WO1993005065A1

WO1993005065A1 - Preparation of peptides by a solid-phase synthesis and intermediates therefor

Info

Publication number: WO1993005065A1
Application number: PCT/GB1992/001567
Authority: WO
Inventors: Ram Prakash Sharma
Original assignee: Porton Developments Limited
Priority date: 1991-08-30
Filing date: 1992-08-26
Publication date: 1993-03-18
Also published as: GB9118669D0; AU2464992A; EP0600996A1

Abstract

The invention relates to the preparation of polypeptides by solid phase synthesis by attachment of carboxy-protected amino acids to the carboxyl end of a peptide chain bound at its N-terminal to a solid support. The carboxy-protection is by a tertiary alkoxy silyl group. Novel tertiary alkoxy silyl amino acid esters are disclosed which may be used as intermediates.

Description

PREPARATION OF PEPTIDES BY A

SOLID-PHASE SYNTHESIS AND INTERMEDIATES THEREFOR

The present invention relates to the preparation of

polypeptides by solid phase synthesis, in particular synthesis on a solid support, to methods for preparing polypeptides under mild conditions and to protected carbonyl group intermediates for use in such preparations and methods.

It is well known that methods for the preparation of di- and polypeptides in solution phase involve N-protection by blocking of the sensitive amine group to prevent this group reacting with the peptide-forming reagent. In a reaction sequence for forming a polypeptide using a protecting group, the connection and removal of the amine protecting group has to be carried out each time a further amino acid is added, in addition to carrying out steps to form the peptide bond. When the reaction is carried out in solution, the peptide intermediates generally have to be separated from the by-products after the addition of each amino acid. The free polypeptide is liberated by removing the amine protecting group. A tertiary butyl oxycarbonyl group is a typical amine protecting group, for example tert-butyl oxycarbonyl (tBoc), which may be removed either by hydrochloric acid gas in organic solvents or by using trifluoroacetic acid (TFA).

Solid phase polypeptide synthesis methods were devised principally by Merri field & Sheppard. Their methods involved conducting the reactions using solid phase conditions by covalently bonding the N-protected peptide via a terminal carboxyl group to a polymeric support, typically a resin, whilst the peptide is being synthesised. tBoc is a preferred protective group, for Merri field's method. A base labile group such as fluorenylmethoxycarbonyl (Fmoc) is used in Sheppard' s method. These methods allowed a "single pot" synthesis which avoided repeated separations of peptide

intermediates, together with providing easy recovery of the product from by-product. The synthesis was in the C to N direction.

To liberate the desired polypeptide it is necessary to remove the N-protecting group and to cleave the link formed between the terminal carboxyl group and the resin. Both deprotection and final cleavage require the use of strong acids. For example, in

Merrifield's method trifluoroacetic acid is used to deprotect and hydrogen fluoride to cleave the peptide from the resin.. The use of strong acid conditions makes this method for preparing polypeptides especially unsuitable for peptides involving basic amino acids such as lysine, histidine, and arginine without first protecting their side chains with acid-stable moieties (eg. benzyl groups).

Another process is known from WO90/05738, which discloses a procedure for solid phase polypeptide synthesis which involves protecting the carboxyl group of a first amino acid by forming the corresponding trialkylsilyl ester, binding the trialkylsilyl ester of the first amino acid to a solid support via its amino group, removing the trialkylsilyl group to leave the support-bound amino acid, and activating the carboxyl group of the bound amino acid, for example by the use of DCC/HOBt (dicyclohexylcarbodiimide- hydroxybenzotriazole). Then the trialkylsilyl ester of a second amino acid is used to produce a peptide bond by nucleophilic attack on the activated carboxyl group αf the bound first amino acid.

Subsequent amino acids are coupled in a similar way to build up the peptide sequence. The synthesis is in the N to C direction.

The types of trialkylsilyl protecting groups disclosed in this prior art document include trimethylsilyl- (TMS) and

tert-butyldimethylsilyl- (TBDMS) groups.

In order to prepare trimethylsilyl (TMS) or

tert-butyldimethylsilyl- (TBDMS) esters of amino acids it is essential to employ a temporary N-protecting group, for example tBoc, to prevent N-silyl derivatisation. The subsequent removal of the N-protection with acid yields the amino acid trialkylsilyl ester acid salt, which must be neutralised before the amino group can participate in peptide bond formation. The preparation of these amino acid trialkylsilyl esters is thus a three-step process. An example for the production of an amino acid TMS-ester is shown below: tBoc. NH . CHR. COOH + TMSC1 + Base tBoc . NH . CHR. COOTMS . + Base-HC1

HC1

HC1. NH₂. CHR. COOTMS .

