Title of Invention

METHOD FOR PRODUCTION OF PEPTIDES HAVING PROTECTED AMINES OF UNTARGETED SITES BY SPECIFICALLY CONJUGATING WITH PEG

Abstract

A method for preparing peptides having selectively protected amines of untargeted sites, comprising synthesizing the peptides by separately blocking amines of targeted sites of the peptides with either ivDde or Mtt and amines of untargeted sites of the peptides with Boc, and protecting Nα-amine of the peptides with Fmoc or Nsc, wherein the targeted sites are sites to be intended to conjugate with polyethyleneglycol(PEG) and the untargeted sites are sites to be intended to remain as free amine after conjugating targeted sites with PEG. Reference: Figure 1.

Full Text

Peptides having protected amines of untargeted sites, methods for production thereof and of specifically conjugated PEG peptides using the same Technical Field The present invention relates to methods of specifically conjugating at least one PEG(polyethylene glycol)s to amines of targeted sites in synthetic peptide with at least two branched amines, and further relates to synthetic peptides having selectively protected amines of untargeted sites and methods for the production thereof, and to method of specifically conjugating PEGs to targeted sites of the synthetic peptide and purifying by ion exchange chromatography. Background Art Technologies for conjugating PEG to peptides and proteins are based on Davis and Abuchowski study (Abuchowski A. et al., J. Biol. Chem., 252, 3571-3581, 1977; Abuchowski A. et al., J. Biol. Chem., 252, 3582-3586, 1977). PEG is a polymer which is hydrophilic, biocompatible and harmless .having the structure of H(OCH2CH2)nOH and it is known that in case of being conjugated to peptide and protein, it inhibits enzymatic metabolism via steric hindrance as the same way as sugar chain in glycoprotein does, decreases renal glomerular filtration with increased molecular size of peptide and protein having conjugated PEGs, thereby increasing the duration of physiological activity. Covalent bond between polypeptide and PEG was published in USP No. 4179337, and it was described that modification of proteins and enzymes with PEG leads to reduced immunogenicity and antigenicity and increased half life within blood. For covalent bonding of PEG to polypeptide, it is necessary that "activation" process of converting the terminal hydroxyl moiety of PEG to a reactive functional group. As an "activated PEG alkylating agents such as PEG aldehydes, PEG epoxides and PEG tresylates, and acylating agents such as PEG esters can be enumerated, and the representative example is PEG succinimidyl succinate. Poly(ethyleneglycol)-N-succinimide carbonate and producing method thereof was known from Abuchowski et al, Cancer Biochem. Biophys. 7:175-186(1984) and USP No. 512614 etc. PEGs in molecular range of 2,000-40,000 can be employed and PEGs such as -methoxylated PEGs and branched PEGs can be used. The branched PEG can be represented with the structure of R(-PEG-OH)m [herein, R defines central core moiety such as pentaerythritol or glycerol, m defines the number of branching arm in a range of 3-100]. Hydroxyl group can be used for chemical modification. Other branched PEG with the structure of (CH3O-PEG-)PR-X (W096/21469) is employed [herein, p is 2 to 3, R represents central core moiety such as lysine and glycerol, and X defines a functional group such as carboxyl group used for activation. Pendant PEG of another branched PEG has functional group such as carboxyl moiety on PEG backbone, other than terminus of PEG chain. All these branched PEGs can be used via "activation" as described previously. In the reaction of conjugating "activated" PEGs to peptide amines, generally, PEGs bond to one or more amines nonspecifically, thus the core of this technology lies in the method of specifically combining PEG molecules to amines at specific positions. However, in case of peptides containing at least one lysine within the amino acid sequence, as two or more amines exist therein, it is very difficult to specifically conjugate PEGs to amines at specific sites. In case PEGs are conjugated to peptide according to general methods, various conjugates can be generated, from a conjugate having one conjugated PEG to a conjugate having multiple-conjugated PEGs as many as the number of amines, and even in case of the conjugate having one PEG (a mono- PEG conjugate), positional isomers in which conjugation sites of PEG are different one another, can be formed. Usually, as PEG conjugates of peptide differ in activity and enzymatic metabolism according to the number of combined PEGs and combined sites thereof, the method producing such mixture containing various conjugates is disadvantageous for the reason of very low activity yield and being a mixture. To separate and purify specific isomer from the mixture is very complicated, accompanied by very low yield and high cost. To compensate such disadvantages, various methods for site-specific PEGylation have been proposed, however, efficient methods for specific conjugation of PEGs to amines on specific positions has not yet been developed. Chem. Pharm. Bull. 39(12):3373-3375(1991) reported on a conjugate of fibronectin-related tripeptide(Arg-Gly-Asp) and amino-poly(ethyleneglycol) and activity thereof. Herein, a method for preparing PEG conjugate by combining amino PEG to aspartic acid activated with dicyclohexylcarbodiiniide(DCC)/!- hydroxybenzotriazole(HOBt), was proposed. However, this method is defective in that it can be used only for modification of C-terminus and racemization of adjacent amino acids can occur during the C-terminal activation of peptide. Such racemization can occur in every amino acids except glycine, resulting in reduction of activity of peptide. Methods for combining monomethoxy poly(ethyleneglycol)(mPEG) or polyvinylalcohol(PVA) to peptide using synthetic resin were published in Journal of Protein Chemistiy 10(6):623-627(1991). Since only the conjugate in which a single polymer is combined to N-terminus of the peptide from the method, it fails to provide a method for combining PEG to positions that can maximize the effect of PEGylation (maximizing the duration of peptide activity and minimizing enzymatic metabolism). PCT patent application No. PCT/US94/06953(1994) provides methods for preparing site-specific PEG peptide in which PEG is introduced during synthetic process of the peptide. That is, it provides a method for preparing PEG-conjugated peptide in which peptide is partially synthesized until its sequence reaches lysine at targeted position, and then PEG is combined, and finally the remaining portion of the peptide is synthesized to. complete PEG peptide, a method for preparing PEGconjugated peptide by using, as the lysine of targeted site, lysine in which Na-amine thereof was protected with Fmoc(9-fluorenylmethoxycarbonyl) and" branched amine thereof was combined with PEG, and a method wherein peptide fragment having PEGs combined to the amines of targeted sitse and the remaining peptide fragment were separately synthesized and combined to complete the PEG-conjugated peptide. Nevertheless, such methods might bring undesirable result due to the effect of the previously combined PEG on subsequent step of the synthesis. For example, in case PEG is introduced during the peptide synthesis and synthesis of the remaining portion is to be continued, a peptide lacking at least one amino acid of the amino acid sequence might be produced, and such peptide can b harmful to a living body and its purification is almost impossible. Therefore, such method is not desirable as a method for preparing a PEG-conjugated peptide usable as a medicine. PCT patent application No. PCT/EP9S/0774S provides methods for combining PEG to amine of GRF in an organic solvent and purifying the resulting mixture of Lys(PEG)12-GRF, Lys(PEG)2I-GRF, Lys(PEG)12'21-GRF and [Na-PEG-Try1, Lys(PEG)12' 21]-GRF by gel chromatography and reverse phase chromatography. However, this method cannot provide specificity between amine moieties of Na, Lys12 and Lys21 . Said patent also provides methods for synthesis of [Lys(Alloc)12'2I ]- GRF(l-29) and [Na-isopropyl-Try1^ Lys(Alloc)12]-GRF(l-29) and specific PEGylation using them, though, in case of [Lys(Alloc)12'21 ]-GRF(l-29), PEG can be conjugated to only Na amine, and fails to provide a preparing method for Lys21 -PEG conjugate which is most effective as described in said patent. In addition, in case PEG conjugate is prepared by using [Na-isopropyl-Try', Lys(Alloc)12]-GRF(l-29), not PEG conjugate of GRF(l-29)- NH2,but PEG conjugate of [N" -isopropyl-Try1]-GRF(l-29)-NH2, i.e. [Na -isopropyl-Try1, Lys(PEG) 21]-GRF(l-29) is to be formed. Therefore, this method fails to provide completely specific PEGylation for the original peptide having Nterminal amine, GRF(l-29)- NH2. The inventors of the present invention intended to develop specific PEGylation by which product yield can be noticeably raised and efforts and cost needed for separation and purification of the final product can be reduced, by forming the final product in which PEGs are conjugated only to amines at targeted sites while preventing the formation of impurity product having PEGs conjugated to amines of untargeted sites. The present invention provides method for-preparing specific PEG-conjugated peptide in which the effect of PEG conjugation is maximized by allowing PEGs to be conjugated to amines at targeted sites only with complete selectivity. Disclosure of the Invention The present invention relates to synthetic peptides having selectively protected amines of untargeted sites, methods for production thereof, and methods for specifically conjugating PEGs to targeted sites of the synthetic peptide. In more detail, the present invention relates to (A) a method for preparing peptides having selectively protected amines of untargeted sites, comprising synthesizing the peptide by separately blocking amines of targeted sites and amings ofiintargeted sites with different protecting groups which are removed under different deblocking condition {e.g. ivDde[l-(4,4-dimethyl- 2,6-dioxocyclohexylidene)-3-methylbutyl] or Mtt(4-methyltrityl), and Boc(tertbuthoxycarbonyl)} and protecting Na -amine with Fmoc or Nsc, (B) peptides having selectively protected amines of untargeted sites prepared by said method and (C) methods for preparing specific PEG-conjugated peptide in which PEG are specifically conjugated to amines of targeted sites, comprising (1) reacting said peptide with activated PEGs and (2) removing the amine blocking groups of the compound obtained in said step (1) under acid-base deblocking conditions. The present invention further relates to methods for preparing peptides having selectively protected amines of untargeted sites, additionally comprising a step of substituting the protecting groups of the amines at untargeted sites, including Na -amine of the peptide synthesized as described above with final amine protecting groups. As the final amine protecting groups, at least one selected from the group consisting of Fmoc, Nsc, Dde, ivDde and other protecting groups removed under basic conditions, can be used, or at least one selected from the group consisting of Boc and other protecting groups removed under acidic conditions, can be used. One of general methods for synthesizing peptide is to repeat the reaction in which carboxyl moiety of C-terminal amino acid with protected Na -amine is combined to resin, deblocking of the amine is conducted under basic condition, and according to the sequence, next ammo acid with protected Na -amine is combined, thereby reaching N-terminal amino acid. In case lysine having branched amine is contained in the amino acid sequence, lysine in which the branched amine is protected with Boc removed under acidic condition and Na-amine is protected with Fmoc or Nsc[4- nitrophenylsulfonylethoxycarbonyj]., removed under basic condition is used, thereby allowing the branched amine not to undergo deblocking under the