Preparation and characterization of paramagnetic polychelates and their protein conjugates

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).     

Total solid-phase synthesis of 1,4,7,10-tetraazacyclododecane-N,N', N'',N'''-tetraacetic acid-functionalized peptides for radioimmunotherapy

A convenient approach to the functionalization of peptides with the macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) moiety has been developed. Protected components (using tert-butyl or tert-butyloxycarbonyl groups) of both the peptide and the chelate were assembled on the same solid resin support. Deprotection and cleavage of the resin-bound DOTA-peptides were performed in one step using a trifluoroacetic acid cleavage mixture to yield free DOTA-peptide amides.     

Improved synthesis of the bifunctional chelating agent 1,4,7,10-tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N',N'',N'''-tri s(acetic acid)cyclododecane (PA-DOTA)

A concise synthesis of the bifunctional chelating agent 1,4,7,10-tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N',N',N' '-tris(acetic acid)cyclododecane (PA-DOTA) is reported. Difficulties involving the production of partially alkylated products and their removal have been addressed and obviated. After the pure nitro form of PA-DOTA was obtained, conversion to the isothiocyanato form PA-DOTA (1, conjugation to HuCC49 and HuCC49deltaCH2 monoclonal antibodies was achieved. Subsequent radiolabeling with 177Lu was performed, demonstrating a useful bifunctional chelating agent suitable for clinical radioimmunotherapy applications.     

Radiometal-labeled peptide-PNA conjugates for targeting bcl-2 expression: preparation,characterization, and in vitro mRNA binding

A new antisense peptide-peptide nucleic acid (peptide-PNA) conjugate, designed for targeting bcl-2 expression, has been radiolabeled, characterized, and evaluated for bcl-2 mRNA binding in a cell-free system. A PNA complementary to the first six codons of the bcl-2 gene was synthesized by standard solid-phase Fmoc chemistry and conjugated to a new derivative of 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) that allows macrocyclic radiometal chelates to be incorporated into any sequence position of a peptide-PNA conjugate. The DOTA-PNA conjugate was then coupled to a membrane-permeating transduction peptide, PTD-4, designed for intracellular delivery of the radiolabeled PNA. The conjugate was characterized by HPLC and ESI-MS and labeled with (111)In and (90)Y to high specific activities (>1000 Ci/mmol) with high radiochemical purity. Northern blot analysis showed that (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA bound specifically to as little as 50 fmol of bcl-2 mRNA, a result equivalent to that obtained with the analogous (32)P-labeled DNA antisense oligonucleotide. Thus, the mRNA targeting properties of (111)In- and (90)Y-PTD-4-K(DOTA)-anti-bcl-2-PNA demonstrate potential for diagnostic imaging and targeted radiotherapy applications in bcl-2-positive cancers.     

An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.     

Evaluation of 64Cu- and 125I-radiolabeled bitistatin as potential agents for targeting alpha v beta 3 integrins in tumor angiogenesis