The amino acid TMS-ester hydrochlorides produced are unstable and decompose even when stored under an inert atmosphere (e.g. N₂). If the hydrochloride is neutralised with base, for example pyridine, the resulting 'free' amino acid TMS-esters form a translucent "gel" in most solvents commonly used in peptide synthesis (for example in dichloromethane or dimethylformamide). The severe instability and poor solubility of the amino acid TMS-ester hydrochlorides renders them almost useless in solid-phase peptide synthesis. Amino acid TBDMS-esters may be used in place of amino acid TMS-esters, but although the amino acid TBDMS-esters have been shown to be more stable and more soluble than the corresponding TMS-esters, they too have their drawbacks;

a) they cannot be prepared in high yield. For example a typical yield of tBoc. amino acid TBDMS-ester is 25-45%. This low yield seems to be due to the steric hindrance of the N-protection when trying to introduce another group as big as the TBDMS moiety. The steric hindrance noticed in the introduction of the TBDMS group in solution would also be expected to cause prolonged coupling times and a high frequency of difficult couplings if amino acid

TBDMS-esters are used in solid-phase peptide synthesis.

b) the increased stability of the amino acid TBDMS-esters over their TMS counterparts, whilst being advantageous during the preparation of the amino acid trialkylsilyl esters, can be very disadvantageous in peptide syntheses. For a temporary protecting group to be useful in solid-phase peptide synthesis it must be able to be removed totally and rapidly under the chosen conditions.

Under harsh conditions there is the risk that the peptidyl- support bond will also be cleaved, whereas under mild conditions it might require between 1 - 2 hours to slowly achieve complete removal of the trialkylsilyl group.

In the art of amino acid chemistry many substituents are already known as "protective groups" for carboxyl groups. As a particular example, W. Gruszecki, M. Gruscecka and H. Bradaczek, in Peptides 1990 - Proceedings of the 21st European Peptide Symposium, Edited by Ernest Giralt and David Andreu, ESCOM Leiden 1991 page 27-28, have disclosed the specific protection of carboxyl groups (both on the side chain and terminal) of Aspartic acid and Glutamic acid by the formation of stable tri-t-butoxysilyl esters. The direction of the protection to give respectively α, β and γ esters could be controlled to some extent. Some of these tri-t-butoxysilyl esters of aspartic and glutamic acids were also N-protected by the use of a t-Boc substituent. The Gruszecki paper discloses the use of an aspartic acid and a glutamic acid tri-t-butoxysilyl ester in a liquid phase coupling reaction with an amino acid to produce a dipeptide. The tri-t-butoxysilyl carboxy-protecting group is disclosed as being unstable in acidic conditions and the ester is said to be split in seconds in the present of 50% trifluoroacetic acid (TFA) in dichloromethane (DCM).

WO 90/05738 discloses amino acids or peptide derivatives linked to an insoluable resin support via a terminal amino group. An example of such a resin which can bond to the amino group is a polystyrene resin bearing a -CH₂OCOCl substituent.

To create the linkage between the resin and the amino group of the first amino acid, activation of the resin must first occur.

This activation can be difficult to achieve and an accurate measure of the amount of amino acid bound to the resin is not easy to obtain. In addition, this type of linkage is very stable and requires unfavourably harsh acidic conditions, such as liquid HF, for cleavage of the synthesised peptide.

The use of linker compounds forming labile linkages with

N^α-amino groups is known per se in conventional synthetic chemistry (e.g. cf. CARPINO, L.A. & HAN, G.Y. (1972) J. Org. Chem 37 3404) but harsh conditions have precluded the use of such linker compounds in peptide syntheses.

In contrast to all of the synthetic methods of the prior art the present invention can provide a process for preparation of peptides, especially extended chain polypeptides, by a solid phase synthesis under mild conditions. The mild conditions apply both in the chain extension steps and in the cleavage of the finished desired peptide from the solid phase support.