deblocking condition of Na -amine for combining 'subsequent amino acid. However, in case of peptide having at least two branched amines, as distinction between the branched amines is difficult, synthesis of completely selectively protected peptide was" difficult. In synthesizing peptides with at least two branched amines, the inventors of the present. invention synthesized selectively protected peptide in which the branched amines can be differentiated simply based on acid-base deblocking management by using Fmoc or Nsc as protecting groups of Na-amine and two kinds of amine protecting groups removed under different conditions, i.e. either Boc and ivDde or Mtt as protecting groups of the branched amines, and based on this, developed completely specific PEGylation, thereby completing the present invention. The reason for differentiating between branched amines of untargeted sites and N-terminal amine and using individually different protecting groups is because only N-terminal amine should be selectively reacted upon synthesis of peptide. Amines at untargeted sites may be substituted with other protecting groups at final step of the peptide synthesis according to necessity for stability under PEGylation conditions. As this method enables synthesis of selectively protected peptide simply by modifying acid-base conditions, it is advantageous compared to the methods based on reductive reaction or allyl substitution in that the reaction is simple, economical and yielding the peptide with high purity. For example, as specific embodiments of the present invention, the following methods can be enumerated. In case of method hi which branched amines of targeted sites are protected with Boc and branched amines of untargeted sites are protected with ivDde, in the step of introducing lysine of targeted site, a lysine in which Na-amine is protected with Fmoc or Nsc and branched amine is protected with Boc is used, and in the step of introducing lysine of other sites, lysine where Na-amine is protected with Fmoc or Nsc and branched amine is protected with ivDde, is used for the peptide synthesis. - During the procedure for the synthesis, ivDde is not removed under deblocking condition of Fmoc or Nsc, e.g. 1% DBU(l,8-diazabicyclo[5,4,0]undes-7-ene)-20% piperidine/DMF(N,Ndimethylformamide). In case of the synthesized peptide-resin(branched amine of targeted site is protected with Boc, branched amine of untargeted site, with ivDde, and N-terminal amine, with Fmoc or Nsc), after ivDde.on branched amine of untargeted site and Fmoc on N-terminai arnine are removed, thus resulting free amine is protected with Fmoe or Nsc {for instance, by reacting with Fmoc-0su(9-fluorenylmethoxycarbonylsuccinamide) or Nsc-OSu}. At this time, said deblocking can be conducted, for example, by treating with 2% hydrazine solution. This peptide-resin is treated with cleavage solution (for example, triisopropylsilane(TIS):water:trifluoroacetic acid(TFA)= 2.5:2.5:95) to separate peptide from resin and at the same time, to remove Boc on amine of targeted site, thereby obtaining peptide in which amines of untargeted site are i protected with Fmoc or Nsc. In case of method in which branched amine of targeted site is protected with Boc and branched amines of untargeted site are protected with Mtt, in the step of introducing lysine of targeted site, lysine in which Na-amine is protected with Fmoc or Nsc and branched amine is protected with Boc is used, and in the step of introducing lysine .afother sites, lysine where Na-amine is protected with Fmoc or Nsc and branched amine is protected with Mtt is used for the peptide synthesis. During the procedure for the synthesis, Mtt is not removed, under the basic conditions used for removal of Fmoc or Nsc. Thus synthesized peptide-resin (branched amine of targeted site is protected with Boc, branched amine of untargeted site, with Mtt, and N-terminal amine, with Fmoc or Nsc) is subjected to removal of Mtt on branched amine of untargeted site. Said deblocking can be carried out, for exmple, by treating with 1% TFA/MC(methylene chloride). At this time, the protecting group of amine at targeted site, Boc, is not removed. This peptide-resin (branched amine of targeted site is protected with Boc, N-terminal amine is protected with Fmoc or Nsc, and branched amine of untargeted site is exposed) is reacted with Fmoc-OSu or Nsc-OSu to protect branched amines of untargeted sites with Fmoc or Nsc, and then, the protecting group of amine at targeted site, Boc, is removed by cleavage solution (for exmple, TIS:water:TFA=2.5:2.5:95), to obtain selectively protected peptide(amine of targeted site is exposed and amines of untargeted sites are protected with Fmoc or Nsc). Peptide in which amines of untargeted sites are protected with Boc can be prepared by method of protecting amine of targeted site with ivDde and amines of untargeted sites with Boc. In step of introducing lysine of targeted site, lysine in which Na-amine is protected with Fmoc or Nsc and branched amine is protected with ivDde is used, and in step of introducing lysine of other sites, lysine in which Na-amine is protected with Fmoc or Nsc and branched amine is protected with Boc is used for the peptide synthesis. During the synthesis, ivDde is not removed under deblocking condition of Fmoc or Nsc, for example, 1% DBU-20% piperidine/DMF. For the synthesized peptide-resin (branched amine of targeted site is protected with ivDde, branched amine of untargeted site, with Boc and N-terminal amine, with Fmoc or Nsc), removal of the N-terminal amine protecting group, Fmoc is performed (for example, by treatment with 1% DBU-20% piperidine in DMF) and then treated with cleavage f- solution (for example, TIS:water:TFA=2.5:2.5:95) to separate resin and peptide. At this time, a branched amine protecting group at targeted site, Boc is removed. Exposed amine of this peptide (branched amine of targeted site is protected with ivDde, and branched amine of untargeted site and N-terminal amine are exposed) is protected with Boc, and ivDde on amine of targeted site is removed, thereby obtaining peptide in which amines of untargeted sites are protected with Boc. At this time, said protection can be done, for example, by reacting with Boc2O(di-tert-butyl dicarbonate), and said deblocking can be done, for example, by treatment with 2% hydrazine solution. Peptides in which amines of untargeted sites are protected with Boc can be prepared by methods of protecting amine of targeted site with Mtt, and amines of untargeted sites with Boc. In this method, a resin extremely sensitive to acid such as 2-CLTR (2-chlorotrityl resin) should be used, and caution is required since yield and purity can be reduced due to moisture. In this method, in the step of introducing lysine of targeted site, lysine where Na-amine is protected with Fmoc or Nsc and branched amine is protected with Mtt is used, and in the step of introducing lysine of other sites, lysine where Na-amine is protected with Fmoc or Nsc and branched amine is protected with Boc is used for the peptide synthesis. During the synthesis, Mtt is not removed under the basic conditions used for removal of Fmoc or Nsc. Thus in synthesized peptide-resin (branched amine of targeted site is protected with Mtt, branched amine of untargeted site, with Boc, and N-terminal amine, with Fmoc or Nsc), removal of the protecting group of N-terminal amine, Fmoc or Nsc is conducted and protection of exposed Na-amine of N-temiinus is performed with Boc. Said deblocking can be carried out, for example, by treating with 1% DBU-20% piperidine in DMF, and said protection can be conducted, for example, by reacting with Boc20. Thus synthesized peptide-resin (branched amine of targeted site is protected with Mtt, and amine(s) of untargeted sites are protected with Boc and C-terminus is combined to resin) is treated with TFA/MC to separate resin and peptide, and the protecting group of amine at targeted site, Mtt is removed, thereby obtaining peptide in which amine(s) of untargeted sites are protected with Boc. The protected peptide thus obtained is in a state where amines of untargeted sites are protected with Boc and active moiety except amine, i.e. hydroxyl or carboxyl group is protected and these protecting groups are to be removed at one time during the removal of Boc by cleavage solution (for instance, TIS:water:TFA=2.5:2.5:95) after PEGylation. Besides said methods, various methods can be derived from the present invention, and these are also included in the present invention. In addition, the peptide synthesized according to the present invention can be directly used for PEGylation, or can be employed for PEGylation, if necessary, after passing through additional step of protecting amines of untargeted site, including Naamine with final amine protecting group. At this time, the final amine protecting group can be at least one selected from the group consisting of Fmoc, Nsc, Dde[l-(4,4- dimethyl-2,6-dioxocyclohexylidene)ethyl], ivDde or other protecting group removed under basic condition, or at least one protecting group selected from the group consisting of Boc or other protecting group removed under acidic condition. The present invention further includes a method where peptide is separately synthesized by dividing the peptide into two or more fragments to raise efficiency of the peptide synthesis, and these are condensed to produce peptide in which amine(s) of untargeted sites are protected. Even in this case, protection-deblocking of the amine(s) is same as previously described, ^ej;, as racemization may occur during condensation of fragments, thus this method can be effectively used for peptides which contain Gly or Pro that do not bring racemization upon condensation, in its amino acid sequence, and more useful in case of peptide whose synthesis or purification is difficult. In this method, for example, peptide synthesis is conducted by dividing the peptfde into two fragments, i.e. one fragment of from N-terminus to Gly or Pro and the other fragment of from subsequent amino acid to C-terminus and the two fragments are condensed to form the peptide. A resin used for synthesis of fragment having N-terminal region should be extremely sensitive to acid so that TRT series resin is used, and upon completion of the synthesis of fragments, the fragments are separated from the resin under acidic condition while retaining other protecting group. A fragment having C-terminal region can be synthesized using conventional resin and condensed with N-terminal fragment while being combined to resin, and then can be separated from the resin, or Cterminal fragment can be synthesized using an acid-sensitive resin, separated from resin with protecting group being retained, and then subjected to condensation with Nterminal fragment in solution phase. In case the fragment is synthesized using an acidsensitive resin and Mtt as a branched amine protecting group, and then separated from the resin, separation is conducted under the condition, AcOH:TFE(2,2,2- triflubroethanol):MC(l:l:8) or TFE:MC(2:8) in order for Mtt not to be removed. In case branched amine of untargeted site in the N-terminal fragment is protected with ivDde, after condensation is completed, before separating peptide having full sequence from resin, protecting group can be substituted with Fmoc or Nsc. Additionally, in case amine at a targeted site is present on N-terminal fragment, separation from resin should be conducted under condition in which removal of Boc does not occur. Cterminal fragment is synthesized by the same method as in continuous synthesis. As synthetic method for peptide that can be used in the present invention, there is no special limitation as long as it is compatible with the present invention, yet preferably, solid phase peptide synthesis is employed. As the peptide to which the present invention can be applied, there is no special limitation as long as it is peptides having at least two branched amines. The present invention will be useful for peptides, which require extension of biological halflife or reduction of immunogenicity or antigenicity via combining PEGs to specific sites thereof. As an example, calcitonin or GRF(l-29) can be enumerated. Peptides having protected amine(s) of untargeted sites enable completely specific PEGylation to peptide. Reacting peptide in which amine(s) of untargeted sites are protected with Fmoc, Nsc, Boc or other amine protecting groups stable under PEGylation condition, with activated PEGs leads to specific combining of PEGs to only amines at targeted sites and the reaction can almost complete in accordance with their equivalent ratio and reaction condition. Conducting deblocking {for example, 5% piperidine/DMF condition for protecting group, Fmoc or Nsc, and cleavage solution (TIS:water:TFA=2.5:2.5:95) for protecting group, Boc} of thus protected peptide-PEG conjugate (PEG are combined to amines of targeted sites and amines of untargeted sites are protected with protecting groups) leads to formation of peptide in which PEGs are specifically combined to amines at targeted sites. Therefore, another embodiment of the present invention is methods for preparing specific PEG-conjugated peptide in which PEGs are specifically combined to amines of targeted sites, comprising (1) reacting the peptide in which amines at untargeted sites are selectively protected with activated PEGs and (2) amine protecting groups of thus obtained compound are removed under acid-base deblocking {...