Synthesis of tetrazole analogs of gamma- and delta-amino acids.

N-benzyloxycarbonyl-protected alpha- or beta-amino alcohols, easily prepared from alpha- and beta-amino acids, were converted into aldehydes and directly reacted with (triphenyl phosphoranylidene) acetonitrile, leading to unsaturated nitriles. Treatment of nitriles with NaN(3) and ZnBr(2) produced unsaturated gamma- and delta-amino tetrazoles, which were deprotected and converted to the corresponding saturated compounds by catalytic hydrogenation. For the case of delta-amino tetrazole, the methylation of the acidic moiety occurred after treatment with CH(2)N(2), leading to the N(1)- and N(2)-methylated constitutional isomers, which were separated by column chromatography and hydrogenated.     

Cyclic dermorphin tetrapeptide analogues obtained via ring-closing metathesis

The dermorphin-derived cyclic tetrapeptide analogues H-Tyr-c[D-Cys-Phe-Cys]NH(2) and H-Tyr-c[D-Cys-Phe-D-Cys]NH(2) are opioid agonists at the mu and delta receptor. To enhance the metabolic stability of these peptides, we replaced the disulfide bridge with a bis-methylene moiety. This was achieved by solid-phase synthesis of the linear precursor peptide containing allylglycine residues in place of the Cys residues, followed by ring-closing metathesis. In the case of the peptide with L-configuration in the 4-position both the cis and the trans isomer of the resulting olefinic peptides were formed, whereas the cis isomer only was obtained with the peptide having the D-configuration in position 4. Catalytic hydrogenation yielded the saturated -CH(2)-CH(2)- bridged peptides. In comparison with the cystine-containing parent peptides, all olefinic peptides showed significantly reduced mu and delta agonist potencies in the guinea pig ileum and mouse vas deferens assays. The -CH(2)-CH(2)-bridged peptide with l-configuration in the 4-position was equipotent with its cystine-containing parent in both assays, whereas the bis-methylene analogue with D-configuration in position 4 was 10-27-fold less potent compared to its parent. The effect of the disulfide replacements with the -CH=CH- and -CH(2)-CH(2)- moieties on the conformational behavior of these peptides was examined by theoretical conformational analysis which provided plausible explanations in terms of structural parameters for the observed changes in opioid activity.     

Group separation of peptides by ligand-exchange chromatography with a Sephadex containing N-(2-pyridylmethyl)glycine

The synthesis of 2-hydroxy-3[N-(2-pyridylmethyl)glycine]propyl Sephadex ether--a new chelating resin--is described. This resin has been employed in the form of its Cu2+ complex to separate peptides, as a group, from alpha-amino acids and NaCl. Ninety-seven ligands of different structures were separated chromatographically at room temperature. It was shown that two structural parameters of the ligands control the separation process, namely, the presence of ligand donor groups and the possibility for them to form chelate rings of suitable size. Separation of peptides from alpha-, gamma-, delta-amino acids, N-acetyl derivatives of amino acids (except N-Ac-Trp), and NaCl is possible if the peptides fulfill the following structural requirements: the peptide molecule must have a free terminal amino group; a carbonyl group (of the peptide linkage) must be situated in the alpha- or beta-position of the free amino group; and the peptide may not contain an imidazole residue (except Gly-Gly-His). A relationship was found between the log k' and the corresponding pKHHL, log KCuCuL, and log KCuBipyCuBipyL values. Interpretation of the different K' values was possible based on the different basicities of the terminal amino groups and on the structures of the different side chains of the peptides.     

Hydrogen bonding between cytosine and peptides of threonine or serine: is it relevant to the origin of the genetic code?

