WO1996016077A2

WO1996016077A2 - Reagent for quantifying free amine groups

Info

Publication number: WO1996016077A2
Application number: PCT/US1995/015691
Authority: WO
Inventors: Siegfried H. Reich; Shao Song Chu
Original assignee: Agouron Pharmaceuticals, Inc.
Priority date: 1994-11-16
Filing date: 1995-11-16
Publication date: 1996-05-30
Also published as: AU4414396A; WO1996016077A3

Abstract

This invention relates to chemical compounds of formula (I), inwhich the variables are as defined in the claims, useful for quantifying free amines or amino groups, methods of preparing such compounds, and the use of such compounds.

Description

REAGENT FOR QUANTIFYING FREE AMINE GROUPS

Background of the Invention

This invention relates to chemical compounds useful for quantifying free amines or amino groups, methods of preparing such compounds, and the use of such compounds.

During a chemical synthesis, one often desires knowledge of whether a particular reaction step produces the desired coupled reaction product. For example, during solid-phase peptide synthesis, it is useful to know whether a particular amino acid completely reacts with the amino terminal end of the growing peptide prior to proceeding with subsequent amino acid coupling steps.

The solid-phase reaction scheme permits one to determine whether each peptide coupling step is complete by monitoring the presence of free amine. During peptide synthesis, an amino acid with an amino protecting group is linked to a solid support. The protecting group is then removed, creating a free amine. Adding the next amino acid (with an amino protecting group) along with a coupling reagent should result in a peptide bond between the added amino acid and the free amine of the growing peptide.

Accordingly, no free amine should be present if the amide bond forming reaction is complete, since the newly added amino acid should contain an amino protecting group, rather than free amine, and since the newly coupled amino acid replaces the free amine at the amino terminal end of the growing peptide. With incomplete peptide coupling, however, the residual free amine at the amino terminal end of the growing peptide should be present,

Of course, during solid phase synthesis one often synthesizes numerous peptides rather than a single peptide. Accordingly, for each peptide coupling step, one adds numerous identical amino acids to numerous growing peptides in one batch. The quantity of free amines thus indicates the degree of peptide coupling for the entire batch. Several methods for quantifying free amines during peptide coupling have been attempted. Kaiser et al. reported using a ninhydrin color test to quantify free amines in connection with solid phase peptide synthesis. Anal Biochem. 34. 595 (1970) . Picric acid was used by Gisin to monitor peptide coupling reactions by measuring free amines on a solid support. Anal . Chim. Acta J58., 248 (1972). Hancock and Battersby reported using 2,4,6-trinitrobenzene-sulfonic acid that reacts with the amino resin to form an orange-red trinitrophenylated derivative. Anal. Biochem. , 1_1 , 260 (1976) .

Sarin et al. altered the Kaiser test, reporting a quantitative ninhydrin procedure for measuring the progress of the coupling reaction. Anal Biochem. , 117, 147 (1981) . The Reddy and Voelker protocol involves tritylation of the uncoupled amino groups with an active tritylating agent 4,4'- dimethoxytrityl chloride in the presence of tetra-n- butylammonium perchlorate, followed by detritylation by a mild acid. Reddy and Voelker stated that the cleaved trityl cation produced a measurable chromophore that is proportional to the extent of coupling. Int . J. Peptide Protein Res . , 31, 345 (1988) .

Young et al. reported using a counterion distribution system that monitors the total population of amino groups on the solid support using the distribution of a minute quantity of an anionic reporter dye between protonated amino groups and cations in solution. The inert anionic dye apportions itself between all available cations and, as the acylation reaction proceeds, will be progressively discharged from the support matrix into solution. One then measures spectrometrically the absorbance of the solution. Biochem . Soc . Trans . , !8_, 1311 (1990).

The monitoring methods above, however, include certain disadvantages. For example, using the picric acid method, one will measure not only primary and secondary free amines, but also tertiary free amines, since the picric acid method is not specific for primary and secondary free amines. The presence of tertiary free amines, however, does not indicate whether or not coupling has occurred, so the picric acid method can provide inaccurate measurements of the degree of coupling.

Also, certain methods above are incapable of determining the presence of free amines of certain compounds or amino acids.

A desirable method for quantifying free amines would be very sensitive, general, and easy to carry out. One also desires a method that specifically quantifies only primary and secondary free amines without quantifying other groups such as tertiary amines or alcohols.

Such a method also should not be limited to quantifying free amines during solid phase peptide synthesis. A chemist reacting a wide variety of compounds should also benefit from a method of determining the extent of chemical coupling where the presence of free amines indicates incomplete coupling. Accordingly, the method should quantify a wide variety of primary and secondary free amines, including those that are poorly reactive, such as sterically hindered primary or secondary free amines.

It is also desirable that the quantifying method use a minimal sample size and proceed in a reasonable amount of time with minimal complications, so the method does not unduly slow down the entire chemical synthesis process. For example, when performing several sequential coupling reactions, a chemist desires to move on to the next coupling reaction rather than spending time awaiting results from the quantifying method.

Summary of the Invention

The present invention relates to compounds of formula I below that are useful for quantifying free amines. These compounds may be used not only to monitor peptide coupling during peptide synthesis, but also to quantify free or unreacted amine to measure the extent of reaction or coupling in nonpeptide reactions. The novel class of compounds according to this invention is represented by the formula I

wherein:

A is substituted or unsubstituted aryl or partially unsaturated cycloalkyl or heterocycle or is nothing;

X, X. and X_ are independently selected from substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, acyl, sulfonyl, sulfinyl, or NZ, wherein Z is nothing or NZ is amino; or sulfur, oxygen or nothing;

X_ is acyl or nothing, wherein when X- is acyl, -X-,- N=C=X. is bonded directly to an unsaturated atom, and when X- is nothing, -N=C=X. is bonded directly to an unsaturated atom;

X. is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing, ;

R is a substituted or unsubstituted diarylmethyl or triarylmethyl group; and wherein the bonds between X and X and between X and X may be independently a single, double, or triple bond, and the bond between X2 and Y may be a single or double bond. Certain preferred embodiments of the invention include compounds according to the formula II:

wherein:

X, X- and X_ are independently selected from substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, acyl, sulfonyl, sulfinyl, or NZ, wherein Z is nothing or NZ is amino; or sulfur, oxygen or-nothing;

X- is acyl or nothing, wherein when X_ is acyl,

N=C=X₁ is bonded directly to an unsaturated atom, and -wvhen X. iiss nnoothing, -N=C=X is bonded directly to an unsaturated atom;

X. is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing;

R is a substituted or unsubstituted diarylmethyl or triarylmethyl group;

V. -V. are independently selected from hydrogen, hydroxyl, cyano, mercapto, nitro, halogen, substituted or unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfinyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl; and wherein the bonds between X and X. and between X and X may be independently a single, double, or triple bond, and the bond between X_? and Y may be a single or double bond.

