Peptide purification method using novel linker and solid-phase ligand

Disclosed are novel compounds which are used as linkers to bind peptides to solid support. The novel compounds can be used for the purification of synthesized peptides and are represented by the following structural formula: EQU X--NH--(CH.sub.2).sub.n --SO.sub.2 --CH.sub.2 --CH.sub.2 --O--CO--Y; PA1 n is an integer from 1-4; X is a thiol functionalized with a protecting group that is cleavable under acidic conditions; and Y is a leaving group.

INDUSTRIAL UTILITY 
The present invention relates to a method for purifying a peptide produced 
by solid-phase synthesis, and relates to a linker and a solid-phase ligand 
for bonding the linker for this purification method. More particularly the 
invention relates to a method for performing a one-step purification of 
peptides synthesized by a solid-phase method, with high accuracy and high 
yield, and relates to the linker and the solid-phase ligand for bonding 
the linker used for this purification. 
PRIOR ART 
Peptides and proteins are biological molecules existing normally in 
organisms. The elucidation of physiological activities and mechanisms of 
these biological molecules are of much interest to the fields of 
biochemistry, physiology and medicine. The synthesis of peptides and 
proteins having specific amino acid sequences has increased due to the use 
of automated peptide synthesizers. Studies in the above mentioned fields 
are expected to show sharp progress, if peptides or proteins having 
specific amino acid sequences can be synthesized with higher purities. 
However, present peptide synthesis methods produce a relatively large 
number of impurities, as well as target compound. Therefore an important 
objective of a solid-phase peptide synthesis method is to recover the 
target peptide alone from impurities with high speed and high yield. 
Gel filtration, high-performance liquid chromatography, and combination 
thereof are presently used for the purification of peptides or proteins 
synthesized by a solid-phase method (R. B. Merrifield, J. Am. Chem. Soc., 
85, 2149 (1963)). For some special peptides and proteins, affinity 
chromatography may be an effective purification method, but not a perfect 
one. The reason is that some of the amino acid deleted peptides may have 
an affinity (even a low degree) for the supports used in affinity 
chromatography, the amino acid deleted peptides being synthesized as 
impurities during the solid-phase synthesis as part of the resultant 
peptide mixture. 
The peptides are synthesized by a step-wise elongation. For example, in 
case of condensation of a 50 residue peptide with the condensation 
reaction yield of 99%, the theoretical synthetic yield reaches to 60%. 
Condensation reaction yield over 99% can not always be obtained since the 
condensation reaction depends on the sequence of peptides. As a result, 
amino acid deleted peptides pile up as impurities by incomplete 
condensation reactions. 
A capping by acetic anhydride is performed after every condensation 
reaction to terminate further elongation of peptide chains of a non-target 
sequence and to avoid further production of amino acid deleted peptides. 
After the coupling of the final amino acid, only the peptide having a 
target amino acid sequence will have an amino group at its N-terminus. 
Several reports on purification methods using the N-terminus amino group 
have been published. (See, for example, R. Camble, R. Garner and G. T. 
Young, Nature (London), 217, 247 (1968); K. Suzuki, Y. Sasaki and N. Endo, 
Chem. Pharm. Bull., 24, 1 (1976); D. S. Kemp and D. G. Roberts, 
Tetrahedoron Lett., 4269 (1975); T. Weiland, C. Birr and H. Wissenbach, 
Angew. Chem., Int. Ed., Engl., 8, 764 (1969), H. Wissman and R. Geiger, 
Angew. Chem., Int. Ed., Engl., 9, 908 (1970); R. B. Merrifield and A. E. 
Bach, J. Org. Chem. 43, 4808 (1975); T. J. Lobl, R. M. Deibel and A. W. 
Yen, Anal. Biochem., 170, 502 (1988); H. Ball, C. Grecian, S. B. H. Kent 
and P. Mascagni, in "Peptides", J. E. Rivier and G. R. Marshall, Eds., 
ESCOM, Leiden 1990 pp435). However, none of these methods have been able 
to achieve effective one-step separation, instead complicated separation 
processes are required. 
Another method has been developed in which the target peptide alone is 
absorbed to a phenyl-mercury column by attaching cysteine-methionine to 
the N-terminus of the synthesized peptide, and using the SH group of the 
cysteine. Subsequent to the separation, the methionine-peptide bond is 
cleaved by BrCN to yield the target peptide. (D. E. Krieger, B. W. 
Erickson and R. B. Merrifield, Proc. Natl. Acad. Sci. U. S. A., 73, 3160 
(1976)). However, this method has a limitation of being not applicable to 
peptides containing methionine. 
SUMMARY OF THE INVENTION 
An objective of this invention is to offer a method for purifying peptides 
made by solid-phase synthesis, and a linker and a solid-phase ligand for 
bonding the linker used therein so that one-step purification with 
high-accuracy and high-yield can be performed. 
The above objective is achieved by a purification method comprising 
chemically and selectively isolating a target mature peptide having an 
amino acid group at the terminus from a mixture of mature target peptide 
and end-capped immature amino acid deleted peptides by immobilization to 
the solid-phase ligand via the linker having two functional groups at the 
termini. 
The above objective is also achieved by a purification method for 
synthesized peptide comprising: 
a) after adding the final amino acid for a solid-phase peptide synthesis, 
adding a linker to the solid-phase to which both a mature target peptide 
with an amino group at a terminus and immature peptides end-capped by an 
acylating agent such as acetic anhydride or propionic anhydride have been 
bound, thereby selectively modifying the mature peptide with the liner, 
wherein the linker has at one terminus a functional group which is able to 
form a bond with the N-terminus of the mature peptide, the bond being 
stable to the deprotection reaction of the opposite terminus site of the 
linker treated later under acidic condition and being specifically cleaved 
under another condition, and wherein the opposite terminus site of the 
linker is able to be converted into another active functional group by the 
deprotection reaction; 
b) dissociating the mature peptide and the immature peptides from the 
solid-phase support by the cleavage reaction performed as the final 
process of the peptide synthesis; 
c) contacting the resultant freed peptide mixture of above step b) with a 
solid-phase ligand, and thereby selectively immobilizing the mature 
peptide to the solid-phase ligand via the linker, wherein the solid-phase 
ligand is modified by a compound which has a functional group capable of 
forming a stable bond with the functional group of the opposite terminus 
of the linker produced by the deprotection reaction of the opposite 
terminus site of the linker; and 
d) exposing the mature peptide immobilized ligand of above step c) to the 
other condition under which said bond between the mature peptide and the 
linker is selectively cleaved, thereby separating the mature peptide from 
the linker bound ligand. 
