Using internal marker

A method is provided for quantitatively monitoring the deprotection and coupling reactions employed in the solid phase synthesis of peptides. The method entails synthesizing a peptide on a support matrix that has a first marker associated therewith. The amino acids employed in the peptide synthesis procedure have a second marker attached thereto, which can be the blocking group used for the amino acid. After the coupling or deprotection step a portion of the support matrix is processed to release first and second identifers from the first and second markers, respectively. The completeness of the coupling or deprotection step can be determined by comparing the relative amounts of the detected first and second identifiers. Novel compositions of matter are used in or produced during this method, including support matrixes having pyrolyzable markers attached thereto.

BACKGROUND 
The present invention relates to methods for forming a peptide on a support 
matrix. 
The Merrifield method of solid phase peptide synthesis is an extremely 
useful synthetic tool. In the Merrifield method, a peptide attached to a 
support matrix is lengthened by coupling with an amino acid. The 
N-terminus of the amino acid is protected by a blocking group such as a 
BOC group (t-butoxycarbonyl). The lengthened peptide is then decoupled by 
removing the blocking group, and then the coupling reaction is repeated. A 
problem with solid phase peptide synthesis is the determination of when 
the coupling and deprotection are complete. The completeness of these 
reactions is essential to peptide synthesis, since an incomplete coupling 
or deprotection reaction can lead to deletion of an amino acid in the 
desired sequence. These deletions can vary from trace to significant 
amounts. 
Numerous methods for monitoring the completeness of the deprotection and 
coupling reactions have been reported. For example, the Kaiser (ninhydrin) 
test is convenient, rapid (requires about 5 minutes to run) and well 
documented. For these reasons, the Kaiser test is the most widely used of 
the qualitative monitoring methods. In the Kaiser test, a reagent is 
reacted with the unblocked supported peptide to produce a purple product, 
the intensity of the purple color qualitatively indicatng the amount of 
decoupling. 
The Kaiser test has disadvantages. For example, it has been shown to give 
false positive results for complete coupling. In addition, the Kaiser test 
lacks sensitivity with respect to the degree of deprotection of BOC-amino 
acids because the intensity of Ruhemann's purple is noted from about 50 to 
about 100 percent free amino groups. Another drawback to the Kaiser test 
is its lack of good color resolution for the deprotection of secondary 
amino acids. More particularly, the deprotection of proline, 
hydroxyproline, and sarcosine gives a brown color instead of purple. 
Another monitoring method uses chloromil. The chloromil method, like the 
Kaiser method, is inherently inaccurate since it relies on color 
differentiation. The reason for this is that in dilute solutions, faint 
amounts of color are difficult to detect with the naked eye. 
Two common quantitative tests, the picric acid titration and the 
quantitative ninhydrin test, have advantages over the qualitative Kaiser 
and chloromil tests in that they give quantitative information about the 
degree of deprotection or coupling during peptide synthesis. However, both 
of these quantitative methods suffer as synthetic monitoring tools due to 
(1) the length of time (about 2 hours) required to complete the test 
because of the need to dry and accurately weigh the resin peptide samples; 
(2) the need for a highly skilled technician to obtain reproducible 
results; and (3) the lack of sensitivity in determining the completeness 
of the deprotection and coupling reactions. Since peptide chains can have 
30 or more amino acids, a monitoring test that requires in excess of 2 
hours per amino acid added to the support is not commercially feasible on 
a routine basis. 
Accordingly, it would be very desirable to have a quantitative method for 
monitoring the completness of the coupling and deprotection reactions 
employed in solid phase peptide synthesis where the method (1) requires a 
relatively short period of time to perform; (2) does not require the use 
of a highly skilled technician to obtain reproducible results; and (3) is 
sensitive to the completeness of both the deprotection and coupling 
reactions. 
SUMMARY 
The present invention provides a system that satisfies these needs. More 
particularly, a method according to the present invention for monitoring 
the completion of coupling and deprotection reactions employed in solid 
phase peptide synthesis is advantageous in that (1) the length of time 
required for monitoring is short because there is no need to dry or 
accurately weigh samples; (2) the method does not require a highly skilled 
technician in order to obtain reproducible results; and (3) the method is 
sensitive to the degree of completeness of both the deprotection and 
coupling reactions. 
A method embodying features of the present invention comprises the 
following steps: 
A. Selection 
A support matrix is selected, the support matrix having a marker associated 
therewith, and having a group capable of bonding to an amino acid. The 
support matrix can be processed to release a first detectable identifier 
from the marker. The marker is generally attached to the support matrix, 
although it can be attached to a second matrix where a blend of the 
support matrix and the second matrix is used. 
B. Coupling 
A supported blocked amino acid is formed by reacting a plurality of the 
selected support matrix with a blocked amino acid to attach the blocked 
amino acid to the support matrix. The supported amino acid can be 
processed to release a second detectable identifier different from the 
first identifier. The blocked amino acid has an N-terminus blocking group 
attached thereto. 
The supported blocked amino acid can be treated to release the N-terminus 
blocking group and unblock the amino acid thereby forming a supported 
unblocked amino acid for reaction with another blocked amino acid. This 
treatment step can be performed without deleteriously affecting the 
marker, i.e., the marker can still be processed to release the first 
detectable marker. The unblocked amino acid is available to react with 
another N-terminus protected amino acid to form a peptide. 
C. Processing 
At least a portion, and usually only a small portion, of the coupling step 
reaction product is processed to release the first and second identifiers. 
Preferably the marker is chosen so that processing is effected by 
pyrolyzing the coupling step reaction product. Processing is effected 
without deleteriously affecting the peptide forming on the support. 
D. Detection and Comparison 
The first and second identifiers are detected. The respective amounts of 
the detected identifiers are compared to determine whether the coupling 
step is complete, i.e., whether the support matrix has any group which is 
capable of and available to react with an N-terminus blocked amino acid. 