The present invention also relates to methods of making compounds of formula I and compounds of formula II. The present invention also relates to methods of using compounds of formula I and of formula II to quantify free amines. Brief Description of the Figure

Figure 1 is a graph of data of a kinetics study set forth in Example 11 of this application. The graph plots the reaction percentage of certain amines with certain testing reagents against time. Detailed Description of the Invention

The present invention provides for new chemical compounds falling within formula I, as described above, that are useful for quantifying free amine groups. Certain preferred compounds of the present invention fall within the formula II, as described above.

All temperatures stated herein are in degrees Celsius ( C) . All units of measurement employed herein are in weight units except for liquids which are in volume units.

The term "alkyl" as used herein refers to straight or branched chain groups, preferably, having one to eight, more preferably having one to six, and most preferably having from one to four carbon atoms.

The term "cycloalkyl" represents a saturated or partially saturated, mono- or poly-carbocylic ring, preferably having 5-12 ring carbon atoms. Exemplary cycloalkyls include monocyclic rings having from 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. An exemplary cycloalkyl is a C..-C cycloalkyl, which is a saturated hydrocarbon ring structure containing from five to seven carbon atoms.

The term "alkoxyl" represents -0-alkyl. An example of an alkoxyl is a C,-C₈ alkoxyl, which represents a straight or branched alkyl chain having from one to six carbon atoms attached to an oxygen atom. Exemplary C, -C- alkoxyl groups include methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, sec-butoxyl, t-butoxyl, pentoxyl, hexoxyl, and the like. C - Cg alkoxyl includes within its definition a C1-C4 alkoxyl. The term "alkenyl" as used herein refers to a class of acyclic unsaturated hydrocarbons having one or more double bonds.

The term "cycloalkenyl" as used herein refers to a class of partially unsaturated carbocyclic compounds.

The term "alkynyl" as used herein refers to a class of acyclic unsaturated hydrocarbons having one or more triple bonds.

The term "aryl" as used herein refers to a carbocyclic or heterocyclic, aromatic, 5-12 membered monocyclic or polycyclic ring. Exemplary aryls include phenyl, naphthyl, anthryl, phenanthryl, thienyl, pyrrolyl, imidazolyϊ, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, benzo[b]thienyl, naphtho[2,3-b]thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxyalinyl, quinzolinyl, benzothiazolyl, benzimidazolyl, tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, and phenoxazinyl.

The term "heterocycle" means an aromatic or a saturated or a partially saturated, 5-14 membered, monocylic or polycyclic ring, such as a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic ring, having from one to three heteroatoms selected from nitrogen, oxygen and sulfur, and wherein any nitrogen and sulfur heteroatoms may optionally be quaternized. The heterocyclic ring may be attached at any suitable heteroatom or carbon atom. Examples of such heterocycles include decahydroisoquinolinyl, octahydro-thieno[3,2-c]pyridinyl, piperidinyl, piperazinyl, azepinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, isobenzofuranyl, furazanyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, thianthrenyl, triazinyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, chromenyl, xanthenyl, isoquinolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, benzoazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, benzo[b] thienyl, naphtho[2,3-b] thienyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazolyl, triazolyl, tetrahydroquinolinyl, tetrahydriso- quinolinyl, phenoxathienyl, indolizinyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxyalinyl, quinzolinyl, tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, and phenoxazinyl.

The term "aryloxyl" represents -0-aryl.

The term "thioether" includes S-aryl, such as phenylthio and naphthylthio; S-heterocycle where the heterocycle is saturated or partially saturated; S- (C^-C ) -cycloalkyl; and S-alkyl, such as C,-C₈ alkythio. In the thioether, the - aryl, the -heterocycle, the -cycloalkyl and the -alkyl can optionally be substituted. An example of a thioether is "C - C_g alkythio, " which represents a straight or branched alkyl chain having from one to six carbon atoms attached to a sulfur atom. Exemplary C. -C, alkylthio groups include methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, t-butylthio, pentylthio, hexylthio, and the like.

The term "mercapto" represents -SH. The term "cyano" represents -CN. The term "nitro" represents NO..

The term "amino" represents -N- or -NL_{1 2}—

^Ll ^Q aM wherein L and L_ are preferably independently selected from oxygen, carbocycle, heterocycle, alkyl, alkenyl, sulfonyl, and hydrogen, wherein one of L. or L₂ can be nothing, and Q is hydrogen or nothing; or NC(0)L_, wherein L-. is preferably alkyl, alkoxyl, hydrogen or

-N- or -N ₁L₂—

^Ll ^Q ^L2

The aryl, alkyl and alkoxyl groups can optionally be substituted.

The term "acyl" represents L_gC(0)L., wherein L_ is a single bond, -O or -NQ, wherein Q is hydrogen or nothing, and further wherein L. is preferably alkyl, amino, hydroxyl, alkoxyl or hydrogen. The alkyl and alkoxyl groups can optionally be substituted. An exemplary acyl is C -C. alkoxycarbonyl, which is a straight or branched alkoxyl chain having from one to four carbon atoms attached to a carbonyl moiety.

The term "sulfinyl" represents -SO-Lb.., wherein L.b. is preferably alkyl, amino, aryl, cycloalkyl or heterocycle. The alkyl, aryl, cycloalkyl and heterocycle can all optionally be substituted.

The term "sulfonyl" represents -SO„-L , wherein L is preferably alkyl, aryl, cycloalkyl, heterocycle or amino. The alkyl, aryl, cycloalkyl and heterocycle can all optionally be substituted. An example of a sulfonyl is a C - C. alkylsulfonyl, which is a straight or branched alkyl chain having from one to four carbon atoms attached to a sulfonyl moiety.

As indicated above, many of the groups are optionally further substituted. Examples of further substituents are hydrogen, hydroxyl, cyano, mercapto, nitro, halogen, substituted or unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfinyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl. Some compounds of the invention may possess one or more asymmetrically substituted carbon atoms and therefore exist in racemic and optically active forms. The invention is intended to encompass the racemic forms of the compounds as well as any of the optically active forms thereof.

Preferably, the compounds of the present invention are substantially pure, i.e. over 50% pure. More preferably, the compounds are at least 75% pure. Even more preferably, the compounds are more than 90% pure. Even more preferably, the compounds are at least 95% pure, more preferably, at least 97% pure, and most preferably at least 99% pure.

According to certain embodiments the compounds of the present invention are in a solid form.

Throughout this specification, the term -DMTr represents the following:

Preferred compounds include

3-Isothiocyano-benzyl alcohol 4,4' -dimethoxytritylate (PITC)

2-Nitro-5-isothiocyano-benzyl alcohol 4,4' -dimethoxytritylate (NPIT)

Other compounds that may be in a solid form include compounds of the formula:

wherein one or two of V through V. is an election withdrawing group such as nitro or acyl, and each of the remaining V through V substituents is hydrogen.

