Bioactive Analogues Research Articles

A new sulfoxide analog of 1,2,3,6-tetrahydrophenylpyridine and antimicrobial activity.

Bioactivities of thiotetrahydropyridines were previously described. Herein, a novel bioactive sulfoxide analog; N-acetyl-2-(1-adamantylsulfoxo)-3-acetoxy-4-phenyl-6-hydroxy-1,2,3,6-tetrahydropyridine (3) from the deoxydative substitution of 4-phenylpyridine 1-oxide is reported. Its structure was elucidated using spectral data including 2D-NMR, MS, IR and UV. The sulfoxide 3 exhibited antibacterial activity against Moraxella catarrhalis and Streptococcus pyogenes with minimum inhibitory concentration of 128 and 256 µg/mL, respectively.

E-2-[3-(3,4-Dichlorophenyl)-1-oxo-2-propenyl]-3-methylquinoxaline-1,4-dioxide: A lead antitubercular agent which alters mitochondrial respiration in rat liver

A series of 2-(3-aryl-1-oxo-2-propenyl)-3-methylquinoxaline-1,4-dioxides 1a– l and 2-acetyl-3-methylquinoxaline-1,4-dioxide 2 were evaluated against Mycobacterium tuberculosis H 37Rv. With the exception of the 4-nitro analog 1k, significant antitubercular potencies were observed in series 1 and 2 which have IC 50 values in the range of 1–23 μM. Negative correlations were noted between the IC 50 values of 1a– j, l towards M. tuberculosis and both the σ and π constants of the substituents in the benzylidene aryl ring. In particular, 1h emerged as a lead compound having IC 50 and IC 90 figures of 1.03 μM and 1.53 μM, respectively. This molecule affected respiration in rat liver mitochondria which is likely one way that 1h and the bioactive analogs exert their antitubercular properties. The quinoxaline 2, which lacks an α,β-unsaturated group, has no effect on mitochondrial respiration using concentrations which inhibit the growth of M. tuberculosis.

ChemInform Abstract: An Asymmetric Synthesis Protocol to Prepare Topographically Constrained β-Substituted Aromatic Amino Acids

The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor/acceptor binding and for signal transduction (1). Therefore, complete understanding of the stereochemical requirements of side chain groups important in peptide ligand-receptor/acceptor interactions plays a crucial role in the rational design of bioactive peptides and nonpeptide mimetics. Topographical considerations provide a major approach to explore the structure-activity relationships of the side chains of the amino acid residues in the bioactive peptide analogues (2). This approach can be realized by incorporation of conformationally constrained unusual amino acids into a peptide or nonpeptide template. Thus, design and synthesis of unusual amino acids with specific biased side chain conformations have tremendous impact on the exploration of the mechanism of peptide ligand-receptor recognition and of activating signal transduction. Our laboratory has been engaged in the design and synthesis of conformationally constrained amino acids for almost a decade. One representative family among the special amino acids we have designed is the β-substituted unusual amino acids, in which substitution of the diastereotopic β-hydrogens of natural α-amino acids provides an approach to topographic control of side chain conformation (3). Incorporation of these β-substituted unusual amino acids into bioactive peptide hormones has produced highly selective and potent peptide hormone analogues and provided new insights into the stereochemical requirements in peptide-receptor interactions (4).

Efficient Total Synthesis of (S)‐14‐Azacamptothecin

An efficient total synthesis of (S)-14-azacamptothecin has been accomplished in 10 steps and 56% overall yield from 5H-pyrano[4,3-d]pyrimidine 8. A mild Hendrickson reagent-triggered intramolecular cascade cyclization, a highly enantioselective dihydroxylation, and an efficient palladium-catalyzed transformation of an O-allyl into N-allyl group are the key steps in the synthesis. This work provides a much higher overall yield than the previous achievement and shows sound flexibility for the further applications that will lead to new bioactive analogues.

Abstract 212: Targeting p73 as a strategy for enhancing anticancer efficiency of chemotherapeutic drugs to triple-negative human breast cancers

