BACKGROUND OF THE INVENTION

I• > Ele.l-l_0-___-L-l_L_I-__-en i.on

This invention relates to a novel class of N-protected amino acid-N-carboxyanhydrides and thiocarboxyanhydrides, namely N- υrethane protected amino acid-N-carboxyanhydrides and N- υrethane protected N-thiocarboxyanhydrides, their preparation and their use in peptide, poly peptide and protein synthesis.

I1• > E£ia£__£_

Classically, polypeptldes of a defined sequence have been prepared by extremely laborious techniques wherein the Intermediates bave been isolated after the addition of each amino acid moiety. This has complicated the synthesis and made the preparation of long chain polypeptldes and proteins nearly impossible because of low yields, racemlzation, and/or other 3ide reactions. In 1963, Merrifield <J. Am. Che . Soc, 85, 21-_9> and Letsinger and Kornet (J. Am. Chem. Soc, 8S, 2045) suggested the use of insoluble polymeric supports for the growing peptide chain. This process, commonly referred to as a solid phase peptide synthesis, permitted the "purification" of the growing peptide chain, without isolating the intermediate.

SUBSTITUTE SHEET

Heretofore, the widely accepted methods for both classical (liquid phase) and solid phase polypeptide synthesis required the use of a coupling or activating agent to react with the carboxyl group of an otherwise N-protected amino acid to give a carboxyl-activated N-protected amino acid. This activated species would then be used in several ways to promote peptide bond formation. For example, the activated protected amino acid is allowed to react directly with the free amino group of an amino acid, amino acid ester, or amino acid amide to form the peptide bond. This has been the procedure of choice for preparing peptides for many years. The activation step can be accompanied by a number of possible side reactions. For example, where dicyclohexylcarbodiimide.DCC) is the activating reagent, the active molecule may "re-arrange" to an inactive N-acyl urea.

Another disadvantage of the carbodii ide procedure is the formation of insoluble ureas. This is particularly troublesome in solid phase synthesis and is virtually unacceptable in solid phase flow systems. These ureas also cause difficult purification problems in solution phase reactions.

Researchers have alleviated some of the problems associated with in situ activation by first reacting the DCC activated N- protected amino acid with an alcohol or phenol .such as p- nitrophenol, pentachlorophenol, N-hydroxy-succlnimide, etc.) to form an "active ester" which may be isolated and purified and then allowed to couple with the free amlne of the next amino

acid. This approach is not without its shortcomings, however, because the liberated alcohol or phenol may be involved in or promote other side reactions and active ester couplings tend to be sluggish and require long reaction times.

Another common procedure is to form a "symmetrical anhydride" by allowing two equivalents of N-protected amino acid to react with one equivalent of DCC, filtering the DCU formed and then allowing the "symmetrical anhydride" to couple with the free amine group of the next amino acid. This procedure has the urea problem in addition to requiring use of twice as much of an expensive N-protected amino acid.

Some researchers have recently begun to use carbodllmldes that form soluble ureas after coupling, but these are still prone to re-arrangement to N-acyl ureas. ^*

Various types of N-protecting groups have been proposed for use in peptide synthesis, but the most widely accepted class of N-protecting groups are the urethanes. Urethanes are broadly acknowledged to provide a high degree of protection, minimize race ization, are readily prepared, and are stable to storage. Urethane-protecting groups can be prepared which are labile to mild acid (i.e. t-butyloxycarbonyl) , strong acid (i.e., benzyloxycarbonyl> , extremely mild acid 2<p-biphenylyl>- lsopropyloxycarbonyl, anhydrous base (i.e., 9-flυorenyl- methyloxycarbonyl) , and so forth.

SUBSTITU ^TE HEE

Urethane-protected amino acids are commonly prepared by reaction of an alkyl, aryl or aralkylchloroformate (or other suitably activated formate or carbonate) with the amino acid in the presence of alkali metal hydroxide or carbonate in a mixed aqueous/organic solvent system (i.e., Schotten-Baumann conditions). After acidification of the reaction mixture, the urethane-protected amino acid is extracted into an organic solvent leaving all side products in the aqueous phase. After crystallization, these compounds are used for peptide bond formation as described above.

A particularly interesting type of reactive derivative of amino acids for use in peptide bond formation are the so-called N-carboxyanhydrides or N-thiocarboxyanhydrides, such as:

wherein R, R' are typically hydrogen or the side chains (or protected side chains) of the common amino acids, and Z is oxygen or sulfur.

Amino acid-N-carboxyanhydrides (it is understood that the term N-carboxyanhydride in this specification and appended claims is to include the N-thiocarboxyanhydrides) are well-known and react readily with most free amines. A primary advantage of

N-carboxyanhydrides (NCA's) and even protected NCA's for use in peptide bond formation is the fact that they are potent acylating agents (see Peptides, Vol. 9, page 83). They also generally give higher yields of peptides than DCC or N- hydroxysucclnimide (OSu) ester coupling procedures. But NCA's have not found widespread use in polypeptide synthesis because of the lack of ability to control or limit the coupling reaction. Once an NCA reacts with the free amine of an amino acid, carbon dioxide is Immediately liberated and a dipeptlde is formed which also contains a free amine. This amine will subsequently react with another NCA to form a tripeptlde, and so on. This reaction has allowed amino acid N-carboxyanhydrides to find extensive use in the formation of poly α-amino acids but has virtually precluded their use in sequential polypeptide formation. Hirschmann, et al (The Controlled Synthesis of Peptides in Aqueous Medium. VIII.) The Preparation and Use of Novel -Amino Acid N-Carboxyanhydrides. J.A.C.S., 93:11, 1971, pg. 27-i6-277--> have succeeded In using amino acid N- carboxyanhydrides for the preparation of di-and tripeptides in aqueous-organic solvent systems by careful control of temperature, pH, 3alt, and organic solvent of the reaction mixture. However, this procedure is limited to small peptides because of the chemistry of NCA's described above. Furthermore, the products obtained from these solution phase reactions must be extensively purified prior to being used for the preparation of larger peptides.

A variety of N-substi _, tuted amino acid N-carboxyanhydrides

B _STI T _U TESHEET

have been reported in the literature such as N-methyl, N- benzyl, N-acetyl, N-nitrophenylsulfenyl, N-xanthyl, 4,4'- dlmethylbenzhydryl, trityl, and the like. Several of these substituted-NCA ^τ s have been proposed for use in sequential peptide synthesis and particularly for solid phase peptide synthesis, but none have gained acceptance for general use by

_^ ^* peptide chemists.

Kricheldorf (Angew. Chem. Acta £35 _^ , 86-87, (1978)) proposed the use of o-nitrophenylsulfenyl (NPS) substituted NCA's for use in sequential polypeptide synthesis. These were prepared by the reaction of o-nitrophenylsulfenylchloride with a N- carboxyanhydride in the presence of trlethylamine. Subsequently, it has been shown that trlethylamine promotes the racemizatlon of NPS- NCA's. In addition, oligomerlzatlon due to the action of trlethylamine on the NCA, requires that very stringent reaction conditions must be employed (i.e. temperature <0 °C. and very slow addition of trlethylamine to the reaction mixture) during NPS-NCA synthesis. Halstrom, et al, (Z. Physiol. Chem. 3S5. 82-84, (1974)) consequently proposed the synthesis of NPS-NCA's by reaction of phosgene with the NPS- amino acid but the yields were very low (about 20%). Once prepared, NPS-NCA*s are difficult to store, and tend to "bleed off" the protecting group during condensation, giving rise to multiple coupling, and other side reactions. Also, the nitrogen of the resulting NPS protected peptide possesses substantial nucleophilicity and may undergo additional condensation reactions.