If coupling is not complete, steps B through D are repeated until 
substantially complete coupling is achieved. 
E. Deprotection 
The remainder of the supported blocked amino acid, i.e., the portion not 
processed in step (C), is treated to remove the N-terminus blocking group 
from the amino acid to form a supported unblocked amino acid. 
F. Processing 
At least a portion, and generally only a small portion, of the supported, 
unblocked amino acid is processed to release the first and second 
identifiers. Processing is effected without deleteriously affecting the 
peptide forming on the support. 
G. Detection and Comparison 
The first and second identifiers are detected and a comparison is made of 
the respective amounts of the detected identifiers. The purpose of this 
comparison is to determine whether the deprotection step is complete, 
i.e., whether any support matrix has any N-terminus protected amino acids 
attached thereto. 
Steps E-G can be repeated until deprotection is compete. In general, steps 
B-G are repeated until the desired peptide is formed on the support 
matrix. 
There are several compositions that can be employed in and can be produced 
by this method. A first composition of matter comprises a support matrix 
having a peptide and a marker attached thereto. The peptide has a terminal 
amino acid with an N-terminus blocking group attached thereto. This 
composition of matter can be treated, without deleteriously affecting the 
marker, to release the N-terminus blocking groups and to thereby make the 
terminal amino acid available to react with another N-terminus blocked 
amino acid to lengthen the peptide. In addition, the composition can be 
processed to release a first detectable identifier from the marker and a 
second detectable identifier from the blocking group. The first and second 
identifiers are different. 
The first composition can have the formula: 
##STR1## 
where: R is the support matrix; 
m and n are positive integers; 
X is a side chain comprising the marker; 
L is a linking group; 
Y is an amino acid; 
AA is the terminal amino acid of the peptide; and 
NTBG is the N-terminus blocking group. 
X can be: 
EQU --(CH.sub.2).sub.a --(NH).sub.q --Z 
where 
a is 0 or a positive integer; 
q=0 or 1; and 
Z is the marker. 
Z can be: 
##STR2## 
Preferred markers where the processing is by pyrolysis have the formula 
##STR3## 
where a beta carbon has at least one hydrogen attached thereto. There can 
be more than one beta carbon. The more hydrogen attached to a beta carbon, 
the more readily does the marker release an identifier by pyrolysis. 
Pyrolysis generally breaks the bond between the oxygen and the alpha 
carbon releasing as the identifier a composition including the alpha 
carbon, the beta carbon(s), and substituents attached thereto. 
A second composition of matter comprises the first composition wherein the 
terminal amino acid is devoid of an N-terminus blocking group, i.e. the 
first composition after the blocking group is removed. Accordingly, the 
terminal amino acid of this second composition of matter is available to 
react with another N-terminus blocked amino acid. 
A third composition of matter is the support by itself with the markers 
attached. This composition has the formula: 
##STR4## 
where R m, and X are as defined above and; Q is H or a linking group; X 
can be selected from the group consisting of: 
EQU --(CH.sub.2)Hd a--NH--Z, --(CH.sub.2).sub.a --Z, and 
EQU --(CH.sub.2).sub.a --S--CH.sub.3 ; 
a is 0 or a positive integer; and Z the marker is selected from the group 
consisting of 
##STR5## 
Alternatively, X can be selected from the group consisting of: 
##STR6## 
wherein a is as defined above and Z is selected from the group consisting 
of --O--trityl, --S--trityl, --O--pixyl, --S--pixyl, and homologs and 
analogs thereof. 
A further composition of matter is a marked amino acid having the formula: 
##STR7## 
wherein T is H or an N-terminus blocking group and X is as defined above. 
These and other features, aspects, and advantages of the present invention 
will become better understood with reference to the following description 
and appended claims. 
DESCRIPTION 
In accordance with the present invention, an internal standard or first 
marker is associated with a support matrix. This support matrix is present 
in a reaction medium where a solid phase peptide synthesis is conducted. 
In addition, a second marker is associated with the amino acids employed to 
snythesize the desired peptide. The first and second markers, upon 
processing, release a first and second identifier, respectively. These 
identifiers are different. 
By comparing the relative amounts of the first and second identifiers, a 
determination can be made as to the completion of the coupling and/or 
deprotection steps employed in solid phase peptide synthesis. The ability 
to perform this comparison obviates the need to either dry or weigh the 
resin peptide sample. The result is that the quantitative monitoring 
procedure of the present invention for monitoring the completion of 
coupling and deprotection reactions is expeditious, does not require a 
highly skilled technician, and is sensitive to the completeness of both 
the deprotection and coupling reactions. 
As noted above, a support matrix having a first marker or internal standard 
associated therewith is employed in the present invention. The first 
marker must be capable of being attached to a matrix and must be stable to 
peptide synthesis conditions. This matrix can be either the support matrix 
or a separate matrix used in association with the support matrix. 
The first and second markers must also be capable of being directly or 
indirectly detected. For example, the markers can be groups which, upon 
pyrolysis, produce detectable product or identifiers. Similarly, the 
markers can be groups which, upon treatment, produces identifiers which 
are chromophoric products. As used herein, chromophoric products include 
substances that can be detected in the visible and non-visible ranges. 
Accordingly, as used herein, the term pyrolytic marker indicates a group 
which upon thermal decomposition, of at least a portion thereof, releases 
a composition or identifier capable of being detected such as by gas 
chromotography. 
In addition, as used herein, the term chromographic marker indicates a 
group, wherein at least a portion thereof upon processing (e.g. by 
treatment with a chemical substance) releases a composition or identifier 
capable of being detected by spectroscopy. 
As also indicated in the above definitions the identifier consists of at 
least a portion of the marker. 