It is also desirable that the compounds of the present invention exhibit favorable reaction kinetics so that free amines can be quantified in as little time as possible. According to certain preferred embodiments, greater than 95% of the free amines are reacted within 10 to 15 minutes. More preferably, greater than 99% of the free amines are reacted within 10 to 15 minutes. The specific compounds listed as preferred compounds above have favorable reaction kinetics. Other compounds that may exhibit favorable reaction kinetics include compounds of the formula:

wherein one or two of V. through V is nitro, cyano, or ester, and each of the remaining V through V substituents is hydrogen.

Compounds of the formula I may be prepared according to reaction scheme I:

in which the variables A, X, X , X , X , X , Y, and R are defined as above and n = 1 or 2, and in which the reaction steps are as follows: a) a compound of the formula 1 is reacted with reagent A under conditions sufficient to obtain compound of the formula 2 ; and b) the compound of the formula 2 is reacted with R L, under conditions sufficient to obtain compound of the formula 3, wherein L is a suitable leaving group. Reagent A is an isocyanate or isothiocyanate forming reagent, such as phosgene or triphosgene. According to certain preferred embodiments reagent A is di-2-pyridyl thionocarbonate or thiophosgene. According to certain preferred embodiments R. of R.L is triphenylmethyl. According to certain preferred embodiments R.L is 4,4'- dimethoxytrityl chloride.

According to certain preferred embodiments the compound

is made by carrying out the following reaction scheme II

in which the reaction steps are as follows: a) the compound 4 is protected with an appropriate acylating agent to form compound 5; b) the compound 5 is nitrated to form compound 6; c) the compound 6 is deprotected to form compound 7; d) the compound 7 is reacted with reagent A under conditions sufficient to obtain compound 8; and e) the compound 8 is reacted with R.L, under conditions sufficient to obtain compound 9, wherein L is a suitable leaving group.

According to certain preferred embodiments, reagent A is di-2-pyridyl thionocarbonate. According to certain preferred embodiments, R of R.L is triphenylmethyl . According to certain preferred embodiments, R L is 4,4' -dimethoxytrity1 chloride.

In certain preferred embodiments, in step a) , compound 4 is reacted with acetic anhydride or benzoic anhydride. According to certain preferred embodiments, in step b) , compound 5 is reacted with HNO_/H_SO.. According to certain preferred embodiments, in step c) , compound 6 is deprotected with aqueous HCl .

Compounds of the formula I can be used in a method for qualitatively detecting or quantitatively determining free amine groups in a sample by performing the following steps: a) combining an excess amount of a compound of the formula I with a sample such that the -N=C=X groups of the compounds of formula I are coupled to free amine groups that may be present in the sample,- b) removing any unreacted compound of the formula I from the sample from step a) ,* c) treating the sample from step b) with an acid sufficient to cleave any Y-R, bonds that are present to release a cleavage product; and d) qualitatively detecting or quantitatively determining the amount of the cleavage product . According to certain preferred embodiments, in step a) , the sample is covalently linked to a solid support. In certain preferred embodiments, in step b) , the unreacted compound is removed by washing with solvent. According to certain preferred embodiments, in step c) , the sample is treated with trifluoroacetic acid and the cleavage product is separated from the solid support by filtration. According to certain preferred embodiments, in step d) , free amine is qualitatively indicated by a visible color. In certain preferred embodiments, in step d) , free amine is quantitatively determined by measuring the UV absorbance of the filtrate. According to certain preferred embodiments, in step d) , the presence of the cleavage product is indicated by a color, and free amine is quantitatively indicated by the relative intensity of the color.

To quantify free amines using the present compounds, the free amines can be on a solid support or may not be on a solid support. In either case, any unreacted testing compound should be removed from the sample being tested. One skilled in the art would be familiar with such removal or separation techniques (e.g., such as aqueous extraction methods based on different solubilities) .

According to the present invention, the extent of coupling or coupling in the entire reaction vessel can be determined based on the quantification of the free amines in only a small sample taken from the reaction vessel. Thus, only a small amount of material needs to be removed from the reaction vessel, leaving the remainder of the material undisturbed.

Quantifying free amines according to this invention not only can provide a measure of unreacted compounds in a given reaction step, but also can be used to indicate the quantity of coupling in a reaction. In one such embodiment, the extent of coupling between compound A and compound B is measured as follows.

Compound A, having a free amine and being fixed to a solid support, is coupled to compound B under suitable reaction conditions. After coupling to compound A at the free amine of compound A, compound B will contain a protected amino group.

After running the reaction, one takes a sample from a reaction vessel and quantifies free amines using the present compounds as set forth above. That quantification indicates the residual measurement.

A new sample from the same reaction vessel is then acylated or capped off with a suitable acylating agent, such as acetic anhydride and quantified using the compound as set forth above. That quantification indicates the background measurement.

Another new sample from the same reaction vessel is then subjected to conditions that deprotect the amino protecting groups present. One then quantifies the free amines using the present compounds as set forth above. That quantification represents the total amine. One quantifies the extent of compound B coupled to compound A using the following equation: Coupling yield % =

1 - Residual amine (*Sub, level)' - control (^*Sub, level)^*** x ,1_n0_n0_β%.

Total amine (Sub. level) - control (Sub. level)

A protocol that can be used to quantify free amines according to the present invention is set forth in Examples 9 and 12B below. One skilled in the art would be familiar with other ways of using absorbance readings in a quantitative or qualitative assay. For instance, the method could be automated by a machine that automatically calculates the results.

Moreover, one skilled in the art would also be familiar with protocols in which one visually compares the degree of the intensity of the color to a precalibrated reference, to provide quantitative or qualitative visual results without the need for absorbance readings.

Accordingly, the present invention is not limited to any specific method of detecting or determining the amount of color signal generated when the present compounds are used to quantify free amines. Likewise, the present invention is not limited to any particular calculation method for determining the amount of free amines.

The following Examples illustrate aspects of the invention. These examples are for illustrative purposes and are not intended to limit the scope of the invention.

Abbreviations for the terms melting point, nuclear magnetic resonance spectra, mass spectra, infrared spectra, and ultraviolet spectra, are, respectively, m.p., NMR, MS, IR, and UV.

The NMR spectra were obtained on a General Electric QE- 300 300 MHz instrument. IR spectra were obtained on a Midac FTIR instrument. UV spectra were obtained on a Cary 118 instrument. Melting points are uncorrected.

Examples 1 through 7 are directed to synthesis of compounds according to the present invention.

Reaction Scheme I shows the structures of compounds in Examples 1 and 2 below.