SUBSTITUTE SHEET

Block and Cox ("Peptides, Proc. of the Sth Europ. Sy p., Oxford, September 1962". Perga on Press 1963, Ed. G.T. Young, pp. 84-87.) proposed the use of N-trityl ammo acid-N- carboxyanhydrides for use in peptide synthesis, although they were only able to prepare the simplest N-tπtylamino acid NCA's <ie. glycine and alanme). These compounds were prepared by the reaction of a N-trityl-amino acid with phosgene. By this procedure, they were also able to prepare N-acetyl-glycine-NCA. These researchers recognized the potential usefulness of t- butyloxycarbonyl glycine N-carboxyanhydride and benzyloxycarbonyl glycine N-carboxyanhydπde but were unsuccessful in their attempts to prepare them and concluded that urethane-protected amino acid N-carboxyanhydrιdβ3 could not be made! Even if all the N-trityl-NCA's could be prepared it is well known that the use of trityl protection of amino acids in various condensation methods produces low yields because of the considerable ster c hindrance imposed by the trityl group. The trityl group is also extremely sensitive to acid which makes the preparation of the trityl-NCA difficult and tends to "bleed off" during normal solid phase manipulations.

Halstroem & Kovacs (A_Lt___Che.ml.£a_Sc:andinavl.£aj Sgrj._B_198,6,

BYQlil-—- _. 62-46S and U.S. Patent No. 4, 267, 344 ⁾ also recognized the advantages and the potenti ^' al usefulness of N-protected amino acid N-carboxyanhydrides and were able to prepare several N- substituted NCA's which they felt would fulfill all the requirements necessary for use in peptide synthesis. They were able to prepare a number of 9-xanthyl (and related) substituted

SUBSTITUTE SHEET

a ino acid N-carboxyanhydrides. These compounds were claimed to be preparable by the direct condensation of xanthydrol with the appropriate NCA in refluxing benzene, toluene, xylene or other alkyl benzene. The water formed during condensation was removed azeotropically. This procedure suffers from the instability of NCA's to heat and to water, consequently- leading to low yields and potentially impure products. These compounds may also be prepared by the reaction of phosgene (or phosgene equivalent) with the corresponding 9-xanthyl-amino acid and, in fact, most of the substituted NCA's in this class have been prepared by this procedure.

When useά in peptide synthesis the 9-xanthyl-NCA's have been found to react sluggishly requiring as long as 5 hours at 50°C in solution and 24 hours at 25 °C in solid phase synthesis. This is likely due to the steric hindrance of the 9-xanthyl group and/or the deactivating effect. Another problem associated with 9-xanthyl protection of amine groups is that the nitrogen atom of the 9-xanthyl amino acid formed after the coupling reaction is still nucleophilic and capable of undergoing subsequent condensation reactions. These groups will also tend to bleed off during manipulation. Consequently, to date, substituted amino acid N-carboxyanhydrides of this or any other type have not found widespread use in peptide synthesis, particularly in solid phase peptide synthesis.

richeldorf, (Hakromol. Chem. Vol. 178, pp 905-939, 1977) has described a method for the preparation of methoxycarbonyl

SHEET

glyclne NCA and ethoxycarbonyl glycine NCA. However, Krlcheldorf also reports that this procedure was incapable of producing urethane-protected NCA's of amino acids having a side chain other than hydrogen because of steric hindrance.

It is an object of the invention, therefore, to provide the heretofore unobtainable urethane-protected N-carboxyanhydrides and N-thiocarboxyanhydrides of the higher amino acids.

Another object of the invention Is to provide procedures for the preparation of pure, crystalline, stable urethane-protected amino acid N-carboxyanhydrides and urethane-protected N- hiocarboxyanhydrides.

Yet another object of the Invention is to provide a -aethod for the synthesis of polypeptldes utilizing pure, crystalline urethane-protected amino acid-N-carboxyanhydrides, which synthesis offers the following major advantages over conventional methods of polypeptide synthesis:

1) Pre-activatlon of the carboxyl group to be coupled is unnecessary, thus eliminating side products generated by conventional activating molecules.

2) No additives such as N-hydroxybenzotriazole are needed to inhibit racemization.

UTE SHEE

3) The only co-product from the coupling reaction is carbon dioxide.

4) These N-protected carboxyl activated amino acids are stable, storable, crystalline materials and, therefore, facilitate and simplify both solid and liquid phase peptide synthesis, especially in automated peptide synthesizers, by eliminating the need for activations, flltrations, and couplings prior to the peptide bond forming reaction. The purification of peptides prepared in solution is greatly facilitated by the use of these novel compounds because of the lack of by-products produced by coupling agents.

5) The procedure will provide, after coupling, the widely accepted urethane-protectlng groups on the amino function of the growing peptide chain which may then be manipulated by conventional techniques.

SUBSTITUTESHEET

SUMMARY_OF_THE_INVgNXIQN

These objects are obtained by a urethane-protected ammo acid-N-carboxyanhydride or N-thiocarboxyanhydride having the structure:

wherein R and R' are hydrogen, alkyl, cycloalkyl, substituted alkyl substituted cycloalkyl, aryl, or substituted aryl and at least one of R and R' is other than hydrogen; R" is alkyl, aryl, substituted alkyl or substituted aryl; Z is oxygen or sulfur; and n is 0, 1, or 2.

The preferred R, R' and R" groups are alkyl groups, Including cycloalkyl groups, of 1 to 12 carbon atoms or more, aryl groups of 6 to 20 carbon atoms or more including aralkyl and alkaryl groups of 7 to 20 carbon atoms or more. Exemplary of suitable alkyl groups are methyl, ethyl, propyl, isopropyl, isobutyl, butyl, t-butyl , hexyl , cyclopentyl, cyclohexyl, heptyl, octyl, and the like. Illustrative of suitable aryl groups are phenyl, methylphenyl , ethylphenyl, naphthyl, methylnaphthyl , anthracyl and the like. Examples of suitable aralkyl groups include benzyl, p-methoxybenzyl , 9- fluorenyl ethyl, phenylethyl, and the like. Suitable alkaryl

SUBSTITUTE SHEET

groups include tolyl, ethylphenyl, isopropylphenyl and the like, The R, R' and R" groups may also be substituted with non- interfering groups such as fluoro, mεthoxy, t-butoxy, carboxyl, amido, benzyloxy, hydroxy, substituted amino, substituted hydroxy, sulfur, substituted sulfur, chloro, bromo, and the like. R and R' are typically the protected or unprotected groups attached to the α-carbon atom (side chains) of amino acids or analogues thereof.

In most instances, one of R or R' is usually H while the other is the side chain on the α-carbon atom of of an amino acid such as lysine, leucine, arginlne, serine, aspartic acid, alanine, asparagine, cysteine, cystlne, glutamic acid, histidlne, glutaainβ, isoleueine, methionine, norleυcine, ornithine, phenylalanine, threonine, tryptophan, tyrosine, vallne, β-alanine, ho oserine and the like. Exemplary of such side chains are:

-(CH ₂ > ₄ -NH ₂ -(CH ₂ >2-COOH

—CH—CH ₂ —CHg

CH

-CH ₂ -COOH

SUBSTITUTESHEET

-CH ^* CH ₂ — CH ⁴ } — S— CH

- ⁽ CH ₂ ⁾ ₃ -NH ₂

-CH ₂ -SH

NH

-CH ₂ -S-S-CH ₂ -C IH-C00H -CH-CH ₃

I

OH

-CH ₂ -CH ₂ -OH

=CH Ό- OH

-CH-CH ₂ CH ₃

CH .