Support matrixes having a first marker attached thereto can have the 
formula I 
##STR8## 
wherein R is a support matrix; m is a positive integer; Q is H or a 
linking group; and X is selected from the first group consisting of 
--(CH.sub.2).sub.a --NH--Z, --(CH.sub.2).sub.a --Z, and 
##STR9## 
a is 0 or a positive interger; and Z is selected from the group consisting 
of 
##STR10## 
Exemplary support matrixes include, but are not limited to styrene and 
divinylbenzene copolymers, polyamides, polyacrylate, 
polymethylmethacrylate, polysaccharides, phenoliic resins, silica, porous 
glass, and polyacrylamides. A preferred support matrix is 
copoly(styrene-1%-divinylbenzene) resin. 
Various linking groups, designated as L, can be used in the support matrix 
of formula I. These linking groups include, but are not limited to, 
##STR11## 
wherein T is H or an amino blocking group and b is 0 or 1. Because the 
extent of its coupling can be determined, it is preferred that the linking 
group present in the support matrix of formula I have the formula. 
##STR12## 
In order to react with an N-terminus blocked amino acid, T must be H. 
In the support matrix of formula I, X can also be selected from a second 
group consisting of --(CH.sub.2).sub.a --Z, --(CH.sub.2).sub.a --Z, 
--(CH.sub.2).sub.a --CHZ--(CH.sub.2).sub.a --CH.sub.3, and 
##STR13## 
wherein as is as defined above and Z is selected from the group consisting 
of --O-trityl, --S-trityl, --O-pixyl, --S-pixyl, and homologs and analogs 
thereof. 
Homologs and analogs of trityl include, but are not limited to, 
tris(4-hydroxyphenyl)methyl, tris(4-aminophenyl)methyl and 
dimethoxytrityl. 
Exemplary amino blocking groups include, but are not limited to, (BOC), 
2-(4-biphenylyl)-2-propyloxy carbonyl, alpha-2,4,5,-tetramethylbenzyloxy 
carbonyl, 2-phenyl-2-propyloxycarbonyl, 
2-(3,5-dimethoxyphenyl)-2-propyloxy carbonyl, fluorenyl methyloxy 
carbonyl, 3-nitro-2-pyridine sulfenyl, and homologs and analogs thereof. 
When X is selected from the first group consisting of --(CH.sub.2).sub.a 
--NH--Z, --(CH.sub.2).sub.a --Z, and 
##STR14## 
the side chain can have a pyrolytic group attached thereto. When the side 
chain has a pyrolytic group attached thereto, it is preferred that the 
amino blocking group be capable of yielding a distinguishable detectable 
product upon pyrolysis. Such amino blocking groups include but are not 
limited to t-butyloxycarbonyl, 2-(4-biphenylyl)-2-propyloxycarbonnyl, 
alpha-2,4,5-tetramethylbenzyloxy carbonyl, 2-phenyl-2-propyloxycarbonyl, 
2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl groups. 
When X is selected from the second group consisting of --(CH.sub.2).sub.a 
--NH--Z, --(CH.sub.2).sub.a --Z, --(CH.sub.2)--CHZ--(CH.sub.2).sub.a 
--CH.sub.3, and 
##STR15## 
the side chain can have a chromophoric group attached thereto. When the 
side chain has a chromophoric group attached thereto, it is preferred that 
the amino blocking group be capable of yielding a distinguishable product 
upon treatment. Such amino blocking groups include, but are not limited 
to, flourenyl methoxy carbonyl and 3-nitro-2-pyridine sulfenyl groups. 
When a pyrolytic method embodying features of the present invention is 
used, a preferred marked support matrix of formula I is selected from the 
group consisting of 
##STR16## 
wherein Q, a, and R are as defined above. These marked support matrixes 
are preferred because the --CH--(CH.sub.3).sub.2 group readily yields a 
detectable identifier upon pyrolysis. 
Because of their commercial availability, it is also preferred that a be 1 
to about 6. For the same reason, a is more preferably 1 to about 4. 
The marked support matrix of formula I can be prepared by several 
techniques. In one technique, a blocked, marked amino acid is reacted with 
a support matrix. The blocked marked amino acid can have the formula II 
##STR17## 
wherein X is as defined above and T, which in general can be H or an 
N-terminus blocking group, is an N-terminus blocking group. 
N-terminus blocking groups include, but are not limited to, the same groups 
set forth above with respect to amino blocking groups. 
Preferred blocked, marked amino acids having a pyrolytic group attached 
thereto have the formula 
##STR18## 
These blocked marked amino acids are preferred because of the relative 
ease with which the N-terminus blocking group and the marker pyrolyze to 
yield distinguishable identifiers. 
The blocked, marked amino acid can be prepared by various methods. For 
example, a blocked amino acid having a pyrolytic marker attached thereto 
can be obtained by suitable treatment of a N- and C-terminus protected 
amino acid having a carboxyl group in its side chain. Typical amino acids 
of this type are aspartic acid and glutamic acid. The following 
preparation of BOC--beta--isopropyl asparate exemplifies this procedure: 
##STR19## 
In the above procedure, BOC--alpha--benzyl asparate (BOC is 
alpha-t-butoxycarbonyl) is esterified with isopropyl alcohol (IPA) using 
dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The 
resulting diester II is subjected to hydrogensis to produce the desired 
betaisopropyl ester III. 
Another procedure for preparing BOC-beta-isopropyl asparate can be 
schematically depicted as follows: 
##STR20## 
A blocked amino acid having a pyrolytic marker attached thereto can also be 
obtained by suitable treatment of an amino acid having an amino group in 
its side chain. Typical amino acids of this type are lysine and ornithine. 