According to a first aspect, the present invention provides a process for the production of a peptide of general formula (I) (I)

n+m(x+1) which comprises the following steps:

(a) forming a solid support-bound compound of general formula (IV);

(IV)

(b) removi ng the protecti ng group Z under mi ld aci di c or mi l d basi c condi ti ons to produce a sol i d support-bound carboxyl i c aci d or peptide derivative of general formul a (V) ; (V)

(c) reacting the carboxylic acid or peptide derivative (V) with a carboxyl group activating agent to form an activated solid

support-bound compound of general formula (VI);

(VI)

(d) reacting the activated solid support-bound compound with a second carboxy-protected amino acid or peptide derivative of general formula (VII); (VII)

to form a peptide chain-extended compound of general formula (VIII); (VIII)

(e) repeating step (b)

(f) repeating steps c, d, and e x times to form a compound of general formula (IX); (IX)

(g) and cleaving the resultant chain-extended compound at the N^α-Y covalent bond under mild acidic or mild basic conditions to form the desired compound of formula (I); wherei n

n is a positive integer

m is a positive integer

x is o or a positive integer

Z is a tri-t-alkoxysilyl, alkyl-di-t-alkoxysilyl

or dialkyl-t-alkoxysilyl group

Y is a linker compound linked to the

amino acid or peptide derivative

e is a leaving group

and for each A, which may the same or different:

i) A represents the residue of an amino acid; or

ii) represents a peptide residue; or iii) the structure NH.A represents the residue N<A of an imino acid HN<ACOOH

(wherein N<A represents a heterocyclic group).

The above described process may be varied, so as to provide a process which differs in that after step (f) and before step (g) the terminal carboxyl group of the solid support bound peptide is modified by substitution of or addition to the terminal -OH group, resulting in production by step (g) of a modified polypeptide of formula (I)^1. U (I)

n + m (x + 1 )

Wherein M is any substituent group other than -OH.

According to a second aspect, the present invention provides a process for the production of a peptide of general formula (I) (I)

which comprises the following steps:

(a) reacting a first carboxy-protected amino acid or peptide derivative of general formula (II); (II)

with a linker compound Y' to form a compound of general formula (III);

(III)

having a labile N^α-Y covalent bond;

(b) reacting the compound of formula (III) with a solid support W to form a solid support-bound compound of general formula (IV); (IV)

(c) removing the protecting group Z under mild acidic or mild basic conditions to produce a solid support-bound carboxylic acid or peptide derivative of general formula (V); (V)

(d) reacting the carboxylic acid or peptide derivative (V) with a carboxyl group activating agent to form an activated solid

support-bound compound of general formula (VI);

(VI)

(e) reacting the activated solid support-bound compound with a second carboxy-protected amino acid or peptide derivative of general formula (VII); (VII)

to form a peptide chain-extended compound of general formula (VIII); U (VIII)

(f) repeating step (c)

(g) repeating steps d, e, and f x times to form a compound of general formula (IX); (IX)

(h) and cleaving the resultant chain-extended compound at the N^α-Y covalent bond under mild acidic or mild basic conditions to form the desired compound of formula (I); wherei n

n,m,x,Z,Y,ε and A are as defined above. Accordi ng to a thi rd aspect , the present i nventi on further provides a process for the producti on of a pepti de of general formula (I) (I)

which comprises the following steps;

(a) obtaining W-Y, a solid support having a linker group, for example by reacting a solid support W with a linker compound Y';

(b) reacting the linker group containing solid support W-Y with a first carboxy-protected amino acid or peptide derivative of general formula (II)

(II)

to form a support-bound compound (IV) having a labile N^α-Y covalent bond (IV)

(c) removing the protecting group Z under mild acidic or mild basic conditions to produce a support-bound carboxylic acid or peptide derivative of general formula (V); (V)

(d) reacting the carboxylic acid or peptide derivative (V) with a carboxyl group activating agent to form an activated support-bound compound of general formula (VI); (VI)

(e) reacting the activated support-bound compound with a second carboxy-protected ami no aci d or pepti de deri vative. of general formula (VII) ;

(VII)

to form a peptide chain-extended compound of general formula (VIII); (VIII)

(f) repeating step (c)

n,m,x,Z,Y,ε and A are as defined above

Examples of suitable protecting groups Z are those of formula (X)

(X)

wherein R₁, R₂, R₃ are the same or different saturated or

unsaturated alkyl or t-alkoxy groups containing 1 to 20 carbon atoms, which may be unsubstituted or substituted by one or more groups selected from C₄-alkoxy, nitro, tri(C₁-C₄alkyl)silyl and halogen; and wherein at least one of R₁, R₂ and R₃ is a t-alkoxy group.

