Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine

The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.     

Synthesis and stability of 2'-deoxyguanosine 3'-monophosphate adducts of dimethyl sulfate, ethylene oxide and styrene oxide

Deoxyguanosine 3'-monophosphate (dGMP) was alkylated at the 7-position by dimethyl sulfate, ethylene oxide and styrene oxide in aqueous media and glacial acetic acid, respectively, to yield reasonable quantities of the products, which were purified by HPLC. dGMP adducts are needed as standards for the 32P-postlabelling assay. The stability of the adducts was studied at 37 degrees and neutral pH. The half-lives of disappearance of 7-methyl-dGMP and the beta-isomers of the styrene oxide adducts were about 250 min; 7-hydroxy-ethyl-dGMP and the alpha-isomers of the styrene oxide adducts had respective half-lives of 340 and 440 min. In all cases the main degradation product was the corresponding guanine adduct. The results demonstrate considerable lability of the 7-alkylation products of dGMP which has to be taken into consideration in devising the 32P-postlabelling assay.     

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide as a potential bioreductively activated prodrug of phosphoramide mustard

N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were synthesized stereospecifically from Boc-l-Hse(OBn)-OH and the degradation of the corresponding reduced amine 5a was investigated by UV/vis spectroscopy and LC/MS. The rate of cyclization of 5a was found to increase with decreasing pH, with half-lives ranging from 3.2 to 54 min at pH 4–7.4, suggesting that the cyclization is catalyzed by the hydronium ions. LC/MS analysis of the degradation products of 5a indicates that 4-aminocyclophosphamide is rapidly released from 4 upon reductive activation under acidic conditions and further decomposes into the cytotoxic phosphoramide mustard. These results validated 4-aminocyclophosphamide as a prodrug form of phosphoramide mustard and suggest that compound 4 can potentially be used as a prodrug of phosphoramide mustard for bioreductive activation.     

Reactions of propylene oxide with 2'-deoxynucleosides and in vitro with calf thymus DNA

Propylene oxide (PO) is a direct-acting mutagen and rodent carcinogen. We have studied how PO modifies 2'-deoxynucleosides at pH 7.0-7.5 and 37 degrees C for 10 h. PO reacts as an SN2 alkylating agent by forming the following 2-hydroxypropyl (HP) adducts: N6-HP-dAdo (7% yield), 7-HP-Gua (37%) and 3-HP-dThd (4%). Alkylation at N-3 of dCyd resulted in conversion of the adjacent exocyclic imino group at C-4 to an oxygen (hydrolytic deamination) with the formation of a dUrd adduct, 3-HP-dUrd (14%). Ultraviolet spectroscopy and mass spectrometry were used for the structural determination of these adducts. Confirmation of the unexpected 3-HP-dUrd adduct was provided by an accurate mass measurement technique where diagnostic ions in the mass spectra of 3-HP-dUrd were measured to within 0.0005 atomic mass units of the predicted mass. PO was reacted in vitro with calf thymus DNA (pH 7.0-7.5, 37 degrees C, 10 h) and yielded N6-HP-dAdo (1 nmol/mg DNA), 3-HP-Ade (14 nmol/mg DNA), 7-HP-Gua (133 nmol/mg DNA) and 3-HP-dUrd (13 nmol/mg DNA). A mechanism for the hydrolytic deamination of 3-HP-dCyd to 3-HP-dUrd involving the OH on the HP side chain is proposed. This cytosine to uracil conversion may play a role in the mutagenic and carcinogenic activity of this epoxide.     

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)glutathione. Evidence for a role of S-(2-N7-guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide.

