PROCESS

The present invention relates to a process for the preparation of peptides and proteins by solid phase synthesis, to peptides and proteins obtainable by such a process, and to intermediates useful in such a process.

Peptides and proteins are composed of amino acids. There are about 20 different amino acids commonly available in nature, and they are linked together in long chains to form peptides. Biologically active peptides, consisting of between 2 and 50 amino acids, span a wide range of functions in nature: hormones, chemokines, neurotransmitters, cytokines and immunological agents. They have also been shown to be effective as prophylactic and therapeutic vaccines as well as enzyme inhibitors.

Protein therapeutics has emerged as one of the most promising segments of the pharmaceutical market since the introduction of recombinant insulin in 1982. To produce these important drugs commercially, companies have focused to date on biological approaches such as recombinant-DNA expression methods (microbial fermentation and mammalian cell culture) and native protein isolation. However numerous problems are associated with these methods:

a) limited supply of product is possible b) viral contamination risk c) product heterogeneity d) inability to produce some proteins e.g. those that are toxic to the cell e) non-human post-translational modifications, i.e. incorrect glycosylation or folding f) time-consuming g) structural modifications are limited to the 20 naturally occurring amino acids

Chemical protein synthesis provides a rapid and efficient route for the production of homogenous proteins containing up to 250 amino acids that are free of biological contaminants. In this field the development of solid-phase peptide synthesis (SPPS) by Merrifield in 1963 merited the award of Nobel prize in 1984 (Merrifield, R. B. (1963) J.Amer. Chem. Soc, 85,2149-2154). This method is still widely used. However this method originally only allowed efficient production of small peptides, for example up to

about 10 kDa, such as hormones and cytokines. Another significant limitation of this method is incomplete synthesis and side reactions.

PEG (polyethyleneglycol) conjugated biomolecules are known, and have been shown to possess clinically useful properties (Inada et al., J. Bioact. and Compatible Polymers 5,

343 (1990); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9, 249

(1992); Katre, Advanced Drug Delivery Systems 10, 91 (1993)). Among these are better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life, decreased clearance and enhancing potency. It has been reported that branched PEG conjugates exhibit increased pH and thermal stability and greater stability towards proteolytic digestion than linear PEG conjugates. (Monfardini et al., Bioconjugate Chem. 6, 62 (1995)). Other properties of PEG proteins are reduced immunogenicity and antigenicity, as well as reduced toxicity. A PEG conjugate of interferon-α disclosed in EP-A-0809996 has been commercialised for the treatment of hepatitis C.

N-terminal fixation of a PEG-containing derivatisation reagent to a resin-bound peptide fragment, and subsequent cleavage from the resin gave free N-terminal PEG containing peptides (Lu et al., Peptide Research, 6, 140, (1993)). PEGs have been attached to N- protecting groups, and employed in side-chain N-protection in solid-phase peptide synthesis using Fmoc chemistry (Mutter et al., Tetrahedron Lett, 35, 1039-1042 (1994)).

The present invention provides novel techniques for the synthesis of peptides and proteins without the limitations and disadvantages of previous methods.

According to a first aspect, the invention provides a process for the preparation of a solid support bound peptide of formula (I)

(I)

wherein: n is a positive integer greater than 1 m is a positive integer

W is a solid support

Y and Y' are independently selected from linker groups or a chemical bond

Q is a soluble polymer

R ¹ is hydrogen or a substituent and for each A, which may be the same or different, i) A represents an amino acid residue; or ii) A, taken together with R ¹ and N, forms a heterocycle (for instance in the case of proline) comprising reacting an activated peptide of formula (II) with solid support bound peptide of formula (III)

Q- -Y ¹ - NR ¹ - A — CO- -LG H- NR ¹ - A — CO- -Y- -W n m

(H) (III)

wherein n, m, W, Y, Y', Q, R ¹ and A are as defined above, and LG is a leaving group.

Surprisingly, it has beep found that the reaction of (II) and (III) proceeds rapidly and in good yield. This is despite the fact that it is known that fragment condensations are often difficult to accomplish, proceed in low yield, or are rather slow.