A preferred embodiment is to isolate the target mature peptide by the 
following steps: 
a) after introducing the final amino acid for a solid phase peptide 
synthesis, adding a linker to the solid-phase to which both a mature 
target peptide with an amino group at a terminus and immature end-capped 
peptides have been bound, thereby selectively modifying the mature peptide 
with the linker, wherein the linker has at one terminus a functional group 
which is able to form a bond with the N-terminus of the mature peptide, 
the bond being stable to the deprotection reaction of the opposite 
terminus site of the linker treated later under an acidic condition and 
also being specifically cleaved under an alkaline or basic condition, and 
wherein the opposite terminus site of the linker is able to be converted 
into an SH group by the deprotection reaction; 
b) dissociating the mature peptide and the immature peptides from the 
solid-phase support by the cleavage reaction performed as the final 
process of the peptide synthesis; 
c) contacting the resultant freed peptide mixture of step (b) with a 
solid-phase ligand, and thereby selectively immobilizing the mature 
peptide to the solid-phase ligand via the linker, wherein the solid-phase 
ligand is modified by a compound immobilized thereon and which has a 
functional group capable of forming a stable bond with the SH-group of the 
opposite terminus of the linker produced by the deprotection reaction; and 
d) exposing the mature peptide immobilized ligand of step (c) to an 
alkaline condition under which the bond between the mature peptide and 
said one terminus of the linker is cleaved selectively, thereby separating 
the mature peptide from the linker-bound ligand. 
Another embodiment of the method is to isolate the mature target peptide by 
the following steps: 
a) after introducing the final amino acid for a solid-phase peptide 
synthesis, adding a linker to the solid-phase to which a mature target 
peptide with an amino group at a terminus and immature end-capped peptides 
have been bound, thereby selectively modifying the mature peptide, wherein 
the linker has at one terminus a carbonate group which can be converted to 
a urethane bond reacting with the N-terminus of the target peptide, the 
urethane bond being stable to the deprotection reaction of the opposite 
terminus site of the linker treated later under an acidic condition and 
being specifically cleaved under a basic condition, and wherein the 
opposite terminus site of linker is able to be converted into a thiol 
group by the deprotection reaction; 
b) dissociating the mature peptide and the immature peptides from the 
solid-phase support by the cleavage reaction performed as the final 
process of the peptide synthesis, while cleaving a thiol protecting group 
existing at the opposite terminus site of the linker in order to produce 
the thiol group; 
c) contacting the resultant freed peptide mixture of step (b) with 
solid-phase ligand under a neutral condition, wherein the solid-phase 
ligand is modified by a compound selected from the group consisting of 
iodo- and bromo-substituted aliphatic carboxylic acid derivatives 
immobilized thereon, thereby selectively immobilizing the mature peptide 
to the solid-phase ligand via the linker with a stable covalent bond, the 
covalent bond resulting from deiodination and debromination; and 
d) exposing the mature peptide immobilized ligand of step (c) to alkaline 
condition in order to effect the beta-elimination reaction on the urethane 
bond between the mature target peptide and the linker, thereby cleaving 
the mature peptide from the linker-bound ligand. 
A linker having the structural formula (III) is preferably used for the 
method of this invention: 
##STR1## 
wherein n is an integer of one to four, X is one of 
##STR2## 
The above linkers can be easily synthesized from intermediates which are 
represented by the following structural formula (I) or intermediates by 
the following structural formula (II): 
##STR3## 
wherein n is an integer of one to four, X is one of H, hydrochloride 
thereof (H.HCl), 
##STR4## 
wherein n in an integer of one to four, X is one of 
##STR5## 
A preferred solid-phase ligand for binding the linker, comprises molecular 
chains having one of the following structural formulae are bound to the 
surface thereof: 
##STR6## 
n=1, 2, m=2-6, R is H or an alkyl group from one to six carbons. 
A method is provided to rapidly and effectively separate the mature target 
peptide from a mixture of mature peptide and many immature peptides 
produced in the solid-phase peptide synthesis. The method includes a 
selective modification of the mature peptide by a linker which has a 
functional group at one terminus that is capable of bonding with the 
N-terminus of the mature peptide. This bond is stable to the deprotection 
reaction of the opposite terminus site of the linker under acidic 
condition in the post-treatment of the peptide synthesis process and is 
able to be cleaved specifically under another condition. The opposite site 
of the linker is able to be converted into another active functional group 
by the deprotection reaction. When binding the linker to the N-terminus of 
the mature peptide after the final amino acid coupling step is performed, 
only the mature peptide has at the opposite terminus the active functional 
group (resulting in -SH after treatment under acidic conditions). When the 
mixture is contacted with another solid-phase ligand where a compound 
having the functional group capable of forming the stable bond with the 
active functional group of the mature peptide is immobilized thereon, this 
solid-phase ligand can selectively immobilize the mature peptide alone. 
This results in the separation of the target mature peptide from the 
mixture containing immature peptides. The target mature peptide may be 
cleaved from the linker-bound ligand, when the ligand immobilized to the 
mature peptide is exposed to other conditions that specifically cleave the 
urethane bond between the mature peptide and the linker. 
The method of this invention can be used to rapidly purify the target 
mature peptide prepared by the solid-phase synthesis in high yield. 
Therefore, the method will lead to the elucidation of physiological 
activities and mechanism of peptides or proteins, and will lead to the 
rapid development of synthesized peptides and proteins.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is hereby described in detail. 
FIG. 1 illustrates an embodiment of the method according to this invention 
for purifying peptides prepared by the Fmoc (9-fluorenylmethyloxycarbonyl) 
method of solid-phase synthesis. 
Peptides are synthesized, as shown in FIG. 1, by the repetition of step 1 
to step 3 in the conventional way. 
Step 1 involves the following procedure: Many first amino residue 
##STR7## 
groups whose N-terminus is protected by Fmoc group are bound to 
solid-phase support beads P; the support beads P are put into a reaction 
column of a peptide-synthesizer, where the mixed solution of 20% 
piperidine and the dimethylformamide (DMF) is added in order to deprotect 
the N-terminal Fmoc group. 
Step 2 involves: N-terminal Fmoc blocked second amino acid 
##STR8## 
and a condensation agent is added to the column in order to bond the 
second amino residue group next to the first one bound to the support 
beads P. A condensation agent, such as N,N'-di-isopropylcarbodiimide 
(DIPCD)+N-hydroxybenzotriazole (HOBT), Benzotriazole-1-yl-oxy-tris 
(dimethylamino)-phosphonium hexafluorophosphate (BOP)+HOBT, or 
Pentafluoro-phenylester of these amino acids+HOBT is used. 
Step 3 involves: The chains of the first amino acid that are not bound to 
the second amino acid should not be elongated in further synthetic steps; 
the mixture of acetic anhydride/pyridine is added to the column to let the 
terminus of non-reacted chains end-cap by acetyl bonding. On the support 
beads there are two types of peptide chains, i.e., one group contains the 
first and the second amino acid residues bound together whose N-terminus 
is protected by an Fmoc group, and the other group contains the first 
amino residue whose N-terminus is bound to an acetyl group. 
The process continues back to step 1. The N-terminal Fmoc group of the 
chains where the first and the second amino residues are bound together is 
deprotected. As in step 2, the third amino acid and the condensation agent 
are added to let the third one bond to the second. The end-capping, as in 
step 3, results in chains where the third amino acid residue is not bound. 
