Alma Mater Studiorum – Università di Bologna

Dipartimento di Chimica Organica “A. Mangini”

DOTTORATO DI RICERCA IN

Scienze Chimiche

Ciclo XXIV

Settore Concorsuale di afferenza: 03/C1

Settore Scientifico disciplinare: CHIM/06

Sulfanyl Radical Addition to Alkynes: Revisiting an Old Reaction to Enter the Novel Realms of Green Chemistry, Bioconjugation, and Material Chemistry

Presentata da: Alessandro Monesi

Coordinatore Dottorato Relatore Prof. Adriana Bigi Prof. Daniele Nanni

Correlatori Prof. Piero Spagnolo Dott. Mauro Boero, CNRS Strasburgo Dott. Carlo Massobrio, CNRS Strasburgo Dott. Alessandro Massi, Università di Ferrara

Esame finale anno 2012 2 Abstract In the last decade considerable attention has been devoted to the rewarding use of Green Chemistry in various synthetic processes and applications. Green Chemistry is of special interest in the synthesis of expensive pharmaceutical products, where suitable adoption of “green” reagents and conditions is highly desirable. Our project especially focused in a search for new green radical processes which might also find useful applications in the industry. In particular, we have explored the possible adoption of green solvents in radical Thiol-Ene and Thiol-Yne coupling reactions, which to date have been normally performed in “ordinary” organic solvents such as benzene and toluene, with the primary aim of applying those coupling reactions to the construction of biological substrates. We have additionally tuned adequate reaction conditions which might enable achievement of highly functionalised materials and/or complex bioconjugation via homo/heterosequence. Furthermore, we have performed suitable theoretical studies to gain useful chemical information concerning mechanistic implications of the use of green solvents in the radical Thiol-Yne coupling reactions.

3 4 Table of Contents

CHAPTER 1! 7

Introduction! 7 1.1 Green and Click chemistry : concept 7 1.2 Basics of the radical chemistry 16 1.3 Thiol radical coupling 37 1.4 Theoretical approach 45

CHAPTER 2! 55

Radical Additions of Thiols to Alkenes and Alkynes in Ionic Liquids! 55 2.1 Introduction 55 2.2 Result and discussion 56 2.3 Conclusions 63 2.4 Experimental Section 63

CHAPTER 3! 71

Thiol/Yne Coupling for Peptide Glycosidation! 71 3.1 Introduction 71 3.2 Result and Discussion 71 3.3 Conclusions 82 3.4 Experimental Section 83

CHAPTER 4! 95

Theorical approach for radical additions Computational Methods! 95 4.1 Theoretical approach 95 4.1.1 Density Functional Theory 95 4.1.2 Car-Parinello Method 102 4.1.3 Blue Moon 105 4.2 Introduction of our problem 106 4.3 Computational Section 107 4.4 Result and Discussion 109 4.5 Conclusion 122

List of Publications! 127

5 6 CHAPTER 1 Introduction 1.1 Green and Click chemistry : concept In 1998 Warner and P. Anastas first coined the concept of “Green Chemistry”, in a the seminal book “Green Chemistry: Theory and Practice1”, where a precise definition of Green Chemistry was provided together with the Twelve Principles which were taken as the basis of the Green Chemistry itself (Table 1). The original guidelines of Green Chemistry have become generally accepted and the field has since seen a rapid expansion, with numerous innovative scientific developments associated with the production and utilization of chemical products. The concept and philosophy of green chemistry now go beyond chemistry and actually involve different fields ranging from energy to societal sustainability. The main topic is “efficiency’’2 and “security”, including material, energy, work force and property efficiency; any waste is to be addressed through innovative green chemistry means. Low efficiency in organic synthesis causes great challenges in resource conservation and draws environmental and health concerns related to the chemical wastes. The majority of the processes that make use of chemicals cause a potential negative impact on the environment. Therefore, it is extremely important to eliminate, or, at least, reduce any possible risk to an acceptable level. Risk can be expressed as:

Risk = Hazard * Exposure

Traditionally, the risks connected with chemical processes have been minimized by controlling the so-called “circumstantial” factors, such as the use, handling, treatment, and disposal of chemicals. In contrast with the tradition, the Green Chemistry aims to minimize the risk by minimizing the hazard. It therefore shifts control from circumstantial to intrinsic factors, such as the design or selection of chemicals with reduced toxicity and the use of reaction pathways that tend to eliminate by-products or ensure that they are benign.

7 To improve the security of a synthetic process, it is necessary to operate on different directions such as feedstock of chemical(s), type of reaction and solvent, and product separation. The main feedstock of chemical products comes from nonrenewable petroleum that is being depleted rapidly both for chemical and energy needs. However, nature provides a vast amount of biomass in the renewable forms of carbohydrates, amino acids, and triglycerides to obtain organic products3, but a major obstacle to using renewable biomass as feedstock is the need for novel chemistry able to transform large amounts of biomass in its natural state, without the need of extensive functionalisation or protection.

Table 1. The most widely accepted definition of green chemistry (1) is “the design, development and implementation of chemical processes and products to reduce or eliminate substances hazardous to human health and the environment.” This definition has been expanded into 12 principles listed in the table. Green chemistry principles 1. It is better to prevent waste than to treat or clean up waste after it is formed. 2. Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product. 3. Wherever practicable, synthetic methodologies should be designed to use and generate substances that possess little or no toxicity to human health and the environment. 4. Chemical products should be designed to preserve efficacy of function while reducing toxicity. 5. The use of auxiliary substances (e.g., solvents, separation agents, and so forth) should be made unnecessary wherever possible and innocuous when used. 6. Energy requirements should be recognized for their environmental and economic impacts and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure. 7. A raw material or feedstock should be renewable rather than depleting wherever technically and economically practicable. 8. Unnecessary derivatization (blocking group, protection/deprotection, temporary modification of physical/chemical processes) should be avoided whenever possible. 9. Catalytic reagents (as selective as possible) are superior to stoichiometric reagents. 10. Chemical products should be designed so that at the end of their function they do not persist in the environment and break down into innocuous degradation products. 11. Analytical methodologies need to be developed further to allow for real-time in-process monitoring and control before the formation of hazardous substances. 12. Substances and the form of a substance used in a chemical process should be chosen so as to minimize the potential for chemical accidents, including releases, explosions, and fires.

Solvents represent the largest amount of auxiliary wastes in most chemical productions (approximately 80%)4, they are used extensively for dissolving reactants, extracting and washing products, separating mixtures, cleaning reaction apparatus and dispersing products for practical applications; efficient control can produce a substantial improvement in the environmental impact of a

5 process . The primary function of solvents in classical chemical syntheses is to facilitate mass transfer to modulate chemical reactions in terms of reaction rate, yields, conversions, and selectivity. They do this by dissolving the reactants in dilute homogeneous mixtures; but, after the reaction, the final products have to be separated from the solvent through energy-intensive means. The

8 development of Green Chemistry redefines the role of a solvent: An “ideal solvent facilitates the mass transfer but does not dissolve!”2a In addition, an ideal green solvent should be natural, non-toxic, cheap and also readily available. Furthermore, it should have the additional ability of favoring reaction, product separation, and/or catalyst recycling. The most elegant way to avoid any problem with solvents is avoiding their use, which approach has been widely exploited in the paints and coatings industries and also, in most recent years, in the arduous sphere of organic chemistry6. Most reactions, however, require solvent: a green chemical process must therefore involve an environmentally acceptable solvent, but, unfortunately, there is no green solvent suitable for every need. Among the most widely explored greener solvents are ionic liquids7,

8 9 supercritical CO2 , and water . Ionic liquids are salts of highly asymmetrical organic ions with melting points below or close to room temperature10, while supercritical fluids are gases that are nearly as dense as liquids11. A supercritical fluid is a gas that has been brought above its critical temperature and pressure. At the critical point all the properties of the two phases are equal and the two phases become a single continuous phase. The diffusivity, while being lower than that of a gas, is significantly higher than that of a liquid. The combination of these two properties makes supercritical fluids much better solvents than they would be expected to be. This is particularly true when the temperature and pressure are not too far above the critical temperature and pressure. It is therefore possible to tune the density, and as a consequence the solvent power (Figure 1).

9 Figure 1.Carbon dioxide phase diagram

Processes that make use of supercritical fluid solvents occur necessarily at elevated pressures. In certain cases, they may also occur at elevated temperatures. Solvents are often chosen on the basis of the suitability of the temperatures and pressures corresponding to the supercritical region. However, other factors are important as well. These include toxicity and other hazards, cost, availability, and environmental friendliness (or lack thereof) of the solvent itself. On these bases, carbon dioxide is the most preferred solvent: in fact, besides being the cheapest natural solvent, it is renewable, nonflammable, and highly volatile. Moreover, it has solvent properties similar to light hydrocarbons,

12 apart from an unusually high affinity with fluorocarbons . Supercritical CO2 has found numerous applications in synthesis, and, more interestingly, in certain industrial processes. The first example of its industrial use is offered by the extraction of caffeine from coffee, by SFE in the 1970s,13 which led to an explosive interest in the 1980s for food treatment (tea decaffeination, extraction of fats from foods and of essential oils and spices from plants). The recent literature reflects the wide variety of current applications of CO2 supercritical fluid: dry cleaning14, precision-cleaning of inorganic surfaces15, deposition media for the production of metallic powders and thin films16 and other numerous applications. In a recent book Leitner and Jessop and Anastas17 highlighted the versatile use of this medium in chemical and industrial processes.

10 In addition to natural solvents, other non-natural ones have been extensively adopted as green solvents. Ionic liquids18 are probably the most studied ones. Ionic liquids are organic salts What’s an with melting points Ionic Liquid? below ambient (or reaction) temperature: such solvents by generally Keith E. Johnson have very low vapor “When I use a word,” Humpty Dumpty said, in rather a scornful tone, pressures and hence present much “it means lower just what toxicity I choose it to than mean – low-boiling neither more nor less.” solvents, which fact makes them “The especially question is,” said safe Alice, for “whether microwave you can make words synthesis mean so many methods. different things.”19 LEWIS CARROLL, Through the Looking Glass Importantly, ionic liquids present high thermal and chemical stability (in Table 2 Introduction to the Phrase “Ionic Liquids” scheduling of groups of papers. One property that we emphasized EUCHEM 2006 dealt with Molten recently is the molarity of the liquid6 are summarized the he main Structure properties and Properties of Salts a modern and Ionic Liquids ionic with liquid). the An (a straightforward attractive quantity except of Ionic Melts” was the content of each session usually for mixed systems such as a basic Ttitle of a Faraday Society mixed. chloroaluminate containing both Cl- “ - feature of ionic liquids is that, depending on the nature of their organic cations Discussion held in Liverpool in 1961; and AlCl4 in significant amounts). it dealt exclusively with molten Modern Ionic Liquids The molarity is important regarding inorganic salts.1 “Ionic Liquids” kinetic measurements, including and inorganic counter was the title anions, of Chapter 6 they of the can be readily The properties separated from of a modern conductivities. organics as textbook Modern Electrochemistry ionic liquid are summarized in Table II indicates a range of by Bockris and Reddy, published in Table I. Particularly significant molarities of many liquids from 1 well from water; 1970: moreover, it discussed liquids their peculiar properties ranging are (i.) the low vapor enable ready solubility of pressures to 60, with water at 55, liquid alkali from alkali silicates and halides to in most instances which contrast halides up to 35 (LiCl) and most even complex systems like carbohydratestetraalkylammonium salts.2 The the20 environmental or polymer problems21. For this reason, ionic of organic salts less than 10. Specific modern era of ionic liquids stems volatile organic solvents and (ii.) conductivities span a far greater range from the work on alkylpyridinium moderate specific conductivities, from the metal sodium through liquids have been termed "designer solvents".and dialkylimidazolium salts in usually in the same range as molten inorganic salts in the Scm-1 region to organic salts (the modern ionic liquids) and aqueous solutions Table I. Modern ionic liquids. in the mScm-1 region and finally A salt Cation and or anion quite large to the near non-conducting but ionizing acetic acid and water at Freezing point Preferably below 100°C µScm-1. Combining these data into molar conductances is illuminating. Liquidus range Often > 200°C We see comparable values for simple Thermal stability Usually high inorganic salts alone and in aqueous solutions* but much smaller values Viscosity Normally < 100 cP, workable for the low temperature semi-organic Dielectric constant Implied < 30 and organic systems. Thus these modern ionic liquids must consist of Polarity Moderate IONS AND ION PAIRS, (undissociated molecules), while liquid alkali halides Specific conductivity Usually < 10 mScm-1, “Good” are purely IONIC and aqueous Molar conductivity < 10 Scm2 mol-1 electrolytes behave as a mixture of hydrated ions and the molecular Electrochemical window > 2V, even 4.5 V, except for Brønsted acidic systems solvent water. Figure 1 attempts to Solvent and/or catalyst Excellent for many organic reactions picture these differences.

Vapor pressure Usually negligible Acid-Base Properties and Water Interactions 3 Colorado in the late 1970s. The term those of aqueous electrolytes. It is While simple salts such as KCl can Table 2. Properties of a modern ionic liquid4 ionic liquids was introduced to cover found that many such systems are be thought of as the product of an systems below 100°C, one reason excellent solvents or catalysts for electron transfer between elements, being to avoid the words “molten organic reactions3 and some simple organic salts can be traced to a proton Ionic liquid has been used in different research fields: chemical engineering salts” in phrases such as “ambient processes such as electrodeposition.5 transfer between an acid and base. temperature molten salts,” another Unfortunately, one finds reports of Cations such as emim+ and n-bupy+ to create an impression of freshness new “ionic liquids” without data on result from the22 alkylation of the bases (extraction and separation process, and a third, perhaps, for patent ands also build selective membranes)conductivities, which would establish , for purposes. The first “Conference on that they are dissociated to some Ionic Liquids” took place in Salzburg extent at least into ions. normal organic synthesis (like chiral synthesis, polymerization, in 2005, Molten Salts 7 in Toulouse nano materials, in 2005 had one of ten sessions Liquids Comparisons * Extrapolation of aqueous solution surfactants)23 and in biotechnology (biocatalysis and purification of proteins)devoted to Ionic Liquids; but the molar or equivalent24 conductances. to International Symposia on Molten How do these ionic liquids infinite dilution, at which ion pairing is Salts of ECS from 1976 to the present eliminated, gives many values in the 100 compare with other liquids, especially to 150 Scm2 mol-1 range,11 the exceptions have not shown discrimination on The only natural solvent on earth is those water. which Water is the most inexpensive, conduct electricity? Table involving H+ and OH- for which the the basis of temperature, beyond II presents some illustrative data. Grotthus mechanism operates.2 safe and green 38 solvent in nature. This points have attracted the The Electrochemical attention Society of Interface • Spring 2007 scientists for many years: the first use of water for an organic reaction could be dated back to Wohler’s synthesis of urea from ammonium cyanate25. Water possesses unique physical and chemical properties: a large temperature window in which it remains in the liquid state, extensive hydrogen

11 bonding, high heat capacity, large dielectric constant, and optimum oxygen solubility (it could to maintain aquatic life forms). These distinctive properties are the consequence of the unique structure of water. However, unfortunately, water presents limited chemical compatibility: in fact, many (organic) reactants and reagents, including most organometallic compounds, are totally incompatible with water. Hydrophilicity can be properly enhanced by introduction of polar functional soluble26, but the need of possible manipulations can seriously reduce the advantages (low cost, simplicity of reaction conditions, ease of workup and product isolation) connected with the use of water in place of the traditional organic solvents. Since the solubility of any reacting species and product in water can be dramatically changed, leading to homogeneous or heterogeneous reaction mixtures, recent publications describe reactions in water under very different conditions27. The green solvents so far studied complement one another in properties and applications: perhaps one day, already known and others new green solvents will become the most used ones in synthesis. Green Chemistry seems to be based on new exotic solvents and new technologies; however, running reactions in the latest green solvents or water instead rather than in organic solvents does not necessarily improve the environmental impact of a synthetic sequence. A measure of the efficiency of a process is furnished by the so-called E- factor, which is actually due to the ratio of the amount of waste produced with respect to that of the desired material which is achieved.

1392 M. J. EARLE AND K. R. SEDDON

- - NN+ [PF6] + - [NO ] N [BF4] NN+ 3

C6H13

Fig. 1 Examples of simpleFigure 2. room-temperatureTipical Ionic liquid used for organic synthesis. ionic liquids.

show simple melting E-factor = mass of waste ÷ mass of productbehavior. For the binary systems, the melting point depends upon composition,*

and this complex behavior has been studied extensively for the archetypal system, [emim]Cl-AlCl3 ([emim]+ = 1-ethyl-3-methylimidazolium) [9]. Since Ionic it liquids is not have been easy described to perform as designer solvents a direct [1], and this determination means that their properties of the can waste be adjusted to suit the requirements of a particular process. Properties such as melting point, viscosity, generated, it is usual to measure the quantity of material put into the system and density, and hydrophobicity can be varied by simple changes to the structure of the ions. For example, the melting points of 1-alkyl-3-methylimidazolium tetrafluoroborates [10] and hexafluorophosphates [11] are a function of the length of the 1-alkyl group, and form liquid crystalline phases for alkyl chain lengths over 12 carbon atoms. Another important property that changes with structure is the miscibility of water in these ionic liquids. For example, 1-alkyl-3-methylimidazolium tetrafluoroborate salts are miscible with water at 25 °C where the alkyl chain length is less than 6, but at or above 6 carbon atoms, they form a separate phase when mixed with water.12 This behavior can be of substantial benefit when carrying out solvent extractions or product separations, as the relative solubilities of the ionic and ex- traction phase can be adjusted to make the separation as easy as possible.

REACTIONS IN CHLOROALUMINATE(III) IONIC LIQUIDS

The chemical behavior of Franklin acidic chloroaluminate(III) ionic liquids (where X(AlCl3) > 0.50) is that of a powerful Lewis acid. As might be expected, it promotes reactions that are conventionally promoted by aluminum(III) chloride, without suffering the disadvantage of the low solubility of aluminum(III) chloride in many solvents. Indeed, chloroaluminate(III) ionic liquids are exceptionally

powerful solvents, being able to dissolve kerogen [12], C60 and many polymers [13]. The preparation of these ionic liquids is straightforward. Simply by mixing the appropriate organic halide salt with aluminum(III) chloride results in the two solids melting together to form the ionic liquid. However, this synthesis must be performed in an inert atmosphere. A classical reaction promoted by Lewis acids is the Friedel-Crafts reaction, which was found to work efficiently in chloroaluminate(III) ionic liquids [14]. A number of commercially important fra- grance molecules have been synthesized by Friedel-Crafts acylation reactions in these ionic liquids [15]. Traseolide® (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindane) and Tonalid® (6-acetyl-1,1,2,4,4,7-

hexamethyltetralin) have been made in high yield in the ionic liquid [emim]Cl-AlCl3 (X = 0.67) (Fig. 2). In the acylation of naphthalene, the ionic liquid gives the highest known selectivity for the 1-position [15]. Cracking and isomerization reactions occur readily in acidic chloroaluminate(III) ionic liquids. A remarkable example of this is the reaction of polyethylene, which is converted to a mixture of gaseous

alkanes with the formula (CnH2n+2, where n = 3–5) and cyclic alkanes with a hydrogen to carbon ratio of less than two (Fig. 3) [16]. The distribution of the products obtained from this reaction depends upon the reaction temperature and differs from other polyethylene recycling reactions in that aromatics and

*The composition of a tetrachloroaluminate(III) ionic liquid is best described by the apparent mole fraction of AlCl3 {X(AlCl3)} - – present. Ionic liquids with X(AlCl3) < 0.5 contain an excess of Cl ions over [Al2Cl7] ions, and are called “basic”; those with – – X(AlCl3) > 0.5 contain an excess of [Al2Cl7] ions over Cl , and are called “acidic”; melts with X(AlCl3) = 0.5 are called ‘neutral’.

