Tetrafluorophenyl Ester Research Articles

A novel borinate ester copolymer for Poly(Frustrated Lewis pair) gels

The versatile chemistry of boronic acid and boronate ester containing polymers has led to key applications in drug delivery, chemical sensing, and dynamic materials. However, their use in polymeric Frustrated Lewis pairs (FLP) remains unreported. In this paper we report the synthesis of a novel fluorinated borinate ester copolymer which, when combined with a suitable Lewis basic copolymer, generates polymeric FLP networks capable of responsive gelation. The borinate ester moiety, bis(pentafluorophenyl)borinic acid tetrafluorophenyl ester, was affixed to a polystyrene copolymer by a two-step post-polymerisation modification, which can be easily upscaled and purified. The Gutmann-Beckett method suggests these fluorinated borinate ester moieties are amongst the most Lewis acidic boron-containing polymers reported to date. Upon mixing with a Lewis basic copolymer poly(styrene-co-p-diphenylphosphino styrene), gelation cannot be achieved due to steric hindrance around the Lewis centres. However, network formation can be triggered by addition of small molecules such as diethylazodicarboxylate (DEAD) and cyclic ethers. The physical parameters and nature of the crosslinks of the gels prepared from different crosslinkers were probed by rheology. All networks demonstrated strong elastic behaviour, corresponding to covalently crosslinked materials. DEAD crosslinked gels displayed different behaviour to those triggered by cyclic ethers, indicating that mechanism of small molecule activation also plays an important role in the mechanical properties of poly(FLP) gels.

Investigation of Monocarbon Carboranes as Pendant Groups for Labeling Small Molecules with Astatine-211

The dianionic boron cage molecule closo-decaborate(2-) is being used as a pendant group to label monoclonal antibodies with the alpha-emitting radionuclide, astatine-211 (211At). While this works very well for antibody labeling, it appears that application of the same moiety for labeling small molecules can significantly alter blood clearance of biomolecule conjugates. As an example, in a murine model an 211At-labeled biotin derivative used in a cancer pretargeting approach clears the blood so rapidly that it fails to reach the antibodystreptavidin conjugate bound with tumor cells. Our hypothesis is that the dianionic nature of the astatinated pendant group facilitates rapid excretion of the biotin conjugate. Therefore we are investigating the use of another boron cage moiety, a monocarbon carborane, closo-1carba-nonadecaborate(1-) {referred to as closo-1-CB9H10} as an alternative 211At-labeling moiety. Two closo-CB9H10(1-) derivatives, 1-carboxyl-1-CB9H9, 1, and 1-p-benzoate-1-CB9H9, 2, were prepared following literature reports (Chem. Commun. 328-329,2004 & Dalton Trans. 3552-3561, 2004). Tetrafluorophenyl esters of the carboxylates in 1 & 2 were prepared and were reacted with an amine in a biotin-sarcosine derivative to provide biotin derivatives that contain the two anionic closo-CB9H9(1-) moieties. Electrophilic astatination reactions of the derivatives containing closo-CB9H9(1-) were found to be low yielding, even at elevated temperatures. To facilitate the astatination reactions, 10-substituted phenyl iodonium salts of 1 & 2, compounds 3 & 4, were prepared and astatination of those derivatives were evaluated using [211At]sodium astatide. While astatination occurred (up to 50%), it was noted that a new less lipophilic product was formed in the reaction, particularly at elevated temperatures. The labeling reactions of 1-p-benzoate-10-phenyl-iodonium-closo-CB9H8, 4, had fewer side products than 3, so that reagent was selected for our subsequent studies. Reactions conducted under the same conditions, but without 211At present, provided an opportunity to isolate and characterize the reaction side product. NMR and mass spectral data supported characterization of the compound as the 10-hydroxyl-1-p-benzoate-closoCB9H8, 5. Compound 5 is readily prepared from 4. Electrophilic astatination of 5 provided a single compound in ∼80% radiochemical yield, making it an attractive pendant group for small molecule labeling of 211At. Astatination studies are continuing with compounds 2, 4 and 5 to optimize the radiochemical yields, as are astatinations of biotin derivatives containing these moieties. Tissue biodistribution studies are planned to determine the in vivo stability of the 211At-labeled compounds. It should be noted that the use of the phenyliodonium intermediate allows introduction of 211At under nucleophilic non-oxidizing conditions, so this labeling method can be used for 211At-labeling of compounds that are sensitive to oxidizing (electrophilic) conditions.