Step 1 to step 3 are repeated until the desired number of amino acid 
residues are bound. 
After the coupling of the final amino acid, two types of peptides are bound 
to the solid support (solid-phase support beads P), namely, the mature 
target peptide having an amino group at the N-terminus, and the immature 
peptides having termini modified by an acetyl-group from the capping with 
acetic anhydride. The target peptide is mixed with many kinds of 
impurities. 
FIG. 1 describes the method to end-cap the terminus of immature peptides 
with an acetyl group. However, any effective capping reagent can be used 
for the capping of the termini of immature peptides. Capping reagents such 
as a propionyl group, a 4-nitrophenyl group, a 2,4-dinitrophenyl group, or 
a 2,6-dinitrophenyl group are usable. 
For the purification of synthesized peptides, a suitable linker is added to 
the solid-phase support after the coupling of the last amino acid. The 
linker should have a functional group at one terminus, which forms with 
the N-terminus of the mature peptide a bond, such as urethane bond, which 
is stable under the acidic condition of the deprotection reaction of the 
opposite terminus site of the linker and cleavable specifically under 
other conditions, such as basic conditions. The linker should also form an 
active functional group at the opposite terminus site when the 
deprotection reaction is performed. 
The Fmoc group (9-fluorenylmethyloxycarbonyl) and the Msc group 
(methylsulphonylethyloxycarbonyl) (shown below) are suitable amino 
protection groups because they are stable to the deprotection reaction of 
the opposite terminus site of the linker performed under acidic conditions 
and are deleted under basic conditions. These were developed respectively 
by Carpino et al., (L. A. Carpino and G. Y. Han, J. Am. Chem. Soc., 92, 
5478 (1970) and J. Org. Chem., 37, 3404 (1972)) and by Tesser et al., (G. 
I. Tesser and I. C. Balvert-Geers, Int. J. Peptide Protein Res., 7, 295 
(1975)). 
##STR9## 
Both amino protection groups can be deleted in a short time, undergoing a 
.beta.-elimination reaction by an alkaline treatment. Therefore a suitable 
linker for the present invention can be preferably constructed based on 
these amino protection groups. The Msc group by Tesser et. al., is more 
preferred because of the ease of the synthesis and the length of its 
molecular chain. A preferred linker compound is represented by the 
structural formula (III) shown below. 
##STR10## 
wherein n is an integer of one to four, X is one of 
##STR11## 
Y is one of 
##STR12## 
The linker comprises the Msc group as its basic structure, active alkyl 
aryl carbonate at one terminus site which can be coupled to the N-terminal 
amino group of the peptide, and a precursor of a thiol group (SH group) as 
an active functional group at the opposite terminus (S is in the state of 
being protected by a protection group). One terminus site of the linker 
forms a bond with the N-terminus of the peptide. The bond is stable to any 
kind of acid used as a deprotection reagent, and is easily cleaved by an 
alkaline reagent causing a .beta.-elimination reaction, such a 0.2N NaOH 
(50 w/w % in methanol, 5 sec.) or 5 w/w % in NH.sub.4 OH (50 w/w % in 
methanol, 5 min.). The SH group at the opposite terminus site of the 
linker reacts with an iodoacetic acid derivative, permitting the formation 
of a stable thioether type covalent bond under neutral or basic 
conditions. 
The synthetic pathway to the linker represented by structural formula (III) 
is not subject to any limitation. For example, the linker is preferably 
synthesized from the intermediate represented by the structural formula 
(I) involving the Msc group as a basic structure. 
##STR13## 
wherein n is an integer of one to four; X is one of H, hydrochloride 
thereof (H.HCl), 
##STR14## 
The hydroxyl group at one end of the intermediate represented by structural 
formula (I) can easily be converted to an active carbonate which can 
couple with an amino group of a peptide or protein. An amino group or an 
amino group as shown in Structural Formula I by a suitable protection 
group at the other terminus of the intermediate (I) can easily couple with 
the compound having another functional group, e.g. compound having a 
protected SH group. 
An intermediate represented by structural formula (II) is a compound which 
may be obtained by introducing a thiol group protected with a protection 
group to the other terminal amino group of the intermediate represented by 
structural formula (I). 
##STR15## 
wherein n is an integer of one to four, m is 1 or 2, X is one of 
##STR16## 
When the linker as mentioned above is added, as explained in FIG. 1, the 
linker will only couple with the mature target peptide, since only the 
mature peptide has the N-terminal amino group. Therefore, the active group 
included in the linker such as the SH group is introduced in the form of 
precursor to only the mature peptide at the other terminus thereof. 
According to the conventional method, the deprotection reaction is effected 
under acidic conditions. The mature peptide coupled to the linker and the 
immature peptides end-capped, for example by an acetyl group, are cleaved 
from the solid-phase support. The linker is maintained stably bound to the 
N-terminus of the mature peptide in the deprotection reaction as explained 
before. When the compound represented by structural formula (III) is used 
as the linker, the p-methoxybenzyl group which is the protection group for 
the SH group of the linker is removed by the deprotecting reagent to allow 
the SH group to be exposed at the terminus. The ligand is then allowed to 
contact with the mixture of mature and immature peptides. The ligand is 
modified by a compound which has another functional group forming a stable 
bond with the functional group at the opposite terminus site of the linker 
immobilized thereon. In the case where SH is the functional group, the 
ligand which contains an immobilized iodo-substituted aliphatic carboxylic 
acid or bromo-substituted aliphatic carboxylic acid on its surface is 
used. Especially preferable is an iodo-substituted aliphatic carboxylic 
acid derivative shown by the following general formula: 
##STR17## 
wherein R is H or an alkyl group of C.sub.1 to C.sub.6, n is an integer of 
one to six; 
or a bromo-substituted aliphatic carboxylic acid derivative shown by the 
following general formula: 
##STR18## 
wherein R is H or an alkyl group of C.sub.1 to C.sub.6, n is an integer of 
one to six. 
Derivatives of iodoacetic acid (I--CH.sub.2 COOH) or bromoacetic acid 
(BrCH.sub.2 COOH) are more preferred. 
Specifically, a ligand having one of the structural formulae below is 
contacted with the peptide mixture under near neutral conditions: 
##STR19## 
wherein R is H or an alkyl group of C.sub.1 to C.sub.6, n is 1 or 2 and m 
is 2 to 6. 
Preferably the reaction is allowed to proceed at around pH=7 to 9 and in an 
inert buffer so as not to disturb the expected reaction. More preferably, 
a denaturing agent such as guanidine hydrochloride or urea is added in 
order to increase the solubility of the peptides. 
As an example, 0.4M phosphate buffer or 0.4M tris 
(hydroxymethyl)aminomethane hydrochloride buffer (abbreviated as 
trishydrochloride buffer) is used. Guanidine hydrochloride of 4-7M, 
preferably ca.6M guanidine hydrochloride, or urea of 4-8M, preferably 
ca.6M urea is used as a denaturing agent. Other buffers and denaturing 
agents can be used on a case-by-case basis. 