*- condition. Said reaction mixture is confirmed not having other peptide-derived substance, and specifically conjugated PEG-peptide with high purity can be obtained by removing uncombined PEG and released protecting groups through ionic exchange chromatography, removing salts via reverse phase resin (e.g. C-18 Sep-Pak catridge), and subjecting to lyophilization. Activated PEG that can conjugate with the peptide having selectively protected amines of untargeted sites, prepared according to the present invention, has no particular limitation and includes all the above mentioned PEG, yet, it is preferably linear or branched hydroxy- or methoxy-type alkylating or acylating PEG of 1,000 to 40,000, and more preferably, at least one selected from the group consisting of monomethoxy poly(ethyleneglycol)succinimidyl succinate, mono-methoxy poly(ethyleneglycol)succinimidyl propionate, mono-methoxy poly(ethyleneglycol)succinimidyl carbonate, mono-methoxy poly(ethyleneglycol)succinimidyl carbamate and mono-methoxy poly(ethyleneglycol)tresylate, can be used. The specifically conjugated PEG-peptide of the present invention can be formulated into pharmaceutical dosage forms containing the peptide in therapeutically effective amount. Formulations such as injection, infusions, depot for injections, inhalations can be available, and buffering agents, tonicity regulating agents, stabilizers, surfactants, thickeners, preservatives, coloring agents and flavoring agents can be added. Brief Explanation of Drawings Fig. 1 represents reverse phase chromatograms for LysIS-PEG 2K-salmon calcitonin in Example 20(B) ,and mono-PEG 2K-salmon calcitonin in Comparative Example 1(A). Fig. 2 shows MALDI-TOF mass spectra for Lys-C enzyme-treated fragments of salmon calcitonin(A) and Lys18-PEG 2K-salmon calcitonin in Example 20(B). Fig. 3 represents MALDI-TOF mass spectra for salmon calcitonin(A) and Lys18-PEG 2K-salmon calcitonin in Example 20(B). Fig. 4 shows reverse phase chromatograms for GRF(1-29)(A) and Lys21-PEG 5K-GRF(1 -29) in Example 23(B). Fig. 5 represents MALDI-TOF mass spectrum for Lys2I-PEG 5K-GRF(l-29) in Example 23. Fig. 6 represents MALDI-TOF mass spectra Lys-C enzyme-treated fragments of GRF(1-29)(A) and of Lys21-PEG 5K-GRF(l-29) (B) in Example 23. Best mode for carrying out the invention In the below, detailed methods of the present invention are exemplified by Examples. However, the Examples are not intended to limit the scope of the present invention. Abbreviations used in the present Examples define the following meaning. Ac=acetyl, Ac2O= acetic anhydride, AcCN= acetonitrile, AcOH= acetic acid, Bop= benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate, Clt= 2- chlorotrityl, DCM= dichloromethane, DIC= 1,3-diisopropylcarbodiimide, DIPEA= N,N-diisopropylethylamine, DMSO= dimethylsulfoxide, EDT= 1,2-ethanedithiol, EtOAc= ethyl acetate, HBTU= N-[(lH-ben20triazol-l-yl)(dimethylamino)methylene]- t N-methylmethaneammonium hexafluorophosphate N-oxide, HOBt= 1- hydroxybenzotriazole, Pbf=2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Pmc= 2,2,5,7,S-pentamethylchroman-6-sulfonyl, PyBop= benzotriazol-1- yloxytri(pyrrolidino)phosphonium hexafluorophosphate, SPPS= solid phase peptide synthesis, TBTU=N-[(lH-benzotriazol-l-yl)(dimethylamino)methylene]-Nmethylmethaneammonium tetrafluoroborate N-oxide, tBu=tert-butyl, TEA=triethylamine, and Trt=trityl Example 1. Continuous synthesis of 1,11-diFmoc-salmon calcitonin using ivDde as a protecting group for branched amine of untargeted site Structure: Fmoc-Cys'-Ser-Asn-Leu-Ser-Thr-Cys'-Val-Leu-Gly^-LysCFmoc)- Leu-Ser-Glri-Glu-Leu-His-Lys-Leu-Gln20-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser- Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 1 ] (Table Removed) * indicates the amount used per coupling cycle Peptide was synthesized according to the following reaction order. Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, Nsc- Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), Nsc-Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Nsc-Lys(Boc), Nsc-His(Trt), Nsc-Leu, Nsc-Glu(tBu), Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Fmoc-Lys(ivDde), Nsc- Gly, Nsc-Leu, Nsc-Val, Nsc-Cys(Trt), Nsc-Thr(tBu), Nsc-Ser(tBu), Nsc-Leu, Nsc-Asn(Trt), Nsc-Ser(tBu), Nsc-Cys(trt) 2.0 g of link-amide resin was put in 50 ml peptide reaction vessel, 20 ml of DCM was poured and left for 30 min to allow the resin to sufficiently expand. The solution was removed via filtration. 20 ml of deblocking solution (1% DBU-20% piperidine in DMF) was added and reacted for 5 min (this process was repeated twice) to remove Fmoc, and washed 5 to 7 times with 20 ml DMF. Removal of remained piperidine and dibenzofulvene was confirmed with chloranil test [the test can be used to determine whether the removal of Nsc or Fmoc-related adducts and residual piperidine is complete. The test solution is prepared by adding a drop of a saturated solution of chloranil in toluene to about 1 ml of acetone. The DMF washing may be tested by adding a drop of the washing solution to the chloranil test solution. A blue or violet color is a positive indication for the presence of secondary amine]. To introduce C-terminal amino acid of calcitonin, Pro, Nsc-Pro-OH(1.5 eq), Bop(1.5 eq), HOBt(1.5 eq) and DIPEA(1.5 eq) were dissolved in 4 ml DMF and 8 ml DCM and put to said reactor containing the resin. The reaction solution was further washed with 8 ml DCM, added to the reactor, additional DIPEA(1.5 eq) was added, and reacted at 35°C to 40°C for 1 hr while mixing with an adequate blender. End point of the reaction was determined by Kaiser test [To check completion of the reaction using the qualitative ninliydrin test, a 2-10 mg sample of the resin is withdrawn and washed with ethanol. To the sample, 2 drops of 80% phenol solution, 2 drops of 0.02 mM KCN in pyridine and 2 drops of 5% ninhydrin in ethanol are added. The sample is left in a heat block at about 120°C for 4 to 6 min. A blue or violet color is a positive indication of the presence of free amine], and in case the reaction did not reach completion, the reaction solution was further reacted for 30 min to 1 hr. Upon completion of the reaction, the reaction solution was removed by filtration, washed with 3x20 ml DMF, reacted with deblocking solution (2x 20 ml) for 5 min in each time, and thoroughly washed with DMF. Continuous introduction of amino acids was performed according to the method described above, and the reaction started from Pro32 of C-terminus of calcitonin and proceeded in order, and after the introduction of protected amino acid, removal of Nsc or Fmoc was repeatedly conducted using deblocking solution. After Nsc-Cys(Trt) was introduced, the peptide was reacted with 3x 20 ml of 2% hydrazine solution for 10- 30 min to remove ivDde and Nsc, and then the resin was filtered and sufficiently washed with 4x20 ml DMF, 4x20 ml DCM and 4x20 ml DMF. Fmoc-OSu(5.0 eq) dissolved in 20 ml of DMF was put to 'the reactor, thoroughly mixed for 1 to 2 hr to allow Fmoc to be introduced to the amines on 1,11-positions, respectively. Progress of the reaction was checked by Kaiser test, and when determined as being completed, the reaction solution was removed via filtration, sufficiently washed with 4x20 ml DMF and 4x20 ml DCM, dried under-nitrogen stream to obtain peptide-attached resin 8.5g. 1,7 disulfide bond could be formed by Ij oxidation. The peptide resin obtained as above was put in 150 ml filterable peptide reactor, 50 ml DMF was poured and allowed to maintain equilibrium for 30 min. 0.1M IT in 50ml DMF was further added to the reactor, mixed well for 2 hrs to perform oxidation. Progress of the reaction was monitored with HPLC, and upon completion of the reaction, filtration and washing with 5x50 ml DMF and 5x50 ml ascorbic acid for 5 min in each time, was conducted to completely remove the residual k Additionally, the peptide resin was washed with 3x20 ml DMF, 3x20 ml DCM, 3x20 ml MeOH and 3x20 ml heptane, dried under nitrogen, vacuum dried for 6 hrs to obtain dried peptide-attached resin 8.1g. Said dried resin was reacted with 80 ml cleavage solution (2.5:2.5:95=TIS:water:TFA) at ordinary temperature for 1.5 hr, the resin was removed and resulting peptide was washed with about 10 ml of TFA. The washing and filtrate were collected and added to 500 ml of ether, subjected to centrifuge to precipitate formed floating material, and additionally washed with 3x200 ml ether via centrifuge. The precipitate was dried under nitrogen to obtain dried peptide mixture 4.8g, and this mixture was purified by prep-HPLC, and subjected to lyophilization to obtain 896mg of 1,11-diFmoc-salmon calcitonin. Targeted site of the peptide prepared in this Example is the amine of Lys . [Purification conditions for preparative HPLC] Vydac protein & peptide-C1S) 20x250 mm, 5 u., 300 A TFA buffered AcCN and water gradient [Peptide analysis condition 1] Instrument: Waters Alliance Flow rate: 1.0 ml/min Gradient: 0-45 min, B 0-100% (A: 0.1% TFA in water, B: 0.1% TFA in AcCN) Column: Nova-Pak-Cis, 3.9 mm x 150 mm, 5 \i, 100 A [Mass analysis condition] Instrument: Voyager DE-STR Maldi Tof Mass(Perspective) Mode of operation: Reflector Extraction mode: Delayed Polarity: Positive Matrix: cc-cyano-4-hydroxycinnamic acid Maldi Tof; 3877.43, (M+l=3877.44) HPLC; 98% up (peptide analysis condition 1) Example 2. Continuous synthesis of 1,18-diFmoc-salmon calcitonin using ivDde as a protecting group for branched amine of untargeted site Structure: Fmoc-Cys1-Ser-Asn-Leu-Ser-Thr-Cys7-Val-Leu-Glyl°-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(Fmoc)-Leu-Gln20-Thr-Tyr-Pro-Arg-Tlir-Asn-Thr-Gly-Ser-Gly30- Thr-Pro-NH2 (1,7-disulfide bond). [ Table 2] (Table Removed) Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, Nsc-Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), Nsc- Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Fmoc-Lys(ivDde), Nsc-His(Trt), Nsc-Leu., Nsc- Glu(tBu), Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Nsc-Lys(Boc), Nsc-Gly, Nsc-Leu, Nsc- Val, Nsc-Cys(Trt), Nsc-Thr(tBu), Nsc-Ser(tBu), Nsc-Leu, Nsc-Asn(Trt), Nse-Ser(tBu), Nsc-Cys(trt). Reaction was conducted according to the above Table and the reaction order of ammo acid, as described in Example 1. When removal of ivDde and introduction of diFmoc and \i oxidation were performed, the peptide-attached resin was 8.2g, and was treated with TFA-cleavage solution (2.5:2.5:95=TIS:water:TFA) as disclosed in Example 1 and purified by prep-HPLC to obtain 892 mg of 1,18-diFmoc-salmon calcitonin. Maldi Tof; 