EXAMPLE 1

3-Isothiocvano-benzγl alcohol

A mixture of 2.46 (20 mmol) 3-aminobenzyl alcohol (purchased from ALDRICH) and 4.64 g (20 mmol) of di-2- pyridylthionocarbonate in 50 ml of CH_CN was stirred at room temperature for 30 minutes. The solvent was removed under reduced pressure and the residue was dissolved in 200 ml of EtOAc. The ethyl acetate solution was washed with saturated NaHC0₃ (3 x 50 ml) , 0.1 N HCl (3 x 50 ml), washed with brine, and then was dried with MgSO.. Silica column chromatography purification afforded 2.51 g product in 76% yield. The structure was confirmed by NMR, IR, and MS.

EXAMPLE 2

MTr

3-Isothiocvano-benzγl alcohol 4.4' -dimethoxytitritylate (PITC)

A mixture of 1.65 g (10 mmol) 3-isothiocyano-benzyl alcohol, 3.38 g (10 mmol) 4,4' -dimethoxytrityl chloride, 3.41 g (10 mmol) tetrabutylammonium percholorate and 1.01 g (10 mmol) triethylamine in 100 ml CH₂C1₂ was stirred at room temperature for 2 hours. The solvent was removed and the residue was dissolved in 150 ml of ethyl ether. The ethyl ether solution was washed with saturated NaHCO- (3 x 100 ml) , washed with brine, and then dried with MgSO.. Silica column chromatography purification (hexane/CH₂Cl₂=3/2) afforded 3.6 g product in 77% yield. The structure was confirmed by NMR, IR, MS, and elemental analysis.

Reaction Scheme II shows the structures of compounds in Examples 3 through 7 below.

EXAMPLE 3

3-Amino-benzyl alcohol diacetate

Acetic anhydride 20.7 g (203 mmol) was added dropwise to a solution of 10 g (81.2 mmol) 3-amino-benzyl alcohol in 250 ml pyridine at 0 °C with stirring over 30 minutes. The ice bath was removed and the reaction was stirred at room temperature for two hours. Pyridine was removed under reduced pressure and the residue was dissolved in ethyl ether (500 ml) . The ethyl ether solution was washed with 0.1 N HCl (3X100 ml) , 0.1 N NaOH (3X100 ml) , washed with brine (200 ml) , and then dried with MgSO.. Ethyl ether was removed and the residue was crystallized in hexane. The product (15.6 g) was obtained by filtration in 92.7% yield. The structure was confirmed by NMR.

EXAMPLE 4

2-Nitro-5-amino-benzyl alcohol diacetate

1 .5 g (70 mmol) 3-amino-benzyl diacetate was dissolved in 100 ml H₂S0₄ (98%) and cooled to -10°C, and 4.87 ml HNO_ (70%, 75 mmol) was added slowly. The reaction mixture was stirred at -10°C for 30 minutes, at 10°C for 30 minutes, and at room temperature for another 60 minutes. The mixture was then poured into 1000 ml ice/H₂0 and extracted with 500 ml ethyl acetate. The ethyl acetate solution was then washed with 0.1 N NaOH, washed with brine, and then dried with MgSO.. The solvent was removed under reduced pressure and 6.17 g product was obtained after silica column chromatography purification (hexane/EtoAc/MeOH=4/4/l) in 35% yield. The structure was confirmed by NMR.

EXAMPLE 5

2-Nitro-5-amino-benzyl alcohol

5.04 g 2-Nitro-5-amino benzyl alcohol diacetate was dissolved in 50 ml of 3N HCl and refluxed for 60 minutes. The solvent was removed and the residue was evaporated twice with isopropanol under reduced pressure. The product (3.0 g) was obtained after silica column chromatography purification (EtOAc/hexane/MeOH=4/2/l) in 90% yield. The structure was confirmed by NMR (m.p. =141-143°C, lit. reported m.p.=143°C) . EXAMPLE 6

2-Nitro-5-isothiocvano-benzyl alcohol

A mixture of 2.5 g (15 mmol) 2-nitro-5-amino-benzyl alcohol and 4.6 g (20 mmol) di-2-pyridyl thionocarbonate in 100 ml acetonitrile was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was dissolved in 250 ml ethyl acetate. The ethyl acetate solution was washed with 0.1 N HC1 (3 x 100 ml) , saturated NaHO , (3 x 100 ml) , washed with brine (100 ml), and then dried with MgSO.. Silica column chromatography purification (hexane/EtOAc=2/l) afforded 2.3 g product in 66% yield. The structure was confirmed by NMR and IR.

EXAMPLE 7

2-Nitro-5-isothiocvano-benzyl alcohol 4,4' -dimethoxytritylate (NPIT)

A mixture of 2.08 g (10 mmol) 2-nitro-5-isothiocyano- benzyl alcohol, 3.38 g (10 mmol) 4,4' -dimethoxytrityl chloride and 1.01 g (10 mmol) triethylamine in 100 ml CH₂C1₂ was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and the residue was dissolved in 160 ml ethyl ether. The ether solution was washed with water (3 x 100 ml), washed with brine and dried with MgSO.. Silica column chromatography purification (hexane/EtOAc=3/l) afforded 2.0 g product in 40% yield. The structure was confirmed by NMR, IR, MS and elemental analysis.

Examples 8 through 13 are provided to illustrate certain methods of using compounds of the present invention to quantify free amines.

EXAMPLE 8

(A) General method for the preparation of

N-t-Boc-amino acid residues (Scheme III)

The amino acid residues 1-amino-l-cyclopentane carboxylic acid (Acp) , 3-aminophenyl acetic acid (Apa) , and N-cyclohexyl-0-alanine (Cha) were purchased from ALDRICH. A solution of 12 g (55 mmol) di-butyl-dicarbonate [ (Boc) ₂0] in 100 ml of THF was added dropwise during one hour to a well-stirred solution of 50 mmol of each amino acid residue and 2 g (50 mmol) NaOH in 100 ml water and 100 ml THF to form a reaction mixture.

After stirring the reaction mixture at room temperature overnight, the reaction mixture was concentrated under reduced pressure to remove THF and another 100 ml water was added. The aqueous solution was then adjusted to pH 3 with concentrated HCl and extracted with 300 ml ethyl acetate. The ethyl acetate solution was washed with water followed by brine and dried with MgSO..

After recrystallization from hexane/ethyl acetate, the product was obtained in a yield of 45-80%. The structure of the corresponding N-t-Boc protected amino acid residue was confirmed by NMR, MS and elemental analysis.

Scheme I I I

(B) Synthesis of N-t-Boc-N- (4-ethyl morpholine) -glycine (Scheme IV)

To a solution of 25 g (0.2 mol) 4- (2-aminoethyl) morpholine and 10. 1 g (0.1 mol) triethylamine in 100 ml CH-CN, a solution of 22.9 g (0.1 mol) benzyl 2-bromoacetate and in 200 ml CH3CN was added dropwise during 60 minutes at o

0 C with vigorous stirring. The reaction mixture was then stirred at room temperature for another 3 hours. The solvent was removed under reduced pressure and the residue was dissolved in 500 ml ethyl acetate. The ethyl acetate solution was washed with 0.1 NaOH followed by brine and was dried with MgSθ₄. N- (4-ethyl morpholine) glycine benzyl ester (20.4 g) was obtained after removal of the solvent in 77.3%.