A solid-phase support, especially a hydrophilic solid-phase support, is the 
preferred ligand to which a compound having functional groups is 
immobilized. Kieselguhr ligand (solid-phase support beads) to which a 
suitable hydrophilic solid-phase support is absorbed so that it is usable 
in an aqueous system is shown as an example. 
By the above-process, the selective immobilization of the mature peptide to 
the ligand can be attained. The ligand is washed to remove immature 
peptides which do not bond to the ligand to remove scavengers used for 
deprotection. The mature peptide is finally cleaved from the linker-bound 
ligand under conditions to selectively cleave the bond between the linker 
and the mature peptide. A benzyl group bonded to either oxygen of sulfur 
is needed in order for cleavage to occur at either site. For example, the 
mature peptide is cleaved from the linker-bound ligand under a basic 
condition, if the linker is a compound shown by structural formula (III). 
If 5% ammonium solution is used for this basic treatment, some peptides 
which are not dissolved in 5% NH.sub.4 OH may remain as precipitates in 
the column. If the 50% ammonium treatment is followed by an additional 
washing process with 50% acetic acid, the precipitates can be eluted from 
the column and recovered effectively. The washing of the ligand with 
acetic acid following the base treatment is also preferable from the 
viewpoint that ammonia is neutralized and may be sublimed by 
freeze-drying. The compound finally obtained is the mature peptide alone. 
FIG. 2 is a flow-chart showing one embodiment of the peptide purification 
method of the present invention. In the figure the mature target peptide 
is shown as A-B-C-D-E-F-G, and A-B-C, A-B-C-D, A-B-C-D-E, and A-B-C-D-E-F 
are impurities. Step 1 of FIG. 2 shows the targeted mature peptide mixed 
with many impurities. Both the mature peptide having an N-terminal amino 
group and immature peptides (impurities) having an acetyl-group modified 
terminus (produced by end-capping with acetic anhydride) are bound to the 
solid-phase support (solid-phase support beads). The linker of the present 
invention is added to the solid-phase support, and the linker selectively 
bonds to the terminus of the mature peptide A-B-C-D-E-F-G alone. The final 
deprotection treatment in the peptide synthesis method cleaves the target 
peptide and immature peptides from the solid-phase support, as shown in 
step 2. This treatment allows the formation of an active functional group 
(SH group) at the other end of the linker bound to the target peptide. In 
step 3 the mixture of the mature peptide and immature peptides is 
contacted with a ligand of this invention packed in a column that can bond 
to the linker. The ligand bonds to the mature peptide alone via the 
linker, and immature peptides and other reagents are eluted from the 
column. Step 4 involves exposing the column to basic condition under which 
the urethane bond between the linker and the mature peptide is selectively 
cleaved. This results in the cleavage of the mature peptide from the 
linker-bound ligand and the elution of the mature peptide from the column. 
EXAMPLES 
The present invention is described in more detail by the following 
non-limiting examples. However, it will be understood that the present 
invention is not limited to such a few disclosed examples. 
Example I 
Synthesis of the Linker (A) 
This example describes a method for synthesizing linker, where the 
synthesis is performed according to steps shown in FIG. 3. 
1-1 Synthesis of the Compound (I) 
Compound (I): 2-[2-(N-Phthaloyl)ethylthio]ethanol 
##STR20## 
N-(2-Bromoethyl)phthalimide (50.8 g, 0.2 mol) and .beta.-mercaptoethanol 
(14.3 ml, 0.2 mol) were dissolved in dimethylformamide (400 ml). After the 
further addition of dicyclohexylamine (39.9 ml, 0.2 mol) under cooling by 
ice, the solution was stirred for 4 hr. at room temperature. The produced 
salt (dicyclohexylamine hydrobromide) was removed by filtration. The 
filtrate was mixed with the washing solution that was obtained by the 
washing of the salt with a small amount of dimethylformamide. It was 
followed by the evaporation of the dimethylformamide under reduced 
pressure. The residue was dissolved in ethyl acetate and transferred to a 
separatory funnel. The ethyl acetate phase was washed with saturated brine 
three times. The ethyl acetate phase, being transferred to a Mayer's 
flask, was dried over anhydrous sodium sulfate. Subsequent to the removal 
of sodium sulfate by filtration, ethyl acetate was evaporated under 
reduced pressure to obtain 45.9 g of oily white compound (yield 91%). 
1-2 Synthesis of Compound (II) 
Compound (II): 2-[2-(4-Methoxybenzyloxycarbonylamino)-ethylthio]ethanol 
##STR21## 
Compound I (43.5 g, 0.17 mol) obtained by the above procedure was dissolved 
in methanol (250 ml). The solution was stirred for 16 hr. after the 
addition of hydrazine hydrate (9.3 ml, 0.18 mol). The produced crystals 
were removed by filtration. Methanol was evaporated under reduced 
pressure. The residue was crystallized by the addition of acetonitrile, 
and followed by recrystallization from methanol/acetonitrile to obtain 
45.9 g of white crystals (yield 86%). The crystals were dissolved in a 
mixture of water (100 ml) and triethylamine (23.6 ml). 
The crystals were added to an acetronitrile solution (100 ml) of 
4-methoxybenzyl azidoformate (34.7 g, 0.16 mol from Watanabe Chemical 
Kogyo Ltd.) under cooling by ice. The solution was then stirred for 10 hr. 
Subsequent to the evaporation of water/acetonitrile under reduced 
pressure, the oily residue was dissolved in ethyl acetate and transferred 
to a separatory funnel. The ethyl acetate phase was washed with 5% citric 
acid three times, followed by washing 3 times with saturated brine. After 
the ethyl acetate phase was transferred into a Mayer's flask, it was dried 
over anhydrous sodium sulfate. Subsequent to the removal of sodium sulfate 
by filtration, ethyl acetate was evaporated under reduced pressure. 
N-hexane was added to the residue to obtain crystals. The crystals were 
further recrystallized by ethyl acetate/n-hexane to obtain 37.12 g of 
white crystals (yield 93%). 