3877.33, (M+l=3877.44) HPLC; 97% up (peptide analysis condition 1) Example 3. Continuous synthesis of 11,18-diFmoc-salmon calcitonin using ivDde as a protecting group for branched amine of untargeted site Structure: H-Cys'-Ser-Asn-Leu-Ser-Thr -Cys7-Val-Leu-Gly10-Lys(Fmoc)- Leu-Ser-Gln-Glu-Leu-His-Lys(Fmoc)-Leu-Ghr°-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 3] (Table Removed) Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, Nsc- Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), Nsc- Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Fmoc-Lys(ivDde), Nsc-His(Trt), Nsc-Leu, Nsc- Glu(tBu), Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Fmoc-Lys(ivDde), Nsc-Gly, Nsc-Leu, Nsc-Val, Nsc-Cys(Trt), Nsc-Thr(tBu), Nsc-Ser(tBu), Nsc-Leu, Nsc-Asn(Trt), Nsc- Ser(tBu), Boc-Cys(trt). Reaction was conducted according to the above Table and the reaction order of amino acid, as described in Example 1. When removal of ivDde and introduction of diFmoc and ^ oxidation were performed, the peptide-attached resin was 8.2g, and was treated with TFA-cleavage solution (2.5:2.5:95=TIS:water:TFA) as disclosed in Example 1 and purified by prep-HPLC to obtain 890 mg of 11,18-diFmoc-salmon calcitonin. Maldi Tof; 3877.23, (M+l=3877.44) HPLC; 98% up (peptide analysis condition 1) Example 4. Continuous synthesis of l,12-diFmoc-GRF(l-29) using ivDde as a protecting group for branched amine of untargeted site Structure: Fmoc-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr10-Arg-Lys(Fmoc) -Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 [ Table 4] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tBu), Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc), Nsc-Arg(Pbf), Nsc-Ala, Nsc-Ser(Trt), Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(ivDde), Nsc-Arg(Pbf), Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ala, Nsc-Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) 2.0g of link-amide resin was put in 50 ml peptide reaction vessel, 20 ml DCM was poured and left for 30 min to allow resin to sufficiently expand. The solution was removed via filtration. 20 ml of deblocking solution (1% DBU-20% piperidine in DMF) was added and reacted for 5 min to remove Fmoc (this process was repeated twice), and washed 5-7 times with 20 ml DMF. Removal of remaining piperidine and dibenzofulvene was confirmed with chloranil test. To introduce C-terminal amino acid of GRF(l-29). Arg; Nsc-Ars(Pbf)- OH(1.5 eq), Bop(1.5 eq), HOBt(1.5 eq) and DIPEA(1.5 eq) were dissolved in 2ml DMF and 8ml DCM and put in the reactor containing the resin. The reaction solution was washed with additional 8 ml DCM and pour to the reactor, DIPEA(1.5 eq) was further added, and the reaction was proceeded at 35 °C to 40 °C for 1 hr while mixing with an adequate blender. End point of the reaction was determined by Kaiser test, and in case the reaction did not complete, the reaction solution was further reacted for 30 min to 1 hr. Upon completion of the reaction, the reaction solution was removed by filtration, washed with 3x 20 ml DMF, reacted with deblocking solution (2x 20ml) for 5 min in each time and thoroughly washed with DMF. Continuous introduction of amino acids was performed as described above, and the reaction started from Arg of C-terminus of GRF(l-29) and proceeded in order, and after the introduction of protected amino acid, removal of NSc or Fmoc was repeatedly conducted using deblocking solution. After Nsc-Tyr(tBu) was introduced, reacted with 3x20 ml of 2% hydrazine solution for 10-30 min in each time to remove ivDde and Nsc, and then the resin was filtered and sufficiently washed with 4x20 ml DMF, 4x20 ml DCM and 4x20 ml DMF. Fmoc-OSu(5.0 eq) dissolved in 20 ml of DMF was put in the reactor, thoroughly mixed for 1 to 2 hr to allow Fmoc to be introduced to the amines on 1,12-positions, respectively. Progress of the reaction was checked by Kaiser test, and when determined completion of the reaction, the reaction solution was removed via filtration, sufficiently washed with 4x20 ml DMF and 4x20 ml DCM, dried under nitrogen stream to obtain peptide-attached resin 8.5 g. 1 g of said dried resin was withdrawn and reacted with 10 ml of cleavage solution (2.5:2.5:95=TIS:water:TFA) at ordinary temperature for 1.5 hr, and further reacted with TMS-Br and EDT for 15 min. The resin was removed via filtration, and the resin was washed with 2 ml TFA. The washing and filtrate were collected and added to 100ml of ether, subjected to centrifuge to precipitate formed floating material, and additionally washed with 3x50 ml ether via centrifuge. The precipitate was dried under nitrogen to obtain 690 mg of dried peptide mixture, and this mixture was purified by prep-HPLC, subjected to lyophilization to obtain 116 mg of l,12-diFmoc-GRF(l-29). Maldi Tof; 3803.39, (M+l=3803.46) HPLC; 96% up (peptide analysis condition 1) Example 5. Continuous synthesis of l,21-diFmoc-GRF(l-29) using ivDde as a protecting group for branched amine of untargeted site Structure: Fmoc-Tyr-Ala-Asp-Ala-Ile-Phe-Tlir-Asn-Ser-Tyr10-Arg-Lys-Val- Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys(Fmoc)-Leu-Leu-Gln-Asp:Ile-Met-Ser-Arg-NH2 [ Table5] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tBu), Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Fmoc-Lys(ivDde), Nsc-Arg(Pbf), Nsc-Ala, Nsc- Ser(Trt), Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Nsc-Lys(Boc), Nsc- Arg(Pbf), Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ala, Nsc-Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) The reaction was conducted as described in Example 4 by using the reagents and solvent given in the above Table. Introduction of DiFmoc could be achieved by use of Fmoc-Osu (3.37g in 20 ml DMF) after the removal of ivDde with 2% hydrazine in DMF according to the method as in Example 4, and after the reaction, 7.9 g as dried resin was obtained. 1 g of thus obtained peptide resin was withdrawn, mixed and reacted with 10 ml of cleavage solution (2.5:2.5:95= TIS/water/TFA) for 1.5 lir, and additional EDT(0.130 ml) and TMS-Br(0.157 ml) were added to the reaction solution and further stirred for 15 min. The reaction solution was treated with ether as in Example 4 to obtain 710 mg of peptide mixture, and through prep-HPLC purification, 86 mg of 1,21-diFmoc GRF(l-29) was obtained. Maldi Tof: 3803.59, (M+l=3803.46) HPLC; 98% up (peptide analysis condition 1) Example 6. Continuous synthesis of 12,21-diFmoc-GRF(l-29) using ivDde as a protecting group for branched amine of un targeted site Structure: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr'°-Arg- Val-Leti-Gly-Gln-Leu-Ser-Ala-Arg20-Lys(Fmoc)-Leu-Leu-Gln-Asp-Ile-Met- NH2 Table 6. (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met. Nsc-Ile, Nsc-Asp(tBu), Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(ivDde), Nsc-Arg(Pbf), Nsc-Ala, Nsc- Ser(Trt), Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(ivDde), Nsc- Arg(Pbf), Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Tlir(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ala, Nsc-Asp(tBu), Nsc-Ala, Boc-Tyr(tBu) The reaction was conducted as described in Example 4 by using the reagents and solvent given in the above Table. Introduction of DiFmoc could be achieved by use of Fmoc-OSu(3.37 g in 20 ml DMF) after the removal of ivDde with 2% hydrazine in DMF according to the method as in Example 4, and after the reaction, 8.4 g as dried resin was obtained. 1 g of thus obtained peptide resin was withdrawn, mixed and reacted with 10 ml of cleavage solution (2.5:2.5:95= TIS/water/TFA) for 1.5 hr, and additional EDT(0.130 ml) and TMS-Br(0.157 ml) were added to the reaction solution and further stirred for 15 min. The reaction solution was treated with ether as in Example 4 to obtain 710 mg of peptide mixture, and through prep-HPLC purification, 86 mg of 12,21-diFmoc GRF(l-29) was obtained. Maldi Tof; 3803.39, (M+l=3803.46) HPLC; 96% up (peptide analysis condition 1) Example 7. Continuous synthesis of l,12-diNsc-GRF(l-29) using ivDde as a protecting group for branched amine of untargeted site Structure: Nsc-Tyr-Ala-Asp-Ala-Ile-Phe-Tlir-Asn-Ser-Tyr10-Arg-Lys(Nsc)- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 [ Table] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tBu), Nsc-GIn(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc) Nsc-Arg(Pbf), Nsc-Ala, Nsc-Ser(Trt), Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(ivDde), Nsc-Arg(Pbf), Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ala, Nsc-Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) The synthesis was conducted as described in Example 4 by using the reagents and solvent given in the above Table. Introduction of DiNsc could be achieved by use of Nsc-OSu(3.72 g in 20 ml DMF), after the removal of ivDde with 2% hydrazine in DMF according to the method as in Example 4, and after the reaction, 8.1 g as dried nd on in an, sis resin was obtained. 1 g of thus obtained peptide resin was withdrawn, mixed reacted with 10 ml of cleavage solution (2.5:2.5:95= TIS:water:TFA) for 1.5 hr, additional EDT(0.130 ml) and TMS-Br(0.157 ml) were added to the reaction solu and further stirred for 15 min. The reaction solution was treated with ether a Example 4 to obtain 700 mg of peptide mixture, and through prep-HPLC purificat 136 mg of 1,12-diNsc GRF(l-29) was obtained. Maldi Tof; 3874.46, (M+1=3S74.39) HPLC; 98% up (peptide anal condition 1) Example 8. Continuous synthesis of l,12-di(ivDde)-GRF(l-29) using ivDde protecting group for branched amine of untargeted site Structure: ivDde-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr'°-Arg-Lys (ivDde)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Ar NH2 [ Table] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tBu Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc), Nsc-Arg(Pbf), Nsc-Ala, Nsc-Ser(Trt Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(ivDde), Nsc-Arg(Pbf) Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ai Nsc-Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) The synthesis was conducted as described in Example 4 by using the reagen and solvent given in the above Table. After reaction with Nsc-Tyr(tBu) an subsequent removal of the Nsc, introduction of ivDde to the N-terminus could achieved by reaction with 2-isovaleryldimedone(1.0 g in 20 ml DMF) for 12 hrs, and a a result of the reaction, 8.1 g as dried resin was obtained. 