These side chains may be protected as required, using common techniques and protecting groups well known to one skilled in the art, such as the commonly employed amino, hydroxy, thiol and carboxy protecting groups.

The compounds of the invention also include instances where both R and R' are side chains attached to the α-carbon of an amino acid as, for example, In the case of Isovallne where one of R or R' is -CH ₂ CH ₃ and the other is methyl.

The compounds of the invention also Include instances where R and R' are part of a cyclic structure as, for example, in the

SUBSTITUTESHEET

case of 1-amino-l-cyclohexane carboxylic acid.

The compounds of the invention also include examples such as ortho-amino benzoic acid or i-amino-2-carboxy cyclohexane wherein carbon atoms from the R or R' groups are part of a cyclic ring.

Another aspect of the invention involves an improvement in the synthesis of a polypeptide chain wherein a N-protected amino acid component is deprotected and the deprotected amino acid component is allowed to react with a second similar or dissimilar activated N-protected amino acid component and the process repeated until the desired polypeptide is obtained, said improvement comprising using as the activated N-protected amino acid component in at least one of said reactions a compound having the structure:

wherein R, R' , R", Z and n are as designated above.

Yet another aspect of the invention involves an Improvement in the solid phase synthesis of a polypeptide chain on an insoluble solid support wherein a N-protected amino acid

co ponent Is coupled by condensation reaction to an insoluble solid support containing substltuent groups reactive with the carboxyl terminus end of said amino acid component, the coupled N-protected amino acid component Is deprotected, a second similar or dissimilar activated N-protected amino acid component is coupled to said deprotected amino acid compound, and the process repeated until the desired polypeptide Is obtained, said improvement comprising using as the activated N-prctected amino acid component in at least one of said reactions a compound having the structure:

wherein R, R' , Z and n are designated above.

Included as a further embodiment of the invention is the method of preparing the urethane-protected amino acid-N- carboxyanhydrides of the invention which comprises the reaction of an amino acid N-carboxyanhydrlde having the structure:

S _U BST _IT UTESHEET

43 '

-16- wherein R, R', Z and n are as designated above, with a haloformate (or other suitably reactive formate, such as azido formate) having the structure:

0 ^"

R"—0 —C II —X

wherein X is chlorine, bromine, fluorine, azide-, or the like, and R ^,r is alkyl, aryl, or aralkyl, in an inert diluent, under anhydrous conditions and in the presence of N- ethylmorpholine. It has been surprisingly found that utilizing an inert diluent, anhydrous conditions and selecting N- ethylsorpholine as the base in this reaction avoids polymerization of N-carboxyanhydrides and otherwise enables the production of the heretofore unobtainable urethane-protected NCA's arid NTA's of higher amino acids.

DETAILED DESCRIPTION OF THE INVENTION

The amino acid N-carboxyanhydrides (NCA's) and N- thiocarboxyanhydrldes (NTA's) which serve as starting materials for the preparation of the N-urethane protected NCA's and NTA's of the invention may be prepared by a number of procedures well known to one skilled in the art. See for example: Fuller et. al. Biopolymers IS, No. 9, 1869-1871 (1976); Kricheldorf, Chem. Ber. 104. 87-91 (1971); and Halstro and Kovacs, Acta Chemica Scandinavica, B40, 462-465 (1986).

While urethanes in general may be used as protecting groups for nucleophillc atoms, only a few have found widespread use in peptide synthesis, for example, t-butyloxycarbonyl (Boc); benzyloxycarbonyl (Cbz); and 9-fluorenomethyloxycarbonyl (FMOC). Consequently, amino acid N-carboxyanhydrides or N- thiocarboxyanhydrideβ substituted with these protecting groups are of particular interest. Accordingly, very useful molecules for peptide synthesis are the NCA'a of L-α-amino acids protected by one of the abovementioned protecting groups, such as:

SUB _S TITUTE SHEET

wherein R is the side chain of an α-amino acid, Z is 0 or S, and X is methoxy, chloro or the like.

As aforementioned, the N-urethane protected NCA's of the invention are unobtainable by the reaction of phosgene with the N-urethane protected amino acid as described by Block and Cox ⁽ "Peptides, Proe. of the Sth Europ. Sy p., Oxford, September 1962". Pergamon Press 1963, Ed. G.T. Young, pp. 84-87.); nor are N-urethane protected NCA's of the higher amino acids obtainable by the synthesis described by Kricheldorf (Makro ol. Chem., Vol. 176, pp 905-939, 1977). It has been found that urethane-protected NCA's and NTA's may be prepared by the reaction of a previously synthesized NCA or NTA with the appropriate haloformate in an anhydrous, non-interfering solvent with the use of a tertiary amine as base. The reaction is preferably carried out below room temperature. Useful solvents for the reaction are tetrahydrofuran, ethyl acetate, methylene chloride, toluene, benzene, dioxane, and the like.

Thus, the novel urethane-protected amino acid N- carboxyanhydrides and N-thiocarboxyanhydrides of the invention may be prepared by dissolving an NCA in a non-interfering

SUBSTITUTESHEET

solvent (such as toluene) and cooling the resulting solution with stirring. The desired haloformate (e.g. benzylchlorofor ate) is then added all at once. To this mixture is added a tertiary amine base (such as trlethylamine, diisopropylethylamine, N-methylmorpholine , etc.) which promotes condensation and scavenges hydrochloric acid formed during the reaction. Surprisingly, we have found that certain tertiary amine bases such as N-methylmorphollne and N- ethylmorphollne do not promote polymerization of NCA's. Such preferred bases are those with a p low enough not to promote NCA polymerization but high enough to catalyze the reaction of an NCA with a haloformate. Consequently, when one of these preferred bases is used In the condensation reaction, polymerization is not initiated. Since there is no fear of polymerization, the base can be used in excess and the resulting urethane protected NCA's are easily isolated by crystallization.

As a result of these discoveries, virtually any urethane protected NCA (or NTA) can be prepared easily in high yield with only minimal precaution for exclusion of moisture. The process Is readily scaled up and provides products which are highly crystalline, are readily purifiable by simple techniques (i.e. crystallization), and are stable to storage (completely stable at 25 °C for at least 6 months and probably much longer). Thus, these materials can be weighed, shipped, and stored for use in peptide synthesis without fear of decomposition.

SUBSTITUTE SHEET

_ _2fJ _

The major advantage that the urethane-protected NCA's offer over other N-substituted NCA's is that after they are used to form a peptide bond, the resulting peptide is protected on the N-terminus by one of the widely accepted urethane protecting groups commonly used in peptide synthesis. These protecting groups are well known by those skilled in the art to provide the best available protection to the amine group of a growing peptide chain.