The formation of new blood vessels (angiogenesis) is a feature common to all solid tumors. The integrin receptor alpha(V)beta(3), which is found on endothelial cells lining newly growing blood vessels at a higher density than on mature blood vessels, is being explored as a marker for tumor angiogenesis. Bitistatin, a member of the disintegrin family of polypeptides, has affinity for alpha(V)beta(3) integrins. To determine whether radiolabeled bitistatin could target tumors, its biodistribution was tested in tumor-bearing mice. For initial validation studies, (125)I-bitistatin was injected into BALB/c mice bearing EMT-6 mouse mammary carcinoma tumors, a model that is highly vascular but which lacks alpha(V)beta(3) directly on tumor cells. Tumor uptake reached maximal values (11.7 +/- 4.6 %ID/g) at 2 h. Co-injection of 200 microg of unlabeled bitistatin reduced tumor uptake 62%, suggesting that the binding of (125)I-bitistatin is receptor-mediated. This work was extended to include the beta(+)-emitting radionuclide (64)Cu, which was attached to bitistatin via 1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (DOTA). This modification did not significantly alter receptor binding in vitro. MicroPET images obtained with (64)Cu-DOTA-bitistatin showed that the tumor could easily be identified 4 h after administering the radiopharmaceutical. The biodistribution of (64)Cu-DOTA-bitistatin differed from the (125)I analogue, in that maximum tumor uptake was nearly 8-fold lower and took at least 6 h to reach maximal binding (1.6 +/- 0.2 %ID/g). As with (125)I-labeled bitistatin, the (64)Cu conjugate showed a 50% reduction in tumor uptake with the co-injection of 200 microg of unlabeled bitistatin (0.8 +/- 0.2 %ID/g). Competition studies with integrin-specific peptides indicated that the tumor uptake was related to both alpha(v)beta(3) and alpha(IIb)beta(3) integrin binding. To see if tumor uptake could be improved upon, (64)Cu was tethered to bitistatin using bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (BAD). Tumor uptake for (64)Cu-BAD-2IT-bitistatin was higher than the DOTA conjugate at all time points, reaching a maximum at least 6 h postinjection (5.2 +/- 0.6 %ID/g); however, this was accompanied by higher uptake in nontarget organs at all time points. Radiolabeled ligands of this type may be useful in the targeting of tumor angiogenesis, but the choice of radiolabeling approach has a significant impact on the in vivo properties of the radioligand.     

Stability assessment of different chelating moieties used for elemental labeling of bio-molecules

Integrating elemental labeling in quantitative LC-ICP-MS based bio-analysis requires fundamental experiments concerning the stability of complexes during analysis. In a competitive approach complex stability of the chelating moieties 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraaceticacid (DOTA), 1,4,7-triazacyclononane-N,N',N'-triacetic acid (NOTA) and diethylenetriaminepentaacetic dianhydride (DTPA) in combination with 11 different lanthanides was investigated under typical chromatographic conditions. Measurements were carried out via LC-ICP-QMS using a novel mixed mode separation method. The influence of chromatographic separation, pH and temperature on complex stability constants was assessed regarding further applications of multiplexing in bio-analytical assays. The limit of detection (LOD) for LC-ICP-QMS was 0.03 nM for all investigated Tm complexes (0.15 fmol absolute). Quantification of the complexes was performed via external, flow injection based calibration. For all investigated complexes the stability was significantly decreased by the chromatographic conditions. Moreover, complexation by DOTA revealed two different signals suggesting the presence of a stable intermediate product. Ln(3+)-DOTA and Ln(3+)-NOTA complexes provided high stability at 5 °C and 37 °C over a time of 12 hours, whereas Ln(3+)-DTPA complexes showed significant degradation at 37 °C.     

Element-coded affinity tags for peptides and proteins

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.     

90Y-DOTA-D-Phe1-Try3-octreotide in therapy of neuroendocrine malignancies

Somatostatin receptors type 2 (sst(2)) are expressed in high concentration on numerous neudoendocrine tumors. The successful use of radiolabeled somatostatin analogs in imaging promoted further studies in utilizing them in radiopeptide therapy. The somatostatin analog [(90)Y-DOTA-D-Phe(1)-Try3]octreotide (DOTATOC) (DOTA: 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid) possesses favorable characteristic for its therapeutic use; shows high affinity for sst(2), moderately high affinity for sst(5), and intermediate affinity for sst(3); high hydrophilicity; stable and facile labeling with (111) In and (90) Y. In this article we report our experience with (90)Y-DOTATOC in neuroendocrine tumors. Eighty-seven patients with neuroendocrine tumors were treated with a cumulated activity ranging from 7.4 to 20.2 GBq. Most patients responded with stabilization of disease (48%); however, objective responses were observed in 28% of patients (5% complete response). No major acute reactions were observed up to the activity of 5.55 GBq per cycle. The dose limiting was bone marrow toxicity and the maximal tolerated dose was defined as 5.18 GBq.     