By use of the term "solid support" we mean the support onto which the amino acids are linked, optionally through a linker. The supports include solid and soluble solid materials

or matrixes, and resins. Preferably, the solid support is insoluble in the solvents in which the desired reactions take place.

The term "soluble polymer" refers to a polymeric moiety that confers solubility on a peptide to which it is linked, when that peptide would otherwise be insoluble. Solubility is assessed in the solvent in which the reaction of (II) and (III) occurs. Preferably, the solvents are dimethylsulphoxide, dimethylformamide, and dichloromethane.

In a preferred embodiment, the soluble polymer Q is selected from polymers comprising at least a portion of polyether.

In a particularly preferred embodiment, Q comprises at least a portion of polyethylene glycol (PEG). More preferably, Q is PEG.

The number average molecular weight of PEG may range from about 200 Da to about 100,000 Da, and preferably about 1 ,000 Da to about 50,000 Da, and more preferably about 2,000 Da to about 20,000 Da. Most preferably, the PEG is PEG 2000, ie PEG having a number average molecular weight of about 2000.

Preferably, the PEG has one terminal alkyl group. More preferably, the PEG is a polyethylene glycol monomethyl ether or mPEG.

By "leaving group" it is meant any chemical moiety that is capable of detachment from the acyl group of the amino acid with the concomitant formation of a new amide bond. Many suitable leaving groups will be known to those skilled in the art. Examples of particularly suitable leaving groups include:

i) derivatives of carbodiimides of formula (IV)

(IV)

wherein R ² and R ³ are independently C _1-10 hydrocarbyl groups, preferably cyclohexyl or isopropyl;

ii) derivatives of pentafluorophenol, hydroxybenzatriazole, hydroxysuccinimide, 1- hydroxy-7-azabenzotriazole, carbonyldiimidazole, 3-hydroxy-3,4-dihydro-4-oxo-1 ,2,3- benzotriazine, N-ethyl-5-phenylisoxazolium-3'-sulphonate;

iii) halides, particularly fluoride;

iv) derivatives of carboxylic acids (ie acid anhydrides).

A particularly preferred leaving group is oxybenzotriazole (-OBt).

Preferably, the activated peptide of formula (II) is prepared by treatment of the corresponding peptide of formula (V) with an activating agent.

(V)

By "activating agent" it is meant any reagent or combination of reagents that is capable of converting the free carboxylic acid group of an amino acid or peptide fragment to an activated form, in which the acyl carbon bears a leaving group LG as defined above. Many activating agents have proved useful in this capacity, and the skilled man will have little difficulty in selecting an appropriate one.

Preferred activating agents are selected from: i) carbodiimides, including 1.S-dicyclohexylcarbodiimide (DCC); 1- ethyl-3-(3'- dimethylaminopropyl)carbodiimide hydrochloride, (EDCI), N,N'-Diisopropylcarbodiimide (DIC) optionally with base;

ii) aminium / uronium based reagents, including 1-benzotriazol-1-yloxy- bis(pyrrolidino)uronium hexafluorophosphate, 5-(1 H-benzotriazol-1-yloxy)-3,4-dihydro-1- methyl 2H-pyrrolium hexachloroanitimonate, benzotriazol-1-yloxy-N,N- dimethylmethaniminium hexachloroantimonate, O-(7-azabenzotriazol-1-yl)-1 ,1 ,3,3- tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)- 1 ,1 ,3,3- bis(tetramethylene)uronium hexafluorophosphate, O-(benzotriazol-1 -yl)-1 ,1 ,3,3- tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)- 1 ,1 ,3,3- bis(pentamethylene)uronium tetrafluoroborate, 2-(3,4-dihydro-4-oxo-1 ,2,3-benzotriazin-3- yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate, 2-(5-norbornene-2,3-dicarboximido)- 1 ,1 ,3,3-tetramethyluronium tetrafluoroborate, 2-(2-oxo-1(2H)-pyridyl-1 , 1 ,3,3- tetramethyluronium tetrafluoroborate, 2-succinimido-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate, optionally in combination with base;

