N,N'-bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl); a superb reagent for coupling at and with iminoacid residues

Analytically pure dipeptides containing two iminoacids [Pro, MeAla, MePhe, MeVal and SPip (γ-thiapipecolinic acid)] were prepared with BOP-Cl [N,N]-bis(2-oxo-3-oxazolidinyl) phosphinic chloride] at normal temperature (0° — r.t.) in high yields (80–100%) and with excellent optical purity (below noise limit in ¹H n.m.r.). Maximum yields were obtained with Zervas' preactivation procedure, using, for example, an excess of BOP-Cl followed by the addition of (an equal excess of) the aminocomponent. Racemization suppressors (HOBt, imidazole, etc.) are of no use in these difficult couplings. Z-Me Val-Me Val-OBu^t (yield: 89%, without racemization (!)) and Z-SPip-SPip-OMe (yield: 96%, less than 5% stereomutation) were prepared for the first time, whereas all other coupling reagents have failed. Peptide acids with N-methylaminoacids as the COOH-terminal residues were coupled with excellent yields (70–90%), but unfortunately chiral integrity could not be preserved. Pronounced chiral induction was observed during the preparation of Z-Pro-MePhe-MeAla-OBu^t, and an influence of the penultimate protecting group was detected. In contrast, Z-MeVal-MeVal-MeVal-MeVal-OBu^t was formed with probably only 5% stereomutation (yield: 52%). Some side reactions with BOP-Cl are observed: e.g. symmetrical anhydride and DKP formation.     

Anchoring of Fmoc-amino acids to hydroxymethyl resins

A method to anchor Fmoc-amino acids to p-alkoxybenzyl ester resins without using DMAP is described. Esterification takes place with 3–4 equiv. of Fmoc-amino acid plus DCC and a slightly lower excess of HOBt in good yields, without racemization or dipeptide formation. Addition of N-methylmorpholine to the reaction medium enhances the reaction yield but is accompanied by a small amount of racemization and dipeptide formation. Coupling via Fmoc-amino acid chlorides has also been evaluated.     

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method

In the carbodiimide mediated coupling of Z-Gly-l -Val-OH with H-l -Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leuenkephalin. In the 4+1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.     

Practical protocols for stepwise solid‐phase synthesis of cysteine‐containing peptides

Abstract: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62 , 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH₂, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH₂Cl₂/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.     

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO), a promising new reagent for racemization-free couplings

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO) was synthesized, and its ability to effect racemization-free couplings and cyclization of a peptide and its C-terminal epimer was examined. MPTO showed good reactivity in aprotic polar solvents such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone. In reactivity MPTO resembles DPPA and dimethylphosphinothioyl azide (MPTA) previously developed by us, but it is much better than these reagents because the side reactions specific to the azide method could be avoided. In coupling of Z-Gly-Val-OH with H-Val-OMe in DMF at 0°C, no racemization was observed without use of racemization-suppressing additives. Slight racemization observed at room temperature could be completely suppressed by addition of HOBt but not by HOSu. The utility of MPTO was demonstrated by the synthesis of cyclo-(d -Trp-d -Glu(OBzl)-Ala-D-Val-Leu), an intermediate for an endothelin-binding inhibitor BE 18257A. In a comparative study using DPPA, MPTA and MPTO, no racemization was observed for MPTA or MPTO, while DPPA caused considerable racemization. When MPTO was used in the presence of HOBt rapid cyclization (3 h at RT) occurred to give the optically pure cyclic product.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids

When L-proline or O-benzyl-trans-4–hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the Cα of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Histidine racemization in the synthesis of an analog of the lutenizing hormone releasing factor

To investigate histidine racemization in the synthesis of a LHRH analog, (D-Trp)⁶-LHRH^2–10 was built up by stepwise elongation of the sequence 3–10 using the solid phase technique on a 1% cross-linked chloromethyl polystyrene. For the whole synthesis the tert.-butyloxycarbonyl (BOC) group was used for temporary N-terminal protection. To protect the π-nitrogen in histidine the benzyloxymethyl (BOM) group was utilized. The condensation position in the (D-Trp)⁶-LHRH analog was chosen so as to be able to investigate the racemization of histidine. We coupled BOC-His(BOM) with the (D-Trp)⁶-LHRH^3–10 fragment using three different activating agents, mixed anhydride, carbonyldiimidazole (1 equiv.)/1-hydroxybenzotriazole (2 equiv.) and dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The racemization was investigated by enzymatic digestion and by HPLC. For HPLC, (D-His(BOM)²-(D-Trp)⁶-LHRH^2–10 was also synthesized. It could be proved that practically no racemization occurs during the actual peptide synthesis. The small amount (1%) of D-histidine found is due to racemization in the synthesis of BOC-His(BOM).     

