AIMS:Polymyxins are generally recommended to be used in combination with other antibiotics for improving their antibacterial effects. As for treating carbapenem-resistant Acinetobacter baumannii infections, which antibiotic is the best option for polymyxin B-based combination therapy is still under debate. Resveratrol, a natural compound that can be well-tolerated by the human body, may have potential value in the polymyxin B-based combination therapy against carbapenem-resistant A. baumannii infections. METHODS AND RESULTS:With checkerboard assays and time-killing studies, we have found that resveratrol increased the sensitivity of carbapenem-resistant A. baumannii to polymyxin B and enhanced the bactericidal activity of polymyxin B against carbapenem-resistant A. baumannii. With biofilm formation studies, we have confirmed that resveratrol potentiated the antibiofilm activity of polymyxin B and improved its bactericidal activity against the bacterial cells with established biofilms. CONCLUSIONS:Resveratrol potentiates the antibiofilm and antibacterial activity of polymyxin B against carbapenem-resistant A. baumannii. Polymyxin B used in combination with resveratrol may have better clinical efficacy than polymyxin B monotherapy in the treatment of carbapenem-resistant A. baumannii infections.

Sodium channel Nav1.7 triggers the generation of nociceptive action potentials and is important in sending pain signals under physiological and pathological conditions. However, studying endogenous Nav1.7 currents has been confounded by co-expression of multiple sodium channel isoforms in dorsal root ganglion (DRG) neurons. In the current study, slow-repriming (SR) and fast-repriming (FR) tetrodotoxin-sensitive (TTX-S) currents were dissected electrophysiologically in small DRG neurons of both rats and mice. Three subgroups of small DRG neurons were identified based on the expression pattern of SR and FR TTX-S currents. A majority of rat neurons only expressed SR TTX-S currents, while a majority of mouse neurons expressed additional FR TTX-S currents. ProTx-II inhibited SR TTX-S currents with variable efficacy among DRG neurons. The expression of both types of TTX-S currents was higher in Isolectin B4-negative (IB4−) compared to Isolectin B4-positive (IB4+) neurons. Paclitaxel selectively increased SR TTX-S currents in IB4− neurons. In simulation experiments, the Nav1.7-expressing small DRG neuron displayed lower rheobase and higher frequency of action potentials upon threshold current injections compared to Nav1.6. The results suggested a successful dissection of endogenous Nav1.7 currents through electrophysiological manipulation that may provide a useful way to study the functional expression and pharmacology of endogenous Nav1.7 channels in DRG neurons.

Radix angelicae pubescentis (RAP) has been used in Chinese traditional medicine to treat painful diseases such as rheumatism and headache. A previous study has reported that columbianadin (CBN), a major coumarin in RAP inhibits acute and inflammatory pain behaviors. However, the effects of CBN on neuropathic pain behaviors, and the potential underlying mechanism have not been reported. In the present study, the effects of CBN, compared to another major coumarin of RAP osthole (OST), on oxaliplatin-induced neuropathic pain behaviors and on the voltage-gated calcium currents in small dorsal root ganglion (DRG) neurons were studied in mice. It was found that CBN and OST inhibited both mechanical and cold hypersensitivity induced by oxaliplatin. Moreover, CBN and OST might preferentially inhibit T- and L-type calcium currents (Ica). The inhibitory effects of CBN and OST on the oxaliplatin-induced mechanical allodynia were prevented by gabapentin. These results suggest that CBN, as well as OST might inhibit neuropathic pain behaviors through an inhibition of T- and L-type calcium currents in nociceptive DRG neurons.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need for chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analoging. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in linker, peptide loading, and antibody attachment site. We found conjugates with 100x improved in vitro potency relative to complementary GpTx-1 analogs, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold whilst retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analog with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss in peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.

