WO2008089487A2 - Carboxyethylpyrrole compounds and methods of their production - Google Patents

Abstract

The present invention involves methods of anchoring carboxyethylpyrrole (CEP) haptens to carriers via various linkers. Provided herein are 9-fluorenylmethyl (Fm) masked-CEP-linker acids and Fm masked-CEP-linker esters that are useful for the preparation of CEP-linker- carrier derivatives. Also provided are methods of preparing said Fm masked-CEP-linker acids, Fm masked-CEP-linker esters and CEP-linker-carrier derivatives.

Description

CARBOXYETHYLPYRROLE COMPOUNDS AND METHODS OF THEIR

PRODUCTION

RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of US Provisional Application No. 60/881,252, filed January 19, 2007, the entire contents of which are incorporated herein by reference.

GOVERNMENT RIGHTS

[0002] This work was supported at least in part by grants GM21249 and HL53315 from the National Institutes of Health. The Government of the United States of America has certain rights in this invention.

BACKGROUND

[0003] Although it is relatively scarce in most tissues, docosahexaenoic acid (DHA) is essential for the growth, functional development and maintenance of the brain. Phospholipids containing DHA are major structural elements of phororeceptor cell membranes in the retina. Owing to the presence of the six homoconjugated C=C bonds in DHA, it is exquisitely sensitive to oxidative damage. Oxidative cleavage of retinal phospholipids containing DHA produces reactive electrophilic 4-hydroxy-7-oxohept-5- enoates that convert the primary amino group of protein lysyl residues into 2-(ω- carboxyethyl)pyrrole (CEP) derivatives. CEPs are especially abundant in the retinas of individuals with age-related macular degeneration (AMD), a slow, progressive disease of the eye that is the major cause of untreatable loss of vision among the elderly in developed countries. Roughly 11 % of people in the United States have AMD, and owing to increases in human life span, AMD is expected to nearly double in the next 25 years. [0004] Proteomic characterization of drusen, deposits that accumulate in the back of the retina, have revealed that CEP derivatives are more abundant in AMD than in normal eyes and are associated with drusen proteins. CEPs and autoantibodies against CEPs are elevated in the blood of individuals with AMD. CEPs are not simply benign markers of oxidative damage. A recent report showed that CEPs promote the growth of capillaries into the retina (choroidal neovascularization), and destruction of the photoreceptor cells. Such neovascularization is responsible for 90 % of the loss of vision associated with AMD.

[0005] CEP derivatives produced in vitro are useful for various assays, including in diagnostic assays, as antigens to raise anti-CEP antibodies, and for the study of the pathogenesis of AMD. We previously reported a method of producing CEP derivatives of proteins by reacting 4,7-dioxoheptanoic acid (DOHA) with proteins, and showed that these derivatives can also be generated, albeit less efficiently, through the reaction of (E)-4- hydroxy-7-oxohept-5-enoic acid (HOHA)-phospholipds with proteins (U.S. Pat No. 7,172,874).

[0006] It is desirable to develop a more efficient method of synthesizing CEPs, as well as methods of producing alternative CEP derivatives, which can be used for various applications, such as for example: (1) as biomarkers for clinical diagnosis and prognosis of AMD, (2) for the study of the role of CEPs in promoting AMD, (4) for raising anti-CEP antibodies, and (3) as "bait" to capture human ScFv antibodies from a phage display library.

SUMMARY

[0007] hi accordance with the present invention, provided herein ia an Fm rnasked-

CEP-linker acid compound having the general structure:

wherein n is from 1-12. [0008] Also provided is an Fm masked-CEP-linker active ester compound having the general structure:

wherein X is an active ester group.

[0009] In another aspect, the invemtion relates to a method of making an Fm masked-

CEP-linker acid compound, comprising:

reacting an ω-amino acid with the general formula HOOC(CH₂)_nNH₂, wherein n is from 1 to 12, with a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA) (DOHA-Fm) that has the following structure:

wherein the resultant Fm masked-CEP-linker acid compound has the general structure:

wherein n is from 1-12

[0010] The invebtion also relates to a method of making an Fm masked-CEP-linker ester compound, comprising:

[0011] a. reacting an ω-amino acid with the general formula HOOC(CH₂)_nNH₂, wherein n is from 1 to 12, with a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA) (DOHA-Fm) that has the following structure:

to provide an Fm masked-CEP-linker compound that has the general structure:

[0012] b. converting the Fm-masked CEP-linker of step (a) into an Fm-masked-CEP- linker ester having the general structure:

where X is an active ester group.

[0013] Also provided are isolated CEP-linker-carrier derivatives having the following structure:

wherein n is from 1 to 12.

[0014] In one embodiment, the carrier is a protein comprising a plurality of available lysyl amino groups.

[0015] In another embodiment, the carrier is biotin. [0016] In yet another embodiment, the carrier is a detectable label comprising one or more primary amine groups.

[0017] The invention also relates to a method of making a CEP-linker-carrier derivative, comprising the steps of:

[0018] a. reacting an Fm masked CEP-linker ester, described above, with a carrier comprising one or more primary amine groups to provide an Fm masked CEP-linker- carrier derivative in which the carrier primary amine groups are linked through an amide bond to the Fm masked CEP-linker ester;

[0019] b. removing Fm from the Fm-masked CEP-linker-carrier derivative to provide a CEP-linker-carrier derivative;

[0020] wherein the method provides a CEP-linker-carrier derivative having the following structure:

wherein n is from 1 to 12.

[0021] In certain embodiments if this method, the carrier is a protein, biotin, or a detectable label having one or more primary amine groups.

BRIEF DESCRIPTION OF FIGURES

[0022] Figure 1 shows an example of a reaction scheme showing the treatment of proteins with the fluorenemethyl (Fm) ester of 4,7-dioxohepatnoic acid followed by hydrolysis of the ester under mild conditions to generate protein derivatives that incorporate lysyl ε-amino groups into carboxyethyl pyrroles (CEPs). [0023] Figure 2 depicts the compound DOHA, which exists in equilibrium with the corresponding spiroacylal hemiacetal.

[0024] Figure 3 shows an example of a reaction scheme for the synthesis of a 9- fluorenylmethyl (Fm) ester of DOHA (DOHA-Fm).

[0025] Figure 4 shows an example of a reaction scheme for the synthesis of a CEP dipeptide derivative using DOHA-Fm.

[0026] Figure 5 shows an example of a reaction scheme for the synthesis of CEP-

HSA or -MSA derivatives using DOHA-Fm.

[0027] Figure 6 shows an example of a reaction scheme for synthesis of a biotinylated

CEP derivative.

[0028] Figure 7 shows another example of a reaction scheme for synthesis of a biotinylated CEP derivative.

[0029] Figure 8 shows an example of a reaction scheme for the synthesis of an ethanolamine phospholipid CEP derivative.

[0030] Figure 9 shows an example of a reaction scheme for synthesis of a CEPH-

BSA using DOHA-Fm.

[0031] Figure 10 illustrates inhibition curves showing cross-reactivity of the anti-

CEP-KLH antibody for CEP-HSA (λ) and CEPH-BSA (σ) against CEP-HSA as coating agent.

[0032] Figure 11 shows an example of a reaction scheme for preparation of CEP- linker-biotin.

[0033] Figure 12 shows an example of a reaction scheme for preparation of CEP- linker- Fluorescein. [0034] Figure 13 shows an example of a reaction scheme for the synthesis of an active pentafluorophenyl ester of a lysyl CEP using CEP-Fm modified lysine.

[0035] Figure 14 shows a general schematic of synthesizing a labeled CEP peptide using a CEP-Fm modified lysine compound.

DETAILED DESCRIPTION

[0036] The present invention will now be described with occasional reference to some specific embodiments disclosed herein. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting . As used in the description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0038] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical properties set forth in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurements. [0039] ABBREVIATIONS

[0040] AMD= age-related macular degeneration;

[0041] BCA= bicinchoninic acid;

[0042] BOP = benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexa- fluorophosphate;

[0043] BSA= bovine serum albumin;

[0044] CEP= 2-(.omega.-carboxyethyl)pyrrole;

[0045] COA= chicken ovalbuman;

[0046] DBF= dibenzofulvene;

[0047] DBU= l,8 -Diazabicyclo[5.4.0]undec-7-ene;

[0048] DCC= dicyclohexylcarbodiimide;

[0049] DHA= docosahexaenoic acid;

[0050] DMAP= dimethlamino pyridine;

[0051 ] DMF= N,N-dimethylformamide;

[0052] DOHA= 4,7-dioxoheptanoic acid;

[0053] DOHA-Fm= 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA);

[0054] EDC = l~Ethyl-3-[3-dimemylaminopropyl]carbodiimide Hydrochloride;

[0055] EDTA= ethyl enediaminetetraacetate;

[0056] ELISA= enzyme-linked immunosorbent assay; [0057] Fm= 9-fluorenylmethyl;

[0058] HBTU = O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro- phosphate;

[0059] HOAt = l-hydroxy-7-aza-benzotriazole;

[0060] HOBt = 1-hydroxybenzotriazole;

[0061] HSA= human serum albumin;

[0062] MSA= mouse serum albumin;

[0063] NHS = N-hydroxysuccinimide;

[0064] NMR= nuclear magnetic resonance;

[0065] PBS= phosphate buffered saline;

[0066] PE= Phosphatidylethanolamine;

[0067] pFP = perfluorophenyl;

[0068] pNP = para-nitrophenyl;

[0069] TBTU = O-(Benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate;

[0070] THF= tetrahydrofuran;

[0071 ] TLC= thin layer chromatography.

EMBODIMENTS

[0072] The present invention involves compounds and methods of producing compounds that relate to efficient synthesis of carboxyethylpyrroles (CEP) derivatives. [0073] We found that a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid

(DOHA) reacts with primary amines to provide esters of CEPs that can be deprotected without causing protein degradation or denaturation. The introduction of multiple CEPs into proteins is readily achieved using this strategy. (See Figure 1) Accordingly, various CEP- derivatives and their preparation are described.

[0074] The DOHA-Fm Compound

[0075] In one aspect, the invention relates to a DOHA-FM compound having the following structure:

wherein DOHA-Fm is a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA).

[0076] The DOHA-Fm compound is useful for synthesis of CEP derivatives, such as those described below. In general, the terms "CEP derivative" or "CEP-modified" are used interchangeably and refer to a molecule which includes CEP bound to a carrier wherein one or more amine groups of the carrier have been incorporated into CEP moieties. A carrier is any molecule that has a primary amine group. Examples of carriers include a protein, a peptide, an amino phospholipid, an amino sugar, a native or non-native amino acid (e.g. lysine), biotin, a detectable label or any of the aforementioned molecules with detectable label moieties (e.g. a radioisotope, biotin, chromophore, fluorophore and chemiluminiscent moiety), or an analog, derivative, or metabolic product of these molecules.

[0077] An example of a method of synthesizing DOHA-Fm is presented in Example

1.

[0078] CEP-carrier derivatives [0079] Also provided is a method of preparing a CEP-carrier derivative. The method includes:

[0080] a. reacting a carrier or carrier precursor comprising one or more primary amine groups with a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA) (DOHA-Fm) having the structure:

provide an Fm-masked CEP-carrier derivative; and

[0081] b. removing Fm from the Fm-masked CEP-carrier derivative. The method provides a CEP-carrier derivative in which the primary amine groups of the carrier or carrier precursor are incorporated into 2-(ω-carboxyethyl)pyrrole (CEP) moieties.

[0082] This method involves the novel step of removing an Fm group from an ester bound to a carrier in a "deprotection" step (step b). The deprotection step is performed by addition of a base.

[0083] The base used in the deprotection step may be organic or inorganic. In some embodiments, the base is a secondary amine. In other embodiments, the base is a tertiary amine.

[0084] In certain embodiments, the carrier is a protein, or a phosphatidyl- ethanolamine (PE) (see below).

[0085] CEP-protein derivatives

[0086] In another aspect, the invention relates to an isolated CEP-protein derivative, wherein at least 10%, 25%, 50%, or 75% of the available lysyl amino groups of the protein are incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) moiety. [0087] The number of "available lysyl amino groups" in a protein can be identified by a TNBS assay as outlined in Example 8. Therefore, "available lysyl amino groups" are the TNBS-reactive lysyl amino groups in a protein.

[0088] CEP -protein derivatives, are useful for immunoassays that measure levels of

CEPs or anti-CEP autoantibodies in vivo. CEP -protein derivatives can also be used as antigens to immunize animals to serve as a model to test whether immune responses against CEP -protein derivatives generated in the retina contribute to the pathogenesis of AMD. Using the DOHA-Fm compound described above, we found that it is possible to synthesize CEP-protein derivatives with a higher ratio of pyrrole to protein than was previously possible, indicating that a higher percentage of the protein's available lysyl amino groups are incorporated into the CEP moieties.

[0089] In another aspect, a method of preparing a CEP-protein derivative is provided.

The method includes the steps of: (a) reacting a protein having available lysyl amino groups with a DOHA-Fm compound described above; and (b) removing Fm from intermediate Fm esters produced in step (a) to obtain a CEP-protein derivative in which at least 10%, 25%, 50% or 75% of the available lysyl amino groups of the protein are incorporated into 2-(ω- carboxyethyl)pyrrole (CEP) moieties.