© 2000 IUPAC, Pure and Applied Chemistry 72, 1391–1398 PERSPECTIVE

Green chemistry for chemical synthesis

Chao-Jun Li*† and Barry M. Trost†‡ *Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 2K6; and ‡Department of Chemistry, Stanford University,subtract the mass of material Stanford, CA 94305-5080 output from the system. In the simplest case, the

Edited by Jack Halpern, Universityonly of Chicago, output Chicago, from the IL, and system approved would July 3, be 2008 the (received final product. for review If May materials 5, 2008). (e.g. solvents or catalysts) are recycled, however, then the output of the system Green chemistry for chemical synthesis addresses our future challenges in working with chemical processes and products by invent- ing novel reactions that can maximizewould also include those materials. the desired products and minimize by-products, designing new synthetic schemes and appa- rati that can simplify operations inIn chemical parallel productions, of the green and chemistry seeking concept, greener Kolb, solvents Finn that and are Sharpless, inherently in environmentally and ecologically benign. 200128 have published a review where was introduced the “Click Chemistry” atom economy ͉ synthetic efficiencyconcept. The click chemistry’s principles overlaps in different sections the green ͉ sustainable chemical feedstocks ͉ green solvents chemistry: atom economy, simple purification procedures, and benign solvents ver the past two centuries, quantity of product isolated addition incorporates all atoms of the and side products.Reaction Yield = x100% fundamental theories and re- theoretical quantity of product starting materials into the final product. activities in chemistry have Recognizing this fundamental phenome- been soundly established. molecular wt. of desired product non, in 1991 (4) Trost presented a set of O Atom Economy = x100% coherent guiding principles for evaluat- Such theories and reactivities have pro- molecular weight of all products vided the foundations for the chemical ing the efficiency of specific chemical enterprise that generates critical living Fig. 1. Definition of the fundamental difference processes, termed the atom economy, Click chemistry is based on the philosophy that the any reaction must be needs such as food for the world’s popu- in the manner in which the reaction and the atom which has subsequently been incorpo- lation, achieves various medicalmodular, wonders wide in economy scope, give yields very are generated. high yields, generate only inoffensive rated into by- the ‘‘Twelve Principles of Green Chemistry’’ and has altered the that save millions of lives andproducts, and also be stereospecific (but not necessarily enantio-selective). The improve way many chemists design and plan their people’s health, and produces materials feedstocks, reactions, solvents, and features of a click process also include simple reaction conditions syntheses. (ideally, the Atom economy seeks to maxi- essential to the present and future needs separations. of mankind. Just less than twoprocess centuries should be insensitive to oxygen and water), readily available mize the starting incorporation of the starting ago, organic compounds were believed Chemical Feedstocks materials into the final product of any materials and reagents, the use of benign solvents (green solvents and/or water) given reaction. The additional corollary to be only accessible through biological Presently, the main feedstock of chemi- is that, if maximum incorporation can- processes under the influenceas of well ‘‘vital as simple cal product products isolation. comes from Moreover, nonrenewable product purification (when not be achieved, then ideally the quanti- forces’’ (1). Today, many moleculesrequired) should entail of petroleum crystallization or distillation (not chromatography) and that is being depleted rapidly great complexity can be synthesized both for chemical and energy needs. ties of side products should be minute Abstract: Supramolecular chemistry andthe ensuing product mechanostereo- wards should be stable under the creation of advanced functional physiological conditions. supramolecular and environmentally Finally, a innocuous. There is chemistryreadily. have The been total major syntheses beneficiaries of of naturalthe concepts andHowever, mechanostereochemical nature provides systems. a vast In this amount Focus afundamentaldifferenceinthemanner andproducts reactions pioneered with extremely under the “click highclick chemistry” complexity process phi- should have a high thermodynamic Review,of biomass recent advances in the in renewable the use of click forms driving chemistry of force, usually greater in losophy. The success of the copper(I) 1,3-dipolar cycload- these fields are highlighted. in which a reaction yield and the atom such as vitamin B12 (2) and palytoxin carbohydrates, amino acids, and triglcer- dition between azides and alkynes, resultingthan 20 kcal/mol. Most common examples of click reaction are: in the triazole economy yield is calculated (Fig. 1). ring(3) has in inspired the laboratory the application are of other testimonials emerging click Keywords:ides to obtainclick chemistry organic· cycloaddition products· (9),mechanical but 29 The reaction yield is only concerned reactions,of achievements for example, Diels–Alder comparable cycloadditions, to the• Cycloadditions thiol–con- bondamajorobstacletousingrenewable· supramolecular of unsaturated species, Huisgen 1,3-dipolar chemistry · thiol additions ene/yne chemistry, and nitrile N-oxide cycloadditions, to- biomass as feedstock is the need for with the quantity of the desired product struction of the great pyramidscycloaddition at the reactions of azides and alkynes, and hetero-Diels-Alder molecular scale. However, despite such novel chemistry to transform the large that is isolated, relative to the theoreti- enormous achievements, we aretransformation. facing amounts of biomass selectively and effi- cal quantity of the product. Atom econ- great challenges1. Introduction in future chemical syn- ciently, in its natural state, without ex- omy takes all used reagents and unwanted side products into account along with the thesis. The present state-of-the-art pro- tensive functionalization, defunctional- In a day and age when petroleum feedstocks are dwindling, desired product. For example, substitu- andcesses the idea for that synthesizingwe are approaching, chemical or have already products sur- ization, or protection. [1] tions and eliminations represent the vast passed,are peak highly oil production inefficient.is relatively The concept widespread—all of Reactions majority of uneconomical classical reac- of whichatom is intimately economy tied (4, to the 5) growing was created threat ofThe classical [3+2] cycloaddition of unactivated alkynes and azides requires toa global energy crisis[2]—perhaps it is not so surprising that the phi- Reactions play the most fundamental tions in which inherent wastes are un- losophyemphasize of “click chemistry”, the importance only introduced ofhigh temperatures or pressures and was relatively ignored for decades after its this in 2001ineffi- by [3] role in synthesis. The ideology of Green avoidable (Scheme 1). Simple additions or Sharplessciency. et al., Thehas E takenfactor such (6) a provided strong foothold a quan- in so cycloadditions and rearrangements repre- manytifiable fields of measure scientific research. of such In inefficiency comparisondiscovery by Huisgen with and the Chemistry30. When Sharpless calls for the31 and Meldal development32 independently reported of philosophy of green chemistry[4] and its 12 tenets,[5] it is clear new chemical reactivities and reaction sent desired modes of reactivities (Scheme showed that, for every kilogram of fine that the two schools of thought—click anda green Cu(I)-catalyzed chemis- conditions variant that that proceeds can potentially rapidly provide at room temperature 1). Reaction to Mass Efficiency (RME) try—havechemical emerged and with pharmaceutical a few overlapping principles, products that is, benefits for chemical syntheses in terms and Mass Intensity (MI) are additional atomproduced, economy, simple 5–100 purification times procedures, that amount and benign of of resource and energy efficiency, prod- concepts to evaluate the efficiency of solventschemical and side waste products. is From generated. the perspective Such of substan- low tial overlap between these two philosophies, the importance uct selectivity, operational13 simplicity, synthetic reactions to take into account of theefficiency click chemistry in state-of-the-art philosophy for many chemistsorganic can be Schemeand 1. health Generic examples and environmental of a) a 1,3-dipolar cycloaddition safety. between the reaction yield (10). expectedsyntheses to continue presents to grow great in popularity challenges throughout in the azides and terminal alkynes, leading to the formation of 1,4-disubstitut- ed-1,2,3-triazole rings, b) a Diels–Alder cycloaddition, c) a double addi- Recently, innovative reactions with early years of this new century. resource conservation and draws envi- tion of a thiol across a triple bond, and d) a cycloaddition between an The phrase click chemistry has come to be all but synony- alkyneAtom and Economy. an nitrile N-oxide.Conventionally, Especially in the cases of attaining b), c), and d), it such inherent advantages have been de- mousronmental with copper(I)-catalyzed and health 1,3-dipolar concerns cycloadditions related be- shouldthe be highest noted that theyield alkyne and can be product replaced with selectivity an alkene. veloped with the aid of chemical and tweento azides the chemical and terminal wastes. alkynes (CuAAC) to yield 1,4- were the governing factors of chemical disubstituted-1,2,3-triazoleSince its birth rings. over Although a decade this ago click the reac- synthesis. Little consideration was given tion is arguably the most widespread and popular one to be to the different supramolecular species and mechanically in- field of Green Chemistry has been spe- Author contributions: C.-J.L. and B.M.T. wrote the paper. found in the literature to date, it is by no means the one and terlockedto the molecules, usage of which multiple will be addressed reagents individually. in stoi- onlycifically star player. designed It is our intention to meet in this such Focus chal- Review to chiometric quantities, which often were The authors declare no conflict of interest. highlight some of the recent creative advances in the litera- lenges in chemical synthesis (7, 8). To not2. 1,3-Dipolar incorporated Cycloadditions into the of Azides target with mole- Alkynes This article is a PNAS Direct Submission. ture on click chemistry (Scheme 1), not only the CuAAC re- address these challenges, innovative and cule and would result in significant side †To whom correspondence may be addressed. E-mail: action, but also thiol–ene/yne, Diels–Alder, and nitrile N- The CuAAC reaction has proven to be highly efficient when [email protected] or [email protected]. oxidefundamentally chemistries towards novel the chemistry production of is functional needed employedproducts. in the However, final step leading in a balancedto the formation chemi- of the supramolecularthroughout species the and synthetic mechanically processes: interlocked mole- mechanicalcal reaction, bond[6] aduring simple the construction addition of or mechanically cyclo- © 2008 by The National Academy of Sciences of the USA cules. This Focus Review has been organized according to interlocked molecules (MIMs), in particular, in the case of the types of click reactions employed and has been related rotaxanes[7]and, to a lesser extent, in the case of catenanes.[7] www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804348105 PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13197–13202 The success of the CuAAC reaction in the synthesis of MIMs is, in part, a consequence of the fact that the reaction [a] A. C. Fahrenbach, Prof. J. F. Stoddart occurs efficiently at room temperature; an observation Department of Chemistry which means that the stabilities of the supramolecular spe- Northwestern University 2145 Sheridan Road, Evanston, IL 60208 (USA) cies, which are the immediate precursors, are better main- Fax: (+1)847-491-1009 tained with higher stabilities at lower rather than at higher E-mail: [email protected] temperatures. Not only does the copper(I) metal center Homepage: http://stoddart.northwestern.edu serve as a catalyst, but it can also act simultaneously as a [b] Prof. J. F. Stoddart template in the so-called “active-metal template” strategy Graduate School of EEWS (WCU) aimed at the formation of MIMs, pioneered early on in the Advanced Institute of Science and Technology (KAIST) [8] 373-1, Guseong Dong, Yuseong Gu, Daejeon 305-701 Leigh group. For a review on active-metal templation, see Republic of Korea (Korea) reference [8].

Chem. Asian J. 2011, 6, 2660 – 2669  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org 2661 Click Chemistry, A Powerful Tool for Pharmaceutical Sciences 2217

400 purpose. In this review, important aspects of click chemistry Click350 Chemistry, A Powerful Tool for Pharmaceutical Scienceswill be discussed, along with its emerging applications in drug2217 delivery and nanomedicine. We will also provide some of our 300 400 Abstract: Supramolecular chemistry and mechanostereo-concernswards and the creation vision forof advanced its future functional development supramolecular in pharma- chemistry have been major beneficiaries of the conceptspurpose.and Inmechanostereochemical this review, important systems. aspects In of this click Focus chemistry 250 ceutical sciences. Several reviews on click chemistry have 350 and reactions pioneered under the “click chemistry” phi-will beReview, discussed, recent along advances with in theits emerging use of click applications chemistry in in drug regiospecifically already form been 1,4-disubstituted 1,2,3-triazoles published (1–3). The readers may the reaction back find them to-life. 200 losophy. The success of the copper(I) 1,3-dipolar cycload-deliverythese and fields nanomedicine. are highlighted. We will also provide some of our 300 helpful in understanding other aspects of click chemistry or in dition between azides and alkynes, resulting in the triazoleconcerns and vision for its future development in pharma- 150 The success obtaining of the CuAAC more background reaction is information. now well recognised by the chemical 250 ring has inspired the application of other emerging clickceuticalKeywords: sciences.click Several chemistry reviews· cycloaddition on click· mechanical chemistry have reactions, for example, Diels–Alder cycloadditions, thiol– bond · supramolecular chemistry · thiol additions 100 community, but its biological applications remain seriously limited by toxicity of already been published (1–3). The readers may find them Number of Publications 200 ene/yne chemistry, and nitrile N-oxide cycloadditions, to-CLASSIFICATIONhelpful in understanding OF CLICK other aspects REACTIONS of click chemistry or in 50 the copper catalyst. This fact has led to the recent discovery of copper-free 150 obtaining more background information. 33 0 cycloadditions , As which nonetheless occur at comparably fast rates. already implicated, click chemistry encompasses a The groups 100 Number of Publications 1999 2000 2001 2002 20031. Introduction 2004 2005 2006 2007 group of powerful linking reactions that are simple to CLASSIFICATION34 OF CLICK REACTIONS 50 Year of Fokin and perform, Jia have used high a yields, ruthenium(II) require no catalyst, or minimal but purification, under more severe In a day and age when petroleum feedstocks are dwindling, conditions. and areAs versatile already implicated, in joining diverseclick chemistry structures encompasses without the a Fig. 1.0Numberand of thepublications idea that we containing are approaching, the key or words have already“click sur- 1999 2000 2001 2002 2003[1] 2004 2005 2006 2007 prerequisitegroup of powerful of protection linking steps. reactions To date, that four are major simple classi- to chemistry” or “clickpassed, reaction peak” oilfrom production 1999–2007.is The relatively literature widespread—all search Year ficationsperform, of have click high reactions yields, have require been no identified or minimal (Fig. purification,3). was performed viaof SciFinder which is intimately Scholar® tied on Dec. to the 31, growing 2007 and threat included of a global energy crisis[2]—perhaps it is not so surprising that the phi-and are versatile in joining diverse structures without the journal articles, abstracts, preprints, dissertations, patents, and & Cycloadditions—these primarily refer to 1,3-dipolar cyclo- Fig. 1. Numberlosophy of publications of “click chemistry”, containing only the introduced key words in“click 2001 byprerequisite of protection steps. To date, four major classi- reviews. The years 1999 and 2000[3] each contain one publication. additions, but also include hetero-Diels-Alder cycloadditions chemistry” or “Sharplessclick reaction et al.,” fromhas 1999 taken–2007. such The a strong literature foothold search in sofications of click reactions have been identified (Fig. 3). (2). was performedmany via SciFinder fields of Scholar® scientific on research. Dec. 31, In 2007 comparison and includedThe Diels-Alder reaction is legendary in organic chemistry with the 35. The solid-state istry.journal For articles, instance,philosophy abstracts, they of are green preprints, typically chemistry dissertations,[4] performedand its 12 patents,tenets, in water.[5] it and is clear&&NucleophilicCycloadditions ring-openings—these primarily—these refer refer to to 1,3-dipolar the openings cyclo- of Furthermore,reviews. The years thethat reaction 1999 the two and conditions schools 2000 each of thought—click contain are preferred one publication. and toDiels–Alder proposed green be mild chemis-strainedadditions, heterocyclic butby Kochi also include electrophiles, ten years hetero-Diels-Alder ago such36, besides as aziridines, cycloadditions providing epox- 100% atom so that biologicalstry—have and other emerged fragile with structuresa few overlapping present principles, will not that is,ides,(2). cyclic sulfates, aziridinium ions, episulfonium ions, etc. lossistry. their For functions. instance,atom economy, Protections they are simple typically and purification deprotections performed procedures,economy also in and water.need benign with (2). no solvent required, and affords a single product with no solvents and side products. From the perspective of substan-& Nucleophilic ring-openings—these refer to the openings of to be considered to avoid unwanted side reactions. Click & Carbonyl chemistry of the non-aldol type—examples Furthermore,tial the overlap reaction between conditions these two are philosophies, preferredpurification. The Diels–Alder reaction is certainly modular and wide in its scope: to the be importance mild strained heterocyclic electrophiles, such as aziridines, epox- chemistry,so that biologicals through andits unique other fragile features, structures easily presentsatisfies will all notof includeides,Scheme cyclic the 1. formations sulfates,Generic examples aziridinium of ureas, of a) a 1,3-dipolarthioureas, ions, episulfonium cycloaddition hydrazones, between ions, oxime etc. of the click chemistry philosophy for manyits chemists ability can beto function as a click reaction in different fields is being well theseloss constraints.their functions.expected It has Protections to repeatedly continue to and shown grow deprotections in to popularity serve the alsothroughout needs need the(2). azides and terminal alkynes, leading to the formation of 1,4-disubstitut- ed-1,2,3-triazole rings, b) a Diels–Alder cycloaddition, c) a double addi- ofto the be pharmaceutical consideredearly years to communityavoid of this newunwanted century. exceedingly side reactions. well, despite Click Cycloadditions recognized. & Carbonyltion of a thiol chemistry across a triple of bond, the and non-aldol d) a cycloaddition type between—examples an thechemistry, fact that through it wasThe phrase intendedits unique click tochemistry features, serve has an easily come entirely tosatisfies be different all but all synony- of alkyne and an nitrile N-oxide. Especially in the cases of b), c), and d), it include the formations of ureas, thioureas,I R hydrazones,' N oxime mous with copper(I)-catalyzed 1,3-dipolar cycloadditions be- should be noted that the alkyne can be replacedCu with an alkene.N these constraints. It has repeatedly shown to serve the• needsNucleophilic ring-openings, R N3 + R' these refer to the openings of strained tween azides and terminal alkynes (CuAAC) to yield 1,4- N of the pharmaceutical community exceedingly well, despite Cycloadditions R heterocyclic electrophiles, such as aziridines, epoxides, cyclic sulfates, the40% fact thatdisubstituted-1,2,3-triazole it was intended to serve rings. an Although entirely this different click reac- tion is arguably the most widespread and popular one to be to the(Husigen different 1,3-dipolar supramolecular cycloaddition speciesof azidesI andand terminal mechanicallyR' N alkynes) in- Cu N 35% aziridinium ions, episulfonium ionsR N3 + R' found in the literature to date, it is by no means the one and terlocked molecules, which will be addressed individually.N Nucleophilic Ring-Openings only star player. It is our intention in this Focus Review to R 30%40% highlight some of the recent creative advances in the litera- X + HX 2.(Husigen 1,3-Dipolar 1,3-dipolar CycloadditionsH cycloaddition of azides Azides and with terminalX = O Alkynes, NR alkynes), +SR, +NR ture on click chemistry (Scheme 1), not only the CuAAC re- :Nu 2 25%35% Nu action, but also thiol–ene/yne, Diels–Alder, and nitrile N-NucleophilicThe CuAAC Ring-Openings reaction has proven to be highly efficient when 20%30% oxide chemistries towards the production of functional employed in the final step leading to the formation of the Non-AldolX Carbonyl Chemistry[6] + HX supramolecular species and mechanically interlocked mole- mechanical bond duringH the construction ofX mechanically= O, NR, +SR, +NR • B carbonyl chemistry of the “non-aldol” type, such as formation of ureas, :Nu 2 15%25% Nu cules. This Focus Review has been organized according to interlockedO molecules (MIMs),XR in3 particular, in the case of R3XNH2 N [7] + H O X = O, NR[7] 10%20% the types of click reactions employed and hasthioureas, aromatic heterocycles, oxime ethers, hydrazones, and amides; been related rotaxanes and, to a lesser extent, in the case2 of catenanes. R1 R2 Non-AldolThe Carbonyl success Chemistry of the CuAACR1 R reaction2 in the synthesis of Percent of Total Publications 5%15% MIMs is, in part,(Hydrazone/oxime a consequence ether of the formation) fact that the reaction O XR3 [a] A. C. Fahrenbach, Prof. J. F. Stoddart R3XNH2 N occurs efficiently at room temperature;+ H anO observationX = O, NR 0%10% Department of Chemistry 2 whichR1 meansR2 O that theR NH stabilitiesR R ofO the supramolecular spe- Drug DiscoveryNorthwestern Pharmaceutical University Polymer Other 3 2 1 2 Percent of Total Publications cies, which are the immediate precursors, are+ betterHR2 main- 5% 2145 Sheridan Road, Evanston, IL 60208 (USA) R1 R2 R1 N R3 Research Areas (Hydrazone/oxime etherH formation) Fax: (+1)847-491-1009 tained with higher stabilities at lower rather than at higher E-mail: [email protected] 0% temperatures.O Not(Amide/isourea only does the formation) copper(I) metal center R3 NH2 O Fig. 2. MajorDrug classifications DiscoveryHomepage: Pharmaceutical http://stoddart.northwestern.edu of the applications Polymer of click chemistry. Other serve as a catalyst, but it can also act simultaneously+ HR as a R R 2 [b] Prof. J. F. Stoddart 1 2 R1 N R3 Analysis was performed based onResearch a literature Areas search via SciFinder Carbon templateMultiple Bond in Additions the so-called “active-metalH template” strategy Graduate School of EEWS (WCU) Scholar® on Dec. 31, 2007. The search included journal articles, aimed at the formation of MIMs, pioneered early on in the Advanced Institute of Science and Technology (KAIST) [X] (Amide/isoureaX formation) R R [8] + + abstracts,Fig. 2. Major preprints, classifications373-1, dissertations, Guseong of Dong, thepatents, Yuseong applications and Gu, reviews.Daejeon of click 305-701 The chemistry. field of 1 Leigh2 group. For a review on+ active-metalByproduct templation,X = O, NR see, SR, NR2 R R drugAnalysis discovery was was performedRepublic considered ofbased Korea separate on (Korea) a literature from pharmaceutical search via SciFinder and the Carbonreference Multiple Bond [8]. Additions1 2 categoryScholar®“other on” Dec.contains 31, 2007. miscellaneous The search applications included thatjournal cannot articles,Additions be to carbon - (Formation carbon of various multiple three-member bonds, rings) especially oxidative cases • [X] X grouped into the other three categories, such as applications in R R + + abstracts, preprints, dissertations, patents, and reviews. The field of 1 2 + Byproduct X = O, NR, SR, NR2 Chem. Asian J. 2011, 6, 2660 – 2669  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemasianj.org EWG 2661 materialsdrug discovery sciences, was certain considered reviews, separate and novel from methods pharmaceutical for improvedsuch and the as epoxidation, dihydroxylation, R1 R2 aziridination, and 3 sulfenyl halide EWG1 EWG3 catalysts.category Many“other applications” contains miscellaneous are subjective applications in that they that can cannot fall into be + (Formation of various three-memberEWG 1rings) moregrouped than one into category the other (i.e. three the synthesis categories, of certain such blockas applications copolymersaddition, but also Michael additions of Nu-H reactants. in EWG2 EWG2 EWG couldmaterials be considered sciences, certain both pharmaceutical reviews, and novel and methods polymer-related). for improved In EWG 3 EWG1 (Certain3 Michael Additions) thesecatalysts. cases, Many the applications applications were are subjective categorized inthat as pharmaceutical they can fall into if + EWG1 andmore only than if their one category corresponding (i.e. the abstracts synthesis specifically of certain block mentioned copolymers that EWG2 14 EWG2 thecould involved be considered compound(s) both could pharmaceutical play a role and in polymer-related). a pharmaceutical In Fig. 3. Major classifications of click chemistry reactions, along with setting.these cases,It is intended the applications for this chart were to categorized represent aas general pharmaceutical overview if corresponding examples.(CertainNu Nucleophile; Michael AdditionsEWG) electron withdraw- andand should only if not their be takencorresponding literally. abstracts specifically mentioned that ing group. the involved compound(s) could play a role in a pharmaceutical Fig. 3. Major classifications of click chemistry reactions, along with setting. It is intended for this chart to represent a general overview corresponding examples. Nu Nucleophile; EWG electron withdraw- and should not be taken literally. ing group. Click Chemistry, A Powerful Tool for Pharmaceutical Sciences 2217