The following schematic preparation of 
##STR21## 
exemplifies this procedure: 
##STR22## 
A blocked amino acid having a pyrolytic marker attached thereto can also be 
obtained by suitable treatment of an amino acid having a hydroxyl group in 
its side chain. Typical amino acids of this type are serine, threonine and 
tyrosine. The following schematic preparation of 
##STR23## 
exemplifies this procedure: 
##STR24## 
A blocked amino acid having a chromophoric marker attached thereto can be 
obtained by suitable treatment of an amino acid having a (a) hydroxyl 
group, (b) amino group, or (c) thiol group in its side chain. Typical 
amino acids of this type are (a) serine, threonine, tyrosine, (b) 
ornithine, lysine, and (c) cysteine, respectively. The following 
discription exemplifies this procedure. 
Trityl chloride can be treated in a polar, aprotic solvent (e.g., dioxane, 
tetrahydrofuran) with an N-terminus and C-terminus protected amino acid. 
The N-terminus protecting group can be, for example, fluorenyl methoxy 
carbonyl (FMOC) and 3-nitro-2-pyridine sulfenyl (NPYS ). The C-terminus 
protecting group can be, for example, benzyl ester, methyl ester, ethyl 
ester, and phenacyl ester. Phenacyl ester is preferred because it can be 
relatively easily removed from the amino acid. This treatment procedure is 
conducted in a mild base (e.g., pyridine, leutidine) at a temperature of 
about 50.degree. to about 60.degree. C. for a minimum of four hours. Thin 
layer chromotograply (TLC) can be used to monitor the reaction. 
The above reaction medium is evaporated to dryness and the resulting 
residue is extracted with ethyl acetate (EtOAc) or methylene chloride 
(CH.sub.2 Cl.sub.2). The extract is washed with a dilute acid, (e.g., 
about 0.1 to about 1N HCl). This is followed by a wash with water. The 
organic layer is first dried with anhydrous sodium sulfate and then 
evaporated to dryness. The dried product has the trityl group attached to 
the side chain of the N- and C-terminus blocked amino acid. The free acid 
can be obtained by saponifying the dried product with a mild base (e.g., 
about 1N NaOH). However, when phenacyl ester is used, the free acid is 
obtained by treating the dried product with thiophenol. 
Procedures analogous to those set forth above for producing blocked, marked 
amino acids can be employed to produce other amino acids having different 
markers and/or diferent side chain groups and/or different N-terminus 
blocking groups attached thereto. 
The support matrix, with which the blocked amino acid of formula II is 
reacted, has a functional group attached thereto which is capable of 
reacting with the C-terminus of the blocked amino acid. For example, in 
the case of support matrixes having a hydroxyl functionality (e.g., 
phenolic resins, polysaccharides, hydoxymethyl polystyrene), the blocked 
amino acid is activated by DCC in the presence of an activator (e.g., 
DMAP) and the support matrix. 
This yields a blocked marked amino acid attached to the support matrix. The 
number of such attachments will depend, in part, upon the number of 
available hydroxyl groups. 
The reagents and by-products are then drained from the support matrix. The 
support is washed with a solution (e.g., CH.sub.2 Cl.sub.2, dimethyl 
formamide (DMF), dioxane). 
Unreacted hydroxyl groups on the support matrix are now blocked with a 5% 
solution of phenylisocyanate in CH.sub.2 Cl.sub.2. This blocking step is 
conducted for about 30 minutes at about room temperature. The reacting 
mixture is then drained and the support matrix is washed 3 times with 
CH.sub.2 Cl.sub.2. The N-terminus protecting group is removed from the 
blocked, marked amino acid that is attached to the support matrix. 
In the case of support matrices having an amino functionality (e.g., amino 
functionalized polystyrene divinlybenzene copolymer, and amino 
functionalized polyamide), the attachment procedure is the same as that 
set forth above with the exception that the blocked, marked amino acid 
does not require an activator to be present in order to react with amio 
functionalized support matrixes. 
The linking group can be attached to N-terminus of the unblocked, marked 
amino acid, via convential procedures. 
When Q of the support matrix of formula I is H, the initial amino acid of 
the desired peptide (i.e., the C-terminus amino acid of the peptide to be 
synthesized) is first attached to the linking group. In such instance, the 
resulting compound has the formula III 
##STR25## 
wherein L is a linking group, X.sub.sc is an amino acid side chain, and 
NTBG is the N-terminus blocking group. 
In the above formula III, L can be, for example, 
##STR26## 
wherein b is 0 to 1. For the reasons set forth above, the first of the 
above linkers is preferably attached to the support matrix without the 
initial amino acid being attached thereto. Accordingly, in formula III, L 
is preferably selected from the group consisting of 
##STR27## 
wherein b is as defined above. 
NTBG can be any N-terminus protecting group. Exemplary N-terminus 
protecting groups include, but are not limited to, t-butyloxycarbonyl, 
2-(4-biphenylyl)-2-propyloxy carbonyl alpha-2,4,5,-tetramethylbenzyloxy 
carbonyl, 2-phenyl-2-propyloxycarbonyl, 
2-(3,5-diemthoxyphenyl)-2-propyloxy carbonyl, fluorenyl methyloxy 
carbonyl, 3-nitro-2-pyridine sulfenyl, and homologs and analogs thereof. 
Because ether linking groups, such as: 
##STR28## 
are acid labile, these linkers can only be employed in solid phase peptide 
synthesis emloying neutral or basic conditions. Since some N-terminus 
blocking groups are removed from N-terminus blocked amino acids under 
acidic conditions, the N-terminus blocking group employed when an ether 
linkage is used preferably is capable of being removed under basic or 
neutral conditions. Exemplary linkers that can be removed under basic or 
neutral conditions include, but are not limited to, fluorenyl methoxy 
carbonyl, 3-nitro-2-pyridine sulfenyl, and homologs and analogs thereof. 