Gating modifier toxins (GMTs) from spider venom can inhibit voltage gated sodium channels (NaVs) involved in pain signal transmission, including the NaV1.7 subtype. GMTs have a conserved amphipathic structure that allow them to interact with membranes and also with charged residues in regions of NaV that are exposed at the cell surface. ProTx-II and GpTx-1 are GMTs able to inhibit NaV1.7 with high potency, but they differ in their ability to bind to membranes and in their selectivity over other NaV subtypes. To explore these differences and gain detailed information on their membrane-binding ability and how this relates to potency and selectivity, we examined previously described NaV1.7 potent/selective GpTx-1 analogues and new ProTx-II analogues designed to reduce membrane binding and improve selectivity for NaV1.7. Our studies reveal that the number and type of hydrophobic residues as well as how they are presented at the surface determine the affinity of ProTx-II and GpTx-1 for membranes and that altering these residues can have dramatic effects on NaV inhibitory activity. We demonstrate that strong peptide-membrane interactions are not essential for inhibiting NaV1.7 and propose that hydrophobic interactions instead play an important role in positioning the GMT at the membrane surface proximal to exposed NaV residues, thereby affecting peptide-channel interactions. Our detailed structure-activity relationship study highlights the challenges of designing GMT-based molecules that simultaneously achieve high potency and selectivity for NaV1.7, as single mutations can induce local changes in GMT structure that can have a major impact on NaV-inhibitory activity.

a composition of matter which comprises an isolated polypeptide which is an inhibitor of NaV1.7 peripherally restricted described. In some embodiments disclosed, the isolated polypeptide is an inhibitor of NaV1.7. Other embodiments are conjugated embodiments of the composition of matter of the application and pharmaceutical compositions containing the composition of matter of the application. Disclosed are isolated nucleic acids encoding some embodiments of polypeptides and expression vectors of the application, as well as recombinant host cells containing them. It also discloses a method for treating or preventing pain. Claim 1: A composition of the matter, comprising an isolated polypeptide comprising the amino acid sequence of formula: // Xaa¹Xaa²Xaa³Xaa⁴Xaa⁵Xaa⁶Xaa⁷Xaa⁸Xaa⁹Xaa¹⁰Xaa¹¹Asp¹²Xaa¹³Xaa¹⁴Xaa¹⁵Xaa¹⁶Xaa¹⁷Xaa¹⁸Xaa¹⁹Xaa²⁰Leu²¹Xaa²²Xaa²³Xaa²⁴Xaa²⁵Xaa²⁶Xaa²⁷Xaa²⁸Xaa²⁹Xaa³⁰Xaa³¹Xaa³²Xaa³³Xaa³⁴ SEQ. ID No. 590 or a pharmaceutically acceptable salt thereof, wherein: Xaa¹Xaa² is absent; or Xaa¹ is any amino acid residue and Xaa² is any amino acid residue; or it is absent and Xaa² Xaa¹ is any amino acid residue; or Xaa¹ is absent and Xaa² is absent; Xaa³ is any amino acid residue; Xaa⁴ is Cys, if Xaa¹⁸ is Cys; or Xaa⁴ is SeCys, if Xaa¹⁸ is SeCys; Xaa⁵ is any neutral or basic hydrophilic amino acid residue; Xaa⁶ is any basic or neutral hydrophilic amino acid residue; Xaa⁷ is Trp, 5-bromoTrp, 6-bromoTrp, 5-cloroTrp, 6-cloroTrp, 1-Nal, 2-Nal, tioTrp, BhPhe, 2-BrhF, 2-ClhF, 2-FHF, 2-MEHF, residue 2-MeOhF, 3-BrhF, 3-ClhF, 3-FHF, 3-MEHF, 3-MeOhF, 4-BrhF, 4-ClhF, 4-FHF, 4-me-F, 4-MEHF, 4-MeOhF; Xaa⁸ is Met, Nle, Nva, Leu, Ile, Val, or Phe residue; Xaa⁹ is Trp, 5-bromoTrp, 6-bromoTrp, 5-cloroTrp, 6-cloroTrp, 1-Nal, 2-Nal, or tioTrp residue; Xaa¹⁰ is a basic or neutral hydrophilic amino acid residue, or Ala residue; Xaa¹¹ is Cys if Xaa²³ is Cys; or Xaa¹¹ is SeCys if Xaa²³ is SeCys; Xaa¹³ is any amino acid residue; Xaa¹⁴ is a basic or acid residue or an Ala residue; Xaa¹⁵ is Arg or Cit residue; Xaa¹⁶ is any amino acid residue; Xaa¹⁷ is Cys if Xaa²⁷ is Cys; or Xaa¹⁷ is SeCys if Xaa²⁷ is SeCys; Xaa¹⁸ is Cys or SeCys; Xaa¹⁹ is any amino acid residue; Xaa²⁰ is Gly, Asp or Ala residue; Xaa²² is an acidic, basic or neutral hydrophilic amino acid residue, or an Ala or Val residue; Xaa²³ is a Cys or SeCys residue; Xaa²⁴ is a basic or neutral hydrophilic amino acid residue or an Ala residue; Xaa²⁵ is an aliphatic hydrophobic residue; Xaa²⁶ is Trp, 5-bromoTrp, 6-bromoTrp, 5-cloroTrp, 6-cloroTrp, 1-Nal, 2-Nal, tioTrp, 5-fenilTrp, 5-iPrTrp, 5-etilTrp, or 5-MeTrp residue; Xaa²⁷ is a Cys or SeCys residue; Xaa²⁸ is a hydrophilic amino acid residue basic or neutral; Xaa²⁹ is a basic amino acid residue, or Tyr or Leu residue; Xaa³⁰ is Ile, Trp, Tyr, 5-bromoTrp, 6-bromoTrp, 5-cloroTrp, 6-cloroTrp, 1-Nal, 2-Nal, tioTrp, 1-Nal or 2-Nal residue, if Xaa²² is a residue acid amino acid; or is an amino acid residue Xaa³⁰ acid or Pro residue, if Xaa²² is a basic or neutral hydrophilic amino acid residue or an Ala or Val residue; Xaa³¹ is Ile, Trp, Phe, Cha, Tyr, 5-bromoTrp, 6-bromoTrp, 5-cloroTrp, 6-cloroTrp, 1-Nal, 2-Nal, tioTrp, or 4-tBu-F residue; each Xaa³², Xaa³³ and Xaa³⁴ is independently absent or is independently an amino acid residue or hydrophobic acid or Ser or Gly residue; and wherein: if Xaa⁴ and Xaa¹⁸ are both Cys, a disulfide bond exists between the residue and Xaa¹⁸ Xaa⁴ residue; or Xaa⁴ and Xaa¹⁸ are each residues SeCys, there is a diselenide bond between the residue and Xaa¹⁸ Xaa⁴ residue; if Xaa¹¹ and Xaa²³ are both Cys, a disulfide bond exists between the residue and Xaa²³ Xaa¹¹ residue; or Xaa¹¹ and Xaa²³ are each residues SeCys, there is a diselenide bond between the residue and Xaa²³ Xaa¹¹ residue; if Xaa¹⁷ and Xaa²⁷ are both Cys, a disulfide bond exists between the residue and Xaa²⁷ Xaa¹⁷ residue; or Xaa¹⁷ and Xaa²⁷ are each residues SeCys, there is a diselenide bond between the residue and Xaa²⁷ Xaa¹⁷ residue; the amino terminal residue is optionally acetylated, biotinylated or 4-pentinoilado, or PEGylated; and the carboxy terminal residue is optionally amidated.

The Ala(1)-Gly(28) glycopeptide fragment (28) of EPO was prepared by chemical synthesis as a single glycoform. Key steps in the synthesis include attachment of a complex dodecasaccharide (7) to a seven amino acid peptide via Lansbury aspartylation, native chemical ligation to join peptide 19 with the glycopeptide domain 18, and a selective desulfurization at the ligation site to reveal the natural Ala(19). This glycopeptide fragment (28) contains both the requisite N-linked dodecasaccharide and a C-terminal (alpha)thioester handle, the latter feature permitting direct coupling with a glycopeptide fragment bearing N-terminal Cys(29) without further functionalization.

A synthesis of EPO 22–37 glycopeptide (1), presenting the N-linked dodecasaccharide of erythropoietin, is described.

A synthesis of EPO 114–166 glycopeptide (1), presenting the O-linked glycophorin of erythropoietin, is described.

