in each case for 0.5 minutes. The title 
compound (6), dried at 45.degree. C. under a high vacuum, has a Fmoc 
content of 0.26 mmol/g. 
EXAMPLE 9 
O-[Fmoc-.beta.-Ala]-hydroxy resin (7) 
The title compound (7) is manufactured analogously to (6) and has an Fmoc 
content of 0.33 mmol/g. 
EXAMPLE 10 
O-[Fmoc-Leu-Pro-Glu(OBut)-Gly-Ser(But)-Pro-Val-Thr(But)-Leu-Asp(OBut)-Leu-A 
rg(Mtr)-Tyr(But)-Asn-Arg(Mtr)-Val-Arg(Mtr)-Val-.beta.-Ala]-hydroxy resin 
(side chain-protected eglin fragment-[.beta.-Ala.sup.55 ]-eglin 
C(37-55)-nonadecapeptide bonded to hydroxy resin), (8) 
1.10 g of (7) (0.36 mmol) from Example 9 are reacted in a peptide 
synthesiser with washing processes and Fmoc removal processes analogously 
to Example 6. Amino acids 10 to 18 (that is 46 to 54 of eglin C) are 
coupled in the form of 2,4,5-trichlorophenyl ester (1.08 mmol) in 1.8 ml 
of DMA, with the addition of 1.89 ml of 0.57M HOBt (1.08 mmol) and 0.285M 
diisopropylethylamine (0.54 mmol) in DMA, for two hours at room 
temperature. This process is repeated for amino acids 11, 12, 16 and 18 
(that is 47, 48, 52 and 54 eglin C). Amino acids 1 to 9 (that is 37 to 45 
of eglin C) are used for the coupling in the form of symmetrical 
anhydrides (1.08 mmol): 2.16 mmol of Fmoc-amino acid are dissolved in 10 
ml of dichloromethane (in the case of Fmoc-Gly-OH with the addition of 1 
ml of DMA), 245 mg of DCCI (1.12 mmol) are added at room temperature while 
stirring, the whole is maintained at room temperature for 15 minutes, the 
precipitated dicyclohexylurea is filtered off, 2.5 ml of DMA (distilled) 
are added to the filtrate and the dichloromethane is removed in vacuo. 
This mixture is added in each case to the synthetic resin and, after the 
addition of 58 .mu.l of diisopropylethylamine (0.36 mmol), maintained at 
room temperature for 1 hour. When the synthesis is complete and the 
terminal Fmoc group has been removed in the manner described in Example 5, 
the resin is washed 3 times with 5 ml of isopropyl alcohol each time and 
dried at room temperature under a high vacuum. 
EXAMPLE 11 
Side chain-protected [.beta.-Ala.sup.55 ]-eglin C(37-55)-nonapeptide 
1.55 g of (8) (approximately 83 .mu.mol) from Example 10 are stirred for 
1.5 hours at room temperature with 20 ml of dichloromethane/acetic acid 
(9:1 vol.), the resin is filtered off and washed as follows wih 10 ml of 
solvent each time: 3.times.dichloromethane, 4.times.methanol, 
4.times.dichloromethane. The original filtrate is combined with the 
washing solutions and concentrated in vacuo, approximately 5 ml of acetic 
acid are added and the whole is lyophilised to dryness yielding the title 
compound in the form of a colourless powder. HPLC on Nucleosil 5C.sub.18, 
25.times.4.6 mm, gradient: 100% A/0% B.fwdarw.0% A/100% B for 60 minutes, 
A=water 0.1% TFA, B=acetonitrile 0.1% TFA, retention time 52 mins. 
Preparative HPLC: 10 mg are dissolved in 500 .mu.l of 
trifluoroethanol/acetonitrile (1:1), separation is carried out with the 
above gradient and column and the main fraction is collected and 
concentrated by evaporation. In this manner 6.8 mg of a product are 
obtained which, according to HPLC, contains more than 90% of the title 
compound. 