X.sub.sc can be any natural or unnatural amino acid side chain. In 
addition, X.sub.sc can have the second marker attached thereto. Amino 
acids having the second marker attached to X.sub.sc can be prepared by 
methods analogous to those employed to prepare the amino acids of formula 
II. In general, the first and second markers are preferably chosen from 
the same class of markers (i.e. pyrolytic, chromophoric, etc.). The reason 
for this is that the same type of detection procedure can be employed to 
determine the first or second markers, respectively. However, the markers 
are chosen so that the first and second identifiers are different. 
Pyrolytic and chromophoric markers attached to X.sub.sc can be removed from 
the synthesized peptide with the same procedure employed to cleave the 
peptide from the solid support. 
Compounds of formula III can be prepared by reacting an N-terminus blocked 
amino acid with a linking group via conventional procedures. Exemplary 
linking groups have the formulas 
##STR29## 
Similarily, when the linking group is attached to the support matrix, as 
shown in formula I wherein Q is the linking group, an N-terminus blocked 
amino acid can be reacted with the linking group via conventional 
procedures employed to couple an amino acid to a solid support. 
The thus formed support matrix having the initial amino acid attached 
thereto has the formula IV 
##STR30## 
wherein NTBG, X.sub.sc, L, X, R and m are defined above. In the case of 
the linking group, L can be more precisely represented by the modified 
formulas 
##STR31## 
wherein b is as defined previously. 
A portion or sample of the support matrixes present after the coupling 
reaction is then processed to release the first and second identifiers 
from the first and second markers, respectively. The particular procedure 
employed depends on the type of first and second markers present. 
When both markers are pyrolytic markers, the process employed entails 
heating the sample to a temperature sufficient to release the first and 
second identifiers without deleteriously affecting the remainder of the 
composition. In general any temperature can be employed to release these 
identifiers provided that the sample does not decompose to release any 
substance which can interfere with the detection of the first and second 
identifiers. To prevent such decomposition, the temperature employed is 
preferably below about 750.degree. C. 
Generally, a temperature of about 450.degree. to about 600.degree. C. is 
satisfactory. However, the optimal temperature employed depends upon the 
particular first and second pyrolytic markers used. 
When both markers are chromophoric markers, the process employed entails 
chemically treating the sample to release the first and second 
identifiers. The specific chemical treatment employed depends upon the 
particular first and second chromophoric markers used. For example, type 
group, the sample is treated with an appropriate acid (e.g., 
dichloroacetic acid) to remove the trityl or pixyl type group. The 
effluent of this acid treatment step is collected. 
When the second marker is FMOC, the sample can be treated under basic 
conditions (e.g., with about 20% piperidine in DMF) to remove the FMOC 
group. 
When the second marker is NPYS, the sample can be treated under neutral 
conditions (e.g., with a triphenyl phosphine reagent). 
In both of the latter two examples, the effluent is also collected. 
The first and second identifiers are then detected. The particular 
detection technique employed depends upon the type of first and second 
identifiers present. 
When the identifiers are obtained by thermal decomposition, the identifiers 
can be detected by gas chromatography. Exemplary of the identifiers 
obtained by thermal decomposition include, but are not limited to, the 
following: 
##STR32## 
When the identifiers are chromophoric substances, the identifiers can be 
detected by determining the absorbance or transmittance or fluoresence of 
each collected effluent. (For example, FMOC can be detected at 310 nm and 
dimethoxytrityl can be detected at 490 nm). The effluents can be either 
checked separately or can first be mixed together before being evaluated. 
Because a combined effluent normalizes the results, it is preferred to 
first combine the effluents, if collected separately, prior to determining 
the absorbance or transmittance of the identifiers. 
Once a determination of the relative amounts of first and second 
identifiers has been made, a comparison or ratio of these results 
indicates the completeness of the coupling reaction. The coupling of the 
initial amino acid can be assumed to be 100% complete and the ratio of 
second identifier to first identifier can be taken as the standard by 
which to determine whether the subsequent coupling steps are complete. If 
the ratio after coupling a subsequent amino acid is less than that of the 
initial ratio, then the subsequent coupling step is not complete. The 
subsequent coupling step can be repeated until the ratio obtained is the 
same as or as close to the initial ratio as desired. 
After this comparison procedure, the composition of formula IV can be 
deprotected via conventional solid phase peptide systhesis procedures to 
yield a composition having the formula V 
##STR33## 
wherein X.sub.sc, L, X, R, and m are as defined above. 
To employ a method embodying features of the present invention to evaluate 
the completeness of the deprotection step, the second marker must be 
either the N-terminus blocking group of the initial amino acid or of a 
subsequent amino acid or a group is also removed from the amino acid 
during the deprotection step. 
When the second marker is the N-terminus protecting group, a direct 
determination is made of its presence or absence. In addition, when the 
second marker is the N-terminus protecting group, there is no need to 
further modify the amino acid prior to its coupling to the support matrix 
because the N-terminus protecting group is present on the amino acid 
during the coupling step. For these reasons, it is preferred that the 
second marker be the N-terminus protecting group. 
After the deprotection step, a portion or sample of the support matrices is 
again taken. This sample can be processed in the same manner described 
with respect to the sample taken after the coupling step. 
If the resulting ratio is zero, then the deprotection step is complete. 
However, if the resulting ratio is greater than zero, the deprotection 
step is incomplete. In this latter case, the deprotection step can be 
repeated until the ratio is zero or as close to zero as desired. 
After this latter comparison procedure, subsequent N-terminus protected 
marked amino acids can be added following coupling/deprotection protocols 
employed in the solid phase synthesis of peptides. After each subsequent 
coupling and deprotection step, the above processing, detection, and 
comparison procedures can be employed to monitor the completion of each 
subsequent coupling and deprotection step. 