The Specifications of Compound (II) above were as follows: 
Rf.sub.1 =0.74; Melting point: 41-41.5.degree. C.; The theoretical values 
of C.sub.13 H.sub.19 NO.sub.4 S: C=54.72; H=6.71; N=4.91; The measured 
values: C=54.79; H=6.88; N=4.98; NMR (CDCl.sub.3): .delta.2.08(br.s, 1H); 
2.73-2.76 (m,4H), 3.39(q,J=6, 1 Hz, 2H), 3.73 (t, J=5.7 Hz, 2H), 3.80 
(s,3H), 5.04 (s, 2H), 5.12 (br. s, 1H); 6.88, 7.30 (AA' BB' pattern, 
J.sub.ortho =8.8). FAB Mass Spectroscopy: 286.1 (M+H.sup.+); (calculated 
on C.sub.13 H.sub.19 NO.sub.4 S: 285.1) 
1-3 Synthesis of Compound (III) 
Compound III: 4-Methoxybenzyl 2-(2-hydroxyethylsulfonyl ethylcarbamate 
aminoethylsulphonylethanol 
##STR22## 
Compound II (18.2 g, 65 mmol) obtained by the above procedure was dissolved 
in water (100 ml) and methanol (100 ml). After the addition of sodium 
tungstenate (65 mg), hydrogen peroxide (16.3 ml, 114 ml/mol) was added 
dropwise to the solution while stirring. This was an exothermic reaction 
and the reaction temperature was controlled to 60.degree. C. After the 
two-hour stirring, additional stirring was made with the addition of 5% 
palladium/carbon (0.5 g) until the foaming due to decomposition of excess 
hydrogen peroxide had ceased. 5% palladium/carbon was removed by 
filtration, and the solvent was evaporated under reduced pressure. The 
oily residue was dissolved in ethyl acetate and transferred to a 
separatory funnel. The ethyl acetate phase was washed with 5% citric acid 
three times, 5% sodium bicarbonate one time and saturated brine three 
times. After the ethyl acetate phase was transferred into a Mayer's flask, 
it was dried over anhydrous sodium sulfate. Subsequent to the removal of 
sodium sulfate by filtration, ethyl acetate was evaporated under reduced 
pressure. Diethyl ether was added to the oily residue to obtain crystals. 
The crystals were further recrystallized by ethyl acetate/diethyl ether to 
obtain 16.7 g of white crystals (yield 81%). 
The specifications of Compound (III) obtained by the above were as follows: 
RF.sub.1 =0.52; Melting point 65-66.degree. C.; The theoretical values of 
C.sub.12 H.sub.19 NO.sub.6 S: C=59.20; H=6.03; N=4.41; The measured 
values: C=59.03; H=5.97; N=4.41; NMR(CDCl.sub.3): .delta.2.09(br.s.1h), 
3.20 (t, J=5.4 Hz, 2H). 3.34(t, J=5.9 Hz, 2H). 3.72 (q, J=5.9 Hz, 2H), 
3.80 (s, 3H), 4.08 (t, J=5.4 Hz, 2H), 5.04 (s, 2H). 5.46 (br. t like, 1H), 
6.88, 7.29 (AA' BB' pattern, J.sub.ortho =8.8). FAB Mass Spectroscopy: 
340.1 (M+Na.sup.+); (calculated on C.sub.13 H.sub.19 NO.sub.6 SNa: 340.1) 
1-4: Synthesis of Compound IV 
Compound IV: Succinimidyl 4-methoxybenzylthioglycolate 
##STR23## 
S-(p-methoxybenzyl)thioglycolic acid (1.49 g, 7 mmol) and 
1-hydroxysuccinimide (0.81 g, 7 mmol) were dissolved in tetrahydrofuran 
(10 ml). After the further addition of N,N'-dicyclohexylcarbodiimide (1.59 
g, 7.7 mmol) with ice cooling, the solution was stirred for 5 hr. The 
resultant N,N'-dicyclohexylurea was separated by filtration, and 
tetrahydrofuran was evaporated under reduced pressure. Isopropyl alcohol 
was added to the residue to obtain crystals. The crystals were further 
recrystallized from tetrahydrofuran/iso-propyl alcohol to obtain 1.25 g of 
white crystals (yield 58%). 
The specifications of compound (IV) obtained by the above were as follows: 
Rf.sub.2 =0.86; Melting point 88-89.degree. C.; The theoretical values of 
C.sub.14 H.sub.15 NO.sub.5 S: C=54.36; H=4.88; N=4.53; The measured 
values: C=54.07; H=4.85; N=4.48; FAB Mass Spectroscopy: 332.1 
(M+Na.sup.+); (calculated on C.sub.14 H.sub.15 NO.sub.5 SNa: 332.1) 
1-5: Synthesis of Compound V 
Compound V: 
N-[2-(2-Hydroxyethylsulfonyl)ethyl]-4-methoxybenzylthioacetamide 
##STR24## 
Compound III mentioned above (5.0 g, 15.8 mmol) was treated with 
trifluoracetic acid (20 ml) for 60 min. under the presence of anisole (5 
ml) with ice cooling. Trifluoracetic acid was evaporated under reduced 
pressure. The residue was dissolve in diethyl ether to produce an oily 
residue. The oily residue dissolved in dimethylformamide (50 ml) with ice 
cooling was stirred for 12 hr. at room temperature after the addition of 
triethylamine (4.5 ml). N-hydroxybenzotriazole (2.42 g) and Compound IV 
(5.86 g, 19.0 mmol). Dimethylformamide was evaporated under reduced 
pressure to obtain an oily residue. The obtained oily residue was 
dissolved in ethyl acetate, and transferred to a separatory funnel. The 
ethyl acetate phase was washed with 5% citric acid 3 times, 5% sodium 
bicarbonate 3 times, and saturated brine. The ethyl acetate phase was 
transferred into a Mayer's flask and dried over anhydrous sodium sulfate. 
The removal of anhydrous sodium sulfate by filtration was followed by the 
evaporation of ethyl acetate under reduced pressure. The obtained oily 
residue was crystallized by the addition of diethyl ether, and the 
obtained crystals were recrystallized from ethyl acetate/diethyl ether to 
obtain 4.31 g of white crystals (yield 78%). 
The specifications of compound (V) obtained by the above were as follows: 
Rf.sub.1 =0.61; Melting point 73-75.degree. C.; The theoretical values of 
C.sub.14 H.sub.21 NO.sub.5 S.sub.2 : C=48.40; H=6.09; N=4.03; The measured 
values: C=48.60; H=6.29; N=4.11; NMR (CDCl.sub.3): .delta.2.29 (br.s,1H), 
3.13 (s, 2H), 3.31-3.23 (m, 4H), 3.71 (s,2H), 3.72 (q, J=6.0 Hz, 2H), 3.80 
(s, 3H), 4.13 (t, J=5.2 Hz, 2H), 6.86, 7.22 (AA' BB' Pattern, J.sub.ortho 
=8.7); 7.31 (br. t like, 1H). FAB Mass Spectroscopy: 348.1 (M+H.sup.+); 
(calculated on C.sub.14 H.sub.21 NO.sub.5 S.sub.2 : 347.1) 
1-6: Synthesis of Compound VI (linker) 
Compound VI: 2-[2-(4-Methyoxybenzylthiomethylcarbonyl 
amino)ethylsulfonyl]ethyl 4-nitrophenyl carbonate 
##STR25## 
The compound V mentioned before (4.00 g, 11.5 mmol) was dissolved in 
anhydrous pyridine (30 ml). After the addition of para-Nitrophenyl 
chloroformate (2.32 g, 11.5 mmol) under ice cooling, the solution was 
stirred for 4 hr. Pyridine was evaporated under reduced pressure. The 
produced oily residue was crystallized by the addition of 1N hydrochloric 
acid (50 ml) and diethyl ether. The obtained crystals are recrystallized 
from hydrochloric acid/diethyl ether to obtain 4.07 g of white crystals 
(yield 69%). 