[0090] In some embodiments, it is desirable that the deprotection step occur under conditions that do not modify the protein except for the incorporation of lysyl ε-amino groups into CEPs. This is achieved by using a mild base. The term "conditions that do not modify the protein," is used hereinafter to refer to conditions that do not modify the protein to such an extent that it loses its intended functionality.

[0091] In one embodiment, the intended functionality is defined as the ability of the CEP-linker-protein to bind to its respective antibody in an antigen-antibody assay, such as a diagnostic assay. It may also be desirable for the CEP-linker-protein to be coated onto a solid support, such as a plastic assay plate. In the case of diagnostic assays, the antibody of interest may be one that is found in a subject having, or being at risk of developing, AMD. So it is desirable that the naturally occurring antibody react with the CEP-protein of the invention. [0092] For example, in some embodiments, it is desirable that the majority (i.e. more than 50%) of the CEP -protein derivative be soluble in an aqueous buffer solution so that it can be homogeneously and reproducibly deposited onto a solid substrate (e.g. a plastic assay plate). Therefore, conditions that do not cause denaturation and concomitant precipitation are desirable. Any minor amounts of insoluble material may be removed from the solution (e.g. by centrifugation or other methods). Accordingly, in some embodiments, the deprotection step comprises reacting the intermediate Fm ester with a mild base under conditions that result in a CEP-protein derivative that can remain soluble in an aqueous solution.

[0093] In another embodiment, the intended functionality of the CEP-protein is to mimic the naturally occurring (i.e., in vivo) CEP-proteins found in a subject having, or being at risk of developing, AMD. Proteins are rarely denatured in vivo and therefore, it is undesirable to create and inject denatured proteins into in vivo models, e.g., animal models of AMD. Therefore, it is desirable that the protein does not degrade.

[0094] For example, one type of protein degradation is denaturation, i.e. loss of tertiary structure. This loss of tertiary structure is often accompanied by precipitation, i.e., loss of solubility and hence aggregation. Therefore, in some embodiments, it is desirable to use mild bases that do not substantially denature the protein, i.e. change its tertiary structure to the point that it becomes insoluble in aqueous buffer (i.e. foπns aggregates or precipitates out of solution). Denatured proteins are recognized as damaged and consequently removed from organisms. Denaturation can be caused by variations in pH, so strongly basic or acidic conditions can cause denaturation.

[0095] Another type of protein degradation is hydrolysis. For example, if the base used in the deprotection step causes hydrolysis of the protein, the resultant CEP-linker- protein would not bind to the solid support and so will loose its intended functionality.

[0096] In some embodiments, it is also desirable that during the above described method, the protein is not modified from its original structure except for the addition of CEP moieties. [0097] In some embodiments, the mild bases do not react with water. In some embodiments, the mild bases are epitomized by non-nucleophilic and/or hindered organic bases.

[0098] Examples of mild bases include, but are not limited to: 1,8-

Diazabicyclo[5.4.0]undec-7-ene (DBU), l,5-Diazabicyclo[4.3.0]non-5-ene (DBN), Morpholine, Dicyclohexylamine, Dimethylaminopyridine, Piperazine,

Diisopropylethylamine, Tris(2-aminoethyl)amine (TAEA), Lutidine, Pyrrolidine, Pyridine, Triethylamine, Dimethyamine, Diethylamine, Dipropylamine, Dibutylamine, Hexamethylenimine, 1-Methyl-piperazine, 4-Piperidinemethanol, 4-Piperidinopiperidine, Piperidine, 1-Methylpyrrolidine, 4-Phenylpiperidine, 3-Methylpiperidine, 4- Methylpiperidine, 2,6-Dimethylpiperidine, Aminomethylpiperidine, and

Tetrabutylammonium Flouride; cesium, sodium, or potassium carbonate; diisopropyl amine, diisopropyl ethylamine, N-alkyl morpholine.

[0099] In some embodiments, the deprotection step is conducted by the addition of

DBU or morpholine.

[00100] Methods of preparing representative CEP-peptide and CEP-protein derivatives are presented in Figure 1 and Example 2.

[00101 ] Biotinylated CEP derivatives

[00102] Using the DOHA-Fm compound described above, it is possible to make biotinylated CEP derivatives that are useful in a number of applications.

[00103] For example, biotinylated-CEP derivatives can be used to explore the utility of monoclonal scFv antibodies as competitive inhibitors of the choroidal neovascularization that is promoted by CEPs. Immunoglobins and B-cells bind divalently through their variable (Fv) regions with haptens such as CEP. This can cause aggregation of proteins and/or cells displaying CEP modifications, and can activate B-cells. Immunoglobins also possess a constant (Fc) effector region that activates immune responses. In contrast, monoclonal scFv antibodies are monovalent constructs that contain only a single Fv binding region and no constant Fc region, and are therefore not expected to cause protein aggregation and deposition. Furthermore, monoclonal scFv antibodies are likely to favor penetration of the internal limiting membrane and access the subretinal space when injected intravitreously because of their relatively low molecular weight. Therefore, a monoclonal anti-CEP scFv antibody will be useful as a competitive inhibitor of CEP induced neovascularization in the eye.

[00104] Specific human scFv antibodies can be selected from engineered libraries of phage that display the antibody protein on their exterior. Selection is accomplished by using the corresponding hapten as "bait" to catch phage that produce (with the help of E. colϊ) and display antigen specific scFv on their exterior. One approach involves anchoring the hapten, through a biotin linker, to streptavidin-coated agents, such as streptavidin-coated magnetic beads.

[00105] Thus, provided herein are isolated biotinylated CEP derivatives having the following general structure:

where m is from 0 to 5, n is from 2 to 4, p is from 0 to 5, and q is from 0 to 5.

[00106] An example of a biotinylated CEP derivative and methods of its production are presented in Example 3.

[00107] PE-CEP derivatives

[00108] Phosphatidylethanolamines (PEs) are major components of certain membranes in the brain cells and in the photoreceptor cells of the retina. Because levels of PEs are strictly regulated, they are believed to have unique functional importance. In view of the reactivity of the primary amino group of PEs and the abundance of DHA in brain and retina, we anticipate that DHA-derived oxidatively truncated phospholipids containing reactive electrophilic 4-hydroxy-7-oxohept-5-enoates convert the primary amino group of PEs into CEP-PE derivatives. Accordingly, synthesized PE-CEPs would facilitate their detection and identification in vivo.

[00109] Thus, another aspect of the invention relates to isolated phosphatidylethanolamine (PE)-CEP derivatives having the following structure:

where R] is H or a fatty acyl group, and R₂ is a fatty acyl group.

[00110] hi some embodiments of the isolated PE-CEP derivative, R₂ is any saturated fatty acyl = CH₃(CH₂)_nCO- where n = 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20.

[00111] In one embodiment, R2 is palmityl (n=14). In another embodiment, R2 is stearoyl (n = 16).

[00112] Also provided is a method of preparing a phosphatidylethanolamine (PE)-CEP derivative. The method includes the steps of: (a) reacting a l,2-diacyl-sn-glycero-3- phosphoethanolamine or a l-acyl-sn-glycero-3-phosphoethanolamine with a DOHA-Fm compound described above; and (b) deprotecting the intermediate Fm esters produced in step (a). The CEP-PE derivative prepared by the method has the following structure:

[00113] where Ri is H or a fatty acyl group, and R₂ is a fatty acyl group. In one embodiment, R₂ is palmityl.

[00114] In one embodiment of the method, the deprotecting step (b) is performed by addition of l,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[00115] An example of an PE-CEP derivative and its method of production is presented in Example 4. [00116] CEP linked to a carrier with a linker (CEPX-carrier)

[00117] In another aspect, a CEP-linker-carrier compounds and methods for preparing the same are provided. These methods serve as a means of anchoring CEP haptens to carrier via various linkers.

[00118] Thus, provided herein is an Fm ester of a CEP derivative of an ω-amino acid (that will be referred to hereinafter as an Fm-masked CEP-linker) having the general structure:

where n is from 1-12. The Fm masked CEP linker can be an acid, wherein X is H. hi other embodiments, the Fm masked CEP linker is an active ester and X is an "active ester group." Examples of active ester groups include, but are not limited to, N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), l-hydroxy-7-aza-benzotriazole (HOAt), perfluorophenyl (pFP), para-nitrophenyl (pNP), N-hydroxysulfosuccinimidyl (NHSS), sulfotetrafluorophenyl (STP), pentachlorophenyl (pCP), iV-hydroxy-5-norbornene-endo-2,3- dicarboximide ester (HNb), 3 -Hydroxy- 1,2,3 -ben zotriazin-4(3H)-one (HOOBt), 1- Hydroxy- IH-1, 2,3 -Triazole-4-carboxylate (HOCt), etc. An active ester is more reactive than other esters due to its increased ability as a leaving group and an increase in nucleophilicity of the ester carbonyl, allowing for acylation under milder conditions.

[00119] The Fm-masked CEP-linker acids or esters can be used as reagents to produce various CEP-linker-carrier molecules, as described below. The Fm-masked CEP-linker acid can be activated to an active ester by reaction with a coupling agent, such as, but not limited to, DCC, EDC, HBTU, TBTU, BOP, benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium (pyBOP), diisopropylcarbodiimide (DIC), Azabenzotriazol-l-yloxytris(dimethyamino) phosphonium hexafluorosphate (AOP), Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), Bromotris(dimethylamino)phosphonium hexafluorophosphate (BrOP), 1,1'- Carbonyldiimidazole (CDI), 6-Chloro- 1 -hydroxibenzotriazol (Cl-HOBt), 3- (Diethoxyphosphoryloxy)- 1 ,2,3-benzotriazin-4(3H)-one (DEPBT),

Diethylcyanophosphonate (DEPC), 2-Fluoro-l ,3-dimethyliraidazolidinium hexafluorophosphate(DFEH), N,N'-Disuccinimidyl carbonate (DSC), O-(7-Azabenzotriazol- l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(6-Chloro-l- hydrocibenzotriazol-1-yl)- -1,1,3,3-tetramethyluroniumhexafluorophosphate (HCTU), 2- (3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HDBTU), 2-(endo-5-norbornene-2,3-dicarboxymido)- 1 , 1 ,3,3- tetramethyluronium hexafluorophosphate (HNTU), O-(N-Succinimidyl)-1, 1,3,3- tetramethyluronium hexafluorophosphate (HSTU), O-(7-Azabenzotriazole-l-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TATU), or any other uronium tetrafluoroborate or phosphonium-hexafluorophosphate compound. The active ester is purified and can be stored for use in coupling with any carrier or label, such as, but not limited to, any chromophore, fluorophore, chemiluminescent moiety, or radiolabel, (e.g. biotin, fluorescein, acridinium) for use in diagnostic assays.

[00120] Although these CEP-linker-carrier derivatives do not occur in nature, they are useful reagents for immunoassays because they can cross react with the same antibodies that bind with the naturally occurring CEP derivatives.

[00121] In one embodiment, the carrier is a protein. Therefore provided herein are isolated CEP-linlcer-protein derivatives, in which CEP moieties are covalently linked to one or more lysine residues in a protein through an ω-amino acetyl, propionyl, butyryl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, or dodecanoyl, linker. Such an isolated CEP -protein derivative has the following structure:

where n is from 1 to 12.

[00122] In this compound, the CEP-linker is preformed and then appended to the protein through attachment to lysyl residues.

[00123] In another embodiment, the carrier is biotin and the CEP-linker-biotin has the following structure:

[00124] In other embodiments, the carrier can be any detectable label which has a primary amine group.

[00125] A detectable label may be any chromophore, fluorophore, chemiluminescent moiety, or a radiolabel for use in a diagnostic assay. Examples of detectable labels include, but are not limited to, fluorescein compounds including fluoresceinamine isomer I or II, 5- (aminoacetamido)fluorescein, 4-(aminoacetamido)methylfluorescein, other fluorophores including 5-aminoeosin and eosin derivatives, 7-amino-4-methylcoumarin and coumarin derivatives, rhodamine derivatives, pyrenes and derivatives, acridinium compounds and derivatives, N-alkyl acridinium and derivatives, dansyl and derivatives. [00126] In one embodiment, the label is fluorescein and the CEP-linker- fluorescein has the following structure:

[00127] Also provided are methods of making the isolated CEP-linker-carrier derivatives described above. The methods include:

[00128] (a) reacting the Fm-masked CEP-linker ester, described above, with a carrier to provide Fm esters of CEP derivatives of ω-amino acid amides of carrier amine residues; (i.e, to provide an Fm masked CEP-linker-carrier in which the carrier amine groups are linked to the Fm masked CEP-linker);

[00129] (b) removing the Fm protecting groups from the Fm-masked CEP-linker- carrier to provide a CEP-linker amide derivatives of the carrier;

[00130] In some embodiments, the acylating agent of step (b) is an active ester derived from the CEP derivative of the ω-amino acid (i.e., the Fm-masked CEP-linker), as described above.

[00131] hi one embodiment, step (b) is performed by the addition of any base. The base may be organic or inorganic. In some embodiments, the base may be a secondary amine. In other embodiments, the base may be a tertiary amine.

[00132] In one embodiment of the method, the carrier is a protein.

[00133] In another embodiment, the carrier is a biotin. [00134] In another embodiment, the carrier is a detectable label containing an amine, as described above.

