The invention relates to urethane-protected amino acid-N-carboxyanhydride and N-thiocarboxyanhydride compounds which are useful in peptide, polypeptide and protein synthesis. Disclosed herein is the preparation and use of these novel compounds.

BACKGROUND OF THE INVENTION 
(I) Field of the Invention 
This invention relates to a novel class of N-protected amino 
acid-N-carboxyanhydrides and thiocarboxyanhydrides, namely N-urethane 
protected amino acid-N-carboxyanhydrides and N-urethane protected 
N-thiocarboxyanhydrides, their preparation and their use in peptide, poly 
peptide and protein synthesis. 
(II) Prior Art 
Classically, polypeptides of a defined sequence have been prepared by 
extremely laborious techniques wherein the intermediates have been 
isolated after the addition of each amino acid moiety. This has 
complicated the synthesis and made the preparation of long chain 
polypeptides and proteins nearly impossible because of low yields, 
racemization, and/or other side reactions. In 1963, Merrifield (J. Am. 
Chem. Soc., 85, 2149) and Letsinger and Kornet (J. Am. Chem. Soc., 85, 
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. 
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 carbodiimide 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-succinimide, etc.) to form an "active ester" which may be 
isolated and purified and then allowed to couple with the free amine 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 carbodiimides 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 racemization, 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)isopropyloxycarbonyl, anhydrous base (i.e., 
9-fluorenylmethyloxycarbonyl), and so forth. 
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: 
##STR1## 
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-hydroxysuccinimide 
(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 dipeptide is 
formed which also contains a free amine. This amine will subsequently 
react with another NCA to form a tripeptide, and so on. This reaction has 
allowed amino acid N-carboxyanhydrides to find extensive use in the 
formation of poly .alpha.-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 .alpha.-Amino Acid N-Carboxyanhydrides. J.A.C.S., 93:11, 1971, pg. 
2746-2774) 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, salt, 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-substituted amino acid N-carboxyanhydrides have been 
reported in the literature such as N-methyl, N-benzyl, N-acetyl, 
N-nitrophenylsulfenyl, N-xanthyl, 4,4'-dimethylbenzhydryl, trityl, and the 
like. Several of these substituted-NCA'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 85, 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 
triethylamine. Subsequently, it has been shown that triethylamine promotes 
the racemization of NPS- NCA's. In addition, oligomerization due to the 
action of triethylamine on the NCA, requires that very stringent reaction 
conditions must be employed (i.e. temperature &lt;0.degree. C. and very slow 
addition of triethylamine to the reaction mixture) during NPS-NCA 
synthesis. Halstrom, et al, (Z. Physiol. Chem. 355, 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. 
Block and Cox ("Peptides, Proc. of the 5th Europ. Symp., Oxford, Sept. 
1962". Pergamon Press 1963, Ed. G. T. Young, pp. 84-87.) proposed the use 
of N-trityl amino acid-N-carboxyanhydrides for use in peptide synthesis, 
although they were only able to prepare the simplest N-tritylamino acid 
NCA's (i.e. glycine and alanine). 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-carboxyanhydride but 
were unsuccessful in their attempts to prepare them and concluded that 
urethane-protected amino acid N-carboxyanhydrides could not be made ! Even 
if all the N-trityl-NCA's could be prepared it is well known that the use 
or trityl protection of amino acids in various condensation methods 
produces low yields because of the considerable steric 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 (Acta Chemica Scandinavica, Ser. B 1986, BYO(6), 462-465 
and U.S. Pat. No. 4,267,344) also recognized the advantages and the 
potential 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 amino 
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 used in peptide synthesis the 9-xanthyl-NCA's have been found to react 
sluggishly requiring as long as 5 hours at 50.degree. C. in solution and 
24 hours at 25.degree. 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. 
Kricheldorf, (Makromol. Chem. Vol. 178, pp 905-939, 1977) has described a 
method for the preparation of methoxycarbonyl glycine NCA and 
ethoxycarbonyl glycine NCA. However, Kricheldorf 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-thiocarboxyanhydrides. 
Yet another object of the invention is to provide a method for the 
synthesis of polypeptides utilizing pure, crystalline urethane-protected 
amino acid-N-carboxyanhydrides, which synthesis offers the following major 
advantages over conventional methods of polypeptide synthesis: 
(1) Pre-activation 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. 
(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, filtrations, 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-protecting groups on the amino function of the growing peptide 
chain which may then be manipulated by conventional techniques. 