One-pot synthesis and biodistribution of fluorine-18 labeled serum albumin for vascular imaging

IntroductionEquilibrium single-photon radionuclide imaging methods for assessing cardiac function and the integrity of the vascular system have long been in use for both clinical and research purposes. However, positron-emitting blood pool agents that could provide PET equivalents to these (and other) clinical procedures have not yet been adopted despite technical imaging advantages offered by PET. Our goal was to develop a PET blood pool tracer that not only meets necessary in vivo biological requirements but can be produced with an uncomplicated and rapid synthesis method which would facilitate clinical translation. Herein, albumin labeled with fluorine-18 was synthesized using a one-pot method and evaluated in vitro and in vivo in rats. MethodsA ligand (NODA-Bz-TFPE), containing NODA attached to a tetrafluorophenylester (TFPE) via a phenyl linker (Bz), was labeled with aluminum fluoride (Al[18F]F). Conjugation of the serum albumin with the ligand (Al[18F]F-NODA-Bz-TFPE), followed by purification (size exclusion chromatography), yielded the final product (Al[18F]F-NODA-Bz-RSA/HSA). In vitro stability was evaluated in human serum albumin by HPLC. Rat biodistributions and whole-body PET imaging over a 4 h time course were used for the in vivo evaluation. ResultsThis synthesis exhibited an overall radiochemical yield of 45 ± 10% (n = 30), a 50-min radiolabeling time, a radiochemical purity >99% and apparent stability up to 4 h in human serum. Blood had the highest retention of Al[18F]F-NODA-Bz-RSA at all times with a blood half-life of 5.2 h in rats. Al[18F]F-NODA-Bz-RSA distribution in most rat tissues remained relatively constant for up to 1 h, indicating that the tissue radioactivity content represents the respective tissue plasma volume. Dynamic whole-body PET images were in agreement with these findings. ConclusionsA new ligand has been developed and radiolabeled with Al[18F]F that allows rapid (50-min) preparation of fluorine-18 serum albumin in one-pot. In addition to increased synthetic efficiency, the construct appears to be metabolically stable in rats. This method could encourage wider use of PET to quantify cardiac function and tissue vascular integrity in both research and clinical settings.

Amyloid-β42 protofibrils are internalized by microglia more extensively than monomers

One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescently-labeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils.

2-(3-{1-Carboxy-5-[(6-[18F]Fluoro-Pyridine-3-Carbonyl)-Amino]-Pentyl}-Ureido)-Pentanedioic Acid, [18F]DCFPyL, a PSMA-Based PET Imaging Agent for Prostate Cancer

We have synthesized and evaluated in vivo 2-(3-{1-carboxy-5-[(6-[(18)F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [(18)F]DCFPyL, as a potential imaging agent for the prostate-specific membrane antigen (PSMA). PSMA is upregulated in prostate cancer epithelia and in the neovasculature of most solid tumors. [(18)F]DCFPyL was synthesized in two steps from the p-methoxybenzyl (PMB) protected lys-C(O)-glu urea precursor using 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) for introduction of (18)F. Radiochemical synthesis was followed by biodistribution and imaging with PET in immunocompromised mice using isogenic PSMA PC3 PIP and PSMA- PC3 flu xenograft models. Human radiation dosimetry estimates were calculated using OLINDA/EXM 1.0. DCFPyL displays a K(i) value of 1.1 ± 0.1 nmol/L for PSMA. [(18)F]DCFPyL was produced in radiochemical yields of 36%-53% (decay corrected) and specific radioactivities of 340-480 Ci/mmol (12.6-17.8 GBq/μmol, n = 3). In an immunocompromised mouse model [(18)F]DCFPyL clearly delineated PSMA+ PC3 PIP prostate tumor xenografts on imaging with PET. At 2 hours postinjection, 39.4 ± 5.4 percent injected dose per gram of tissue (%ID/g) was evident within the PSMA+ PC3 PIP tumor, with a ratio of 358:1 of uptake within PSMA+ PC3 PIP to PSMA- PC3 flu tumor placed in the opposite flank. At or after 1 hour postinjection, minimal nontarget tissue uptake of [(18)F]DCFPyL was observed. The bladder wall is the dose-limiting organ. These data suggest [(18)F]DCFPyL as a viable, new positron-emitting imaging agent for PSMA-expressing tissues.