Abstract Successful treatment of p53-mutant, triple-negative breast cancer (TNBC) remains a daunting challenge. Chemotherapeutic drugs such as doxorubicin (DOX) and cisplatin (CDDP) remain the first line treatments for TNBC; however, chemotherapy eventually fails due to acquired drug resistance and toxicity. Therefore, new treatments for overcoming drug resistance and toxicity in TNBC are badly needed. Here we report on the reconstitution of the p53 tumor suppressor pathway in a p53-independent manner via p53 homolog p73 as an exciting possibility for improving cancer therapy in p53 mutant breast cancers. α-TEA, a small bio-active lipid analog of vitamin E, exhibits potent anticancer actions in human breast and murine mammary cancer both in vitro and in vivo. Treatments with combinations of α-TEA plus DOX or CDDP enhances in vitro apoptotic killing of two p53-mutant TNBC cells; namely, MDA-MB-231 and BT-20 cells. Sub-apoptotic doses of α-TEA plus non-apoptotic doses of DOX or CDDP significantly enhanced caspase-8 and 9 mediated apoptosis in both cell lines. The combination treatments enhanced p73 protein expression, enhanced levels of death receptors-5 (DR5), CD95/APO-1 (Fas), pro-apoptotic Bax and Noxa proteins, and reduced pro-survival Bcl-2 protein levels, all of which are p53 regulated genes. The combination treatments induced enhanced levels of phospho-c-Ab1 and JNK, upstream mediators for p73. Furthermore, combination treatments activated Yes-associated protein (Yap), a transcriptional coactivator which has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Taken together, our data suggest that combination of α-TEA with clinically relevant chemotherapeutic drugs (DOX or CDDP) enhances apoptosis of MDA-MB-231 and BT-20 p53-mutant TNBC cells via p73, and that c-Abl/JNK and Yap may contribute to the enhanced anti-cancer efficacy of the combination treatments. These data provide the rationale for conducting pre-clinic TNBC xenograft studies testing the safety and efficacy of this new therapeutic approach. Supported by DOD concept grant W81XWH-09-1-0564 (Yu). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 212.

Enzymatic transformation of flavonoids and terpenoids: Structural and functional diversity of the novel derivatives

Flavonoids and terpenoids are promising candidates for use as functional foods and novel therapeutics due to their prominent biological activities. However, the use of several bioactive plant compounds is limited by low stability and solubility problems. This review underlines the feasibility of enzymatic synthesis of novel bioactive analogs of selected flavonoids (silybin, rutin) and terpenoids (perillyl alcohol, POH) in nonconventional reaction systems. The effect of various parameters on the enzymatic acylation and/or glucosylation of these phytochemicals was studied. Also, the role of the structure of the novel molecules in relation to their biological function was investigated in various cancer cell lines.

Potential of mean force of the hepatitis C virus core protein–monoclonal 19D9D6 antibody interaction

Antigen–antibody interactions are critical for understanding antigen–antibody associations in immunology. To shed further light on this question, we studied a dissociation of the 19D9D6-HCV core protein antibody complex structure. However, forced separations in single molecule experiments are difficult, and therefore molecular simulation techniques were applied in our study. The stretching, that is, the distance between the center of mass of the HCV core protein and the 19D9D6 antibody, has been studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. Our simulations indicate that the 7 residues Gly70, Gly72, Gly134, Gly158, Glu219, Gln221 and Tyr314, the interaction region (antibody), and the 14 interprotein molecular hydrogen bonds might play important roles in the antigen–antibody interaction, and this finding may be useful for protein engineering of this antigen–antibody structure. In addition, the 3 residues Gly134, Gly158 and Tyr314 might be more important in the development of bioactive antibody analogs.

The structure of bioactive analogs of the N-terminal region of gastrin-17

Gastrin-17 (G17) processing intermediates bind to non-CCK receptors which mediate growth of the colonic mucosa but also the formation and development of colonic cancers. In previous studies, we removed the C-terminal region of G17 to form G17(1–12) and considerably shorter C-terminally amidated and non-amidated analogs. Peptides as short as G17(1–4) continued to bind to a single site on DLD-1 human colonic carcinoma cells, while only the G17(1–6)-NH 2 and G17(1–12) peptides retained the ability to activate the receptor and stimulate cell proliferation in vitro. In this report, we studied the structure of these analogs, using a combination of ECD and VCD spectroscopy and replica exchange molecular dynamics (REMD) simulations in water, TFE, and membrane-mimicking environments, in order to determine preferred conformations that may have importance in promoting the biological activities. Mostly random meander structures, punctuated by a β-turn at residues 1–4, were found in most peptides by REMD simulations. G17(1–3)-NH 2, which cannot form a β-turn, failed to bind the non-CCK receptor, suggesting the importance of this feature for binding. Additionally, the β-turn appeared more frequently in longer sequences, possibly explaining the higher affinity of the non-CCK receptor for these peptides seen previously. Finally, C-terminally amidated peptides generally showed greater formation of turn structure than their non-amidated counterparts as shown by ECD spectra, suggesting the importance of peptide length in stabilizing turn structure in N-terminal sequences, and perhaps explaining the ability of G17(1–6)-NH 2 to activate the non-CCK receptor where as the non-amidated G17(1–6) and shorter peptides do not.