Synthesis and evaluation of two new bifunctional carboxymethylated tetraazamacrocyclic chelating agents for protein labeling with indium-111

The synthesis of two new N- and C-functionalized tetraazamacrocyclic ligands intended to be covalently linked to biomolecules like monoclonal antibodies and to bind the gamma-emitting isotope indium-111 in a thermodynamically and/or kinetically inert way is described. 12-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraa cetic acid (L1) was synthesized by means of bimolecular cyclization with the appropriate malonic acid diethyl ester and triethylenetetraamine, followed by reduction with diborane and alkylation of the cyclic tetraamine with bromoacetic acid. The corresponding triscarboxymethylated ligand L2 was made by statistical alkylation of the tetraamine. Both ligands fulfill the criteria for antibody labeling using the bifunctional chelate approach, namely fast chelate formation, high radiochemical yield, and high stability under physiological conditions. Surprisingly the heptadentate ligand L2 confers higher stability to In3+ and exhibits faster complex formation than octadentate L1. 13C NMR spectra in solution indicate that the difference in stability is not due to incomplete coordination of all four carboxylate groups in In-L1.     

DOTA derivatives for site-specific biomolecule-modification via click chemistry: synthesis and comparison of reaction characteristics

Due to the high stability of its complexes with many M(2+) and M(3+)-ions, DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N?-tetraacetic acid) is the most commonly used chelator for the derivatization and radiolabeling of bioactive molecules. Most of the currently used DOTA derivatives comprise amine-reactive functionalities, limiting their application to the derivatization of fully protected molecules or otherwise resulting in randomly distributed conjugation sites of undefined number. Click chemistry reactions are a valuable alternative to this unspecific conjugation as they proceed efficiently and chemoselectively under mild conditions allowing a site-specific derivatization of unprotected biomolecules. In this work, we describe straightforward syntheses of DOTA derivatives containing thiol, maleimide, aminooxy, aldehyde, alkyne, and azide functionalities, amenable to the currently most often used click chemistry reactions. Furthermore, the efficiency of the respective click reactions introducing DOTA into bioactive molecules was investigated. For each of the synthesized DOTA synthons, the site-specific and efficient conjugation to Tyr(3)-octreotate could be shown. Among these, the addition and oxime formation reactions proceeded fast and without side reactions, giving the products in high yields of 64-83% after purification. The copper-catalyzed triazole formation reactions produced some side-products, giving the desired products in lower, but still reasonable overall yields of 19-25%. All synthesized peptide-DOTA-conjugates were labeled with (68)Ga in high radiochemical yields of 96-99% and high specific activities providing compounds of high purity, demonstrating the applicability of all synthons for biomolecule modification and subsequent radiolabeling.     

Simplified method for conjugating macrocyclic bifunctional chelating agents to antibodies via 2-iminothiolane

A one-step method for conjugating macrocyclic chelators to antibodies using the protein modification reagent 2-iminothiolane controls aggregation, maintains immunoreactivity, and produces consistent chelate/antibody ratios. Conjugation conditions have been investigated with the macrocyclic chelates 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N ',N",N"'-tetraacetic acid and 2-[p-(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N',N ",N"'-tetraacetic acid, with three different monoclonal antibodies. The bifunctional chelating agents are prepared by bromoacetylation of their amine precursors using a two-phase H2O/CHCl3 system, which improves product purity.     

A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaacetic acid and their coordination chemistry with yttrium(III)

The bifunctional chelating agents N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)methyl]-diethylenetriamine and N,N,N',N',N'-pentakis(carboxymethyl)-1-[(4-aminophenyl)methyl]-4- methyldiethylenetriamine were prepared in six-step syntheses in overall yields of 38% and 31%, respectively. The use of bromoacetate esters in the synthesis allowed large-scale flash chromatographic purification of reaction products. The synthesis of N,N,N',N',N'-pentakis(carboxymethyl)-1- [(4-aminophenyl)-methyl]-4-methyldiethylenetriamine resulted in a mixture of two diastereomers. Chelation of yttrium-(III) with these bifunctional chelating agents resulted in 1:1 chelates. In the case of N,N,N',N',N'-pentakis(carboxymethyl)-1-[4- aminophenyl)methyl]diethylenetriamine, two diastereomers were observed upon chelation, as expected. In the case of N,N,N',N',N'- pentakis(carboxymethyl)-1-[(4-aminophenyl)-methyl]-4- methyldiethylenetriamine, only three of the four anticipated diastereomers were observed.     