Preferred are tri-t-alkoxysilyl groups wherein R₁, R₂, R₃ are the same or different t-alkoxy groups containing 1 to 20 carbon atoms.

Preferred alkoxysilyl groups are tri-t-butoxysilyl,

di-t-butoxymethylsilyl, di-t-butoxyethylsilyl, and tri-t-isopropyl-oxysilyl.

Removal of the protecting group, step (c), may be carried out in aqueous solution at pH<4, for base-labile solid supports.

Step (c) is preferably carried out in dilute TFA

(trifluoroacetic acid), DAC (dichloroacetic acid) or chloroacetic acid solution at pH< 4 in the presence of organic solvents.

Still preferably, step (c) is carried out in about 5% TFA in the presence of DCM (dichloromethane).

Alternatively, for acid-labile solid supports step (c) may be carrried out in aqueous solution at pH>8.

For such conditions step (c) is preferably carried out in the presence of 5% piperidine in N,N-di methyl acetamide.

Examples of suitable linker compounds Y' are:

A) 9-hydroxymethylfluorene-2-acetic acid;

B) 4-hydroxymethylphenoxyacetic acid;

C) p-(2-hydroxymethyl sul phonyl ) benzoate, al so referred to as the "C.A.S. E.T. " l i nker .

For syntheses according to the first and second aspects of the invention, an amino acid or peptide derivative may be linked to these compounds via an "oxycarbonyl" group following

chloroformylation of the hydroxy! moiety of such linker compounds with phosgene. These linkers may also be attached to any solid support W bearing a terminal amino function. A) allows release of a newly synthesised peptide under mild conditions such as 15% piperidine in dimethyl formamide. For B) the conditions for peptide release are slightly more rigorous, requiring 90% trifluoroacetic acid (TFA). Cleavage from C) can be achieved under mild basic conditions, such as 0.1N sodium hydroxide.

Preferably the solid support W is a resin having a terminal amino group, for example commercially available resins MBHA., BHA., or aminomethyl ated resins. W and the linker compound are preferably reacted to form a W-Y bond in the presence of DCC/HOBt.

For the processes according to the first and third aspects of the invention, formation of the W-Y bond can be avoided by using linker group containing solid supports W-Y, for example:

D) O-chlorotrityl resin

which can react directly with the first amino acid or peptide derivative;

E) p-benzyloxybenzylalcohol resin;

the hydroxy group of which can be linked to the amino acid or peptide derivative via an "oxycarbonyl" bond as for linker compounds A), B) and C). However, the oxycarbonyl bond cannot be formed using phosgene due to the acid lability of this linkage and a suitable reaction sequence to form the linkage is :

Again, the amino acid or peptide derivative may be linked via an oxycarbonyl bond following functionalisation of the resin hydroxy moiety. The subsequent peptidyl-resin bond may be cleaved with tetrabutyl ammonium fluoride or by hydrazine in chloroform/methanol;

G) 4(2',4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin

This is a super-acid labile resin which can be linked to an amino acid or peptide derivative via a reaction sequence as used for E). Release of a synthesised peptide from this resin-linker is possible in very mild acidic conditions, for example 2% TFA.

Examples of suitable leaving groups ε introduced by carboxyl group activating agents are those of general formula (XI)

R⁴-NH-C=N-R⁵ (XI) wherein R⁴ and R⁵, which may be the same or different, represent C₁-C₁₀ hydrocarbyl groups. In a well studied example both are cyclohexyl groups. Other suitable leaving groups include

pentafluorophenoxy

and the group

made by reacting the amino acid or peptide derivative with pentafluorophenol or 1-hydroxybenzotriazole, respectively. Other suitable examples of activating agents are DCC/HOBt

(dicyclohexylcarbodiimide-hydroxybenzotriazole); BOP, otherwise known as CASTRO's reagent (benzotriazole-1-yl-oxy-tris

(dimethyl-amino) phosphonium hexafluorophosphate); TBTU; and the like.

If BOP is used, activation of the carboxyl group and formation of the peptide bond with the next carboxy-protected amino acid or peptide derivative (Steps (c) or (d) and (d) or (e), respectively) can take place simultaneously.

