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Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.

Solid phase synthesis of Ψ[CH2NH] pseudopeptide 3 was accomplished by reductive amination of peptidyl aldehyde 1 with resin bound amino peptide 2. No epimerization took place during the aldehyde preparation or the reductive amination step. This convergent strategy should facilitate the synthesis of analogues of the neuropeptide PACAP.

A cyclic analog of the natural antiproliferative compound dolastatin 10 was synthesized by introducing an ester link between the N- and C-terminal residues which were modified accordingly. The final macrolactonization was performed by using isopropenyl chloroformate and DMAP as reagents. This analog exhibits submicromolar antiproliferative activity against the L1210 and HT29 cell lines and inhibits in vitro tubulin polymerization (IC50, 39 μM).

Peptidyl aldehydes were synthesized using thiazolidine peptides as precursors. Hydrolysis of the heterocycles is easily accomplished using cupper salts to furnish the peptidyl aldehydes with very good yield and with no epimerization. © 1997 Published by Elsevier Science Ltd.

A solid phase synthesis of a phosphonopeptide is described, in which a type 1 aminophosphonate with a free phosphonic acid was coupled just like an amino acid, using BOP as reagent.

The potent antineoplasic pseudopeptide dolastatin 10 isolated from the sea hare Dolabella auricularia was examined by a combination of one and two dimensional NMR techniques (DQF-COSY, HOHAHA, ROESY, HMQC, and HMBC) followed by restrained molecular-dynamics (MD) simulations to elucidate its conformation in solution (DMSO). The study showed that dolastatin 10 exists in two different conformations corresponding to a cistrans isomerisation of the Dil-Dap amide bond.

Dolastatin 15 1(4S,7S) is a potent cytostatic depsipeptide of marine origin. Analysis of the effects of the stereochemistry in the non-peptide moiety on activity revealed that chirality inversion in the aromatic terminal region preserves the antiproliferative activity.

A stepwise synthesis of dolastatin 10 starting from the dolaphenine residue is described. The chiral dola isoleuine and dolaproine residues were obtained by 5-step procedures from the corresponding N-Boc amino acid. The key steps are respectively the NaBH4 reduction of an allylic ketone and the addition of an achiral Z-crotylboronate on the N-Boc- L -prolinal. Peptidic couplings were efficiently realised with reagents developed in the laboratory.

The absence of a coupling reaction between a phosphinic acid and an amino ester during activation with the reagents BOP or PyBOP allowed the synthesis of phosphinopeptides from phospho-analogues of dipeptides, unprotected on the phosphinic acid.

The Mitsunobu reaction was efficiently used to introduce oxazolines and thiazolines in the peptide backbone. The reaction proceeded from β-hydroxy α-amino acid-containing peptides at room temperature in 58-72% isolated yields.

Efficient synthesis of dolastatin 15 (1) was accomplished following a convergent strategy. The pyrrolidinone cycle of 5 was obtained by thermic cyclization of the corresponding Meldrum's adduct 4. The methylation of the enol function was performed under Mitsunobu conditions. On the other hand, the peptide part 10 was elongated by using PyCloP as coupling reagent. The ester linkage between both segments was efficiently performed under conditions we previously described by using a mixed anhydride activation with isopropenyl chlorocarbonate. Final compound 12 was found to exhibit physical properties slightly different from those recently reported by Pettit and co-workers for the natural dolastatin 15 and their synthesis product.

The main product of N-methylated amino acid coupling, when oxybenzotriazole is present in the reagent (BOP, PyBOP, HBPyU) or as an additive (DCC/HOBt), is the weakly reactive benzotriazolyl ester. On the other hand, the corresponding new halogenated reagents PyBroP, PyCloP and PyClU give very good results and can be recommended.

The stereocontrolled synthesis of the N,O-dimethylated-γ-amino-β-hydroxy acid (R)-MeVal-ψ(CHOMe)-Gly ( 3b ) is reported; the key step is the formation of the nonmethylated allylic precursor with the anti configuration 20 by reduction of the keto analogue 16 .

Monoesters of N-protected α-amino phosphonic acid were prepared from phosphonic acid using BroP or TPyClU as activating agent. This reaction proceeds with good yield, even with hindered alcohols.

Mixed phosphonate diesters of a N-protected α-amino phosphonic acid are prepared from phosphonate monoesters using BOP or PyBOP as activating agent. This reaction proceeds without racemisation.

The steric hindered Aib-Aib-Aib tripeptide was synthesized in mild conditions, using PyBroP for the coupling of two Aib and Fmoc-Aib-NCA for the generation of the tripeptide, which was obtained with an excellent yield in THF. The coupling of Gln was carried out in the presence of BOP.

Activation of Boc-amino acids and Boc-N-methyl amino acids leads to the corresponding NCA. This side reaction explains the low yields obtained when coupling N-methyl amino acids. It was not observed with the Z-or Fmoc-protective groups.

Activation of N-protected aminoacids by isopropenyl chloroformate leads to esters of primary, secondary and tertiary alcohols with 4-dimethylamino-pyridine as catalyst.

Activation of N-protected α-amino acids and O-protected α-hydroxy acids with 2,2'-carbonylbis (3,5-dioxo-4-methyl-1,2,4-oxadiazolidine) 6 provide a stable activated intermediate 7 suitable for the synthesis of β-keto ester 8 . This activation was used in the preparation of the non-proteogenic units 1st 1 and Hip 4 present in the cyclodepsipeptides didemnins isolated from the tunicates Didemnidae.

Esterification of N-protected α-amino acids was achieved via isopropenyl chlorocarbonate (IPCC) activation. In situ alcoholysis of the unstable mixed anhydride intermediate was catalyzed by 4-(dimethylamino)pyridine (DMAP). Competing isopropenyl ester formation was negligible when using methylene chloride as the solvent. A variety of esters from primary and secondary alcohols were obtained with good yields (60 to 96 %), and even the more hindered tertiobutyl alcohol gave acceptable yields under more drastic conditions. The improvement in depsipeptide synthetic methodology is illustrated by preparation of the antibiotic valinomycin, using IPCC for ester bond formation, and BOP reagent for peptidic coupling and the last-step cyclization.

A comparative study of isodideimnine-1 and didemnin B is presented using spcctroecopic methods, partial degradation and partial synthesis. This leads to the conclusion of the presence of a single depsipeptide, namely didemnin B, with (3S,4R,5S) isostatine instead of the previous statine residue. An attempt to determine the whole conformation in solution of didemnin B by using 2D-NMR is also described.

Mono-esters of N-protected di-acids aspartic and glutamic are prepared by a one-pot activation with alkyl chloroformates or isopropenyl chloroformate and an additionnal alcohol. This process involves the intermediate internal anhydride formation.

Tert-butyl fluorocarbonate (Boc-F) is efficiently used for the synthesis of tert-butyl esters of N-protected amino acids. The reaction proceeds at room temperature and under mild conditions in the presence of triethylamine and 4-dimethylamino-pyridine.