Synthesis of a novel 'smart' bifunctional chelating agent 1-(2-[beta,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and its Gd(III) complex

A new synthetic pathway to 1-(2-[beta,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and 1-(2-[beta,D-galactopyranosyloxy]ethyl)-4,7,10-tris(carboxymethyl)-1, 4,7,10-tetraazacyclododecane (Gal-DO3A) chelating agents was developed involving full hydroxyl- and carboxyl-group protection in precursors to product. Two sequences of cyclen-N-functionalisation were subsequently investigated, one successfully, towards synthesis of the novel 'smart' bifunctional Gal-PA-DO3A-NH2 chelate. The longitudinal proton relaxivities of the neutral [Gd-(Gal-PA-DO3A-NH2)] and [Gd-(Gal-DO3A)] complexes were increased by 28% and 37% in the presence of beta-galactosidase, respectively.     

Synthesis of aminooxy-functionalized lanthanide(III) chelates for carbonyl-group conjugation

The syntheses of three new aminooxy-tethered lanthanide(III) chelates, compounds 1-3, incorporating DOTA (= 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA (= diethylenetriaminepentaacetic acid), or a substituted terpyridine (2,2',2',2'-[2,2': 6',2'-terpyridine-6,6'-diylbis(methylenenitrilo)]tetraacetic acid), respectively, are described. Reagents 1-3 can be used for carbonyl 'labeling', as shown by the formation of the corresponding oxime-ether bioconjugates of naltrexone (16) and 2-deoxy-beta-D-glucose (17) (Scheme 4).     

Conjugation of DOTA using isolated phenolic active esters: the labeling and biodistribution of albumin as blood pool marker

A convenient method for the functionalization of proteins with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) has been developed. For this purpose DOTA was converted into a series of different monoreactive activated phenolic esters. The esters were prepared in a single step from commercially available DOTA, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide or 1,3-dicyclohexylcarbodiimide as coupling agent. The resulting activated esters were isolated by HPLC, lyophilized, and stored for future applications. In solid form the compounds exhibit high hydrolytic stability. The reactions with proteins proceeded in good yields. The conjugation and subsequent radiolabeling of the 4-nitrophenol ester of DOTA with 67Ga was investigated with rat serum albumin. A time-dependent biodistribution study in tumor bearing rats was conducted to demonstrate the integrity of the albumin conjugate. These results suggest that phenolic esters of DOTA represent versatile reagents to conjugate DOTA with proteins and other biomolecules in high yields.     

Functional and structural study on chelator-induced suppression of S2/S1 FTIR spectrum in photosynthetic oxygen-evolving complex

