Steven D. Townsend

Advances in Proline Ligation

Application of native chemical ligation logic to the case of an N-terminal proline is described. Two approaches were studied. One involved incorporation of a 3R-substituted thiyl-proline derivative. Improved results were obtained from a 3R-substituted selenol function, incorporated in the context of an oxidized dimer.

At last: erythropoietin as a single glycoform.

Given the central role of erythrocytes in enabling life, it is not surprising that there has evolved a highly sophisticated system for their production and orchestration. Broadly speaking, the multifaceted erythrocyte management process is encompassed in the term “erythropoiesis.”[1] In the early mammalian fetus, erythropoiesis begins in mesodermal cells in the yolk sac. With further development, the spleen and liver become the venues of erythrocyte production. Ultimately, the bone marrow becomes the “plant site” of erythropoiesis.[2] Upon sensing decreased oxygen in circulation, the kidneys secrete a hormone called erythropoietin (EPO). Contact of EPO with its receptor initiates signaling routines, which trigger erythropoiesis. Thus, EPO is clearly a major participant in erythropoiesis, which is central to life itself. The history of erythropoietin and the intellectual milestones leading to its recognition, demonstration, purification, sequencing, expression, production, and multifaceted medical ramifications (including applications to anemia induced by dialysis, and cancer chemotherapy) are continually being updated in various review forums.[3] Notwithstanding its celebrated status in biology and medicine, the term “erythropoietin” – insofar as it implies a chemically discrete entity – is a misnomer. Erythropoietin (EPO), as encountered by researchers and employed by physicians, is actually a large family of entities. The primary protein structure is highly conserved, as are its sites of glycosylation. Indeed, its sole O-linkage (to glycophorin) at Ser126 is substantially conserved. By contrast, the remaining three oligosaccharide domains (i.e. the N-linkages at asparagines 24, 38 and 83) are not under tight genetic supervision. This uncharacteristically permissive biomanagement leads to a highly complex medley of non-separable EPO glycoforms,[4] which has defied diligent efforts at separation.[5] To our knowledge, erythropoietin as a homogeneous chemical entity, containing a defined unitary array of N-linked carbohydrate domains, was unknown prior to our study. Curiously, our laboratory first became interested in erythropoietin from its presumably less functionally critical carbohydrate sectors. These complicated domains posed seemingly daunting problems from the perspective of organic synthesis. As we developed strategies and methods to deal with assembling suitably complex carbohydrate domains,[6] we began, in ca. 2002, to fantasize about the possibility of generating homogeneous erythropoietin itself, solely through the resources of organic chemistry. This paper describes a major advance in the realization of this goal. A prime reason that the EPO-directed venture gained increasing fascination in our chemistry–centered laboratory was the perception that, powerful as it was, the “state of the art” of protein synthesis was not then up to the task of solving the problem. It seemed that new methods, and conceptual advances would be necessary to synthesize EPO by chemical means. The two vital field resources, then available toward the synthesis of an EPO-sized protein, were step-by-step solid phase peptide synthesis (SPPS)[7] and possible ligations for merging polypeptides. While there were, and still are, no reliable rules limiting the size of a polypeptide which is accessible by linear reiterative SPPS, the size of the EPO protein we were after (166-mer), in the context of its seriously hydrophobic stretches, not to speak of its four carbohydrate domains, seemed to place it out of the range of SPPS, per se. Rather, SPPS, properly employed, could hopefully provide for the synthesis of useful (i.e. combinable) fragments of EPO. It would then be necessary to ligate judiciously selected subunits, bearing N- and O-oligosaccharide domains to reach target EPO, itself. Of the ligation methods then available, the seminal native chemical ligation (NCL) protocols of Kent and associates were certainly the most powerful.[8] However, the NCL method requires an N-terminal cysteine at the N→C ligation site (see Figure 2). Examination of the primary structure of EPO protein reveals that the positioning of its four cysteine residues is such that NCL per se would be of likely value only for the Cys29 (or Cys33) site. Figure 2 New Methods for the synthesis of proteins and glycoproteins. GP = glycopeptide. In Figure 2, we briefly summarize a menu of new methods that were developed for EPO and related protein-based synthesis projects. Of particular interest to this program early on, was the development of the ortho-mercaptoaryl ester rearrangement (OMER) methodology.