EXAMPLE 12 
O-[Fmoc-Asp(OBut)-Arg(Mtr)-Gly-Phe-Tyr(But)-Phe-Ser(But)-Arg(Mtr)-Pro-Ala-S 
er(But)-Arg(Mtr)-Val-Ser(But)-Arg(Mtr)-Arg(Mtr)-Ser(But)-Arg(Mtr)-Gly]-hydr 
oxy resin (side chain-protected IGF-2 (23-41)-nonadecapeptide bonded to 
hydroxy resin), (9) 
1.20 g of (6) (0.31 mmol) from Example 8 are reacted in a peptide 
synthesiser with washing processes and Fmoc removal processes analogously 
to Example 6. All the amino acids are used for the coupling (1 hour) in 
the form of symmetrical anhydrides (3 equivalents, manufacture as in 
Example 10). With the amino acids 14, 17 and 18 (that is 36, 39 and 40 of 
IGF-2) the coupling process is repeated (1 hour). When the synthesis is 
complete, the resin is washed 3 times with 5 ml of isopropyl alcohol each 
time and dried under a high vacuum. 
EXAMPLE 13 
Fmoc-Asp(OBut)-Arg(Mtr)-Gly-Phe-Tyr(But)-Phe-Ser(But)-Arg(Mtr)-Pro-Ala-Ser( 
But)-Arg(Mtr)-Val-Ser(But)-Arg(Mtr)-Arg(Mtr)-Ser(But)-Arg(Mtr)-Gly-OH 
[Fmoc-protected IGF-2 (23-41)-nonadecapeptide] 
1.65 g of (9) (approximately 154 .mu.mol) from Example 12 are cleaved with 
33 ml of dichloromethane/acetic acid (9:1 vol.) and washed, analogously to 
Example 11. Lyophilisation yields a colourless powder. For purification, 
this product is partitioned in an automatic countercurrent partitioning 
apparatus (5 ml/phase) with a methanol/water/chloroform/carbon 
tetrachloride mixture (2700:675:900:1575) over 1050 stages. Fractions 
46-75 contain the pure title compound (TLC, HPLC) and are together 
concentrated by evaporation, and the residue is triturated with ether and 
filtered. According to TLC (4 systems on silica gel) and HPLC (system as 
in Example 11; retention time 57 mins.) the resulting colourless powder is 
homogeneous. 
FAB-MS (fast atom bombardment mass spectrum): correct mass peak (2480). 
EXAMPLE 14 
Fmoc-amino resin (10) and amino resin 
[4-(2,4-dimethoxyphenyl-amino-methyl)-phenoxymethylpolystyrene] 
4.1 g of hydroxy resin (3) (approximately 1.89 mmol) are suspended in 80 ml 
of dioxan. 1.35 g of 9-fluorenylmethylcarbamate (5.65 mmol) (manufactured 
in accordance with Carpino, L. A. et al., J. Org. Chem. 48, 661 (1983)) 
and 0.47 ml of 1M benzenesulphonic acid in 1,2-ethane dichloride (EDC) 
(0.47 mmol) are added and the mixture is maintained at 50.degree. C. for 3 
hours. After the addition of a further 0.47 ml of 1M benzenesulphonic 
acid, the whole is reacted for a further 17 hours at 50.degree. C. The 
resin is filtered and washed with 30 ml portions of the following 
solvents: 3.times.isopropyl alcohol, 3.times.EDC, 
1.times.dimethylacetamide (DMA), 6.times.isopropyl alcohol, 3.times.DMA. 
The resin is then maintained at room temperature for 2 hours with 5.0 g of 
benzoic acid anhydride in 25 ml of DMA and 5 ml of pyridine in order to 
block unreacted hydroxy groups, and washed as described above. The last 
wash is carried out with 6.times.isopropyl alcohol. The resulting 
Fmoc-amino resin (10) is then dried under a high vacuum until a constant 
mass is reached. After cleaving a sample in accordance with Example 5, the 
spectrophotometric determination of Fmoc gives a specific loading of 
approximately 0.35 mmol of Fmoc/g of resin. 