400 purpose. In this review, important aspects of click chemistry 350 will be discussed, along with its emerging applications in drug delivery and nanomedicine. We will also provide some of our 300 concerns and vision for its future development in pharma- 250 ceutical sciences. Several reviews on click chemistry have already been published (1–3). The readers may find them 200 helpful in understanding other aspects of click chemistry or in 150 obtaining more background information.

100 Number of Publications CLASSIFICATION OF CLICK REACTIONS 50

0 As already implicated, click chemistry encompasses a 1999 2000 2001 2002 2003 2004 2005 2006 2007 group of powerful linking reactions that are simple to Year perform, have high yields, require no or minimal purification, and are versatile in joining diverse structures without the Fig. 1. Number of publications containing the key words “click prerequisite of protection steps. To date, four major classi- chemistry” or “click reaction” from 1999–2007. The literature search fications of click reactions have been identified (Fig. 3). was performed via SciFinder Scholar® on Dec. 31, 2007 and included journal articles, abstracts, preprints, dissertations, patents, and & Cycloadditions—these primarily refer to 1,3-dipolar cyclo- reviews. The years 1999 and 2000 each contain one publication. additions, but also include hetero-Diels-Alder cycloadditions (2). istry. For instance, they are typically performed in water. & Nucleophilic ring-openings—these refer to the openings of Furthermore, the reaction conditions are preferred to be mild strained heterocyclic electrophiles, such as aziridines, epox- so that biologicals and other fragile structures present will not ides, cyclic sulfates, aziridinium ions, episulfonium ions, etc. loss their functions. Protections and deprotections also need (2). to be considered to avoid unwanted side reactions. Click & Carbonyl chemistry of the non-aldol type—examples chemistry, through its unique features, easily satisfies all of include the formations of ureas, thioureas, hydrazones, oxime these constraints. It has repeatedly shown to serve the needs of the pharmaceutical community exceedingly well, despite Cycloadditions the fact that it was intended to serve an entirely different R' N CuI R N + R' N 3 N R 40% (Husigen 1,3-dipolar cycloaddition of azides and terminal alkynes) 35% Nucleophilic Ring-Openings 30% X + HX H X = O, NR, +SR, +NR :Nu 2 25% Nu

20% Non-Aldol Carbonyl Chemistry 15% O XR3 R3XNH2 N + H O X = O, NR 10% 2 R1 R2 R1 R2 Percent of Total Publications 5% (Hydrazone/oxime ether formation)

0% O R NH O Drug Discovery Pharmaceutical Polymer Other 3 2 + HR2 R1 R2 R1 N R3 Research Areas H (Amide/isourea formation) Fig. 2. Major classifications of the applications of click chemistry. Analysis was performed based on a literature search via SciFinder Carbon Multiple Bond Additions Scholar® on Dec. 31, 2007. The search included journal articles, [X] X R R + + abstracts, preprints, dissertations, patents, and reviews. The field of 1 2 + Byproduct X = O, NR, SR, NR2 drug discovery was considered separate from pharmaceutical and the R1 R2 category “other” contains miscellaneous applications that cannot be (Formation of various three-member rings) grouped into the other three categories, such as applications in Highly efficient reactions of thiols with reactive carbon–carbon double/ materials sciences, certain reviews, and novel methods for improved• EWG3 EWG1 EWG3 catalysts. Many applications are subjective in that they can fall into + triple bonds (TEC-TYC) EWG1 more than one category (i.e. the synthesis of certain block copolymers EWG2 EWG2 could be considered both pharmaceutical and polymer-related). In R (Certain Michael Additions) these cases, the applications were categorized as pharmaceutical if 1 R1 R2 R2 SH and only if their corresponding abstracts specifically mentioned that S the involved compound(s) could play a role in a pharmaceutical Fig. 3. Major classifications of click chemistry reactions, along with setting. It is intended for this chart to represent a general overview corresponding examples. Nu Nucleophile; EWG electron withdraw- S and should not be taken literally. ing group.R3 R2 SH R3 R2

15 1.2 Basics of the radical chemistry 37 38 Heterolysis ‘versus’ homolysis “Common” organic reactions are envisioned to occur by displacement of electron pairs: this dissociative process entails unsymmetrical cleavage of covalent bonds (heterolysis), in which both bonding electrons remain with one partner and there is consequent formation of charged species (ions). The alternative possibility is a symmetrical bond breaking (homolysis), in which case each atom carries away one of the two original bonding electrons. In this way, neutral species bearing an unpaired electron (radical intermediates) are generated. Most radical reactions involve homolytic cleavage (Scheme 1), but exceptions are usually encountered with electron-transfer processes.

X Y X + Y X Y X. + Y. heterolysis homolysis

Scheme 1. Heterolysis vs. Homolysis

It is worth noting that, at least in gas phase, more familiar heterolysis is generally less feasible than homolysis. However, under more usual solution conditions, especially when (highly) polar solvents are employed, ionic solvatations become so important as to allow most reactions to proceed through heterolytic pathways. Since free radicals are often electrically neutral, solvent stabilization on those species are usually meaningless: consequently, only bonds with (particularly) low Bond Dissociation Energy (BDE) such as, for instance, Sn- H bonds (≈310 kJ mol-1 at 300 K in gas-phase), can be involved in alternative radical pathways. A main solvent effect is that “solvatated” ions have a low tendency to recombine and, therefore, can occur in somewhat high concentrations; on the other side, radical intermediates tend to recombine very quickly to non-radical products and so they usually occur in very low concentrations. This is the main reason why radical intermediates are often not easily detectable even by spectroscopic methods and why free radical reactions have long been believed to be very poorly selective. Nevertheless, through a careful choice of the experimental conditions (solvent, temperature, reagent ratio and

16 reagent concentration), radical reactions can allow highly useful synthetic transformations both in terms of product yield and by-product minimization. Structure and stability of radicals The structure and stability of free (carbon) radicals roughly resemble those of the corresponding (carbo)cations. In general, lowering of BDE values favour production of radical species which are therefore regarded as being more stable (Table 3). Progressive substitution at the radical centre can usually enhance radical stability owing to inherent hyperconjugation and/or resonance effects (carbon radical stability follows the order: benzyl > tertiary > secondary > primary > methyl).

Table 3. Approximate dissociation energies of selected bonds (kJ mol-1 at 300 K in gas-phase)

CH3-H 439 Cl3C-H 402 ((CH3)3Si)3Si-H 331

CH3CH2-H 423 F-H 569 (CH3)3Sn-H 310

(CH3)2CH-H 410 Cl-H 431 C2H5-Cl 339

(CH3)3C-H 397 Br-H 366 C2H5-Br 289

CH2=CH-H 431 I-H 297 C2H5-I 222 HC≡C-H 544 HO-H 498 RO-OR 155

C6H5-H 464 HOO-H 368 CH3-CH3 372

CH2=CHCH2-H 364 CH3O-H 439 CH3CH2-CH3 364

C6H5CH2-H 372 C6H5O-H 360 (CH3)2CH-CH3 360

RC(=O)-H 364 R2NO-H 310 (CH3)3C-CH3 351

EtOCH(CH3)-H 385 CH3S-H 385 Cl-Cl 243

N≡CCH2-H 360 C6H5S-H 343 Br-Br 192

CH3COCH2-H 385 (CH3)3Si-H 377 I-I 151

The unpaired electron can occupy a p orbital or a hybrid orbital having some s-character: the corresponding radicals are referred to as π- and σ-radicals, respectively. Ethyl radical 1 as well as primary, secondary and tertiary alkyl radicals usually are “planar” π-radicals, but their geometry is affected by the presence of strongly electronegative substituents like fluorine or alkoxyl. In fact, trifluoromethyl 2 is a pyramidal σ-radical bearing its electron in a sp3-orbital. Benzyl radical 3 also is a π-radical where the unpaired electron is delocalized onto the adjacent aromatic ring (Figure 3). Similar to benzyl radical 3, other π-

17 radicals bearing an adjacent unsaturated substituent like the vinyl, cyano or carbonyl group are stabilized by conjugation with the adjacent π bond.

. H CH2 CH2 CH2 CH2 H . . H . . H F F H F . hyperconjugation pyramidal, σ resonance in planar, π benzyl radical in planar, π ethyl radical trifluoromethyl radical 1 2 3

Figure 3. Stucture and stabilisation effects in radicals

Stabilization can additionally arise from overlapping of the orbital of the π- radical with the lone pair of a vicinal heteroatom including inter alia oxygen, nitrogen and, to a lesser extent, halogen atom; such interaction in fact leads to a resonance-stabilized ‘three-electron bonding’ where there is a separation of charge since an electron is donated from the heteroatom (Scheme 2). Consequently, the extent of such lone pair stabilization is expected to decrease with increasing the electronegativity of the heteroatom involved. The concomitant presence of an electron-donating and electron- withdrawing group brings about a synergistic effect between the two groups and radical stabilization becomes greater than might be expected based on the sum of the effects of the two separate groups. The effect has been referred to as ”captodative” stabilization, where the electron-withdrawing group is the “capto” group and the electron-donating group is the “dative” one. For instance, such effect is responsible for the very low BDE displayed by the α-C–H bond of glycine (318 kJ mol–1, Scheme 2)39

.. . . R . .. R .. . H2N . H2N .. H2N X X H H H R' R' HO2C HO2C HO C O. resonance with lone pairs glycine radical

Scheme 2 . Stabilization by lone pairs and captodative

18 ‘Stability’ versus ‘persistence’ There is a further, important point concerning “radical stability” which is worth note: the difference between thermodynamic stability (or “stability”) and lifetime (or “persistence”) of a radical species. Stability largely depends on electronic effects such as the mesomeric and inductive ones discussed above. On the other side, persistence is a general result of steric factors: the more congested is the radical centre the greater will be the radical lifetime. This is reflected in relatively high concentrations of radicals species occurring in solution and/or in self-reactions significantly less than the diffusion limit. For example, methyl radical 4 has a half-life of 0.2 × 10−3 s at a 10−6 M concentration, whereas tri(iso-propyl)methyl radical 5 (a relatively ‘persistent’ radical), at the same concentration, has a half-life of 21 h! The longer the lifetime, the less reactive a radical intermediate will be, since its persistency will slow down the reaction rate with itself and other species. It is worth note that benzyl 6, even though being ‘stabilized’ by resonance delocalization, undergoes diffusion-controlled self-reaction and hence is not at all a “persistent’ radical (Figure 4).40

Me Me Me H H H H H H Me Me Me 4 5 6

Figure 4. Stability vs. persistence: benzyl radical 6 is thermodynamically more ‘stable’ than 5, but kinetically less persistent

In certain cases of noteworthy persistency (due to both thermodynamic and kinetic factors), the lifetimes of radical species can be so long that they can survive for a very long time (like some biologically or industrially important phenoxyls) or even almost indefinitely as do some commercially available free- radicals such as TEMPO and DPPH) (Figure 5).

19 Me O. NO2 .O R R Me Me . Ph C H O N N N 16 33 N 2 Me O Me Me Ph Me O. Me R NO2 2,4,6-trisubstituted Vitamin E phenoxyls TEMPO DPPH

Figure 5. Stable and persistent free-radicals

The idea of persistency is hence strongly related to the mechanism of action of popular antioxidants which play a crucial role in the fields of food conservation and cell protection. Moreover, the concept of radical persistency is also very important in organic synthesis as a radical generation method coupled with efficient trapping control: infact, radical persistency is exploited in the achievement of living-radical polymerization.41 ‘Philicity’ of radicals and ‘Hydrogen Abstraction’‘ The presence of electron-donating (EDG) and electron-withdrawing (EWG) substituents also affects the ‘philicity’ of radicals species with important consequences for their chemical behaviour. Indeed, although radical reactions have long been considered to be independent of polar effects owing to the intervention of neutral intermediates, more recent studies have clearly established that those reactions can be largely governed by an appropriate choice of radical/molecule partners having opposite philicity. This concept can be simply accounted for by considering that EDG groups can actually enhance the electron density on the radical centre, whereas EWG groups can behave in an opposite fashion. In fact, the oxidation/reduction potentials of radicals are largely affected by the electronic properties of their substituents: the presence of an EDG group will lower the oxidation potential of a radical centre, thus favoring possible occurrence of a corresponding (carbo) cation, while the presence of an EWG will lower the reduction potential, thus encouraging alternative occurrence of a corresponding (carb)anion. In the former cases ‘electrophilic’ radicals will arise and in the latter ‘nucleophilic’ ones (Scheme 3).

20 electron density electron density

R . .. R .. . R . O R .O X X R' R' R' R" R' R" nucleophilic radical electrophilic radical R . .. -e- R .. R R . O +e- R O R O X X X R' R' R' R' R" R' R" R' R"

Scheme 3. Radical philicity

The radical philicity can be explained more rigorously in terms of a frontier molecular orbital (FMO) approach.42,43 For a radical having a vicinal EDG substituent with a lone pair, interaction of the p-orbital containing the unpaired electron (SOMO) with the adjacent lone pair gives rise to two new molecular orbitals: two of the three electrons occupy the new lower energy orbital, while the third electron occupies the new SOMO which is higher in energy than that of the original one (Scheme 4a). On the contrary, a radical bearing an unsaturated EWG substituent has a SOMO that is very close in energy to the antibonding π*-orbital (LUMO) of the substituent: in this case, the interaction between the SOMO and the empty LUMO gives rise to a new SOMO that is lower in energy than the original one (Scheme 4b). Accordingly, unlike ions, radicals are stabilized by both EDG and EWG substituents. Furthermore, the FMO approach also explains the high selectivity of most radical reactions. Since in fast reactions small energy differences are required in SOMO-LUMO or SOMO-HOMO interactions, radicals with a high-energy SOMO have a tendency to interact with unfilled LUMO orbitals (such as those of electron-deficient olefins), and hence behave as electron-donating (nucleophilic) species. On the contrary, radicals with a low-energy SOMO tend to interact with SOMO filled orbitals (like those of electron-rich alkenes) and then behave as electrophilic species (Scheme 4c,d).