During the course of the solid phase peptide synthesis, compositions having 
the formula VI 
##STR34## 
are synthesized, wherein NTBG, L, X, R, and m are as defined above and AA 
is the terminal amino acid of the peptide, Y is an amino acid, and n is a 
positive integer. When n is greater than 1, each Y can be the same or a 
different amino acid. Exemplary compositions of formula VI having 
pyrolytic first and second markers attached thereto are as follows 
##STR35## 
wherein AA, Y, L, R, m, and n are as defined above. 
Deprotection of the composition of formula VI yields a composition having 
the formula VII 
##STR36## 
wherein AA, Y, L, X, R, m, and n are as previously defined. Exemplary 
compositions of formula VII having pyrolytic markers attached thereto are 
as follows 
##STR37## 
wherein AA, Y, L, R, m, and n are as defined above. 
In another method embodying features of the present invention, the first 
marker is attached to a separate matrix which is used in association with 
a support matrix. More particularly, these marked, separate matrixes are 
present during the coupling and deprotection procedures employed in solid 
phase peptide synthesis. The samples taken after each coupling and 
deprotection step contain a mixture of the support matrix and the marked 
matrix. These samples are processed to release the first and second 
identifiers via the same techniques as set forth above. The detection and 
comparison steps in this method can be also the same as those employed 
above. 
Exemplary marked matrixes which can be used in this latter described method 
include but are not limited to, those having the formula 
##STR38## 
wherein R is a matrix; Z is a pyrolytic substituent; Y is selected from 
the group consisting of 
##STR39## 
acetyl; and m is at least 1. 
Exemplary pyrolytic substituents include but are not limited to, those 
having the following formulas 
##STR40## 
Marked matrixes having the formulas 
##STR41## 
can be prepared by reacting a composition having the formulas 
##STR42## 
respectively, with an alcohol in the presence of DCC and DMAP. An 
emexplary alcohol is isopropanol.

EXAMPLES--INTRODUCTION 
In the examples, melting points were taken on a Buchi melting point 
apparatus with a Cole-Parmer empeller and are uncorrected. Nuclear 
magnetic resonance (NMR) spectra were taken on a Varian EM360A brand 
spectrophotometer with tetramethylsilane as the internal standard. 
Pyrolysis of the resin peptide samples was carried out in a Chemical Data 
Systems Pyroprobe 150 brand thermal processing system. Gas chromatography 
(GC) of the pyrolyzed samples was carried out on a Beckman GC-45 brand 
flame ionization gas chromatograph fitted with a Supelco 2 m.times.1/8" 
Carbopack C (0.19 percent picric acid) brand column. Pyrograms were 
recorded and integrated on a Hewlett-Packard 3390A brand integrator. 
Peptide synthesis was performed on a Beckman Instrument model 990B peptide 
synthesizer. Amino acid analysis was performed on a Beckman Instrument 
model 121B amino acid analyzer. Hydrogenations were carried out on a Parr 
low-pressure shaker type reaction apparatus. Analytical thin layer 
chromatography (TLC) was performed with E. Merck precoated F-254 brand 
silica gel 60 (0.25 mm.times.5 cm.times.20 cm) (TLC) plates and was 
visualized under ultra violet (UV) light at 25 nm. Boc-amino acids unless 
otherwise specified were puchased from the Protein Research Foundation 
(Japan). Benzhydrylamine resin (BHA resin) hydrochloride (0.69 meq/gm) was 
obtained from Beckman Instruments. All solvents and bulk chemicals were 
reagent grade and were not further purified except for 
dimethylaminopyridine (DMAP), which was purchased from Aldrich and was 
recrystallized from ethyl acetate (EtOAc) prior to use. 
EXAMPLE 1 
Preparation of BOC-(Beta-isopropyl) Aspartic Acid-alpha-benzyl ester (II) 
BOC-aspartic acid-alpha-benzyl ester (100 g, 0.31 mol) was dissolved in 120 
ml of methylene chloride (CH.sub.2 Cl.sub.2) and to this solution was 
added a solution of 57.8 g (0.28 mol) of dicyclohexylcarbodiimide (DCC) in 
280 ml of CH.sub.2 Cl.sub.2 over a period of 10 min followed by the 
addition of 34.2 g (0.28 mol) of DMAP in 50 ml of CH.sub.2 Cl.sub.2. Then 
to the stirred reaction mixture 19.0 g (0.31 mol) of isopropyl alcohol 
(IPA) were added, the stirring taking place for 48 hours. 
The reaction mixture was filtered to remove precipitated dicyclohexylurea 
(DCU), and the resulting cake was washed with CH.sub.2 Cl.sub.2. The 
combined filtrates were evaporated to a residual oil. The residue was 
dissolved in 400 ml of diethyl ether (Et.sub.2 O) and the solution washed 
in a separatory funnel with the following sequence of washes at 100 ml per 
wash: 2 times with aqueous 10% sodium carbonate (Na.sub.2 CO.sub.3); 2 
times with H.sub.2 O; 2 times with aqueous 0.5M hydrochloric acid (HCl); 3 
times with H.sub.2 O; and 2 times with saturated aqueous sodium chloride 
(NaCl). The organic layer was dried over anhydrous magnesium sulfate 
(MgSO.sub.4). The drying agent was removed by filtration and the filtrate 
evaporated. The weight of the resulting product was 79 g which represents 
a 70% yield. TLC (95/5/3; (CH.sub.2 Cl.sub.2 /MeOH/HOAc)) gave a major 
spot at R.sub.f 0.85. NMR deuterochloroform (CDCl.sub.3): gamma 7.34 (s, 
5) 5.52 (m, 1), 5.16 (s, 2), 5.05 (m, 1), 4.8 (m, 1), 2.88 (m, 2), 1.4 (s, 
9), 1.15 (d, 6). 