In order to produce the free amino resin, 2.0 g of Fmoc-amino resin (10) in 
30 ml of 20% piperidine in dimethylacetamide (DMA) are stirred at room 
temperature for 1 hour, filtered, treated again with a further 30 ml of 
20% piperidine in DMA for 1 hour, and washed as follows with 30 ml 
portions: 3.times.DMA, 3.times.isopropyl alcohol, 3.times.DMA, 
6.times.isopropyl alcohol. The resin is dried under a high vacuum until a 
constant mass is reached. 
EXAMPLE 15 
N-[Met-His(Trt)-Glu(OBut)-Gly-Asp(OBut)-Glu(OBut)-Gly-Pro-Gly]-amino resin 
(11) 
0.50 g of Fmoc-amino resin (10) (approximately 175 .mu.mol) from Example 14 
are reacted in an automatic peptide synthesiser by means of the following 
process by successive alternate removal of the Fmoc group and condensing 
on of Fmoc-Gly, Fmoc-Pro, Fmoc-Gly, Fmoc-Glu(OBut), Fmoc-Asp(OBut), 
Fmoc-Gly, Fmoc-Glu(OBut), Fmoc-His(Trt) and Fmoc-Met, unreacted amino 
groups in each instance being blocked by acetylation: 
Washing and removal (deblocking) of Fmoc at room temperature with 
approximately 10 ml each time: 1.times.isopropyl alcohol (1 min.), 
4.times.DMA degassed (0.5 min.), 6.times.20% piperidine in DMA (2 mins.), 
2.times.DMA degassed (0.5 min.), 1.times.isopropyl alcohol (1 min.), 
4.times.DMA degassed (0.5 min.), 3.times.DMA dist. (0.5 min.). 
Coupling: 0.52 mmol of protected amino acid 2,4,5-trichlorophenyl ester and 
0.92 ml of 0.57M 1-hydroxybenzotriazole (HOBt)/0.57M diisopropylethylamine 
in DMA (0.52 mmol) with the addition of 0.88 ml of DMA (30 mins. at room 
temperature). 
Washing and acetylation of the remaining free amino groups with: 
1.times.acetic anhydride/pyridine/DMA (1:1:8 vol.) (5 mins.), 3.times.DMA 
degassed (0.5 min.), 1.times.isopropyl alcohol (1 min.), 3.times.DMA 
degassed (0.5 min.). The terinal Fmoc group is removed in the manner 
described above. The resin is washed with isopropyl alcohol and dried 
under a high vacuum at room temperature. 
EXAMPLE 16 
Met-His-Glu-Gly-Asp-Glu-Gly-Pro-Gly-NH.sub.2 (MIF-related protein 14 
(94-102)-amide 
0.65 g of (11) (approximately 150 mmol) from Example 15 are washed in a 
column for 60 mins. with approximately 30 ml of 2% trifluoroacetic acid 
(TFA) in dichloromethane, the eluate is concentrated in vacuo to 
approximately 0.3 ml, 1 l of TFA/water (95:5 vol.) is added and the whole 
is left at room temperature for 15 mins. for complete removal of the 
protecting groups. The peptide amide is precipitated by the addition of 5 
ml of diisopropyl ether. The precipitate is filtered off and dried in 
vacuo. The product is dissolved in 5 ml of water, the turbid portion is 
centrifuged off and the supernatant is lyophilised. A colourless powder is 
obtained which, according to HPLC, is more than 90% title compound. HPLC: 
retention time 6.8 mins., Nucleosil column 7C.sub.18, 120.times.4.6 mm, 
gradient: 100% A/0% B.fwdarw.10% A/90% B in 30 l mins., A=0.1% TFA/water, 
B=0.1% TFA/acetonitrile, 1.5 ml/min., detection at 215 nm. FAB-MS: mass 
peak 927 (M+H.sup.+). 