The N-t-Boc-N- (4-ethyl morpholine) glycine benzyl ester was prepared following the general method described above in Example 8 (A) . The benzyl group was then removed by hydrogenolysis of the ester at 20 psi in the presence of 2 g 10% Pd/C at room temperature for 2 hours in methanol. After recrystallization from methanol/ethyl acetate 12 g of final product was obtained in 55.5% yield and the structure was confirmed by NMR and elemental analysis.

Scheme IV

(C) Coupling of protected amino acid residues to the solid support

First Pam-|3-Ala(N-t-Boc) resin (purchased from ADVANCED CHEMTECH) was deprotected as follows to provide Pam-0-Ala with free amino groups. (PAM resin is phenylacetoamidomethyl polystyrene.) The resin was pre-swollen with excess CH^Cl- for 20 minutes. The resin was then washed with 30% TFA/ CH Cl₂ followed by treatment with 30% TFA/CH₂Cl₂ for 30 minutes. The deprotected resin was washed with CH_?Cl₂ and neutralized with 10% DIEA/CH₂C1 twice.

Separate samples of the above deprotected resin were then reacted with 3 eq each of the t-Boc protected residues, 3 eq of PyBop and 1 eq of HOBT (based on the substitution level of resin) in 10% DIEA/DMF for 60 minutes. After the reaction, each of the four substituted PAM resins were completely washed with DMF, CH₂C1₂.

To confirm coupling of the protected residue to the Pam- 0-Ala resin, a "Kaiser test" was performed on a sample of the reaction material . The Kaiser test was performed according to the protocol set forth in Kaiser, Anal Biochem. 34 : 595-598 (1970) . A positive Kaiser test indicates the presence of free amino groups. Accordingly, since successful coupling of the protected residues should result in no free amino groups of the resin (and the amino groups of the resins are protected on the coupled residues) , a negative Kaiser test qualitatively confirms coupling of protected residue to the Pam-3-Ala resin.

A negative Kaiser test indicated the completion of coupling. The remainder of each of the four substituted PAM resins were further treated with 5% Ac₂0 in 10% DIEA/CH₂C1₂ solution for 30 minutes to acylate or cap off any possible unreacted free amine of the Pam-jS-Ala resin (resin not coupled to a protected residue) in the coupling step.

The four substituted Pam resin components synthesized were : a. Pam-j(J-Ala-Acp(N-t-Boc) b. Pam-/?-Ala-Apa(N-t-Boc) c. Pam-β-Ala-Emg(N-t-Boc) d. Pam-3-Ala-Cha(N-t-Boc) .

The components were washed with CH₂Cl₂ and dried under vacuum and used for the determination of the substitution levels following the assay protocol in Example 9.

EXAMPLE 9 (A) General method of quantifying primary and secondary amines on a solid support

The protecting group (t-Boc or Fmoc) on the substituted Pam resin was first removed by 50% TFA/CH C1₂ or 30% Piperidine/DMF, respectively. After coupling with 0.1 M NPIT in DMF, DMTr⁺ was cleaved using 2% TFA/CH₂Cl₂, which has an extinction coefficient of 76000. The amount of amine was then calculated based on the UV absorption of cleavage solution at 498 nm as the substitution level of amine on the solid support in mmol/g or meq/g.

Reaction Scheme V shows the general method for quantifying amines on a solid support used in Example 9 through 13.

Scheme V

Deprotection 50% TFA or 1 N piperidine

(B) General protocol for the determination of the substitution level of amine on solid support

To quantitatively measure the substitution level of amine on the solid support, an exact amount by weight (about 5 mg) of dried substituted Pam resin was placed in a small fritted disc funnel and the general protocol in Table 1 was carried out.

Table 1

Step Operation Volume Time

(ml) (Min.)

1 Wash with CH₂C1₂ 1 3 X 1

2 Prewash with 50% TFA/CH₂C1₂ 1 1 x 1

3 Deprotection with 50% TFA/CH₂C1₂ 1 1 x 30

4 Wash with CH₂C1₂ 1 3 x 1

5 Neutralization with 10% DIEA/CH₂C1₂ 1 3 x 2

6 Wash with DMF 1 2 x 1

7 Coupling with 0.1M NPIT/DMF 0 . 5 1 x 10

8 Wash with DMF 1 2 x 1

9 Wash with CH₂C1₂ 1 3 x 1

10 Cleavage with 2% TFA/CH₂C1₂ 1 3 x 2

11 Wash with CH₂C1₂ 1 5 x 1

12 Combine the cleavage solution of step 10 and the wash fraction of step 11 and dilute to 250 ml with 0.2% TFA/CH₃CN

13 Measure UV absorption at 498 nm As a control or background measurement, another exact amount by weight (about 5 mg) of dried sample was placed in a small fritted funnel and step 4 through step 13 of the above protocol in Table 1 were carried out .

The substitution (Sub.) level of the sample was calculated using Equation I as follows:

Equation I

Sub. Level (mmol or meq/g)

Sub. Level (sample) - Sub. Level (control)

Abs. 4.9_O8_Q nm (samp ^cle) x 250000/76000 x Weig³htmg

(sample)

- Abs.4.3_no_o nm x 250000/76000 x Weightmg (control)

(C) Determination of substitution levels of Pam-3-Ala, Pam-/3-Ala-Acp,

Pam-/3-Ala-Apa, Pam-j8-Ala-Emg, and

Pam-/3-Ala-Cha

Table 2 below contains exemplary data obtained when the NPIT method set forth above in Examples 9 (A) and (B) was performed in triplicate on Pam-J-Ala-Acp(N-t-Boc) obtained in Example 8. Table 2

Pam-jS-Ala-Acp (N-t-Boc) Assay Control

Weight (mg) 4.74 4.96 4.73 4.62 4.51 4.23 Abs. (498 nm) 0.646 0.655 0.649 0.002 0.000 -0.001 Sub. level 0.449 0.434 0.451 0.001

Mean of Substitution Level 0.446 ± 0.009 mmol/g The substitution levels of the four substituted Pam-j8- Ala resins synthesized in Example 8 and Pam-/3-Ala(N-t-Boc) were determined using the NPIT method as set forth above in Examples 9 (A) and (B) as well as using three separate literature methods (picric acid, TrCl, and Kaiser test assays) . Specifically, an assay using picric acid was performed according to the procedure in Gisin, Anal. Chim. Acta 5_8: 248-249 (1972) , an assay using TrCl was performed according to Reddy and Voelker, Int. J. Peptide Protein Res . , 31: 345-348 (1988) , and an assay using the Kaiser test was performed according to Kaiser, Anal Biochem. .34.: 595-598 (1970) . The results are listed in Table 3 below.