1 g of the peptide resin wa withdrawn, mixed and reacted with 10 ml of cleavage solution (2.5:2.5:95 TIS:waterTFA) for 1.5 hr, and additional EDT(0.130 ml) and TMS-Br(0.157 ml) wei added to the reaction solution and further stirred for 15 min. The reaction solution we treated with ether as in Example 4 to obtain 680 mg of peptide mixture, and through prep-HPLC purification, 97 mg of l,12-di(ivDde)-GRF(l-29) was obtained. Maldi Tof; 3771.39, (M+l=3771.55) HPLC; 94% up (peptide analys condition 1) Example 9. Continuous synthesis of l,12-diBoc-GRF(l-29) using ivDde as protecting group for branched amine of untargeted site Example 9-1. Synthesis of 21-Nsc-GRF(l-29) Structure: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr10-Arg-Lys-Val-L GIy-Gln-Leu-Ser-Ala-Arg20-Lys(Nsc)-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 [ Table 9] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tB4), Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Fmoc-Lys(ivDde), Nsc-Arg(Pbf), Nsc-Ala, Ns Ser(Trt), Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Nsc-Lys(Boc), Nsp- Arg(Pbf), Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Pher Nsc-IJe, Nsc-Ala, Nsc-Asp(tBu), Nsc-Ala, Boc-Tyr(tBu) The synthesis was conducted as described in Example 4 by using the reagerlts and solvent given in the above Table. Introduction of Nsc could be achieved according to method as in Example 4 by use of Nsc-OSu(3.72 g in 20 ml DMF), after removal bf ivDde with 2% hydrazine in DMF, and after the reaction, 7.5 g as dried resin \Mas obtained. Thus obtained peptide resin was reacted with 80 ml of cleavage solution (2.5:2.5:95= TIS:water:TFA) for 1.5 lir, and additional EDT(1.26 ml) and TMS-Br(1.04 ml) were added to the reaction solution and further stirred for 15 min. The reaction solution was treated with ether as in Example 4 to obtain 4.1 g of peptide mixture, ahd through prep-HPLC purification, 520 mg of 21-Nsc-GRF(l-29) was obtained. Maldi Tof; 3616.39, (M+l=3616.17) HPLC; 98% up (peptide analysis condition 1) Example 9-2. Synthesis of l,12-diBoc-GRF(l-29) Structure: Boc-Tyr-Ala-Asp-Ala-Ile-Phe-Thi--Asn-Ser-Tyr10-Arg-L\'s(Bo( Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Ai-g-NH2 [ Table 10] (Table Removed) 360 mg of the synthesized 21-Nsc-GRF(l-29) and 5 ml of DMF were put 25ml-single neck reactor with magnetic stirrer and dissolved. The reactant allowed to place under air-blocked environment by use of nitrogen gas, temperature lowered to 0 to 5°C with ice bath. To the reaction solution, Boc20 (1 g/5 ml was added dropwise 0 to 5°C, the temperature was maintained for about lOmin, the bath was removed to allow the temperature to slowly reach to ordinary temperature While keeping stirring for 3 to 4 hrs, progress of reaction was monitored with HP (peptide analysis condition 1) by collecting sample every hour. Upon completion reaction, 5% NaiCOa 10ml was added dropwise to the reaction solution with stirring to cause precipitation. The reaction was proceeded for about 30 min and progress the removal of Nsc was checked with HPLC. The reaction solution was concentra ed to 1/4 fold, washed with 150 ml of hexane to obtain tacky oil. To this oil, 20 ml of MTBE was poured and left for 12 hrs to lead to precipitation, and this precipitation tyas separated from centrifuge. It was further washed with 2x20ml MTBE and dried under nitrogen to obtain 330 mg of l,12-diBoc-GRF(l-29). Maldi Tof; 3559.21, (M+l=3559.20) HPLC; 92% up (peptide analysis condition 1) Example 10. Continuous synthesis of l,12-di(Dde)-GRF(l-29) using Dde a; protecting group for branched amine of untargeted site Structure: Dde-Tyr-Ala-Asp-Ala-Ile-Phe-Tlir-Asn-Ser-Tyr10-Arg-Lys(Dde)- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 of hot of [ Table 11] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc), Nsc-Arg(Pbf), Nsc-Ala, Nsc-Se Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(Dde), Nsc-An Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Ns ;tBu), (Trt), (Pbf), -Ala, Nsc-Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) The synthesis was conducted as described in Example 4 by using the reagents and solvent given in the above Table. After reaction with Nsc-Tyr(tBu) and removal of the Nsc, introduction of Dde to the N-terminus could be achieved by reaction with acetyldimedone(1.0 g in 20 ml DMF) for 6 hrs, and after the reaction, 8.8 g as dri resin was obtained. 1 g of the peptide resin was withdrawn and reacted with 10 ml cleavage solution (2.5:2.5:95= TIS/water/TFA) for 1.5 hr, and additional EDT(0.130 r and TMS-Br(0.157 ml) were added.to the reaction solution and further stirred for min. The reaction solution was treated with ether as in Example 4 to obtain 590 mg peptide mixture, and through prep-HPLC purification, 104 mg of 1,12-diDde-GRF 29) was obtained. Maldi Tof; 3687.69, (M+l=3687.55) HPLC; 95% up (peptide analy condition 1) Example 11. Continuous synthesis of 1,11-diFmoc-salmon calcitonin using Mtt a protecting group for branched amine of untargeted site Structure: Fmoc-Cys1-Ser-Asn-Leu-Ser-Thr-Cys7-Val-Leu-Glyl°-Lys(Fmo Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln20-Thr-Tyr-Pro-Arg-Tlir-Asn-Thr-Gly-Ser- Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 12] (Table Removed) Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, Nsc Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), Nsc Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Nsc-Lys(Boc), Nsc-His(Trt), Nsc-Leu, Nsc-Glu(tBu) Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Fmoc-Lys(Mtt), Nsc-Gly, Nsc-Leu, Nsc-Va Nsc-Cys(Trt), Nsc-Thr(tBu), Nsc-Ser(tBu), Nsc-Leu, Nsc-Asn(Trt), Nsc-Ser(tBu Fmoc-Cys(trt) 2.0 g of link-amide resin was put in 50 ml peptide reactor, 20 ml DCM wa poured and left for 30 min to allow resin to sufficiently expand. The solution wa removed via filtration. Fmoc was removed by reaction with 2x20ml deblocking solution (1% DBU-20% piperidine in DMF) for 5 min in each time, and washed 5- times with 20 ml DMF. Removal of remaining piperidine and dibenzofulvene checked with chloranil test. To introduce C-terminal amino acid of calcitonin, Pro, Nsc-Pro-OH(1.5 Bop(1.5 eq), HOBt(1.5 eq) and DIPEA(1.5 eq) were dissolved in 4 ml DMF and was eq), ml DCM and put in the reactor containing the resin. The reaction solution was further t •' •*' washed with 8 ml DCM, added to the reactor, and additional DIPEA(1.5 eq) was added, reacted at 35 °C to 40°C for about 1 hr under thoroughly mixing with an adequate blender. End point of the reaction was determined by Kaiser test, and in case reaction did not complete, the reaction solution was further reacted for 30 min to Upon completion of the reaction, the reaction solution was removed by filtra washed with 3x20 ml DMF, reacted with deblocking solution (2x 20 ml) for 5 m each time, and thoroughly washed with DMF. Continuous introduction of amino acids was performed according to method described above, and the reaction started from Pro32 of C-terminus of calcitonin and proceeded in order, and coupling reaction and deblocking was repeated according to the order. Repeated reaction as explained above, coupling reaction and deblocking; can be conducted by using automatic instrument. Lastly, after Fmoc-Cys(Trt) was introduced to the N-terminus, the resin was washed with DCM. The resin was reacted with 3x20 ml 1% TFA solution for 10 to 30 min in each time to remove Mtt at 11- position. The removal of Mtt was confirmed by TLC analysis based on reactivity of the reaction solution to UV and TFA fume in comparison with blank. Upon completion of reaction, the resin was filtered and sufficiently washed with 4x20 ml DMF, 4x 20 ml DCM and 4x20 ml DMF. Fmoc-OSu(5.0 eq) dissolved in 20 ml :DMF was put into the reactor, thoroughly mixed for 1 to 2 hr to allow Fmoc to be introc.uced the hr. ion, n in the to Lys at 11-position. Progress of the reaction was checked by Kaiser test, wien determined as being completed, the reaction solution was removed via filtration, sufficiently washed with 4x20 ml DMF and 4x20 ml DCM, and dried under nitrogen stream to obtain peptide-attached resin 8.5g. 1,7 disulfide bond could be formed by k oxidation. The peptide rejsin obtained as above was put in 150 ml filterable peptide reactor, 50 ml DMF was pou and allowed to maintain equilibrium for 30 min. Additional 0.1M \i in 50 ml D was added to the reactor, mixed well for 2 hrs to perform oxidation. Progress of reaction was monitored with HPLC, and upon completion of the reaction, filtration washing with 5x50 ml DMF and 5x50 ml ascorbic acid for 5 min in each time, w conducted to completely remove residual IT. Additionally, the resulting product A washed with 3x20 ml DMF, 3x20 ml DCM, 3x20 ml MeOH and 3x20 ml heptane, dijied under nitrogen, vacuum-dried for 6 hrs to obtain dried peptide-attached resin 8.1 g. The dried resin was reacted with 80 ml cleavage solution (2.5:2.5:95=TIS:water:TFA) at ordinary temperature for 1.5 hr, the resin was removed and washed with about 10 ml of TFA. The washing and filtrate were collected ;md added to 500ml of ether, subjected to centrifuge to precipitate the formed precipitate and additionally centrifuged and washed with 3x200 ml ether. The precipitate dried under nitrogen to obtain dried peptide mixture 4.8g, and this mixture was puri by prep-HPLC, subjected to lyophilization to obtain 896 mg of 1,11-diFmoc-salmon calcitonin. Maldi Tof; 3877.43, (M+l=3877.44) HPLC; 98% up (peptide anal condition 1) ed vlF the nd ere vas vas led 'SIS Example 12. Continuous synthesis of l,12-diFmoc-GRF(l-29) using Mtt as a protecting group for branched amine of untargeted site Structture:Fmoc-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr10-Arg-Lys(Fn|oc) -Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH2 [ Table 13] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(t^u), Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc), Nsc-Arg(Pbf), Nsc-Ala, Nsc-Ser(Ti Nsc-Leu, Nsc-Gln(Trt), Nsc-Gly, Nsc-Leu, Nsc-Val, Fmoc-Lys(Mtt), Nsc-Arg(Pb Nsc-Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-A Nsc-Asp(tBu), Nsc-Ala, Fmoc-Tyr(tBu) 2 g of link-amide resin was put in 50 ml peptide reactor, 20 ml DCM w poured and left 30 min to allow the resin to sufficiently expand. The solution w removed via filtration. Fmoc was removed by reaction with 2x 20 ml deblocki solution (1% DBU-20?/o piperidine in DMF) for 5 min in each time, and washed 5 times with 20 ml DMF. Removal of remaining piperidine and dibenzofulvene w confirmed with chloranil test. To introduce C-terminal amino acid of GRF(l-29), Arg, Nsc-Arg(Pb OH(1.5 eq), Bop(1.5 eq), HOBt(1.5 eq) and DIPEA(1.5 eq) were dissolved in 4 DMF and 8 ml DCM and put in the reactor containing the resin. The reaction soluti was further washed with 8 ml DCM, added to the reactor, and additional DIPEA(1.5 e was added, reacted at 35°C to 40°C for about 1 hr under thoroughly mixing with adequate blender. End point of the reaction was .determined by Kaiser test, and in ca the reaction did not complete, the reaction solution was further reacted for 30 min to hr. Upon completion of the reaction, the reaction solution was removed by filtrati washed with 3x 20 ml DMF, reacted with deblocking solution (2x 20 ml) for 5 min each time, and thoroughly washed with DMF. Continuous introduction of amino acids was performed according to t method described above, and the reaction started from Arg of the C-terminus of GRF 29) and proceeded in order, and coupling reaction and deblocking were repeated 7 n an 1 n n accordance with the order. Repeated reaction as explained above, coupling reaction and deblocking can be conducted by using automatic instrument. After Fmo Tyr(tBu) was introduced to the N-terminus, the resin was washed with DCM and soaked in 20 ml DCM for about 15 min. After DCM was removed, the resin was reacted wi 3x20 ml 1% TFA solution for 10 to 30 min in each time, to remove Mtt at 12-positio The removal of Mtt was confirmed by TLC analysis based on reactivity of the reaction solution to UV and TFA fume in comparison with blank. Upon completion of reaction, the resin was filtered and sufficiently washed with 4x 20 ml DMF, 4x20 ml DCM and 4x20 ml DMF. Fmoc-OSu(5.0 eq) dissolved in 20 ml DMF was put into the reactc thoroughly mixed for 1 to 2 hr to allow Fmoc to be introduced to Lys at 12-positio Progress of, the reaction was checked by Kaiser test, when determined as beirj completed, the reaction solution was removed via filtration, sufficiently washed wi 4x20 ml DMF and 4x20 ml DCM, dried under nitrogen stream to obtain peptid attached resin 8.5g. 1 g of the dried resin was withdrawn and reacted with 10 ml cleavage soluti (2.5:2.5:95=TIS: water: TFA) at ordinary temperature for 1.5 hr, and additional reacted with TMS-Br and EDT for 15 min. The resin was removed via filtratioi, washed with about 2 ml TFA. The washing and filtrate were collected and added 100 ml of ether, subjected to centrifuge to precipitate the formed floating material, a s additionally centrifuged and washed with 3x50 ml ether. The precipitate was dri under nitrogen to obtain dried peptide mixture 690 mg, and this mixture was purified prep-HPLC, subjected to lyophilization to obtain 116 mg of U2-diFmoc-GRF(l-29). Maldi Tof; 3803.39, (M+l=3803.46) HPLC; 96% up (peptide analy n y o IS conditionl) Example 13. Frgament condensation synthesis of 1,11-diFmoc-salmon using ivDde as a protecting group for branched amine of untargeted site Example 13-1. synthesis of Nsc-Gly-2-ClTrt-resin I Table 14] (Table Removed) 10 g of 2-CLTR resin was put into 250 ml peptide reactor, 100 ml DCM was poured and allowed the resin to sufficiently expand for 30 min, and then the solution was removed via filtration. Nsc-Gly-OH(3.32 g) and DIPEA(5.22 ml) were dissolv in 100 ml DCM, the solution was put to the reactor containing the resin, mixed at ordinary temperature for about 1 hr to allow the reaction to be proceeded. The reaction solution was removed via filtration, washed with 100 ml DCM, reacted with !!0 ml DCM:MeOH:DIPEA(17:2:l) for 20 min to remove active moiety of the resin substituting with MeOH. After washing with 4x100 ml DCM, subjected to drying under nitrogen to obtain Nsc-Gly-CLTR 13.1 g {UV-spectrum assay (instrument: BioRad Smart Spec 3000, Extinction coefficient of Nsc: 2000 crrf'M'1 at 300 m|i; capacity 0.61 mmol/g)}. Example 13-2. Synthesis of Nsc-AAsCt(l-10)-OH Structure: Nsc-Cys1-Ser(tBu)-Asn(Trt)-Leu-Ser(tBu)-Thr(tBu)-Cys7-Val-Leii GlyIO-OH (1,7-disulfidebond) [ Table 15] (Table Removed) 8.2g of Nsc-Gly-2-CLTR (capacity 0.61 mmol/g) synthesized as above was put in 200 ml peptide reactor, 50 ml of DCM was poured and left for 30 min to allow the reaction mixture to reach equilibrium. DCM was removed via filtration, and 2x50 ml deblocking solution (1%DBU-20% piperidine in DMF) was reacted for 5 mir in each time, and washed thoroughly with 5x50 ml of DMF. According to the sequence of peptide, coupling and deblocking was repeatedly conducted as described in Example 1 starting from C-terminus by using reagents shown in the above Table. The reaction order was Leu, Val, Cys(Trt), Thr(tBu), Ser(tBu), Leu, Asn(Trt), Ser(tBu) and Cys(Trt), and after condensation of Nsc-Cys(Trt)-OH, the Nsc was not removed for subsequent reaction. 