SUMMARY OF THE INVENTION 
These objects are obtained by a urethane-protected amino 
acid-N-carboxyanhydride or N-thiocarboxyanhydride having the structure: 
##STR2## 
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-fluorenylmethyl, phenylethyl, and the like. Suitable 
alkaryl groups include tolyl, ethylphenyl, isopropylphenyl and the like. 
The R, R' and R" groups may also be substituted with noninterfering groups 
such as fluoro, methoxy, 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 .alpha.-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 .alpha.-carbon atom of an amino acid such as lysine, leucine, 
arginine, serine, aspartic acid, alanine, asparagine, cysteine, cystine, 
glutamic acid, histidine, glutamine, isoleucine, methionine, norleucine, 
ornithine, phenylalanine, threonine, tryptophan, tyrosine, valine, 
.beta.-alanine, homoserine and the like. Exemplary of such side chains 
are: 
##STR3## 
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 .alpha.-carbon of an amino acid as, for 
example, in the case of isovaline where one of R or R' is --CH.sub.2 
CH.sub.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 case of 
1-amino-1-cyclohexane carboxylic acid. 
The compounds of the invention also include examples such as ortho-amino 
benzoic acid or 1-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: 
##STR4## 
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 component is coupled by condensation 
reaction to an insoluble solid support containing substituent 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-protected amino acid component in at least one of said 
reactions a compound having the structure: 
##STR5## 
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-carboxyanhydride 
having the structure: 
##STR6## 
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: 
##STR7## 
wherein X is chlorine, bromine, fluorine, azide, or the like, and R" is 
alkyl, aryl, or aralkyl, in an inert diluent, under anhydrous conditions 
and in the presence of N-methylmorpholine. It has been surprisingly found 
that utilizing an inert diluent, anhydrous conditions and selecting 
N-methylmorpholine as the base in this reaction avoids polymerization of 
N-carboxyanhydrides and otherwise enables the production of the heretofore 
unobtainable urethane-protected NCA's and NTA's of higher amino acids. 
DETAILED DESCRIPTION OF THE INVENTION 
The amino acid N-carboxyanhydrides (NCA's) and N-thiocarboxyanhydrides 
(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 15, No. 9, 1869-1871 (1976); 
Kricheldorf, Chem. Ber. 104, 87-91 (1971); and Halstrom and Kovacs, Acta 
Chemica Scandinavica, B40, 462-465 (1986). 
While urethanes in general may be used as protecting groups for 
nucleophilic 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-thiocarboxyanhydrides substituted with these 
protecting groups are of particular interest. Accordingly, very useful 
molecules for peptide synthesis are the NCA's of L-.alpha.-amino acids 
protected by one of the abovementioned protecting groups, such as: 
##STR8## 
wherein R is the side chain of an .alpha.-amino acid, Z is O 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, Proc. of the 5th 
Europ. Symp., Oxford, Sept. 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 (Makromol. 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 N-methylmorpholine 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 solvent (such as toluene) and cooling the 
resulting solution with stirring. The desired haloformate (e.g. 
benzylchloroformate) is then added all at once. To this mixture is added 
N-methylmorpholine which scavenges the hydrochloric acid formed during 
condensation thus promoting the condensation reaction. Under these 
conditions, 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.degree. 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. 
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 CO.sub.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 polypeptides 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 the term "polypeptides" 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 amino 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 polypeptides. Typical of such polymeric resins are 
crosslinked polystyrene resins, glass beads, clays, celite, crosslinked 
dextran, polyacrylamides, polyamide 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. Pat. No. 
4,192,798, hereby incorporated by reference, but the use of 
superatmospheric pressures is not essential. 
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 
amino 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 can be carried out by 
contacting the protected amino acid residue with an appropriate 
deprotecting 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-fluorenylmethyloxycarbonyl, basic conditions such as piperidine in DMF 
will be the preferred method. The concentrations of the particular 
deprotecting agent in the solvent will vary depending again upon the 
particular protecting agent employed but will ordinarily range from about 
5 to 50% 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 triethylamine in a solvent such as 
dichloromethane. Any excess triethylamine 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 amino acid is a urethane-protected amino acid 
N-carboxyanhydride 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 activated N-protected amino acid component is 
employed in the reaction. 
After the coupling of the second protected amino acid component to the 
first amino acid component, the attached protected dipeptide 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 byproducts (CO.sub.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, aminolysis, 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.