The major DNA adduct (greater than 95% total) resulting from the bioactivation of ethylene dibromide by conjugation with GSH is S-(2-(N7-guanyl)ethyl)GSH. The mutagenic potential of this adduct has been uncertain, however, because the observed mutagenicity might be caused by other adducts present at much lower levels, e.g. S-(2-N1-adenyl)ethyl)GSH. To assess the formation of other potential adducts, S-(2-(N3-deoxycytidyl)ethyl)GSH, S-(2-(O6-deoxyguanosyl)ethyl)GSH, and S-(2-(N2-deoxyguanosyl)ethyl)GSH were prepared and used as standards in the analysis of calf thymus DNA modified by treatment with [1,2-14C]ethylene dibromide and GSH in the presence of rat liver cytosol; only minor amounts (less than 0.2%) were found. A forward mutation assay in (repair-deficient) Salmonella typhimurium TA100 and sequence analysis were utilized to determine the type, site, and frequency of mutations in a portion of the lacZ gene resulting from in vitro modification of bacteriophage M13mp18 DNA with S-(2-chloroethyl)GSH, an analog of the ethylene dibromide-GSH conjugate. An adduct level of approximately 8 nmol (mg DNA)-1 resulted in a 10-fold increase in mutation frequency relative to the spontaneous level. The spectrum of spontaneous mutations was quite varied, but the spectrum of S-(2-chloroethyl)GSH-induced mutations consisted primarily of base substitutions of which G:C to A:T transitions accounted for 75% (70% of the total mutations). All available evidence implicates S-(2-(N7-guanyl)ethyl)GSH as the cause of these mutations inasmuch as the levels of the minor adducts are not consistent with the mutation frequency observed in this system. The sequence selectivity of alkylation was determined by treatment of end-labeled lac DNA fragments with S-(2-chloroethyl)GSH, cleavage of the DNA at adduct sites, and electrophoretic analysis. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. We suggest that the mechanism of mutagenesis involves DNA sequence-dependent alterations in the interaction of the polymerase with the (modified) template and incoming nucleotide.     

Synthesis of aromatic nitrogen mustard agents and analysis of their alkylation activity at physiological pH and temperature

A Structure Activity Relationship (SAR) study was accomplished with six aromatic compounds which have a nitrogen mustard (N-mustard) substituent. N-mustard agents are very important for the clinical treatment of many types of cancers. All N-mustard agents synthesized alkylated a nucleophilic primary amine (p-chloroaniline) in aqueous solvent at pH 7.4 and 37 degrees C. Rate constants and rate equations were determined for the alkylation reactions by monitoring the formation of a fluorescent complex formed when fluorescamine complexes the unreacted p-chloroaniline. Fluorescamine complexation of the unreacted primary amine halts the alkylation reaction and allows the determination of remaining unreacted primary amine, which in turn permits the determination of rate constants and rate equations. The fluorescamine-amine complex shows a strong absorbance peak at a wavelength of 400 nanometers in aqueous solvent. The molar absorptivity (epsilon) was calculated to be 18.37 L/(mole x cm). First order rate constants ranged from 0.513E-2 minute(-1) to 1.32E-2 minute(-1) with second order rate constants from 2.85E-2 (M x minute)(-1) to 4.78E-2 (M x minute)(-1). The aromatic compounds included benzoic acid, m-chlorobenzoic acid, hydrocinnamic acid, m-toluic acid, and 3,4-dimethoxybenzoic acid. The synthesis procedure produced the N-mustard agents at > or = 90% yield and high purity. Partition coefficient Log(Kow)Log P values ranged from 2.66 to 4.18. The N-mustard agents consisted of greyish-yellow crystals which retained alkylation activity for more than ten weeks when stored at -10 degrees C and were soluble in water at 25 degrees C and 37 degrees C.     

Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N₇ guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N₇ position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N₇ guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 μM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3′-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.     

Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells

Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.     

Characterization of the carbamino adducts of insulin.