Any reactive side chains on residues A are protected and subsequently deprotected, as necessary by known procedures. The choice of side-chain protecting groups may be adjusted to suit the method of removal of the protecting group Z.

The invention still further provides compounds of general formula (IV);

(IV)

for use as intermediates in the process of the invention wherein W, Y, A, Z and n are as previously defined. The invention also provides compounds of general formula (III); (III)

for use as intermediates in the process of the invention wherein Y, A, Z and n are as previously defined.

The invention further provides compounds of general formula (II); (II)

for use as intermediates in the process of the invention wherein A, Z and n are as previously defined, with the exception of compounds wherei n

i) A is either Asp or Glu;

and ii) Z is

-Si (O-t-Bu)₃

Brief Description of the Figures

Figure 1 is an HPL Chromatograph of Leucine-Enkephalin synthesised using tert-butoxysilyl (TBOS) esters;

Figure 2 is a FIB Mass Spec Analysis of Leu-Enkephalin synthesised using L-amino acid-TBOS esters;

Figure 3 is an HPL Chromatograph of Leucine Enkephalin Alcohol;

Figure 4 is a FIB Mass Spec of Leucine Enkephalin Alcohol;

Figure 5 is a FIB Mass Spec of Leucine Enkephalin Diol; and

Figure 6 is a FIB Mass Spec of Leucine Enkephalin Chloromethyl Ketone. The invention will now be illustrated by way of example with reference to the following description.

Preparation of Compound (III )

The compound of general formula III may be 9-(methoxycarbonyl amino acid t-al koxysi lyl ester)-2-Fluorene acetic aci d:

This compound may be produced by a process which comprises the following steps:

The carboxy-protected compound 9-(methoxy carbonyl amino acid t-alkoxysilyl ester)-2-Fluorene acetic acid (9) can couple to a solid support resin W via the "free" carboxylic acid group (*). Coupling with a resin carrying a terminal amino group is preferred.

Solid Phase Synthesis: Compound (I)

A synthesis can be carried out using compound (9) according to the following procedure:

N

0

x)

Preparation of Resin-Bound Amino Acid, Compound (V)

Example: Formation of the Benzyloxycarbonyl resin - L-leucine Linkage.

Benzyl acetate resin. Chioromethylated co-polystyrene-2% divinylbenzene (5g. 1mmol./g.) suspended in 2-methoxyethanol

(40mls.) was treated with potassium acetate (1.4g.; 14.26 mmol.) and heated at 130°C for 64 hours. Following this the reaction mixture was allowed to cool to room temperature, filtered and the resin washed with 100mls. of water, methanol and diethyl ether

respectively, then dried under vacuum (i.r. spectrum = Amax. = 1740 cm.^-1).

Hydroxymethyl resin. The acetate resin from above was stirred in 0.5M. sodium hydroxide (40mls.) at room temperature for 48 hours. Filtration, washing and drying as described above was followed by analysis by its i.r. spectrum where some of the

1740cm.^-1 ester band could still be detected. The treatment with the 0.5M sodium hydroxide was repeated for a further 24 hours after which time no ester could be detected.

Methylchloroformate resin. The anhydrous hydroxymethyl resin was treated with 20% Phosgene in toluene (40mls.; 30mmol.) at room temperature for 4 hours after which time the resin was washed thoroughly with anhydrous diethyl ether and its i.r. spectrum recorded. (Amax. = 1785 cm.^-1).

Benzyloxycarbonyl resin L- leucine ethyl ester. To a portion of the methylchloroformate resin (500mg.) were added L-leucine ethyl ester hydrochloride (0.294g.; 1.5mmol.) and triethylamine (0.21mis.; 3.75mmol.) in dry DMF, and the suspension shaken at room temperature for 2.5 hours. The liquid phase was drained away and the resin washed thoroughly with DCM (i.r. spectrum: Amax. = 1750 cm^-1).

Benzyloxycarbonyl resin L- leucine. The ethyl ester in the above product was hydro!ysed by shaking the resin with 0.8M. sodium hydroxide (2mls.) in methanol (3mls.) and acetone (3mls.) at room temperature for 3 hours, by which time the ester band had

di sappeared from its i.r. spectrum. Preparation of the Benzyl oxycarbonyl Resin Amino Acid

RESIN-C₆H₄.-CH₂C1 ------------------------------> RESIN-C₆H₄-CH₂-O-CO-CH₃

RESIN-C₆H₄-CH₂-O-CO-C1 <------------------------------RESIN-C₆H₄-CH₂-OH

RESIN-C₆H₄-CH₂-O-CO-NH-CHR-CO-OCH₂+CH₃

RESIN-C₆H₄-CH₂-O-CO-NH-CHR-COOH

Preparation of Synthetic Polypeptide, compound (I)

Example: Synthesis of Leucine - Enkephalin.