iii) phosphonium based reagents including O-(7-azabenzotriazol-1-yl)- tris(dimethylamino)phosphonium hexafluorophosphate benzotriazol-1-yl diethylphosphate, 1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate (Castro's Reagent), 7- azobenzotriazolyoxytris(pyrrolidino)phosphonium hexafluorophosphate, 1 - benzotriazolyoxytris(pyrrolidino)phosphonium hexafluorophosphate, optionally in combination with base;

iv) other peptide coupling reagents including 2-bromo-3-ethyl-4-methyl thiazolium tetrafluoroborate, bis(2-oxo-3-oxazolidinyI)phosphinic chloride, bromotris(dimethylamino)phosphonium hexafluorophosphate, bis(tetramethylenefluoroformamidinium) hexafluorophosphate, 2-chloro-1 ,3- dimethylimidazolidinium hexafluorophosphate, 3-(diethoxyphosphoryloxy)-1 ,2,3- benzotriazin-4(3W)-one, diphenylphosphinic chloride, 2-ethoxy-1-ethoxycarbonyl-1,2- dihydroquinoline, pentafluorophenyl diphenylphosphinate, S-(1-oxido-2-pyridinyl)-1 ,1 ,3,3- tetramethylthiouronium hexafluorophosphate, bromotris(pyrrolydino)phophonium hexafluorophosphate, chlorotris(pyrrolydino)phophonium hexafluorophosphate,

tetramethylfluoroformamidinium hexafluorophosphate, S-(1-oxido-2-pyridinyl)-1 ,1 ,3,3- tetramethylthiouronium tetrafluoroborate, optionally in combination with base.

Optionally, the activating agent includes at least one activating additive. Preferred activating additives include pentafluorophenol, hydroxybenzatriazole, hydroxysuccinimide, 1 -hydroxy-7-azabenzotriazole, carbonyldiimidazole, 3-hydroxy-3,4- dihydro-4-oxo-1 ,2,3-benzotriazine or N-ethyl-5-phenylisoxazolium-3'-sulphonate.

A preferred activating agent is a combination of N-N'-diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HOBT).

The skilled person will readily appreciate that the activation of (V) above to give (II) above, and the coupling of (II) with (III) can be carried out in one operational step; for instance, the peptide (V) may be activated in the presence of (III), without the necessity of isolating activated form (II).

Preferably, the reaction of (II) with (III) to give (I) occurs in DMSO or DMF.

The skilled person will moreover appreciate that before and after each step it may be necessary to swell the resin with a suitable solvent to enable reagent(s) to permeate fully and react completely with the bound peptide. Furthermore, after each step it may be necessary or expedient to wash the resin to remove excess reagent, byproducts and impurities.

The amino acids can be natural, unnatural or modified. The residues A of the amino acids may incorporate protected functional groups. Preferably, the amino acids are α amino acids, although β and other amino acids may also be employed.

Preferred solid supports W are derivatised Merrifield resins, that is resins based on chloromethylstyrene / divinylbenzene copolymers. Particularly useful resins are PEG-PS e.g. Tentagel (obtained from Novabiochem) and PEGA supports, co-polymers of PEG and acrylamide monomers (obtained from Polymer Laboratories), both of which have increased tolerance to aqueous media.

The linker group Y is a chemical bond, or chemical moiety capable of forming a covalent bond to both the solid support W and the carboxylic acid group of an amino acid. Many suitable linker groups are known. Preferably, the Y-C bond of compound (I) above is cleavable to yield a soluble polymer-bound peptide of formula (Vl)

(Vl)

or a salt form thereof.

The linker group Y ¹ is a chemical bond, or chemical moiety capable of forming a covalent bond to both the soluble polymer group P and the amine group of an amino acid. Many suitable linker groups are known. Preferably, the Y'-N bond of compound (I) above is cleavable to yield a solid support-bound peptide of formula (VII).

(VlI)

In a preferred embodiment, the linker groups Y and Y' are selected such that the bond N-Y ¹ can be cleaved under conditions to which the C-Y bond is stable. In this context, "stable" means that the C-Y bond undergoes less than 20 % cleavage; preferably less than 10 % and most preferably less than 5 %.