Chelating agents have been shown to induce characteristic changes in the light-minus-dark Fourier transform infrared (FTIR) difference spectrum for the S(2)/S(1) difference in the oxygen-evolving complex (OEC). Addition of various ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA)-type chelators, such as EDTA, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), to Ca(2+)-depleted PS II membranes resulted in the suppression of typical S(2)/S(1) vibrational features, including the symmetric (1365(+)/1404(-) cm(-1)) and the asymmetric (1587(+)/1566(-) cm(-1)) carboxylate stretching vibrations, as well as the amide I and II modes of the backbone polypeptides. In contrast, the addition of ethylenediamine-N,N'-diacetic acid (EDDA) showed less inhibitory effects. The effects of the chelators depended on the number of the carboxylate groups; chelators with more than two carboxymethyl groups were effective in altering the FTIR spectrum. The bridging structure that connects the two nitrogen atoms also influenced the inhibitory effects. However, the effects were not necessarily correlated with the stability constants of the chelators to Mn(2+). The vibrational modes that were suppressed by EDTA were almost completely restored by subsequent washing with Chelex-treated Ca(2+)-free buffer medium, indicating that the spectral changes are attributable to the reversible association of chelators with the Ca(2+)-depleted OEC. Nevertheless, prolonged incubation with chelators led to the impairment of the O(2)-evolving capability, with differences in the effectiveness, in the order that is consistent with that for the suppression effects on FTIR spectra. Chelators with carboxylate and/or carboxymethyl groups bound to a single nitrogen [nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)] or carbon (citric acid) were relatively ineffective for the suppression. A chelator that includes four phosphate groups, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic) acid (EDTPO), also showed suppression effects on both the carboxylate and amide modes. Based on these findings, a possible mode of interaction between the chelators and the Mn cluster is discussed.     

Novel gadolinium(III) polyaminocarboxylate macrocyclic complexes as potential magnetic resonance imaging contrast agents

Two novel Gd(III) complexes with functionalised polyaminocarboxylate macrocycles, 1,4,7-tris(carboxymethyl)-9,24-dioxo-14,19-dioxa-1,4,7,10,23- pentaazacyclododecane (L(1)) and 1,4,7-tris(carboxymethyl)-9,25-dioxo-14,17,20-trioxa-1,4,7,10,23- pentaazacyclotridecane (L(2)), were prepared in good yield. Their potential use as magnetic resonance imaging (MRI) contrast agents (CAs) was evaluated by investigating their relaxation behaviour as a function of pH, temperature and magnetic field strength. The 1/T(1) proton relaxivities at 20 MHz and 25 degrees C of GdL(1) (5.87 mM(-1) s(-1)) and GdL(2) (6.14 mM(-1) s(-1)) were found to be significantly higher than the clinically used Gd 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd(DOTA)(-)) and Gd diethylenetriaminepentaethanoic acid (Gd(DTPA)(2-)). The complexes possess one water molecule in the inner coordination sphere whose mean residence lifetime was estimated to be 1.1 and 1.5 micros at 25 degrees C by variable temperature (VT) (17)O NMR spectroscopy.     

Large-scale synthesis of the bifunctional chelating agent 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetr aacetic acid, and the determination of its enantiomeric purity by chiral chromatography.

The attachment of radiometals to monoclonal antibodies for medical applications requires extreme stability under physiological conditions, with no significant release of metal. Chelators that can hold radiometals like 111In, 67Ga, and 90Y with high stability under these conditions are essential for radiotherapy or immunoscintigraphy. 2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane- N,N',N',N'-tetraacetic acid (nitrobenzyl-DOTA) is one of the most promising bifunctional chelating agents. A large-scale synthesis of nitrobenzyl-DOTA is described. The overall yield for the nine-step synthesis sequence starting from nitrophenylalanine is 5.6%. Synthesis of nitrobenzyl-DOTA according to the new procedure yields up to approximately 10 g without special apparatus. Both enantiomers of the chiral chelate nitrobenzyl-DOTA have been prepared, and their enantiomeric purity has been checked by chiral chromatography.     

Amide and ester derivatives of N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid: the selective inhibitor of glucosamine-6-phosphate synthase

Several amide and ester derivatives of a glutamine analogue, N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) (1-8), were synthesized and evaluated for the inhibitory activity in regard to glucosamine-6-phosphate synthase from Candida albicans. The syntheses were accomplished by the reaction of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (BocFMDP) with the corresponding amines to give the FMDP amides (1-4) or with alkyl halides to give corresponding esters of FMDP (5-8). Among the synthesized compounds, the acetoxymethyl ester of FMDP was the most active inhibitor of the enzyme. Its IC50 value compared to that of FMDP (4 microM) was equal to 11.5 microM. The methyl and allyl esters and the N-hexyl-N-methyl-amide of FMDP exhibited a moderate enzyme inhibitory activity.