[00135] In some embodiments, where the carrier is a protein, it may be desirable that the deprotection step occur under conditions that do not modify the protein except for the incorporation of lysyl ε -amino groups into CEPs. This is achieved by using a mild base. The teπn "conditions that do not modify the protein," is used hereinafter to refer to conditions that do not modify the protein to such an extent that it loses its intended functionality.

[00136] In one embodiment, the intended functionality is defined as the ability of the CEP-linker-protein to bind to its respective antibody in an antigen-antibody assay, such as a diagnostic assay. It may also be desirable for the CEP-linker-protein to be coated onto a solid support, such as a plastic assay plate. In the case of diagnostic assays, the antibody of interest may be one that is found in a subject having, or being at risk of developing, AMD. So it is desirable that the naturally occurring antibody would react with the CEP-linker- protein of the invention.

[00137] For example, in some embodiments, it is desirable that the majority (i.e. more than 50%) of the CEP-linker-protein derivative be soluble in an aqueous buffer solution so that it can be homogeneously and reproducibly deposited onto a solid substrate (e.g. a plastic assay plate). Therefore, conditions that do not cause denaturation and concomitant precipitation are desirable. Any minor amounts of insoluble material may be removed from the solution (e.g. by centrifugation or other methods). Accordingly, in some embodiments, the deprotection step comprises reacting the intermediate Fm ester with a mild base under conditions that result in a CEP-linker-protein derivative that can remain soluble in an aqueous solution.

[00138] In other embodiments, it may be desirable that the protein does not degrade. For example, one type of protein degradation is denaturation, i.e. loss of tertiary structure. This loss of tertiary structure is often accompanied by precipitation, i.e., loss of solubility and hence aggregation. Therefore, in some embodiments, it is desirable to use mild bases that do not substantially denature the protein, i.e. change its tertiary structure to the point that it becomes insoluble in aqueous buffer (i.e. forms aggregates or precipitates out of solution). Denaturation can be caused by variations in pH, so strongly basic or acidic conditions can cause denaturation.

[00139] Another type of protein degradation is hydrolysis. For example, if the base used in the deprotection step causes hydrolysis of the protein, the resultant CEP-linker- protein would not bind to the solid support and so will loose its intended functionality.

[00140] In some embodiments, it is also desirable that during the above described method, the protein is not modified from its original structure except for the addition of CEP moieties.

[00141] In some embodiments, the mild bases do not react with water. In some embodiments, the mild bases are epitomized by non-nucleophilic and/or hindered organic bases.

[00142] Examples of mild bases include, but are not limited to: 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU), l,5-Diazabicyclo[4.3.0]non-5-ene (DBN), Morpholine, Dicyclohexylamine, Dimethylaminopyridine, Piperazine,

Diisopropylethylamine, Tris(2-aminoethyl)amine (TAEA), Lutidine, Pyrrolidine, Pyridine, Triethylamine, Dimethyamine, Diethylamine, Dipropylamine, Dibutylamine, Hexamethylenimine , 1 -Methyl-piperazine, 4-Piperidinemethanol, 4-Piperidinopiperidine, Piperidine, 1-Methylpyrrolidine, 4-Phenylpiperidine, 3-Methylpiperidine, 4- Methylpiperidine, 2,6-Dimethylpiperidine, Aminomethylpiperidine, and

Tetrabutylammonium Flouride; cesium, sodium, or potassium carbonate; diisopropyl amine, diisopropyl ethylamine, N-alkyl morpholine.

[00143] In some embodiments, the deprotection step is conducted by the addition of DBU, morpholine, or Piperidine.

[00144] An example of the above described CEP-linker-protein and a method of preparing the same is presented in Example 5. Examples of schemes for preparing a CEP- linker-biotin and CEP-linker fluorescein are provided in Example 6.

[00145] CEP-Fm modified lysine [00146] In another aspect, a CEP-Fm modified lysine compound is provided having the following structure:

where Fmoc is 9H-fluoren-9-ylmethoxycarbonyl and Fm is 9-fluorenylmethyl.

[00147] The CEP -modified lysine compound is useful in a method of producing CEP- modifled derivatives. In one example, such CEP-modified derivatives are labeled and can be used as ligands that could detect CEP receptors. For example, the labeled (e.g. fluorescent or radioactive) CEP -peptides can be used to detect and characterize receptors that promote angiogenesis when they bind CEP -peptide or CEP -protein derivatives.

[00148] Thus, another aspect relates to a method of preparing a CEP-modified carrier.

The carrier can be an amino acid, polypeptide, a derivative of an amino acid or polypeptide, or any primary or secondary amine containing a fluorophore, chromophore, chemiluminescent or radioactive label. Examples of detectable labels include, but are not limited to, fluoresceinamine or aminomethylcoumarin. Examples of radiolabels include, but are not limited to, radioactive isotopes of carbon, nitrogen, oxygen or hydrogen.

[00149] In one embodiment the primary amine is dansylhydrazine and the product is a florescent acyl hydrazide.

[00150] The carriers that can be used in the present method have an Fm protecting group, and may have other protecting groups. The protecting groups mask other nucleophilic groups in the molecule that might compete with the amine as a nucleophile that would react with the acylating agent. Examples of nucleophiles are the alpha amino group of the n-terminal amino acid, the epsilon amino group of lysyl residues, the thiol group of cysteine residues, the hydroxyl group of serine and threonine residues, the guanidino group of arginine residues, and the imidazole group of histidine residues. Examples of protectoing groups include, but are not limited to, Fmoc and t-butyloxycarbonyl (t-boc).

[00151] The method includes the steps of: (a) reacting an acylating reagent derived from the CEP-Fm modified lysine with the alpha-amino group of a carrier containing protecting groups to provide an amide incorporating a CEP-Fm modified lysine; and (b) reacting the amide incorporating the CEP-Fm modified lysine with a deprotecting reagent or reagents to remove the protecting groups and provide a CEP -modified carrier.

[00152] In one embodiment, the carrier is an amino acid or a derivative of an amino acid, including labeled amino acids. In another embodiment, the carrier is a peptide, or labeled peptide, that has one or more primary amino groups. In yet another embodiment, the carrier is a primary or secondary amine containing a label. Examples of detectable labels used to produce the labeled carriers include any fluorophore, chromophore, chemiluminescent, or radiolabels known in the art.

[00153] In the embodiments where the carrier is labeled, reaction with a labeled amine (H2N-L*) would deliver a labeled amide from which a labeled CEP -modified carrier is obtained by removal of all protecting groups (Fig. 14).

[00154] An example of a pentafluorophenyl ester of a CEP-Fm modified lysine, which in turn can be used for construction of CEP modified peptides, is presented in Example 7.

[00155] Also provided herein are diagnostic kits comprising DOHA-Fm, one or more of the CEP-derivative compounds, or any intermediate compounds described herein.

[00156] The present invention will be better understood by reference to the following examples which are offered by way of illustration not limitation.

EXAMPLE 1- DOHA-Fm

[00157] Paal-Knoor synthesis using 4,7-dioxo-heptanoic acid is ineffective for the preparation of CEPs. The reaction of γ-keto aldehydes with primary amines, the Paal- Knoor synthesis, is generally an efficient method for the preparation of pyrroles. This reaction was successfully applied to the generation of carboxyheptylpyrrole and carboxypropylpyrrole derivatives through the reactions of 9,12-dioxododecanoic or 5,8- dioxooctanoic acid with proteins. However, attempts at preparing the corresponding carboxyethylpyrrole derivatives of proteins by treatment with 4,7-dioxoheptanoic acid (DOHA) frequently caused precipitation, and in the instances that precipitation did not occur, the ratio of pyrrole to protein, e.g. 1.6:1 for human serum albumin (Gu, X.; et al., Journal of Biological Chemistry 2003, 278, 42027-42035) was much lower than we had obtained previously for the longer chain carboxyalkylpyrroles (Kaur, K.; et al., Chemical Research in Toxicology 1997, 10, 1387-1396). Another distinguishing feature of DOHA was the nearly complete absence of a signal for the aldehydic hydrogen in its ¹H NMR spectrum. We postulated that the unusual H NMR spectrum aberrant reactivity of DOHA is a consequence of the proximity of the carboxyl group to the γ-ketoaldehyde array and that DOHA exists in equilibrium with the corresponding spiroacylal hemiacetal (Figure 2). To obviate complications engendered by the carboxyl group, we sought a masked derivative that could be deprotected under conditions that would not lead to denaturation and consequent precipitation of proteins. This excluded acidic conditions. Therefore, we opted for a 9-fluorenylmethyl ester.

[00158] Synthesis of a 9-fluorenylmethyl (Fm) ester of DOHA. Coupling of 3- carbomethoxypropionyl chloride with the Grignard reagent derived from 2-(2-bromoethyl)- 1,3-dioxolane provides access to the methyl ester 1 (Figure 3). The desired 9H-fiuoren-9- ylmethyl ester 4,7-dioxo-heptanoic acid (DOΗA-Fm, 4) was then obtained through saponification to afford the carboxylic acid 2, esterificaton with 9-fluorenylmethanol, followed by hydrolysis of ethylene ketal in 3.

[00159] Experimental Procedures

[00160] 6-[l,3]Dioxolan~2-yl-4-oxo-hexanoic acid methyl ester (1). A solution of 2- (2-bromoethyl)-l,3-dioxolane (1 g, 5.5 mmol) in anhydrous TΗF (2 mL) was added dropwise to a flame dried 100 mL flask with Mg turnings (150 mg, 6.25 mmol) and 4 mL TΗF and a small piece of I₂ under argon at room temperature to initiate the reaction. After adding a few drops, the reaction started as evidence by disappearance of the red-brown I₂ color. Then 5 mL TΗF was added to the flask. After completion of the addition, more TΗF (15 mL) was added. The reaction mixture was stirred for another 1 h and then cooled to -78 ⁰C followed by slow addition of 3-carbomethoxypropionyl chloride (710 mg, 4.7 mmol) dissolved in 2.5 niL dry THF. The resulting mixture was stirred for another 40 min, then quenched with 30 mL of a saturated aqueous solution Of NH₄Cl, and extracted with EtOAc (4 x 15 mL). The combined organic phase was washed with brine, dried with MgSO₄, and evaporated to obtain the crude product. The crude compound was purified by silica gel chromatography (30 % ethyl acetate in hexane, TLC: Rf = 0.3) to give 714 mg (60 %) of pure 1. ¹H NMR (CDCl₃, 200 MHz) £4.85 (t, J= 4.3 Hz, IH), 3.7-3.90 (4H), 3.62 (s, 3H), 2.67 (m, 2H), 2.56 (t, J = 7.48 Hz, 4H), 1.93 (dt, J= 4.3, 7.48 Hz, 2H). ¹³C NMR (CDCl₃, 50 MHz, APT) £ 207.81 (+) (CO), 173.09 (+) (COO), 103.06 (-) (CH), 64.83 (+) (CH₂), 51.61 (-) (CH₃), 36.87 (+) (CH₂), 36.30 (+) (CH₂), 27.62 (+) (CH₂), 27.41 (+) (CH₂). HRMS (FAB) (m/z) calcd for Ci₀H₁₇O₅ (MH⁺) 217.1076, found 217.1081.

[00161] In a modification of this procedure, a dioxane starting matertial was used instead of a dioxolane. Accordingly, 6-[l,3]Dioxan-2-yl-4-oxo-hexanoic acid methyl ester was made using a solution of 2-(2-bromoethyl)-l,3-dioxane (1 g, 5.5 mmol) in anhydrous.

[00162] The corresponding 6-[l,3]Dioxan-2-yl-4-oxo-hexanoic acid methyl ester can be prepared similarly by substituting 2-(2-bromoethyl)-l,3-dioxane for 2-(2-bromoethyl)- 1,3-dioxolane in the above procedure and the resulting 6-[l,3]Dioxan-2-yl-4-oxo-hexanoic acid methyl ester can be substituted for 6-[l,3]Dioxolan-2-yl-4-oxo-hexanoic acid methyl ester (1) in the subsequent procedures.

[00163] 6-[l,3]Dioxolan-2-yl-4-oxo-hexanoic acid (2). Ester 1 (390 mg, 1.8 mmol) in 10 mL of H₂O/MeOH/THF (2:5:3, v/v/v) was stirred for 3 h with NaOH (367 mg, 9.2 mmol) at room temperature. The reaction mixture was then acidified with 3 N HCl to pH 3.0 and extracted with EtOAc (3 x 15 mL). The combined organic phase was washed with brine, dried with MgSO₄, and evaporated to give acid acetal 2 (360 mg, 90%). ¹H NMR (CDCl₃, 200 MHz), £ 4.85 (t, J = 4.3 Hz, IH), 3.7-3.90 (4H), 2.5-2.64 (6H), 1.93 (dt, J = 4.3, 7.48 Hz, 2H). ¹³C NMR (CDCl₃, 100 MHz,) £ 207.81 (CO), 178.03 (COOH), 103.17 (CH), 64.97 (CH₂), 36.76 (CH₂), 36.36 (CH₂), 27.71 (CH₂), 27.51 (CH₂). HRMS (FAB) (m/z) calcd for C₉H]₅O₅ (MH⁺) 203.0919, found 203.0917. [00164] 6-[l,3]Dioxolan-2-yl-4-oxo-hexanoic acid 9H-fluoren-9-ylmethyI ester (3).