The missing link: Reiterative ligations based on non-cysteine and cysteine-based acyl acceptors have been used to prepare synthetic polypeptides and proteins with multiple sites of glycosylation. Highly complex positionally defined glycopolypeptides corresponding to single glycoforms can be formed from simple components. The development of increasingly efficient and general methods for the merging of complex peptidic fragments remains a central objective in the field of polypeptide and glycopolypeptide synthesis. A number of traditional native chemical ligation (NCL) techniques have been applied to the problem of polypeptide assembly through convergent ligation.1 Nearly all of these require the presence of an N-terminal cysteine residue to function as the acyl acceptor. Given the relative scarcity of cysteine residues in nature, a clear impetus arises for the realization of new NCL capabilities. Our laboratory has a major interest in the development of methods for the preparation of homogeneous, fully synthetic polypeptides, and even proteins, that display multiple sites of glycosylation. A particularly relevant glycoprotein, which serves to coordinate and focus our efforts in this field, is the naturally occurring erythropoietin alpha (EPO; Figure 1).2 This multiply glycosylated protein has found widespread therapeutic application in the treatment of anemia. Despite the clear-cut clinical importance of this compound, attempts to rigorously evaluate the role of glycosylation on the activity and stability of erythropoietin have thus far been complicated by the daunting difficulties associated with isolating significant quantities of homogeneous EPO.3 On the basis of our long-term involvement in the arena of carbohydrate and glycopeptide total synthesis,4 we reasoned that if the considerable powers of chemical synthesis were brought to bear on this problem in a principled and focused fashion, it might be possible to gain access to fully synthetic, homogeneous erythropoietins. Needless to say, such a capability would likely enable access to a range of EPO analogues for structure–activity-relationship investigations. Broadly speaking, an undertaking of this magnitude could well lead to the development of generally useful strategies and protocols of utility to the entire field of glycoprotein synthesis. Structure of erythropoietin. Erythropoietin is a 166-residue protein possessing four sites of glycosylation. Three of these are N linked to asparagine residues and one is O linked to a serine residue. In considering a strategy for the de novo synthesis of erythropoietin, we took note of the paucity of cysteine residues on the molecule. A maximally convergent route to erythropoietin would involve the preparation of the four different glycopeptide fragments, which would subsequently be joined through some form of ligation to furnish the fully glycosylated protein backbone. Thus, in light of the fact that the four cysteine residues of erythropoietin do not segregate into nearly equal-sized carbohydrate-bearing domains, we were led to consider glycopeptide ligation methods that do not require cysteine-based acyl acceptors. The development of a repertoire of ligation methods could well be critical to the success of our EPO-directed venture. Scheme 1 a shows, in the most general terms, a scenario wherein two differentially glycosylated peptide fragments (1 and 2) would be temporarily engaged through an auxiliary linker5 such that the C-terminal coupling fragment would be activated as a thioester (3). Having been coaxed into proximity, the N-terminal coupling partner would attack the thioester, thus forming the key amide bond (4). Removal of the auxiliary would provide the doubly glycosylated peptide 5. In this way, the limitation of a cysteine acyl acceptor situated at the N-terminus would have been removed. Cysteine-free ligation strategy. See text for details. One conceivable manifestation of this general strategy would commence with the covalent appendage of a sulfur-displaying auxiliary of the type 6 to the N terminus of an appropriate peptide fragment (7) through reductive amination.6 In this early feasibility demonstration, we benefited from a recently disclosed clever idea of Dawson et al. for a non-cysteine-based ligation, albeit by a different organizing step.7 The resultant intermediate would be advanced to the glycopeptide 9. At this point, we envisioned joining the two fully functionalized glycopeptide fragments under conditions analogous to those that we had previously developed in the context