Thus, the use of urethane-protected N-carboxyanhydrides will offer all the advantages of the unsubstituted NCA's (high reactivity, freedom from formation of undesired rearrangement products, and C0 ₂ as the only by product) but with none of the disadvantages of the unsubstituted NCA's (i.e. instability, polymerization, and multiple condensations) which have limited their use to carefully controlled aqueous conditions. Consequently, the invention provides a storable, yet highly reactive, preactivated reagent, which yields minimal side products during peptide bond formation. The invention also provides the widely accepted, well understood, urethane protection on the nitrogen of the N-terminus of the peptide after the condensation reaction.

While the urethane-protected amino acid-N-carboxyanhydrides of the invention can be used in the synthesis of polypeptldes by classical methods using a series of deprotection and coupling reactions, they undoubtedly will find more extensive use in solid phase polypeptide synthesis. It should be understood that

SUBSTITUT SHEET

the term "polypeptldes" as used in the specification and appended claims is meant to include peptides and proteins. Also, it should be understood that the present invention contemplates sequential peptide synthesis wherein N-protected amino acids other than the urethane-protected amino acid-N- carboxyanhydrides are employed as well as at least one urethane- protected NCA of the invention. In practice, however, the N- protected am o acid component used in each sequence will more than likely be the urethane-protected NCA's of the invention.

In solid phase polypeptide synthesis, an insoluble solid support or matrix, advantageously in bead form, is used. Such solid supports can be any of the solid-phase polymeric substrates conventionally employed for the synthesis of polypeptldes. Typical of such polymeric resins are crosslinked polystyrene resins, glass beads, clays, celite, crosslinked dextran, polyacrylamldes, polyamlde resins and similar insoluble solid supports which either naturally contain reactive sites for coupling with the amino acid components or which can be provided with such reactive sites.

If desired, the solid phase polypeptide synthesis of the invention can be carried out in a flow reactor under pressure as described in U.S. Patent No. 4,192,798, hereby incorporated by reference, but the use of superatmospheric pressures is not essential.

SUBSTITUTE SHEET

Several preliminary operations are necessary before the solid phase synthesis of a peptide can be started. First, the supporting resin containing the C-terminal amino acid component of the proposed peptide chain must be prepared. This can be accomplished by any of a number of procedures known to one skilled in the art. Many of these N-protected a ino acids, linked to various solid supports, are articles of commerce and may be purchased as desired.

The remaining synthesis to form the desired polypeptide sequence is carried out as follows. Before coupling of the second amino acid residue can take place, the first residue already on the support must be deprotected. Deprotection of the first amino acid residue on the resin as well as of each of the subsequently coupled amino acid residues cf.n be carried out by contacting the protected amino acid residue with an appropriate deprotectlng agent. The deprotecting agents employed for this purpose are well known to those of ordinary skill in the art of peptide synthesis and the particular deprotecting agent employed in any given instance will depend, of course, upon the protecting group on the amino acid/resin. For example, if the protecting group Is t-butyloxycarbonyl, trifluoroacetic acid in dichloromethane or hydrochloric acid in a suitable solvent such as dioxane may be used. On the other hand, if the protecting group is 9- luorenylmethyloxycarbonyl, basic conditions such as piperidine in DMF will be the preferred method. The concentrations of the particular deprotectlng agent in the solvent will vary depending again upon the particular protecting

agent employed but will ordinarily range from about 5 to SOZ by volume.

After the deprotecting step, the resin is washed with a suitable solvent in order to remove excess deprotecting agents. If the deprotecting agent is an acid the peptide on the resin must be neutralized by washing with an appropriate base such as trlethylamine in a solvent such as dichloromethane. Any excess trlethylamine and triethylammonium chloride or trifluoroacetate formed may be removed by repeated washings with a suitable solvent such as dichloromethane or dimethylformamide. The free amine, thus prepared, is now ready for coupling with the next N- protected amino acid.

If the next N-protected ammo acid is a urethane-protected amino acid N-carboxyanhydrlde of the Invention, it need not be activated and can be reacted directly with the support now containing an unprotected resin bound amino acid. If, however, the N-protected amino acid component is to be coupled by more conventional procedures, it will be necessary to first activate, that is, convert it into a reactive form, for instance, by converting the amino acid into an anhydride or by activation with dicyclohexylcarbodiimide, carbonyldiimidazole or other activating agents. In general, an excess of the accivated N- protected amino acid component Is employed in the reaction.

After the coupling of the second protected amino acid

UBSTITUTE SHEET

/08643 _-2-.4,- PCT/US89/0087

component to the first amino acid component, the attached protected dipeptlde is then deprotected, neutralized if necessary, and washed as described above before coupling of the next amino acid derivative is effected. This procedure is repeated until the desired sequence of amino acids has been assembled on the Insoluble support.

Because of the lack of undesirable side reactions and by¬ products (C0 ₂ being the only one) in the urethane protected NCA coupling, and because of their stability, the excess urethane protected NCA used in the coupling reactions may be easily recovered, recrystallized and re-used, thus markedly increasing the cost effectiveness of these materials.

The completed peptide can be removed from the insoluble support by any of the standard methods as, for instance, by cleavage with anhydrous hydrogen fluoride, transesterification, amiπolysis, etc.

After cleavage, the resulting peptide is found to be remarkably homogeneous and to require no or minimal purification. Because of the very low contamination of byproducts overall yields are found to be surprisingly high and whatever purification is necessary can be carried out with relative ease. Such purifications are preferably carried out by partition chromatography, ion exchange chromatography or a combination of both. Such procedures are well-known to one skilled in the art of peptide synthesis. STITUTE SH

EXAMPLE I

N £ £b_!__χafl ϊ_-£id-!_B_L£&mE£ _S i _t i--Ω__-ϊ_

A. Valiπe N-carboxyanhydπde (72 ) was dissolved In dry, distilled tetrahydrofuran (2 L) and trlethylamine (30 μl) added. The disappearance of the NCA was followed by infrared spectroscopy.

B. Vallne N-carboxyanhydrlde (72 mg) was dissolved in tetrahydrofuran (2 mL) and N-methylmorpholine <2S μl) added. The disappearance of the NCA was followed by infrared spectroscopy.

The results of A and B are shown in the graph of Figure 1:

EXAMPLE II

__i2__ElH--£--B_£iffi----_l-ii___ϊ£--£--≥fi^

A. A mixture of L-alanine (40.3 g, 0.45 mol) and phosgene (275 mL of 3.3 M solution in tetrahydrofuran, 0.90 mol) was stirred at 62-64 °C for _ hours. The resulting solution was allowed to cool to room temperature, filtered, and the volatlles removed under reduced pressure. The resulting oil was dissolved in 100 mL of tetrahydrofuran and 300 L of hexane was added with stirring, followed by cooling to -20 °C. The yield of L-alanine N-carboxyanhydride was 35.79 g (69Z).

SUBSTITUTE SHEET

B. A solution of N-methylmorpholine (8.15 g, 80.5 mmol) in toluene (50 mL) was added to a 0 °C mixture of L-alanine-N- carboxyanhydride (8.84 g, 76.8 mmol) and 9- fluorenylmethyloxycarbonyl chloride (19.9 g, 76.8 mmol) in toluene (200 mL)- The reaction mixture was stirred at O °C for

2 hours and filtered. The volume of solvent was reduced to 20 L and crystallization occurred upon addition of 100 L of hexane to give 21.4 g (82%) of crude 9- fluorenylmethyloxycarbonyl-L-alanlne-N-carboxyanhydrlde. The product was purified by trituratlon with cold diisopropyl ether followed by recrystallization from ethyl acetate/hexane: mp 106-

107 ^O C; IR (CH ₂ C1 ₂ > 1870, 1801, 1740 cm ^"1 ; NMR (CDCI3) $ 6.90-

7.80 (m, 8 H>, 3.9S-4.SS ( , 4 H) , 1.35 (d, J = 7 Hz, 3 H) .