EXAMPLE 17 
O-[Fmoc-Ala-Tyr(But)-Arg(Mtr)-Pro-Ser(But)-Glu(OBut)-Thr(But)-Leu-Cys(Trt)- 
Gly-Gly-Glu(OBut)-Leu-Val-Asp(OBut)-Thr(But)-Leu-Gln-Phe-Val-Cys(SBut)-Gly] 
-hydroxy resin (side chain-protected IGF-2 (1-22)-docosapeptide bonded to 
hydroxy resin), (12) 
1.00 g of O-[Fmoc-Gly]-hydroxy resin (6) (approximately 0.36 mmol) are 
reacted in a peptide synthesiser with washing processes and Fmoc-removal 
processes analogously to Example 15. 
Coupling: Val, Gln, Gly, Leu and Arg are coupled in the form of Fmoc-amino 
acid 2,4,5-trichlorophenyl ester (0.72 mmol) in 1.20 ml of DMA together 
with 1.44 ml of 0.5M HOBt/0.5M diisopropylethylamine in DMA at room 
temperature for 1 hour. The other amino acid derivatives are coupled in 
the form of symmetrical anhydrides (1.08 mmol) (preparation analogous to 
Example 10), for 1 hour at room temperature. In the case of amino acids 
Nos. 3, 8, 10, 11, 18, 20 and 21, the process is repeated (recoupling). 
When the synthesis is complete, the resin is washed 5 times with isopropyl 
alcohol, the terminal Fmoc group being retained, and dried under a high 
vacuum. 
EXAMPLE 18 
Fmoc-Ala-Tyr(But)-Arg(Mtr)-Pro-Ser(But)-Glu(OBut)-Thr(But)-Leu-Cys(Trt)-Gly 
-Gly-Glu(OBut)-Leu-Val-Asp(OBut)-Thr(But)-Leu-Gln-Phe-Val-Cys(SBut)-Gly-OH 
(side chain-protected IGF-2 (1-22)-docosapeptide) 
1.80 g of (12) (approximately 0.19 mmol) from Example 17 are mixed for 1.5 
hours at room temperature with 35 ml of dichloromethane/acetic acid (9:1 
vol.). The resin is filtered off and the difficultly soluble fragment is 
extracted at 60.degree. C. as follows: 3.times.50 ml dimethyl sulphoxide 
(DMSO), 3.times.trifluoroethanol/dichloromethane (1:1 vol.), 
1.times.N-methylpyrrolidone/DMSO (8:2 vol.). The combined filtrates are 
concentrated by evaporation under a high vacuum and the residue is stirred 
for 1 hour at 50.degree. C. with 5 ml of water/methanol (5:95 vol.). The 
undissolved material is filtered off and dried at room temperature under a 
high vacuum. The extremely difficultly soluble fragment cannot be analysed 
by the HPLC technique. TLC (2 systems): the product is more than 90% 
strength (dissolved in DMSO/N-methylpyrrolidone (2:8)). FAB-MS: mass peak 
3539 (M+Na.sup.+). 
EXAMPLE 19 
O-[Fmoc-Ala-Tyr(But)-Arg(Mtr)-Pro-Ser(But)-Glu(OBut)-Thr(But)-Leu-Cys(Trt)- 
Gly]-hydroxy resin (side chain-protected IGF-2 (1-10)-undecapeptide bonded 
to hydroxy resin), (13) 
1.00 g of O-[Fmoc-Gly]-hydroxy resin (6) (approximately 0.36 mmol) is 
reacted in a peptide synthesiser with washing processes and Fmoc-removal 
processes analogously to Example 15. 
