Reflections on a Copenhagen-Minneapolis Axis in Bioorganic Chemistry.

The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

Preparation, Characterization, and Mechanistic Considerations for 1,1,1-Tri(thioacetyl)ethane and 1,1-Di(thioacetyl)ethene.

Two new compounds, 1,1,1-tri(thioacetyl)ethane and 1,1-di(thioacetyl)ethene, arose during reactions of acetyl methoxy(thiocarbonyl) sulfide with potassium methyl xanthate. Relevant mechanisms were elucidated, which in turn suggested novel streamlined routes to these same compounds. Several further transformations of the title compounds were demonstrated, suggesting their potential synthetic utility.

Crystal structures of three (trichloromethyl)(carbamoyl)disulfanes.

The present paper reports crystallographic studies on three related compounds that were of inter-est as precursors for synthetic and mechanistic work in organosulfur chemistry, as well as to model nitro-gen-protecting groups: (N-methyl-carbamo-yl)(tri-chloro-meth-yl)disulfane, C3H4Cl3NOS2, (1), (N-benzyl-carbamo-yl)(tri-chloro-meth-yl)disulfane, C9H8Cl3NOS2, (2), and (N-methyl-N-phenyl-carbamo-yl)(tri-chloro-meth-yl)disulfane, C9H8Cl3NOS2, (3). Their mol-ecular structures, with similar bond lengths and angles for the CCl3SS(C=O)N moieties, are confirmed. Compounds (1) and (3) both crystallized with two independent mol-ecules in the asymmetric unit. Classical hydrogen bonding, as well as chlorine-dense regions, are evident in the crystal packing for (1) and (2). In the crystal of (1), mol-ecules are linked via N-H⋯O hydrogen bonds forming chains along [110], which are linked by short Cl⋯Cl and S⋯O contacts forming sheets parallel to (001). In the crystal of (2), mol-ecules are linked via N-H⋯O hydrogen bonds forming chains along [001], which in turn are linked by pairs of short O⋯Cl contacts forming ribbons along the c-axis direction. In the crystal of (3), there are no classical hydrogen bonds present and the chlorine-dense regions observed in (1) and (2) are lacking.

Crystal structure of O-ethyl N-(eth-oxy-carbon-yl)thio-carbamate.

The title compound, C6H11NO3S, provides entries to novel carbamoyl disulfanes and related compounds of inter-est to our laboratory. The atoms of the central O(C=S)N(C=O)O fragment have an r.m.s. deviation of 0.1077 Šfrom the respective least-squares plane. While several conformational orientations are conceivable, the crystal structure shows only the one in which the carbonyl and the thio-carbonyl moieties are anti to each other across the central conjugated C-N-C moiety. Pairs of 2.54 ŠN-H⋯S=C hydrogen bonds between adjacent mol-ecules form centrosymmetric dimers in the crystal.

Crystal structures of (N-methyl-N-phenyl-amino)(N-methyl-N-phenyl-carbamoyl)sulfide and the corresponding disulfane.

The title compounds, (N-methyl-N-phenyl-amino)(N-methyl-N-phenyl-car-bam-oyl)sulfide, C15H16N2OS, (I), and (N-methyl-N-phenyl-amino)-(N-methyl-N-phenyl-carbamo-yl)disulfane, C15H16N2OS2, (II), are stable derivatives of (chloro-carbon-yl)sulfenyl chloride and (chloro-carbon-yl)disulfanyl chloride, respectively. The torsion angle about the S-S bond in (II) is -92.62 (6)°, which is close to the theoretical value of 90°. In the crystal of (II), non-classical inter-molecular C-H⋯O hydrogen bonds form centrosymmetric cyclic dimers [graph set R 2 (2)(10)], while inter-dimer C-H⋯S inter-actions generate chains extending along the b axis.

Unexpectedly Stable (Chlorocarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, and Related Compounds That Model the Zumach-Weiss-Kühle (ZWK) Reaction for Synthesis of 1,2,4-Dithiazolidine-3,5-diones.

The Zumach-Weiss-Kühle (ZWK) reaction provides 1,2,4-dithiazolidine-3,5-diones [dithiasuccinoyl (Dts)-amines] by the rapid reaction of O-ethyl thiocarbamates plus (chlorocarbonyl)sulfenyl chloride, with ethyl chloride and hydrogen chloride being formed as coproducts, and carbamoyl chlorides or isocyanates generated as yield-diminishing byproducts. However, when the ZWK reaction is applied with (N-ethoxythiocarbonyl)urethane as the starting material, heterocyclization to the putative "Dts-urethane" does not occur. Instead, the reaction directly provides (chlorocarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, a reasonably stable crystalline compound; modified conditions stop at the (chlorocarbonyl)[1-ethoxy-(N-ethoxycarbonyl)formimidoyl]disulfane intermediate. The title (chlorocarbonyl)(carbamoyl)disulfane cannot be converted to the elusive Dts derivative, but rather gives (N-ethoxycarbonyl)carbamoyl chloride upon thermolysis, or (N-ethoxycarbonyl)isocyanate upon treatment with tertiary amines. Additional transformations of these compounds have been discovered, providing entries to both known and novel species. X-ray crystallographic structures are reported for the title (chlorocarbonyl)(carbamoyl)disulfane; for (methoxycarbonyl)(N-ethoxycarbonylcarbamoyl)disulfane, which is the corresponding adduct after quenching in methanol; for [1-ethoxy-(N-ethoxycarbonyl)formimidoyl](N'-methyl-N'-phenylcarbamoyl)disulfane, which is obtained by trapping the title intermediate with N-methylaniline; and for (N-ethoxycarbonylcarbamoyl)(N'-methyl-N'-phenylcarbamoyl)disulfane, which is a short-lived intermediate in the reaction of the title (chlorocarbonyl)(carbamoyl)disulfane with excess N-methylaniline. The new chemistry and structural information reported herein is expected to contribute to accurate modeling of the ZWK reaction trajectory.