Anal. Calcd for C _l9 H ₁₅ NO _s . C, 67.65; H, 4.48,- N, 4.15. Found:

C, 67.73; H, 4.65; N, 4.19.

EXAMPLE III

__i-___Ii--_-_-ϋ__ifi-_!ffi≥-_--∑l£X^

A. L-Leuclne-N-carboxyanhydride was prepared 'from L-leucine in 78% yield by the procedure outlined in Example IIA.

B. A mixture of L-leuclne N-carboxyanhydride (9.2 g, 58.4 mmol) and 9-fluoroxylmethyloxycarbonyl chloride (15.1 g, 58.4 mmol) in toluene (125 mL) was cooled to 0 °C and a solution of N-methylmorpholine (6.S g, 64 mmol) in 20 mL of toluene was added dropwise. The reaction mixture was stirred at 0 °C for

SUBSTITUTESHEET

2.5 h, filtered, and the volume of solvent reduced to 20 mL.

Hexane (480 mL) was added, and the solution was cooled to -20 <>C overnight, to give 18.8 g (85%) of N-( 9-fluorenyl ethyloxy carbonyl>-L-leucine-N-carboxyanhydride. An analytical sample was obtained by recrystallization from ether/ ethylene chloride/hexane: mp 118-120 °C; NMR (CC1 ₄ ) 6 7.35-7.91 (m, 8

H); 4.72 <t, J = 7 Hz, 2 H); 4.58 <m, 3 H) ; 4.37 <t, J = 7 Hz, 1

H); 2.05 ( , 2 H); 1.09 <t, J = 6 Hz, 6 H>. Anal. Calcd for

C ₂₂ H ₂₁ N0 ₅ : C, 69.64; H, S.58; N, 3.69. Found: C, 69.08; H,

5.97; N, 3.70.

EXAMPLE IV

_______El__-££2 _∑ l!£_-]l∑l&-_X£-i£--£Ωϊl2~N-ε-^

L__sine-N-£arbax2anhϊdride

A. A mixture of N-e-t-butyloxycarbonyl-L-lysine (1.23 g, 5.0 mmol) and chlorotrimethylsilane (1.08 g, 10.0 mmol) in tetrahydrofuran (50 mL) was cooled to 0 °C and a solution of trlethylamine (1.01 g, 10.0 mmol) in S mL of tetrahydrofuran added dropwise. The mixture was stirred at 0 °C for 2.5 hours, filtered, and added to a solution of phosgene (10 mmol) in 15 L of tetrahydrofuran. The temperature was raiβed to 60 °C and the solution was stirred for 2.0 hours, then overnight at ambient temperature. The volatlles were removed by rotary evaporation to give 0.79 g <S8%) of N-ε-t-butyloxycarbonyl-L-lysine N- carboxyanhydride: IR(CH ₂ C1 ₂ > 1860, 1795, 1710 cm ^"1 .

SUBSTITUTE SHEET

3

-28-

B. A mixture of N-ε-t-butyloxycarbonyl-L-lysine N- carboxyanhydride (0.79 g, 2.90 mmol) and 9- fluorenyl ethyloxycarbonyl chloride (0.75 g, 2.90 mmol) in toluene C25 ml) was cooled to 0 °C and a solution of N- methylmorpholine (0.32 g, 3.2 mmol) in toluene (5 ml) added dropwise. The reaction was worked up as in Example IIIB to give 0.88 g (66%) of N-α-(9-fluorenylmethyloxycarbonyl)-N-ε-t- butyloxycarbonyl-L-lysine-N-carboxyanhydride: mp 81-85 °C (ethyl acetate/hexane); NMR (CDC1 ₃ ) δ 7.3-7.7 (m, 8 H) , 4.11- 4.58 (m 5 H), 2.9S-3.20 (m, 2 H> 1.90-1.98 (m, 2 H); 0.9-1.4 (m, 13 H). Anal. Calcd for C ₂₇ H ₃₀ N ₂ O ₇ : C, 65.57; H, 6.11; N, 5.67. Found C, 66.33; H, 6.38; N, 5.67.

EXAMPLE V

N-Benzyloxycarbonyl-L-AIanlne N-Carboxyanhydrlde

• A solution of N-methylmorpholine (1.06 g, 10.5 mmol) in ethyl acetate (20 mL) was added dropwise to a mixture of L- alanine-N-carboxyanhydride (from IIA) (0.81 g, 7.0 mmol) and benzyloxycarbonyl chloride (1.89 g, 10.5 mmol) in ethyl acetate (80 mL) at 0 °C. The reaction mixture was stirred for l.S h at 0 °C, filtered, and the volume of the solution was reduced to 7S mL. Hexane (75 mL) was added with stirring, followed by cooling to -20 °C, to give 1.20 g (71%) of N-benzyloxycarbonyl-L-alanine- N-carboxyanhydride: mp 101-104 «C; NMR (CDC1 ₃ ) 6 7.33 (s, 5 H), 5.27 (s, 2 H>, 4.60 (q, J = 7 Hz, 1 H) , 1.61 (d, J = 7 Hz, 3 H). Anal. Calcd for C^H^NOg: C, 57.83; H, 4.45; N, S.62.

ET

Found: C, 57.60; H, 4.50; N, 5.53.

EXAMPLE VI

—^&IiΑϊλθ}LZS.&L 2.Rϊλz. z &£.lIk^~ &L 2.}iϊ&Iϊ Z j ii§.

A solution of N-methylmorpholine (0.76 g, 7.50 mmol) in ethyl acetate (10 mL) was added dropwise to a solution of L- leucine N-carboxyanhydride (0.79 g, 5.0 mmolMfrom IIIA) and benzyloxycarbonyl chloride (1.35 g, 7.50 mmol) in ethyl acetate (50 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1.25 h, filtered and the volume of the solution was reduced to 5 mL. Hexane (50 mL) was added, followed by cooling to -20 °C, to give 0.89 g (61%) of N-benzyloxycarbonyl-L-leucine-N- carboxyanhydride: mp 72-73.S °C (ether/hexane) ; NMR (CDC1 ₃ ) δ 7.40 (s, S H), S.33 (s, 2 H>, 4.71 (t, J = 6 Hz, 1 H>, 1.80-2.04 (m, 3 H), 0.91 (m, 6 H). Anal. Calcd for C _ls H ₁₇ NO _s : C, 61.84; H, S.88; N, 4.81. Found: C, 61.64; H, 6.02; N, 4.90.

EXAMPLE VII

H~E -_Qϊl2-_--___-£-_£fiZl-k-:_--iiπ-l-:N-C £b_ ____nhydrid£

A. L-Valine-N-carboxyanhydrlde was prepared from L-valine in 75% yield by the procedure described in Example IIA.

B. Example V was repeated substituting phenylchloroformate for benzylchloroformate and valine N-carboxyanhydride for

SUBSTITUTE SHEET

leucine N-carboxyanhydride. The result was a 78% yield of N-phenyloxycarbonyl-L-valine-N-carboxyanhydride: mp 105-106 °C (chlorofor /hexane) ; NMR (CDC1 ₃ ) δ 7.30 (m, 5 H) , 4.70 (d, J = 3.5 Hz, 1 H>, 2.60 ( , 1 H), 1.22 ( , J - 7 Hz, 3 H) , 1.07 (d, J = 7 Hz, 3 H), 1.07 (d, J = 7 Hz, 3 H) . Anal. Calc for C ₁₂ H ₁₃ N0 ₅ : C, 59.31 H, 4.98; N, 5.32. Found: C, S9.09; H, 4.91; N, 5.49.