(9H-Fluoren-9-yl)-methanol (373 mg, 1.9 mmol) in 3 mL dry CH₂Cl₂ was slowly added to the solution of dicyclohexylcarbodiimide (DCC, 295 mg, 1.425 mmol), dimethlamino pyridine (DMAP, 58 mg, 0.475 mmol) and the acid 2 (96 mg, 0.475 mmol) in 5 mL dry CH₂Cl₂. The resulting mixture was stirred for 72 h at room temperature. The solvent was removed. Flash chromatography of the residue (30 % ethyl acetate in hexane, TLC: Rf = 0.3) gave the ester 3 (153 mg, 95 %). ¹R NMR (CDCl₃, 200 MHz) (57.77 (d, J = 6.74 Hz, 2H), 7.60 (d, J= 7.7 Hz, 2H), 7.42 (dd, J = 6.74, 7.7 Hz, 2H), 7.29 (dd, J = 6.74, 7.7 Hz, 2H), 4.90 (t, J= 4.3 Hz, IH), 4.40 (d, J= 7.38 Hz, 2H), 4.21 (t, J= 7.38 Hz, IH), 3.80-3.96 (4H), 2.62-2.73 (4H), 2.56 (t, J = 7.3 Hz, 2H), 1.98 (td, J = 7.3, 4.3 Hz, 2H). ¹³C NMR (CDCl₃, 50 MHz, APT), δ 207.76 (+) (CO), 172.62 (+) (COO), 143.69 (+) (C), 141.19 (+) (C), 127.69 (-) (CH), 127.04 (-) (CH), 125.00 (-) (CH), 119.92 (-) (CH), 103.12 (-) (CH), 66.45 (+) (CH₂), 64.90 (+) (CH₂), 46.67 (-) (CH), 36.89 (+) (CH₂), 36.35 (+) (CH₂), 27.90 (+) (CH₂), 27.48 (+) (CH₂). HRMS (FAB) (m/z) calcd for C₂₃H₂₄O₅ (M⁺) 380.1624, found 380.1614; calcd for C₂₃H₂₅O₅ (MH⁺) 381.1702, found 381.1711.

[00165] 4,7-Dioxo-heptanoic acid 9H-fluoren-9-ylmethyl ester (DOHA-Fm, 4).

Ester 3 (94 mg, 0.25 mmol) in 10 mL of AcOH/H₂O (3:1, v/v) was stirred at 50 ⁰C for 5 h. TLC (100 % CHCl₃): Rf = 0.5 showed the completion of the reaction.The solvent was removed by rotary evaporation. Flash chromatography of the residue (25 % ethyl acetate in hexane) gave 4 (73 mg, 88 %). ¹H NMR (CDCl₃, 400 MHz), δ 9.78 (s, IH), 7.77 (d, J = 6.74 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.42 (dd, J= 6.74, 7.7 Hz, 2H), 7.30 (dd, J = 6.74, 7.7 Hz, 2H), 4.39(d, J= 7.38 Hz, 2H), 4.20 (t, J = 7.38 Hz, IH), 2.67-2.79 (8H). ¹³C NMR (CDCl₃, 100 MHz), (5206.56 (CO), 200.27 (CHO), 172.53 (COO), 143.70 (C), 141.23 (C), 127.73 (CH), 127.07 (CH), 124.98 (CH), 119.96 (CH), 66.47 (CH₂), 46.70 (CH), 37.42 (CH₂), 36.87 (CH₂), 34.53 (CH₂), 27.93 (CH₂). HRMS (FAB) (m/z) calcd for C₂₁H₂₀O₄ (M⁺) 336.1362, found 336.1361.

EXAMPLE 2: Synthesis of CEP-peptide and CEP-protein derivative by Paal-Knoor synthesis with DOHA-Fm.

[00166] Paal-Knoor synthesis the Fm ester 5 of a CEP dipeptide was readily achieved in 80 % yield by the condensation of methyl 6-amino-2-((2- acetylamino)acetyl)amino)hexanoate (Ac-Gly-Lys-OMe) with 1 equivalent of DOHA-Fm (Figure 4). For peptide synthesis, the deprotection of 9-fluorenylmethyl esters is normally accomplished with piperidine in DMF. Piperidine serves both as a base to fragment the Fm group and as a scavenger to trap the dibenzofulvene (DBF) released. (Sheppeck, J. E.; Kar, H.; Hong, H. Tetrahedron Letters 2000, 41, 5329-5333.) The removal of an Fm group from an ester bound to a protein has not been reported. Piperidine was generally unsatisfactory, vide infra. This was readily determined by the persistence of a UV absorbtion at 265 nm that is characteristic of the flourene group. Therefore, we examined the efficacy of a stronger base. DBU successfully removed all Fm groups from protein derivatives, vide infra, and this reagent was also applied to deprotection of the dipeptide Fm ester 5. Removal of the Fm protecting group by treatment of 5 with DBU in THF solution delivered the CEP dipeptide 2-(2-acetylamino-acetylamino)-6-[2-(2-carboxy-ethyl)-pyrrol- 1 -yl]-hexanoic acid methyl ester (6) in 86 % yield.

[00167] CEP -modified proteins are needed for a variety of applications, for example, immunoassays that measure levels of CEPs or anti-CEP autoantibodies in vivo. We also required CEP-protein derivatives as antigens to immunize animals as models to test the hypothesis that immune responses against CEP-protein derivatives generated in the retina contribute to the pathogenesis of AMD. We now find that CEP-protein derivatives can be readily made by incubation of DOHA-Fm (4) with protein in 30 % DMF/phosphate- buffered saline (PBS) solution for 5 days at 37 °C followed by deprotection by addition of DBU to the reaction mixture and stirring for an additional 9 h. One equivalent of DOHA- Fm was used for each lysine group present in human serum albumin (HSA) or mouse serum albumin (MSA) (Figure 5). Low molecular weight contaminants were removed by dialysis {Mr cutoff 14000) of the reaction mixture against 20% DMF in 10 mM PBS. An advantageous feature of the use of Fm esters of DOHA is the ease with which residual Fm groups can be detected and their complete removal assured by UV spectroscopy. The final protein concentration was determined by Pierce bicinchoninic acid (BCA) protein assay (Smith, P. K., et al., Analytical Biochemistry 1985, 150, 76-85) or Bio-rad protein assay (www.bio-rad.com/LifeScience/pdf/Bulletin_9005.pdfj. The pyrrole concentration was determined by the generation of a characteristic chromophore through reaction with 4- (dimethylamino)benzaldehyde, the Ehrlich reagent (Decaprio, A. P.; Jackowski, S. J.; Regan, K. A. Molecular Pharmacology 1987, 32, 542-548), using the CEP dipeptide 6 as a quantitative standard. In contrast with the preparation of CEP-HSA by direct treatment with DOHA, that delivered a pyrrole to protein ratio of 1.6: 1 for CEP-HAS (Gu, X.; et al, Journal of Biological Chemistry 2003, 278, 42027-42035), the new synthetic method using DOHA-Fm provided CEP-HSA (7) with a much higher pyrrole to protein ratio, 7.6 ± 1.1 to 1, and provided CEP-MSA (8) with a pyrrole to protein ratio of 5.2 ± 1.0 to 1.

[00168] Experimental Procedures

[00169] 2-(2-Acetylamino-acetylamino)-6-{2-[2-(9H^r-fluoren-9- ylmethoxycarbonyl)-ethyl]-pyrrol-l-yl}-hexaiioic acid methyl ester (5). Methyl 6- amino-2-((2-acetylamino)acetyl)amino) hexanoate (Ac-Gly-Lys-OMe, 25.6 mg, 0.08 mmol) in 1 mL methanol was added dropwise to DOHAFm (27 mg, 0.08 mmol) in 1.5 niL methanol. The solution was stirred for 9 h at room temperature under argon. The solvent was removed by rotary evaporation. TLC (4 % methanol in chloroform): Rf = 0.34. The crude compound was purified by silica gel chromatography (4 % methanol in chloroform ) to give 35.8 mg (80%) of pure 5. ¹H NMR (CDCl₃, 400 MHz), £ 7.77 (d, J = 7.6 Hz, 2H), 7.60 (d, J= 7.6 Hz, 2H), 7.36-7.32 (dd, J= 7.6, 7.2 Hz, 2H), 7.27 (dd, J= 7.6, 7.2 Hz, 2H), 6.60 (dd, J = 2.4, 1.6 Hz, IH), 6.54 (m, IH), 6.28 (s, IH), 6.09 (dd, J = 3.6, 2.4 Hz, IH), 5.90 (m, IH), 4.56-4.61 (m, IH), 4.40 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.2 Hz, IH), 3.91 (dABq, J = 5.2, 14 Hz, 2H), 3.82 (t, J= 7.2 Hz, 2H), 3.76 (s, 3H), 2.65-2.86 (4H), 2.01 (s, 3H), 1.86 (m, 2H), 1.66 (m, 2H), 1.36 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz), δ 172.98 (CO), 172.33 (CO), 170.58 (CO), 168.66 (CO), 143.74 (C), 141.31 (C), 130.73(C), 127.80 (CH), 127.12 (CH), 125.01 (CH), 120.37 (CH), 120.04 (CH), 106.99 (CH), 105.26 (CH), 66.47 (CH₂), 52.53 (CH), 51.86 (CH₃O), 46.78 (CH), 46.09 (CH₂), 43.23 (CH₂), 33.22 (CH₂), 31.89 (CH₂), 30.67 (CH₂), 22.92 (CH₂), 22.40 (CH₂), 21.41 (CH₃). HRMS (FAB) (m/z) calcd for C₃₂H₃₈N₃O₆ ⁺ (MH⁺) 560.2755, found 560.2747.

[00170] 2-(2-Acetylamino-acetylamino)-6-[2-(2-carboxy-ethyl)-pyrrol-l-yl]- hexanoic acid methyl ester (CEP-dipep, 6). DBU (75 μL) was added to 5 (27 mg, 0.047 mmol) in 2.5 mL THF. The system was stirred for 6 h under argon. After the removal of solvent, the crude compound was purified by silica gel chromatography (6 % methanol in chloroform) to give 21 mg (86 %) of CEP-dipep. TLC (10 % methanol in chloroform): R_f = 0.3; ¹H NMR (CDCl₃, 400 MHz), £7.18 (d, J= 8 Hz, IH), 6.81 (m, IH), 6.53 (m, IH), 6.03 (dd, J = 3.2, 3.2 Hz, IH), 5.87 (m, IH), 4.53 (dt, J = IH), 3.95 (dABq, J = 5.2, 13.6 Hz, 2H), 3.81 (t, J= 6.8 Hz, 2H), 3.70 (s, 3H), 2.65-2.86 (4H), 2.01 (s, 3H), 1.8-1.62 (4H), 1.36 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz), δ 176.01 (COOH), 172.40 (COO), 171.35 (CO), 169.28 (CO), 131.40 (C), 119.92 (CH), 107.25 (CH), 105.00 (CH), 52.50 (CH), 52.00 (CH₃O), 45.66 (CH₂), 43.19 (CH₂), 32.98 (CH₂), 31.44 (CH₂), 30.90 (CH₂), 22.94 (CH₂), 22.07 (CH₂), 21.37 (CH₃). HRMS (FAB) (m/z) calcd for Ci₈H₂₈N₃O₆ ⁺ (MH⁺) 382.1978, found 382.1986.

[00171] Carboxyethyl pyrrole human albumin derivative (CEP-HSA, 7). A solution of DOHA-Fm (2 mg, 0.006 mmol) in 750 μL DMF was added to 1.5 mL 0.08 mM solution of HSA in PBS. The mixture was stirred under argon for 4 days. 200 μL DBU was added to the system and stirred overnight under argon followed by two successive 12 h dialyses (Mr cutoff 14000) against 500 mL 20 % DMF in 10 mM PBS (pH 7.4) and two additional dialyses (12 h each) against 500 mL 10 mM PBS (pH 7.4) at 4 °C. The final protein concentration (1.80 mg/niL) was determined by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole concentration (187.14 μM) was determined by Ehrlich assay.

[00172] Carboxyethyl pyrrole mouse albumin derivative (CEP-MSA, 8). A solution of DOHA-Fm (18.5 mg, 0.055 mmol) in 8 mL DMF was added slowly to the solution of 100 mg mouse serum albumin in 18 mL 10 mM PBS (pH 7.4). The mixture was stirred under argon for 4 days. 360 μL DBU was added to the system and stirred overnight under argon followed by two successive 24 h dialyses (Mr cutoff 14000) against 1 L 20 % DMF in 10 mM PBS (pH 7.4) and two additional dialyses (24 h each) against I L lO mM PBS (pH 7.4) at 4 ⁰C. The final protein concentration (2.43 mg/mL) was determined by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole concentration (210 μM) was determined by Ehrlich assay.

EXAMPLE 3 - Synthesis of biotinylated CEP derivatives.