of a cysteine-based NCL method.5a Thus, the coupling partner, 8, would be equipped with a C-terminal phenolic ester, as shown in Scheme 1 b. Under disulfide reducing conditions, the C-terminal coupling partner, 8, would be activated to form a thioester with the auxiliary sulfur functionality of the N-terminal fragment, thus providing an intermediate of the type 10. Amide-bond formation followed by auxiliary removal would provide the bifunctional glycopeptide 5. In the end, we elected to equip the sulfur atom of the auxiliary with a PMB protecting group.8 We anticipated that deprotection of this group could be accomplished under mild conditions that are compatible with survival of glycopeptide functionality (see Scheme 2). Thus, aldehyde 11 was prepared through slight modification of a known procedure.7a Reductive amination with hexapeptide 12 provided 13 in 60 % yield. This ability to connect a substantial N-terminal domain to the auxiliary by reductive amination is an important feature of this methodology. Next, the disaccharide 14 was appended to the peptide through reducing-end amination and aspartylation.9 At this stage, we sought to convert the SPMB group into an aromatic disulfide under mild conditions that would leave the sensitive glycopeptide functionality intact. We were pleased to find that, following treatment of 15 with sufenyl chloride 16 in TFE/DCM, compound 17 was obtained.10 Notably, this transformation represents an appealing alternative to previously described conditions that typically require exposure to harsh reagents such as anhydrous HF or Hg(OAc)2 and TFA.6a, 7a Synthesis of compound 17. Reaction conditions, a) NaCNBH3, MeOH/DMF, 60 %; b) 14, HATU, iPr2NEt, DMSO, 62 %; c) 16, TFE/CH2Cl2, 70 %. Ac=acetyl, DMF=N,N-dimethylformamide, DMSO=dimethyl sulfoxide, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N',N′-tetramethyluronium hexafluorophosphate, PMB=para-methoxybenzyl, TFE=2,2,2-trifluoroethanol. With the auxiliary-bearing glycopeptide fragment 17 in hand, we were now prepared to investigate the viability of our cysteine-free coupling strategy. In this context, we first examined a Gly–Ala ligation. As expected, upon exposure to TCEP in PBS buffer solution (pH 8.0), glycopeptides 17 and 18 each underwent reductive disulfide cleavage (see Scheme 3). Presumably, the C-terminal glycopeptide then suffered an O→S acyl transfer of the type previously described to generate the activated thioester (19).11 As anticipated, 19 and 20 were temporarily joined through a thioester exchange reaction. Following intramolecular acyl transfer, the fully functionalized glycopeptide 21 was isolated in 37 % yield.12 Synthesis of 21. Reaction conditions, a) TCEP, PBS (pH 8.0), 37 %. Aux=auxiliary, Dmab=4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzyl, PBS=phosphate-buffered saline buffer solution, TCEP=tricarboxyethylphosphine. Encouraged by this early success, we next sought to explore the generality of the method by attempting ligation at a more challenging Gly–Gln center. Thus, glycopeptides 22 and 23 (Scheme 4) were prepared and subjected to ligation conditions (TCEP in PBS buffer solution; pH 8.0). Unfortunately, the yield of 24 was found to be quite low, and a significant quantity of carboxylic acid arising from the hydrolysis of 22 was observed. We postulated that, although in the previous instance (17+18→21) the rate-determining step of the sequence had been the joining of the two fragments through transthioesterification, in the case at hand, the increased steric hindrance around the reacting center had caused the intramolecular acyl transfer to become rate limiting. Consequently, hydrolysis of the tethered intermediate had the opportunity to intervene as a competitive side reaction. Although we were unable to improve upon the product distribution by adjusting the pH of the system, we did find that, by introducing DMF as a cosolvent with a small amount of Na2HPO4, we were able to isolate the ligation product 24 in a more-acceptable 54 % yield. Ligation of 22 and 23. Reaction conditions, a) TCEP, DMF, Na2HPO4, 32 °C, 54 %. Having demonstrated the capacity of our methods to successfully ligate two N-linked glycopeptide domains, we next sought to investigate its compatibility with O-linked glycodomains. Thus, intermediate 25 was advanced to 26 through a three-step sequence, as shown in Scheme 5.13 The latter was converted into the N-terminal coupling partner, 27, according to previously developed reaction conditions. We were pleased to find that, upon exposure to TCEP and