EXAMPLE VIII

N-Ethyloxycarbonyl-L-Alanine. N-Carboxyanhydrlde

Example V was repeated substituting ethyl chloroformate for benzyl chloroformate. The result was a 62% yield of N- ethyloxycarbonyl-L-alanine N-carboxyanhydride: mp 72-73.S °C

(ethyl acetate/hexane); NMR (CDC1 ₃ > δ 4.73 (q, J = 7 Hz, 2 H> ,

4.33 (q, J = 7 Hz, 1 H> , 1.70 (d, J = 7 Hz, 3 H> , 1.33 (t, J = 7

Hz, 3 H>. Anal. Calcd for C ₇ H ₉ N0 _S : C, 44.92; H, 4.85; N, 7.49. Found: C, 4S.08; H, 5.03; N, 7.33.

EXAMPLE IX

Benzyloxycarbonyl-L-Phenylalanine-N°Carboxγanhydride

A. L-Phenylalanine-N-carboxyanhydride was prepared from L-phenylalanine in 53% yield by the procedure of Example IIA.

B. To a solution of L-phenylalanine-N-carboxyanhydride (2.5

g, 13 mmol) and benzylchloroformate (3.4 g, 20 mmole) in ethyl acetate (130 mL) was added dropwise a solution of N- methyl orpholine (2.0 g, 20 mmol) in ethyl acetate (10 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 2.5 h and worked up as described in Example V to give 2.0 g (48%) of N- benzyloxycarbonyl-L-phenylalanine-N-carboxyanhydride: mp 108- 109 °C; NMR (CDC1 ₃ > & 7.35 (s, S H), 7.00 (m, 5 H) , S.31 (s, 2 H), 4.83 <m, 1 H>, 3.28 ( , 2 H), Anal. Calcd for C _ιe H ₁₇ N0 ₅ : C, 68.13; H, 5.40; N, 4.42. Found: C, 68.11; H, 5.38; N, 4.20.

EXAMPLE X

Ehen__lo________£__on2i-L-Alanin _£ -N_Thia_La _£ -_2-_χa

A. O-Ethyl-S-methylxanthate. To a solution of potassium ethylxanthate (16.0 g, 100 mmol) in water (50 L > was added dropwise dimethyl sulfate (12.6g, 100 mmol) at 4 * 1 °C. Upon completion of the addition, the reaction mixture was washed with dichloromethane (2 x 40 mL) and the combined organic fractions were dried (MgS0 ₄ ) and concentrated. The oily residue was dissolved in methanol and concentrated to give 0- ethyl-S-methylxanthate of sufficient purity for use in the next step.

B. Ethoxythiocarbonyl-L-alanine. To the O-ethyl-S- methylxanthate prepared above was added a solution of L-alanine (8.9 g, 100 mmol) and NaOH (4.0 g, 100 mmol) in water (100 mL) The solution was heated to 45 °C for 2.3 h. While being purged

SUBSTITUTESHEET

with N ₂ » methanol (50 mL) was added and the mixture stirred at 45 °C an additional 0.7 h. The reaction mixture was allowed to cool to room temperature, washed with dichloromethane (3x25 mL), acidified to pH 2.S with concentrated HC1, and extracted with ethyl acetate (2 x SO mL). The combined organic solutions were dried (MgSO^) and concentrated. Addition of hexane to the resulting oil gave 9.S g (54%) of ethyloxythiocarbonyl-L-alanine as a colorless solid which melted at 74-78 °C. This material was further purified„by recrystallization from ether/hexanes mp 77-79 °C; IR (CC1 ₄ > 3397, 1716 cm ^"1 .

C. L-Alanine—N-thiocarboxyanhydrlde. To a solution of ethyloxythiocarbonyl-L-alanine (3.0 g, 17 mmol) and imldazole (1.2 g, 17 mmol) in THF (20 mL) was added dropwise PBr ₃ (S.4 g, 20 mmol) at 20 °C. Stirring was continued until the solid mass had broken up into a fine suspension. The reaction mixture was poured into a mixture of saturated NaHC0 ₃ (200 L) and ethyl acetate (150 mL). The organic layer was separated, washed with 1 K HCK2 x 100 mL), saturated NaHC0 ₃ (100 mL), and brine (100 mL), dried (MgSO^), and concentrated. The resulting oil solidified on standing. Recrystallization of the solid gave 0.75 g (34%) of L-alanine-N-thiocarboxyanhydride: mp 91- 92 °C; IR (CC1 ₄ ) 17S0, 169S cm ^"1 .

D. Phenyloxycarbonyl-L-alanine-N-thiocarboxyanhydride. To a solution of L-alanine-N-thiocarboxyanhydride (0.49 g, 3.8 mol) in 50 mL of ethyl acetate was added phenyl chloroformate (0.95 g, 6.1 mmol) at 0 °C, followed by dropwise addition of a

SU

solution of N-methylmorpholine (0.57 g, S.6 mmol) in ethyl acetate (10 mL) at 0°C. The resulting mixture was stirred for 3 h at 0 °C, filtered and concentrated to a white semi-solid. The semi-solid material was dissolved in 20 mL of ethyl acetate, hexane (150 mL) was added, and the mixture cooled to -20°C to give 0.S5 g (62%) of phenyloxycarbonyl-L-alanine-N- thiocarboxyanhydride: mp 110-111 °C; NMR <CDC1 ₃ ) δ 7.18 (m, 5 H), 4.83 (q, 1 H, J = 7 Hz), 1.71 (d, 3 H, J = 7 Hz); IR <CH ₂ C1 ₂ ) 1810, 1740 (doublet), ,1715 (Shoulder). Anal. Calcd for C ₁₁ H ₉ N0 S: C, 52.58; H, 3.61; N, 5.58; S, 12.76. Found: C, S2.7S; H. 3.72; N, S.36; S, 12.98.

EXAMPLE XI

___2_Elyo£!£Xlffl-_£h∑!-_-_X£-i£b£BZl2 _-_£ _b __-_2ϋπhχd£i-__!

A. O-t-Butyl-L-threonine N-carboxyanhydride was prepared from O-t-butyl-L-threonine in 57% yield using the trlmethylsllyl procedure described in Example IVA.

B. To a solution of O-t-butyl-L-threonine-N-carboxyanhydride (0.80 g, 4.0 mmol) and 9-fluorenylmethyloxycarbonyl chloride (1.0 g, 4.0 mmol) in toluene (50 L) was added dropwise a solution of N-methylmorpholine (0.49 g, 4.8 mmol) in 8 mL of toluene at 0 °C. The reaction was stirred for 3 h at 0 °C, filtered, and the volatlles removed under reduced pressure. The residue was crystallized from ether/hexane to give 1.0 g (60%) of N-(9-

SUBSTITUTESHEET

fluorenylmethyloxycarbonyl>-0-t-butyl-L-threonine: mp 124-127

°C; NMR (CDC1 ₃ ) δ 7.08-7.78 ( , 8 H) , 4.05-4.61 ( , 4 H) ,

1.18 (s, 9 H), 1.16 (d, 3 H, J= 7 Hz). Anal. Calcd for

C ₂₄ H ₂₅ N0 ₆ : C, 68.07; H, 5.95; N, 3.31. Found: C, 67.89; H, S.96; N, 3.28.