13C, 15N, and 1H nuclear magnetic resonance measurements indicate that chloroform-soluble threonine-containing tripeptide derivatives, such as t-Boc-Thr-Gly-Gly-OBz, form three strong hydrogen bonds to the cytosine moiety of 2',3'-O-isopropylidene-5'-O-t-butyldimethylsilylcytidine. The C = O and NH of the central peptide residue plus the OH of the threonine side chain appear to form bonds to the N(4')H2, N(3), and C(2) = O, respectively, of the pyrimidine. An association constant calculated from the cytidine 15N(4') nuclear magnetic resonance response to added peptide is four times larger than the corresponding cytosine-guanine constant. It is suggested that cytosine-peptide bonding was part of the primitive genetic coding mechanism early in evolution and accounts for the origin of the cytosine-centered codons for the hydroxy amino acids, serine and threonine, in the present code.     

Synthesis of 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-substituted-benzenes: "thymine replacement" analogs of thymidine for evaluation as anticancer and antiviral agents

A group of unnatural 1-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluorobenzenes having a variety of C-5 two-carbon substituents [-C...C-X, X = I, Br; -C...CH; (E)-CH=CH-X, X = I, Br; -CH=CH2; -CH2CH3; -CH(N3) CH2Br], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. The 5-substituted (E)-CH=CH-I and -CH2CH3 compounds exhibited negligible cytotoxicity in a MTT assay (CC50 = 10(-3) to 10(-4)M range), relative to thymidine (CC50 = 10(-3) to 10(-5)M range), against a variety of cancer cell lines. In contrast, the C-5 substituted -C...C-I and -CH(N3)CH2Br compounds were more cytotoxic (CC50 = 10(-5) to 10(-6)M range). The -C...C-I and -CH2CH3 compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B) and HSV-1 TK+ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines expressing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK+). This observation indicates that expression of the viral TK enzyme did not provide a gene therapeutic effect. The parent group of 5-substituted compounds, that were evaluated using a wide variety of antiviral assay systems [HSV-1, HSV-2, varicella-zoster virus (VZV), vaccinia virus, vesicular stomatitis, cytomegalovirus (CMV), and human immunodeficiency (HIV-1, HIV-2) viruses], showed that this class of unnatural C-aryl nucleoside mimics are inactive and/or weakly active antiviral agents.     

Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the P1 position

Through the development of a new chemical strategy, aminophosphinic peptides containing a pseudoglutamyl residue (Glu Psi(PO2-CH2)Leu-Xaa) in the N-terminal position were synthesized and evaluated as inhibitors of aminopeptidase A (APA). The most potent inhibitor developed in this study, Glu Psi(PO2-CH2)Leu-Ala, displayed a Ki value of 0.8 nM for APA, but was much less effective in blocking aminopeptidase N (APN) (Ki = 31 microM). The critical role of the glutamyl residue in this phosphinic peptide, both in potency and selectivity, is exemplified by the P1 position analogue, Ala Psi(PO2-CH2)Leu-Ala, which exhibited a Ki value of 0.9 microM toward APA but behaved as a rather potent inhibitor of APN (Ki = 25 nM). Glu Psi(PO2-CH2)Leu-Xaa peptides are poor inhibitors of angiotensin converting enzyme (Ki values higher than 1 microM). Depending on the nature of the Xaa residue, the potency of these phosphinic peptides toward neutral endopeptidase 24-11 varied from 50 nM to 3 microM. In view of the in vivo role of APA in the formation of brain angiotensin III, one of the main effector peptides of the renin angiotensin system in the central nervous system, highly potent and selective inhibitors of APA may find important therapeutic applications soon.     