Coupling: the amino acid derivatives are coupled in the form of symmetrical 
anhydrides (1.08 mmol) (preparation analogous to Example 10) for 1 hour at 
room temperature. In the case of amino acids Nos. 3, 8 and 9 the coupling 
process is repeated with 2 equivalents of 2,4,5-trichlorophenyl ester. 
When the synthesis is complete the resin is washed 5 times with isopropyl 
alcohol, the terminal Fmoc group being retained, and dried under a high 
vacuum. 
EXAMPLE 20 
Fmoc-Ala-Tyr(But)-Arg(Mtr)-Pro-Ser(But)-Glu(OBut)-Thr(But)-Leu-Cys(Trt)-Gly 
(side chain-protected IGF-2 (1-10)-undecapeptide) 
1.50 g of (13) (approximately 0.19 mmol) from Example 19 are mixed for 1.5 
hours at room temperature with 30 ml of dichloromethane/acetic acid (9:1 
vol.). The resin is filtered off and the filtrate is concentrated in vacuo 
and lyophilised under a high vacuum. The resulting colourless powder is 
for the purposes of purification partitioned in an automatic 
countercurrent partitioning apparatus (5 ml/phase) with the same mixture 
as in Example 13 over 1330 stages. Fractions 65-111 contain the pure title 
compound (TLC, HPLC) and are together concentrated by evaporation. The 
residue is triturated with ether and filtered. According to TLC (5 systems 
on silica gel) and HPLC, the colourless powder is homogeneous. Retention 
time 24.1 mins. on Nucleosil column 5C.sub.18, 4.6.times.250 mm, gradient 
50% A/50% B.fwdarw.0% A/100% B in 30 mins., A=0.1% TFA/water, B=0.1% 
TFA/acetonitrile, 1.0 ml/min., detection at 215 nm. 
EXAMPLE 21 
O-[Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Gaba]-hydroxy resin (side 
chain-protected somatostatin-D-Trp(8)-4-aminobutyric 
acid(12)-(5-12)-octapeptide bonded to hydroxy resin), (14) 
1.00 g of O-[Fmoc-Gaba]-hydroxy resin (produced analogously to Example 8 
with 4-aminobutyric acid instead of glycine) (approximately 0.31 mmol) is 
reacted in a peptide synthesiser with washing processes and Fmoc-removal 
processes analogously to Example 15. 
Coupling: the amino acid derivatives are coupled in the form of symmetrical 
anhydrides (0.93 mmol) (preparation analogous to Example 10) for 1 hour at 
room temperature. Phe and Asn are coupled in the form of 
2,4,5-trichlorophenyl ester (2 equivalents). Recoupling is carried out 
with amino acid 1 (Asn). When the synthesis is complete, and after the 
terminal Fmoc group has been removed, the resin is washed 5 times with 
isopropyl alcohol and dried under a high vacuum. 
EXAMPLE 22 
H-Asn-Phe-Phe-D-Trp-Lys(Boc)-Thr(But)-Phe-Gaba-OH (side chain-protected 
somatostatin-D-Trp(8)-4-aminobutyric acid(12)-(5-12)-octapeptide) 
1.38 g of (14) (approximately 0.26 mmol) from Example 21 are mixed for 1 
hour at 50.degree. C. with 5 ml of 5% pyridine hydrochloride in DMA. The 
resin is filtered off and washed with DMSO, the filtrate is concentrated 
under a high vacuum and lyophilised once from DMSO and twice from 
tert.-butyl alcohol. According to HPLC the resulting colourless powder is 
more than 95% pure and in HPLC and TLC is identical to an authentic 
specimen that has been produced by synthesis in solution. HPLC: retention 
time 20.6 mins., Nucleosil column 5C18, 4.6.times.250 mm, gradient 100% 
A/0% B.fwdarw.10% A/90% B in 30 mins., A=0.1% TFA/water, B=0.1% 
TFA/acetonitrile, 1.0 ml/min., detection at 215 nm.