Carbon-13 (13C) nuclear magnetic resonance spectroscopy (NMR) is performed to characterize the formation of carbamino adducts between insulin and (13C) carbon dioxide over a range of pH values in the presence of a physiological concentration (23 mM) of sodium bicarbonate. The peaks from two of the carbamino adducts resonate at higher frequencies than the signal from bicarbonate, at 164.6 and 165.3 ppm, and are attributed to the adducts with the terminal amino groups of phenylalanine B1 and glycine A1. The intensities of these signals vary with the pH, with unique patterns. Over 6% of each terminal amino group exists as the carbamino adduct at the optimum pH values of 7.8 and 8.3. A unique third adduct resonates at 159.3 ppm, and is attributed to lysine B29. This adduct is present on 2% of the insulin molecules at pH 8.2, but has minimal intensity at pH 7.4. No signals from adducts are detected below pH 6.2, where the amino groups exist predominantly in the protonated form. Creation of the adducts is rapid and they are stable for over 4 wk at 37 degrees C. The narrow bandwidth of the resonance of the adduct (4.0-4.5 Hz) relative to the irreversible cyanate adduct is consistent with molecular forms of the carbamino adduct smaller than the 2-Zn-hexamer which is the preponderate form of clinically utilized U-100 insulin (i.e., 100 U/ml).     

Styrene oxide-induced 2'-deoxycytidine adducts: implications for the mutagenicity of styrene oxide

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.4 h for the alpha- and beta-isomers, respectively. The N(4)-alkylated products were stable at neutral pH. The O(2)-alkylated products were unstable being prone to depyrimidation and to isomerisation between alpha- and beta-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysis allowed the identification of the beta3-deoxyuridine and alphaN(4)-deoxycytidine adducts (1.9 and 0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts. The 3-substituted uracil may have implications for the mutagenicity of SO.     

Gas chromatographic-mass spectrometric assay for N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in rat plasma

A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml (n=6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% (n=6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n=6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37°C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37°C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37°C. The generated CEA was very stabble in the culture media with a degradation half-life of 265 h.     

Structure of the adduct of 16 alpha-hydroxyestrone with a primary amine: evidence for the Heyns rearrangement of steroidal D-ring alpha-hydroxyimines.

16 alpha-Hydroxyestrone, a product of estrogen 16 alpha-hydroxylation in humans that is suspected to be implicated in cell transformation, has been found to form stable adducts with nuclear components. The stable covalent adduct formed from 16 alpha-hydroxyestrone with 2-methoxyethylamine via the Heyns rearrangement of the alpha-hydroxyimine was identified as 3-hydroxy-17 beta-(2-methoxyethylamino)estra-1,3,5(10)-trien-16-one. Since the same product was obtained from 16 beta-hydroxyestrone with the amine, the alpha-hydroxyenamine is the most likely intermediate of the Heyns rearrangement. The adduct was fairly stable at 37 C in phosphate buffer (pH 7.4)/methanol (1:1 v/v), while the adduct formed from 16-oxoestradiol was disrupted reversely and completely within 6 hours. The evidence suggests that N-(3-hydroxy-16-oxoestra-1,3,5(10-trien-17 beta-yl)amine is the partial structure of the stable adducts formed from D-ring alpha-ketol estrogens with proteins.     

The reaction of melphalan with deoxyguanosine and deoxyguanylic acid.

The reaction of melphalan (phenylalanine mustard, I) with 2'-deoxyguanosine, followed by removal of the sugar in acid, yielded two products. The major product was identified as 4-(N-(2-guanin-7-ylethyl)-N-(2-hydroxyethyl)amino)phenyl- alanine (II) by ultra-violet absorption, mass and NMR spectroscopy. The minor product has already been identified as the corresponding bis-guaninyl adduct III (Tilby et al., Chem.-Biol. Interact., 73 (1990) 183-194). The reaction of melphalan with 5'-deoxyguanylic acid yielded the deoxyribonucleotide of II and products resulting from reaction with the phosphate group. The initial products, which were formed with a half-life of approximately 40 min at 37 degrees, still had a reactive chloroethyl group; this was displaced more slowly, by reaction with water or with another molecule of dGMP. The products of reaction of melphalan with DNA were released by treatment with acid (0.1 M HCl, 70 degrees, 30 min) and separated from each other on a cation exchange column. They were identified as II, III and an adenine adduct, in a ratio of approximately 3:1:2.     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

Formation of a phospholipid-linked pyrrolecarbaldehyde from model reactions of D-glucose and 3-deoxyglucosone with phosphatidyl ethanolamine