[9] Thus, an incipient C-terminal thioester is generated from a phenyl ester following TCEP mediated cleavage of an ortho positioned disulfide bond (Figure 2b). The thioester, thus generated, participates as the C-terminus in an NCL ligation, or in HOBT–mediated ligations with other N-terminal protected but non-cysteine containing peptides. Moreover, it was shown that NCL could be applied to the coupling of glycopolypeptide fragments.[10a] This complements an earlier report of a NCL between two O-linked glycopeptides.[10b] Another advance, which was stimulated by EPO and related projects, involved hindered isonitrile mediated N-terminal elongation of a polypeptide chain with an N-protected thioacid (Figure 2c).[11] Finally, and most critically for the purpose at hand, we were able to accomplish major extensions of NCL, occasioned by the discovery of metal-free dethiylation (MFD)[12] (Figure 2d). This finding has had a huge enhancing effect on the reach of NCL. Our first application of MFD was to enable N-terminal alanine ligations.[12] NCL logic has been extended, from our laboratory and others, to embrace ligations at N-terminal valine,[13] leucine,[14] lysine,[15] threonine,[16] proline,[17] and phenylalanine.[18] Cumulatively, these capabilities served to change the landscape of retrosynthetic analysis in the polypeptide field, by building into planning exercise, options for using non-cysteine containing N-terminal fragments, which would reenter the world of proteogenic amino acids through MFD.[12a] The first demonstration of the consequences of this capability in the context of building therapeutic-sized proteins arose from our recently reported synthesis of the human parathyroid hormone (hPTH),[19a] as well as truncated versions thereof.[20] Following this demonstration, the combined NCL/MFD logic for the synthesis of glycoproteins has been reported by other laboratories.[19b,c] As it turned out, our earlier engagements in trying to synthesize erythropoietin preceded some of these enabling discoveries. Our most advanced point previous to this disclosure, using largely OMER technology, as well as applications to glycopeptide synthesis, brought us to three fragments – EPO(1-28),[21] EPO(29-77),[22] and EPO(78-166)[23] – which formally corresponds to erythropoietin in need of two ligations (Figure 3). Unfortunately, the weak acyl donor and acyl acceptor reactivity of the various fragments, in the face of solubility problems with the partially protected substrates, and serious aggregation tendencies, served to frustrate all attempts to join EPO(78–166) with EPO(29–77) in a meaningful yield. Figure 3 Earlier synthetic routes from our laboratory toward EPO. Fortunately, at the time that these major limitations were surfacing, the MFD method, which vastly extended the logic of NCL, came into use. At that difficult juncture, we could undertake a revised plan for the total synthesis of erythropoietin, the success of which we are pleased to present below. A central question that we were addressing in the first instance was the ability to implement the new technologies described above to reach a homogeneous, “wild-type” erythropoietin. Unlike concurrent and illuminating programs in other laboratories, which were also exploiting the possibilities of EPO retrosyntheses based on MFD technology,[24] we set as a non-compromisable condition, that our target would be strictly of the wild-type, rather than contain artificial mutants to simplify handling and isolation. Furthermore, we adopted as a sine qua non that all three wild-type asparagine sites, and the one serine site be glycosylated. In so doing, we were asking whether we could obtain indications for erythropoietic activity from a homogeneous, but more simply, glycosidated EPO, lacking the “high mannose” and sialic acid containing sectors (see Figure 1). We would hope to determine whether a structure of that sort would be foldable and would manifest both activity and stability. Finally, we hoped to compare the properties of such a homogeneous synthetically derived construct with those of wild-type “aglycone protein”. In this fashion, we would be providing the scientific basis need to address the fascinating question as to why nature glycosidates many of its most precious proteins. Figure 1 Ribbon structure of erythropoietin containing a consensus sequence of N-linked carbohydrate domains.