21 a) E c) LUMO

LUMO SOMO NEW SOMO SOMO HOMO non-bonding, n [lone pair] orbital HOMO

R . .. R . .. OR" OR" CN R' R' nucleophilic radical nucleophilic radical / electron-deficient olefin (SOMO-HOMO interaction) (SOMO-LUMO interaction)

b) E d)

LUMO

LUMO antibonding, π* SOMO orbital SOMO HOMO NEW SOMO HOMO

R . O R . O OMe R' R" R' R" electrophilic radical electrophilic radical / electron-rich olefin (SOMO-LUMO interaction) (SOMO-HOMO interaction)

Scheme 4 . Molecular orbitals and radical philicity

An analogous explanation can account for the very high selectivity often observed in hydrogen abstractions. These reactions are known to be highly affected by polar factors and substituents capable to stabilize a partial charge separation in the transition state increase the reaction rate (Scheme 5). For example, nucleophilic alkyl radicals abstract a hydrogen atom from a thiol with a kinetic constant of ≅ 107 − 108 M−1 s−1, whereas the same reaction with a silicon hydride has a kinetic constant of ≅ 103 M−1 s−1 only. Since the sulfur−hydrogen bond (370 kJ mol−1) has a similar strength to the silicon−hydrogen one (375 kJ mol−1), the reaction is clearly not influenced by thermodynamic factors. Indeed, the reaction with thiols is fast because the nucleophilic alkyl radical rapidly abstracts the thiol hydrogen through a transition state with partial charge separation in which the carbon radical donates an electron to sulfur. With nucleophilic silanes analogous charge separation in the transition state is

22 evidently discouraged (Scheme 5a). Of course, the opposite applies to for electrophilic radicals, which can rapidly abstract hydrogen from nucleophilic silanes but not from electrophilic thiols (Scheme 5b).

R R a) R' H R' H OR" OR" . δ- δ+ δ+ δ- . R' δ- R δ+ H SiR3 R' R H SR R' δ+ R δ- H SiR3 . H SR OR" OR" OR" unfavoured nucleophilic radical favoured

b) δ- δ+ δ+ δ- δ- . δ+ . δ- R' R H SiR3 R' R H SR R' δ+ R H SiR3 . H SR R" O R" O R" O favoured electrophilic radical unfavoured

R R R' H R' H

R" O R" O

Scheme 5. Polar effects in hydrogen abstraction

We can obtain the same rationale by the frontier molecular orbital approach. The hydrogen abstraction reaction is the result of the interaction between the SOMO of the radical and either the HOMO (σ) or the LUMO (σ*) of the C−H bond. Electron-withdrawing groups attached to the C−H bond will lower the HOMO and LUMO energies, whereas electron-donating groups will have the opposite effect. Therefore, electrophilic radicals will react faster with the electron-rich C−H bond of silanes through SOMO-HOMO interaction (see Scheme 4d), whereas nucleophilic radicals will prefer to react with the electron-deficient C−H bond of thiols through SOMO-LUMO interaction (see Scheme 4c). Mechanism of radical transformations: “chain reactions” and unit steps Many radical reactions are “chain reactions” and they are characterized by three fundamental steps: initiation, propagation and termination of chain. For better understanding these concepts, we shall examine the radical reaction of

23 methane with molecular chlorine leading to formation of methyl chloride upon photochemical irradiation (Reaction 1).

hν CH4 + Cl2 CH3Cl + HCl

Reaction 1. Chlorination of methane

The first step of this reaction is the initiation of a radical chain: suitable irradiation causes initial fragmentation of molecular chlorine to chlorine atoms (Reaction 2).

h Cl Cl ν 2 Cl ((1)1)

Reaction 2. Initiation step of radical chain

Like in this case, an initiation step generally entails generation of some radical species from non-radical precursors (initiators)44. The second step involves propagation of a radical chain: an initially-formed chlorine abstracts a hydrogen atom from methane to form hydrochloric acid and a methyl radical (CH3•) which then reacts with molecular chlorine to form chloromethane with regeneration of a chlorine atom (Scheme 6).

Cl + H CH3 HCl + CH3 (2)

CH3 + Cl Cl CH3Cl + Cl (3)

Scheme 6 . Propagation step of radical chain

The repetitive character of Scheme 6 is referred to as propagation of a “radical chain”: through radical chain propagation the starting reagents (molecular chlorine and methane) are consumed and chloromethane and hydrogen chloride products are formed. The third and last step, occurring in competition with the propagation step, entails termination of a radical chain: pairs of radical species formed in Equation 2 (Cl• and CH3•) interact to form non-radical products45 according to Scheme 7.

24 Cl + Cl Cl Cl (4)

CH3 + Cl CH3Cl (5)

CH3 + CH3 H3C CH3 (6)

Scheme 7. Termination step of radical chain

We will now analyze more in details these three processes which are very important for successful applications of radical reactions in organic synthesis. Radical Initiation Radical reactions are commonly initiated by thermolysis or photolysis of suitable precursors, but radicals can also be produced by redox reactions (oxidations and reductions).46 Also dioxygen can initiate radical processes, since the ground state of O2 is a triplet with two unpaired electrons one of which is accommodated into two degenerate π*-orbitals. Dioxygen is therefore a stable biradical and can, for instance, abstract hydrogen atoms from weak carbon −hydrogen bonds or act as a radical scavenger: these processes are of basic importance for example in lipid and polymer autoxidations.47 Radical generation by thermolysis can occur in solution at relatively low temperatures from molecules having suitably low BDEs (125-165 kJ mol−1). Low BDEs are usually encountered with carbon−heteroatom, heteroatom −heteroatom, or metal−metal bonds, but carbon−carbon or carbon−hydrogen bonds are normally too strong to be thermally broken. Suitable molecules include diacyl and dialkyl peroxides, where O−O bonds are broken (125-150 kJ mol−1), and azo-compounds, which undergo cleavage of two N−C bonds with concomitant formation of molecular nitrogen. Peroxides like di-tert-butyl, dilauroyl, or dibenzoyl peroxide and azobis-iso-butyronitrile (AIBN) are the most commonly used radical initiators, also employed in industrial applications (e.g. polymerisations) (Scheme 8a). The decomposition temperature of a radical initiator largely depends on structural features and electronic properties of substituents: its decomposition can range from room temperature (hyponitrites, peroxyoxalates, or V-70 [azobis(4-methoxy-2,4-dimethylvaleronitrile]) up to 140-150 °C.

25 ! ! a RO OR 2 RO. RN NR 2 R . + N or h" or h" 2

h" h" h" b RO NO RO . + . NO 2 RI 2 R . + I2 R3M MR3 2 R3M.

e e RX R . + X RO OR(H) RO . + (H)RO c + e ArN2 ArN2. Ar . + N2

-e + -e + ROH RO . + H ArCH3 ArCH2 . + H d O OH Mn(III) O +. OH O O + H+ R R' -Mn(II) R R' R . R'

Scheme 8. Radical initiation methods

Visible or UV light can promote low-energy or non-bonding electrons to antibonding orbitals, giving rise to excited states characterized by weaker bonds with respect to the ground states. Light can therefore be used to break bonds, providing that the molecule is able to absorb it. Under mild thermal conditions Photolysis can cleave even strong bonds, which would be otherwise broken at elevated temperatures, in a very selective way through selected choice of light of requisite energy. However, photolysis (especially UV irradiation) often requires special apparatus, reactors, and vessels, and is hence not as practical as other usual experimental procedures. Substrates that can be broken photolytically include peroxides, azo-compounds, halides (especially iodides), nitrites, and organometallics (to give metal-centred radicals) (Scheme 8 a,b). Carbonyl compounds absorb UV light (270-300 nm) yielding singlet excited states, through n → π* transitions, that next form triplet states through intersystem crossing processes: the resulting di-radicals can participate in many radical reactions. Neutral molecules can also be converted into radicals by electron-transfer routes: addition of an electron gives a radical anion that usually fragments to a radical and an anion, whereas extrusion loss of an electron generates a radical cation that commonly breaks into a radical and a cation. Alternatively, radicals can be directly generated from anions or cations by removal or addition of an electron, respectively. These redox reactions are usually carried out by means of metal ions that change their oxidation state and thus behave like one-electron

26 oxidizing or reducing agents. These processes normally occur under very mild conditions and are very selective. Single-electron transfer can also take place in electrolytic cells, where neutral molecules can be either reduced to radical anions at the cathode or oxidized to radical cations at the anode (in the same way, cations can add an electron at the cathode or anions can lose an electron at the anode). Radical generation by one-electron reduction can be easily accomplished with halides (which fragment to carbon radical and halide anion), peroxides or hydroperoxides (which break into alkoxyl radical and alkoxide or hydroxide anion) as well as with arenediazonium salts giving rise to diazenyl radicals that rapidly extrude dinitrogen (Scheme 8c). Radical generation by one- electron oxidation can be achieved with carboxylic acids and alcohols (whose radical cations lose a proton to give acyloxyl or alkoxyl radicals, respectively), alkylarenes (which give rise to benzylic-type radicals by loss of a proton), and carbonyl compounds (mainly di-carbonyls, whose enolic form can be especially oxidized with Mn(III) salts), to give resonance-stabilized α–carbonyl or α,α- dicarbonyl radicals (Scheme 8d)48. Radical propagation Propagation reactions are processes in which a radical intermediate is converted into a new radical by means of either unimolecular (rearrangements and fragmentations) or bimolecular processes (intermolecular reactions with non-radical molecules); a number of these reactions can take place in sequence until a termination step pairs all of the electrons49. Unimolecular propagation reactions encompass rearrangements and fragmentations. The former involve transformation of a precursor radical into a more stable (or more reactive!)50 radical intermediate and usually occur through migration of atoms (e.g. 1,5-H shifts) or groups (e.g. 1,2-aryl shift), or by intramolecular addition51 to C-C double and triple bonds and carbonyl moieties (Scheme 9). Atom transfers are typically limited to 1,5 and 1,6 shifts, since lower-order migrations require strained transition states, whereas group translocations may occur also between vicinal atoms, like in the cases of neophyl rearrangement (1,2-aryl shift) and 1,2-acyloxy migration.

27 Fragmentations occur by β– (or α–) elimination of a radical species with concomitant formation of an unsaturated molecule. The driving force, besides the obvious increase in entropy, can be due to generation of a stable radical, formation of a strong π–system like a carbonyl group or an aromatic ring, or release of a very stable molecule (CO2, CO, N2). Alkyl radicals can suffer β– fragmentation only in the presence of a weak σ–bond in the β–position, since the resulting alkene bond is not enough strong as to provide a suitable driving force (suitable β–substituents are halogen atoms, sulfanyl and stannyl groups). On the contrary, the fragmentation of alkoxyl or cyclohexadienyl radicals is strongly favoured by the formation of strong carbonyl groups or aromatic rings, respectively. Acyloxyl and diazenyl radicals have a high propensity to extrude carbon dioxide and nitrogen, respectively: with these radicals even aryl radicals can be generated in an efficient fashion. As far as bimolecular propagation reactions are concerned, they include atom abstractions (SH2 reactions) and addition reactions to unsaturated moieties (alkenes, alkynes, carbonyls and their derivatives) and aromatic compounds (Scheme 9).

28 radical propagation reactions unimolecular bimolecular

R H(X). R .(X)H RO . + H R' RO H + R' .

R3Sn . + X R' R3Sn X + R' . hydrogen (or group) migrations SH2 reacti ons . R' R' R . . . R. R R' R' R R R . neophyl rearrangements additio ns to al kenes/al kynes . Y Y . Y X X X Y .X R' R" cyclisations R. R' R" O . O R + R. additio ns to carbonyl deri vatives Ar R Ar R' R' . X O H CO2 + R. R. . X R O R fragmentations additio ns to aro matics

Scheme 9. Intra- and intermolecular radical propagation reactions

Atom abstractions are analogous to the ionic SN2 reactions: indeed, SH2 stands for Substitution Homolytic Bimolecular (2) and the unimolecular analogue discussed above is often called SHi (Substitution Homolytic intramolecular). They are concerted displacement reactions where a hydrogen or halogen atom (but sometimes sulfanyl, stannyl, and other groups) is transferred from a molecule to a radical through the involvement of a linear transition state in which the radical orbital overlaps with the vacant σ* orbital of the bond being broken. Hydrogen abstractions are typically performed with alkoxyl radicals, which form alcohols having a strong O–H bond, whereas halogen abstractions are usually achieved with tin or silicon radicals in which cases the driving force arises from the formation of a strong metal-halogen bond at the expense of a weaker carbon-halogen bond. Radical addition to unsaturated moieties is very common in the case of alkenes (e.g. in polymerisations), since the formation of a new C–C σ-bond (≅ 370 kJ mol−1) at the expense of a weaker π–bond (≅ 235 kJ mol−1) is usually a

29 good driving force. With mono-or unsymmetrically-substituted alkenes, addition takes place preferentially at the less hindered position: such regiochemical outcome is probably a result of steric factors rather than stability of the ensuing radical adduct. The most popular reaction of this type is the anti-Markovnikov, peroxide-mediated addition of hydrobromic acid to alkenes (Scheme 10). Unlike charged intermediates, radicals can add to both electron-rich and electron-poor multiple bonds, although the addition rate strongly depends on polar effects (see above) and bulkiness of the reactants. Alkenes and alkynes exhibit analogous regioselectivity, but alkyne reactions are usually slower due to ensuing generation of less stable vinyl radical adducts.

Br . H Br ! R' R' RO OR 2 RO. or h" . . RO + HBr ROH + Br HBr . Br R'

Scheme 10. Anti-Markovnikov radical addition of HBr to olefins.

Addition to carbonyl compounds is generally unfavourable, since the C=O π– bond is stronger than the alkene double bond by ca. 80 kJ mol−1; therefore, most of the reported cases involve intramolecular examples. Carbon-centred radicals, especially the nucleophilic ones, show a certain propensity to attack electrophilic carbonyl carbons in a reversible fashion, whereas heteroatom- centred radicals like stannyls and silyls prefer to add to the carbonyl oxygen, due to alternative formation of a stronger metal–oxygen σ–bond. Very useful reactions, but largely confined to intramolecular examples, are those involving addition of nucleophilic radicals to the electrophilic carbon of nitriles, oximes, and hydrazones: these reactions are usually very fast and substantially irreversible, since fairly stable radical adducts are formed and in such cases there is no efficient driving force for the reverse reaction to compete. Radical addition to aromatic groups is rather slow, due to the great stability of the aromatic π–system. The resulting cyclohexadienyl radicals can either

30 revert to the starting reactants or be oxidized, by formal loss of a hydrogen atom, to give substitution products in a process comparable to the electrophilic aromatic substitution. The addition rate strongly depends again on polar factors, hence nucleophilic and electrophilic radicals will react preferentially with electron-poor and electron-rich aromatics, respectively. The addition is often regioselective, since radicals prefer to attack the ring positions that give rise to cyclohexadienyls more stabilized by conjugative and/or inductive effects. Radical Cyclisations as a powerful synthetic tool

Among the great number of useful radical transformations, ring closures reactions play undoubtedly a pivotal role in organic synthesis. Radical cyclisations can occur in sequence (tandem cyclisations) and can show high levels of regio- and even stereoselectivity, being probably the most popular, distinctive contribute of free-radical chemistry to the world of synthesis.

According to Baldwin’s rule52, radical (and also non-radical) cyclisations can be classified into exo and endo modes, depending on whether ring closure occurs on either the inside or the outside of the unsaturated moiety, respectively. In other words, exo- or endo-cyclisations are those leading to new radicals that are exocyclic or endocyclic with respect to the newly-formed rings. The exo or endo term is usually preceded by a number indicating the ring size and followed by an additional term representative of the hybridisation of the (carbon) atom at the reaction site. Hence, cyclisations are called tet, trig, or dig when the reaction sites are tetrahedral sp3, trigonal sp2, or digonal sp carbons, respectively (Scheme 10). As an example, a “5-exo-trig” cyclisation (one of the most common radical ring-forming reactions) is a ring-closure reaction that gives rise to a 5-membered ring through attack of the starting radical to the inner carbon atom of an alkene moiety, with consequent formation of a new exocyclic radical.

31 endo exo-tet . exo X endo-tet X. sp3 (- X . ) . endo exo-trig . exo endo-trig sp2 . . endo exo-dig . exo endo-dig sp . .

R R 5-exo-trig . cyclisation !

Scheme 11. Baldwin’s rule for radical cyclisations.

At this stage it is worth noting that most useful radical reactions are exothermic processes. According to Hammond Postulate, the transition states of exothermic reactions – and thence of the radical reactions – are generally reagent-like (Figure 6).

Energy T.S.

A

B

Reaction coordinate

Figure 6. Generalized reaction profile for an exothermic reaction: the transition state (T.S.) is closer to the reagent (A) than to the products (B) along both coordinate axes.

In light of Hammond Postulate for two similar exothermic processes it is possible to envision a reaction profile like that shown in Figure 7, termed “crossing”, where the process thermodynamically less favored (A→B) has however a transition state of lower energy, and is therefore kinetically more favored.

32 !

"#$%&'! !" #"

$"

($)*+,-#!*--%.,#)+$!

Figure 7. “Crossing” reaction profiles

The concepts just discussed can help to explain a very particular behaviour that occurs in the field of the radical cyclisations. For radical reactions to be fast, the single occupied orbital (SOMO) of the reacting radical must overlap efficiently with a suitable orbital of the radicophilic partner. For intermolecular reactions this condition is easily fulfilled, since the reactants can rotate freely to ensure maximum overlap. On the other hand, intramolecular reactions, namely cyclisations, can be strongly affected by so-called “stereoelectronic effects”, because not all the conformations are energetically favoured and the reaction could be kinetically controlled by the process triggered by the most favoured interaction. Cyclisation of the hex-5- en-1-yl radical is an emblematic example of this behaviour (Scheme 12 ). For cyclisation of this radical, at 25 °C, the 5-exo mode (kexo = 2.3 × 105 s−1) is about 60 times faster than the 6-endo competitive ring closure (kendo = 4.1 × 103 s−1).53

33 . ~109° . (2) 5-exo cyclisation (1) . . 6-endo .

(1) cyclisation (2) !

Scheme 12. Anti-Markovnikov radical addition of HBr to olefins.