The specifications of compound (VI) obtained by the above were as follows: 
Rf.sub.2 =0.37; Melting point 81-82.degree. C.; The theoretical values of 
C.sub.21 H.sub.24 N.sub.2 O.sub.9 S.sub.2 : C=49.21; H=4.72; N=5.47; The 
measured values: C=49.30; H=4.72; N=5.25; NMR (CDCl.sub.3): .delta.3.13 
(s, 2H), 3.28 (t, J=6.0 Hz, 2H), 3.47 (t, J=5, 7 Hz, 2H), 3.75 (q, J=6.0 
Hz, 2H), 3.79 (s, 3H), 4.74 (t, J=5.7 Hz, 2H), 6.84, 7.20 (AA' BB' 
pattern, J.sub.ortho =8.7); 7.25 (m, 1H), 7.40, 8.28 (AA' BB' pattern, 
J.sub.ortho =9.3). FAB Mass Spectroscopy: 513.2 (M+H.sup.+); (calculated 
on C.sub.21 H.sub.24 N.sub.2 O.sub.9 S.sub.2 : 512.1) 
Example II 
Synthesis of the Linker (B) 
This example describes another synthesis of the linker, according to steps 
shown in FIG. 4. 
2-1 Synthesis of the compound (VII) 
Compound (VII): 4-Methoxybenzyl 2-bromoethylcarbamate 
##STR26## 
Hydrobromide of 2-bromoethylamine (100.0 g, 0.49 mol) was dissolved in 
tetrahydrofuran (THF 500 ml). After the further addition of 
para-methoxybenzylazido formate (111.2 g, 0.54 mol) and triethylamine 
(148.9 ml, 1.07 mol) under ice cooling, the solution was stirred for 10 
hr. The produced salt (triethylamine hydrobromide) was removed by 
filtration. The filtrate was mixed with the washing solution that was 
obtained by the washing of the salt with a small amount of THF. The 
evaporation of THF under reduced pressure was followed. The residue was 
dissolved in ethyl acetate and transferred to a separatory funnel. Ethyl 
acetate phase was washed with 5% citric acid three times and with 
saturated brine three times. The ethyl acetate phase, being transferred to 
a Mayer's flask, was dried over anhydrous sodium sulfate. Subsequent to 
the removal of anhydrous sodium sulfate by filtration, ethyl acetate was 
evaporated under reduced pressure. The residue was crystallized by the 
addition of n-hexane followed by recrystallization from ethyl 
acetate/n-hexane to obtain 102.5 g of white crystals (yield 73%). 
The specifications of Compound (VII) above were as follows: 
Rf.sub.4 =0.3; Melting point 44.5-45.5.degree. C.; The theoretical values 
of C.sub.11 H.sub.14 NO.sub.3 Br: C=45.85; H=4.90; N=4.86; The measured 
values: C=45.59; H=4.84; N=4.80 
2-2 Synthesis of Compound (II) 
Compound VII (100 g, 0.35 mol) obtained by the above method and 
.beta.-mercaptoethanol (25.0 ml, 0.35 mol) were dissolved in 
dimethylformamide (DMF, 700 ml). After the further addition of 
dicyclohexylamine (70 ml, 0.35 mol) under ice cooling, the solution was 
stirred for 4 hr. at room temperature. The produced salt 
(dicyclohexylamine hydrobromide) was removed by filtration. The filtrate 
was mixed with washing solution obtained by the washing of the salt with a 
small amount of DMF. DMF was evaporated under reduced pressure. The 
residue dissolved in ethyl acetate, was transferred to a separatory 
funnel. The ethyl acetate phase was washed with 5% citric acid three 
times, followed by saturated brine three times. The ethyl acetate phase 
was transferred to a Mayer's flask, and subsequently ethyl acetate was 
dried over anhydrous sodium sulfate. Subsequent to the removal of 
anhydrous sodium sulfate by filtration, ethyl acetate was evaporated under 
reduced pressure. N-hexane was added to the residue for crystallization. 
The obtained crystals were further recrystallized from ethyl 
acetate/n-hexane to obtain 90.5 g of white crystals (yield 91%). The 
melting point of compound II obtained was 41-41.5.degree. C. 
2-3 Synthesis of Compound (VI) (linker) 
Compound (III) was produced from Compound II, obtained as above, according 
to the procedure described in 1-3. Compound V was synthesized according to 
the procedure described in 1-5 and compound VI (linker) was produced 
according to the procedure in 1-6. 
Example III 
Synthesis of the Linker (C) 
This example describes another method for the synthesis of the linker, 
according to steps shown in FIG. 5. 
3-1 Synthesis of the Compound (VIII) 
Compound (VIII): t-Butyl 2-bromoethylcarbamate 
##STR27## 
2-Bromoethylamine hydrobromide (10 g, 49 mmol) was dissolved in 
dimethylformamide (DMF 100 ml). After the further addition of 
di-t-butyldicarbonate ((Boc).sub.2 0) (11.8 g, 54 mmol, 1.1 equivalent) 
and triethylamine (10.3 ml, 74 mmol, 1.5 equivalent) under ice cooling, 
the solution was stirred for 2 hr. at room temperature. The produced salt 
(triethylamine hydrobromide) was removed by filtration. The filtrate was 
mixed with the solution obtained by washing the salt with a small amount 
of DMF. Rf.sub.2 of this compound is 0.90. 
3.2 Synthesis of Compound IX 
Compound (IX): t-Butyl 2-(2-hydroxyethylthio)ethyl carbamate 
##STR28## 
DMF solution (filtrate and washing solution) of Compound VIII (49 mmol) 
obtained above was mixed with .beta.-mercaptoethanol (3.5 ml). After the 
further addition of dicyclohexylamine (9.8 ml, 49 mmol) under ice cooling 
the solution was stirred for 20 hr. at room temperature. The produced salt 
(dicyclohexylamine hydrobromide) was removed by filtration. The filtrate 
was mixed with the solution obtained by the washing of the salt with DMF. 
DMF was evaporated under reduced pressure. The residue was dissolved in 
ethyl acetate, and the ethyl acetate solution was washed with 5% citric 
acid three times and with saturated brine three times. The washed ethyl 
acetate solution was dried over anhydrous sodium sulfate. Subsequent to 
the removal of anhydrous sodium sulfate by filtration, ethyl acetate was 
evaporated under reduced pressure to obtain a transparent oil compound 
(Compound IX). 
3-3 Synthesis of Compound (X) 
Compound X: 2-(2-Hydroxyethylsulfonyl)ethylamine hydrochloride 
##STR29## 
Compound (IX) (49 mmol) obtained by the procedure described in 3-2 was 
dissolved in methanol (85 ml). Water (40 ml) followed by sodium tungstate 
(49 mg) were added, and aqueous hydrogen peroxide (11.3 ml) was added 
dropwise with stirring of the solution. This was an exothermic reaction 
and the reaction temperature was controlled to remain below 60.degree. C. 
After stirring for one-hour and a half 5% paladium/carbon (0.38 g) was 
added with additional stirring to decompose the excess hydrogen peroxide. 