Cyclic analogs of substance P. III. Cyclo (6(sup)gamma----oligomethylenediamine----11) substance P(6-11)-hexapeptides]

Cyclic analogues of substance P of the formula cyclo-[Glu-Phe-Phe-Gly-Leu-Met-NH(CH2)nNH-], where n = 3-10, 12, and open-chain analogues (XVIIIa, b) H-Glu.(NHR)-Phe-Phe-Gly-Leu-Met-NHR, where R = -CH3, -CH2CH2CH3, were synthesized. By NMR spectroscopy it was found that cyclo-compounds with n = 3-8 have regularly arranged structures, stabilized by intramolecular hydrogen bonds. Substances of this type showed less than or equal to 0.1% of the substance P activity on the guinea pig ileum, but some of them antagonize the natural peptide (for compound with n = 5 IC50 = 3.2.10(-6) M). The open-chain compounds proved to have rather high myotropic activity, viz., 22% (R = -CH3) and 8% (R = -CH2CH2CH3) of the substance P activity.     

Poly (delta-L-ornithine)

Poly (delta-L-Orn) is an example of an iso-polypeptide (i.e. variant of the usual poly a-peptide chain), where the a-carboxyl and delta-amino groups of ornithine are used in polymerization while the a-amino groups form the side chain. A procedure for the synthesis of this iso-polypeptide is described. Circular dichroism studies of poly (delta-L-Orn) and its Na-Boc derivative suggest that these polymers might adopt a conformation in solution similar to the beta-pleated sheet.     

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.     

Synthesis of a bicyclic δ-amino acid as a constrained Gly-Asn dipeptide isostere

Delta-amino acids are very attractive in drug discovery, especially in the peptidomimetic area, because of their capability to act as dipeptide isosteres and reverse turn mimetics. Herein we report the synthesis of a rigid delta-amino acid constrained by a 3-aza-6,8-dioxabicyclo[3.2.1]octane-based scaffold, which can be considered as a Gly-Asn dipeptide mimetic. Key steps are the condensation of glycidol and tartaric acid derivatives, and the intramolecular trans-acetalization of the oxidized adduct to give the bicyclic delta-amino acid. Starting from L-tartaric acid derivative, it was achieved the corresponding Gly-D-Asn isostere, whereas from the enantiomeric D-tartaric acid derivative the corresponding Gly-L-Asn isostere could be obtained, thus giving access to both enantiomeric dipeptide sequences.     

Polar group burial contributes more to protein stability than nonpolar group burial

On the basis of studies of Asn to Ala mutants, the gain in stability from burying amide groups that are hydrogen bonded to peptide groups is 80 cal/(mol A(3)). On the basis of similar studies of Leu to Ala and Ile to Val mutants, the gain in stability from burying -CH(2)- groups is 50 cal/(mol A(3)). Thus, the burial of an amide group contributes more to protein stability than the burial of an equivalent volume of -CH(2)- groups. Applying these results to folded proteins leads to the surprising conclusion that peptide group burial makes a larger contribution to protein stability than nonpolar side chain burial. Several studies have shown that the desolvation penalty for burying peptide groups is considerably smaller than generally thought. This suggests that the hydrogen bonding and van der Waals interactions of peptide groups in the tightly packed interior of folded protein are more favorable than similar interactions with water in the unfolded protein.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Amino acid deletion products resulting from incomplete deprotection of the Boc group from Npi-benzyloxymethylhistidine residues during solid-phase peptide synthesis.

In peptide synthesis, the use of N(alpha)-tert-butyloxycarbonyl-N(pi)-benzyloxymethylhistidine [Boc-His(pi-Bom)] raises the problem of the Bom group generating formaldehyde during the hydrogen fluoride (HF) cleavage reaction. This can lead to modification of the functional groups on amino acids in the peptide chain. Besides this side reaction, the failure of N(alpha)-Boc deprotection from the His(pi-Bom) residue occurs during TFA treatment for the standard solid-phase peptide synthesis (SPPS) even in the case of a non 'difficult sequence'. This gives amino acid deletion products generated at the N-terminus of the His(pi-Bom) residues. Reviewing the removability of the Boc group on amino acid derivatives showed that the group on the His(pi-Bom) residue was much more resistant under the deprotecting conditions than expected. To circumvent this problem, special precautions, i.e. prolonged deprotection steps and/or increased concentrations of TFA, should be taken for a successful SPPS.     