[00173] We prepared a biotinylated CEP Fm ester (15) from DOHA-Fm and 4~amino butylbiotin (Figure 6). Deprotection of the intermediate 3-(l-{4-[5-(2-Oxo-hexahydro- thieno[3,4-(i]imidazol-4-yl)-pentaiioylamino]-butyl}-lH-pyrrol-2-yl)-propionic acid 9H- fiuoren-9-ylmethyl ester 15 by treatment with DBU in TΗF generated 3-(l-{4-[5-(2-Oxo- hexahydro-thieno[3,4-<fjimidazol-4-yl)-pentanoylamino]-butyl}-lH-pyrrol-2-yl)-propionic acid (16). [00174] AU of the functionality in biotinylated CEP derivative 16 survived treatment with sodium hydroxide ethanol solution. This observation suggested the feasibility of simpler synthesis for preparing CEP derivatives of substrates that are stable to strong base. Thus, we prepared 3-(l-{4-[5-(2-oxo-hexahydro-thieno[3,4-J]imidazol-4-yl)- pentanoylamino]-hexyl}-l//-pyrrol-2-yl)-propionic acid (20) by reacting 4,7-dioxo- heptanoic acid methyl ester (DOHA-Me, 17) with biotinyl-l,6-diaminohexane (18) followed by hydrolysis of the methyl ester 19 with ethanolic sodium hydroxide (Figure 7).

[00175] Experimental Procedures

[00176] {4-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoyIamino]- butyl}-carbamic acid tert-butyl ester (13). N-Boc-l,4-butanediamine (20 mg, 0.106 mmol) in 2 nxL CH₂Cl₂ was added in J-biotin p-nitrophenyl ester (38 mg, 0.10 mmol) in 2 mL CH₂Cl₂. The system was stirred for 10 h. TLC (30 % ethyl acetate in hexane): Rf= 0.25. After removal of the solvent by rotary evaporation, the crude compound was purified by flash chromatography (30 % ethyl acetate in hexane) to give 28 mg (80 %) 13. ¹H NMR (CD₃OD, 400 MHz), δ 4.47-4.50 (m, IH), 4.28-4.31 (m, IH), 3.2 (m, IH), 3.16 (t, J = 7.2 Hz, 2H), 3.03 (t, J= 6.8 Hz, 2H), 2.92 (dd, J= 12.4, 4.8 Hz, IH), 2.68-2.71 (d, J= 12.8 Hz, IH), 2.19 (t, J= 7.6 Hz, 2H), 1.44-1.7 (10H), 1.42 (s, 9H).

[00177] 5-(2-Oxo-hexahydro-thieno[3,4-rf]iraidazol-4-yI)-pentanoic acid (4-amino- butyl)-amide (14). 100 μL trifluoroacetic acid was added in 6 mg 13 in 400 μL CH₂Cl₂. After stirring for 3 h, TLC (20% methanol in chloroform, Rf = 0.1) showed the reaction was completed. The solvent was removed by rotary evaporation. The yellowish residue was neutralized by IN NaOH to pH 8.5. After removal of the solvent, the residue was treated with methanol and the methanol extraction was concentrated and dried to give 4 mg (90%) 14. ¹H NMR (CD₃OD, 400 MHz), «5 4.46-4.51 (m, IH), 4.28-4.31 (m, IH), 3.2 (m, IH), 3.16 (t, J= 7.2 Hz, 2H), 2.9 (dd, J= 12.4, 4.8 Hz, IH), 2.68-2.71 (d, J= 12.4 Hz, IH), 2.65 (t, J= 7.2 Hz, 2H), 2.19 (t, J= 7.6 Hz, 2H), 1.34-1.7 (10H).

[00178] 3-(l-{4-[5-(2-Oxo-hexahydro-thieno[3,4-</]imidazoI-4-yl)- pentanoylamino]-butyl}-lH-pyrrol-2-yl)-propionic acid 9i/-fluoren-9-yI-methyI ester

(15). Amine 14 (4 mg, 0.012 mmol) in 150 μL methanol was added in DOHA-Fm (4 mg, 0.0119 mmol) in 150 μL methanol. The solution was stirred for 9 h at room temperature under argon. The solvent was removed by rotary evaporation. TLC (5 % methanol in CHCl₃): R/ = 0.2. The crude compound was purified by silica gel chromatography (5 % methanol in chloroform ) to give 6 mg (84 %) of pure 15. ¹H NMR (CDCl₃, 400 MHz), δ 7.70 (d, J= 7.6 Hz, 2H), 7.50 (dd, J= 7.6, 1.2 Hz, 2H), 7.34 (dd, J= 7.6, 7.2 Hz, 2H), 7.27- 7.23 (ddd, J= 7.6, 7.2, 1.2 Hz, 2H), 6.51 (dd, J= 1.6, 2.8 Hz, IH), 5.98 (dd, J= 2.8, 3.6 Hz, IH), 5.79 (m, IH), 5.74 (m, IH), 5.58 (s, IH), 4.82 (s, IH), 4.40 (m, IH), 4.36 (d, J = 7.2 Hz, 2H), 4.20 (m, IH), 4.15 (t, J= 7.2 Hz, IH), 3.76 (X, J= 7.2 Hz, 2H), 3.15 (m, 2H), 3.05 (m, IH), 2.84-2.79 (dd, J = 4.8, 12.8 Hz, IH), 2.78-2.76 (m, 2H), 2.71-2.66 (m, 2H), 2.64- 2.61 (d, J = 12.8 Hz, IH), 2.06-2.10 (dt, J = 3.6, 7.2 Hz, 2H), 1.72-1.36 (10H). HRMS (FAB) (m/z) calcd for C₃₅H₄₃N₄O₄S⁺ (MH⁺) 615.3005, found 615.2995.

[00179] 3-(l-{4-[5-(2-Oxo-hexahydro-thieno[3,4-rf]imidazol-4-yl)- pentanoylamino]-butyI}-lH-pyrrol-2-yl)-propionic acid (16). 5 μL DBU was added to 15 (6 mg, 0.01 mmol) in 500 μL TΗF and stirred for 2 h. TLC (10 % methanol in chloroform, Rf = 0.3) showed the completion of the reaction by disappearance of the UV active starting spot. The corresponding acid was purified by flash chromatography (7 % methanol in chloroform) to provide 3.7 mg (85 %) acid 16. ¹H NMR (CD₃OD, 400 MHz), δ 6.57 (dd, J= 2.8, 1.6 Hz, IH), 5.92 (dd, J= 3.2, 2.8 Hz, IH), 5.79 (m, IH), 4.60 (3H), 4.48 (m, IH), 4.27 (m, IH), 3.86 (t, J= 7.2 Hz, 2H), 3.19-3.15 (3H), 2.94-2.89 (dd, J= 4.8, 12.8 Hz, IH), 2.86-2.82 (m, 2H), 2.71-2.67 (d, J= 12.8 Hz, IH), 2.60-2.56 (m, 2H), 2.20-2.17 (t, J = 7.2 Hz, 2H), 1.72-1.36 (10H). HRMS (FAB) (m/z) calcd for C₂₁H₃₃N₄O₄S⁺ (MH⁺) 437.2222, found 437.2193; calcd for C₂₎H₃iN₄O₃S⁺ (M⁺-OH) 419.2117, found 419.2102.

[00180] 4,7-Dioxo-heptanoic acid methyl ester (DOHA-Me, 17). Ester 1 (22.5 mg, 0.104 mmol) in 5 mL of AcOH/H₂O (3:1, v/v) was stirred at 50 ⁰C for 5 h. TLC (CHCl₃): R₁ = 0.45 showed the completion of the reaction.The solvent was removed by rotary evaporation. Flash chromatography of the residue (25 % ethyl acetate in hexane) gave 17 (15.6 mg, 87 %). ¹H NMR (CDCl₃, 200 MHz), (59.78 (s, IH), 3.67 (s, 3H), 2.56-2.81 (8H); HRMS (FAB) (m/z) calcd for C₈H₁₁O₄ ⁺ (M⁺-H) 171.0658, found 171.0656; calcd for C₈H₁₃O₅ ⁺ (M⁺+ OH) 189.0763, found 189.0783. [00181] 5-(2-Oxo-hexahydro-thieno[3,4-</]imidazol-4-yl)-pentanoic acid (6-amino- hexyl)-amide (18). 1 ,6-diaminohexane (270 mg, 2.324 mmol) in 10 niL pyridine-H₂O (9:1, v/v) was added slowly to J-biotin /?-nitrophenyl ester (Sigma) (100 mg, 0.274 mmol) in 20 ml, pyridine-H₂O (9:1, v/v). The clear yellow solution was stirred for 24 h at room temperature. The solvent was removed by rotary evaporation. TLC (30% NH₃-saturated methanol in CHCl₃, v/v), R/ = 0.24. The crude compound was purified by silica gel chromatography (30 % methanol saturated with NH₃ in chloroform) to give 84 mg (90 %) of pure 18. ¹H NMR (CD₃OD, 400 MHz), δ 4.46-4.51 (m, IH), 4.28-4.31 (m, IH), 3.20 (m, IH), 3.16 (t, J= 7.2 Hz, 2H), 2.9 (dd, J= 12.4, 4.8 Hz, IH), 2.68-2.71 (d, J= 12.4 Hz, IH), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.34-1.7 (14H). ¹³C NMR (CD₃OD, 100 MHz), δ 175.97 (CO), 166.10 (CO), 63.36 (CH), 61.60 (CH), 57.01 (CH), 42.3 (CH₂), 42.0 (CH₂), 40.2 (CH₂), 36.8 (CH₂), 33.2 (CH₂), 30.50 (CH₂), 29.8 (CH₂), 29.5 (CH₂), 28.8 (CH₂), 28.6 (CH₂), 28.0 (CH₂). HRMS (FAB) (m/z) calcd for Ci₆H₃₁N₄O₂S⁺ (MH⁺) 343.2168, found 343.2165.

[00182] 3-(l-{6-[5-(2-Oxo-hexahydro-thieno[3,4-rf]imidazol-4-yl)- pentanoyIamino]-hexyl}-lH-pyrrol-2-yl)-propionic acid methyl ester (19). Amine 18 (45 mg, 0.13 mmol) in 1 mL methanol was added to DOHA-Me (23 mg, 0.13 mmol) in 1.5 mL methanol. The solution was stirred for 9 h at room temperature under argon. The solvent was removed by rotary evaporation. The crude compound was purified by silica gel chromatography (5 % methanol in chloroform, TLC: R_j = 0.18) to give 47 mg (75 %) of pure 19. ¹H NMR (CD₃OD, 400 MHz), £6.52 (dd, J= 2.0, 2.8 Hz, IH), 5.9 (dd, J= 3.2, 2.8 Hz, IH), 5.75 (m, IH), 4.46-4.49 (dd, J = 8.0, 4.0 Hz, IH), 4.29 (dd, J = 8.0, 4.8 Hz, IH), 4.28-4.3 (m, IH), 3.82 (t, J= 7.6 Hz, 2H), 3.65 (s, 3H), 3.1-3.2 (3H), 2.9 (dd, J= 12.4, 4.8 Hz, IH), 2.85 (t, J = 7.2 Hz, 2H), 2.68-2.71 (d, J= 12.4 Hz, IH), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t, J= 7.6 Hz, 2H), 1.34-1.7 (14H).

[00183] 3-(l-{4-[5-(2-Oxo-hexahydro-thieno[3,4-rf]imidazol-4-yl)- pentanoylamino]-hexyl}-lH-pyrrol-2-yI)-propionic acid (20). Sodium hydroxide (16 mg, 0.4 mmol) was added to 19 (47 mg, 0.1 mmol) in 1 mL absolute ethanol and stirred for 4 h at room temperature. TLC (10 % methanol in chloroform, Rf = 0.3) showed the completion of the reaction by disappearance of the starting spot. After removal of the solvent, the residue was neutralized with 3 N HCl to pΗ = 3 and extracted with ethyl acetate. The ethyl acetate extract was washed with brine, dried with MgSO₄, and concentrated to afford 38 mg (84 %) 20. ¹H NMR (CD₃OD, 400 MHz), £6.57 (dd, J= 2.0, 2.4 Hz, IH), 5.93 (dd, J= 2.4,

3.6 Hz, IH), 5.80 (m, IH), 4.46 (dd, J = 7.6, 5.2 Hz, IH), 4.29 (dd, J = 4.8, 7.6 Hz, IH), 3.84 (t, J = 7.6 Hz, 2H), 3.1-3.2 (3H), 2.9 (dd, J = 12.4, 4.8 Hz, IH), 2.85 (t, J = 7.2 Hz, 2H), 2.68-2.71 (d, J = 12.4 Hz, IH), 2.61 (t, J= 7.2 Hz, 2H), 2.19 (t, J= 7.2 Hz, 2H), 1.34-

1.7 (14H). ¹³C NMR (CD₃OD, 100 MHz), £ 176.82 (CO), 175.97 (CO), 166.10 (CO), 132.00 (C), 121.22 (CH), 107.52 (CH), 106.08 (CH), 63.36 (CH), 61.60 (CH), 57.01 (CH), 47.19 (CH₂), 41.03 (CH₂), 40.22 (CH₂), 36.82 (CH₂), 34.48 (CH₂), 32.54 (CH₂), 30.30 (CH₂), 29.78 (CH₂), 29.51 (CH₂), 27.62 (CH₂), 27.44 (CH₂), 26.95 (CH₂), 22.55 (CH₂). HRMS (FAB) (m/z) calcd for C₂₃H₃₇N₄O₄S⁺ (MH⁺) 465.2530, found 465.2524.