The recorded results in Table 3 reflect a calculated average for each assay performed in triplicate. The number in parenthesis is the theoretical value calculated from the substitution level of Pam-/3-Ala determined by picric acid, TrCl and NPIT, respectively, using equation II below: Equation II

Theoretical value =

Sub, level (Pam-β-Ala)

1 + Sub. level (Pam-j8-Ala) x MW (residue)/1000 For example, for Acp using the picric acid test the theoretical value is:

0.53/(1 + 0.53 x 112/1000) = 0.50 mmol/g.

Table III

P. Acid 0.53 0.50 (0.50) 0.40 (0.49) 0.70 (0 48) 0 . 4 8 ( 0 . 4 9

TrCl 0.48 0.41 (0.45) 0.44 (0.45) 0.46 (0 44) 0 . 01 ( 0 . . 4 5

NPIT 0.49 0.45 (0.46) 0.45 (0.46) 0.46 (0 45) 0 . 4 7 ( 0 . . 46

KT dark blue dark brown light brown brown brown

P. Acid = Picric Acid

KT = Kaiser Test

The Kaiser Test was used only qualitatively

The results indicate that the NPIT reagent was the only reagent that accurately quantified free amine (primary and secondary amine) for all five resin-residues. Only the NPIT reagent effectively quantified all five types of amines within about 5% of theoretical value.

The picric acid test is inferior for the anilinic amine (Apa) and dibasic amine (Emg) . (The measurement for the Emg substituted resin in the picric acid test was significantly larger than the theoretical value since the picric acid test does not distinguish between primary and secondary amines and tertiary amines. This is another disadvantage of the picric acid test.)

The TrCl assay is not very effective for the sterically hindered primary and secondary amines (see the results for Acp and particularly Cha) . The Kaiser test is only visibly effective (dark blue) with regular primary amine (j8-Ala) , but not with the majority of amines tested. EXAMPLE 10

Qualitative assay for free amine on solid support To detect the free amine on the solid support qualitatively, a small amount of the resin having amine groups is placed in a small fritted disc funnel and treated with 0.1 M NPIT/DMF for 10 minutes. The resin is then washed with DMF twice, followed by CH₂Cl₂ five times, and is then treated with 10% TFA/CH₂C1_ . Any visible red color indicates the presence of residual free amines. EXAMPLE 11

Kinetic study of NPIT and PITC methods To study the kinetics of the NPIT method, the assay protocol in Example 9 was performed as follows on each of the four substituted Pam-/3-Ala resins of Example 8, as well as on Pam-S-Ala resin. Exact amounts of several portions of each of the resins were placed in small fritted disc tunnels and step 1 through step 6 of the protocol shown in Table I were carried out on each portion.

At step 7 of the protocol, NPIT was added to the resin samples at the same time and counted as time 0. The coupling reaction was terminated at various times by filtering the resin followed by immediately washing with DMF (step 8 of the protocol) . The substitution levels were then measured using steps 9 through 13 of the protocol.

The percentage of reaction was then calculated based on the determined substitution levels. The experimental results for Pam-3-Ala-Apa(N-t-Boc) are listed in Table 4.

Table 4

Time (min.) 1 5 10 20 40 control

Weight (mg) 4.87 4.44 4.34 4.29 4.00 4.28

Abs. (498 nm) 0.463 0.580 0.582 0.5671 0.541 0.001

Sub. Level 0.313 0.409 0.441 0.438 0.445 0.000

Reaction % 70 91.9 99.1 98.4 100

(The substitution level at time 40 was considered to be 100% of the reaction and was used for the reaction % calculation. The control used is defined in Example 9 and the coupling reaction for the control was terminated at 40 minutes.)

The same experimental procedure described above was performed for Pam-0-Ala(N-t-Boc) , Pam-J-Ala-Acp(N-t-Boc) , Pam-/3-Ala-Emg(N-t-Boc) , and Pam-0-Ala-Cha(N-t-Boc) .

The reaction rate of PITC with Pam-3-Ala-Apa(N-t-Boc) was also determined using the same protocol as described in Table 1 except that PITC was used rather than NIPT.

The results of these studies with NPIT and PITC are summarized in Figure 1. EXAMPLE 12

Sensitivity study of NPIT method

To examine the sensitivity of the NPIT method, a mixture of substituted Pam resin was prepared such that a predetermined ratio of unprotected to protected amines was present. Specifically, the mixture contained a substituted PAM resin in which 1 % of the terminal amine was protected with Fmoc and the remaining 99% was protected with t-Boc. By selective deprotection of Fmoc, the amount of "residual amine" (unprotected amine) should be about 1% of the total amine. The NPIT method was used to determine if the 1% free amine in the created mixture could be detected. (A) Synthesis of Pam-/?-Ala-Apa(N-t-Boc) / Pam-/3-Ala-Apa(N-Fmoc) resin mixture, and Pam-3-Ala-Cha(N-t-Boc) /Pam-3-Ala Cha(N-Fmoc) resin mixture

The same general procedure as in Example 8 (C) was employed except that a mixture of N-t-Boc-Cha and N-Fmoc-Cha (or N-t-Boc-Apa and N-Fmoc-Apa ) in a molar ratio of 99 to 1 was used in the coupling step instead of pure N-t-Boc-Cha (or pure N-t-Boc-Apa) . Specifically, Pam-β-Ala(N-t-Boc) (0.2 g, 0..108 meq) was deprotected with 30% TFA/CH₂C1₂ for 30 minutes. The resin was neutralized with 5% DIEA/CH„C1₂ and washed with DMF, CH₂C1₂.

In a separate vessel, a solution of N-t-Boc-Cha (313.94 mg, 1.157 mmol), N-Fmoc-Cha (4.61 mg, 0.0012 mmol) and PyBop (1.2 g, 2.32 mmol) in 10 ml of 10% DIEA/DMF was first stirred for 10 minutes at room temperature. The activated solution was then transferred to the above resin and coupled for 60 minutes. After the coupling reaction, the resin was thoroughly washed with DMF and further treated with 5% Ac„0 in 10% DIEA/CH^Cl- for 30 minutes. The resin was then washed with CH„C1 and dried under vacuum.

A Pam-J-Ala-Apa(N-t-Boc) /Pam-3-Ala-Apa(N-Fmoc) resin mixture was prepared in substantially the same way as the Pam-jS-Ala-Cha(N-t-Boc) /Pam-3-Ala-Cha(N-Fmoc) resin mixture.

(B) Assay

For the sensitivity assay, a similar procedure as described in Example 9 (B) (and in Table 1) was used except for the following modifications of that protocol.