Carbonyldiimidazole/1-hydroxybenzotriazole activation in polymer phase synthesis of the arginine rich proalbumin hexapeptide extension

The synthesis on different polymer phases of Arg-Gly-Val-Phe-Arg-Arg, the proalbumin extension, is reported. The peptide was prepared both on a 0.5% cross-linked polystyrene gel containing 2-oxoethyl bromide anchor functions and on a 1% cross-linked chloromethyl polystyrene. For temporary blocking of the amino groups we utilized the 2-(3, 5-dimethoxyphenyl)propyl-(2)-oxycarbonyl (Ddz). The guanidino groups of the three arginine moieties were protected by the 4-toluenesulfonyl (Tos) group. As coupling procedure we used 1 equiv. carbonyldiimidazole (CDI)/2 equiv. 1-hydroxybenzotriazole (HOBt). The C-terminal activation of the Ddz-amino acids with CDI/HOBt made it possible to recover the excess Ddz-amino acids in 60–80% yield. We also investigated different procedures to cleave the 2-oxoethyl ester bond between the peptide and the polymer. This bond was completely stable against trifluoro-methanesulfonic acid and was split by 1 N triethylamine in methanol/dioxane 1:1 (v/v) + 1 vol% 1 N NaOH. The optical rotation and HPLC properties of Arg-Gly-Val-Phe-Arg-Arg from this synthesis are identical to the product from a different synthesis published earlier.     

(1H‐Benzotriazol‐1‐yloxy)‐N,N‐dimethylmethaniminium hexachloroantimonate (BOMI), a novel coupling reagent for solution and solid‐phase peptide synthesis

Abstract: A HOBt‐based immonium‐type compound,(1H‐benzotriazol‐1‐yloxy)‐N,N‐dimethyl methaniminium hexachloro‐antimonate (BOMI), was synthesized and successfully applied to the synthesis of various oligopeptides with good yields. The estimation of racemization and the influence of several reaction parameters such as solvents, bases and temperature were studied by HPLC using a model reaction. It was found that the reactivity of BOMI was much higher than that of HOBt‐based phosphonium‐ and uronium‐type coupling reagents. Moreover, its racemization was lower than that of other HOBt‐derived coupling reagents. The effectiveness of BOMI was also demonstrated by the synthesis of Leu‐enkephalin both in solution and in the solid‐phase.     

Applications of BOP reagent in solid phase synthesis Advantages of BOP reagent for difficult couplings exemplified by a synthesis of [Ala 15]-GRF(1–29)-NH2

The BOP reagent [benzotriazol-l-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate] introduced by Castro et al. [Tetrahedron Lett. (1975) 14, 1219–1222] is ideally suited for solid phase peptide synthesis. The rate of coupling using BOP compared favorably to DCC and other methods of activation including the symmetrical anhydride and DCC/HOBt procedures. BOP couplings using the solid phase procedure proceeded more rapidly and to a greater degree of completion for peptide bond formations that were previously determined to be very slow using the conventional DCC method. Stepwise solid phase peptide synthesis using BOP was successfully utilized for the preparation of the (22–29) and (13–29) fragments of [Ala¹⁵]-GRF(1–29)-NH₂. Single couplings with 3 equiv. BOP and Boc-amino acids and 5.3 equiv. of diisopropylethylamine in DMF were used for each cycle. The yields of the fragments were superior and the purities comparable using the BOP procedure (single couplings) to those observed using multiple couplings via the DCC coupling method. A total synthesis of [Ala¹⁵]-GRF(1–29)-NH₂ was also carried out using the BOP procedure (single couplings and 3 equiv. BOP and Boc-amino acids and 5.3 equiv. diisopropylethylamine in DMF for each cycle). Multiple couplings were only required for Boc-Asn-OH due to the proposed formation of Boc-aminosuccinimide during activation. The resultant GRF(1–29) analog was comparable to a control prepared with multiple DCC couplings under optimized conditions. In a parallel study, unprotected Boc-(hydroxy)-amino acids were successfully coupled with the BOP reagent. However, the number of coupling cycles after the introduction of unprotected hydroxy-amino acid must be minimal (<10). The use of the BOP reagent with unprotected Tyr in solid phase peptide synthesis was also clearly established.     

BOP reagent for the coupling of pGlu and Boc-His(Tos) in solid phase peptide synthesis

The model peptide TRH was successfully synthesized using benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.     