DMF with Na2HPO4, 27 and 28 readily underwent cysteine-free native chemical ligation to provide glycopeptide 29, which possesses both N- and O-linked carbohydrate domains. Notably, no carbohydrate decomposition products were observed. Ligation of O-linked glycodomains. Reaction conditions, a) TFA, PhOH, H2O, TESH; b) 0.1 n NaOH, MeOH; c) H2NNH2, MeOH, 61 % over 3 steps; d) 16, TFE, 67 %; e) TCEP, DMF, Na2HPO4, 65 %. TFA=trifluoroacetic acid, TESH=triethylsilane. We next validated our methodology in the context of more-complex glycan fragments, including those containing characteristic non-reducing and sialic acid moieties. The extent of sialidation is apparently a determinant of EPO stability.14 Thus, the coupling of two glycopeptide fragments, each displaying an N-linked core pentasaccharide, was found to proceed smoothly to provide the bifunctional glycopeptide 30 (Scheme 6). Bifunctional glycopeptide. The final phase of this investigation would be the development of appropriately mild conditions for the cleavage of the thiol auxiliary. In this context, TFA with a scavenger has been used in similar types of systems. However, in our hands, the treatment of the ligation product with 95 % TFA with a triisopropyl silane (TIPS) scavenger resulted in a mixture of the desired native glycopeptide along with another compound of the same molecular weight as the starting glycopeptide. The latter was tentatively assigned to be the thioester intermediate, arising from acid-mediated intramolecular N→S acyl transfer.15 Presumably, the otherwise endothermic step is driven by irreversible protonation of the benzylic amine. In light of this finding, a two-step sequence (Scheme 7) was devised for the removal of the auxiliary. First, intermediate 24 was treated with methyl p-nitrobenzene sulfonate.16 This step accomplished selective methylation of the sulfur on the aromatic ring to provide intermediate 31. The latter was not purified, rather, it was exposed to the action of 95 % TFA, thereby providing the native glycopeptide 32, free of any observable thioester by-product. Synthesis of 32. Reaction conditions, a) methyl p-nitrobenzene sulfonate; b) 95 % TFA. Having successfully field tested our novel cysteine-free ligation protocol in the context of a convergent bis-domainal glycopeptide synthesis, we next turned to the challenge of synthesizing longer peptide chains that contain more than two sites of glycosylation. Indeed, to consider the total synthesis of a complex glycoprotein, such as EPO, it would be critical to be able to couple, in a reiterative fashion, multiple glycopeptide fragments. Along these lines, we recently disclosed a method to generate differentially glycosylated trifunctional glycopeptides based on a cysteine-dependent native chemical ligation protocol.17 As a demonstration of the applicability of our new non-cysteine NCL technology to complex targets, we next sought to apply a combination of our cysteine-dependent and cysteine-free NCL protocols to the preparation of the multiply glycosylated peptide, 33. Under our synthetic plan, we would first prepare each of the three glycopeptide fragments (34, 35, and 23) according to glycal assembly and glycopeptide synthesis protocols that have been validated and optimized over the course of many years in our laboratory (Scheme 8).4 Fragments 23 and 35 would then be joined according to our newly developed cysteine-free ligation method to form the Gly–Gln junction. Next, following deprotection of the N-terminal cysteine residue, the bifunctional peptide would be merged with glycopeptide 34 through cysteine-based ligation to afford the fully functionalized target compound. Trifunctional glycopeptide. Thus, polypeptides 36 and 37 were prepared for the cysteine-free ligation event. It is noted that the termini have been suitably equipped in anticipation of the reiterative sequence. Thus, peptide 37 bears the requisite N-terminal auxiliary for the cysteine-free coupling, whereas fragment 36, which will serve as the middle glycopeptide component, incorporates the C-terminal phenolic ester for the first cysteine-free ligation as well as a 1,3-thiazolidine-4-carboxo (Thz)-protected N-terminal cysteine residue, which is unmasked prior to the second, cysteine-based ligation event.18 Each peptide fragment was subjected to glycosylation with disaccharide 14, and, following conversion of the N-terminal auxiliary SPMB group to the requisite disulfide, glycopeptides 35 and 23 were in hand. As hoped, the coupling of the two fragments proceeded readily in the presence