EXAMPLE 4: Synthesis of ethanolamine phospholipid CEP derivatives.

[00184] Reaction of DOHA-Fm with l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE) or l-palmitoyl-2-hydroxy-sn-glycero-3- phosphoethanolamine (lysoPE) followed by deprotection of the intermediate Fm esters 21 or 23 with DBU (Figure 8) delivered the CEP-PEs 22 and 24.

[00185] Experimental Procedures

[00186] Octadec-9-enoic acid 2-[(2-{2-[2-(9if-fluoren-9-ylmethoxycarbonyl)- ethyl]-pyrrol-l-yl}-ethoxy)-hydroxy-phosphoryloxy]-l-hexadecanoyloxymethyl-ethyl ester (21). Triethylamine (TEA) (25 μL, 0.247 mmol) was added to POPE (50 mg, 0.065 mmol) in 500 μL CHCl₃, then DOHAFm (22 mg, 0.065 mmol) in 500 μL CHCl₃ was added to the mixture. The system was stirred 20 h under Argon. After evaporation of solvent, the crude product was purified by silica gel chromatography (10 % methanol in chloroform, TLC: Rf= 0.2) to give 50 mg (76 %) of pure 21. ¹H NMR (CD₃OD:CDC1₃ = 1 :1, 400 MHz), £7.74 (d, J= 7.6 Hz, 2H), 7.53 (d, J= 7.6 Hz, 2H), 7.36 (dd, J= 7.6, 7.6 Hz, 2H), 7.27 (dd, J= 7.6, 7.6 Hz, 2H), 6.63 (dd, J= 2.8, 1.6 Hz, IH), 5.94 (dd, J= 3.2, 2.8 Hz, IH), 5.77 (m, IH), 5.28 (m, 2H), 4.36 (d, J = 6.8 Hz, 2H), 4.28 (m, IH), 4.17 (t, J = 6.8 Hz, IH), 4.05- 3.98 (6H), 3.68 (t, J = 6.4 Hz, 2H), 2.86-2.82 (t, J = 7.2 Hz, 2H), 2.73-2.69 (t, J= 7.2 Hz, 2H), 2.25-2.21 (4H), 2.0-1.94 (4H), 1.57 (4H), 1.21 (44H), 0.84 (t, J = 6.4 Hz, 6H). ¹³C NMR (CD₃OD:CDC1₃ = 1 :1, 50 MHz, APT), £ 175.21 (+) (CO), 174.84 (+) (CO), 174.81 (+) (CO), 145.08 (+) (C), 142.68 (+) (C), 132.30 (+) (C), 131.24 (-) (CH), 130.95 (-) (CH), 129.13 (-) (CH), 128.53 (-) (CH), 126.31 (-) (CH), 121.30 (-) (CH), 121.22 (-) (CH), 108.49 (-) (CH), 106.77 (-) (CH), 71.86 (-) (CH), 67.96 (+) (CH₂), 66.62 (+) (CH₂), 64.60 (+) (CH₂), 64.05 (+) (CH₂), 48.10 (+) (CH₂), 47.82 (-) (CH), 35.47 (+) (CH₂), 35.34 (+) (CH₂), 34.66 (+) (CH₂), 33.27 (+) (CH₂), 31.03 (+) (CH₂), 30.85 (+) (CH₂), 30.70 (+) (CH₂), 30.66 (+) (CH₂), 30.61 (+) (CH₂), 30.49 (+) (CH₂), 28.49 (+) (CH₂), 26.24 (+) (CH₂), 26.20 (+) (CH₂), 23.98 (+) (CH₂), 22.64 (+) (CH₂), 15.14 (-) (CH₃). HRMS (FAB) (m/z) calcd for C₆₀H_9INNa₂Oi₀P⁺ [(M-H)Na₂ ⁺] 1062.6177, found 1062.6.

[00187] Octadec-9-enoic acid 2-({2-[2-(2-carboxy-ethyl]-pyrrol-l-yl}-ethoxy)- hydroxy-phosphoryloxy]-l-hexadecanoyloxymethyl-ethyl ester (PE-CEP, 22). DBU (20 μL, 0.13 mmol) was added to 21 (24 mg, 0.025 mmol) in 500 μL CHCl₃. After 3 h, the reaction was completed and the system was diluted by 1.5 mL CHCl₃. The solution was washed with 2 mL phosphate buffer of pH 5.5. The organic phase was washed with brine, dried over magnesium sulfate, then vacuum-filtered, concentrated, purified by flash chromatography (15 % methanol in chloroform, TLC: R_f = 0.18) to yield 19 mg (90 %) of pure 22. ¹H NMR (CD₃OD:CDC1₃ = 1 :1, 400 MHz), δ 6.56 (m, IH), 5.96 (m, IH), 5.83 (m, IH), 5.30 (m, 2H), 5.15 (m, IH), 4.33 (m, IH), 4.05-3.98 (6H), 3.81 (m, 2H), 2.83 (m, 2H), 2.58 (m, 2H), 2.27 (t, J= 7.2 Hz, 4H), 2.0-1.94 (4H), 1.57 (4H), 1.21 (44H), 0.84 (t, J= 6.4 Hz, 6H). ¹³C NMR (CD₃OD:CDC1₃ = 1:1, 100 MHz), δ 180.36 (COOH), 174.49 (CO), 174.08 (CO), 133.54 (C), 130.46 (CH), 130.14 (CH), 120.98 (CH), 107.26 (CH), 105.05 (CH), 71.00 (CH), 65.84 (CH₂), 64.03 (CH₂), 63.11 (CH₂), 47.29 (CH₂), 34.70 (CH₂), 34.56 (CH₂), 32.40 (CH₂), 32.42 (CH₂), 30.23 (CH₂), 30.17 (CH₂), 30.00 (CH₂), 29.80 (CH₂), 29.63 (CH₂), 27.66 (CH₂), 25.40 (CH₂), 23.13 (CH₂), 14.31 (CH₃). HRMS (FAB) (m/z) calcd for C₄₆H₈₂NNaOi₀P⁺ (MNa⁺) 862.5574, found 862.5550; calcd for C₄₆H₈₁NNa₂Oi₀P⁺ [(M-H)Na₂ ⁺] 884.5394, found 884.5291.

[00188] Hexadecanoic acid 3-[(2-{2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]- pyrroI-l-yl}-ethoxy)-hydroxy-phosphoryloxy]-2-hydroxy-propyl ester (23).

Triethylamine (TEA) (12 μL, 0.116 mmol) was added to lyso-PE (42 mg, 0.093 mmol) in 500 μL CHCl₃, then DOHAFm (26 mg, 0.077 mmol) in 500 μL CHCl₃ was added to the mixture. The cloudy system was stirred 20 h under Ar. After evaporation of solvent, the crude product was purified by silica gel chromatography (10 % methanol in chloroform, TLC: Rf= 0.2) to give 40 mg (69 %) of pure 23. ¹E NMR (CDCl₃, 400 MHz), £7.70 (d, J= 7.6 Hz, 2H), 7.52 (d, J= 7.6 Hz, 2H), 7.35 (dd, J= 7.6, 7.6 Hz, 2H), 7.25 (dd, J= 7.6, 7.6 Hz, 2H), 6.58 (m, IH), 5.94 (m, IH), 5.74 (m, IH), 5.28 (m, 2H), 4.31 (d, J= 6.8 Hz, 2H), 4.14 (t, J = 6.8 Hz, IH), 4.03-3.60 (9H), 2.82-2.78 (t, J= 7.2 Hz, 2H), 2.69-2.65 (t, J = 7.2 Hz, 2H), 2.21-2.17 (t, J= 7.2 Hz, 4H), 1.54-1.45 (m, 2H), 1.23-1.19 (24H), 0.86 (t, J= 6.8 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz), δ 173.69 (CO), 173.42 (CO), 143.69 (C), 141.22 (C), 131.07 (CH), 127.77 (CH), 127.09 (CH), 125.02 (CH), 120.97 (CH), 119.98 (CH), 107.20 (CH), 105.34 (CH), 77.20 (CH) 69.07 (CH₂), 66.66 (CH₂), 65.37 (CH₂), 64.30 (CH₂), 46.65 (CH₂), 46.35 (CH), 33.97 (CH₂), 33.11 (CH₂), 31.92 (CH₂), 29.73 (CH₂), 29.67 (CH₂), 29.58 (CH₂), 29.37 (CH₂), 29.23 (CH₂), 24.83 (CH₂), 22.69 (CH₂), 21.23 (CH₂), 14.11 (CH₃). HRMS (FAB) (m/z) calcd for C₄iH₅₈NNa0₉P⁺ (MNa⁺) 776.3904, found 776.3939; calc. for C_4IH₅₇NNa₂O₉P⁺ (MNa₂ ⁺) 798.3724, found 798.3717.

[00189] Hexadecanoic acid 3-(pp-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester

(lysoPE-CEP, 24). DBU (20 niL, 0.13 mrnol) was added to 23 (24 mg, 0.03 mmol) in 500 rnL CHCl₃. After 3 h, the reaction was completed and the system was diluted by 1.5 mL CHCl₃. The solution was washed with 2 mL phosphate buffer of pH 5.5. The organic part was washed with brine, dried over magnesium sulfate and vacuum-filtered, concentrated, purified by flash chromatography (CHCl₃ :MeOH:H₂O = 65:25:4, v/v, TLC: R₁ = 0.18) to yield 16 mg (95 %) of pure lysoPE-CEP. ¹H NMR (CD_.3OD:CDC1₃ = 1:1, 400 MHz), £6.60 (m, IH), 5.96 (m, IH), 5.83 (m, IH), 4.10-4.0 (5H), 3.74-3.6 (m, 2H), 3.6-3.5 (m, 2H), 2.8- 2.9 (m, 2H), 2.68-2.65 (m, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.57 (m, 2H), 1.23 (24H), 0.85 (t, J = 7.2 Hz, 3H). ¹³C NMR (CD₃OD:CDC1₃:D₂O = 50:50:1, 100 MHz), δ 175.20 (CO), 133.00 (C), 121.23 (CH), 107.36 (CH), 105.56 (CH), 70.80 (CH), 66.05 (CH₂), 65.63 (CH₂), 47.17 (CH₂), 34.61 (CH₂), 33.29 (CH₂), 32.48 (CH₂), 30.22 (CH₂), 30.05 (CH₂), 29.90 (CH₂), 29.88 (CH₂), 29.72 (CH₂), 27.04 (CH₂), 25.42 (CH₂), 23.21 (CH₂), 14.38 (CH₃). HRMS (FAB) (m/z) calcd for C₂₇H₄₈NNaO₉P⁺ (MNa⁺) 598.3121, found 598.3053.

EXAMPLE 5 - CEP linked to proteins with an ω-aminohexanoyl tether.

[00190] Direct coupling of DOHA-Fm to proteins results in a high yield of CEP modifications of lysyl residues. As an alternative approach for anchoring CEP haptens to proteins for use as coating agents to capture anti-CEP antibodies, we examined the utility of CEPs anchored to proteins through hexanoyl amides of protein lysyl residues. An Fm masked 2-carboxyethylpyrrole 9 was generated through the reaction of DOHA-Fm with 6- aminocaproic acid. After purification, 9 was activated by conversion into an N- hydroxysuccinimide ester 10. Incubation of the active ester 10 with bovine serum albumin (BSA) followed by deprotection in situ by addition of DBU to the reaction mixture, delivered a 6~(2-carboxyethyl-l-pyrrolyl)hexanoyl amide derivative of BSA, CEPH-BSA (11, Figure 9). Low molecular weight impurities were readily removed by dialysis (Mr cutoff 14000) with 20% DMF in 10 mM PBS 2 x 12 h and then with 10 mM PBS 2 x 12 h. The protein concentration was determined by a modified Lowry protein assay (www.piercenet.comj using the Lowry protein assay reagent and Folin-Ciocalteu reagent. The pyrrole concentration was determined using Ehrlich assay. The pyrrole to BSA ratio in CEPH-BSA (11) was 5.4 ± 0.9 to 1.

[00191] The CEPH-BSA (11) was also prepared with an alternative method that yielded a higher CEP to BSA incorporation ratio. Incubation of 20 mg of active ester 10 with 50 mg bovine serum albumin (BSA) in 50 ml 10 mM PBS buffer with 16% DMF for 24 hours followed by deprotection in situ by addition of morphilne (for a final morpholine concentration of 2%, v/v) for 48 hours delivered a 6-(2-carboxyethyl-l-pyrrolyl)hexanoyl amide derivative of BSA, CEPH-BSA. Low molecular weight impurities were readily removed by dialysis (MW cutoff 14000) with 15% DMF in 10 mM PBS 2 x 24 h and then with 10 mM PBS 3 x 24 h. The protein concentration was determined by Amino Acid Analysis (AAA). The pyrrole concentration was deteπnined using Ehrlich assay. The pyrrole to BSA ratio in CEPH-BSA (11) was 19.7 to 1. SDS/PAGE analysis of the CEPH- BSA revealed a protein band of slightly higher apparent molecular weight than BSA as the predominant product, with a small percentage (approximately 1-2%) of a protein band of twice the apparent molecular weight of BSA, and no protein bands of less apparent molecular weight than BSA, indicating the protein has not degraded. Subsequent Western blot analysis of the gel, using anti-CEP mouse monoclonal antibody prepared against CEP- BSA showed the predominant band was reactive toward the monoclonal antibody, indicative that the intended functionality of the preparation was not impacted by this method. [00192] The antibody binding affinity of CEPH-BSA (11) was determined by competitive enzyme-linked immunosorbant assay (ELISA) (Sayre, L. M.; et al., Chemical Research in Toxicology 1996, 9, 1194-1201) using an anti-CEP-KLH polyclonal antibody (Figure 10). CEP-HSA was used as a coating agent and standard whose binding was inhibited by CEPH-BSA. The IC₅₀ of CEPH-BSA (1.93 pmol/mL) is lower than the IC₅₀ of CEP-HSA (3.02 pmol/mL) indicating that CEPH-BSA has a slightly higher affinity than CEP-HSA for binding anti-CEP-KLH antibody.