Phospholipid-linked 'advanced glycation end products' (AGEs) are supposed to play an important role for lipid oxidation in vivo. The identification of the pyrrolecarbaldehyde 1-[2-formyl-5-(hydroxymethyl)-1 H-pyrrol-1-yl]-4,10-dioxo-7-(tetradecanoyloxy)-3,5,9-trioxa- 4lambda5-phosphatricosan-4-olate (7) from model reactions of D-glucose or 3-deoxyglucosone (4, 3-DG) with phosphatidyl ethanolamine (PE) is described. A preparation method is given for 1-(2-hydrox?ethyl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (8). Independent syntheses as well as unequivocal structural characterization are reported for the substitution products of 8 1-(2-hydroxyethyl)-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (9a) and 5-(ethoxymethyl)-1-(2-hydroxyethyl)-1H-pyrrole-2-carbaldehyde (9b). For all these compounds, chromatographic and spectroscopic data were established by GLC-MS and HPLC with diode array detection (DAD). PE and D-glucose or 3-DG 4 were either incubated at pH 7.4, 100 degrees C for 3 h or at pH 7.4, 37 degrees C for 5 weeks in neat buffer or ethanol buffer mixtures. The phospholipid fraction was purified on a C18 solid-phase extraction column and cleaved with ethanolic potassium hydroxide. The carbaldehyde 8, released in this process, was identified bs GLC-MS and quantified by HPLC-DAD. Formation of 7 is favored in the ethanol buffer reactions relative to those in buffer solution only although the amounts determined from the 37 degrees C incubations generally are very low. It seems likely, therefore, that phospholipid-linked pyrrolecarbaldehydes, such as 7, are biomarkers rather than effectors of membrane damage in vivo.     

Peptide conjugates of 4-aminocyclophosphamide as prodrugs of phosphoramide mustard for selective activation by prostate-specific antigen (PSA)

In our continued effort to develop prodrugs of phosphoramide mustard, conjugates of 4-aminocyclophosphamide (4-NH₂-CPA) with three PSA-specific peptides were synthesized and evaluated as substrates of PSA. These include conjugates of cis-(2R,4R)-4-NH₂-CPA with a tetrapeptide Succinyl-Ser-Lys-Leu-Gln-OH, a hexapeptide Succinyl-His-Ser-Ser-Lys-Leu-Gln-OH, and a pentapeptide Glutaryl-Hyp-Ala-Ser-Chg-Gln-OH. These conjugates were cleaved by PSA efficiently and exclusively after the expected glutamine residue to release 4-NH₂-CPA, the activated prodrug form of phosphoramide mustard. The cleavage was most efficient for the pentapeptide conjugate 3 (Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-CPA), which showed a half-life of 55 min with PSA, followed by the hexapeptide conjugate 2 (Succinyl-His-Ser-Ser-Lys-Leu-Gln-NH-CPA) and the tertrapeptide conjugate 1 (Succinyl-Ser-Lys-Leu-Gln-NH-CPA) with half-lives of 6.5 and 12 h, respectively. These results indicate a potential of the conjugate 3 as an anticancer prodrug of phosphoramide mustard for selective PSA activation.     

Phenylalanyl-aminocyclophosphamides as model prodrugs for proteolytic activation: synthesis, stability, and stereochemical requirements for enzymatic cleavage

4-Aminocyclophosphamide (4-NH2-CPA, 7) was proposed as a prodrug moiety of phosphoramide mustard. Four diastereomers of phenylalanine-conjugates of 4-NH2-CPA were synthesized and their stereochemistry was assigned based on chromatographic and spectroscopic data. All diastereomers were stable in phosphate buffer but only the cis-(4R)-isomer of 15 was efficiently cleaved by alpha-chymotrypsin with a half-life of 20 min, which is much shorter than the 8.9h to >12h half-lives found for the other diastereomers. LC-MS analysis of the proteolytic products of cis-(4R)-15 indicated that 4-NH2-CPA was released upon proteolysis and further disintegrated to phosphoramide mustard. These results suggest the feasibility of using peptide-conjugated cis-(4R)-4-NH2-CPA as potential prodrugs for proteolytic activation in tumor tissues.