Synthesis and reductive desulfurization of chiral non-racemic benzothienoindolizines. An efficient approach to a novel bioactive tylophorine alkaloid analogue and 6-phenylindolizidine

Enantiopure di- and tetrahydro[1]benzothieno[3,2- f]indolizines 3, 4, 9 and the benzothieno analogue 5 of the bioactive alkaloid tylophorine 10 were synthesized easily from the readily available benzo[ b]thiophene-3-carboxaldehyde 1 and ( S)-glutamic acid 2 in good overall yields. Applying a diastereoselective reductive desulfurization of benzo[ b]thiophene 3, 4 and 5 were readily converted into the enantiopure alcohols 11a–d, alcohols 11c,d and the target 6( R)-phenyl-8a( R)-indolizidine 6 (7 steps, 14%). During these reduction studies, the results obtained including the stereoinduction of the reaction are also discussed.

Complete Characterization of the Seventeen Step Moenomycin Biosynthetic Pathway

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

Synthesis of a COMC–estradiol conjugate for targeted, tissue-selective cancer chemotherapy

Guided by a revised mechanism of action for the antitumor agent 2-crotonyloxymethyl-(4 R,5 R,6 R)-4,5,6-trihydroxy-2-cyclohexenone (COTC) recently advanced by our laboratory, an estradiol conjugate of the bioactive COTC analog, COMC, has been synthesized.

Bioactive peptides: signaling the future

Natural processes within the body are modulated almost exclusively by the interaction of specific amino acid sequences, either as peptides or as subsections of proteins. With respect to skin, proteins and peptides are involved in the modulation of cell proliferation, cell migration, inflammation, angiogenesis, melanogenesis, and protein synthesis and regulation. The creation of therapeutic or bioactive peptide analogs of specific interactive sequences has opened the door to a diverse new field of pharmaceutical and active cosmetic ingredients for the skincare industry. Here, we describe the origin of such sequences, their role in nature, their application to dermatology, as well as the advantages and challenges posed by this new technology.

Application of FITC-labeled Ternatin on Its Cellular Localization in 3T3-L1 Murine Preadipocytes

Abstract The cellular localization of a potent fat-accumulation inhibitor (−)-ternatin was investigated. FITC-labeled ternatin, a chemical probe for current study, was synthesized from a highly bioactive ternatin analogue using Click Chemistry, which was found to be bioactive. The treatment of 3T3-L1 murine preadipocytes with synthetic probe revealed that (−)-ternatin is localized in a specific organelle of 3T3-L1 cells.

Enzymatic synthesis of perillyl alcohol derivatives and investigation of their antiproliferative activity

The monoterpene perillyl alcohol (POH), an intermediate in the plant terpenoid biosynthetic pathway, has well-established tumor chemopreventive and chemotherapeutic potential. We have previously shown that the primary hydroxyl group of POH is essential for its antitumor and anti-angiogenic activities. In the current study we present the enzymatic synthesis of two POH derivatives with different polar and hydrophobic characteristics, namely perillyl glucoside and perillyl glucoside fatty ester, through a two-step modification. Initial glucosylation of POH on its active hydroxyl group with D-(+)-glucose and subsequent esterification of the perillyl glucoside product with vinyl laurate were carried out using almond β-glucosidase and lipase B from Candida antarctica, respectively, in a low-water system. Optimization of enzymatic reactions was performed to achieve the highest possible conversion yields. The antitumor cell proliferation activity against mouse Lewis lung carcinoma cells was retained in both derivatives, although the perillyl glucoside ester showed greater inhibition than perillyl glucoside. Our results underline the feasibility of enzymatically producing novel bioactive analogs of phytochemicals displaying useful physicochemical properties.

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA Pyl for amber decoding as pyrrolysine. PylS and tRNA Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-(( R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.