In a preferred embodiment, the linker groups Y and Y' are selected such that the bond C-Y can be cleaved under conditions to which the bond N-Y ¹ is stable. In this context, "stable" means that the N-Y bond undergoes less than 20 % cleavage; preferably less than 10 % and most preferably less than 5 %.

Suitable linker groups Y include (a), (b), (c), (d) and (e).

(e) (f)

Preferably, the linker Y is chlorotrityl (c).

Preferred linker groups Y' are (VIII) and (IX)

wherein A' is an amino acid residue or H, and Q is as defined above.

Particularly preferred linker groups Y' are (X) and (Xl)

Conditions for the cleavage of the Y'-N and C-Y bond will depend on the nature of the groups Y and Y'. For example, when Y is chlorotrityl and Y ¹ is (VIII) or (IX), then the C-Y bond is selectively cleaved under mild acid conditions, without cleavage of the Y'-N bond. Alternatively, treatment with piperidine cleaves the Y'-N bond without cleavage of the C-Y bond.

Many suitable methodologies for the synthesis of solid support-bound peptides (III) are described in the art, and in particular in Barany, G. and Merrifield, R.B. (1979) in The Peptides (Groaa, E. and Meienhofer, J. eds.), vol 2, pp.1-284, Academic Press, New York, and Atherton, E. and Sheppard, R.C. (1989) in Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford. Particularly preferred methods are described in the examples herein.

Activated peptides (II) are preferably prepared as described above by treatment of the corresponding peptide (V) with an activating agent. Peptide (V) is suitably prepared by assembling solid support-bound peptide (XII) using conventional solid phase peptide synthesis methods described above;

(XII) wherein R ¹ , A and n are as defined above, Y is a linker and W is a solid support; reacting compound (XII) with a suitable polymer containing reagent to give a solid support-bound polymer linked peptide (XIII);

(XIII)

wherein Q and Y are as defined above; and cleaving the C-Y bond to give a peptide of formula (V).

Preferably, R ¹ is hydrogen, hydrocarbyl, or A, taken together with R ¹ and N, forms a heterocycle. More preferably, R ¹ is hydrogen, C _1-6 alkyl, or C _1-6 acyl, or A, taken together with R ¹ and N, forms a heterocycle. A preferred heterocycle is pyrrolidine.

Preferably, n is less than 100. More preferably, n is less than 25. Still more preferably, n is between 5 and 20. Most preferably, n is about 15. For soluble polymer-bound peptides wherein n is of these values, couplings of (II) and (III) proceed particularly effectively, whilst still allowing for a convergent synthesis of the target peptide. Moreover, such peptides are easily purified by HPLC.

The term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to

those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non- limiting example of a hydrocarbyl group is an acyl group. Preferred hydrocarbyl groups are those comprising 1 to 10 carbon atoms.

A typical hydrocarbyl group is a hydrocarbon group. Here the term "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.

According to a second aspect, the invention relates to a process for the preparation of a compound of formula (XIV)

(XIV)

wherein W, Y, Q, Y', R ¹ , A, n and m are as defined above and x is a positive integer;

comprising the steps of

(a) reacting a solid support-bound activated peptide of formula (II) with a solid support-bound peptide of formula (III);

Q- -Y ¹ - NR ¹ - A — CO- -LG H- NR ¹ - A — CO- -Y- -W n m

(H) (III)

to give a solid support-bound peptide of formula (I)

Q- -Y'- -NR ¹ - A — CO- -Y- -W n+m

(I)

(b) cleaving the peptide (I) from the support W and linker Y to give a peptide of formula (XV)

(XV)

(c) treating the peptide of formula (XV) with an activating agent to give a solid support-bound activated peptide of formula (XVI)

(XVI)

wherein LG is a leaving group

(d) repeating steps (a), (b) and (c) x times.

According to a third aspect, the invention relates to a process for the preparation of a compound of formula (A)

(XVII)

wherein W, Y, Q, Y', R ¹ , A, and m are as defined above and x and n are positive integers;

comprising the steps of

(a) reacting a solid support-bound activated peptide of formula (II) with a solid support-bound peptide of formula (III);

Q- -Y ¹ - NR ¹ - A — CO- -LG H- -NR ¹ - A — CO- -Y- -W n m

(H) (III)

to give a solid support-bound peptide of formula (I)

Q- -Y ¹ - -NR ¹ - A — CO- -Y- -W n+m

(I)

(b) cleaving the peptide (I) from the soluble polymer Q and linker Y' to give a solid support-bound peptide of formula (VII)

(VII)

(c) repeating steps (a) and (b) x times.