At first sight, this fact is unexpected at least for two well-founded reasons: i) the cyclopentane ring is more strained than the cyclohexane ring, and ii) the radical arising from 5-membered cyclisation is a primary radical and should be therefore thermodynamically less stable than the secondary radical formed in the 6-membered ring closure. Nevertheless, at 25 °C, the 5-exo and 6-endo products are formed in a 98:2 ratio! Actually, stereo-electronic effects operate and the reaction is under kinetic rather then thermodynamic control. This is explained by the fact that the singly occupied orbital overlaps more favourably with the alkene π* orbital at the inner carbon atom, through a chair-like transition state (Beckwith model) that has been proved to be less strained and energetically favoured with respect to the 6-membered analogue: indeed, this overlap is characterised by an attack angle very close to 109 °C (i.e. the bond angle that is required in the product radical). Since radicals are typically quite unstable, very reactive species, radical cyclisations are usually irreversible and kinetically controlled, and the product ratios (and even the stereochemistry!) strictly depend on stereo-electronic effects and can be often predicted on the basis of Beckwith models. However, if the cyclisation is reversible, due to the stability of the starting radical, the reaction can afford the thermodynamically favoured product, since ring opening of the less stable radical product can be faster than, for example, the hydrogen abstraction process involved in the reaction of Scheme 13.54

34 .

(2) 5-exo EtO2C EtO2C

cyclisation NC NC EtO2C . (1)

6-endo . CN (76%) (1) EtO C EtO C cyclisation 2 2 (2) NC NC

Scheme 13. Termodinamically controlled reaction due to reversible cyclisation

Radical Termination The termination reactions are processes in which radical intermediates are destroyed as a result of coupling (or dimerisation), disproportionation, or one- electron exchanges. Coupling or dimerisation (heterocoupling or homocoupling) reactions55 involve combination of two radical species to yield a non-radical molecule as a result of the formation of a covalent bond (Scheme 14). These reaction are very fast and essentially diffusion controlled. Coupling products are though usually observed in very small amounts, since their formation requires fairly high radical concentrations, a condition rarely achieved due to the very short radical lifetimes. Disproportionation entails transfer of a β–hydrogen between two carbon radicals with formation of a C=C π–bond and a C–H σ–bond. Also these reactions are very fast and the greater the number of β–hydrogens and the bulkier the radical centre, the more likely it is that a disproportionation will occur. Electron transfer reactions convert radical species into anions and cations by one-electron reduction and oxidation processes, respectively. The ‘redox reagent’, as in the case of radical generation, can be a transition metal ion or an electrode. The reaction rate depends of course on the redox potential of the radicals: tertiary alkyl radicals are easily oxidized to carbocations, due to the stabilising +I inductive effect of the alkyl groups. Other less substituted carbon radicals can also be readily oxidized when bearing electron donating groups like OR and NR2 which can play an important role in cation stabilisation. On the contrary, primary alkyls prefer reduction to carbanions, and the process is

35 strongly favoured by electron-withdrawing groups (e.g. NO2, COOR), which can stabilise the resulting negative charge by –M mesomeric effect (Scheme 14).

radical termination reactions

2 R. R R R . + R'. R R' homocoupling (dimerisation) heterocoupling (coupling)

H H H H H H H + + R C. H2 R C. H2 R CH3 R CH2 disproportionation O O e -e O O + R C. H2 R CH2 R C. H2 R CH2 one-electron reduction one-electron oxidation

Scheme 14. Radical termination reactions

Starting from these bases, we can now penetrate in the ‘world’ of the radicals and explore their use in synthetic organic chemistry.

36 1.3 Thiol radical coupling Growing interest has been devoted to the use of quick reactions that meet the main criteria of Click and Green Chemistry for an ideal synthesis, namely efficiency, versatility, and selectivity. The existing click reactions, like those discussed above, can offer many interesting applications, but, however, they present several intrinsic drawbacks such as, for example, removal of toxic heavy metal impurities from the final products, explosive nature of azido compounds, limited reactivity of Diels-Alder reagents and retro cycloaddition reactions at high temperatures.56 In the past years Thiol–Ene coupling (TEC, Eq. 1), and, more recently, Thiol-Yne coupling (TYC, Eq. 2) have encountered renewed interest, after the original patent gained by Charles Goodyear at the beginning of the last century for his process concerning vulcanization of natural rubber by sulphur57. Those coupling reactions have been shown to be very versatile, and in fact have found valuable use in the production and modification of (bioconjugated) polymers,58,59 , 60 and surfaces,61 synthesis of star polymers, dendrimers62 and disaccharides.63

R1 SH

SR1 R2 R2 (Eq. 1) Init.

R1 SH

SR1 (Eq. 2) R2 R2 Init.

TEC/TYC can actually proceed under a variety of conditions through radical processes or nonradical processes mediated by nucleophiles, acids, bases,64 in the apparent absence of an added catalyst in highly polar solvents, such as water or DMF.65 We focused our attention to the radical thiol-click reactions owing to long interest of our group in free radical chemistry and, more importantly, to the fact that the radical thiol-click reactions, besides being modular, occur at room temperature with high efficiency and fast kinetics even in the presence of oxygen or water; moreover, they do not require any (toxic) catalyst, and are highly tolerant of a wide range of functional groups. Additionally, thiol-click reactions

37 S N OR 19 - RO N N S N S N S S N S 20 + + + 1 21 22 23 24 Scheme 1-4

1.3.1 S-centred radicals

Amongst the rich and abundant literature on S-centred radicals, the two most

important types for preparative organic chemistry are the thiyl and sulfonyl radicals.30,31

Convenient methods for generating thiyl radicals consists of homolytic cleavage of the S–S

bond in their corresponding disulfides (25) using photolysis (eq. 4), and H-atom abstraction

from their thiols RSH, 26 (eq. 5).22,30,31 Details will be given in Section 1.3.2. For sulfonyl

radicals, sulfonyl halides 27 can undergo halogen (Hal)-abstraction from an initiator, or the are orthogonal to a wide range of chemistries.9A Like any radical thiol addition, the radical thiol-click reactions entail tree steps including initiation, propagation S–Hal bond in 27 can be cleaved photochemically (eq. 6).22,31-33 In the case of Hal = iodine, and termination. the weak S–I bond could cleave under mild conditions such as room temperature in an Generation of thiyl radical (RS⋅) is the initiation of the process. There are overnight reaction34 or UV irradiation.32 Sulfonyl radicals can also be generated through different way to generate the radical: -Homolytic cleavage of the S–S bond in their corresponding disulfides using reversible addition of an alkyl radical R• to sulfur dioxide (eq. 7); conversely, sulfonyl photolysis (Eq. 3);radicals are prone to unimolecular !-scission, which is responsible for the decomposition of -H-atom abstraction from their thiols RSH (Eq. 4); alkanesulfonyl halides to its alkylhalide and sulfur dioxide.22,30,31 Although the unpaired -Autoxidation of RSH with oxygen, very slow; -Anodic oxidation, of thiols, in which cases where thiyl radicals are formed electron of sulfonyl radical is delocalized over the oxygen atoms, sulfonyl radicals add to " as intermediate. systems exclusively through sulfur to form new C–S bonds.30,32

O O h! h! RS SR 2 RS (eq. 4)(Eq. 3) R S Hal R S (eq. 6) O or initiator O (1)25 Thiyl radical 27 Sulfonyl radical Hal = Cl, Br or I

initiator O RS H RS (eq. 5) (Eq. 4) R + SO R S (eq. 7) T(°C) or h 2 26 v "-scission (2) O

The S–S bond dissociation energy (BDE) in diphenyl disulfide (PhSSPh), - 6 - 214.2 kJ/mol, is considerably lower than that of dialkyl disulfides (RSSR), which are reported to be in the range of 277 – 301 kJ/mol.66 In fact, homolysis of the S- S bond is much more feasible with PhSSPh than with dialkyl disulfides (RSSR); owing to resonance-stabilization of ensuing phenylsulfanyl radicals. A more useful method for RS⋅ generation entails hydrogen-abstraction from corresponding thiols in the presence of radical initiators including photoinitiators like (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TMDPO), 2,2-dimethoxy-2-phenyl acetophenone (DMPA), benzophenone (BP), thioxanthone (TX), and camphorquinone (CQ), or typical thermal initiators like azobis(isobutyronitrile) (AIBN) at 80 °C. H-abstraction from thiols in the presence of thermal/photochemical initiators provides a general method for sulfanyl radical production, but recent studies have established that the use of photoinitiators is especially effective to promote certain polymerization processes mediated by sulfanyl radicals.67

38 Thiol-ene coupling(TEC)

S

R2 R1 SH R1

SH Initiator S R S 1 R2 R1 R1

R2

Scheme 15.The mechanism for the hydrothiolation of a C=C bond

Radical thiol additions to alkenes are generally very fast, even at ambient temperature and pressure, are tolerant of the presence of oxygen/air, and give rise to nearly quantitative formation of thio-ether products often in a regioselective fashion. After the initial formation of a thiyl radical, RS⋅, the overal addition process takes place through two distinct steps (Scheme 15): primary addition of the thiyl radical to either carbon of the alkene double bond yielding an intermediate carbon-centered radical and subsequent H-transfer from the thiol reagent to the derived carbon radical with concomitant regeneration of a thiyl radical for chain propagation. Generally, terminal alkenes are significantly more reactive towards radical hydrothiolation than internal alkenes and give rise to sulfide adducts in strictly regioselective anti Markovnikov fashion. Hoyle at al. reported that 1-hexene is eight-fold more reactive than trans-2-hexene and 18-fold more reactive than trans-3-hexene in radical hydrothiolations, thus discovering that steric effects play a significant role in affecting the radical alkene reactivity with thiols. However, it is worth note that an additional role can be played by usual reversibility of the primary addition of sulfanyl radical to the double bond.

39 Thiol-Yne coupling(TYC)

S

R2 R1 SH R1

S SH Initiator S R1 R2 R1 R1

R2

Scheme 16 The mechanism for the hydrothiolation of a C≡C bond

The radical thiol-yne processes (TYC) (Scheme 16) occur in analogous conditions to those of TECs and also involve two analogous radical steps. Indeed, an initially-formed thiyl radical, RS⋅, adds to either carbon of the alkyne triple bond yielding an intermediate β-sulfanyl-substituted vinyl radical, which then undergoes H-abstraction reaction with the thiol present to give the appropriate vinyl sulfide adduct with concomitant regeneration of a thiyl radical. The whole hydrothiolation process, though being regioselective, at least with the terminal alkynes, is usually scarcely stereoselective, since the vinyl sulfide adducts are often formed as mixtures of E/Z geometrical isomers. As a consequence, TYC reactions often fail to fulfill one of the major requirements of click-reactions, i.e. stereoselectivity. TYCs are usually more effective with terminal than internal alkynes owing to lesser steric constraints; moreover, they are also more effective with electron-rich alkynes since these encourage primary addition of sulfanyl radicals known to possess somewhat electrophilic character.

Stereoselectivity and Regioselectivity

Thiyl radical addition to C=C bonds generally occurs in a reversible fashion68 and is often accompanied by some isomerization process: in fact, thiyl radicals are usefully employed to bring about cis/trans isomerization of alkenes (Scheme 16 and Eq. 5).69 Although the β-scission rate constants for β sulfanylvinyl radicals (k-2) have not been actually determined, the reversibility

40 of RS⋅ addition to alkyne C≡C triple bonds has been clearly proved (Eq. 6). The

rate constants for sulfanyl radical additions to alkenes and alkynes, k1 and k2, are in the range of 103–108 M-1s-1 and 103–106 M-1s-1 at at ambient temperature: 43,47-49 demonstratedsuch rate (eq. constants 12). normally In addition, exhibit when a R high is an dependence aryl group, on thiyl the radical nature addition of generallysubstituents attached to the vinylic or ethynylic carbons. has a greater reversibility than for an alkyl group.43,50

k 1 RS R S + (eq. 11) R' (Eq. 5) k-1 R'

k 2 RS R S R' + (eq. 1(Eq. 6)2) k-2 R'

TheDuring various an methods addition for thiyl if the radical R is generation, an aryl70 group, thiyl radical addition which were utilized throughout this

71 project,generally include has the homolytic a greater cleavage reversibility of disulfides than for (eq. an 13), alkyl H group.-abstraction Cramer of thiols, et al, RSH postulate that the ratio of propagation to chain transfer kinetic parameters (kp/ (eq. 14), autoxidation of RSH (eq. 15) and anodic oxidation (eq. 16). The background or kCT it is the same to say k1/K-1) and polymerization rates are correlated to the mechanismselectron density of the vinyl groups and the carbon radical stability. for each of these methods will be discussed in detail in the following72 The rate pages.

constant as a consequence depends h! by all components of our reaction, thiyl RS SR 2 RS (eq. 13) radical group, substituent, electron density of vinyl group and the radical initiator stability. RS H RS (eq. 14) or h!

R1 R2 Y R1 OR22 R1 R4 Y R1 R4 RS H rotationRS (eq. 15) Y (slow) Y - R3 R4 R3 - Re4 R3 R2 R R2 RS H RS (eq. 16) anode Scheme 17.Reversible step of C=C bond hydrothiolation. Cis Trans isomerization

TheIn consequence to all point discussed before, S–S bond dissociation energy (BDE) in result understandable why it diphenyl disulfide (PhSSPh), 214.2is kJ necessary an excess mol-1,51 is considerably of one reagents. lower than that73 To of complete our reagent dialkyl disulfides (RSSR), we have which to are shift the equilibrium towards the sulfide product. It be in contrast with the idea reported to be in the range of 277 – 301 kJ mol-1.51-53 Thus, the homolytic S–S bond scission of Click Chemistry. to produceThe resonance addition stabilized of a sulphanyl thiyl radicals radical from to PhSSPh an alkyne requires gives less rise energy to a thanvinylic from RSSR.carbon More radical. importantly, This the intermediate can energies required be for “σ-bent”, the excitation in which of singlet case the unpaired to triplet states electron is located in an sp2 orbital, or “π-linear” with the unpaired electron and the dihedral angles (C–S–S–C) of the respective disulfides54,55 determine the ease of S–S placed in a p-orbital (Scheme 17). π-Linear vinyl radicals usually occur when the bond homolysis. In addition, when RSSR is irradiated at highly energetic wavelengths, C–S R2 α-substituent is sterically bulky (e.g. t-Bu) or when this latter is able to bond cleavage does occur instead of S–S bonds,45,55,56 since the BDE of C–S bonds in dialkyl

(with ! four carbon atoms) disulfides is ~ 235 kJ mol-1,52 which is less than the associated 41 energy for its S–S bonds. It has also been shown that photolysis of derivatives of PhSSPh

- 8 - stabilize the unpaired electron through resonance delocalisation (e.g. Ph). The

74 TYC rate constants (K2) are very sensitive to steric constrains and hence internal alkynes react more slowly than the terminal ones; moreover, electron- poor alkynes are less reactive than the electron-rich counterparts owing to the known fact sulfanyls are somewhat electrophilic species

R1 S R1 SH R2 Init. R2

Scheme 18. π-linear vinyl radical

The σ-bent vinyl radicals can undergo fast inversion yielding syn/anti conformers in a fashion (highly) dependent on the bulkiness of the R2 α- substituent, (Scheme 19)

Interconversion is very slow when R2 is a heteroatom.

R1 S R1 S R2 R1 S

R2

R2

Scheme 19: σ-bent vinyl radical, fast inter-convention between Z-isomer to E-Isomer

With terminal alkynes radical hydrothiolation usually occurs in a regioselective anti-Markovnikov fashion. Indeed, with the aliphatic alkynes sulfanyl radical attack preferentially occurs at the terminal carbon since this is the site less sterically hindered; with the aromatic ones sulfanyl radical attack at the terminal carbon is even more preferred owing to resonance stabilization of ensuing vinyl radical by the attached aromatic ring.

42 Thiol-Yne / Thiol- Ene Reactions (TYC-TEC)

Using appropriate alkyne substrates and thiol reagents tandem adoption of TYC and TEC chemistry can enable successful achievement of a broad range of simple/complex bis-sulfide bis-adducts which are of significant interest in polymer or material chemistry (Scheme 20).

1,2-bis-sulfide derivatives), giving rise to notable regioselectivity problems. This product 6 alkyne thiol issue was already observed in the earliest studies and has been reagent reagent 45 dealt with in our recent methodological study on the main factors H 14 R2 R1 influencing thiol-yne couplings. On the other hand, the S R2 R1 SH possibility of a TYC/TEC sequence is the feature of the thiol-yne S R1 reaction that has contributed most to its popularity amongst materials chemists, since, when performing TYCs in such

. 50 conditions as to optimise formation of bis-adducts, it allows for R2 R1 2 1 TYC TEC S R R R1 S. the efficient, straightforward construction of highly cross-linked . S chain chain S R1 or hyperbranched polymers and highly functionalised materials such as dendrimers:15 it might be avowed that this is the result of the 'double' functionality of the alkyne moiety under thiol radical

2 1 55 addition conditions. 1 R R R SH S A couple of methodological studies have recently appeared H thiol dealing with the influence of substrates structure and reaction reagent conditions (e.g. temperature and solvent) on the TYC outcome, in vinyl sulfide product terms of both substrate reactivity (kinetics) and product 16 Scheme 1 Mechanism of TYC and TEC radical chains. 60 distribution. In the first one, a kinetic investigation allowed for Scheme 20. Mechanism of TYC and TEC radical chains. reporting a reactivity scale following (to some extent) what 8 was catalysed by organic peroxides or UV light. should had been expected on the basis of the general reactivity of Patton Since et al. the have very beginning, demonstrated all authors thiol-yne realised chemistry what makes as a modular sulfanyl radicals with alkynes:11c like most radical reactions, TYC 5 radical TYC different from the TEC counterpart and hence its is very sensitive to steric hindrance and hence internal alkynes platform for rapid and practical fabrication of highly functional, multicomponent potential advantages and drawbacks. As shown in Scheme 1, the 65 react more slowly with respect to terminal ones; in addition, surfaces,75 and recently a versatile post-polymerization modification strategy to first reaction step is again addition of a sulfanyl radical to the sulfanyls are electrophilic in nature and react more readily with carbon-carbon triple bond to afford a !-sulfanyl-substituted vinyl electron-rich alkynes. However, the reactivity scale is not synthesize multifunctional polymer brush surfaces via combination of surface- radical: hydrogen transfer from the starting thiol affords a vinyl completely in line with the electron-density properties of the initiated photopolymerization and orthogonal 10 sulfide and a new sulfanyl radical that thiol-click reactionssustains the chain. The76 (Figure 8). employed alkynes and also the unexpected results obtained with whole process, although regioselective, at least with terminal 70 N-methyl-N-propargylamine have not been accounted for yet. This approach alkynes, has demonstrate is usually scarcely very stereo interesting selective, since for the material vinyl sulfide with model More and interestingly, it was found that thiols react with cyclooctyne commercially products available thiols. are often The main idea will formed as mixtures of be extended to both E- and Z fabrication of - very rapidly and also without any initiation: this is most likely the stereoisomers: one of the main requirements of click-chemistry- result of relief of ring strain by dissolution of one "-bond by multiplexed biomolecule.15 reactions, i.e. stereoselectivity, is therefore lost. Conversely, it is radical addition, a behaviour already encountered for the copper- 17 worth pointing out that radical additions to alkenes are generally 75 free strain-promoted azide-alkyne cycloaddition (SPAAC). 9 faster than those to alkynes, but the former usually occur in a Unfortunately, the unreactivity of the resulting cyclooctenyl 10 reversible fashion, whereas the latter are substantially sulfide, i.e. its inability to undergo further thiol addition, makes 11 irreversible, at least with alkanesulfanyl radicals. This means this alkyne not suitable as a monomer for network 20 that radical TECs often require a significant excess of photopolymerisations. Furthermore, the high, spontaneous 12 alkene/thiol and/or high thiol concentrations to shift the 80 reactivity of cyclooctyne with thiols suggests limitations to the equilibrium towards the sulfide products. As a consequence, orthogonality of TYC in the presence of azide groups. additional workup is necessary to get rid of that excess, hence In our recent paper,14 we focussed instead our attention on the loosing one of the main advantages of click-reactions. On the influence of the experimental conditions, i.e. thiol/alkyne molar 25 contrary, most of radical TYCs are normally much more efficient ratio, temperature, and, primarily, solvent, on the TYC reaction

when carried out with equimolar amounts of reagents, since the 85 outcome, showing that a proper choice of those conditions can intermediate vinyl radicals are formed in a virtually irreversible favour highly selective occurrence of either mono- or bis-sulfide 43 manner and abstract a hydrogen atom from the thiol reagent more coupling products. This is not a crucial issue in the field of rapidly than their alkyl counterparts. materials chemistry, where obtaining cross-linked polymers or 30 Anyway, the most important point is that, contrary to the highly functionalised materials by one-pot complete addition of

products of TEC (alkyl sulfides), the products of TYC (vinyl 90 two equivalents of the same thiol to the alkyne of interest (by sulfides) are reactive species that can undergo a subsequent what we can call a TYC/TEC homosequence) is the only addition of another sulfanyl radical to afford bis-sulfide bis- attractive target. It could be instead an extremely appealing adducts through the intermediacy of !,!-disulfanyl-disubstituted matter in the domain of bioconjugation, where selective 13 35 alkyl radicals (TYC/TEC sequence, Scheme 1). From a click- formation of a vinyl sulfide mono-adduct can, on one hand, be a

chemistry point of view, this additional process brings about extra 95 valid alternative to TEC for functionalisation of biomolecules drawbacks related to both regio- and stereoselectivity. Indeed, the under true click-chemistry conditions, and, on the other hand, bis-sulfides possess a new chiral centre that is created without pave the way to bis-functionalisations with two different sulfide any stereoselectivity; more crucially, although 1,2-bis-addition is moieties through TYC/TEC heterosequences performed in 40 the rule of thumb and it is observed, for example, with all succession with two different thiols. The results obtained will be

terminal alkyl acetylenes, 1,1-bis-addition can compete with 100 described below in the section dedicated to bioconjugation, but certain alkynes (especially arylacetylenes and arylpropiolic acid