EXAMPLE 2 
Preparation of BOC-(Beta-isopropyl)-aspartic acid (III) 
II (79 g, 0.22 mol) was dissolved in 260 ml of methanol (CH.sub.3 OH) and 
the solution was placed in a Parr reactor vessel. The vessel was flushed 
with nitrogen while adding 29.75 g of 10% palladium catalyst (Pd-C) 
premoistened with HOAc). The vessel was placed in the Parr instrument and 
flushed with nitrogen (3 times at 15 psi) and with hydrogen (3 times at 60 
psi) and agitated with hydrogen at 60 psi for 24 hours. The vessel was 
flushed with nitrogen and the reaction mixture filtered through Celite 
brand cellulose filtration aid. The filtrate was evaporated and the 
residue dissolved in 200 ml of ethyl acetate (EtOAc). This solution was 
extracted with two 200 ml portions of aqueous 10% Na.sub.2 CO.sub.3. The 
aqueous layers were combined and acidified with aqueous 1M HCl to pH 2. 
The solution was then extracted with three 300 ml portions of (EtOAc). The 
combined organic layers were washed with 100 ml of H.sub.2 O, and dried 
over anhydrous MgSO.sub.4. The drying agent was removed by filtration and 
the filtrate was evaporated. The residue was dissolved in Et.sub.2 O and 
hexane was slowly added to the solution until it became turbid. The 
product precipitated upon standing (16 hours). Filtration yielded a white 
solid. TLC (CH.sub.2 Cl.sub.2 /MeOH/HOAc; 95/5/3) showed a single spot 
with R.sub.f 0.05. NMR (CDCl.sub.3): gamma 2.9 (m, 3), 1.49 (s, 9), 1.25 
(d, 6). Elemental analysis: Actually Found (Theoretically present): C, 
52.40 (52.36); H, 7.87 (7.63); N, 5.06 (5.09). 
EXAMPLE 3 
Preparation of BOC-(Beta-isopropyl)aspartyl-benzyhydrylamine (BHA resin 
(IV) 
BHA resin hydrochloride (26.93 g, 18.58 mmol) was placed in the reaction 
vessel of a Beckman 990B brand peptide synthesizer and subjected to the 
following standard protocol to obtain IV. 
It was stirred two times for five minutes each with 250 ml of CH.sub.2 
Cl.sub.2, two times for five minutes each with 10 percent triethylamine 
(TEA) in CH.sub.2 Cl.sub.2, and four times for two minutes each with 250 
ml of CH.sub.2 Cl.sub.2. III (7.7 g, 27.9 mmol) in 200 ml of CH.sub.2 
Cl.sub.2 was mixed with the resin and then hydroxybenztriazole (HOBT) (2.5 
g, 18.6 mmol) was added. DCC (27.9 mmol) was added as a 0.5 molar solution 
in CH.sub.2 Cl.sub.2 The mixture was stirred for six hours. The reaction 
mixture was filtered and the coupling reaction was repeated for 12 hours 
using the same amount of reagents. The filtered resin was then stirred two 
times, two minutes each, with 250 ml of 33% EtOH in CH.sub.2 Cl.sub.2, two 
times, for two minutes each, with 250 ml N-methylpyrrolidone (NMP), three 
times, for two minutes each, with 250 ml of CH.sub.2 Cl.sub.2 and finally 
two times for two minutes each, with 250 ml EtOH. The Kaiser ninhydrin 
test was negative. The resin was dried in a vacuum over pentoxide P.sub.2 
O.sub.5, wt. 31.08 g. GC pyrolysis data (area percent) isobutene 67.97; 
propene 32.03. 
EXAMPLE 4 
Preparation of-Alpha-BOC-valyl-4-(oxymethyl) Phenylacetic Acid (VII) 
VII was prepared by a procedure previously described by Tam et al., 
Synthesis, 12: 955-957 (1979). A white solid was obtained, melting point 
(m.p.) 75.degree.-78.degree. C. (literature 74.degree.-78.degree. C.) The 
NMR spectrum (CDCl.sub.3): gamma 7.3 (s, 4), 5.1 (s, 2), 4.2 (m, 1), 3.6 
(s, 2), 2.05 (m, 1), 1.4 (s, 9), 0.9 (d, 6). 
EXAMPLE 5 
Preparation of N-[BOC-valyl-4-(oxymethyl)phenylacetyl]-(beta 
isopropyl)aspartyl (N-BOC-val-PAMPRO) BHA Resin (VI) 
IV (3.0 g, 1.2 mmol) was placed in the automated synthesizer and the BOC 
group removed according to the following protocol. 
The resin (filtering after each treatment) was stirred for three minutes 
with 60 ml of a 50% trifluoroacetic acid (TFA) CH.sub.2 Cl.sub.2 solution. 
The 50% TFA/CH.sub.2 Cl.sub.2 treatment was repeated for 30 minutes. The 
resin was then stirred four times with 60 ml of CH.sub.2 Cl.sub.2 for two 
minutes each wash, neutralized by stirring two times, 10 minutes each 
time, with 60 ml of a 10% TEA solution in CH.sub.2 Cl.sub.2, and stirred 
five times, for two minutes each, with 60 ml of CH.sub.2 Cl.sub.2. 
The deprotected resin (V) obtained by the above procedure was mixed with a 
solution of VII (1.1 g, 3.0 mmol) in 50 ml of CH.sub.2 Cl.sub.2 and 3.0 
mmol of DCC (6 ml of a 0.5M solution in CH.sub.2 Cl.sub.2) and the 
reaction mixture was stirred for six hours. After filtering the reaction 
mixture the coupling reaction was repeated for 12 hours using the same 
proportion of reactants. After filtration the resin was washed two times 
with 60 ml of 33% ethanol (EtOH) in CH.sub.2 Cl.sub.2, two times with 60 
ml of NMP, four times with 60 ml of CH.sub.2 Cl.sub.2, and two times with 
60 ml of EtOH. The resin was dried in a vacuum over P.sub.2 O.sub.5. The 
Kaiser ninhydrin test was negative. GC pyrolysis data (area percent): 
isobutene 58.41; propene 41.59. These results are set forth in Table II. 