[00193] Experimental Procedures

[00194] 6-{2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-l-yl}-hexanoic acid (9). 6-aminocaproic acid (10.8 mg, 0.082 mmol) in 400 μL water was slowly added to DOΗA-Fm (21 mg, 0.0625 mmol) in 600 μL methanol. The solution became cloudy with the addition. The heterogeneous system was stirred for 48 h under argon at room temperature and became homogenous. The solution was extracted with CH₂Cl₂, washed with brine, dried with Na₂SO₄ and evaporated, the yellowish residue was loaded to the silica gel in a filter with chloroform and washed with 15 mL chloroform, 15 mL 10 % ethyl acetate in hexane, 15 mL 20 % ethyl acetate in hexane and 60 mL 50 % ethyl acetate in hexane successively. The 50 % ethyl acetate/hexane washed fractions were collected and dried to give 21.8 mg (81 %) acid 9. TLC (ethyl acetate/hexane, 2:3 v/v): R_f = 0.22. ¹H NMR (CDCl₃, 400 MHz), £7.77 (d, J= 6.74 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H), 7.42 (dd, J = 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74, 7.2 Hz, 2H), 6.58 (dd, J= 3.2, 3.6 Hz, IH), 6.06 (dd, J= 3.2, 3.2 Hz, IH), 5.87 (m, IH), 4.42 (d, J= 7.2 Hz, 2H), 4.22 (t, J= 7.2 Hz, IH), 3.79 (t, J = 7.2 Hz, 2H), 2.72-2.84 (m, 4H). 2.34 (t, J = 7.2 Hz, 2H), 1.75-1.34 (6H). ¹³C NMR (CDCl₃, 100 MHz), c5 177.40 (COOH), 172.90 (COO), 143.73 (C), 141.20 (C), 130.66 (C), 127.78 (CH), 127.10 (CH), 125.00 (CH), 120.34 (CH), 120.04 (CH), 106.88 (CH), 105.20 (CH), 66.40 (CH₂), 46.79 (CH₂), 46.28 (CH), 33.45 (CH₂), 33.20 (CH₂), 31.01 (CH₂), 26.23 (CH₂), 24.26 (CH₂), 21.44 (CH₂). HRMS (FAB) (m/z) calcd for C₂₇H₃₀NO₄ (MH⁺) 432.2175, found 432.2190.

[00195] 6-{2-[2-(9H-fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-l-yl}-hexanoic acid 2,5-dioxo-pyrrolidin-l-yl ester (CEPFmSu, 10). Acid 9 (15 mg, 0.035 mmol) and N- hydroxysuccinimide (4.5 mg, 0.039 mmol), DCC (7.5 mg, 0.036 mmol) were dissolved in dry CH₂Cl₂ (7.5 niL) under Argon. The clear solution became cloudy after 15 minutes. The reaction mixture was stirred for 3.5 h. Solvent was removed by evaporation. The crude product was purified by silica gel chromatography with ethyl acetate/hexane (2:3, v/v) to deliver 16.6 mg (90 %) active ester 10. TLC (ethyl acetate/hexane, 2:3): R_f= 0.25. ¹H NMR (CDCl₃, 400 MHz), £7.77 (d, J= 6.74 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H), 7.42 (dd, J= 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74, 7.2 Hz, 2H), 6.58 (dd, J = 3.2, 3.6 Hz, IH), 6.06 (dd, J = 3.2, 3.2 Hz, IH), 5.87 (m, IH), 4.42 (d, J= 7.2 Hz, 2H), 4.22 (t, J= 7.2 Hz, IH), 3.79 (t, J= 7.2 Hz, 2H), 2.72-2.86 (m, 8H), 2.60 (t, J = 7.2 Hz, 2H), 1.81-1.70 (m, 4H), 1.48-1.40 (m, 2H). ¹³C NMR (CDCl₃ 100 MHz), δ 172.79 (COO), 169.10 (COO), 168.39 (CO), 143.75 (C), 141.29 (C), 130.69 (C), 127.77 (CH), 127.10 (CH), 125.01 (CH), 120.34 (CH), 120.01 (CH), 106.92 (CH), 105.24 (CH), 66.36 (CH₂), 46.79 (CH₂), 46.21 (CH), 33.31 (CH₂), 30.86 (CH₂), 30.72 (CH₂), 25.87 (CH₂), 25.55 (CH₂), 24.25 (CH₂), 21.44 (CH₂). HRMS (FAB) (m/z) calcd for C₃iH₃₃N₂O₆ (MH⁺) 529.2338, found 529.2340.

[00196] 6-(2-carboxyethyl-l-pyrrolyl)-hexanoyl BSA amide (CEPH-BSA, 11) A solution of 1.4 mg CEPFmSu in 150 μL DMF was added to 1 mL of 10 πiM pH 7.4 PBS containing 3 mg/mL of BSA. The cloudy solution became homogenous with overnight stirring. After 2 days, 25 μL DBU was added and the resulting mixture stirred overnight under argon followed by two successive 12 h dialysis (Mr cutoff 14000) against 500 mL 20 % DMF in 10 mM PBS (pH 7.4) and two additional dialysis (12 h each) against 500 mL 10 mM PBS (pH 7.4) at 4 ⁰C. The final protein concentration (1.34 mg/mL) was determined by modified Lowry protein assay. The pyrrole concentration (134 μM) was determined by Ehrlich assay.

[00197] Competitive ELISA for inhibition of anti-CEP antibody by CEPH-BSA.

CEP-HSA was used as a coating agent and a standard, CEPH-BSA was used as an inhibitor. A blank, a positive control containing no inhibitor, and up to 8 serial dilutions of the inhibitor and 8 serial dilutions of the CEP-HSA standard were run. Each well of the ELISA plate was coated with CEP-HSA solution (100 μL), prepared by diluting a solution containing 187.14 nmol/niL HSA-bound CEP in PBS to 187.14 pmol/mL with pH 7.4 PBS (10 mM). The plate was incubated at 37 ⁰C for 1 h, then washed with 10 mM PBS (3 x 300 μL), and then blocked by incubating 1 h at 37 ⁰C with 300 μL of 1 % chicken ovalbuman (COA) in 10 mM PBS. The plate was then rinsed with 0.1 % COA in 10 mM PBS (300 μL). Eight serial dilutions of CEPH-BSA inhibitor or CEP-HSA standard (120 μL each with a dilution factor of 0.2) were preincubated at 37 ⁰C for 1 h with anti-CEP-KLH antibody solution (120 μL) that was prepared by adding 5 μL of protein G column-purified antibody (1.8 mg/mL) in PBS to 10 mL of 0.2% COA in 10 mM PBS. The initial inhibitor and standard concentrations were 1162 pmol/niL and 935 pmol/mL, respectively. These were prepared by diluting a CEPH-BSA solution (116.2 nmol/mL) or CEP-HSA solution (187.14 nmol/mL) with 10 mM PBS, respectively. Blank wells were filled with 0.1 % COA (100 μL). Positive control wells were filled with the diluted antibody solution (50 μL) and PBS (50 μL). The antibody-antigen complex solutions (100 μL) were then added in duplicate to their respective halves of the plate, which was then incubated at room temperature with gentle agitation on a shaker for 1 h. After the supernatant was discarded, the wells were washed with 0.1 % COA (3 x 300 μL), and then 100 μL of goat anti-rabbit IgG-alkaline phosphatase solution (Boeliringer-Mannlieim, Indianapolis, Indiana) which was prepared by adding 10 μL of the commercial enzyme-linked secondary antibody in 10 mL of 1 % COA was added. The plate was then incubated at room temperature with gentle agitation for 1 h and washed with 0.1 % COA (3 x 300 μL). 100 μL of a solution of 1.0 mg/mL of p- nitrophenyl phosphate in 0.2 M Tris buffer (Sigma-Aldrich, Milwaukee, WI, Cat. Sigma N 1891) was added. The plate was then incubated at room temperature for 20 min until the maximum absorbance reached 0.6-0.8. The development was terminated by adding 3 N NaOH (50 μL) to each well before measuring the final absorbance values. The absorbance in each well was measured with a dual-wavelength Bio-Rad 450 microplate reader with detection at 405 nm relative to 655 nm. Absorbance values for duplicate assays were averaged and scaled to make the maximum curve fit value close to 100 percent. The averaged and scaled percent absorbance values were plotted against the log of concentration. Theoretical curves for each plot were fit to the absorbance data with a four parameter logistic function, f(x) = (a-d)/[l+(x/c)^Λb]+d using SigmaPlot 9.0 (Jandel Scientific Software, San Rafael, CA). Parameter a = the asymptotic maximum absorbance, b = slope at the inflection point, c = the inhibitor concentration at the 50 % absorbance value (IC₅₀), and d = the asymptotic minimum absorbance.

EXAMPLE 6 - CEP-Iinker-carrier synthesis [00198] Figure 11 shows an example of a scheme for the preparation of a CEP-linker- biotin. Figure 12 shows an example of a scheme for the synthesis of a CEP-linker- Fluorescein.

[00199] The CEP-linker-biotin can be prepared by reaction of active ester 10 with a biotin derivative containing an amine in an aprotic solvent, such as methylene chloride or DMF, in the presence of a non-nucleophilic mild organic base, such as triethylamine or diisopropylethylamine and/or an acyl transfer reagent, such as DMAP or imidazole. In this scheme, any label containing an amine can be substituted for the amine-containing biotin.

[00200] The CEP-linker -label can be prepared by reaction of active ester 10 with a derivative of the label containing an amine in an aprotic solvent, such as methylene chloride or DMF, in the presence of a non-nucleophilic mild organic base, such as triethylamine or diisopropylethylamine and/or an acyl transfer reagent, such as DMAP or imidazole.

EXAMPLE 7 - Synthesis of an active pentafluorophenyl ester of a lysyl CEP.

[00201] Pentafluorophenyl esters of protected amino acids are widely used in peptide synthesis. We are interested in testing the ability of CEP modified peptides bound to major histocompatibility proteins to elicit an immune response to antigen specific T-cells. Li another example, complexes of CEP modified peptides bound to constructs called "dimer X", that have major histocompatibility proteins fused to immunoglobin Fc constant regions, can be used to fluorescently label antigen specific T-cells, and consequently enable their quantitation by fluorescence activated cell sorting.

[00202] A pentafluorophenyl ester 26 of a CEP-Fm modified lysine was synthesized as a building block for construction of CEP modified peptides. Reaction of DOHA-Fm with 6- amino-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid (Fmoc-Lys-OΗ) delivered 25. The latter was then coupled with pentafluorophenol using the traditional DCC, DMAP method (Figure 13).

[00203] The CEP -modified lysine can be used to prepare CEP -modified carriers. The carriers can be an amino acid or a derivative of an amino acid, including labeled amino acids; a peptide, or labeled peptide, that has one or more primary amino groups; or a primary or secondary amine containing a label. Figure 14 shows a general schematic of synthesizing a labeled CEP peptide using a CEP-Fm modified lysine compound. The scheme shows an acylating reagent which is an active ester, such as a pentafluorophenyl (PFP) ester, derived from a CEP-Fm modified lysine. Reaction with a labeled amine (H2N- L*), such as a radiolabeled amino acid or peptide, would deliver a labeled amide from which a labeled CEP peptide is obtained by removal of protecting groups.