In a fourth aspect, the invention relates to a compound which is:

wherein LG is a leaving group (preferably chlorine), and r is on average between 4 and 120 (that is, the PEG fragment has a number average molecular weight of about 200 to about 5000 daltons), preferably between 4 and 50.

The present invention will be described in more detail with reference to the following non- limiting examples.

EXAMPLES

Abbreviations: DCC, ty/V-dicyclohexylcarbodiimide; SPPS, solid-phase peptide synthesis; RP-HPLC, reversed-phase high-performance liquid chromatography; HOBt, 1- hydroxybenzotriazole; DIC N.N'-Diisopropyicarbodiimide; BOP, benzotriazole-1- yloxytris(dimethylamino)phosphonium hexafluorophosphate; TLC, thin-layer chromatography; FIB-MS, fast ion bombardment mass spectrometry; ES-MS, electrospray mass spectrometry; MALDI-TOF, matrix assisted laser desorption ionisation-time of flight; DIPEA, diisopropylethylamine; DMAP, N,N- 'dimethylaminopyridine; Boc, terf-butyloxycarbonyl; Fmoc, 9-fluorenylmethoxycarbonyl; DCM, dichloromethane; EDT, ethanedithiol; TFMSA, trifluoromethane sulfonic acid; DMF, /V,/V-dimethylformamide; HVTLE, high voltage thin-layer electrophoresis; Tbos, tri- te/t-butoxysilyl; TFA, trifluoroacetic acid; HF, hydrogen fluoride; PEG2000-OH, Polyethylene glycol 2000 monomethyl ether; HCTU ₁ O-(1H-6-Chlorobenzotriazole-1-yl)- 1 ,1,3,3-tetramethyluronium hexafluorphosphate; TBTU, ©-(BenzotriazoM-yO-N.N.N'.N ¹ - tetramethyluronium tetrafluoroborate; TIS, tri-isopropylsilane. Other abbreviations correspond to standard nomenclature used for naturally occurring amino acids. All amino acids except glycine are of the L-configuration unless otherwise specified. [Standard abbreviations for amino acids, peptides and protecting groups follow the

recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (1984) Eur. J. Biochem. 138, 9-37.]

1. Synthesis of 2-amino-9-fluorenyl-methanol

2-amino-9-fluorenyl methanol was synthesised according to literature methods (Rabanal, F., Giralt, E., and Albericio, F. Tetrahedron, 51 ,1449-1458 (1995)). The intermediates 2- (t-Boc)-amino-fluorene and 2-(tBoc)-amino-9-f!uorenol were purified by reverse-phase HPLC, along with product following the tBoc removal with HCI. All compounds gave the expected mass spectral data and were over 90% pure by UV monitoring at 290 nm on HPLC.