Crystal structure of bis-(N-methyl-N-phenyl-amino)-tris-ulfane.

The title compound, C14H16N2S3, crystallized with two independent mol-ecules [(1 a ) and (1 b )] in the asymmetric unit. Both mol-ecules display a pseudo-trans conformation. The two consecutive S-S bond lengths of the tris-ulfane unit of mol-ecule (1 a ) are 2.06 (3) and 2.08 (3) Å, and 2.08 (3) and 2.07 (2) Šfor mol-ecule (1 b ). Torsion angles about each of the two S-S bonds are 86.6 (2) and 87.0 (2)° for (1 a ), and -84.6 (2) and -85.9 (2)° for (1 b ). The core atoms, viz. the N-S-S-S-N moiety, of the two mol-ecules superimpose well if one is inverted on the other, but the phenyl groups do not. Thus, the two units are essentially conformational enanti-omers. In mol-ecule (1 a ), the two phenyl rings are inclined to one another by 86.7 (3)°, and in mol-ecule (1 b ), by 81.1 (3)°. In the crystal, mol-ecules are linked via C-H⋯π inter-actions, forming sheets lying parallel to (010).

The N-terminal zinc finger and flanking basic domains represent the minimal region of the human immunodeficiency virus type-1 nucleocapsid protein for targeting chaperone function.

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is a chaperone that facilitates nucleic acid conformational changes to produce the most thermodynamically stable arrangement. The critical role of NC in many steps of the viral life cycle makes it an attractive therapeutic target. The chaperone activity of NC depends on its nucleic acid aggregating ability, duplex destabilizing activity, and rapid on-off binding kinetics. During the minus-strand transfer step of reverse transcription, NC chaperones the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nucleotide TAR RNA-DNA annealing was probed by using chemically synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(1-28), NC(1-35), NC(29-55), NC(36-55), and NC(11-55). Most of these peptides displayed significantly reduced annealing kinetics, even when present at concentrations much higher than that of wild-type (WT) NC. In addition, these truncated NC constructs generally bind more weakly to single-stranded DNA and are less effective nucleic acid aggregating agents than full-length NC, consistent with the loss of both electrostatic and hydrophobic contacts. However, NC(1-35) displayed annealing kinetics, nucleic acid binding, and aggregation activity that were very similar to those of WT NC. Thus, we conclude that the N-terminal zinc finger, flanked by the N-terminus and linker domains, represents the minimal sequence that is necessary and sufficient for chaperone function in vitro. In addition, covalent continuity of the 35 N-terminal amino acids of NC is critical for full activity. Thus, although the hydrophobic pocket formed by residues proximal to the C-terminal zinc finger has been a major focus of recent anti-NC therapeutic strategies, NC(1-35) represents an alternative target for therapeutics aimed at disrupting NC's chaperone function.

On the role of NMR spectroscopy for characterization of antimicrobial peptides.

Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structure-function paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based solely on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid- and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs.

Synthesis and characterization of the 47-residue heterodimeric antimicrobial peptide distinctin, featuring directed disulfide bridge formation.

Distinctin, a 47-residue heterodimeric peptide with potent antimicrobial activity, comprises two monomeric units linked covalently by a disulfide bond between Cys19 from the 22-residue A chain and Cys23 from the 25-residue B chain. Previous synthetic strategies involved assemblies of the two individual chains, followed by their co-oxidation to form the connecting disulfide bridge, and resulted in a mixture of three species: two homodimers and one heterodimer. Here, we report synthesis of exclusively heterodimeric distinctin, using recently developed tactics for directed disulfide bridge formation. Material prepared this way was characterized and found to be suitable for more detailed structural studies.

Bis[(methyl-sulfan-yl)carbon-yl]disulfane.

The title compound, C(4)H(6)O(2)S(4), was prepared by repeating, with subtle improvements, a multi-step route originally described by Mott & Barany [J. Chem. Soc. Perkin Trans. 1 (1984) ▶, pp. 2615-2621]. The title compound was obtained for the first time as a crystalline material. The two [(methyl-sulfan-yl)carbon-yl]sulfenyl moieties are essentially perpendic-ular to each other, each approximately planar (r.m.s. deviations of 0.02 and 0.01 Å) and with a C-S-S-C torsion angle = 90.99 (6)°, which compares well with the theoretical value of 90°.