EXAMPLE XII

E ^* yi∑l£-_X£__£__£____T___ϊ:_________£__ϊ_______ __X^

A. α-Aminoisobutyric acid N-carboxyanhydride was prepared in 67% by the procedure described in Example IIA.

B. Example VIIIB was repeated substituting α- aminoisobutyric acid N-carboxyanhydride for L-alanine-N- carboxyanhydride to give a 16% yield of ethyloxycarbonyl- - aminoisobutyric acid N-carboxyanhydride: mp 68-70 °C (chloroform/hexane); NMR (CC1 ₄ ) δ 4.S9 (q, 2 H, J = 7 Hz), 2.00 (s _r 6 H), 1.6S (t, 3 H, J = 7 Hz). Anal Calcd for ^H^ ϋs. C, 47.76; H, 5.51; N, 6.96. Found: C, 47.67; H, 5.51; N, 7.14.

EXAMPLE XIII

N-t-Butyloxyearbonyl-L-Alanine N-Carboxyanhydride

To a solution of t-butyl alcohol (1.25 g, 16.9 mmol) and phosgene (3.4 L of a 5 M solution In dioxane, 17 mmol) in 80 mL of ethyl acetate was added dropwise N-methylmorpholine (3.4 g,

34 mmol) at -50 °C. The reaction mixture was stirred for 0.S h. L-alanine-N-carboxyanhydride (0.23 g, 2.0 mmol) in ethyl acetate (10 mL) was added and the mixture stirred at -50 °C for an additional 0.75 h. N-Methylmorphollne (1.0 g, 10 mmol) was added, and stirring was continued for another 0.75 h at -SO °C. The solids were removed by filtration,- the solution was concentrated, and the product was obtained after trituratlon with hexane. Recrystallization from toluene gave 0.28 g (6SZ) of N-t-butyloxycarbonyl-L-alanine-N-carboxyanhydride. mp 103- 104.S °C; NMR (CDC1 ₃₎ δ 4.71 <q, 1 H, J = 7 Hz), 1.80 (d, 3 H, J = 7 Hz), 1.70 <s, 9 H). Anal. Calcd for C ₈ H ₁₃ N0 ₅ : C, SO.23; H, 6.09; N, 6.51. Found: C. SO.66; H, 6.36; N, 6.38.

EXAMPLE XIV

H~i__-_BH£Xl£x_---a£bon∑I!-- --£__n^

A. O-Benzyl-L-serine-N-carboxyanhydride was prepared In 68% yield by the procedure of Example IIA.

B. Example XIII was repeated substituting O-Benzyl-L- serine-N-carboxyanhydride for L-alanine-N-carboxyanhydride to give N-(t-butyloxycarbonyl)-0-benzyl-L-serlne-N-carboxyanhydrlde in S2Z yield: mp 98-99.S °C; NMR <CC1 ₄ > δ 7.30 ( , 5 H), 4.64 (m, 3 H, benzyl CH ₂ and NCA ring proton), 4.09 (dd, 1 H, J = IS, S Hz), 3.88 (dd, 1 H, J = 15, S Hz), 1.6S (s, 9 H> Anal. Calcd for C ₁₅ H ₁₉ NOg: C, S9.80; H, S.96; N, 4.36. Found: C, 59.71; H, 6.25; N, 4.OS.

SUBSTITUTESHEET

EXAMPLE XV

Phenyloxycarbgxyl„Derlvation of 1-Aminq-l-Cyclohexanecarbqxylic Acld.N-Carboxyanhydride

A. 1-Amino-l-cyclohexanecarboxyllc acid-N-carboxyanhydride was prepared from 1-amino-l-cyclohexanecarboxyllc acid in 50% yield by the procedure described in Example IIA.

B. To a solution of the N-carboxyanhydride prepared in A (0.85 g, 5.0 mmol) and phenyl chloroformate (1.2 g, 7.S mmol) in ethyl acetate (30 mL) at 0 °C was added a solution of N- methylmorpholine (0.76 g, 7.5 mmol) in 8 mL of ethylacetate.

The reaction mixture was stirred for 2 h at 0 °C, filtered, and concentrated. The white, semi-solid residue was recrystallized from ethyl ether/methylene chloride/hexane to give 0.92 g (66%) of the N-carboxyanhydride: mp 156.S-158 °C; NMR (CDC1 ₃ ) δ 7.28 <m, S H> , 1.20-3.10 ( , 10 H> . Anal. Calcd for C _1S H ₁₅ N0 ₅ : C, 62.27; H, 5.23; N, 4.84. Found: C, 62.03; H, S.22; N, 4.77.

•EXAMPLE XVI

H_ii_zEi ££--2Xiffi£--_!∑l££X£-_E--£fiX_-2.~L~L£

A. L-Leueine-N-carboxyanhydride was prepared from L- leucine in 78% yield by the procedure outlined in Example IIA.

B. A mixture of L-leucine N-carboxyanhydride (9.2 gm, S8.4

m ole) in dry, distilled ethyl acetate (150 L) was cooled o to -10 C and a solution of trlethylamine (9mL, 64 mmol) in 20 L of ethyl acetate was added dropwise over S minutes. Cooling was discontinued and the reaction was allowed to stir for IS minutes. A email amount of anhydrous hydrochloric acid in dioxane (4.4M, 5 ml) was added in order to ensure that all of the trlethylamine had been neutralized. The reaction was filtered and the solvent removed m Y.ϋ£__°* The resulting crude product was taken up In ethyl ether, filtered,and the product obtained by crystallization after addition to hexane.

After three additional, careful crystallizations, N-(9- fluorenyl-methyloxycarbonyl)-L-leucine-N-carboxyanhydride was obtained in 16% yield (3.6 grams). Analytical data was essentially identical to that obtained in Example III.

EXAMPLE XVII

L-_k----£_--_---__--___IA-l--

9-Fluorenylmethyloxycarbonyl-L-valine esterified to p- alkoxybenzyl alcohol derivitized 2% crosslinked polystyrene (0.25 gm, 0.13 mmol vallne) was placed In a solid phase peptide synthesis vessel. Dimethylformamide (5 mL) was added and the slurry was shaken for 30 min. The dimethylformamlde was removed and the swollen resin treated twice with 10% plperidine in dimethylformamlde (S L for S min followed by 5 mL for IS min) to remove the 9-fluorenylmethyloxycarbonyl protecting group.