For determining the "total amine" of the substituted resin of Example 12 (A) , an exact amount of the resin (about 5 mg) was first treated with 30% piperidine/DMF for 30 minutes to deprotect the Fmoc protected residues. After this treatment, the resin was then subjected to the same protocol described in Table 1 of Example 9 (B) from steps 1 through 13.

For determining the "residual amine," an exact amount of the resin prepared in Example 12 (A) (about 5 mg) was treated with 30% piperidine/DMF for 30 minutes to remove protecting groups (creating the residual amine) . The resin was then subjected to the protocol from steps 4 through 13 of Table 1.

For the control (background) measurement, an exact amount of the resin (about 5 mg) from Example 12 (A) was subjected to the protocol of Table 1 from step 4 through step 13.

The percentage of residual amine was calculated based on the determined substitution level using Equation III. Equation III

Residual amine % =

Residual amine (Sub, level) - control (^*Sub, level) x .1 _n0_n0_c%

Total amine (Sub. level) - control (Sub. level)

The actual experimental results are shown in Tables 5 and 6.

Table 5

Pam-0-Ala-Cha (N- t -Boc ) /Pam-0-Ala-Cha (N-Fmoc)

Total amine Residual amine Control

Weight (mg) 4.42 4.64 4.40 4.71 4.66 5.06

Abs. (498 nm) 0.591 0.632 0.024 0.028 0.005 0.005

Sub. level 0.440 0.448 0.007 0.007 0.001 0.001

Residual amine % 1.3

Theory residual amine % 1.0 (based on the predetermined mixture)

Table 6

Pam-3-Ala-Apa(N-t-Boc) /Pam-β-Ala-Apa(N-Fmoc)

Total amine Residual amine Control

Weight (mg) 4.21 4.53 4.75 4.78 4 . 33 4 . 52

Abs. (498 nm) 0.585 0.624 0.029 0.029 0 . 003 0 . 003

Sub. level 0.457 0.453 0.008 0.008 0 . 001 0 . 001

Residual amino % 1. .5

Theory residual amine % 1.0 (based on the predetermined mixture)

The results obtained from above experiments demonstrated that NPIT method is readily able to detect free amine at a level of about 1 % of the total amine on the solid support.

EXAMPLE 13

Determination of the coupling yield in solid phase synthesis of polyamides by NPIT method

(A) Synthesis of Pam-β-Ala-Emg-Apa

One example of the use of the NPIT method is to monitor the coupling efficiency in the solid phase synthesis of polyamides involving natural or unnatural amino acids . For example, in the synthesis of Pam-/3-Ala-Emg-Apa, the coupling yield of Apa to Emg could be effectively determined by this method as follows.

Pam-β-Ala-Emg (N-t-Boc) obtained in Example 8 (C) was first deprotected with 30 % TFA/CH₂Cl₂ followed by the coupling with N-t-Boc-Apa as described with the coupling of N-t-Boc-Apa with Pam-jS-Ala in Example 8 (C) . In other words, the N-t-Boc-Apa is coupled to the free amine of Emg residue in the same way that the N-t-Boc-Apa was coupled to the free amine of the Pam-/3-Ala in Example 8 (C) . After coupling, the resin (without an acetylation step or capping off step) was dried and used in the following assay.

(B) Coupling yield assay

In these assays, the "residual amine" refers to the free amine of the preceding residue (Emg) unreacted in the coupling reaction. The "total amine" refers to the "residual amine" and the amine (with the N-t-Boc protecting group removed) of the newly coupled residue (Apa) . The "control" refers to the background signal observed when resin is capped with acetic anhydride.

For the residual amine measurement, an exact amount of the dried resin made in Example 13 (A) (about 5 mg) was subjected to the protocol described in Example 9 (B) (Table 1) from step 4 through 13. For the total amine measurement, an exact amount of the dried resin made in Example 13 (A) (about 5 mg) was subjected to the protocol of Table 1 from step 1 through step 13.

For the control measurement, an exact amount of the dried resin made in Example 13 (A) (about 5 mg) was treated with 10% Ac-0 in 10% DIEA/CH2CL2 for 20 minutes. The resin was then subjected to the protocol of Table 1 from step 4 through step 13.

The coupling yield was calculated using Equation IV based on the substitution levels obtained. Equation IV

Coupling yield % = l - Residual amine (Sub, level) - control (^*Sub, level)^*- x ■1,„0„0.%_>

Total amine (Sub. level) - control (Sub. level)

The experimental results are shown in Table 7.

Table 7

Total amine Residual amine Control Weight (mg) 5.9 5.3 4.7 Abs. (498 nm) 0.745 0.009 0.007 Sub. level 0.415 0.006 0.004

Coupling yield % 99.5

Coupling yield _χ _ 0 . 006 - 0 . 004 _{χ 1 (J 0 % = g 9 5} ,

0 . 415 - 0 . 004

Claims

WE CLAIM :

1. A compound of the formula I:

wherein:

A is substituted or unsubstituted aryl or partially saturated cycloalkyl or heterocycle or is nothing,*

X, X- and X₂ are independently selected .from substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, acyl, sulfonyl, sulfinyl, or NZ, wherein Z is nothing or NZ is amino; or sulfur, oxygen or nothing;

X_ is acyl or nothing, wherein when X.. is acyl, -X 3. N=C=X is bonded directly to an unsaturated atom, and when X_ is nothing,

-N=C=X. is bonded directly to an unsaturated atom;

X is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing, ;

R is a substituted or unsubstituted diarylmethyl or triarylmethyl group; and wherein the bonds between X and X.. and between X and X_ may be independently selected from a single, double, or triple bond, and the bond between X_ and Y may be a single or double bond.

2. A compound of claim 1, wherein A is a monocyclic aromatic ring. 3. A compound of claim 2, wherein A has 0 to 4 further substitutions.

4. A compound of claim 3, wherein each of the further substitutions is independently selected from hydrogen, hydroxyl, cyano, mercapto, nitro, halogen, substituted or unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfinyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl .

5. A compound of claim 2, wherein A is phenyl.

6. A compound having the formula II

wherein :

X_ is acyl or nothing, wherein when X_ is acyl, - ^- N=C=X. is bonded directly to an unsaturated atom, and when X_ is nothing, -N=C=X. is bonded directly to an unsaturated atorn,-

X is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing; R. is a substituted or unsubstituted diarylmethyl or triarylmethyl group;

V. -V. are independently selected from hydrogen, hydroxyl, cyano, mercapto, nitro, halogen, substituted or unsubstituted alkoxyl, aryloxyl, thioether, acyl, sulfinyl, sulfonyl, amino, alkyl, cycloalkyl, saturated and partially saturated heterocycle, and aryl; and wherein the bonds between X and X. and between X and X, may be independently selected from a single, double, or triple bond, and the bond between X₂ and Y may be a single or double bond.