Bis(N-methyl-N-phenyl-carbamo-yl)disulfane.

The title compound, C(16)H(16)N(2)O(2)S(2), has been synthesized by several different high-yield routes, and has been encountered as a co-product in a number of reaction pathways, ever since it became of inter-est to our research program over 30 years ago. We now confirm the proposed mol-ecular structure in which the mol-ecule exhibits a twofold axis of symmetry through the mid-point of the S-S bond and the two planes defined by the (carbamo-yl)sulfenyl moieties are essentially perpendicular to each other [dihedral angle = 81.55 (14)°].

Deciphering structural elements of mucin glycoprotein recognition.

Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates, we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues, are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.

Synthetic routes to, transformations of, and rather surprising stabilities of (N-methyl-N-phenylcarbamoyl)sulfenyl chloride, ((N-methyl-N-phenylcarbamoyl)dithio)carbonyl chloride, and related compounds.

The title compound classes, (carbamoyl)sulfenyl chlorides and ((carbamoyl)dithio)carbonyl chlorides, have been implicated previously as unstable, albeit trappable, intermediates in organosulfur chemistry. The present work reports for each of these functional groups: (i) several routes to prepare it in the N-methylaniline family; (ii) its direct structural characterization by several spectroscopic techniques; (iii) its rather unexpected stability and its ultimate fate when it decomposes; (iv) a series of further chemical transformations that give highly stable derivatives, each in turn subject to thorough characterization. Relevant kinetic and mechanistic experiments were carried out, including some with p-methyl- and 2,6-dimethyl-substituted N-methylanilines. Given that the title compounds can be isolated and are relatively stable, they may find applications in the preparation of thiolyzable and/or photolabile protecting groups for the sulfhydryl function of cysteine and for the development of new protein synthesis and modification reagents.

Intramolecular glycan-protein interactions in glycoproteins.

Glycoproteins are a major class of glycoconjugates displaying a variety of mutual interactions between glycan and protein moieties that ultimately affect molecular organization. Modulation of the pendant glycan structures is important in tuning the functions of glycoproteins. Here we discuss structural aspects and some of the challenges to studying intramolecular interactions between carbohydrate and protein elements in several forms of O-linked as well as N-linked glycoproteins. These illustrate the importance of the relationship of context to function in protein glycosylation.

Solid-phase synthesis and evaluation of glycopeptide fragments from rat epididymal cysteine-rich secretory protein-1 (Crisp-1).

Three 18-residue peptides with the sequence Glp-Asp-Thr-Thr-Asp-Glu-Trp-Asp-Arg-Asp-Leu-Glu-Asn-Leu-Ser-Thr-Thr-Lys, taken from the N-terminus of the rat epididymal cysteine-rich secretory protein (Crisp-1) that is important in the fertilization process, were prepared by Fmoc solid-phase synthesis using a convergent strategy. These peptides were the parent sequence, plus two possible α-O-linked T(N) antigen-containing glycopeptides with a Thr(α-D-GalNAc) residue in place of either Thr3 or Thr4. During chain assembly, two deletion peptides [des-Asp2 and des-Thr(Ac(3)-α-D-GalNAc)] and one terminated peptide [N-acetylated 14-mer] arose, as did several peptides in which aspartimide formation had occurred at each of the four possible positions in the sequence. These by-products totaled ~20% of the desired product; they were recognized by HPLC and ESI-MS and removed during the intermediate purifications. Final products, obtained in 15-21% overall yields, were characterized by HPLC purities and ESI-MS. Circular dichroism (CD) spectra for all three purified peptides, recorded in pure water and in trifluoroethanol-H(2)O (1:1), revealed that the presence of a sugar moiety does not significantly impact the sampled conformations. Future biological evaluation could elucidate the nature and locus of sugar modification of Crisp-1, and provide insight as to why Crisp-1 protein E binds sperm irreversibly, in contrast to protein D that lacks a sugar near the N-terminus and only binds sperm loosely.

On-resin conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) congeners.

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg(2+)) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids.

Investigation of the sequence and length dependence for cell-penetrating prenylated peptides.

Cell penetrating peptides are useful delivery tools for introducing molecules of interest into cells. A new class of cell penetrating molecules has been recently reported-cell penetrating, prenylated peptides. In this study a series of such peptides was synthesized to examine the relationship between peptide sequence and level of peptide internalization and to probe their mechanism of internalization. This study revealed that prenylated peptides internalize via a non-endocytotic pathway regardless of sequence. Sequence length and identity was found to play a role in peptide uptake but prenylated sequences as short as two amino acids were found to exhibit significant cell penetrating properties.

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach.

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of approximately 24 degrees . The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.

Multifunctional prenylated peptides for live cell analysis.

Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated, and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full "CAAX box" sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid-sensitive functionalities.