of TCEP to afford the ligated product 38 in 58 % yield, along with 11 % of the thioester 40. The latter could be converted into 38 upon treatment with thiophenol or MesNa.19 At this stage, the thiol auxiliary could be removed according to the two-step sequence shown in Scheme 9 (38→39); alternatively, the auxiliary could also be maintained in the subsequent ligation event without causing detriment. Synthesis of 40. Reaction conditions, a) 14, HATU, iPr2NEt, DMSO, 72 %; b) 14, HATU, iPr2NEt, DMSO, 83 %; c) TFE, DCM, 16, 69 %; d) TCEP, DMF, Na2HPO4, 58 % (38) + 11 % (40); e) methyl p-nitrobenzene sulfonate; f) 95 % TFA; g) PhSH or MesNa, PBS. DCM=dichloromethane, Fmoc=9-Fluorenylmethoxycarbonyl, MesNa=2-mercaptoethane sulfonic acid, sodium salt. As we had previously demonstrated, the cysteine residue was readily unmasked through exposure of 38 to 10 % morpholine in DMF (to remove the Fmoc group) followed by treatment with an aqueous solution of MeONH2⋅HCl (Scheme 10). Native chemical ligation between 41 and 34 was carried out in the presence of MesNa and TCEP and afforded the multifunctional glycopeptide 42 in 57 % yield. Synthesis of 42. Reaction conditions, a) (i) 10 % morpholine in DMF; (ii) 0.4 M MeONH2⋅HCl, 60 %; b) MesNa, TCEP, PBS (pH 8.0), 57 %. Finally, the viability of our newly developed cysteine-free ligation protocol was demonstrated in another area of peptide chemistry of great interest to those at the forefront of chemistry and glycobiology, that is, the synthesis of cyclic peptides.20 Cyclic peptides often possess enhanced biological specificity, activity, and metabolic stability in comparison to their linear counterparts. This is as a consequence of their constrained conformations and their enhanced levels of resistance to protease digestion. Although traditional strategies for cyclic-peptide formation are restricted to macrolactam or disulfide formation, Tam and co-workers21 have disclosed that cyclic peptides can be accessed through native chemical ligation.22 Recently, our research group reported on a newly modified protocol for native chemical ligation that allows for formation of cyclic peptides possessing a cysteine residue.23 However, given the scarcity of cysteine residues in nature, the applicability of standard cysteine-based ligation methods may be somewhat limited. Clearly, the development of a broadly useful, cysteine-independent ligation protocol could well have profound ramifications for the field of cyclic-peptide synthesis. The linear polypeptide 43 was prepared through solid-phase peptide synthesis. Reductive amination with aldehyde 11 served to introduce the N-terminal auxiliary (44). The C terminus was functionalized through HATU-mediated esterification with phenol 45, providing 46. Following protecting-group removal and exposure to 3-nitro-2-pyridinesulfenyl chloride, the requisite disulfide cyclization precursor was in hand. Thus, the linear peptide bis-disulfide (47) was treated with TCEP and Na2HPO4 in DMF to provide the desired cyclized peptide (48) in good yield. Importantly, no dimers or oligomers were observed in liquid chromatograpy–MS analysis (see Scheme 11). a) 11, NaCNBH3, MeOH, DMF, 66 %; b) 45, HATU, DIPEA, DMF; c) TFA, PhOH, TESH, H2O, 57 % for 2 steps; d) 16, TFE, 60 %; e) TCEP, Na2HPO4, DMF, 78 %. DIPEA=N,N-diisopropylethylamine, Pbf=2,2,4,6,7-pentymethyldihydrobenzofuran-5-sulfonyl. Obviously, in undertaking a target of the complexity of erythropoietin, opportunities for complications and even failure are always inherent. However, we feel that in principle the basis of a realistic total synthesis of homogeneous erythropoietin has been set forth above. Always mindful of the risks, we remain busily engaged in pursuing this goal. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2006/z600538_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A synthesis of the protected biantennary N-glycan of the naturally occurring glycoprotein, erythropoietin, is described.

A pathway has been devised, wherein a phenolic ester of a C-terminal peptide is ligated with an N-terminal peptide through two consecutive acyl migrations. In the first transacylation, the C-terminus is transferred from a phenol to a newly liberated ortho-thiol function. Subsequently, the acyl group is transported to a proximal benzylamine through a six-membered transition state.

Reiterative approaches in the fashioning of erythropoietin-directed, polyglycosylated polypeptides are disclosed.

The synthesis of cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters is described.