2. Synthesis of monomethoxy-polvethyleneglycol-acetate

According to the method of Royer, G. P. et al., J. Am. Chem. Soc, 101, 3394-3396, (1979), Polyethylene glycol 2000 monomethyl ether (PEG2000-OH) (Fluka No. 81321) (5 g) and 100 ml of a 1 molar solution of potassium tert-butoxide (11.2 g) were dissolved in 50 ml of tert-butanol at 35-40° over a period of 20 min. This was followed by the dropwise addition of 5.6 ml (50 mmol) ethyl bromoacetate over 10 min. The mixture was stirred for 2.5 hr at room temperature and evaporated under reduced pressure to give a cream-coloured slurry. To this was added 100 ml aqueous solution of 1N sodium hydroxide and left 2 hours at ambient temperature. The pH was adjusted to 1 - 2 with 6N HCI and extracted with 2 x 200 ml portions of chloroform. The organic phase was washed with 100 ml cold water. The brown organic layer was dried over anhydrous sodium sulfate. After evaporation and drying under vacuum, the solid was dissolved in ~ 250 ml warm (40-45°) isopropanol to give an orange-brown solution which was left at 4° C overnight. The white precipitate was filtered, washed with cold isopropanol and vacuum dried to give 4.23 g (84%) of a white fluffy solid (PEG2000-CH2-COOH) Melting point - 48-50° C. ES-MS, after deconvolution of raw data, gave a cluster of peaks separated by 44 mass units (PEG ethoxy subunits) at the indicated masses, with the peaks in parentheses corresponding in the PEG2000-OH starting material: 1763.7 (1706.3), 1807 (1749.7), 1852.6 (1793.8), 1896.6 (1837.9), 1940.1 (1882.0), 1984.9 (1926.1), 2028.3 (1970.2), 2072.4 (2014.3), 2116.5 (2058.4), 2161.3 (2102.5), 2204.7 (2146.6), 2248.8 (2190.7), and 2292.9 (2234.1). The mass increases of approximately 58 daltons reflect the addition of the acetate group to the PEG primary hydroxyl.

Base titration of 9.6 mg PEG2000-CH2-COOH resulted in consumption of .00480 milliequivalents, yielding an average molecular weight of 2000. A duplicate titration of 11.6 mg consumed .00481 milliequivalents of base, giving an average molecular weight of 2407. These predicted molecular weights are in good agreement with the polydispersity values for PEG2000 and suggest nearly a complete conversion to the carboxylate derivative. A 10 mg sample of the original PEG2000-OH control titrated no base, indicating the absence of carboxyl groups.

3. Preparation of the PEG2000-CH2-COOH amide derivative of 2-amino-9- fluorenyl methanol.

0.1 mmoles (210 mg) of PEG2000-CH2-COOH were dissolved in 1 ml DMF. To this was added a mixture of 0.1 mmol (42 mg) HCTU, 0.1 mmol (15 mg) HOBT, and 0.2 mmol (0.034 ml) DIPEA. After 5 minutes, 0.15 mmol (37 mg) of the hydrochloride salt of 2- amino-9-fluorenyl methanol was added and the mixture allowed to agitate for 1.5 hr. Following the addition of 1 ml water, the mixture was desalted and separated by size exclusion chromatography on a bed (2.5 x 35 cm) of Sephadex G-25 (100-300 microns) packed in water. Fractions were monitored at 290 nm and by reversed phase HPLC. Those containing pure PEG2000-CH2-CO-amino-9-fluorenyl methanol were pooled and lyophilised. Yield was 179 mg (75%).

ES-MS following deconvolution of the raw data gave sets of peaks in the mass range of 1900 - 2400. These were approximately 193 mass units greater than peaks corresponding to the underivatized PEG2000-CH ₂ -COOH, indicating conversion to the amide of the 2-amino-9-fluorenyl methanol. This material was used without further purification.

4. Preparation of the PEG2000-CH2-2-carboxamido-9-fluorenyl-methyl- (p- nitrophenvDcarbonate

87 mg (420 micromoles) of p-nitrophenylchlorocarbonate (MW 201.6) was dissolved in 3 ml dry dichloromethane, and the solution was cooled in an ice bath. 300 mg (app. 130-

145 micromoles) of PEG2000-CH ₂ -CO-2-amido-9-fluorenylmethanol were dissolved in 4 ml DCM and 0.10 ml (600 micromoles) of DIPEA was added. The PEG2000 solution

was added dropwise to the cold p-nitrophenylchlorocarbonate and let stir at room temperature overnight. Ethyl ether was added to the mixture to give a cloudy solution, which was put on ice. The resulting precipitate was centrifuged and washed with cold ether. After drying, the cream-coloured solid weighed 247 mg (65% yield). It was checked for nitrophenol content following hydrolysis with 2N sodium hydroxide (molar extinction coefficient for p-nitrophenolate anion at 402 nm is 18,300). The value found was 76% of theoretical.