1,7 disulfide bond could be formed by \i oxidation. The peptide obtained as above was put in 800 ml filterable peptide reactor, 250 ml DCM was po and allowed to maintain equilibrium for 30 min. Additional 0.1M IT in 250 ml DCM was added to the reactor, mixed well for 2 hrs to perform oxidation. Progress o esm ured the reaction was monitored with HPLC (peptide analysis condition 2), and upon completion of the reaction, filtration and washing with 5x100 ml DMF for 10 min in each time, was conducted to completely remove residual I?. Additionally, the reaction product was washed with 3x100 ml DMF, 3x100 ml DCM, 3x100 ml MeOH and 3x100 ml heptane, dried under nitrogen, and vacuum-dried for 6 hrs to obtain 16.9g of dried peptideattached resin. The peptide was released under mild acidic condition with protecting group being maintained, the peptide was treated with 1% TFA in 150 ml DCM for 2 min and filtered, and thus obtained solution was treated with 0.5% TFA in 100 ml DCM for 1 min, and this solution was immediately neutralized by pyridine in an amount equal to the consumed TFA. This solution was vacuum concentrated to 1/4 volume, treated with 50 ml ethanol, and subjected to re-concentration to final volume of about 5C Water (100ml) was added to this solution to precipitate, and the precipitate separated via centrifuge, and the precipitate was washed with 2x50 ml water. ml. was The precipitate was sufficiently dried under vacuum to obtain 6.8 g of Nsc-AAsCt(l-10)i-OH (93% HPLC purity). [Peptide analysis condition 2] Instrument: Waters Alliance Flow rate: 1.0 ml/min Gradient: 0-50 min, B 40-100% (A: 0.1% TFA in water, B: 0.1% TFA in AcCN) Column: Nova-Pak-Ci8, 3.9mm x 150 mm, 5 u , 100 A Maldi Tof; 1663.23, (M+l=1662.09) HPLC; 93% up (peptide analysis condition 2) Example 13-3. Synthesis of ll-Na-Fmoc-ll-ivDde-18-Boc- AAsCt(ll-32)-link amide resin Structure: Fmoc-Lys(ivDde)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-:Leu-His(Trt)- Lys(Boc)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-TlTr(tBu)- Gly-Ser(tBu)-Gly-Thr(tBu>Pro-link amide resin [ Table 16] (Table Removed) 2.0 g of link amide resin was put in peptide reactor, 20 ml of DCM was poured and left for 30 min to allow the reaction to reach equilibrium. Removal of Fmoc attached to the resin was conducted by reacting with 2x20 ml of deblocking solution (1%DBU-20% piperidine in DMF) for 5 min in each tune, and washed the resin thoroughly with 5x20 ml of DMF. According to the sequence of peptide, condensation and deblocking was repeatedly conducted as described in Example 1 starting from C-terminus by using reagents shown in the above Table. The reaction order was Pro, Thr(tBu), Gly, Ser(tBu), Gly, Thr(tBu), Asn(Trt), Thr(tBu), Arg(Pbf) Pro, Tyr(tBu), Thr(tBu), Gin (Trt), Leu, Lys(Boc), His(Trt), Leu, Glu(tBu),,Gln (Trt) Ser(tBu), Leu and Lys(ivDde). After carrying out condensation of Fmoc-Lys(ivDde)- OH, while allowing the Fmoc to be maintained, the resin was washed with 4x20 m DMF and 4x20 ml DCM, dried under nitrogen to obtain 6.5 g of peptide-attached resir (UV spectrum assay; capacity 0.14 mmol/g). Example 13-4. Synthesis of 1,11-diFmoc-saImon calcitonin based on condensation of Nsc. AAsCt(l-10)-OH and Fmoc- AAsCt(ll-32).-link amide resin Structure: Fmoc-Cys'-Ser-Asn-Leu-Ser-Thr -Cys7-Val-Leu-GIyIO-Lys(Fmoc) - Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln20-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser- Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 17] (Table Removed) Fmoc- AALs Ct(ll-32)-link amide resin (3.85g) prepared as described ah )ve was put into peptide reactor, allowed to sufficiently expand with 30 ml DCM. Solution within the reactor was removed, and treated with 2x20 ml deblocking solution (1% DBU-20% piperidine in DMF) for 5 min in each time to remove Fmoc, iind sufficiently washed 7-9 times with 20 ml of DMF while checking removal of residual by chloranil test. The peptide fragment Nsc- AAsCt(l-10)-OH (1.25 g) prepared in the abbve was dissolved in 4 ml DMF, HOBt(0.108 g) and DIPEA(0.160 ml) were put and the temperature of the reaction solution was lowered to 0-5 °C using an ice bath. Urjder nitrogen, Bop(0.332 g) was put to the reaction solution and mixed well for 10 min while the temperature was .maintained at 0-5°C. The reaction solution was poured to the peptide reactor in which the resin was placed and mixed by shaking for 10 min, and DIPEA(0.100ml) was added. Reaction was proceeded at ordinary temperature for about 12 hr and progress of reactipn was determined based on Kaiser test and analytical HPLC(peptide analysis condition 1). Upon completion of reaction, the resin was washed with 3x20 ml DMF, treated with 2% hydrazine 3x 20 ml for 10-30 min to remove base-labile Nsc and ivDde. The removal of ivDde was confirmed from TLC analysis for which the treated solution and 2% hydrazine solution were spotted, respectively, on TLC plate and their UV absorbance was compared. Upon completion of removal, the resin was filtered and washed with 4x20 ml DMF, 4x20 ml DCM, 3x20 ml DMF, and Fmoc-Osu (3.37 g in 20 ml DMF) was added and reacted for 2 hrs to introduce diFmoc. The introduction of Fmoc was checked by qualitative Kaiser test, and the resin was washed with 4x20 ml DMF and 4x20 ml DCM, dried under nitrogen to obtain 4.3 g of peptide attached resin. Reaction solution obtained by reacting the resin obtained as above with 40 ml of TFA-cleavage solution (2.5:2.5:95=TIS:water.TFA) at ordinary temperature for about 2 hrs, was filtered, added to 300ml of cold ether to induce precipitation of peptide. The precipitate was separated via centrifuge, further washed with 2x100 ml of ethur and dried to obtain 2.6 g of peptide mixture. The peptide mixture was purified by prep- HPLC, concentrated and dried with a freeze dryer to obtain 1036 mg of 1,11-diFmocsalmon calcitonin. Maldi Tof; 3877.41, (M+l=3877.44) HPLC; 98% up (peptide analysis condition 1) Example 14. Fragment condensation synthesis of 1,11-cHNsc-saImon calcitomn using ivDde as a protecting group for branched amine of untargeted site Structure: Nsc-Cys'-Ser-Asn-Leu-Ser-Thr -Cys7-Val-Leu-Gly10-Lys(>jsc)- Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln20-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Serr Gly30-Tlu-Pro-NH2 (1,7-disulfide bond) [ Table 18] (Table Removed) Fragment condensation was conducted according to the method as in Exam 13 by using Fmoc-AAsCt(ll-32')-lmk amide resin (3.57g) and Nsc-AAssCutt(/ l-10)-( prepared in Example 13 and the solvents and reagents shown in the above Table, diNsc was introduced by reaction with Nsc-OSu(3.72 g in 20 ml DMF) to obt peptide attached resin 4.8 g. Peptide mixture obtained from said peptide attached re and TFA cleavage solution (2.5:2.5:95=TIS:water:TFA) was separated and purified obtain 1228 mg of 1,11-diNsc salmon calcitonin. Maldi Tof; 3947.40, (M+l=3947.38) HPLC; 98% up (peptide analy condition 1) Example 15. Fragment condensation synthesis of 1,18-diFmoc-salmon calcito using ivDde as a protecting group for branched amine of untargeted site Example 15-1. Synthesis of ll-Na-Nsc-ll-Boc-18-ivDde- AAsCt(ll-32)-link am le nd in in to sis resin Structure: Nsc-Lys(Boc)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-His(Trt)- Lys(ivDde)-Leu-Gln(Trt)-Tlir(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr(tE Gliy-Ser(tBu)-Gly-Thr(tBu)-Pro-link amide resin [ Table 19] (Table Removed) Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, N: Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), N; Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Fmoc-Lys(ivDde), Nsc-His(Trt), Nsc-Leu, N Glu(tBu), Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Nsc-Lys(Boc) Synthesis was conducted according to the same method as in synthesis Fmoc-AAsCt(ll-32)-link amide resin of Example 13, and as a result, 6.8 g of wellresin was obtained and peptide capacity of the resin was determined as 0.13 mmol/g UV spectrum analysis (UV-spectrum assay; capacity 0.13 mmol/g). Example 15-2. Synthesis of 1,18-diFmoc-salmon calcitonin based on condensat of Nsc-AAsC'(l-10)-OH with Nsc-AAsCt(ll-32)-link amide resin Structure: Fmoc-Cys'-Ser-Asn-Leu-Ser-Thr -Cys7-Val-Leu-Gly10-Lys-L Ser-Gln-Glu-Leu-His-Lys(Fmoc)-Leu-Gln20-Tlor-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser- Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 20 ] (Table Removed) Fragment condensation was conducted according to the method the same as that in Example 13 by using Nsc- AAsCt(ll-32)-link amide resin (3.85g) prepared in the above and Nsc-AAsC'(l-10)-OH (1.2g) synthesized in Example 13 and the solvent and reagents shown in the above Table, and diFmoc was introduced by use of Fmoc-OSu to obtain 4.2 g of peptide attached resin. Peptide mixture obtained by reacting! said peptide attached resin with TFA cleavage solution (2.5:2.5:95=TIS:water:TFA) was separated and purified to obtain 949 nag of 1,18-diFmoc salmon calcitonin. Maldi Tof; 3877.41, (M+l =3877.44) HPLC; 98% up (peptide analysis condition 1) Example 16. Fragment condensation synthesis of 1,18-diNsc-saImon calcitonin using ivDde as a protecting group for branched amine of untargeted site Structure: Nsc-Cys'-Ser-Asn-Leu-Ser-Thr -Cys7-Val-Leu-Gly10-Lys-Leu-S^er- Gln-Glu-Leu-His-Lys(Nsc)-Leu-Gln20-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GIy-Ser-Gly30- Thr-Pro-NH2 (1,7-disulfidebond) [ Table 21] (Table Removed) Fragment condensation was conducted according to the method as in Ex 13 by using Nsc-AAsCt(ll-32)-link amide resin(3.85g) prepared iri Example 15, AAsCt(l-10)-OH (1.2g) prepared in Example 13 and the solvents and reagents shoivn the above Table, and diNsc was introduced by use of Nsc-OSu (3.72 g in 20 ml to obtain 4.2 g of peptide attached resin. Peptide mixture obtained by reactin; peptide attached resin with TFA cleavage solution (2.5:2.5:95=TIS:water:TFA separated and purified to obtain 941 mg of 1,18-diNsc salmon calcitonin. Maldi Tof; 3947.41, (M+l=3947.38) HPLC; 98% up (peptide analysis condition 1) mple Nscin DMF) said was Example 17. Fragment condensation synthesis of 11,18-diFmoc-salmon calc using ivDde as a protecting group for branched amine of untargeted site tonin Example 17-1. Synthesis of ll-Na-Fnioc-ll,18-di(ivDde)-AAsCt(ll-32)-link Jimide resin Structure: Fmoc-Lys(ivDde)-Leu-Ser(tBu)-Gln(Trt)-Glu(tBu)-Leu-Hi Lys(ivDde)-Leu-Gln(Trt)-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pbf)-Thr(tBu)-Asn(Trt)-Thr Gly-Ser(tBu)-Gly-Thr(tBu)-Pro-link amide resin (Trt)- tBu)- [ Table 22] (Table Removed) Reaction order: Nsc-Pro, Nsc-Thr(tBu), Nsc-Gly, Nsc-Ser(tBu), Nsc-Gly, N Thr(tBu), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Arg(Pbf), Nsc-Pro, Nsc-Tyr(tBu), Thr(tBu), Nsc-Gln(Trt), Nsc-Leu, Fmoc-Lys(ivDde), Nsc-His(Trt), Nsc-Leu, Glu(tBu), Nsc-Gln(Trt), Nsc-Ser(tBu), Nsc-Leu, Fmoc-Lys(ivDde). Synthesis was conducted according to the method as in Example 13, and result, well-dried resin 6.1 g was obtained, and peptide capacity of the resin determined to be 0.15 mmol/g by UV spectrum analysis (UV-spectrum assay; capa 0.15 mmol/g). Example 17-2. Synthesis of 11,18-diFmoc-salmon calcitonin by condensatior Nsc-AA, ssCctt(/l-10)-OH with Nsc-AAsCl(ll-32)-link amide resin Structure: H-Cys'-Ser-Asn-Leu-Ser-Thr-Cys7-Val-Leu-Gly10-Lys(Fm Leu-Ser-Gln-Glu-Leu-His-Lys(Fmoc)-Leu-Gln20-Thr-Tyr-Pro-Arg-Tlir-Asn-Thr-Gl) scscs a as bity of .30 Ser-GlyJ-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 23] (Table Removed) Fragment condensation was conducted according to method as in Example by using Fmoc-AAsCt(ll-32)-link amide resin (3.33 g) synthesized previously and Nsc- AAL sCt(I-IO)-OH (1.2 g) prepared in Example 13 and the solvents and reagents showr the above Table. After the condensation, the removal of Nsc was performed reacting with 2x20 ml deblocking .solution (1% DBU-20% piperidine in DMF) fo min in each time, and washed with 4x20 ml DMF and reacted with excess amount Boc2O (0.55 g in 20 ml DMF) to protect N-terminal end with Boc, and washed with 5x20 ml DMF thoroughly. Then, the reaction product was treated with 2% hydrazine 3x20 ml for 10 to 30 min to remove base-labile ivDde, and the removal of ivDde was confirmed by TLC analysis based on comparison of the UV-absorbance between the reaction solution and 2% hydrazine. Upon completion of removal of ivDde, the resin was filtered and washed with 4x20 ml DMF, 4x20 ml DCM, 4x20 ml DMF, Fmoc- OSu(3.37 g in 20 ml DMF) was added and reacted for 2 hrs to introduce diFmoc. introduction of diFmoc was checked by qualitative Kaiser test, the resin was washed with 4x20 ml DMF and 4x20 ml DCM, and dried under nitrogen to obtain 4.5 g peptide attached resin. Reaction solution obtained by reacting the resin obtained above with 40 m TFA-cieavage solution (2.5:2.5:95=TIS:water:TFA) at ordinary temperature for about 2 hrs, was filtered, added to 300ml of cold ether to induce precipitation of peptide. precipitate was separated by centrifuge, further washed with 2x100 ml of ether 13 in by 5 of he of of 'he and dried to obtain 2.6g of peptide mixture. The peptide mixture was purified by prepi HPLC. concentrated and subjected to lyophilization to obtain 769 mg of 11,18-diFrr.ocsalmon calcitonin. Maldi Tof; 3877.41, (M+1=3S77.44) HPLC; 98% up (peptide analysis condition 1) Example IS. Fragment condensation synthesis of 11,18-diNsc-salmon calcitonin using ivDde as a protecting group for branched amine of untargeted site Structure: H-Cys'-Se^Asn-Leu-Ser-Thr -Cys7-Val-Leu-Gly'°-Lys(Nsc)-L^ Ser-Gln-Glu-Leu-His-Lys(Nsc)-Leu-Gln20-Tlir-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser- Gly30-Thr-Pro-NH2 (1,7-disulfide bond) [ Table 24] (Table Removed) Fragment condensation was conducted according to the method as in Example 13 by using Fmoc-AAsCt(ll-32)-link amide resin (3.33 g) synthesized in .Example and Nsc-AAsCt(l-10)-OH (1.2g) synthesized in Example 13 and the solvents £ reagents shown in the above Table, and Boc (0.55 g in 20 ml DMF) was introduced described in Example 17, ivDde was removed, and diNsc was introduced by use of N OSu (3.72 g in 20 ml DMF) to obtain 4.3 g of peptide attached resin. Peptide mixtiire obtained by reacting said peptide attached resin' with TFA cleavage solution v/as separated and purified to obtain 942 mg of 11,18-diNsc salmon calcitonin. Maldi Tof; 39.47.39, (M+1=3947.3S) HPLC; 98% up (peptide analyjsis condition 1) Example 19. Fragment condensation synthesis of l,12-diFmoc-GRF(l-29) us ivDde as a protecting group for branched amine of untargeted site ng Example 19-1. Synthesis of Nsc- AAGRF(1-15)-OH Structure: Nsc-Tyr(tBu)-Ala-Asp(tBu)-Ala-Ile-Phe-Thr(tBu)-Asn(I):t)- Ser(Trt)-Tyr10(tBu)-Arg(Pbf)-Lys(ivDde)-Val-Leu-Gly-OH [ Table 25] (Table Removed) Reaction order: Nsc-Leu, Nsc-Val, Fmoc-Lys(ivDde), Nsc-Arg(Pbf), Nsc- Tyr(tBu), Nsc-Ser(Trt), Nsc-Asn(Trt), Nsc-Thr(tBu), Nsc-Phe, Nsc-Ile, Nsc-Ala, rfsc- Asp(tBu), Nsc-Ala, Nsc-Tyr(tBu) Nsc-Gly-2-CLTR synthesized as in Example 13 was put in 200 ml pep ide reactor, 50 ml DCM was poured and left to allow the resin to sufficiently expjind. DCM was removed by filtration and it was treated with 2x50 ml deblocking solui (1% DBU-20% piperidine in DMF) for 5 min in each time to remove Nsc. Then, the next reaction, the resulting mixture was washed 5-7 times well with Di Coupling reaction was conducted as described in Example 1 according to the reac order indicated above by using Nsc- or Fmoc-protected amino acid (1.5 eq), HOBt' ion for V1F. ion 1.5 eq), Bop(l.5 eq) and DIPEA(3.0 eq). The reaction was checked by Kaiser test and| the presence of amine in the washing solution was confirmed by chloranil test. Thus obtained peptide attached resin was treated for 3 hrs at condition unlder which the peptide could be separated from 2-CTRL resin with other protecting gr being maintained, that is, 100 ml DCM:TFE:AcOH (8:1:1), the reaction solution then separated by filtration and the resin was washed with 2x100 ml DCM. "he reaction solution and the washings were collected and concentrated to 1/4 volume, diluted with 100 ml ethanol and 100 ml water and left in a refrigerator for 12 1 Solid thus formed was filtered and dried to obtain 12.6 g of Nsc-AAGRF(l-15)-OH. ITS. Maldi Tof; 3144.09, (M+l=3143.95) HPLC; 92% up (peptide anal) condition 2) Example 19-2. Synthesis of Nsc-AAUGKR*F(/16-29)-Iink amide resin Structure: Nsc-Gln(Trt)-Leu-Ser(Trt)-Ala-Arg(Pbf)-Lys(Boc)-Leu-L Gln(Trt)-Asp(tBu)-Ile-Met-Ser(Trt)-Arg(Pbf)-link amide resin [ Table 26] (Table Removed) Reaction order: Nsc-Arg(pbf), Nsc-Ser(Trt), Nsc-Met, Nsc-Ile, Nsc-Asp(tBu) Nsc-Gln(Trt), Nsc-Leu, Nsc-Leu, Nsc-Lys(Boc), Nsc-Arg(Pbf), Nsc-Ala, Nsc-SerqYt), Nsc-Leu, Nsc-Gln(Trt) Coupling and deblocking were conducted as described in Exampl^ 1 according to the indicated reactjon order by using link amide resin 2.0 g and reagents and solvents listed in the above Table. The coupling was checked by Kaiser test, removal of side reactants remaining in the washing was confirmed by chloranil test. Peptide attached resin finally obtained was 6.5 g, and reaction capacity was determined to. be 0.12 mmol/g based on LTV-assay. Example 19-3. Synthesis of l,12-diFmoc-GRF(l-29) by condensation of rffse AAGRF(1-15)-OH with Nsc- AAGRF(16-29)-link amide resin Structure: Fmoc-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr' °-Arg- Lys(Fmoc)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg20-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser Arg-NH2 [Table 27] (Table Removed) Nsc-AA (16-29)-link amide resin(4.17 g) synthesized as above was placed on an adequate peptide reactor. To the reactor, 30 ml DCM was poured to allow the resin to sufficiently expand, and the solution was removed via filtration, treated with 2x20 ml deblocking solution (1% DBU-20% piperidine in DMF) for 5 min in each ime to remove Nsc. The resin was washed thoroughly with DMF, and Nsc-AAGRF(l-15)- OH (2.36 g) prepared previously was put to the reactor by dissolving in 10 ml DMSO. HOBt, DIPEA, Bop etc., listed in the above Table were added to the reaction sohltion and reacted for 12 hrs. Progress of reaction was checked by HPLC every 2 hrs when confirmed to be completed, the reaction solution was thoroughly washed and with DMF. After fragment condensation was completed, the resin was treated with 2% hydrazine in DMF (3x20 ml) for 20 min in each time to remove ivDde and Nsc. The removal of ivDde was confirmed by TLC analysis as described in Example 4, and the resulting product was thoroughly washed with washing solvent. Fmoc was introduced to 1,12-positions by treating with Fmoc-OSu(3.37 g in 20 ml DMF) for 2 hrs, the r^sin was washed well, subjected to vacuum dry to obtain 4.3g of peptide attached resin. 1 g of said dried resjn.was withdrawn and reacted with 10 ml cleavij solution (2.5:2.5:95=TIS:water:TFA) at ordinary temperature for about 90min zmd further reacted with TMS-Br and EDT for 15min. Resin was removed via filtration and the resin was washed with 2 ml TFA. The washing and filtrate were collected, added to 100 ml ether and subjected to centrifuge to allow formed precipitate to be precipitated, and additionally centrifuged and washed with 3x50 ml ether, precipitate was dried with nitrogen to obtain 654 mg of dried peptide mixture, and this mixture was purified with prep-HPLC, lyophilized to obtain 141 nig of 1,12-diFmoc- GRF(l-29). Maldi Tof; 3803.49, (M+l=3803.46) HPLC; 97% up (peptide anah condition 1) Example 20. Preparation of Lys -PEG 2K-salmon calcitonin using 1,11-cliFnioc salmon calcitonin 1,11-diFmoc salmon calcitonin lOmg eq was dissolved in DMF 1 ml, and after 0.2% TEA was added and poly(ethyleneglycol) 2,000 succinimidyl propionale 5 eq was added and reacted at 45°C for 1 hr. Piperidine 50 ul was added and reacted 5 min to remove Fmoc, and then acidified by addition of 10% trifluoroacetic acid/acetonitrile 500 ul This reaction solution was diluted 10 times with 20 mM sodium acetate buffer (pH 4.5) and purified by ionic exchange chromatography urjder 'SIS for the following condition. Effluent was added and allowed to adhere to C-18 Sep-Pak catridge, washed with 20ml distilled water, and eluted with 5ml of 70% acetonitrile. Acetonitrile in the effluent was evaporated under nitrogen and subjected to lyophilization. When determined as described in Experimental Example 1, reverse chromatogram of said sample (Fig. 1-B) showed a single peak, and unreacted s calcitonin, di-PEG 2K-salmon calcitonin, tri-PEG 2K salmon calcitonin etc., b mono-PEG 2K salmon calcitonin were not identified by MALDI-TOF mass spe (Fig. 2). In addition, MALDI-TOF mass spectrum for Lys-C enzyme treated fragment measured as described in Experimental Example 1 was exhibited only the corresponding to the fragment, Lysls-PEG 2K-GRF(l-29) (Fig. 3-B). Whe sample was dissolved in distilled water and determined against salmon calc standard based on reverse phase chromatography as described in Experimental Ex 1, yield was 87.6%. [Ionic exchange chromatography condition] Column: TSK SP-5PW (55 x 200 mm, 15 um)(strong cation- exchange media) Eluent Eluent A: 20 mM sodium acetate(pH 4.5) Eluent B: 300 mM sodium chloride/20 mM sodium acetate(pH 4.5) Eluent C: 1 M sodium chloride/20 mM sodium acetate(pH 4.5) 00-1 Omin: 100% A 10-40 min: 0% A -» 100% B 40-5.0 min: 100%C Flow rate: 3 ml/min hase mon ides trum peak the onin nple Detection: UV 215 nm Comparative Example 1. Preparation of mono-PEG 2K-salmon calcitonin us ng salmon calcitonin Salmon calcitonin 10 mg'was dissolved in 5 ml of 10 mM sodium phosphate buffer (pH 7.5) and poly(ethyleneglycol) 2,000 succinimidyl propionate 1.5 eq was added and reacted at ordinary temperature for 20 min, and then 1M glycine solution 50 ul was added and left for 30 min to complete the reaction. The reaction solution was divided in 5 parts by using size-exclusion chromatography to separate mono-P conjugate. The effluent was collected and allowed to adhere to C-18 Sep-Pak catridge, washed with 20 ml of distilled water and eluted with 5 ml of 70% acetonitijile. Acetonitrile in the effluent was evaporated under nitrogen stream and lyophilized. When determined as described in Experimental Example 1, reverse ph cliromatogram of this sample showed three peaks indicating Cysl-Na-PEG 2K-salmon calcitonin, Lysu-PEG 2K-salmon calcitonin and Lys18-PEG 2K-salmon calcitonin (]:ig. 1 -A) and the ratio of peak area was approximately 1:1:1. The sample was dissolved in ase distilled water and determined based on reverse phase chromatogram as describee Experimental Example 1 against salmon calcitonin standard to obtain yield of 28.4% [Size-exclusion chromatography condition] Column: Superose 12 HR 10/30 (Amersham pharmacia) Eluent: 10 mM PBS (pH 7.4) Flow rate:0.4 ml/min Detection:UV215nm m Experimental Example 1. Identification of Lys -PEG 2K-saImon calcitonin Experimental Example 1-1. Identification and determination of isomers bas reverse phase chromatography Samples obtained in Example 20 and Comparative Example 1 were com by reverse phase chromatography under the following condition according t method described in Pharm. Res., 16(6), 813-818,1999. While the sample in Ex£ 20 exhibited a single peak indicating Lysls-PEG 2K-salmon calcitonin (Fig. 1-B sample in Comparative Example 1 showed peaks corresponding to Cys'-Na-PEG salmon calcitonin, Lys11-PEG 2K-salmon calcitonin and LysIS-PEG 2K-sa calcitonin in a ratio of 1:1:1 (Fig. 1-A). Yield of Example 20 was 87.6% calculated into ratio of peak area of Lys18-PEG 2K-salmon calcitonin to that of sa calcitonin standard. In Comparative Example 1, the overall yield for mono conjugate was 28.4%, when calculated based on the ratio of total peak area of the positional isomers against that of salmon calcitonin standard, and yield for Lys18 2K-salmon calcitonin (peak 2) was 10.2%. [Reverse phase chromatography condition] Column: LichrospherlOO RP-8 (4 mm ID x 250 mm L, 5 |im, Merck) Mobile phase Eluent A: 0.1% TFA/D.W. EluentB:0.1%TFA/AcCN 00-30 min: 36% B -> 44% B Flow rate: 1 ml/min Detection: UV 215 nm on ared the iple the 2Kmon hen non EG iree EG Experimental Example 1-2. Identification of side reactants and isomers MALDI-TOF mass analysis The sample of Example 20, when analyzed under the following MALDI-' mass analysis condition, showed .the only peak corresponding to mono-PEG 2K-salmon calcitonin, and unreacted salmon calcitonin, di-PEG 2K-salmon calcitonin, tri-PEG 2Ksalmon calcitonin etc., were not exhibited (Fig. 2). MALDI-TOF mass spectrum peptide fragment obtained by treating the sample from the Example 20 with L] enzyme was shown in Fig. 3 in comparison with salmon calcitonin standard which was treated identically. The Lys-C enzyme-treated sample was prepared by reacting the sample 10 ul with 5 ul of 50 mM tris buffer (pH 8.5) containing Lys-C enzyme (0.1 |ig/ml) for 1 hr at 37°C and by mixing with matrix solution in a ratio of 1:2. MALDITOF mass spectrum for Lys-C enzyme treated fragment of salmon calcitonin (Fig. • confirmed peaks corresponding to Pro'-Gly10 fragment, Lysll-His17 fragment and L] by 'OF for s-C -A) 'SISpro32 fragment, yet the MALDI-TOF mass spectrum for Lys-C enzyme treated fragment of Example 20 sample (Fig. 3-B) exhibited only the peaks corresponding to Pro'-Gly1 fragment and PEG-attached Lys11 -pro32 fragment, and peaks for Lys "-His17 fragment and Lys18-pro32 fragment were not detected. Based on this result, it could be confiijmed that the sample of Example 20 is Lys18-mono-PEG 2K-salmon calcitonin where PEjG is I 9 combined to only Lys position. [MALDI-TOF mass analysis condition] Ion selection: positive ion Matrix solution: cc-cyano-4-hydroxy cinnamic acid saturated solution (0.1% TFA/|50% AcCN/DW) Mode: linear Sample:matrix =1:2 Accelerating voltage : 25 kV Example 21. Preparation of Lys18-PEG IK-salmon calcitonin, Lys18-PEG 5Ksalmon calcitonin, and Lys18-PEG lOK-salmon calcitonin using 1,11-diFmocsalmon calcitonin According to the method as described in Example 20, Lys -PEG IK-salmicon calcitonin, Lys -PEG 5K-salmon calcitonin, and Lys 18-PEG lOK-salmon calciton n were prepared, respectively, by using 1,11-diFmoc-salmon calcitonin ar.d poly(etyhyleneglycol) 1,000 succinimidyl propionate, poly(ethyleneglycol) 5,0(10 succinimidyl propionante and poly(ethyleneglycol) 10,000 succinimidyl propionate. When individual samples were determined by the procedure described in Example 1, the yield was 86.9%, 81.5% and 82.7%, respectively, and no other peptide-deriv^d substance or isomer except Lys18-PEG conjugate was identified. Example 22. Preparation of Lys18-PEG 2K-salmon calcitonin using 1,11-diN^csalmon calcitonin 1,11-diNsc salmon calcitonin lOmg eq was dissolved in 1 ml DMF and 0.2% TEA was added, and then poly(ethyleneglycol) 2,000 succinimidyl propionate 5 eq was added and reaction was conducted for 1 hr at 45°C. Piperidine 100 ul was added and reacted for 5 min to remove Nsc, and then acidified by 500 (J.1 of 10% trifluoroacptic acid/acetonitrile. This reaction solution was purified under the same condition ap in Example 20, and weighted against salmon calcitonin with the method of Experimental ise :nt or Example 1 to obtain yield of 84.8%. Additionally, in case of both reverse ph chromatogram and MALDI-TOF mass spectrum for Lys-C enzyme treated fragm obtained as described in Experimental Example 1, no peptide-derived substance isomer except Lys18-PEG 2K-salmon calcitonin was identified. Example 23. Preparation of Lys21-PEG 5K-GRF(l-29) using 1,12-diFmoc- GRF(l-29) l,12-diFmoc-GRF(l-29) 10 mg eq was dissolved in DMF 1 ml and 0[2% TEA was added, and then poly(ethyleneglycol) 5,000 succinimidyl propionate 5 eq was added and reaction was conducted at 45°C for 1 hr. Piperidine 50 ul was added and reacted for 5 min to remove Fmoc, and then acidified by 500(j.l of 10% trifluoroacetic acid/acetonitrile. This reaction solution was purified by the same method as Example 20 to obtain PEG conjugated peptide. When this sample was determine described in Experimental Example 2, reverse phase chromatogram of the sample (Fig. 4-B) exhibited a single peak, and MALDI-TOF mass spectrum thereof also exhibited only the peak corresponding to mono-PEG 5K-GRF(l-29) and unreacted GRF(l-29), di-PEG 5K-GRF(l-29) and tri-PEG 5K-GRF(l-29) were not identified (Fig. 5). In addition, MALDI-TOF mass spectrum for Lys-C enzyme treated fragment, which was obtained as described in Experimental Example 2, showed only the peak corresponding to Lys21-PEG 5K-GRF(l-29) fragment (Fig. 6-B). When the sample was dissolved in distilled water and subjected to analysis based on reverse phase chromatograph I described in Experimental Example 2 against GRF(l-29) standard, the yield was 91. Experimental Example 2. Identification of Lys21-PEG 5K-GRF(l-29) in as as According to reverse phase chromatogram obtained under the following condition, the sample of Example 23 showed a single peak corresponding to mono-FfEG 5K-GRF(l-29)(Fig. 4-B), and did not exhibit the peak corresponding to unreacted GRP(l-29) (Fig. 4-A) or other peak. In addition, MALDI-TOF mass spectrum obtained by the method as in Experimental Example 1 did not exhibit other peptidederived peaks except mono-PEG 5K-GRF(l-29) (Fig. 5). Further, MALDI-TOF m ss spectrum for Lys-C enzyme treated fragment obtained by the method as in Experimental Example l(Fig. 6-B) also exhibited only the peaks for Try1 ~Argu fragment and Lys Arg29 fragment to which PEG 5K was combined, yet the peaks for Lys12 ~Arg20 fragment or Lys21 ~Arg29 fragment which were identified in MALDI-TOF mass spectrum obtained for GRF(l-29) identically treated (Fig. 6-A), were not identified. Based on this result, it could be confirmed that the sample of Example 23 is Lys21- mono-PEG 5K-GRF(l-29) in which PEG is combined to only Lys21 position. [Reverse phase chromatography condition] Column: LichrospherlOO RP-8 (4 mm ID x 250 mm L, 5 u-m, Merck) Mobile phase Eluent A; 0.1% TFA/D.W. EluentB;0.1%TFA/AcCN 00-15 min: 34% B -» 55% B Flow rate: 1 ml/min Detection: UV215 nm Example 24. Preparation of Lys21-PEG lK-GRF(l-29) using l,12-diNsc-GRF(l-2^9) l,12-diNsc-GRF(l-29) 10 mg eq was dissolved in 1 ml of DMF and 0.2|% TEA was added, then poly(ethyleneglycol) 1,000 succinimidyl propionate 5 eq was added and reaction was conducted at 45 °C for 1 hr. Piperidine 100 ul was added and reacted for 5 min to remove Nsc, and then acidified by 10% trifluoroacetic acid/acetonitrile 500 μl. This reaction solution was purified under the same condition as in Example 20, and then subjected to analysis by reverse phase chromatography described in Experimental Example 2 against GRF(l-29) standard to obtain yield 92.8%. In addition, MALDI-TOF mass spectrum obtained as described as of in Experimental Example 1 showed only the peak corresponding to mono-PEG IKGRF( l-29), and MALDI-TOF mass spectrum for Lys-C enzyme treated fragment a.so showed only the peak corresponding to Lys21-PEG lK-GRF(l-29) fragment. Industrial applicability The present invention enables synthesis of peptides having selectively protected amine of untargeted sites simply by changing acid-base conditions. Accordingly, when compared to conventional methods for the production of PEG conjugated peptides in which PEG is specifically combined to amine of targeted site, the present invention has following advantages; the final products can be obtained with much higher yield, complicated separation and purification is not necessary, thus the present invention provide economical methods, and formation of side products inevitably accompanied by the conventional process is inhibited, thus, the product according to the present invention is more adequate for clinical use. Therefore, when the PEG conjugated peptide of the present invention is used for clinical use, PEGylation effect can be maximized. We Claim: 1. A method for preparing peptides having selectively protected amines of untargeted sites, characterized in that a. synthesizing the peptides by separately blocking amines of targeted sites of the peptides with either ivDde or Mtt and amines of untargeted sites of the peptides with Boc, and b. protecting Na-amine of the peptides with Fmoc or Nsc, wherein the targeted sites are sites to be intended to conjugate with polyethyleneglycol (PEG) to prepare specifically conjugated PEG-peptide and the untargeted sites are sites to be intended to remain as free amine after conjugating targeted sites with PEG. 2. The method as claimed in claim 1, optionally substituting the amine protecting groups for amines of the untargeted sites and for the N°-amine with at least one final amine protecting group selected from the group consisting of Fmoc, Nsc, Dde and ivDde. 3. The method as claimed in claim 1, optionally substituting the amine protecting groups for amines of the untargeted sites and for the N°-amine with Boc. 4. The method as claimed in claim 1, wherein the synthesis of the peptides is performed by solid phase synthesis. 5. The method as claimed in claim 1, wherein the fragments of the peptides are synthesized separately, and then are condensed to form the peptides. 6. Peptides having selectively protected amines of untargeted sites as and when prepared by the method as claimed in any of the preceding claims 1 to 5. 7. The peptides as claimed in claim 6, wherein said peptides is calcitonin or GRF(1-29). 8. A method as claimed in claim 1, wherein specifically conjugated PEG-peptide in which PEG is specifically conjugated to amines of targeted sites, comprises the steps of: a. reacting the peptide as claimed in claim 6 with activated PEG; and b. removing the amine protecting group of the compound obtained in the step (1) under acid-base deblocking conditions. 9. The method as claimed in claim 8, optionally involves a step of purifying the product of step (2). 10. The method as claimed in claim 9, wherein said purification step comprises separating the product by ionic exchange chromatography, removing salt and then drying. 11. The method as claimed in claim 8, wherein said activated PEG is linear or branched hydroxyl- or methoxy-type alkylating or acylating PEG of molecular weight in a range of 1,000 to 40,000. 12. The method as claimed in claim 11, wherein said activated PEG is at least one selected from the group consisting of mono-methoxy poly(ethyleneglycol)succinimidyl succinate, mono-methoxy poly(ethyleneglycol)succinimidyl propionate, mono-methoxy poly(ethyleneglycol)succinimidyl carbonate, mono-methoxy poly(ethyleneglycol)succinimidyl carbamate and mono-methoxy poly(ethyleneglycol)succinimidyl tresylate.

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2929-DELNP-2005-Abstract-(25-05-2009).pdf

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