7. A compound of claim 6, wherein V. is nitro.

8. A compound of claim 7, wherein each of V -V is hydrogen.

9. A compound of claim 1, wherein X is nothing, X is alkyl, aryl, or alkyl or aryl ether, and X_ is nothing.

10. A compound of claim 9, wherein X_ is alkyl.

11. A compound of claim 10, wherein X is CH .

12. A compound of claim 11, wherein Y is oxygen.

13. A compound of claim 1, wherein R is a substituted or unsubstituted triphenylmethyl group and X_. is nothing.

14. A compound of claim 13, wherein R is a di(4- methoxyphenyl)phenylmethyl group.

15. A compound of claim 6, wherein X_ is nothing and R is a substituted or unsubstituted triphenylmethyl group.

16. A compound of claim 15, wherein R is a di(4- methoxyphenyl)phenylmethyl group.

17. A compound of claim 16, wherein X is nothing, X is CH and Y is oxygen.

18. A compound of claim 16, wherein each of V - is hydrogen 1 199.. A compound of claim 16, wherein V is nitro and each of V2-V4 is hydrogen 20. A compound of claim 17, wherein each of -V is hydrogen.

21. A compound of claim 17, wherein V is nitro and each of ₂~V. is hydrogen.

22. A compound of claim 1, wherein X. is nothing, X_ is alkyl, aryl, or alkyl or aryl ether, and X_ is acyl.

23. A compound of claim 22, wherein X₂ is alkyl.

24. A compound of claim 23, wherein X₂ is CH_?.

25. A compound of claim 24, wherein Y is oxygen.

26. A compound of claim 1, wherein R. is a substituted or unsubstituted triphenylmethyl group and X_ is acyl.

27. A compound of claim 26, wherein R. is a di(4- methoxyphenyl) phenylmethyl group.

28. A compound of claim 6, wherein X_ is acyl and R is a substituted or unsubstituted triphenylmethyl group.

29. A compound of claim 28, wherein R. is a di(4- methoxyphenyl) phenylmethyl group.

30. A compound of claim 29, wherein X is nothing, X is CH and Y is oxygen.

31. A compound of claim 29, wherein each of V, -V is hydrogen.

32. A compound of claim 29, wherein V is nitro and each of V„-V. is hydrogen.

33. A compound of claim 30, wherein each of V -V. is hydrogen.

34. A compound of claim 30, wherein V is nitro and each of V2-V₄ is hydrogen.

35. A method for preparing a compound of the Formula I

wherein:

A is substituted or unsubstituted aryl or partially saturated cycloalkyl or heterocycle or is nothing;

X, X. and X₂ are independently selected from substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, acyl, sulfonyl, sulfinyl, or NZ, wherein Z is nothing or NZ is amino,* or sulfur, oxygen or nothing;

X- is acyl or nothing, wherein when X₃ is acyl, -X - N=C=X. is bonded directly to an unsaturated atom, and when X is nothing, -N=C=X. is bonded directly to an unsaturated atom;

X. is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing;

R. is a substituted or unsubstituted diarylmethyl or triarylmethyl group; and wherein the bonds between X and X. and between X and X₂ may be independently selected from a single, double, or triple bond, and the bond between X₂ and Y may be a single or double bond; which method comprises carrying out the following reaction scheme I

wherein n = 1 or 2 and wherein: a) the compound of the formula 1 is reacted with reagent A under conditions sufficient to obtain compound of the formula 2,* and b) the compound of the formula 2 is reacted with R.L, under conditions sufficient to obtain compound of the formula 3, wherein L is a suitable leaving group.

36. A method of claim 35, wherein reagent A is di-2- pyridyl thionocarbonate.

37. A method of claim 36, wherein R is triphenylmethyl .

38. A method of claim 37, wherein R.L is 4,4'- dimethoxytrityl chloride. 39. A method for preparing a compound

N: C=S

which method comprises carrying out the following reaction scheme II

(9) wherein : a) the compound 4 is protected with an appropriate acylating agent to form compound 5; b) the compound 5 is nitrated to form compound 6; c) the compound 6 is deprotected to form compound 7; d) the compound 7 is reacted with reagent A under conditions sufficient to obtain compound 8; and e) the compound 8 is reacted with R-L, under conditions sufficient to obtain compound 9, wherein L is a suitable leaving group.

40. A method of claim 39, wherein reagent A is di-2- pyridyl thionocarbonate.

41. A method of claim 40, wherein R is triphenylmethyl.

42. A method of claim 41, wherein R L is 4,4'- dimethoxytrityl chloride.

43. A method of claim 39, wherein in step a), the acylating agent is selected from acetic anhydride and benzoic anhydride.

44. A method of claim 39, wherein in step b) , compound

5 is nitrated with HN0₃/H₂SO..

45. A method of claim 39, wherein in step c) , compound

6 is deprotected with aqueous HCl .

46. A method for qualitatively detecting or quantitatively determining free amine groups in a sample using a compound of the formula I

wherein:

X, X and X₂ are independently selected from substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, acyl, sulfonyl, sulfinyl, or NZ, wherein Z is nothing or NZ is amino; or sulfur, oxygen or nothing;

X- is acyl or nothing, wherein when X_ is acyl, -X^- N=C=X is bonded directly to an unsaturated atom, and when X_ is nothing, -N=C=X is bonded directly to an unsaturated atom;

X. is sulfur or oxygen;

Y is oxygen, sulfur or NQ, wherein Q is hydrogen or nothing;

R. is a substituted or unsubstituted diarylmethyl or triarylmethyl group; and wherein the bonds between X and X. and between X. and X„ may be independently selected from a single, double, or triple bond, and the bond between X₂ and Y may be a single or double bond; comprising: a) combining an excess amount of a compound of the formula I with a sample such that the -N=C=X groups of the compound of formula I are coupled to free amine groups that may be present in the sample; b) removing any unreacted compound of the formula I from the sample from step a) ,- c) treating the sample from step b) with an acid sufficient to cleave any Y'-R₁ bonds that are present to release a cleavage product; and d) qualitatively detecting or quantitatively determining the amount of the cleavage product.

47. The method of claim 46, wherein in step a), the sample is covalently linked to a solid support.

48. The method of claim 47, wherein in step b) , the unreacted compound is removed by washing with solvent .

49. The method of claim 48, wherein in step c) , the sample is treated with trifluoroacetic acid and the cleavage product is separated from the solid support by filtration.

50. The method of claim 49, wherein in step d) , free amine is qualitatively indicated by a visible color.

51. The method of claim 49, wherein in step d) , free amine is quantitatively determined by measuring the UV absorbance of the filtrate.

52. The method of claim 49, wherein in step d) , the presence of the cleavage product is indicated by a color, and free amine is quantitatively indicated by the relative intensity of the color.