The stirring was continued until the foaming had ceased. 5% 
paladium/carbon was removed by filtration, and the filtrate was evaporated 
under reduced pressure. The oily residue, dissolved in ethyl acetate, was 
washed with 5% citric acid three times. 5% sodium hydrogencarbonate one 
time and saturated brine three times. After the ethyl acetate phase was 
transferred to a Mayer's flask, it was dried over anhydrous sodium 
sulfate. Subsequent to the removal of anhydrous sodium sulfate by 
filtration, ethyl acetate was evaporated under reduced pressure 11.79 g of 
oily compound (compound X) was obtained. 
3-4: Synthesis of Compound XI 
##STR30## 
Compound XI: 2-(2-Hydroxyethylsulfonyl)ethylamine hydrochloride 
Compound X (49 mmol) obtained by the procedure described above in 3-3 was 
dissolved in 4N-HCl/dioxane (40 ml) and stirred for 30-min. The obtained 
crystals were collected by filtration and washed with ether. The crystals 
were subsequently dried under vacuum. 6.6 g (35 mmol) of crystals 
(Compound XI) were obtained, and the yield was 71%. 
Rf.sub.5 =0.45; Melting point 255.degree. C.; The theoretical values of 
C.sub.4 H.sub.12 NO.sub.3 SCl.sub.12 : C=25.33; H=6.38; N=7.39; The 
measured values: C=25.24; H=6.50; N=7.43 
3-5: Synthesis of Compound V 
Compound V was synthesized from Compound XI, produced by the above method, 
according to the procedure described in 1-5. 
3-6: Synthesis of Compound VI (linker) 
Compound VI (linker) was synthesized from compound V mentioned above 
according to the procedure described in 1-6. 
Example 4 
Synthesis of Resin for Bonding the Linker Synthesis of Iodoacetic Acid 
Resin 
Methylene chloride solution (50 ml) of iodoacetic acid (7.4 g, 40 mmol), 
where dicyclohexylcarbodiimide (5 g, 0.6 eq.) was added, was stirred for 
30 min. under ice cooling. The produced precipitate (dicyclohexylurea) was 
removed by filtration. The filtrate was treated with ethylenediamine. The 
treated filtrate was then added to PepSyn K resin* whose terminal 
aminoethyl group was attached (20 g, amino group content: 0.2 mmol/g), and 
shaken until a Kaiser test gave a negative result (2 hr). The resin was 
washed well with dimethylformamide and methylene chloride, and dried under 
vacuum. 
FNT *produced by Millipore Co., Ltd., Kieselguhr type resin: copolymer of 
dimethylacrylamide, bis acroylethylenediamine and acryloylsarcosine 
methylester was supported by Kieselguhr. 
Example 5 
Purification of Synthesized Pentide (1) 
Polyphemusin II was synthesized and purified in order to clarify the 
availability of this purification of the present invention. Polyphemusin 
II was synthesized by Fmoc-based solid-phase synthesis according to the 
procedure shown in FIG. 1, and purified. Polyphemusin II is a 18-residue 
peptide with C-terminus amide and two disulfide bonds. For the synthesis, 
cys derivative whose thiol group was protected with Acm (acetamidomethyl) 
was used. 
H-Arg-Arg-Trp-Cys(Acm)-Phe-Arg-Val-Cys(Acm)-Try-Lys-Gly-Phe-Cys(Acm)-Tyr-Ar 
g-Lys-Cys(Acm)-Arg-NH.sub.2 
At the final step of the synthesis, linker prepared according to Example 1 
(512 mg, 5 equivalents) and N-Hydroxybenzotriazole (152 mg, 5 equivalents) 
were added to the solid phase resin (0.2 mmol), and then stirred in 
dimethylformamide (10 ml) for 2 hr. in order to attach the linker to the 
N-terminus of Polyphemusin synthesized on the resin. Subsequently the 
deprotection of the solid phase resin was carried out at 0.degree. C. for 
2 hr. with a solution (10 ml) of 1M trimethylsilyl bromide (1.33 ml) 
(TMSBr), 1 m thioanisole (1.2 ml)/trifluoroacetic acid (6.97 ml) (TFA) in 
the presence of m-cresol (0.5 ml) [N. Fujii, A. Otaka, N. Sugiyama, M. 
Hatano, and H. Yajima, Chem. Pharm. Bull., 35, 3880]. TMSBr and TFA were 
evaporated off, and diethyl ether was added to obtain the peptide as a 
powder. The obtained powder was dissolved in 0.4M Tris hydrochloric acid 
buffer (pH7.5) containing 6M guanidine hydrochloride. Subsequently the 
solution was introduced onto a column packed with 1 equivalent of 
iodoacetic acid resin prepared by the procedure described in Example 4., 
and it was recirculated for 2 hr. to allow reaction. This was then treated 
with 1 equivalent of 2-mercaptoethanol for 2 hr. to inactivate iodoacetyl 
groups on the resin. The inactivated iodoacetic acid resin was washed well 
with 0.4M tris hydrochloric acid buffer (pH7.5) containing 6M guanidine 
hydrochloride, 50% acetic acid, and water sequentially. 
The obtained peptide-bound resin was treated with 5% ammonium hydroxide 
solution for 30 min. to cleave the peptide from the resin. The resin was 
further washed with 50% acetic acid and the acetic acid washings were 
combined with the solution obtained from the ammonium hydroxide cleavage 
step. The obtained liquid was freeze-dried to obtain 14.2 mg of the 
peptide powder. Analysis by HPLC showed a single peak (purity: &gt;99%). 
The HPLC analysis of crude Polyphemusin II just after the solid-phase 
synthesis was approximately 69%, and a shoulder peak before the main peak 
was observed. However, the purified peptide obtained by the method of the 
present invention had a single peak. The results of amino acid sequence 
analysis and FAB mass spectroscopy of the purified peptide agreed with the 
theoretical [Acm-Polyphemusin II:2714.1 (M+H.sup.+), calculated on 
C.sub.120 H.sub.185 N.sub.41 O.sub.24 S.sub.4 ]. 
Example 6 
Purification of Synthesized Peptide (2) 
The present invention was used to synthesize human cholecystokinin, a small 
sized protein in order to further exemplify the feasibility of the 
purification procedure. 
Human cholecystokinin was synthesized by Fmoc-based solid-phase synthesis 
according to the procedure shown in FIG. 1, and purified. Human 
cholecystokinin is a 33-residue peptide. 