Systemic Delivery of Nanoliposomal Ceramide Displays Anti-Leukemic Activity in a Rat Model of Large Granular Lymphocytic Leukemia

We have previously demonstrated that enhanced survival rather than increased proliferation accounts for the accumulation of natural killer (NK) cells in large granular lymphocyte (LGL) leukemia patients. To further elucidate the mechanism by which NK survival is enhanced, we analyzed leukemic NK cells isolated either from patient peripheral blood or rat splenic cells for altered expression of members of the inhibitor of apoptosis protein (IAP) family, which act as suppressors of apoptosis in a variety of human solid tumors and hematological malignancies. We now report that the IAP, survivin, was highly expressed in NK cells. In contrast, survivin was barely detectable in NK cells from the blood of normal human donors or the splenic cells from normal rats. We next asked if the lipid-derived second messenger, ceramide, which selectively induces apoptosis in cancer cells would diminish survivin protein expression. Treatment of NKL, a human NK-LGL leukemia cell line, or RNK-16, a rat NK-LGL leukemia cell line, with the cell-permeable, short-chain C6-ceramide (C6) in a pegylated liposomal formulation, led to cell apoptosis and diminished survivin protein expression, in a time and dose dependent manner. We next extended these in vitro studies to an in vivo rat model of NK-LGL leukemia. Systemic i.v. delivery of liposomal ceramide displayed significant anti-leukemia activity in a syngeneic Fischer F344 rat NK-LGL leukemia model that exhibits clonal expansion of CD3-CD8a+ lymphocytes. Over a 6-week treatment period, a well-tolerated dose of 40 mg/kg liposomal-C6, three times a week, elicited a 3 to 10- fold reduction in the weight of various lymphoid and non-lymphoid organs, compared with liposomal formulations that did not contain ceramide (ghost). Untreated or ghost-treated leukemic rats presented with lymphocytosis (LGL counts between 2 × 1011 and 3.5 × 1011/L), anemia and thrombocytopenia. Furthermore, the percentage of NK LGL cells, defined as CD3-CD8a+ by flow cytometry, was drastically elevated in the spleen, lymph node, thymus, bone marrow, blood, liver and lung in these leukemic rats, compared with their normal counterparts. In contrast, leukemic rats treated with liposomal ceramide had undetectable LGL cells in the blood and normal counts of red blood cells and platelets. Additionally, the CD3-CD8a+ NK cells in spleen, thymus and liver were found to be remarkably decreased, whereas the NK cells in bone marrow, blood and lung were within normal range. Collectively, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for anti-leukemic efficacy in a rat model of NK LGL leukemia, possibly via decreased survivin expression or signaling.

In Vitro and in Vivo Abrogation of An Erythroid Iron Restriction Developmental Checkpoint by Isocitrate through a Novel Erythropoietin- Coupled Signaling Mechanism

Erythropoietin (Epo) signaling drives normal erythropoiesis by promoting the survival, proliferation and maturation of committed erythroid progenitor cells. Epo acts at an early stage in erythroid development, prior to the initiation of hemoglobin synthesis. Under conditions of iron restriction, erythroid progenitors become refractory to Epo, resulting in hypoplastic anemia. The resulting iron restriction checkpoint protects iron stores from depletion by preventing Epo-driven erythroid expansion and inappropriate iron utilization for hemoglobin synthesis. In addition to diminished body stores, defects in iron uptake or intracellular trafficking can also activate this checkpoint and contribute to Epo-refractory anemias, e.g. sideroblastic anemias. Our previous work using primary human hematopoietic cultures had implicated aconitase enzymes, which interconvert citrate and isocitrate, as critical regulators of the erythroid iron-restriction checkpoint. In those studies, supplying cells with isocitrate had completely abrogated the block in erythroid development caused by iron restriction. In the current studies, we examine the mechanism for isocitrate rescue of erythropoiesis in iron deprived human progenitors and determine the in vivo effects of isocitrate administration in mice with iron deficiency anemia. Initial experiments addressed whether the activity of isocitrate was due to its catabolism to yield ATP and succinyl CoA, a precursor of heme. Several independent findings argued against such a metabolic mechanism. Firstly, erythroid progenitors showed no changes in cellular [ATP] with iron deprivation −/+ isocitrate. Secondly, a bioactive analog of alpha-ketoglutarate, TaKG, failed to rescue erythropoiesis under iron deprivation. Thirdly, isocitrate promoted erythroid differentiation in progenitors with blockade in mitochondrial biogenesis, induced by chloramphenicol. In these last studies, isocitrate reversed chloramphenicol inhibition of glycophorin A and globin expression; exogenous hemin by contrast reversed only the inhibition of globin expression. The combination of isocitrate and hemin, however, showed strong synergy in the rescue of growth and globin expression in cholaramphenicol treated progenitors. Subsequent experiments tested the hypothesis that isocitrate functions as a second messenger in erythroid development. Accordingly, iron deprived erythroid progenitors exposed to a range of Epo levels (0.05–20 U/ml) underwent isocitrate treatment. Remarkably, isocitrate showed no rescue of iron deprived erythroid cultures with 0.05 U/ml Epo, a dose that still promotes erythroid differentiation in high iron cultures. Partial rescue was obtained with 0.2 U/ml Epo, and complete rescue with > 4.5 U/ml. Previous studies have suggested that Epo-mediated calcium signaling shows a strong dosage dependency, requiring relatively high doses of Epo to activate calcium influx. In human CD34+ progenitors exposed to 4.5 U/ml Epo for either 5 hours or 3 days, iron deprivation induced a drop in steady state intracellular calcium levels. Inclusion of isocitrate in the medium restored the intracellular calcium to the levels seen in the iron-replete cultures. Finally, the in vivo activity of isocitrate was assessed in a murine model of iron deficiency anemia. Strikingly, intraperitoneal injections of isocitrate (200 mg/kg/day for 5 days) significantly augmented the red cell counts in C57BL/6 weanlings subjected to a low iron diet: mean RBC of 9.3 ± 0.35 × 10e12 cells/liter for the isocitrate group compared to an average RBC of 7.4 ± 0.51 × 10e12 cells/liter for the saline control group (P=0.012, N = 6 for each group). Interestingly, this increase accompanied a parallel decrease in red cell mean corpuscular hemoglobin concentration. Taken together, our results suggest a role for isocitrate as a second messenger coupled to Epo signaling and potentially involved in intracellular calcium regulation. In vivo administration abrogates the iron restriction checkpoint on red cell production, as in ex vivo studies, but cannot drive hemoglobin synthesis in the face of limited iron stores. The ability of isocitrate to collaborate with hemin in overriding erythroid mitochondrial defects offers novel therapeutic avenues for sideroblastic anemias.