One Step Radiosynthesis of 6-[18F]Fluoronicotinic Acid 2,3,5,6-Tetrafluorophenyl Ester ([18F]F-Py-TFP): A New Prosthetic Group for Efficient Labeling of Biomolecules with Fluorine-18

The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent complexity of the process renders full automation a challenge and leads to protracted synthesis times. Here we describe a new (18)F-labeled prosthetic group based on nicotinic acid tetrafluorophenyl ester. Reaction of [(18)F]fluoride at 40 degrees C with the trimethylammonium precursor afforded 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) directly in 60-70% yield. [(18)F]F-Py-TFP was conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding assay for the integrin alpha(v)beta(3) was established to explore competition with peptide and peptidomimetic prepared from F-Py-TFP with (125)I-echistatin. The nonradioactive conjugates were found to possess high binding affinities with calculated K(i) values in the low nanomolar range.

Microwave-Assisted Synthesis of Thiophene Fluorophores, Labeling and Multilabeling of Monoclonal Antibodies, and Long Lasting Staining of Fixed Cells

We report the expedient microwave-assisted synthesis of thiophene based 4-sulfo-2,3,5,6,-tetrafluorophenyl esters whose molecular structure was engineered to achieve blue to red bright fluorescence. The reactivity toward monoclonal antibodies of the newly synthesized fluorophores was analyzed in comparison with that of the corresponding N-succinimidyl esters. Single-fluorophore and multiple-fluorophore labeled antibodies were easily prepared with both types of esters. Multiple-fluorophore labeling with blue and orange emitting fluorophores resulted in white fluorescent antibodies. Thiophene based fluorophores displayed unprecedented fluorescence stability in immunostaining experiments. First-principles TD-DFT theoretical calculations helped us to interpret the behavior of fluorescence emission in different environments.

Glycoengineered Acid α-Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

Radioiodine labelling of a small chemotactic peptide, utilizing two different prosthetic groups: a comparative study

AbstractThe use of iodobenzoates as pre‐labelled prosthetic groups in the radioiodination of peptides is becoming increasingly popular. The utilization of an iodovinyl ester unit as an alternative conjugation agent for the preparation of a radioiodinated peptide conjugate (peptide‐[123I]I‐PEA) was investigated in this study. A pre‐labelled unit, containing a tetrafluorophenyl ester group, was purified on a small C18 column and the dimethylformamide eluate was used directly in the conjugation step. Similar methodology was applied for a comparative radiosynthesis of an iodobenzoate analogue (peptide‐[123I]IB), using a succinimidyl ester. The influences of various reaction parameters on conjugation yields were investigated while maintaining a fixed amount of peptide. At high activity levels (more than 200 MBq) the conjugation yield of peptide‐[123I]I‐PEA was very sensitive to relatively large reaction volumes and increased amount of base. By optimizing these parameters, the formation of radiochemical impurities was minimized. Under similar conditions, peptide‐[123I]I‐PEA formed much faster than peptide‐[123I]IB. Sep‐Pak C18 purification afforded conjugates with radiochemical purities both in excess of 98% and free from unreacted peptide. Recovered conjugation yields of peptide‐[123I]I‐PEA in 50% ethanol were in excess of 60%, while those for peptide‐[123I]IB were less than 40%. Copyright © 2008 John Wiley & Sons, Ltd.