Synthesis of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH2

The syntheses of Ac-Asp-Gly-Ser and Ac-Asp-Pro-Leu-Gly-NH₂ are described. The two peptides were prepared in solution by stepwise elongation using the DCC method with additives (HOSu or HOBt). The push-pull method was also used in the synthesis of Ac-Asp-Gly-Ser. A racemization test of the amino acids in this peptide has been performed.     

Studies on sensitivity to racemization of activated residues in couplings of N-benzyloxycarbonyldipeptides

A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa= 15 different residues and R= H, NH₂, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylformamide or dichloromethane at +5°. The accompanying racemization was determined by analysis of the epimeric products by normal phase high-performance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5°, and N,N′-dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(Z) and Asp(OBzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.     

Synthesis of thymosin α1 by fragment condensation using tert.-butyl side chain protection

A novel synthesis of thymosin α₁ by classical methods using seven tert. -butyl side chain protected fragments is described. Optimum conditions were found for the final DCC/HOBt coupling of the two key intermediates; decapeptide and octadecapeptide. Thymosin α₁ was purified by two stages of preparative HPLC (partial purification with C₈ and final purification with C₁₈ reverse phase silica gel) to give a 30% overall yield for the final four stages of synthesis (including catalytic hydrogenation of octadecapeptide, coupling, deprotection and purification). The product was shown to be homogeneous by thin-layer and paper high voltage electrophoresis, isoelectric focusing analysis, thin-layer chromatography and high performance liquid chromatography. Amino acid analysis, optical rotation, ¹ H-n.m.r. spectroscopy, FAB mass spectroscopy and peptide mapping after tryptic digestion confirmed the structure of thymosin α₁. Three minor stereoisomer contaminants were isolated by HPLC and characterized as [D-Lys¹⁴]-thymosin α₁, [D-Lys¹⁷]-thymosin α₁ and [D-Ala³]-thymosin α₁ resulting from racemization at Lys¹⁴, Lys¹⁷ and Ala³ during the coupling of the fragments. A final contaminant, isolated by HPLC, was characterized as N^α-isobutyloxycarbonyl-thymosin α₁ (15–28), which results from “wrong way opening” of an activated mixed anhydride.     

Synthesis of 14C‐labelled EM‐800 (SCH 57050) and EM‐652·HCl (SCH 57068·HCl,acolbifene), pure selective estrogen receptor modulators

EM‐800 (SCH 57050) and EM‐652·HCl (SCH 57068·HCl, acolbifene) are orally active pure selective estrogen receptor modulators. The corresponding ¹⁴C₂‐radiolabelled compounds 1 and 2 were synthesized for metabolic studies with uniform labelling of two carbons within the benzene ring of the 2H‐1‐benzopyran moiety by optical resolution of racemic (±)‐[¹⁴C₂]EM‐343 4 . This pivotal intermediate amine was prepared in 6 steps with 38% yield from commercially available [U‐¹⁴C₂]resorcinol ( 3 ). Resolution by selective crystallization of the diastereomeric mixture of (S)‐(+)‐camphorsulfonates salts gave the desired (+)‐[¹⁴C₂]EM‐652·(+)‐CSA 13 . Moreover, the racemic amine 4 was recovered from mother liquors by basic treatment, and resolved again. We obtained salt 13 , at a 52% yield with 97% diastereomeric excess by repeating the resolution–racemization process. Finally, the corresponding dipivaloate (+)‐[¹⁴C₂]EM‐800 1 and hydrochloride salt (+)‐[¹⁴C₂]EM‐652·HCl 2 were prepared at respective specific activities of 19.7 and 24.5 µCi/mg with 96.3% radiochemical purity. Copyright © 2004 John Wiley & Sons, Ltd.     

Racemization free coupling of peptide segments

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂ has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10-20% of the d -epimer.     

Chemical synthesis of urotensin II,a somatostatin-like peptide in the caudal neurosecretory system of fishes

In the goby, Gillichthys mirabilis, urotensin II (a bioactive neuropeptide present in the urophysis of teleost fish) has the dodecapeptide sequence, H₂N-AGTADC-FWKYCV-OH, which is homologous with mammalian somatostatin at positions 1, 2 and 7–9. The Merrifield solid phase synthesis of Gillichthys urotensin II (UII) was accomplished by stepwise assembly from the carboxy terminus using N^α-tert.-butyloxycarbonyl (Boc) amino acids containing benzyl-derived groups for protection of side-chain functionalities. Coupling of amino acids to the growing peptide was mediated by diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenzotriazole (HOBt). Residual α-amino groups remaining after coupling were blocked by acetylation with 1-acetylimidazole. Crude, synthetic UII was extracted from the HF-treated, protected peptide-resin product, reduced with dithiothreitol (DTT), reoxidized at high dilution with O₂, and separated into its components using a single, preparative, reverse-phase HPLC step. The pure, synthetic UII, obtained in 7.6% yield from oxidized crude UII, was indistinguishable from pure, native UII in specific bioactivity, amino acid sequence, and retention time in each of two different HPLC systems.     