(Tyr-Gly-Gly-Phe-Leu).

Methylchloroformate resin (500mg.) was prepared as described before.

The resin was then swollen in dimethyl acetamide (2 x 10mls.) and treated with L-tryosine methyl ester hydrochloride (1.0mmol.; 0.231g.) and triethylamine (2.0mmol.; 0.112mls.) in

dimethylacetamide (10mls.) for 16 hours. The solution-phase was drained away and the resin washed thoroughly with dichloromethane (6 x 10mls.). Examination of the resin's i.r. spectrum showed all the 1785cm^-1 band to have disappeared. The methyl ester was hydrolysed to yield the free acid by treating the resin with 2.0M sodium hydroxide (5mls.) and DMA (5mls.) for two periods of 2 hours at room temperature, followed by thorough washing with water, DMA, methanol and DCM to yield the benzyloxycarbonyl resin L-tyrosine.

The leucine-enkephalin pentapeptide was assembled by four successive cycles of addition and deprotection to yield the desired peptidyl-resin. A typical addition cycle consisted of treating the deprotected resin with the amino acid TBOS ester (1.0mmol.), HOBt (1.0mmol; 0.135g.) and diisoproplycarbodiimide (1.0mmol.; 0.156mls.) in DCM (10mls.) for 4 hours at room temperature. The resin would then be washed thoroughly with DCM before being deprotected by treatment with 25% TFA in DCM (10mls.) for 10 minutes at room temperature followed by washing with DCM (6 x 10mls.) before commencing the subsequent addition cycle.

After completion of the final deprotection to yield the

Resin-Tyr-Gly-Gly-Phe-Leu-COOH the peptidyl-resin was then treated with TFA (5mls.) containing TFMSA (0.5mls.) at room temperature for 2 hours. The solution-phase was collected and the resin washed with TFA (3 x 5mls.) and the combined filtrates were rotorevaporated at room temperature to afford a brown residue. This residue was taken into water and lyophilised to yield a white solid (45mg.) which was analysed by HPLC and examined by FIB mass spectrometry, and found to be substantially pure as shown in Figures 1 and 2.

The Chromatograph of Fig. 1 was obtained under the following conditions:

C₈ Analytical HPLC. of Leucine - Enkephalin.

Column : C₈ analytical.

Buffer A : 0.1% TFA., H₂O

Buffer B : 0.1% TFA., 80% ACN., 30% H₂O

Flow Rate: 0.1 ml./min.

Chart Speed : 5 mm. /mi n

Wavelength : 222 nm. (0.2 FSD. )

Gradient : 0 - 100% B over 20 mi ns.

The peptides were compared by injecting individual ly and

then co-i njecting the two.

[ YGGFL = Tyr-Gly-Gly-Phe-Leu ]

Solid - Phase Synthesis of Leucine - Enkephalin. Intermediates: Compounds (II)

The preferred carboxy-protected amino acids (II) are the tri-t-butoxysilyl esters. These can easily be produced in a one step process as follows:

SiCl₄

NH₂-A-COOH--------------> NH₂-A-COOSi- (0-t-Bu)₃

tBuOH

Pyridine

The reaction is rapid and typically yields 85-90% product. The simplicity of production contrasts favourably with the prior three step process for production of the silyl esters of W090/05738. It is particularly advantageous that the tri-t-alkoxysilyl esters, alkyl-di-t-alkoxysilyl esters and dialkyl-t-alkoxysilyl esters can be made from free amino acids without the need for N^α-protection (by groups such as tBoc). The free amino acids are abundantly available and cheap.

Since no N^α-protection is required for this reaction the product liberated can be immediately used in peptide synthesis without further manipulation.

The tri-t-alkoxysilyl esters, alkyl-di-t-alkoxysilyl esters and di alkyl-t-alkoxysilyl esters show good stability when stored under anhydrous conditions, hence permitting long term storage. Large scale preparation of these derivatives may be carried out well in advance of the peptide synthesis.