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)‐based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.

Convergent and Sequential Synthesis of Dendritic, Multivalent Complexing Agents.

Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent. 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.

Convergent and Sequential Synthesis of Dendritic, Multivalent Complexing Agents

Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent. 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.

Chemical modification to reduce renal uptake of disulfide-bonded variable region fragment of anti-Tac monoclonal antibody labeled with 99mTc.

The anti-Tac disulfide-bonded variable region fragment (dsFv) is a genetically engineered, 25 kDa, murine monoclonal antibody fragment that recognizes the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The dsFv radiolabeled with the tetrafluorophenyl ester (TFP) of [99mTc]mercaptoacetyltriglycine ([99mTc]MAG3-TFP) showed rapid tumor uptake and fast blood clearance in mice, resulting in high tumor-to-nontumor background ratios. However, its high renal uptake was a problem. In this study, we tested the effect of lowering the isoelectric point (pI) of dsFv to <9.3 on renal and tumor uptake. To lower the pI, dsFv was acylated simultaneously with both [99mTc]MAG3-TFP and TFP-glycolate. The acylation of dsFv decreased its pI and its immunoreactivity inversely proportional to the molar ratio of TFP-glycolate to dsFv, whereas the conjugation of [99mTc]MAG3-TFP alone did not. When biodistribution studies were performed in nude mice, the effect of the lowered pI was reflected primarily in decreased kidney uptake and whole-body retention, with its highest effect seen at the earliest time point (15 min) after injection. In tumor-bearing nude mice, glycolated [99mTc]MAG3-dsFv with a pI range of 4.9 to 6.5 accumulated selectively into IL-2 receptor-positive SP2/Tac tumor similar to that of the control [125I]dsFv labeled by the Iodo-Gen method, whereas its renal uptake was 25% of [125I]dsFv at 15 min. At 90 min, the ratios of tumor to receptor-negative SP2/0 tumor, liver, kidney, stomach, and blood had peaked at 10.9, 8.5, 0.3, 5.0, and 6.2, respectively, for the glycolated [99mTc]MAG3-dsFv. The corresponding ratios for [125I]dsFv were 3.7, 5.0, 0.1, 1.5, and 2.1, respectively.

Synthesis and radioiodination of N-Boc-p-(tri-n-butylstannyl)-L-phenylalanine tetrafluorophenyl ester: preparation of a radiolabeled phenylalanine derivative for peptide synthesis.

An investigation to prepare a phenylalanine derivative which could be radioiodinated and used directly in peptide synthesis was conducted. N-Boc-p-(tri-n-butylstannyl)-L-phenylalanine tetrafluorophenyl ester was targeted and synthesized from N-Boc-p-iodo-L-phenylalanine. The requisite aryl stannylation reaction was found to be best conducted using the phenylalanine methyl ester. Thus, N-Boc-p-iodo-L-phenylalanine methyl ester was prepared and stannylated using bis(tributyltin) and tetrakis-(triphenylphosphine)palladium(0) in refluxing toluene to prepare N-Boc-p-(tri-n-butylstannyl)-L-phenylalanine methyl ester. Demethylation with aqueous base was accomplished without racemization to yield N-Boc-p-(tri-n-butylstannyl)-L-phenylalanine. Preparation of the targeted stannylphenylalanine tetrafluorophenyl ester was then accomplished using 2,3,5,6-tetrafluorophenol and 1,3-dicyclohexyl-carbodiimide in anhydrous THF. Iodination and radioiodination reactions of the targeted compound were conducted in MeOH/1% HOAc to yield 83-95% of the desired N-Boc-p-[*I]iodo-L-phenylalanine tetrafluorophenyl ester.

An improved CPG support for the synthesis of 3'-amine-tailed oligonucleotides.