2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] View Online

Scheme 1 General schematic for dual-functional polymer brushes by one-pot thiol-yne co-click reactions from PgMA brushes (DMPA 2,2- ¼ Dimethoxy-2-phenylacetophenone).Figure 8 General schematic for dual-functional polymer brushes by one-pot thiol-yne co-click reactions Based on similar thiol reactivities, the thiol-yne co-click reaction yields a distribution of 1,2-homo and 1,2-hetero dithioether adducts within the brush surface. from PgMA brushes (DMPA 1⁄4 2,2- Dimethoxy-2-phenylacetophenone). Based on similar thiol reactivities, the thiol-yne co-click reaction yields a distribution of 1,2-homo and 1,2-hetero dithioether

77 1 p(PgMA-TMS) brushes were synthesized by surface-initiatedadducts within the brush surface.respectively. The peak at 2189 cmÀ for the protected alkyne 1 photopolymerization as previously described using the trime- group in Fig. 1a, and the peaks at 2125 and 3280 cmÀ for the thylsilyl-protected alkyne monomer and subsequently depro- deprotected alkyne in Fig. 1b are consistent with our previous tected using KOH/methanolThe most to give the important terminal alkyne. applications 29 Fig. 1 work and of confirm TEC the successful and/or synthesis TYC of the chemistry p(PgMA) brush. (a) and 1(b) show the gATR-FTIR spectra for p(PgMA) brush The deprotected p(PgMA) brush was further functionalized via withappeared pendant alkyne in groups the in protected past and few deprotected years forms, are radical-mediated covered by thiol-yne Nanni click by and exposing Massi the surface in to a UV light in the presence of a photoinitiator and a mixture of desired comprehensive monograph which is being published in Organic & Biomolecular thiols (thiol:THF, 50/50 v/v). All thiol-yne reactions were 2 conducted for 4 h at 40 mW cmÀ . The reaction time of 4 h was Chemistry.77 selected to ensure complete conversion of alkyne groups of p(PgMA) regardless of the fact that the actual time required for complete conversion may be significantly shorter than 4 h. Indeed, quantitative conversion of the alkyne groups was observed following thiol-yne click reactions with equimolar mixtures of various thiols as indicated by the disappearance of 1 the peaks corresponding to deprotected alkyne at 2125 cmÀ and 1 3280 cmÀ (gATR-FTIR spectra in Fig. 1c–e). The p(PgMA) brushes showed an expected increase in thickness after thiol-yne reactions with all of the thiol mixtures due to increase in Downloaded by UNIVERSITA DEGLI STUDI BOLOGNA on 16 February 2012 Published on 14 November 2011 http://pubs.rsc.org | doi:10.1039/C1JM14762E molecular mass of repeat units and was consistent with previous results.29 Fig. 1c shows the gATR-FTIR spectrum for a p(PgMA) brush after thiol-yne click from an equimolar mixture of dodecanethiol and thioglycerol. As shown in Fig. 1(c), the broad 1 peak between 3600 and 3100 cmÀ corresponding to the hydroxyl 1 groups of thioglycerol, and peaks at 2955, 2922 and 2853 cmÀ corresponding to the aliphatic chain of dodecanethiol appear indicating that both thiols simultaneously undergo thiol-yne click reaction with p(PgMA) brush. The concentration of the hydrophilic hydroxyl and hydrophobic aliphatic groups in the clicked brush can be easily controlled by simply varying the concentration of respective thiols to control the wettability of the surface. As a second example, we selected a binary mixture of N- acetyl cysteine, a biologically relevant thiol, and dodecanethiol to perform the one-pot thiol-yne click reaction with a p(PgMA) brush. Fig. 1(d) shows the gATR-FTIR spectrum of a p(PgMA) brush clicked with the equimolar mixture of N-acetyl-cysteine 1 1 and dodecanethiol. Peaks at 1643 cmÀ and 1605 cmÀ for the secondary amine groups of N-acetyl cysteine, and peaks at 2955 Fig. 1 Poly(propargyl methacrylate) brush a) protected, 23.7 nm 1.1 1 1 1 Æ cmÀ , 2922 cmÀ and 2853 cmÀ corresponding to the aliphatic nm; b) deprotected, 11.4 nm 1.1 nm; c) clicked with an equimolar Æ mixture of thioglycerol and dodecanethiol, 26.1 nm 3.0 nm; d) clicked chains of dodecanethiol appear suggesting successful simulta- Æ with an equimolar mixture of dodecanethiol and N-acetyl cysteine, 26.9 neous thiol-yne click reaction of both the thiols. The one-pot nm 2.3 nm; and e) clicked with an equimolar mixture of dodecanethiol, thiol-yne click approach can be further extended to more Æ mercaptopropionic acid and N-acetyl cysteine, 26.8 nm 2.8 nm. complex model systems via use of ternary thiol mixtures. Fig. 1(e) Æ This journal is ª The Royal Society of Chemistry 2012 J. Mater. Chem., 2012, 22, 932–943 | 935

44 1.4 Theoretical approach In recent years, significant progress has been made in understanding at the molecular level the structures, functions and processes involved in many natural and artificial chemical systems thanks to instruments like EPR and LFP and their related analytical methods. Along with these advancements, quantum chemical methods have almost reached a speed and accuracy that make them a valuable tool for this kind of studies. Today we can choose among various Quantum chemical approaches (see below). Ab-initio78: the computation comes directly from theoretical principles, with no use of experimental data. The most common method in ab-initio calculations is Hartree Fock (HF)79; The most accurate methods based on an with HF approach is the Quantum Monte Carlo one80. Another opportunity for an ab-initio approach is a newer ab initio method, the Density Functional Theory (DFT)81, in which the total energy is expressed in terms of the total electron density, instead of the wavefunction. In DFT calculations there is an approximate Hamiltonian and an approximate expression for the total electron density. The accuracy of results from DFT calculations can be poor to fairly good, depending on the choice of basis set and density function. Broadly speaking, ab initio calculations give very good qualitative results and can yield increasingly accurate quantitative results as the involved molecules become smaller. The advantage of ab initio methods is that they eventually converge to the exact solution once all the approximations are made sufficiently small in magnitude. However, this convergence is not monotonic. Sometimes, the smallest calculation gives a very accurate result for a given property. There are four sources of errors in ab initio calculations: the Born-Oppenheimer approximation, the use of an incomplete basis set, incomplete correlation, the omission of relativistic effects.

45 The disadvantage of ab initio methods is that they are expensive. These methods often take enormous amounts of computer CPU time, memory, and disk space Semiempirical82 semi-empirical quantum-mechanical methods developed by Dewar and coworkers83 have successfully reproduced and interpreted molecular energies, molecular structures and chemical reactions84 . . Semiempirical methods are much faster than ab initio calculations. The disadvantage of semiempirical calculations is that the results can be erratic and fewer properties can be reliably predicted They have been successfully applied in organic chemistry, wherethey can provide results accurate enough to be useful, since there are only few elements used extensively and the molecules are of moderate size. These methods are generally good for predicting molecular geometry and energetics. This can be used for predicting vibrational modes and transition structures, but in a less reliable way than ab initio methods. Semiempirical calculations generally give poor results for van der Waals and dispersion intermolecular forces, due to the lack of diffuse basis. Initially, semiempirical methods have been devised specifically for the description of inorganic chemistry. Molecular mechanics85 are the calculation methods that it allows the modeling of enormous molecules, such as proteins and segments of DNA. Molecular dynamic86 examine the time dependent behavior of a molecule.. The application of molecular dynamics to solvent/solute systems allows the computation of properties such as diffusion coefficients or radial distribution functions for subsequent statistical mechanical analysis. The favorable scaling (i.e. the computational effort required for a certain system size) of DFT compared to ab initio methods allows the analysis of far larger systems, and thus it encouraged much wider applications than ever before. Of course, the nearly exponential development in computer power has also had a major impact on this field of studies. Quantum chemistry has gone from being a quite expensive branch of science to quite a cheap one, where many problems can be addressed using a few ordinary personal computers.

46 V. Van Speybroeck et al.

1 w0ð Þ rotating top. The conversion factor containing the speed of light I L01 L02 c, 0 ÁÁÁ the Avogadro constant w 1 T NA, the atomic mass unit m and the Bohr 1 1 T 1 0L I L 1 u T wð Þ 01 1 12 0 ; w1; ; wn A with A ÁÁÁ radius rB reduces to 70.939. ¼ 2 ð Þ ÁÁÁ B . C T T B . C ¼ BL L I C Summary of terminology used in this article for the alternative B . C B 02 12 2 C B C B ÁÁÁC methods for treating the low-frequency torsional modes (IR B w C B C @ n A @ ÁÁÁ A modes) beyond the standard HO approach: In 1D-HR, IR modes 5 are uncoupled from the global rotation and uncoupled from one ð Þ another. For each IR mode a 1D rotational potential is constructed where n is the number of internal rotations present in the mole- that leads to a 1D partition function through use of a self-written cule, w [16] 0, w1, … are the instantaneous angular velocities of the program. The HO contributions of the IR modes in the global vi- global rotation and the various internal rotations, I is the inertial brational partition function must be replaced by the manually con- tensor, the L matrix elements are the coupling terms of the global structed 1D partition functions, so that they are not counted twice. rotation with the IRs or the mutual coupling of internal rotors, and Distinction can be made between the following two methods, de- Ii (i=1,…,n) are the instantaneous moments of inertia of the rotat- pending on the determination of the frequency for the HO contri- ing tops. bution of the IR mode: The classical expression of the partition function corresponding to the global rotation and internal rotations reduces to Equation (6): 1) 1D-HRa (standard method): frequency from identification of IR mode in normal mode spectrum (nontrivial for longer chains). 2p 2p s 3 n 1 sn 2) 1D-HRb (new method): frequency from rotational potential and 1 1 2p þ2 q 2 reduced moment of inertia [Eq. (8)]. rot 3 8p d d bV 1 ; n ¼ s h n b 1 n det A 1; n eÀ ð ÁÁÁ Þ ð þ Þ  Z ÁÁÁZ Â ð ÁÁÁ Þ 0 0 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi In 2D-HR, the multidimensional PES is approximated by a set of 6 ð Þ 2D-PES that describe the coupling of adjacent rotors: n 1 n 1 À À where b=(1/kBT), s is the symmetry number of the molecule, and V  ; ;  In any quantum chemical studies, it is essential to 1 choose n V the i; i 1 models V i . The HO to contributions of ð ÁÁÁ Þ¼ þ s (i=1,…,n) the symmetry numbers of the internal rotations. i 1 ð ÞÀ i 2 ð Þ i the IR modes are removedP¼ from the globalP¼ vibrational partition The multidimensional PES V( ,…, f1 fn) is constructed fully ab initio function via Equation (8) (in correspondence with method 1D- accurately represent the chemical situation.on an n-dimensional grid from 0 to 360 in all torsional angles, 8 HRb). with a grid spacing of 10 8. The potential energy in each point is determined by optimizing all variables except the In the following section we will demonstrate the failure of the 1D- n torsional Free-radical polymerization angles that are kept fixed at the grid is values, an important but symmetry consid- and well-established HRa method for long-chain alkyl radicals. process This implies that the har- erations reduce the number of explicit calculations by a factor of monic frequencies of the internal rotations for such systems for the production of polyethylene. Reyniers et al. applied DFT ab initio density two. Clearly, determination of the PES is the computationally ex- should always be determined by using Equation (8), corresponding pensive step in the calculation. The kinetic energy matrix is then to method 1D-HRb. easily constructed on the basis of the relaxed geometries of the functional theory at the B3LYP/6-31g(d) level to study this process. The applied PES. Recently, it was shown that the 2D-HR scheme gives a good ap- 3. Results and Discussion proximation for the nD-HR model at reasonable computational basis set took into cost.[18] account This study showed the that the high multidimensional degree potential of conformational We will illustrate several flexibility general aspects of the and radical addition energy surface can accurately be described by constructing a sub- reactions for reaction AD7, that is, the addition of an octyl radi- internal rotations during the growing of the radical alkyl chainssequent 2D-PES. This 2D-HR method was used to study the influ- cal to ethylene (Figure87 2). ence of coupling on the kinetics of reactions AD7 and AD9. The In a first section, we discuss several aspects related to the global uncoupled IR partition function of the molecule treatment of internal rotations. In a second part, the behavior IR ! quncoupled q1q2 qm is replaced by the 2D-coupled IR partition function [Eq.¼ (7)].ÁÁÁ Ab Initio Study of Free-Radical Ethylene!"#"$%&'()*+*,&-+.$/0*1&2&.&34$ Polymerization

IR q1;2q2;3 qm 1;m q2D coupled ÁÁÁ À À ¼ q2;3 qm 1 ! 7 ÁÁÁ À byð Þ ab initio molecular calculations. For the studied propagation re- !"#"5"$60+7*,&-$'071+-,8'$&9$9:00$:+2,7+.$(&.4'0:,;+*,&-$Identification of IR Modes: For long chains, the identification of in- actions, the reacting alkyl radical and the corresponding transition ternal rotations in the normal-mode spectrum becomes less trivial state are very similar, and thus many effects will cancel in the ratio because several IR mix in one vibrational mode, so there$ is no q°/qradical in Equation (2) The kinetic parameters were deduced by unique frequency for each IR. It is, however, possible to extract the Figure 1. Addition reactions ADn:C H C+C H C H C. Distinction fitting the results of the TST expression [Eq. (2)] to the Arrhenius $%&!'(&&)(*+,-*.!/0.12&(,3*4,05!/(0-&&+6!7,*!*!-%*,5!2&-%*5,628!frequency correspondingn+1 2n+3 to a2 pure4 ! internaln+3 rotation2n+7 from the rota- is made between propagation series with even and odd numbers of carbon Figure 9. Addition reactions ADn : Cn + 1H2n + 3C + Ctional potential and the reduced moment of inertia2H4 [Eq. !Cn + 3H2n + 7C. Distinction (8)] rate law [Eq. (3)] in a specific temperature range. is made between propagation series with even and odd numbers of carbon atoms in atoms in the9%,-%! radical backbone. :*6,-*..1! -056,646! 0'! '0;(! &.&2&54*(1! (&*-4,056! ,<&

imax the radical backbone.1 i2V =kJ mol 1 2 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffimi À E =RT /(0/*=*4,058!-%*,5!4(*56'&(!*5+!4&(2,5*4,05!>?@A

where kB is Boltzmann’s constant, T the absolute temperature, h the number of stable conformers predicted in the uncoupled Planck’s constant, and V the reference volume in which the transla- scheme is incorrect and, moreover, the energy barriers are seriously tional part of the partition function is evaluated.47 The molecular underestimated. However in reference [17] this 1D-HR approach

partition functions qA and qB relate to the two reactants, and q° is was found to perform surprisingly well for entropies and heat ca-

the molecular partition function of the transition state. DE0 repre- pacities of n-alkanes, due to a fortuitous cancellation of errors. For sents the molecular energy difference at absolute zero between our systems we checked for AD7 and AD9 the effect of the two-di- the activated complex and the reactants, with inclusion of the mensional coupling of internal rotations and global rotation on the zero-point vibrational energies. reaction kinetics. Molecular properties such as geometries, ground-state energies, Generally, the kinetic energy of the multidimensional rotational and frequencies that are required for the evaluation of the parti- problem (both internal and external) can be written as Equa-

tion functions, as well as the reaction barrier DE0, were obtained tion (5):

ChemPhysChem 2006, 7, 131 – 140  2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org 133 non-stop increase to computer power, has now made possible to study the chemically controlled reactions in free-radical polymerization with chemical accuracy. Cote recently did an exhaustive study about this topic 89. About radical addition, it is relatively simple to find papers about polymerisation. Reyniers et al topic90, recently calculated thermochemical data of numerous organosulfur compounds and their radicals. They used CBS-QB3 level theory which proven to be able to yield accurate electronic energies for both hydrocarbons91 and organosulphur92, substrates present in our context. For small organosulphur compound recent works are not so numerous, since the attention in the latest years is more focused on the numerous biological precursors present in living systems.93 Alvarez-Idaboy et al.94 calculated that reaction with SH⋅ is about four times faster than with OH. The study was focused on analyzing atmospheric consequences in areas in which the H2S concentration was significant. The energy calculations were performed with unrestricted ab initio methods. Two different approaches were used to optimize the geometries: density functional methods (DFT) and Moller–Plesset perturbation theory. The calculation showed an excellent agreement with experimental data using the BHandHLYP method. To avoid problems deriving from the basis set superposition error the energy profiles were recalculated using the complete basis set extrapolation CBS-QB3 approach, MP2/6-311++G(d,p). All the calculations were performed with DFT ab initio. No theoretical work on the RS⋅ radical addition to double/triple bonds has been published. Molecular dynamics approach hasn’t been used for radical addition on small system yet. The method is very CPU expensive, thus usually it is used for big system, DNA95, protein, chemical adsorption96, or solvatation97. MD allows to observe the pathway during all the calculations, in particular, for solvated reactions, it allows to study the disposition of molecules depending on the different solvents.