The resin was washed with dimethylformamlde (4 x 5 mL) and reacted with 9-fluorenylmethyloxycarbonyl-L-leuclne-N- carboxyanhydride (145 mg _r 0.38 mmol) in dimethylformamlde (6 mL) for 45 min. The fluorenylmethyloxycarbonyl protecting group was removed as above and the resin washed with dimethylformamlde (3 x 5 mL) and methylene chloride (3 5 mL). The resulting dipeptlde was cleaved from the resin by treatment with methylene chloride/ trifluoroacetic acid (6 L, 1/1) for 45 min. The solution was removed and the resin washed with methylene chloride (3 x 5 mL) and methanol (2 x S mL). The combined solution and washes were evaporated in vacuo to a semi-solid, which was taken up in distilled water and filtered. The aqueous solution was freeze-dried, the resulting solid triturated with ether(3X> to remove resin related contaminants, and dried under reduced pressure to give L-leucyl-L-valine in >90X yield. The identity of the dipeptlde was confirmed by HPLC analysis (flow rate = 1.5 mL/min, detection at 215 nm, 30% methanol in 0.5 M perchloric acid) by co-elution with a known standard (Retn. time 8.49 minutes). The purity was determined to be >97%, with all contaminants being traceable to the resin. No D-leucyl-L-valine (Retn. time 32 minutes) could be detected (detection limits <0.1%).

EXAMPLE XVIII

L*-Leucχl-L-Yalin__

Example XVI was repeated except that 9-

flύorenylmethyloxycarbonyl-L-leucine-N- arboxyanhydride was reacted with the free amine of L-valine on the resin using methylene chloride (5 mL) instead of dimethylformamlde as the solvent. The results were comparable to Example XVI.

EXAMPLE XIX

£-J ∑I~L-:ΔI_L_l∑I_: _:Y Iin£_ FMOC_PriC£dure2

The procedure of Example XVI was used to prepare L-leucyl- L-alanyl-L-valine. After cleavage from the resin and ether washes the tripeptlde was obtained in >88% yield as a white solid. HPLC analysis, using conditions described in Example XVI confirmed the identity of the product which co-eluded with a known standard. (Rtn. time 16.28 min.) Deletion sequences such as L-leucyl-L-valine and L-alanyl-L-valine were not detected (detection limits <0.1%).

EXAMPLE XX

LzLil-l£-ii_-L-:-_i-_β-ii~-- -_-ii-k-l--_i&££_££≥£__-i£-i2

t-Butyloxycarbony-L-valine esterified to methylated 2% crosslinked polystyrene (i.e. Merryfleld resin) (0.S0 gm, 0.23 mmol vallne) was placed In a solid phase peptide synthesis vessel. Methylene chloride (5 mL) was added and the slurry shaken for 30 minutes. The solvent was removed and the resin treated with methylene chloride/trifluoroacetic acid (6 mL of

1/1) for 30 min to remove the t-butyloxycarbonyl protecting group. The resin was washed with methylene chloride (3 x 5 L) neutralized with 10% trlethylamine in methylene chloride (5 mL), washed with methylene chloride (3 x S L) and then reacted with a solution of t-butyloxycarbonyl-L-alanine-N-carboxyanhydride (200 mg, 1.0 mmol) in methylene chloride (5 mL) for 45 min. The resulting protected dipeptlde resin was washed with ^' ethylenechloride (3 x 5 mL). The resin was again deblocked, washed, neutralized and washed as described above, t- Butyloxycarbonyl-L-leucine-N-carboxyanhydrlde (240 mg, 1.0 mmol) in methylene chloride (6 mL) was added to the resin and the mixture shaken for 45 min. The solution was removed and the resin washed with methylene chloride (3 x 5 mL), methanol (3 x 5 L) and methylene chloride (3 x 5 mL) and dried under high vacuum. The t-butyloxycarbonyl protected tripeptlde resin was reacted with liquid hydrogen fluoride at 0 °C for 30 minutes. The hydrogen fluoride was removed and the residue dried under high vacuum. The peptide was taken up in water and the resin removed by filtration. The solution was freeze-dried to give a nearly quantitative yield of L-leucyl-L-alanyl-L-valine. HPLC analysis results were comparable to Example XVIII.

EXAMPLE XXI

L-Alan__i-L-Ph__nχl_ιlanme_ Via_Full _^

A. To L-phenylalanine benzyl ester p-toluenesulfonate (1.07

SUBSTITUTESHEET

g, 2.S mmol) in tetrahydrofuran (20 mL) at 0 °C was added N- methylmorpholine (0.25 g, 2.S mmol). The mixture was stirred 0.5 h at 0 °C and N-benzyloxycarbonyl-L-alanine-N- carboxyanhydride (0.50 g 2.0 mmol) was added. The reaction mixture was stirred 2 h at 0 °C and water (20 mL) and dichloromethane (50 mL) were added. The ^' layers were separated and the aqueous layer washed with dichloromethane (25 mL). The combined organic fractions were washed with 0.5 M HC1 (2 x 50 mL), 10% sodium bicarbonate (50 L), and water (2 x 50 mL), dried (MgS0 ₄ ), and concentrated. Crystallization occurred upon addition of hexane to give 0.66 g (72%) of N-benzyloxycarbonyl- L-alanyl-L-phenylalanine benzyl ester: mp 118.5-119 °C; NMR (CDC1 ₃ ) δ 7.64(s, 1 H>, 6.82-7.39 (m, 6 H), S.04 (s, 2 H), 5.00 <s, 2 H>, 4.58-4.92 <m, 2 H>, 3.07 (d, J = 6 Hz, 2 H) 1.29 <d, J=7 Hz, 3 H>.

B. A mixture of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine benzyl ester (0.50 g, 1.1 mmol) and 10% Pd palladium on carbon (0.1 g) in ethyl alcohol (150 L) was shaken on a Parr hydrogenation apparatus for 6.5 h at 20 °C. The reaction mixture was filtered and the filtrate rinsed with water (lOOmL). The solution was concentrated to give 0.26 g (100%) of L-alanyl- L-phenylalanine. HPLC analysis showed >99% purity and no evidence of racemlzatlon.

SUBSTITUTE SHEET

EXAMPLE XXII

L-AlanylrL-Phenylalanlne .(Via Partial..Protection Procedure)

A. To a solution of L-phenylalanine (0.33 g, 2.0 mmol) in 0.20 M potassium carbonate (20 L) and acetonltrile (30 mL) was added dropwise a solution of N-benzyloxycarbonyl-L-alanine N- carboxyanhydride (0.45 g, 1.8 mmol) in acetonltrile (5 mL) at 0 °C. The mixture was stirred 40 min at 0 °C and diluted with ethyl acetate (SO L) and 1 M hydrochloric acid (10 mL). The layers were separated and the aqueous layer extracted with ethyl acetate (2 x 35 mL). The combined organic fractions were washed with brine (30 mL), 0.5 M hydrochloric acid (2 x 50 mL) and water (2 x 50 mL), dried (MgS0 ₄₎ and concentrated. The residue was recrystallized from chloroform/hexane to give 0.26 g (39%) of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine: mp 121-122 °C; NMR (DMSO-d ₆ ) δ 12.71 (s, 1 H) , 8.06 (m, 1 H), 7.30 (m, S H) , 5.01 (s, 2 H), 4.43 ( , 1 H) , 4.06 <m, 1 H), 2.99 (m, 2 H) , 11.19 (d, 2 H, J = 7 Hz).

B. A mixture of N-benzyloxycarbonyl-L-alanyl-L- phenylalanine (0.208 g, 0.562 mmol) and 10% Pd on carbon (0.1 g) in 95% ethyl alcohol (50 mL) was shaken on a Parr hydrogenation apparatus for 16 h at 20 °C. The reaction mixture was filtered and the filtrate rinsed with water (lOOmL). The combined solutions were concentrated to give 0.122 g (92%) of L-alanyl-L- phenylalanine as a white solid. HPLC analysis showed >99.5% purity and no evidence of racemization.