The esters are stable between pH 4.0 and pH 8.0 and are stable to alcoholic solvents. They can be readily removed at pH values less than 4 or greater than 8. The procedure for the preparation of several tri-t-butoxysilyl amino acid esters is illustrated in more detail in the example below:

Example 1

Preparation of L-Alanine tri-t-Butoxysilyl Ester

To a suspension of L-alanine (50mMol, 4.45g) in t.butanol (40ml) is added anhydrous pyridine (210mMol, 16.8ml). To this magnetically stirred suspension, silicon (iv) chloride (50mMol, 5.73ml) is added dropwise from a syringe. An exothermic reaction occurs immediately and results in the reaction mixture becoming a clear solution. Stirring is continued at room temperature and after approximately 15 minutes a white precipitate of pyridinium

hydrochloride is seen to form. The reaction is allowed to continue in this manner for a further 2 hours.

The pyridinium hydrochloride is removed by filtration, washing the precipitate with ethyl acetate (50ml). All solvents, (i.e.

ethyl acetate, t.butanol and pyridine) are removed under reduced pressure. The resultant oil is then dissolved in ethyl acetate and washed with saturated sodium bicarbonate (50ml) and distilled water (2 x 50ml), respectively. The organic layer is dried over anhydrous sodium sulphate. Filtration removes the drying agent and removal of the solvent under reduced pressure leaves the white crystalline solid in good yield, (M. Pt. =49-51°C.).

The product was characterised by means of its infra-red (I.R.) spectrum, thin layer chromatography (t.l.c.) and Mass Spectrum (M.S.).

I.R. spectrum (thin film in nujol) bands at : 1090 cm^-1, 1200cm^-1, 1250cm^-1, 1750cm ^-1 and 3000cm ^-1.

t.l.c. (solvent: chloroform: methanol 9:1) Rf.= 0.55

M.S. showed M^-1 at 336, as expected.

Other tri-t-butoxysilyl amino esters have been prepared and characterised in the same manner, for details see Table 1.

In summary, the tri-t-alkoxysilyl, alkyl-di-t-alkoxysilyl and dialkyl-t-alkoxysilyl amino acid esters are convenient to prepare, they have suitable stabilities, they are soluble in all commonly used organic solvents and they have exhibited no signs of suffering from problems of steric hindrance. Hence, it would appear that most of the problems encountered with the trimethylsilyl and the t-butyldimethylsilyl amino acid esters do no effect these

derivatives and as a result they seem much more suitable for use in solid-phase peptide synthesis. As well as providing to be less problematic than earlier esters studied, the characteristics of the tri-t-alkoxysilyl, alkyl-di-t-alkoxy and dialkyl-t-alkoxysilyl esters open up a wide array of possibilities in the area of pepti de synthesis.

The abil ity to hydrolyse the esters with base or acid widens greatly the choice of side-chain protections used along with them i n an orthogonal sol i d-phase strategy. This i n turn faci l itates the use of milder conditions for the producti on of a syntheti c peptide. Since the activated carboxyl moi ety wi l l be attached to the solid support, it may not be necessary to have such a high degree of side-chain protection as used i n conventional methods. For example, the -OH group on seri ne i s always masked conventional ly, but si nce site-site interactions on sol i d supports are reported to be extremely low, it may only be necessary to mask the hydroxyl duri ng addition of the seri ne to the growing peptide chain . It should be possibl e to achi eve this temporary protection usi ng a

bi s-(tri -t-butoxysi lyl ) seri ne derivative:

H₂N. CH. COOSi-(OtBut. )₃

CH₂. O. Si-(OtBut . )₃

The mild nature of the synthetic strategy proposed may provide a means of synthesising modified peptides, such as phosphopeptides or glycopeptides, that cannot withstand the conditions used at some stages of conventional synthetic procedures. The reversal of the direction of synthesis from the conventional C- to -N terminal also opens up the possibility of carrying out solid-phase fragment coupling for the production of large peptides.

A further advantage of synthesising in the N to C direction is that the C-terminal remains free and this allows for modifying the C-terminal of peptides whilst they are bound on the solid support. Examples of C-terminal modified peptides which have been prepared are enkephalin-alcohol, -diol and -chloromethylketone. Data on these analogues is shown in Figures 3-6. The products made

according to the invention and modified can be seen to be

substantially pure.

t.l.c solvent was chloroform: methanol (9:1). Bz refers to Benzyl.