H-Lys-Ala-Pro-Ser-Gly-Arg-Met-Ser-Ile-Val-Lys-Asn-Leu-Gln-Asn-Leu-Asp-Pro-S 
er-His-Arg-Ile-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2 
At the final step of the synthesis, the linker prepared according to 
Example 1 (512 mg, 5 equivalents) and N-Hydroxybenzotriazole (152 mg, 5 
equivalents) were added to the solid phase resin (0.2 mmol), and 
subsequently stirred in dimethylformamide (10 ml) for 2 hr. in order to 
attach the linker to the N-terminus of human Cholecystokinin which was 
synthesized on the resin. Deprotection was carried out at 0.degree. C. for 
2 hr. by 1M TMSBr-thioanisole/trifluoroacetic acid (TFA) (0.5 ml) in the 
presence of ethanediol (0.2 ml) and m-cresol (0.5 ml). Following the 
deprotection, TMSBr and TFA were evaporated off, and diethylether was 
added to obtain the peptide as a powder. The obtained powder was dissolved 
in 0.4M Tris hydrochloric acid buffer (pH7.5) containing 6M guanidine 
hydrochloride. Subsequently the powder was introduced onto a column, where 
1 equivalent of iodoacetic resin prepared by the procedure in Example 4 is 
packed, to allow recirculation for 2 hr. By the treatment with 1 
equivalent of 2-mercaptoethanol of the resin for 2 hr., excess iodoacetic 
acid was deactivated. After removal of the solution from the column, the 
resin was washed well with 0.4M tris hydrochloric acid buffer (pH7.5) 
containing 6M guanidine hydrochloride, 50% acetic acid, and water 
sequentially. 
The obtained peptide-bound resin (ligand) was treated with 5% ammonium 
hydroxide solution for 30 min. to cleave the peptide from the resin. After 
the further washing of the resin with 50% acetic acid, the wash solution 
was added to the above reagent for cleaving. The obtained liquid was 
freeze-dried to obtain 7.9 mg of the peptide powder. Analysis by HPLC 
showed a single peak (purity: more than 90%). The purity of human 
cholecystokinin which was produced in its sulfate free form was 29%, when 
calculated from crude peptide just after synthesis by the Fmoc method. The 
retention time by HPLC of non-sulphuric acid cholecystokinin purified by 
the method of this invention was identical to that purified by HPLC. The 
results of amino acid sequence analysis and FAB mass spectroscopy of the 
purified peptide agreed with the theoretical [:3865.2 (M+H.sup.+), 
calculated on C.sub.167 H.sub.263 N.sub.51 O.sub.49 S.sub.3 ]. 
Example 7 
Purification of Synthesized Peptide (3) 
The present invention was employed for the synthesis of another small sized 
protein, human Growth Hormone Releasing Factor (hGRF), in order to clarify 
the availability. hGRF was synthesized by Fmoc-based solid-phase synthesis 
according to the procedure shown in FIG. 1, and purified. hGRF is a 
44-residue peptide. 
H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-A 
la-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu- 
Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH.sub.2 
At the final step of the synthesis, the linker prepared according to 
Example 1 (512 mg, 5 equivalents) and N-hydroxybenzotriazole (152 mg, 5 
equivalents) were added to the solid phase resin (0.2 mmol), and 
subsequently stirred in dimethylformamide (10 ml) for 2 hr. in order to 
attach the linker to the N-terminus of hGRF synthesized on the resin. 
Deprotection was carried out at 0.degree. C. for 2 hr. with 1M 
TMSBr-thioanisole/trifluoracetic acid (TFA) (10 ml) in the presence of 
ethanediol (0.2 ml) and m-cresol (0.5 ml). Following the deprotection, 
TMSBr and TFA were evaporated off, and diethylether was added to obtain 
the peptide as a powder. The obtained powder was dissolved in 0.4M Tris 
hydrochloric acid buffer (pH7.5) containing 6M guanidine hydrochloride. 
Subsequently the powder was introduced onto a column, where 1 equivalent 
of iodoacetic acid resin prepared in Example 4 was packed, to allow 
recirculation for 2 hr. By the treatment with 1 equivalent of 
2-mercaptoethanol of the resin for 2 hr., excess iodoacetic acid resin was 
endcapped. After the removal of the solution from the column, the resin 
was washed well with 6M 0.5), 5.0% acetic acid, and water sequentially. 
The obtained peptide-bound resin (ligand) was treated with 5% ammonium 
solution for 30 min. to cleave the peptide from the resin. After the 
further washing of the resin with 50% acetic acid, the wash solution was 
added to with reagent for cleaving. The obtained liquid was freeze-dried 
and 7.9 mg of the peptide powder was obtained. Analysis by HPLC showed 
almost a single peak (purity: more than 80%). The purity of hGRF was 51%, 
when calculated from crude peptide just after synthesis by the Fmoc 
method. The retention time by HPLC of hGRF purified by the method of this 
invention was identical to that purified by HPLC. 
The results of amino acid sequence analysis and FAB mass spectroscopy of 
the purified peptide agreed with the theoretical [5037.8 (M+H.sup.+), 
calculated on C.sub.215 H.sub.358 N.sub.72 O.sub.66 S]. 
Instruments and reagents used for assay of the above examples are as 
follows: 
Thin Layer Chromatography 
Sorbent: silica gel G (Kiesel gel G, E. Merck, Germany) 
Solvents: 
Rf.sub.1 : Lower phase of CHCl.sub.3 --MeOH--H.sub.2 O (8:3:1) 
Rf.sub.2 : CHCl.sub.3 --MeOH (10:0.5) 
Rf.sub.3 : CHCl.sub.3 --AcOH (19:1) 
Rf.sub.4 : CHCl.sub.3 --Cyclohexane (2:1) 
Rf.sub.5 : n-BuOH--AcOH-Pyridine-H.sub.2 O (4:1:1:2) 
Nuclear Magnetic Resonance 
Bruker AC-300 spectrometer (Bruker Co., Ltd.) Inner standard: 
tetramethylsilane 
FAB Mass spectrocopy 
ZAB SE Mass spectrometer (U.G. Analytical) 
Purity Analysis of Peptide 
1) High Performance Liquid Chromatograph (HPLC) HPLC system Model 600E 
(Millipore Corp.) .mu.-Bondasphere 5C18 column (Millipore Corp.) with 
solvent system of water (0.1% trifluoroacetic acid)-acetonitrile 
2) Amino acid sequence of peptides Peptide sequencer Model 431A (Applied 
Biosystems Co.) and protein sequencer Model 6600 (Millipore Corp.) 
Synthesis of Model Peptides Fmoc peptide synthesis by automated peptide 
synthesizer Model 9050 (Millipore Corp.) 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 3 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Arg Arg Trp Cys Phe Arg Val Cys Tyr Lys Gl - #y Phe Cys Tyr Arg 
Lys 
1 5 - # 10 - # 15 
- - Cys Arg 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Lys Ala Pro Ser Gly Arg Met Ser Ile Val Ly - #s Asn Leu Gln Asn Leu 
1 5 - # 10 - # 15 
- - Asp Pro Ser His Arg Ile Ser Asp Arg Asp Ty - #r Met Gly Trp Met Asp 
20 - # 25 - # 30 
- - Phe 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Ar - #g Lys Val Leu Gly Gln 
1 5 - # 10 - # 15 
- - Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Me - #t Ser Arg Gln Gln Gly 
20 - # 25 - # 30 
- - Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Ar - #g Leu 
35 - # 40 
__________________________________________________________________________