A new controlled-pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotides bearing a 3'-aminohexyl tail. This solid support (AH-CPG) exhibits superior performance as compared to a commercially available 3'-amine CPG. The AH-CPG is prepared from 6-aminohexan-1-ol with a unique protecting group for the amine that also functions as the site of attachment to the CPG. A 3'-amine-tailed oligodeoxynucleotide (ODN) was prepared from this support using standard phosphoramidite coupling and deprotection conditions. The 3'-amine-tailed ODN was subsequently modified with an acridinylpropionic acid tetrafluorophenyl ester. Facile synthesis of the AH-CPG and the stability of the deprotected product makes this functionalized solid support especially useful for preparation of oligonucleotides bearing 3'-amine tails and other modifications.

Acridine- and cholesterol-derivatized solid supports for improved synthesis of 3'-modified oligonucleotides

New solid supports are described which allow the direct synthesis of oligonucleotides bearing either cholesterol or acridine at the 3'-terminus. A stereochemically defined amino diol was prepared by reduction of N-Cbz-hydroxy-L-proline. This linker molecule was first acylated with the desired conjugate molecule, then protected as the dimethoxytrityl ether. The remaining secondary hydroxyl group was succinylated and immobilized on a controlled-pore glass support. 3'-Modified oligodeoxynucleotides (ODNs) were prepared from these supports by using standard phosphoramidite coupling and deprotection conditions. A cholesterol-modified support was prepared from cholesterol chloroformate and the amino diol linker. Two types of acridine-modified solid supports were prepared from acridine tetrafluorophenyl esters with linker arms of different length. In an alternative synthesis of 3'-derivatized ODNs, these active esters were also utilized for acylation of a 3'-amine-modified ODN. A thermal denaturation study was done to determine the effect of the different linker arms on hybridization to a complementary ODN target. Facile synthesis and purification of the 3'-modified ODNs makes these functionalized solid supports especially useful for preparation of oligonucleotides bearing these and other modifications.

Radiolabeling of proteins with radioisotopes of copper using p‐carboxyalkylphenylglyoxal bis‐(4N‐methylthiosemicarbazone) (TSC) bifunctional chelates

AbstractA series of p‐carboxyalkylphenylglyoxal and p‐carboxyalkyl‐1,2‐diketo‐bis‐(N4‐methylthiosemicarbazone) bifunctional ligands (TSC) have been prepared and evaluated for use in binding radioisotopes of copper to antibodies. We have developed an improved synthesis of the requisite α‐ketoaldehyde and 1,2‐diketone substrates used for derivatization to the bis‐TSC bifunctional chelates. This approach utilizes a modified Kornblum method and provides a simple alternative to the usual method for fabrication of the 1,2‐bis‐TSC ligands, which avoids the use of highly toxic selenium dioxide for oxidation of substituted acetophenones to 1,2 dicarbonyl compounds. The overall yields of the bis‐TSC chelates using this procedure were 8‐60%. The effects of the alkyl chain length and substitution on the C‐2 position on p‐carboxyalkylphenyl‐1,2‐diketo bis‐TSC bifunctional chelate for attaching radioisotopes of copper to proteins were studied. Following complexing copper‐64 or copper‐67 to the bis‐TSC chelate, the acid moiety of the TSC chelate was activated as the tetrafluorophenyl ester. The copper‐labeled activated TSC chelate was attached to bovine serum albumin under mild conditions in 3% to 40% yield. These studies have demonstrated that the shorter chain analogues of the TSC chelates from the 1,2‐diketones give the highest radiolabeling yields.

ChemInform Abstract: Protected Amino Acid Tetrafluorophenyl Esters for Peptide Synthesis.

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large kcoupling/kracemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-Nim-benzyl-histidine.

Protected amino acid tetrafluorophenyl esters for peptide synthesis

New 2,3,5,6‐tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc‐Pro‐OH, Boc‐Ile‐OH, and Boc‐Val‐OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second‐order kinetics showed similarly large kcoupling/kracemization ratios for both tetra‐ and pentafluorophenyl esters. In particular, the use of 2,3,5,6‐tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N‐tert.‐butyloxycarbonyl‐Nim‐benzyl‐histidine.