Solid-phase synthesis of protected peptides using new cobalt(III) ammine linkers†

Cobalt(III) ammine complexes of the type cis-[CoL₄(4-AMB)O-AA-Boc](CF₃SO₃)₂, where L₄= bisethylenediamine (en)₂ or tetraammine (NH₃)₄, and 4-AMB = 4-(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid-phase synthesis of protected peptides. Boc/t-Bu-protected [Leu⁵]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC-purified protected [Leu⁵]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and ¹H NMR. Stepwise synthesis on the Co(en)₂-resin was also used in the assembly of Boc-Asp(OcHex)-Arg(Mts)-Gly-Asp(OcHex)-Ala-Pro-Lys(2Cl-Z)-Gly-OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC-purified nonapeptide was characterized by amino acid analysis, ¹H NMR and liquid secondary-ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert-butyloxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) N^α-protecting group strategies used in solid-phase peptide synthesis.     

Absorption,metabolism and excretion of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat and dog

1. The oral no overall adverse effect level (NOAEL) for chronic toxicity of 4-chloro-2-methylphenoxyacetic acid (MCPA) in rat is ~1.3?mg kg^-1 and in dog is 0.2 mgkg^-1. In an attempt to explain the difference in toxicology between these species, rats and dogs were orally dosed with (¹⁴C)-MCPA at 5 or 100?mg kg^-1 and plasma toxicokinetics, rates and routes of excretion and biotransformation were investigated. 2. Elimination of radioactivity in rat plasma was biphasic and in dog was monophasic. Rat eliminated radioactivity from plasma significantly faster than dog (approximate values based on total radioactivity: 5 mgkg^-1 rat: t_½dist 3.5 h, t_½elim 17.2-36.2 h, AUC_(0-∞) 230 µg equiv h g^-1; 5 mgkg^-1 dog: t_½47 h, AUC_(0-∞) 2500 µg equiv h g^-1; 100mg kg^-1 rat: t_½dist 10 h, t_½elim 10.27-25.4 h, AUC_(0-∞) 5400 µg equiv h g^-1; 100?mg kg^-1 dog: t_½41 h, AUC_(0-∞) 20 500µg equiv h g^-1). 3. For both species, the principal route of excretion was in urine but renal elimination was notably more rapid and more extensive in rat. 4. In both rat and dog, excretion of radioactivity was mainly as MCPA and its hydroxylated metabolite hydroxymethylphenoxyacetic acid (HMCPA). In rat, both were mainly excreted as the free acids although a small proportion was conjugated. In dog, the proportion of HMCPA was increased and the majority of both species was excreted as glycine or taurine conjugates. 5. These data, along with previously published accounts, indicate that renal elimination of MCPA in dog is substantially slower than in rat resulting in disproportionate elevation of AUC (based on total radioactivity) in dog compared with rat.     

Use of triphenylmethyl (trityl) amino protecting group in the synthesis of ketomethylene analogues of peptides

The Grignard reagents of 2-(2-bromoethyl)-1,3-dioxane and 2-(2-bromoethyl)-1,3-dioxolane readily reacted with the 2-thiopyridyl ester of N-triphenylmethyl-l -leucine to give the ketone adducts 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxane (8a) and 2-[3-oxo-4(S)-(triphenylmethyl) amino-6-methylheptyl]-1,3-dioxolane (8b) in near quantitative yield. When 1 equiv. of the Grignard reagent of 2-(2-bromoethyl)-1,3 dioxane was used, the desired ketone adduct 8a was formed slowly but quantitatively. In contrast, when 2 equiv. of the Grignard reagent were used, the formation of ketone 8a was instantaneous. The triphenylmethyl protecting group was easily removed from 8a using dilute acid to give the amino ketone 2-[3-oxo-4(S)-amino-6-methylheptyl]-1,3-dioxane oxalate salt (9). This material served as a useful intermediate in the synthesis of the ketomethylene analogues of the peptides, Z-Pro-Leu-Gly-OH and Leu-Gly-Val-Phe-OCH₃.