Discovery of a Novel Chemical Class of mGlu5 Allosteric Ligands with Distinct Modes of Pharmacology

We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu5) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu5, distinct from the classical mGlu5 negative allosteric modulator (NAM) MPEP allosteric site. However, a shallow structure−activity relationship (SAR), poor physiochemical properties, and weak PAM activity at rat mGlu5 limited the utility of CPPHA to explore allosteric activation of mGlu5 at a non-MPEP site. Thus, we performed a functional high-throughput screen (HTS) and identified a novel mGlu5 PAM benzamide scaffold, exemplified by VU0001850 (EC50 = 1.3 μM, 106% Glumax) and VU0040237 (EC50 = 350 nM, 84% Glu Max). An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu5 PAM activity to afford VU0357121 (EC50 = 33 nM, 92% Glumax), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396). Like CPPHA, PAMs within this class do not appear to bind at the MPEP allosteric site based on radioligand binding studies. Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu5, distinct from CPPHA, yet share a functional interaction with the MPEP site.

Halocycloalkenones as Diels-Alder Dienophiles. Applications to Generating Useful Structural Patterns

Halocycloalkenones are demonstrated to function as potent dienophiles in inter- and intramolecular Diels-Alder cycloadditions. We have found 2-brominated cycloalkenone dienophiles to be both highly endo selective and significantly more reactive than their nonhalogenated parent compounds. A method for the facile conversion of brominated cyclobutanone DA adducts to synthetically useful cyclopropyl functional handles is described.

Chemistry as an Expanding Resource in Protein Science: Fully Synthetic and Fully Active Human Parathyroid Hormone‐Related Protein (1–141)

Human parathyroid hormone related protein (hPTHrP), originally isolated from lung cancer cell lines in 1987, [1] is a 141-amino acid polypeptide widely found in both normal and tumor tissue cells. The N-terminal region of hPTHrP possesses a high degree of structural homology with human parathyroid hormone (hPTH), and both hormones effect the elevation of calcium levels in the blood.[2] Although PTH and PTHrP act through binding to the same receptor, the PTH-receptor type-1 (PTHR1), in vitro studies suggest that the two ligands may differ in the precise molecular modes of their receptor interactions.[3,4] Under normal conditions, hPTHrP, which is widely expressed in the tissues of embryos and adults, plays an essential role in a range of functions related to development and growth, including: fostering of the cartilaginous growth plate, [5] bone anabolism, [6] development of mammary gland, [7] transport of calcium ions across the placenta, [8] relaxation of smooth muscle, or vasodilatation, [9] and eruption of tooth.[10] In analogy to the related anti-osteoporosis therapeutic agent, PTH, researchers have found that PTHrP, administered daily, may induce anabolic effects on the skeleton. Interestingly, the risk of hypercalcemia associated with PTH–based therapeutics may be lowered with the use of PTHrP. These findings raise the possibility that PTHrP and/or congeners, thereof could offer an advantage over currently used PTH peptides in therapeutics related to osteoporosis. A growing understanding of the role that hPTHrP may play in mediating the progression of cancer further enhances interest in this polypeptide. An intriguing property of hPTHrP is the finding that it exhibits anti-apoptotic and proliferation–promoting effects on tumor cells.[11–14] Recent studies have shown that antagonists of PTHR1 are able to remarkably inhibit the growth of tumors.[15–17] The development of an efficient synthetic route to homogeneous hPTHrP, and analogs thereof, would facilitate the systematic study of the interaction between hPTHrP and its receptor, PTHR1. Such research would offer important insights into the structure-activity relationship (SAR) of the polypeptide, and could well facilitate the development of practical PTHR1 antagonists, to suppress the growth of tumors, or agonists, for the treatment of osteoporosis.[18] Certainly, one could imagine that a wisely crafted hPTHrP lookalike could have exploitable antiproliferative properties. In our judgment, the synthesis of protein targets offers significant learning opportunities at the interface of chemistry, biology and medicine.[19] The advantage of pursuing chemistry based approaches to protein targets arises from the fact that this forum uniquely allows for the versatile design of unnatural probe structures possessing defined alterations of amino sequence and structure, including the incorporation of non-proteogenic amino acids.[20–22] Notwithstanding impressive accomplishments in protein engineering, which were enabled by spectacular advances in molecular biology, we have felt that chemical based synthesis, in principle, also has much to offer in terms of reaching a specific protein target, in reasonable research–level quantities (usually several milligrams), above all with very high levels of homogeneity. Thus, the purposes of this research were several. First, we hoped to reach hPTHrP by purely chemical means, and to show that it manifests full biological function. With this accomplished, the basis for an SAR program, involving alterations of primary structure (proteogenic and non-proteogenic amino acid substitutions) and molecular constraints, would be solidly in place. More broadly, we would be exploring, albeit in only a preliminary fashion, prospects for using chemistry as a major resource in protein discovery science.[23] The field of protein chemical synthesis was greatly advanced with the discovery of cysteine-based native chemical ligation (NCL), by Kent and co-workers.[24–26] More recently, the scope of NCL has been expanded to encompass a wide range of non-cysteine amino acids, through methods developed in our laboratory and others.[27–33] As outlined in Figure 1, the general non-cysteine based NCL strategy adopted by our group involves the installation of a temporary thiol functionality on the N-terminal amino acid residue at the site of ligation. Following amide bond formation, the polypeptide or glycopeptide is exposed to mild, metal-free dethiylation conditions, resulting in the selective removal of the extraneous thiol functionality. Figure 1 In a demonstration of the applicability of this ligation strategy to the assembly of challenging polypeptides lacking Cys residues, we recently disclosed total syntheses of hPTH, [34] and analogs thereof. Using these methods, we have now achieved the de novo total synthesis of hPTHrP (1–141).[35] We describe herein the synthesis and demonstration of biological activity of our synthetic hPTHrP (1– 141) polypeptide and a truncated analog, hPTHrP (1–37).

Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial pathogen that causes invasive infections in both children and adults. During pregnancy, GBS is a significant cause of infection of the fetal membranes (chorioamnionitis), which can lead to intra-amniotic infection, preterm birth, stillbirth, and neonatal sepsis. Recently, breastfeeding has been thought to represent a potential mode of GBS transmission from mother to newborn, which might increase the risk for late-onset sepsis. Little is known, however, about the molecular components of breast milk that may support or prevent GBS colonization. In this study, we examine how human milk oligosaccharides (HMOs) affect the pathogenesis of GBS. HMOs from discrete donor samples were isolated and profiled by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Growth and biofilm assays show that HMOs from mothers of specific milk groups can modulate the growth and biofilm formation of GBS. High-resolution field-emission gun scanning electron microscopy (SEM) and confocal laser scanning microscopy confirmed the quantitative biofilm assays and demonstrated cell arrangement perturbations in bacterial cultures treated with specific oligosaccharides. These findings demonstrate that HMOs affect the growth and cell biology of GBS. Finally, this study provides the first example of HMOs functioning as antibiofilm agents against GBS.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Infant food applications of complex carbohydrates: Structure, synthesis, and function

Professional health bodies such as the World Health Organization (WHO), the American Academy of Pediatrics (AAP), and the U.S. Department of Health and Human Services (HHS) recommend breast milk as the sole source of food during the first year of life. This position recognizes human milk as being uniquely suited for infant nutrition. Nonetheless, most neonates in the West are fed alternatives by 6 months of age. Although inferior to human milk in most aspects, infant formulas are able to promote effective growth and development. However, while breast-fed infants feature a microbiota dominated by bifidobacteria, the bacterial flora of formula-fed infants is usually heterogeneous with comparatively lower levels of bifidobacteria. Thus, the objective of any infant food manufacturer is to prepare a product that results in a formula-fed infant developing a breast-fed infant-like microbiota. The goal of this focused review is to discuss the structure, synthesis, and function of carbohydrate additives that play a role in governing the composition of the infant microbiome and have other health benefits.

Metal-Free Synthesis of Unsymmetrical Organoselenides and Selenoglycosides

A one-pot, metal-free procedure has been developed to synthesize unsymmetrical organoselenides. In the first step of the reaction, arylation of potassium selenocyanate (KSeCN) with an iodonium reagent proceeds in the absence of a metal catalyst to produce an arylselenocyanate. In the second step, treatment with sodium borohydride unmasks a second selenium nucleophile that engages an aliphatic electrophile, iodonium reagent, or glycosyl halide. The procedure represents an umpolung approach to the synthesis of aryl-selenides.

Synthesis of lacto-N-tetraose

Human milk oligosaccharides (HMOs) are the third largest macromolecular component of breast milk and offer infants numerous health benefits, most of which stem from the development of a healthy microbiome. Characterization, quantification, and chemical derivatization of HMOs remains a frontier issue in glycobiology due to the challenge of isolating appreciable quantities of homogenous HMOs from breast milk. Herein, we report the synthesis of the human milk tetrasaccharide lacto-N-tetraose (LNT). LNT is ubiquitous in human breast milk as it is a core structure common to longer-chain HMOs and many glycolipids.