48 Bibliography

1 Anastas P. T. and Warner J. C., Green Chemistry Theory and Practice, Oxford University Press, New York, 1998.

2 A) Trost BM (1991) The atom economy: A search for synthetic efficiency. Science 254:1471–1477. B) Trost BM (1995) Atom economy-A challenge for organic synthesis: Homogeneous catalysis leads the way.

3 Argyropoulos DS, eds (2007) Materials, Chemicals and Energy from Forest Biomass, ACS Symposium Series 954(American Chemical Society, Washington, DC).

4 A) Jimenez-Gonzales, C.; Curzons, A. D.; Constable, D. J. C.; Cunningham, V. L. Int J. LCA 2004, 114. B) Eissen, M.; Hungerbuhler, K.; Dirks, S.; Metzger, J. Green Chem. 2003, G25

5 DeSimone J. M., Science 297, 799 (2002).

6 Tanaka K., Kaupp G., Solvent-Free organic synthesis, Wiley-VCH, 2009

7 Acc. Chem. Res., 2007, 40(11), special issue on ionic liquids, ed. Robin D. Rogers and Gregory A. Voth.

8 A) Tumas W., DeSimone J.M . Green Chemistry Using Liquid and Supercritical Carbon Dioxide, Oxford University Press, New York, 2003; B) Chemical Synthesis Using Supercritical Fluids, ed. P.G. Jessop and W. Leitner, VCH/Wiley, Weinheim, 1999.

9 C.-J. Li, Chem. Rev., 2005, 105, 3095; C.-J. Li and T.-H. Chan, Comprehensive Organic Reactions in Aqueous Media, Wiley & Sons, New York 2007; Organic Reactions in Water, ed. U. M. Lindstrom, Blackwell, 2007.

10 S. G. Kazarian, B. J. Briscoe, T. Welton, Chem. Com-mun., 2047 (2000).

11 P. G. Jessop, W. Leitner, Eds., Chemical Synthesis Using Supercritical Fluid ( Wiley-VCH, Weinheim, Germany, 1999).

12 Darr J. A., Poliakoff M., Chem. Rev. 99, 495 (1999).

13 12. E. Lack and H. Seidlitz. In Extraction of Natural Products using Near-Critical Solvents, M. B. King and T. R. Bott. (Eds.), pp. 101–139, Blackie, Glasgow (1993).

14 Stewart, Gina (2003), DeSimone J. M. and Tumas W., ed., "Dry Cleaning with Liquid Carbon Dioxide", Green chemistry using liquid and supercritical carbon dioxide (USA: Oxford University Press): 215–227

15 .K.L. Hoy and K.A. Nielsen, “Methods for Cleaning Apparatus Using Compressed Fluids”, US Patent No. 5,306,350 (1994).

16 J.J. Watkins and T.J. McCarthy, “Polymer/Metal Nanocomposite Synthesis in Supercritical CO2

17 Leitner W., Jessop P. G. (Eds), Supercritical Fluids, Vol. 4 of the Handbook of Green Chemistry, Paul Anastas (series editor), Wiley-VCH, 2010. B) P. G. Jessop, "Homogeneous Catalysis using Supercritical Fluids: Recent Trends and Systems Studied," J. Supercrit. Fluids, 2006, 38, 211-231.

18 A) Wasserscheid P, Welton T (2002) Ionic Liquids in Synthesis (Wiley-VCH, Weinheim, Germany). B) Sheldon, R. A.; Lau, R. M.; Sorgedrager, M. J.; Rantwijk, F. v.; Seddon, K. R. Biocatalysis in Ionic Liquids. Green Chem. 2002, 4, 147-151

49 19 Martínez-Palou R., Microwave-assisted synthesis using ionic liquids,Mol Divers (2010) 14:3–25 DOI 10.1007/s11030-009-9159-3

20 M. E. Zakrzewska, E. Bogel-Łukasik and R. Bogel-Łukasik Solubility of Carbohydrates in Ionic Liquids, Energy & Fuels, 2010 24 (2), 737-745

21 N. Winterton, Solubilization of polymers by ionic liquids, J. Mater. Chem., 2006, 16, 4281-4293

22 A)Roger RD, Seddon KR, Volkov S (eds) (2002) Green indus- trial applications of ionic liquids. NATO Science Series. Kluwer, Dordrecht. B) Rogers RD, Seddon KR (eds) (2002) Ionic liquids: industrial applications of green chemistry. ACS, Washington, DC

23 Wasserscheid P, Welton T (2002) Ionic Liquids in Synthesis (Wiley-VCH, Weinheim, Germany)

24 van Rantwijk F, Sheldon RA (2007) Biocatalysis in ionic liquids. Chem Rev 107:2757–2785. doi: 10.1021/cr050946x

25 Narayan, S.; Muldoon, J.; Finn, M. G.; Fokin, V. V.; Kolb, H. C.;Sharpless, K. B. Angew. Chem., Int. Ed. 2005, 44, 3275.

26 Itami K., Yoshida, J.-I. (2003), The Use of Hydrophilic Groups in Aqueous Organic Reactions. ChemInform, 34: no. doi: 10.1002/chin.200327272

27 A) Y. Hayashi,In Water or in the Presence of Water?, Angew. Chem. Int. Ed. 2006, 45, 8103 – 8104. B) Brogan, A. P.; Dickerson, T. J.; Janda, K. D., Enamine-Based Aldol Organocatalysis in Water: Are They Really “All Wet”, Angew. Chem., Int. Ed. 2006, 45, 8100.

28 H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056 – 2075 ; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021.

29 A) Tome, A. C. In Science of Synthesis; Storr, R. C., Gilchrist, T. L., Eds.; Thieme: Stuttgart, New York 2004; Vol. 13, pp 415. B) 1,3-Dipolar cycloaddition chemistry; Padwa, A., Ed.; Wiley: New York, 1984.

30 R. Huisgen, Angew. Chem. Int. Ed. 1963, 75, 604.

31 V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596.

32 C. W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057.

33 Lutz,J.F.(2008)Copper-freeazide-alkynecycloadditions:New insights and perspectives. Angew. Chem., Int. Ed. 47, 2182–4.

34 Zhang, L., Chen, X. G., Xue, P., Sun, H. H. Y., Williams, I. D., Sharpless, K. B., Fokin, V. V., and Jia, G. C. (2005) Ruthenium-catalyzed cycloaddition of alkynes and organic azides. J. Am. Chem. Soc. 127, 15998–9.

35 O. Diels, K. Alder, Liebigs Ann. 1928, 460, 98 – 122.

36 J. H. Kim, S. V. Lindeman, J. K. Kochi, J. Am. Chem. Soc. 2001, 123, 4951 – 4959.

37 Mark ‘Radical Chemistry’, M. John Perkins, Ellis Horwood Limited, 1994.

38 D. Nanni, "Radicals in Organic Synthesis: a Century (Actually much less!) of Playing with Unpaired Electrons", published in Seminars in Organic Synthesis [Monographs of the "A. Corbella" Summer School of Organic Chemistry (Gargnano, Italy), XXXII Edition], E. Licandro, R. Ballini, L. Banfi, M. V. D’Auria, M. Maggini, and C. Morelli, Ed.s; Società Chimica Italiana, Rome, 2007, pgg. 159-182

50 39 Mark The real importance of the captodative effect is however still debated. For a recent review, see: Stella, L.; Harvey, J. N. in Radicals in Organic Synthesis, Renaud, P. and Sibi, M. P., Eds.; Wiley- VCH: Weinheim, 2001, Vol. 1, p. 360. See also: Viehe, H. –G.; Janousek, Z.; Merényi, R.; Stella, L. Acc. Chem. Res. 1985, 18, 148.

40 It should also be underlined that the term “unstable” is inappropriate for many short-lived radicals. It is more usually used in the sense of unimolecular instability. In fact, in the vacuum methyl radical 4 would be long-lived!

41 a) For a discussion on the persistent radical effect, see: Fischer, H. J. Am. Chem. Soc. 1986, 108, 3925 and Fischer, H. Chem. Rev. 2001, 101, 3581. For the use of nitroxides (such as TEMPO) in organic synthesis, see: b) Braslau, R.; Anderson, M. O. in Radicals in Organic Synthesis, Renaud, P. and Sibi, M. P., Eds.; Wiley-VCH: Weinheim, 2001, Vol. 2, p. 127 and c) Studer, A. Angew. Chem. Int. Ed. Engl. 2000, 39, 1108. d) For living-radical polymerisation, see Ref. 4b and: Georges, M. ibid., Vol. 1, p. 479.

42 Mark For comprehensive, recent insights into the world of radical reactions and their applications to organic synthesis, see: a) Crich, D.; Motherwell, W. B. Free Radical Chain Reactions in Organic Synthesis; Academic Press: London, 1991. b) Perkins, M. J. Radical Chemistry; Ellis Horwood: London, 1994. c) Parsons, A. F. An Introduction to Free Radical Chemistry; Blackwell Science: Oxford, 2000. d) Radicals in Organic Synthesis, Vols. 1 and 2, Renaud, P. and Sibi, M. P., Eds.; Wiley-VCH: Weinheim, 2001. e) Togo, H. Advanced Free Radical Reactions for Organic Synthesis; Elsevier; Amsterdam, 2004.

43 Deepened discussions on this subject can be found on the books reported in Ref.s 2 as well as in: Giese, B. Radicals in Organic Synthesis: Formation of Carbon-Carbon Bonds; Pergamon Press: Oxford, 1986, Chp. 2.

44 In the brought example, the initiator is also a starting reagent (Cl2), but it is always not this way.

45 A chain can also finish through processes of electron transfer that oxidizes or reduces the radical to the corresponding ion.

46 Mark Additional, but not very used, radical sources include also radiolysis (with X– or γ–radiation) and sonolysis (with ultrasounds).

47 Mark Porter, N. A. Acc. Chem. Res. 1986, 19, 262.

48 For excellent reviews on these subjects, see, for example: a) Iqbal, J.; Bhatia, B.; Nayyar, N. K. Chem. Rev. 1994, 94, 519. b) Snider, B. B. Chem. Rev. 1996, 96, 339.

49 The reactions reported below in this section are the key processes that characterise the whole radical reactivity. Many famous radical reactions (e.g. the Sandmeyer reaction, the neophyl rearrangement, the Kolbe decarboxylation, the Barton-McCombie reaction, or even the Toray process for cyclohexanone oxime) can simply be regarded as sequences of these key steps.

50 In reversible processes one can isolate products derived from a rearranged, less stable radical if this reacts very fast with a radical trap.

51 Due to their importance in organic synthesis, radical cyclisations will be discussed more thoroughly in the subsequent section.

52 Mark Baldwin, J. E. J. Chem. Soc., Chem. Commun. 1976, 734.

53 Beckwith, A. L. J.; Schiesser, C. H. Tetrahedron 1985, 41, 3925. The whole Volume 41 of Tetrahedron is concerned with selectivity and synthetic applications of radical reactions. Mark Julia, M. Acc. Chem. Res. 1971, 386

54 Julia, M. Acc. Chem. Res. 1971, 386.

51 55 “Dimerisation” (or homocoupling) usually refers to coupling between two identical radical species, whereas “coupling” (or heterocoupling) entails combination of two different radicals.

56 B. R. Pool, J. M. White, Org. Lett. 2000, 2, 3505.

57 United States Patent Office, patent number 3633, June 15, 1844

58 G. J. Chen, S. Amajjahe, M. H. Stenzel, Chem. Commun. 2009, 1198.

59 R. J. Pounder, M. J. Stanford, P. Brooks, S. P. Richards, A. P. Dove, Chem. Commun. 2008, 5158.

60 A) C. N. Bowman and C. E. Hoyle, Thiol–Ene Click Chemistry, Angew. Chem. Int. Ed. 2010, 49, 1540 – 1573 B) Lowe A. B, Thiol-ene “click” reactions and recent applications in polymer and materials synthesis, Polym. Chem., 2010, 1, 17-36 C) A. Dondoni, Angew. Chem. 2008, 120, 9133–9135; Angew. Chem. Int. Ed. 2008, 47, 8995 – 8997.

61 L. A. Connal, C. R. Kinnane, A. N. Zelikin, F. Caruso, Chem. Mater. 2009, 21, 576.

62 J. W. Chan, B. Yu, C. E. Hoyle, A. B. Lowe, Chem. Commun. 2008, 4959.

63 K. L. Killops, L. M. Campos, C. J. Hawker, J. Am. Chem. Soc. 2008, 130, 5062.

64 A) J. W. Chan, C. E. Hoyle and A. B. Lowe, J. Am. Chem. Soc., 2009, 131, 5751–5753 B) J. W. Chan, B. Yu, C. E. Hoyle and A. B. Lowe, Chem. Commun., 2008, 4959–4961.

65 H. Kakwere and S. Perrier, J. Am. Chem. Soc., 2009, 131, 1889–1895.

66 Luo, Y.-R. Handbook of Bond Dissociation Energies in Organic Compounds CRC Press: Boca Raton, London, New York, Washington D.C., 2003.

67 M. Uygun, M. A. Tasdelen, Y. Yagci, Macromol. Chem. Phys. 2010, 211, 103–110

68 A) C. Walling and W. Helmreich, J. Am. Chem. Soc., 1959, 81, 1144– 1148. B) C. Chatgilialoglu, A. Altieri and H. Fischer, J. Am. Chem. Soc., 2002, 124, 12816–12823. C) C. Ferreri, A. Samadi, F. Sassatelli, L. Landi and C. Chatgilialoglu,J. Am. Chem. Soc., 2004, 126, 1063–1072.

69 A) Benson S.W., Egger K.W., Golden D.M. (1965) J Am Chem Soc 87:468 B) Golden D. M., Furuyama S., Benson S. W.,Int. J. Chem. Kinet, 1, 57 (1969).

70 Benati, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1991, 2103-2109.

71 Melandri, D.; Montevecchi, P. C.; Navacchia, M. L. Tetrahedron 1999, 55, 12227-12236.

72 Cramer N. B., Reddy S. K., O’Brien A.K., Bowman N. C., Thiol-Ene Photopolymerization Mechanism and Rate Limiting Step Changes for Various Vinyl Functional Group Chemistries, Macromolecules 2003, 36, 7964-7969

73 A) Griesbaum, K. Problems and possibilities of the free-radical addition of thiols to unsaturated compounds Angew. Chem. Int. Ed., 1970, 9, 273; B) Jarvis, B. B. Free-radical additions to dibenzotricyclo[3.3.0.02,8]-3,6-octadiene J. Org. Chem., 1970, 35, 924; C) Brown, H. C.; Kawakami, J. H.; Liu, K.-T. Additions to bicyclic olefins. V. Effect of 7,7-17dimethyl substituents on the stereochemistry and rates of cyclic additions to norbornenes J. Am. Chem. Soc., 1973, 95, 2209.

74 A) P. C. Montevecchi and M. L. Navacchia, J. Org. Chem., 1997, 62, 5600-5607; B) D. Melandri, P. C. Montevecchi and M. L. Navacchia, Tetrahedron, 1999, 55, 12227-12236. C) B. D. Fairbanks, E. A. Sims, K. S. Anseth and C. N. Bowman, Macromolecules, 2010, 43, 4113-4119.

75 Hensarling R. M., Doughty V. A., Chan J. C., Patton D. L. “Clicking” Polymer Brushes with Thiol-yne Chemistry: Indoors and Out, J. AM. CHEM. SOC. 2009, 131, 14673–14675

52 76 Rahane S. B., Hensarling R. M., Sparks B. J., Staffordb C. M. and Patton D. L., Synthesis of multifunctional polymer brush surfaces via sequential and orthogonal thiol-click reactions, J. Mater. Chem., 2012, 22, 932–943

77 Massi A., Nanni D., Thiol-yne coupling: revisiting old concepts as a breakthrough for up-to-date applications. Advanced article 2012

78 F. Jensen, Introduction to Computational Chemistry John Wiley & Sons, New York (1999).

79 P. Jùrgensen, Ann. Rev. Phys. Chem. 26, 359 (1975).

80 J. B. Anderson, Rev. Comput. Chem. 13, 132 (1999).

81 M. Ernzerhof, J. P Perdew, K. burke, D. J. W. Geldart, A. Holas, N. H. March, R. van Leeuwen, O. V. Gritsenko, E. J. Baerends, E. V. LudenÄa, R. LoÂpez-Boada, Density Functional Theory I Springer, Berlin (1996).

82 J. Sadlej, Semi-Empirical Methods of Quantum Chemistry Ellis Harwood, Chichester (1985).

83 Bingham, R. C., M. J. S. Dewar and D. H. Lo, J. Am. Chem. Soc., Vol. 97, pp. 1285-1293, 1975.

84 Hirst, D. M. A., Computational Approach to Chemistry, Blackwell Scientific Publications, Oxford, 1990.

85 F. Jensen, Introduction to Computational Chemistry John Wiley & Sons, New York (1999).

86 D. C. Rapaport, The Art of Molecular Simulation Cambridge, Cambridge (1997).

87 Van Cauter, K., Van Speybroeck, V., Vansteenkiste, P., Reyniers, M.-F. and Waroquier, M. (2006), Ab Initio Study of Free-Radical Polymerization: Polyethylene Propagation Kinetics. ChemPhysChem, 7, 131–140. doi: 10.1002/cphc.200500249

88 Degirmenci I, A computational approach to the free radical polymerization of acrylates and methacrylates, Ghent University. Faculty of Engineering, 2010

89 Coote M. L., Quantum-Chemical Modeling of Free-Radical Polymerization, Macromol. Theory Simul. 2009, 18, 388–400

90 Vandeputte A. G., Maarten K. Sabbe, Reyniers M-F, and Marin G. B., Chem. Eur. J. 2011, 17, 7656 – 7673

91 M. Saeys, M. F. Reyniers, G. B. Marin, V. Van Speybroeck, M. Waro- quier, J. Phys. Chem. A 2003, 107, 9147 – 9159.

92 A) A. G. Vandeputte, M. F. Reyniers, G. B. Marin, Theor. Chem. Acc. 2009, 123, 391 – 412. B) A. G. Vandeputte, M. F. Reyniers, G. B. Marin, J. Phys. Chem. A 2010, 114, 10531–10549.