The absolute configuration plays an important role in muscarinic activity of BGT-A and its analogs

Both enantiomers of 2, 3, and 4, three bioactive analogs of muscarinic agonist BGT-A were prepared respectively and underwent functional studies and radioreceptor binding assays. 6 S enantiomers of 2, 3, and 4 showed obvious muscarinic activity, while 6 R ones elicited little muscarinic activity by functional studies. Besides, the affinity of 6 S enantiomers of 2, 3, and 4 was greatly larger than that of their 6 R enantiomers respectively. All these pharmalogical results indicated the 6 S configuration was beneficial for the active BGT-A analogs to bind with the muscarinic receptors. The finding was in good agreement with our previous SAR study to BGT-A and its active analogs by computational approach. The understanding to the relationship between muscarinic activity and absolute configuration will provide the basis for successive screening of BGT-A analogs as effective muscarinic agonists or antagonists in clinical use.

Synthesis, structure, and bioevaluation of 2,5-bis(arylmethenyl)cyclopentanones

Curcumin is an excellent lead compound with a variety of bioactivity. Recent articles reported that curcumin's instability and low bioavailability in vivo are mainly due to its easily decomposable β-diketone moiety. With the aim of bioactive curcumin analogs with better pharmacokinetic property, we present here 11 bis(arylmethenyl)cyclopentanones similar to curcumin and without β-diketone moiety by reacting relevant arylaldehydes and cyclopentanones. The analogs were structurally determined by 1HNMR and MS spectra, and the crystal structure of 6 was analyzed by X-ray diffraction. Their antibacterial activities in vitro against seven Gram-positive and Gram-negative bacteria were tested, and their inhibition of TNF-α and IL-6 secretion in LPS-induced mouse macrophages was investigated using enzyme-linked immunosorbent assay. It was observed that several derivatives displayed higher activity when compared with curcumin, and most of the analogs exhibited activities against the ampicillin-resistant Gram-negative Enterobacter cloacae.

Expedient Synthesis of N-Methyl Tubulysin Analogues with High Cytotoxicity

An optimized and highly efficient synthesis of potent, bioactive N-methyl tubulysin analogues 2 and 4 has been achieved with > 40% overall yields. This synthesis represents a significant improvement over previously reported syntheses of these and related tubulysin analogues. The stereoselective synthesis of the unnatural amino acid tubuvaline is accomplished using tert-butanesulfinamide chemistry. N-Alkylation to form N-methyl tubuvaline is performed without protection of the tubuvaline alcohol by implementing a unique N-methylation strategy via formation and reduction of a 1,3-tetrahydrooxazine heterocycle. Acylation of the hindered N-methyl tubuvaline amine utilizes a novel sequence of O-acylation followed by an O- to N-acyl transfer to form the hindered amide bond between N-methyl tubuvaline and isoleucine. This high-yielding synthesis should enable the production of large quantities of material for biological studies.