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.     

He(I) and He(II) photoelectron spectra of Hyprrole-2-CHN′t-Bu and the biscarbonyl Rh(I) and Ir(I) complexes of its anion

The He(I) and He(II) photoelectron spectra of a series of [(LL)M(CO)₂] (LL = pyrrole-2-CHN′ R; R = t-Bu; M = Rh, Ir) complexes are reported. Assignments are proposed based on He(I)/He(II) intensity differences, on molecular orbital calculations of related complexes and of free ligands, and by comparison with the spectra of the free ligands Hpyrrole-2-CHN′t-Bu, Hpyrrole-2-carbaldehyde and Hpyrrole.The electronic structure of the complexes is discussed and conclusions are drawn about the metal-ligand interaction.     

Ligand-presenting assembly: a method for C- and N-terminal antigen presentation.

Achiral dicarboxylic acids were coupled with 2 eq. of the free alpha-amino groups of two fully side-chain protected peptide chains while these were still attached to a synthesis resin. Cleavage from the resin with simultaneous side-chain deprotection afforded two assembled peptide chains with free C-terminals. Suitable functionalization of the achiral dicarboxylic acid alternatively permitted continued peptide synthesis in a C to N orientation leading to a final peptide assembly which, after cleavage from the resin, may have multiple N to C and C to N presentation of one or more epitopes.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Inhibition of chlorophyll biosynthesis by mercury in excised etiolated maize leaf segments during greening: effect of 2-oxoglutarate

Mercury (0.01-1.0 mM) inhibited chlorophyll formation in greening maize leaf segments. However, supplementing incubation medium with 2-oxoglutarate, maintained substantially higher level of chlorophyll in absence of metal after an initial period of 8 hr. On preincubation of leaf segments with HgCl2, per cent inhibition of chlorophyll synthesis by metal was same in the presence and absence of 2-oxoglutarate. Supply of 2-oxoglutarate (0.1-10.0 mM) exerted concentration dependent effect on chlorophyll formation in absence or presence of metal. Increase in delta-amino levulinic acid dehydratase as well as NADH-glutamate synthase activity and decrease in NADH-glutamate dehydrogenase activity by 2-oxoglutarate in the presence of Hg suggested that glutamate for delta-amino levulinic acid synthesis could be made available from NH4+ assimilation via., glutamine synthetase/glutamate synthase pathway during mercury toxicity.     

Orthogonal protecting groups for N(alpha)-amino and C-terminal carboxyl functions in solid-phase peptide synthesis

For the controlled synthesis of even the simplest dipeptide, the N(alpha)-amino group of one of the amino acids and the C-terminal carboxyl group of the other should both be blocked with suitable protecting groups. Formation of the desired amide bond can now occur upon activation of the free carboxyl group. After coupling, peptide synthesis can be continued by removal of either of the two protecting groups and coupling with the free C-terminus or N(alpha)-amino group of another protected amino acid. When three functional amino acids are present in the sequence, the side chain of these residues also has to be protected. It is important that there is a high degree of compatibility between the different types of protecting groups such that one type may be removed selectively in the presence of the others. At the end of the synthesis, the protecting groups must be removed to give the desired peptide. Thus, it is clear that the protection scheme adopted is of the utmost importance and makes the difference between success and failure in a given synthesis. Since R. B. Merrifield introduced the solid-phase strategy for the synthesis of peptides, this prerequisite has been readily accepted. This strategy is usually carried out using two main protection schemes: the tert-butoxycarbonyl/benzyl and the 9-flourenylmethoxycarbonyl/tert-butyl methods. However, for the solid-phase preparation of complex or fragile peptides, as well as for the construction of libraries of peptides or small molecules using a combinatorial approach, a range of other protecting groups is also needed. This review summarizes other protecting groups for both the N(alpha)-amino and C-terminal carboxyl functions.