EXAMPLE 6 
Preparation of 
N-[BOC-Leu-Ala-Gly-Val-4-(oxymethyl)phenylacetyl]-(Beta-isopropyl)aspartyl 
Compound VI (2.0 g, 0.6 mmol) was deprotected with 50% TFA in CH.sub.2 
Cl.sub.2, neutralized with 10% TEA in CH.sub.2 Cl.sub.2 as described in 
the deprotection of IV. The deprotected resin was reacted with BOC-glycine 
according to the following protocol. 
A solution of BOC-glycine (0.32 g, 1.8 mmol) in 15 ml of CH.sub.2 Cl.sub.2 
was stirred for two minutes with the resin in an automated peptide 
synthesizer. To this mixture were added 3.6 ml of a 0.5M solution of DCC 
in CH.sub.2 Cl.sub.2 (1.8 mmol of DCC). The reaction mixture was stirred 
for six hours and the reaction vessel drained. The resin was subjected to 
the following treatment: Stir two times, for two minutes each, with 20 ml 
of a 33% solution of EtOH in CH.sub.2 Cl.sub.2, stir 2 times, for two 
minutes each, with 20 ml of NMP, and stir 4 times, for two minutes each, 
with 20 ml of CH.sub.2 Cl.sub.2. The Kaiser test was negative. The resin 
was deprotected according to the procedure set forth in Example 5. 
BOC-alanine and BOC-leucine were added following the 
deprotection/coupling/deprotection/coupling protocols described in this 
example. Following each deprotection and coupling reaction, 50 to 100 mg 
of resin peptide were removed and examined by the Kaiser ninhydrin test, 
by amino acid analysis (AAA), and by a pyrolytic methol embodying features 
of the present invention. These results are set forth in Tables I and II. 
EXAMPLE 7 
Application of Method-to a Resin Peptide Synthesis 
Monitoring of each reaction step by a method embodying features of the 
present invention was carried out in the following manner. An aliquot 
(about 50 mg) of each washed and drained resin peptide produced in 
Examples 5 and 6 was withdrawn with a 2 mm bore flexible capillary tube, 
placed in a 10 ml test tube and evaporated under vacuum for 5 minutes. 
Several milligrams of dry resin peptide beads were withdrawn with a 1.5 
mm.times.90 mm closed end glass capillary tube, the beads centered at 
about 0.5-1.0 cm from the closed end of the tube, the capilllary cleanly 
broken at 1-2 cm from the closed end, then placed within a spiral platinum 
wire coil at the end of a Chemical Data Systems Pyroprobe 150 brand 
thermal processing system. The sample was first pyrolyzed at 100.degree. 
C. for 40 seconds (two 20 second pulses ) to vaporize trapped methylene 
chloride. Next, the sample was pyrolyzed for 80 seconds by heating the 
wire cell with four 20 second pulses of electrical current at a high end 
temperature setting of 500.degree. C. The resultant gases were injected 
directly from the heating chamber onto a 2 m.times.1/8" carbopack (0.19 
percent picric acid) GC column. The column temperature was set at 
70.degree..+-.5.degree. C. and the nitrogen carrier gas flow rate set at 
40+3 ml/min. Chromatograms (pyrograms) were recorded and peak areas 
directly integrated on a Hewlett-Packard 3390A brand integrator. As a 
second monitoring method the Kaiser ninhydrin test was performed at each 
stage of the synthesis. In addition, amino acid analysis was performed on 
the completed resin peptide VIII (Table I). 
The results for the GC assay and ninhydrin tests are summarized in Table 
II. These results are consistent with the chemical steps of BOC group 
removal by TFA and BOC amino acid coupling that were performed during the 
synthesis of resin peptide VIII. 
TABLE I 
______________________________________ 
Amino Acid Analysis Data 
of BOC--Leu-- Ala--Gly-- Val--PASPRO-- BHA Resin 
Amino Acid Molar Ratio 
Theory 
______________________________________ 
Leu 1.04 1 
Ala 1.01 1 
Gly 1.03 1 
Val 1.00 1 
______________________________________ 
Hydrolysis with propionic acid: 6N HCl (1:1) for 4 hours. 
TABLE II 
__________________________________________________________________________ 
Pyrolysis Results of BOC--Ala--Gly-- Val-- PAMPRO--BHA 
Resin 
Completion 
Isobut. 
Propene Percent 
Depro- 
Peptide 
(area) 
(Area) 
Ratio 
Coupling 
tection 
Kaiser Test 
Resin X Y X/Y Reaction 
Reaction 
(Color) 
__________________________________________________________________________ 
BocVal 58.41 
41.59 
1.4 100.0 Yellow (-) 
H--Val 00.00 
100.00 
0 100.00 
Purple (+) 
BocGly-- 
57.95 
42.05 
1.38 98.51 Yellow (-) 
Val 
H--GlyVal 
00.00 
100.00 
0 100.00 
Purple (+) 
BocAla-- 
57.31 
42.69 
1.34 97.1 Yellow (-) 
GlyVal 
H--AlaGly-- 
00.00 
100.00 
0 100.00 
Purple (+) 
Val 
BocLeu-- 
57.11 
42.89 
1.33 99.25 Yellow (-) 
AlaGlyVal 
__________________________________________________________________________ 
Although the present invention has been described in considerable detail 
with reference to certain versions thereof, other versions are possible. 
For example, first markers can be attached to both the support matrix and 
to a separate matrix that is used in association with the support matrix. 
In addition, the method of the present invention can also be used as a 
qualitative monitoring tool. Therefore, the spirit and scope of the 
appended claims should not be limited to the description of the preferred 
versions contained herein.