The ES-MS spectrum of the precipitated PEG2000-CO-amido-fluorenyl-9- methoxycarbonyl nitrophenolate was compared with that of the monomethoxy PEG2000-

OH before conversion (in parentheses): 2121.6 (1705.6), 2166.4 (1750.4), 2210.4

(1795.2), 2255.2 (1838.4), 2298.4 (1882.4), 2342.4 (1926.4), 2387.2 (1970.4), 2430.4

(2014.4), 2475.2 (2058.4). This correlates with the expected mass gain of approximately

416 units. HPLC analysis showed the product at 17.6' representing 65% of the total material seen at 290 nm.

5. Preparation of the PEGaoOO-Chre-Z-carboxamido-g-fluorenyl-methyl- chlorocarbonate (PEG2000-Fmoc)

420 mg (app. 190 micromoles) of PEG2000-CH2-CO-2-amido-9-fluorenylmethanol were dissolved in 7 ml dry DCM in a stoppered round bottom flask. 3 ml of a solution of 20% phosgene in toluene (5.7 mmol, 30 x excess) was added with stirring. The mixture was stirred overnight at room temperature. The solution was purged with nitrogen for 1 hour, then solvents removed under rotary evaporation to give a brown oil which was used immediately without purification.

6. Synthesis of Salmon Calcitonin 1-14 (Cys(Trt)-Ser(t-butyl)-Asn(Trt)-Leu- Serrt-butvD-Thrrt-butvn-CvsfTrtμVal-Leu-Glv-Lvsft-Bocl-Leu- Serft-butvn-GlnfTrt)- OH

Aminomethyl PL-PEGA resin (Polymer Labs, Cat. No. 1432-1679)(0.15 mmol, 0.4 mmol/g) was washed with DMF three times in a shaker vessel. A solution of 0.45 mmol (108 mg) 4-(4'-hydroxymethyl-3'-methoxyphenoxy)-butyric acid, .45 mmol TBTU(128.4 mg), and 0.45 mmol HOBT (69 mg) in 5 ml DMF was added and the vessel shaken for 1 hour. After washing, a ninhydrin analysis of the resin was slightly positive. The coupling

was repeated for 50 minutes, after which the ninhydrin test was negative. The resin was then loaded with glutamine by dissolving Fmoc (Trt)-Glutamine (.75 mmmol, 460 mg) in 4 ml dry DMF. Dry pyridine (1 ,24 mmol, 0.1 ml) was added along with 2,6-dichlorobenzoyl chloride (.75 mmol, 0.108 ml), and the solution added to the resin and shaken overnight for 18 hours. The resin was washed and a sample was checked for loading by treating with a 20% solution of piperidine in DMF and measuring the absorbance at 290 nm. A loading of .283 mmol/g was found. The remainder of the sequence was assembled by Fmoc chemistry using 5 equivalents of each amino acid (except cysteine) per resin substitution, 5 equivalents HOBT, 4.5 equivalents of TBTU, and 9 equivalents DIPEA in each coupling. After 50 minutes, the couplings were checked by ninhydrin and repeated as necessary until the assembly was complete. To minimize racemization, cysteine at positions 1 and 7 were coupled using DIG pre-activation (30 minutes, 1 equivalent per amino acid) with HOBT in a DCM/DMF mixture (3:1), followed by addition of lutidine (1 equivalent) to the resin mixture. Coupling was repeated until a negative ninhydrin test was achieved.

After removal of the last Fmoc group at residue 1 , the amino terminus was coupled to the PEG2000-Fmoc by first dissolving approximately 190 micromoles PEG2000-Fmoc in 5 ml dry DCM. DIPEA (3x molar excess, 0.094 ml) was added and the mixture added to the resin and shaken for 5 hr. The ninhydrin test was negative and the resin washed with DCM, methanol, and DCM, then dried under vacuum.