[00204] Experimental Procedures

[00205] 2-(9H-Fluoren-9-ylmethoxycarbonylammo)-6-{2-[2-(9H-fluoren-9- ylmethoxy carbonyl)-ethyl]-pyrrol-l-yl}-hexanoic acid (25). 6~amino-2-(9H-fluoren-9- ylmethoxy carbonyl amino)-hexanoic acid (40 mg, 0.1 mmol) was suspended in 10 πiL methanol with DOHAFm (30 mg, 0.089 mmol). Then 20 mL of acetic acid was added. The suspension dissolved gradually and a light yellow oil was generated at the bottom of the flask as the reaction proceeded. The system was stirred 24 h under Ar. After the removal of solvent, the crude compound was purified by silica gel chromatography (4 % methanol in chloroform) to give 45 mg (75 %) of light yellow oil 25. TLC (4 % methanol in chloroform): R₁= 0.15; ¹H NMR (CDCl₃, 400 MHz), £7.75 (d, J= 7.2 Hz, 4H), 7.55 (d, J = 7.6 Hz, 4H), 7.39 (dd, J = 7.2, 7.6 Hz, 4H), 7.30 (dd, J = 7.2, 7.6 Hz, 4H), 6.57 (m, IH), 6.06 (dd, J= 3.2, 3.2 Hz, IH), 5.87 (m, IH), 5.4 (m, IH), 4.41 (d, J= 6.8 Hz, 2H), 4.39 (m, IH), 4.20 (t, J = 6.8 Hz, 2H), 3.78 (t, J = 6.8 Hz, 2H), 2.85-2.73 (4H), 1.91-1.39 (6H). ¹³C NMR (CDCl₃, 100 MHz), δ 176.58 (COOH), 173.08 (COO), 156.04 (CO), 143.75 (C), 143.66 (C), 141.25 (C), 130.58 (C), 127.75 (CH), 127.69 (CH), 127.07 (CH), 127.03 (CH), 125.01 (CH), 124.95 (CH), 119.99 (CH), 106.99 (CH), 105.23 (CH), 67.04 (CH₂), 66.46 (CH₂), 53.49 (CH), 47.07 (CH), 46.71 (CH), 46.04 (CH₂), 33.18 (CH₂), 31.84 (CH₂), 30.73 (CH₂), 22.40 (CH₂), 21.37 (CH₂). HRMS (FAB) (m/z) calcd for C₄₂H₄₁N₂O₆ ⁺ (MH⁺) 669.2959, found 669.2949.

[00206] 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-{2-[2-(9H-fluoren-9- ylmethoxy carbonyl)-ethyl]-pyrrol-l-yl}-hexanoic acid pentafluorophenyl ester (26). 10 mL freshly distilled CH₂Cl₂ was added to the mixture of pentafluorophenol (50 mg, 0.27mmol), dicyclohexylcarbodiiniide (DCC, 41.5 mg, 0.201 mmol), dimethlamino pyridine (DMAP, 8 mg, 0.067 mmol) and acid 25 (45 mg, 0.067 mmol). The resulting mixture was stirred for 72 h at room temperature. The solvent was removed. Flash chromatography of the residue (15 % ethyl acetate in hexane, TLC: Rf = 0.2) gave low melting white solid 26 (50 mg, 90 %). ¹H NMR (CDCl₃, 200 MHz) (57.75 (dd, J= 6.8, 6.8 Hz, 4H), 7.56 (dd, J= 7.2, 7.2 Hz, 4H), 7.38 (dd, J= 7.2, 7.6 Hz, 4H), 7.29 (4H), 6.58 (dd, J = 3.2, 1.6 Hz, IH), 6.06 (dd, J = 3.2, 3.2 Hz, IH), 5.87 (m, IH), 5.38 (d, J = 8.2 Hz, IH), 4.39-4.45 (5H), 4.17-4.22 (m, 2H), 3.84 (t, J= 7.2 Hz, 2H), 2.86 (t, J= 6.8 Hz, 2H), 2.77 (t, J = 6.8 Hz, 2H), 2.0-1.46 (6H). ¹³C NMR (CDCl₃, 100 MHz), δ 173.14 (COO), 168.96 (COO), 156.09 (COO), 143.93 (C), 143.82 (C), 141.57 (C), 141.51 (C), 130.94 (C), 128.03 (CH), 127.32 (CH), 125.22 (CH), 120.57 (CH), 120.25 (CH), 107.41 (CH), 105.62 (CH), 67.46 (CH₂), 66.73 (CH₂), 53.83 (CH), 47.32 (CH), 46.99 (CH), 46.31 (CH₂), 33.40 (CH₂), 32.18 (CH₂), 30.99 (CH₂), 22.72 (CH₂), 21.66 (CH₂). HRMS (FAB) (m/z) calcd for C₄₈H₄₀F₅N₂O₆ ⁺ (MH⁺) 835.2801, found 835.2813.

Example 8-Estimation Of Amino Groups Using TNBS

[00207] Amino groups of proteins are basic groups (pK values near 8 for α-NH₂ and 9.5 for e-NH₂) and are positively charged except at high pH. Only the uncharged form, that which predominates at pH values higher than their pK, is reactive as a nucleophile. Higher pH thus usually enhances their reactivity with most reagents.

[00208] Trinitrobenzenesulfonic Acid. The reaction of trinitrobenzenesulfonic acid (TNBS) with protein amino groups takes place at pH values near 7 or above, and can be used to study the effect of amino~group substitution and to quantitatively determine amino groups. One of the procedures is essentially that first described by Habeeb, A.F.S.A. (1966): Anal. Biochem., 14, 328.:

[00209] To 1 ml of protein solution (0.6 to 1 mg/ml) is added 1 ml of 4% NaHCO₃ (pH 8.5) and 1 ml of 0.1% TNBS in water. The solution is then incubated at 37-40 ⁰C in the dark for a time sufficient to give the desired extent of reaction. Two hours at 40 ⁰C gives near- quantitative reaction. Lower temperatures give slower and more selective reaction. To quantitate the extent of reaction, 1 ml of 10% sodium dodecyl sulfate should be added to solubilize the protein upon addition of 0.5 ml of 1 M HCl. The absorbance of the solution at 335 to 345 mμ is read against a blank containing 1 ml of water instead of protein solution. An extinction coefficient of 1.4 x 104 M^*1 cm^"1 can be used to calculate the number of amino groups present.

[00210] Another procedure taught by Fields, R. (1972), Methods in Enzymology 25:464-469 is as follows:

[00211] MATERIALS:

[00212] Solution A: lOOmls of 0.1M Na₂SO₃ (fresh each week)

[00213] Solution B: 1.01 of 0.1M NaH₂PO₄

[00214] Solution C: 1.01 of 0.1M Na₂B₄O₇ in 0.1M NaOH (make up in acid and ddH₂O- washed glass).

[00215] Trinitrobenzene sulfonate (TNBS) 1Og in 10ml H₂O, heat to dissolve and remove black flecks of oil by centrifugation. Add HCl to 2M and cool to room temp. Wash the crystalline precipitate on a glass filter with IM HCl. Desiccate and store at 4 ⁰C in brown bottle. Make up to 1.1 M fresh daily (lOOmg recrystallized TNBS in 0.2ml H₂O).

[00216] METHOD:

[00217] 1. make fresh daily Solution D: 1.5ml Solution A + 98.5ml Solution B;

[00218] 2. standard curve: BSA at 0.1, 0.2, 0.5, 1.0, and 2.0 mg/ml in 0.25ml H₂O;

[00219] 3. samples: same concentration range as above in same volume;

[00220] 4. add 0.25ml solution C;

[00221 ] 5. add 1 Oμl TNBS (take note of time!) ;

[00222] 6. Incubate exactly 5 min at 23 ⁰C;

[00223] 7. add ImI solution D to stop reaction;

[00224] 8. measure OD₄₂₀. Standard curve should range from about 0.09 to >1.8. [00225] CALCULATION:

[00226] OD₄₂Q = 1.0 = 52nmol amino groups = 78nmol/l .5ml assay mix;

[00227] "1.0mg/ml" BSA sample (0.25mg) = OD₄₂₀ of 0.945 = 73.41nmol NH_3;

[00228] therefore, 0.25 minol BS A = 73.41 nmol NH₃ groups

[00229] 3.73ranol BSA = 73.41 nmol NH₃

[00230] therefore 1 molecule BSA = 19.8 molecules of lysine.

Claims

1. An Fm masked-CEP-linker acid compound having the general structure:

wherein n is from 1-12.

2. An Fm masked-CEP-linker active ester compound having the general structure:

wherein X is an active ester group.

3. The Fm masked-CEP-linker active ester compound of claim 2, wherein X is , N- hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), l-hydroxy-7-aza- benzotriazole (HOAt), perfluorophenyl (pFP), para-nitrophenyl (pNP), N- hydroxysulfosuccinimidyl (NHSS), sulfotetrafluorophenyl (STP), pentachlorophenyl (pCP), or N-hydroxy-5-norbornene-endo-2,3-dicarboximide ester (HNb).

4. A method of making an Fm masked-CEP-linker acid compound, comprising:

reacting an ω-amino acid with the general formula HOOC(CH₂)_nNH₂, wherein n is from 1 to 12, with a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA) (DOHA-Fm) that has the following structure:

wherein the resultant Fm masked-CEP-linker acid compound has the general structure:

wherein n is from 1-12

5. A method of making an Fm masked-CEP-linker ester compound, comprising:

a. reacting an ω-amino acid with the general formula HOOC(CH₂)_nNH₂, wherein n is from 1 to 12, with a 9-fluorenylmethyl (Fm) ester of 4,7-dioxoheptanoic acid (DOHA) (DOHA-Fm) that has the following structure:

to provide an Fm masked-CEP-linker compound has the general structure:

b. converting the Fm-masked CEP-linker of step (a) into an Fm-masked-CEP-linker ester having the general structure:

where X is an active ester group.

6. The method of claim 5, wherein X is , N-hydroxysuccinimide (NHS), 1- hydroxybenzotriazole (HOBt), l-hydroxy-7-aza-benzotriazole (HOAt), perfluorophenyl (pFP), para-nitrophenyl (pNP), N-hydroxysulfosuccinimidyl (NHSS), sulfotetrafluorophenyl (STP), pentachlorophenyl (pCP), or N-hydroxy-5-norbomene- endo-2,3-dicarboximide ester (HNb).

7. An isolated CEP-linker-carrier derivative having the following structure:

wherein n is from 1 to 12.

8. The isolated CEP-linker-carrier of claim 7, wherein the carrier is a protein comprising a plurality of available lysyl amino groups.

9. The isolated CEP-linker-carrier of claim 7, wherein the carrier is biotin and wherein the CEP-linker-biotin has the following structure:

wherein n is from 1 to 12.

10. The isolated CEP-linker-carrier of claim 7, wherein the carrier is a detectable label comprising one or more primary amine groups and wherein the CEP-linker-label has the following structure:

wherein n is from 1 to 12.

11. The isolated CEP-linker-label of claim 10, wherein the detectable label is a fluorophore, chromophore, chemiluminescent or radioactive moiety.

12. The isolated CEP-linker-label of claim 10, wherein the detectable label is fluorescein.

13. A method of making a CEP-linker-carrier derivative, comprising the steps of:

a. reacting an Fm masked CEP-linker ester having the following structure:

wherein n is from 1 to 12 and X is an active ester group, with a carrier comprising one or more primary amine groups to provide an Fm masked CEP-linker-carrier derivative in which the carrier primary amine groups are linked through an amide bond to the Fm masked CEP-linker ester;

b. removing Fm from the Fm-masked CEP-linker-carrier derivative to provide a CEP- linker-carrier derivative;

wherein the method provides a CEP-linker-carrier derivative having the following structure:

wherein n is from 1 to 12.

14. The method of claim 13, wherein the carrier is a protein.

15. The method of claim 13, wherein the carrier is biotin.

16. The method of claim 13, wherein the carrier is a detectable label comprising one or more primary amine groups.

17. The method of claims 13, wherein X is , N-hydroxysuccinimide (NHS), 1- hydroxybenzotriazole (HOBt), l-hydroxy-7-aza-benzotriazole (HOAt), perfluorophenyl (pFP), para-nitrophenyl (pNP), N-hydroxysulfosuccinimidyl (NHSS), sulfotetrafluorophenyl (STP), pentachlorophenyl (pCP), or N-hydroxy-5-norbornene- endo-2,3-dicarboximide ester (HNb).

18. The method of claim 13, wherein the Fm masked CEP-linker ester compound of step (a) is an active ester derived from an Fm-masked CEP-linker acid having the following structure

wherein n is from 1 to 12.

19. The method of claim 13, wherein step b comprises reacting the Fm-masked CEP- linker-carrier derivative with a base.

20. The method of claim 13, wherein the base is an organic base.

21. The method of claim 13, wherein the base is a secondary amine.

22. The method of claim 13, wherein the base is a tertiary amine.

23. The method of claim 13, wherein the base is a mild base.

24. The method of claim 21, wherein the mild base is selected from a group consisting of: l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU); l,5-Diazabicyclo[4.3.0]non-5-ene (DBN); Morpholine; Dicyclohexylamine; Dimethylaminopyridine; Piperazine; Diisopropylethylamine; Tris(2-aminoethyl)amine (TAEA); Lutidine; Pyrrolidine; Pyridine; Triethylamine; Dimethyamine; Diethylamine; Dipropylamine; Dibutylamine; Hexamethylenimine; 1-Methyl-piperazine; 4-Piperidinemethanol; 4- Piperidinopiperidine; Piperidine; 1-Methylpyrrolidine; 4-Phenylpiperidine; 3- Methylpiperidine; 4-Methylpiperidine; 2,6-Dimethylpiperidine;

Aminomethylpiperidine; Tetrabutylammonium Flouride; cesium, sodium, or potassium carbonate; diisopropyl amine; diisopropyl ethylamine; N-alkyl morpholine and combinations thereof.

25. The method of claim 13, wherein the mild base is inorganic

26. The method of claim 13 wherein the mild base is DBU or morpholine.

27. The method of claim 13, wherein the carrier is a protein and step b comprises reacting the Fm-masked CEP-linker protein derivative with a mild base under conditions that do not modify the protein.

28. The method of claim 27, wherein the conditions do not substantially decrease the solubility of the CEP-linker-protein derivative in an aqueous solution.