Claims

1. A process for the production of a peptide of general formula (I)

(I)

which comprises the following steps:

(a) forming a solid support-bound compound of general formula (IV);

(IV)

(b) removing the protecting group Z under mild acidic or mild basic conditions to produce a solid support-bound carboxylic acid or peptide derivative of general formula (V); (V)

(c) reacting the carboxylic acid or peptide derivative (V) with a carboxyl group activati ng agent to form an activated soli d

support-bound compound of general formula (VI) ;

(VI)

(d) reacting the activated solid support-bound compound with a second carboxy-protected amino acid or peptide derivative of general formula (VII); r (VII)

to form a peptide chain-extended compound of general formula (VIII); H (VIII)

(e) repeating step (b)

(g) and cleaving the resultant chain-extended compound at the N^α-Y covalent bond under mild acidic or mild basic conditions to form the desired compound of formula (I); wherein

n is a positive integer

m is a positive integer

x is 0 or a positive integer

Z is a tri -t-alkoxysilyl, alkyl-di-t-alkoxysilyl

or dialkyl-t-alkoxysilyl group

Y is a linker compound linked to the

amino acid or peptide derivative

ε is a leaving group

and for each A, which may the same or different:

i) A represents the residue of an amino acid; or

ii) represents a peptide residue; or iii) the structure NH.A represents the residue N<A of an imino acid HN <ACOOH

(wherein N<A represents a heterocyclic group).

2. A process according to claim 1 in which the solid support-bound compound (IV) is formed by:

with a linker compound Y' to form a compound of general formula (III); U (III)

having a labile N

Y covalent bond;

(b) reacting the compound of formula (III) with a solid support W.

3. A process according to claim 1 in which the solid support-bound compound (IV) is formed by:

a) obtaining W-Y, a solid support having a linker group;

(b) reacting the linker group containing solid support W-Y with a first carboxy-protected amino acid or peptide derivative of general formula (II).

(II)

4. A process according to claim 3, wherein the solid support having a linker group is formed by reacting a solid support W with a linker compound Y'.

5. A process according to claim 3, wherein the solid support is 0-chlorotrityl resin.

6. A process according to any of claims 1 to 4, wherein the solid support is a resin with a terminal amino group.

7. A process according to claim 2 or 4 wherein the W-Y bond is formed in the presence of DCC/HOBt.

8. A process according to any preceding claim wherein the protecting group Z is a group of general formula

wherein each of R₁, R₂, R₃ may be the same or different C₁-C₂₀ alkyl or t-alkoxy groups, which may be unsubstituted or substituted, by one or more groups selected from C₄ -alkoxy, nitro,

tri (C₁-C₄alkyl)silyl and halogen, and wherein at least one of R₁, R₂ and R₃ is a t-alkoxy group.

9. A process according to claim 8 wherein R₁, R₂ and R₃ are the same or different C₁-C₂₀ t-alkoxy groups.

10. A process according to claim 9 wherein the protecting group Z is the tri-tertbutoxysilyl group

-Si-(OtBu)

3

11. Compounds of general formula (IV) (IV)

for use in the process of any preceding claim wherein W, Y, A, Z and n are as previously defined.

12. Compounds of general formula (III); (III)

for use as intermediates in the process of claim 2 wherein Y, A, Z and n are as previously defined.

13. Compounds of general formula (II);

(II)

for use as intermediates in the process of claim 2 or 3 wherein A, Z and n are as previously defined, with the exception of compounds wherein

i) A is either Asp or Glu;

and ii ) Z is

-Si (OtBu)

14. A compound of general formula (III) which is 9-(methoxycarbonyl amino acid al koxysilyl ester)-2-Fluorene acetic acid:

wherein Z is a tri -t-alkoxysilyl, alkyl-di-t-alkoxysilyl or

dialkyl-t-alkoxysilyl group.

15. A process according to claim 2 wherein the compound of general formula (III) is the compound of claim 14.

16. A process for the production of the compound of claim 14 which comprises the following steps:

17. A process according to claim 15 which comprises the following steps.

18. A process according to any of claims 1 to 10 which differs in that after step (f) and before step (g) the terminal carboxyl group of the solid support bound peptide is modified by substitution of or addition to the terminal -OH group, resulting in production by step (g) of a modified polypeptide of formula (I)¹. (I)¹

n + m (x + 1)

Wherein M is any substituent group other than -OH.