7. Cleavage of PEG2000-Fmoc-Calcitonin 1-14 with protected side chains

A solution of 40 ml DCM mixture containing 4% TIS (1.6 ml), 1% EDT (0.4 ml) and 1% TFA (0.4 ml) was made up and the resin treated with 8 ml of this mixture for 2.5minutes, then drained into a solution of 4 ml methanol containing 10.4 mmol (.93 ml) pyridine to neutralize the TFA. The resin was treated four more times in this manner, and the combined filtrates evaporated to a whitish residue. This was dissolved in 10-15 ml of dichloromethane and the PEG2000-Fmoc-side chain protected Calcitonin 1-14 precipitated by the addition of 80 ml diethyl ether. The precipitate was collected by centrifugation and washed twice with ether. The original mother liquor was left to stand for 24 hrs. to yield a second crop of precipitate which was also collected by centrifugation. A sample of each was dissolved in DMF and checked for purity by analytical reversed phase HPLC on a Jupiter C4 (4.6 x 250 mm, 5 micron, 300 angstrom) column, using a gradient of 20-60% ACN in 0.1% TFA (0-4min), 60-95% ACN (4- 20

min), and 95% ACN isocratically (20-32 min). The product eluted at 22.4' for each of the precipitates in greater than 90% purity. The purification was performed on a Jupiter C5 (10x250mm , 300 angstrom, 10 micron) column heated to 35° C to yield over 60 mg material. The product was checked by ES-MS, with the spectrum showing clusters of peaks in the approximate mass ranges 800-880 (MH +6/6), 900-1050 (MH +5/5), and 1150-1300 (MH +4/4). The deconvoluted spectrum gave a cluster of peaks in the 4500- 5300 mass range. The predicted mass range of the PEG2000-Fmoc-protected Calcitonin 1-14 is approximately 4800-5300.

8. Assembly of Salmon Calcitonin 15-32 carboxamide (Glu-Leu-His-Lvs-Leu- Gln-Thr-Tyr-Pro-Arq-Thr-Asn-Thr-Glv-Ser-Glv-Thr-Pro-NH2)

The Calcitonin 15-32 sequence was assembled on a RINK amide resin (0.7 mmol/g substitution) by first loading Fmoc-Proline onto 115 mg resin using TBTU activated coupling. The remainder of the sequence was assembled by standard Fmoc chemistry with TBTU activation. Each coupling was checked by ninhydrin and repeated as necessary until the sequence was complete.

9. Assembly of Calcitonin 1-32 by PEG-assisted segment condensation

The Calcitonin 15-32 resin (69 mg) from above was treated with 20% piperidine to remove the N terminal Fmoc group, washed with DCM, and dried under vacuum. A solution of 36.2 mg (7.2 Dmoles) PEG2000-Fmoc-Calcitonin 1-14 (protected) dissolved in 0.5 ml DMSO and activated with DIC(14 Gmol, 2.2 Dl) and HOBT (14 Dmol, 2.5 mg) was added to the dry resin and shaken for 6 hrs. The ninhydrin test was performed and the coupling repeated until the test was negative. The resin was washed thoroughly with DMF, methanol and DCM, and dried. The PEG-Fmoc group was removed with 20% piperidine in DMF and the resin-bound peptide washed with DMF and DCM, then dried under vacuum. Final cleavage and deprotection from the resin was performed with a solution of TFA (10 ml) containing 4%TIS, 5% phenol, and 2% EDT for 1.5 hours. The mixture was filtered into cold ether, the precipitate collected by centrifugation, and washed with cold ether twice. The product was purified by reversed-phase HPLC and confirmed by ES-MS.

10. Synthesis of a 60 amino acid target protein

A 60 amino acid target protein is assembled from four fragments of 15 amino acids each. Three of the fragments (1-15, 16-30, and 31-45) are made on a chlorotrityl resin and

have a PEG-Fmoc at their N terminus. They are detached from the chlorotrityl resin under mild (1 % TFA, 10% acetic acid) acid conditions. The fourth fragment, 46-60, is assembled on a normal WANG resin and does not have a PEG attached. The peptide is assembled by sequentially linking the soluble PEG-Fmoc-31-45 fragment first to the support-bound 46-60 peptide, removing the PEG-Fmoc group, then coupling the PEG- Fmoc-16-30 fragment. This is repeated for the 1-15 fragment, with removal of the PEG- Fmoc and final cleavage of the target peptide from WANG resin with 90% TFA. In this strategy, PEG-Fmoc peptide segments are relatively short and easy to purify by HPLC if necessary. The growing target protein remains anchored to a solid support throughout, thus minimizing losses in handling and avoiding solubility issues.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.