93 van Gastel M., Lubitz W., Lassmann G., Neese, J. Am. Chem. Soc. 2004, 126, 2237-2246

94 Francisco-Màrquez M., Alvarez-Idaboy J. R., Galano A., Vivier-Bunge A., Quantum chemistry and TST study of the mechanism and kinetics of the butadiene and isoprene reactions with mercapto radicals, Chemical Physics 344 (2008) 273–280

95 Perez A., Luque F. J., Orozco M.,Frontiers in Molecular Dynamics Simulations of DNA, Accounts of Chemical Research Article ASAP doi:10.1021/ar2001217

96 Malek K., Sahimi S., J. Chem. Phys. 132, 014310 (2010); doi:10.1063/1.3284542

97 Pabis A., Szala-Bilnik J., and Swiatla-Wojcik D., Phys. Chem. Chem. Phys., 2011, 13, 9458–9468

53 54 CHAPTER 2 Radical Additions of Thiols to Alkenes and Alkynes in Ionic Liquids 1 2.1 Introduction Has been focused the concepts of the new approach to chemistry. Particular attention has been dedicated to make radical reactions enter the realm of sustainable and 'green' chemistry, exploring the possibilities to carry out radical processes either/both in environmentally friendly solvents or/and under 'green' conditions that could avoid the use of typical (but often highly toxic) radical reagents such as stannanes and metal-based oxidants/reductants.2 Among unconventional solvents, over the last decade ionic liquids have achieved the status of an efficient alternative to conventional volatile organic solvents. They have already proved to be no longer an exotic medium, but instead a valuable, benign vehicle that can improve the outcome of many organic syntheses, both by enhancing separation processes (and safety) and, in many cases, by improving reactions rate and selectivity3. Surprisingly, with the exception of radical polymerization4, which has been quite extensively studied, the number of reported examples of radical synthetic procedures carried out in ionic liquids is extremely low5: the investigated reactions include formation of carbon-carbon bonds through manganese(III) acetate5a and CAN- mediated5c oxidations of 1,3-dicarbonyl precursors, triethyl borane-induced addition (or cyclization)/reduction sequences or atom-transfer cascade reactions5b, and three-component photo-induced atom-transfer carbonylations5d. On this ground, we were prompted to study the feasibility in ionic liquids of a long-known radical reaction, i.e. the addition of thiols to double (or triple) carbon-carbon bonds. Although reported long time ago6, more recently this reaction has moved to the forefront of free-radical research both for its applications in the field of tin-free generation of radical intermediates7,3b,c,e and its relevance in the "click-chemistry" domain: in the latter area, the addition of thiols to alkenes is indeed known as the "thiol-ene" coupling (TEC) reaction and it is widely employed in polymer chemistry, in synthesis of biomaterials, dendrimers, and, in general, molecules of bioorganic interest (glycopeptides, alkylated aminoacids, etc.)8. Here we report some results on the radical addition

55 of a few thiols to double and triple carbon-carbon bonds in ionic liquids under various experimental conditions and radical generation methods.

2.2 Result and discussion The radical addition of thiols to alkenes was initially tested by reacting equimolar amounts (1 mmol) of benzenethiol (2A) and aliphatic (1a,e) or aromatic (1b-d) alkenes in [bmim][PF6] (1 mL) at various temperatures in the presence of suitable radical initiators (triethylborane [Et3B] for T ≤ 40 °C, 1,1'- azo-bis-iso-butyronitrile [AIBN] for T = 80 °C) (Scheme 1 and Table 1). The expected anti- Markovnikov reaction products (3) were recovered by centrifuge- mediated extraction with diethyl ether; the ionic liquid was usually recycled up to 3 times without any significant change in yields and byproducts. The overall results, while being strictly comparable with those previously reported in usual solvents (e.g. benzene)9, sometimes reveal slightly improved yields at enhanced temperatures (e.g. for 3cA and 3dA). It is worth noting that yields are generally far from being quantitative, but this is the common case for radical addition of (electrophilic) thiols to non- activated alkenes: this reaction is indeed known to be a reversible process explained before10 and, to attain the best results, it typically requires high thiol concentrations and/or excess of alkene to shift the equilibrium towards the addition products. Back fragmentation to the starting alkene seems particularly favored in the case of 1,1-disubstituted alkenes such as methylenecyclohexane (1e), which gave a notably low yield of hydrothiolation product (3eA).

56 1 1 3 R Rad. In. R SR + R3 SH R2 [bmim][PF6] R2 12 3

1 2 3 a: R = n-C6H13, R = H A: R = Ph 1 2 3 b: R = Ph, R = H B: R = MeO(CO)CH2 1 2 c: R = p-Cl-C6H4, R = H 1 2 d: R = p-MeO-C6H4, R = H 1 2 e: R ,R = c-C6H10 f: R1 = n-BuO, R2 = H 1 2 g: R = MeC(O)O, R = H !

Scheme 1#!$%&%'()!*+,%-%!./'!'(01+()!(00121/&!/.!2,1/)*!2/!()3%&%*#!

4/-5#! 6(0#!7&#! 89:4! 81-%9,! ;1%)09

3aA =2>?! @! @#"! A@!

3aA =2>?! B@! C! A>!

3bA =2>?! D@! C! B@!

3bA E7?F! G@! C! "@!

3cA =2>?! D@! @#"! H@!

3cA E7?F! G@! C! I@!

3dA =2>?! D@! C! A>!

3dA =2>?! B@! C! I@!

3dA E7?F! G@! C! I@!

3eA =2>?! @! C! >G!

3eA =2>?! B@! C! D"!

Table 1#!6%*J)2*!/.!'(01+()!(00121/&!/.!K%&L%&%2,1/)!2/!()3%&%*!1&!MK-1-NMOPAN#!

8,%! 5/**1K)%!The possible effect of benzene and [bmim][PF6] solvent in the radical %..%+2! /.! K%&L%&%! (&0! MK-1-NMOPAN! */)Q%&2! 1&! 2,%! '(01+()! 2,1/)R%&%! +/J5)1&S! thiol-ene coupling reaction was next examined with two thiols, i.e. benzenethiol '%(+21/&!T(*!&%U2!%U(-1&%0!T12,!2T/!2,1/)*V!1#%#!K%&L%&%2,1/)!2A!(&0!-%2,W)!2,1/S)W+/)(2%!2BV!(&0!

2A2T/!(+21Q(2%0!/)%.1&*V!1#%#! and methyl thioglycolate !"KJ2W)!Q1&W)!%2,%'!2B, and two activated olefins, i.e. n-butyl vinyl ether 1f!(&0!Q1&W)!(+%2(2%!1g#!8,%!'%(+21/&*!T%'%!+(''1%0!/J2! 1f and vinyl acetate 1g. The reactions were carried out at r.t. with equimolar (2!'#2#!T12,!%XJ1-/)('!(-/J&2*!/.!'%(S%&2*!(&0!=2>?! (*! 2,%! '(01+()! 1&121(2/'! %12,%'! 1&! K%&L%&%! /'! amounts of reagents and Et3B as the radical initiator either in benzene or [bmim] MK-1-NMOPAN!(2!01..%'%&2!'%(S%&2*!+/&+%&2'(21/&*#!Y&0%'!C!Z!+/&0121/&*V!K/2,!*/)Q%&2*!(../'0%0!2,%! [PF6] at different reagents concentrations. Under 1 M conditions, both solvents afforded the same results, giving the corresponding adducts (! " 3fA, 3fB, 3gA, and 3gB) in almost quantitative yields. When the reactions were instead carried out under more diluted conditions (0.1 M), benzene gave only trace amounts of the

57 addition products, whereas [bmim][PF6] afforded much more interesting results, producing the aimed sulfides in 60-70% yields. This outcome clearly shows that the radical hydrothiolation of alkenes can be favored by the ionic liquid, a feature that should not be surprising, at least taking the second reaction step into consideration. Indeed, hydrogen transfer reactions are known to be highly affected by polar factors and substituents capable of stabilizing a partial charge separation in the transition state are generally established to increase the reaction rate(will be treat with a theoretical approach in Chaper 4).11 The same effect would be achieved by carrying out the reaction in a polar medium such as an ionic liquid, which could stabilize charge separation, hence lowering the activation energy of H-atom donation from the (electrophilic) thiol to the intermediate (nucleophilic) β-sulfanyl-substituted alkyl radical. Such kind of effect has already been suggested with ionic liquids not only in radical reactions involving metal ions5a,c or radical/polar crossover synthetic sequences5d, but also in typical radical reactions such as chain intermolecular halogen atom transfers5b,12. However cannot be exclude the high cohesive energy density of the ionic liquid, forcing a decrease in the volume of the reactants in analogy with what happens in water1e, could play an additional role by lowering the rate of back fragmentation of β-sulfanyl- substituted alkyl radicals: this effect would facilitate the subsequent hydrogen transfer, hence favoring the formation of the final hydrothiolation product.4a It is worth noting that the hydrothiolation reaction can be performed in [bmim][PF6] even using other radical conditions, i.e. at r.t. under UV irradiation (λ 310-400 nm) in the presence of either AIBN (15%)13 or 2,2- dimethoxy-2-phenylacetophenone (DMPA, 15%) as a photoinitiator14. Electron- rich alkenes such as 1f,g are not suitable for these conditions, since they give rise to extensive polymerization and very complicate reaction mixtures, but non- activated olefins can be efficiently employed: for example, 1-octene 1a underwent photochemical reactions with both thiols 2A,B to give almost quantitative yields of the corresponding adducts 3a,A and 3a,B. Under these conditions the only identifiable byproducts were trace amounts of disulfides and (probably) sulfoxides of adducts 3. The ionic liquid remained substantially

58 unchanged under photolytical conditions: a darkening of its color did not affect the reaction outcome and the ionic liquid was recycled up to 3 times without any significant problem. We subsequently investigated the radical addition of benzenethiol to alkynes in ionic liquids, in view of the known synthetic potential of the expected vinyl sulfide products [18]. Reactions were carried out for ca. 3 h with equimolar amounts (0.1 mmol) of benzenethiol 2A and various alkynes 4a-d, both at r.t. and at 100 °C, in three different ionic liquids (1 mL), i.e. [bmim][PF6], [bmim] [Tf2N], and [bmim][BF4].

Radical initiation was attained with either Et3B or 1,1'-azo-bis- cyclohexanecarbonitrile (VAZO®), depending on the reaction temperature. In almost all cases, after usual workup, nearly quantitative yields of vinyl sulfides 5 were obtained as mixtures of E- and Z-isomers (Scheme 2 and Table 2).

1 Rad. In. Ph R Ph SPh Ph R1 + Ph SH + [bmim][X] H SPh H R1 4 2A E-5 Z-5 a: R1 = H b: R1 = Me c: R1 = Et 1 d: R = Pr !

Scheme 2#!$%&'(%)!%&&'*'+,!+-!./,0/,/*1'+)!*+!%)23,/4#!

5+67#! $%&#!8,#! 9:;5! 4+)!?!"#!@%*'+A!

5aA B*CD! EF! G.6'6HGIJKH! LMN!?E"O"CA!

“ 5aA PQRS ! TFF! G.6'6HGIJKH! LMN!?UVOTKA!

5aA B*CD! EF! G.6'6HG9-EWH! LMN!?ENO"NA!

5aA B*CD! EF! G.6'6HGDJVH! LMN!?NCOV"A!

5bA B*CD! EF! G.6'6HGIJKH! LMN!?ENO"NA!

“ 5bA PQRS ! TFF! G.6'6HGIJKH! LMN!?N"OVCA!

5bA B*CD! EF! G.6'6HG9-EWH! LMN!?NEOVUA!

5bA B*CD! EF! G.6'6HGDJVH! LMN!?NCOV"A!

5cA B*CD! EF! G.6'6HGIJKH! UF!?TKOUVA!

“ 5cA PQRS ! TFF! G.6'6HGIJKH! LMN!?VFOKFA!

5cA B*CD! EF! G.6'6HG9-EWH! LMN!?E"O"CA! 59 5cA B*CD! EF! G.6'6HGDJVH! "F!?C"OKCA!

5dA B*CD! EF! G.6'6HGIJKH! MN!?ENO"NA!

“ 5dA PQRS ! TFF! G.6'6HGIJKH! LMN!?C"OKCA!

5dA B*CD! EF! G.6'6HG9-EWH! "F!?VTONMA!

5dA B*CD! EF! G.6'6HGDJVH! LMN!?CEOKUA!

Table 2#!$/4X)*4!+-!@%&'(%)!%&&'*'+,!+-!./,0/,/*1'+)!*+!%)23,/4!',!<%@'+X4!'+,'(!)'YX'&4#!

$%&'(%)! ','*'%*'+,! Z%4! %**%',/&! Z'*1! /'*1/@! B*CD! +@! T[T\]%0+].'4](3()+1/^%,/(%@.+,'*@')/!

?PQRS“A[!&/7/,&',_!+,!*1/!@/%(*'+,!*/67/@%*X@/#!8,!%)6+4*!%))!(%4/4[!%-*/@!X4X%)!Z+@2X7[!,/%@)3!

! " Comp Rad. In. T/°C Solvent Yeld% (E/Z ratio)

5aA Et3B 20 [bimim][PF6] >95(27:73)

5aA VAZO! 100 [bimim][PF6] >95(84:16)

5aA Et3B 20 [bimim][Tf2N] >95(25:75)

5aA Et3B 20 [bimim][BF4] >95(53:47)

5bA Et3B 20 [bimim][PF6] >95(25:75)

5bA VAZO! 100 [bimim][PF6] >95(57:43)

5bA Et3B 20 [bimim][Tf2N] >95(52:48)

5bA Et3B 20 [bimim][BF4] >95(53:47)

5cA Et3B 20 [bimim][PF6] 80 (16:84)

5cA VAZO! 100 [bimim][PF6] >95(40:60)

5cA Et3B 20 [bimim][Tf2N] >95(27:73)

5cA Et3B 20 [bimim][BF4] 70 (37:63)

5dA Et3B 20 [bimim][PF6] >95(25:75)

5dA VAZO! 100 [bimim][PF6] >95(37:63)

5dA Et3B 20 [bimim][Tf2N] 70 (41:59)

5dA Et3B 20 [bimim][BF4] >95(32:68)

Table 2. Results of radical addition of benzenethiol to alkynes in various ionic liquids. As expected on the basis of a free-radical mechanism, all of the vinyl sulfide products 5aA-dA derived from anti-Markovnikov addition of benzenethiol to the respective alkyne; vinyl sulfides 5bA-dA, in particular, derived from regioselective addition of benzenesulfanyl radicals to the alkyl end of the alkyne triple bond, a result that is consistent with the intermediacy of α-phenyl- substituted vinyl radicals, which are more stable than the alternative, isomeric α-alkyl-substituted counterparts.15 The observed significant (often predominant) formation of the Z- isomers was not unexpected. Indeed, α-phenyl-β-(arylsulfanyl)vinyl radicals, to minimize steric hindrance, are known to approach hydrogen donors from the side trans to the sulfanyl moiety, yielding the corresponding vinyl sulfides in kinetically controlled 1:9 E/Z ratio when the reactions are carried out at 100 °C

60 in neat alkyne15. At the same temperature, as a result of thermodynamic control attained through the use of equimolar amounts of reagents in 0.1 M benzene solutions15 (i.e. concentrations identical to those we presently used in ionic liquids), alkynes 4a-d have been reported to give the corresponding adducts 5aA-dA, at 100 °C, in 9:1, 9:1, 5:5, and 4:6 E/Z ratio, respectively. If we compare these results with those obtained in ionic liquids at the same temperature, it can be seen that [bmim][PF6] favors formation of the Z-isomer to a small extent: this is particularly notable for adduct 5bA. This outcome suggests that hydrothiolation of alkynes is somewhat faster in ionic liquids with respect to aromatic solvents, in line with what suggested above for hydrothiolation of alkenes. Under radical conditions, Z/E isomerization of vinyl sulfides occurs by reversible addition (discussed before in Thiol radical coupling, Chapter 1) of sulfanyl radicals to the carbon-carbon double bond: as a consequence, the more efficient is the hydrothiolation reaction, the lower will be the concentration of sulfanyl radicals and hence the slower the isomerization. Of course, formation of the kinetic product is favored at lower temperatures, although kinetic control was never attained in any ionic liquid. By comparing the various results obtained at r.t., it seems that [bmim][PF6] would be the best solvent for promoting formation of the Z-isomer, and hence the medium in which hydrothiolation of alkynes is faster. Finally, we briefly examined the possible use of ionic liquid solvents in click-chemistry reactions leading to biologically interesting molecules. Our preliminary results include addition of 8 L-N-Fmoc-cysteine tert-butyl ester to 1- hexadecene and to an O-allyl glucoside, and hydrothiolation of phenylacetylene with L-cysteine ethyl ester hydrochloride (Scheme 3).

61 #$!$%&'($()*+,-.,! "#$"$/0+)1! ,*+,2! +'! 3$4,567,(,.,! 6.7! +'! 6.! %$611)1! 810('*-7,9! 6.7!

4)72'+4-'16+-'.!':!;4,.)16(,+)1,.,!<-+4!#$()*+,-.,!,+4)1!,*+,2!4)72'(41'2-7,!=>(4,&,!?@A!

NHFmoc NHFmoc S H29C14 + HS H C CO t-Bu CO2t-Bu 29 14 2 6

OAc OAc NHFmoc O AcO O + HS AcO AcO CO2t-Bu AcO H AcO NHFmoc AcO O O S CO2t-Bu _ 7 + NH Cl 3 NH2 HS Ph + CO Et S 2 Ph CO2Et 8 !

Scheme 3A!B)72'+4-'16+-'.!6*!6!+''1!:'2!+4,!*).+4,*-*!':!/-'1'8-(611)!-.+,2,*+-.8!('&;'0.7*A!

C4,! :-2*+!The first adduct ( 6770(+! =6@9! <4-(4!6 46*!), which has been recently synthesized in 42% yield /,,.! 2,(,.+1)! *).+4,*-D,7! -.! EFG! )-,17! /)! H20.*I,17! 6.7! by J617&6..!+42'084!6!267-(61!+4-'1$,.,9!26(,&-D6+-' Brunsveld and Waldmann through a .$:2,,!;2'(,702,!0*-.8!+4,2&61!('.7-+-'.*9!<6*! radical thiol-ene, racemization-free procedure (4'*,7! 6*! 6! using +628,+! ('&;'0.7!thermal conditions, :'2! +4,! 2,(,.+! -.+,2,*+!was chosed as -.! 6((,**-.8! a 4)72'1)*-*$2,*-*+6.+! target compound for the .'.$.6+0261! recent interest in accessing hydrolysis-resistant non-natural S-alkylated cysteine &$61K)16+,7! ()*+,-.,! 7,2-I6+-I,*! L3MNA! O02! ;2,*,.+! 2,6(+-'.! <6*! (622-,7! '0+! -.! L/&-&NLP%QN! 0.7,2! 13 derivatives . Our present reaction was carried out in [bmim][PF6] under DMPA- RSPT$;2'&'+,7! ;4'+'1)*-*! ('.7-+-'.*! <-+4! ?! ,U0-I! ':! 3$4,567,(,.,! :'2! M! 4! 6.79! 6:+,2! 0*061! promoted photolysis conditions with 3 equiv of 1-hexadecene for 5 h and, after <'2K0;!:'11'<,7!/)!(42'&6+'826;4)9!*-&-1621)!86I,!6770(+!6!/0+!-.!*'&,<46+!1'<,2!)-,17!=?VG@A! usual workup followed by chromatography, similarly gave adduct 6 but in C4,! *,('.7! *01:-7,! =7@! <6*! *,1,(+,7! -.! I-,

62 cysteine ethyl ester hydrochloride is strictly necessary, since the reaction carried out with 'free' cysteine ethyl ester gave only tars.

2.3 Conclusions This report shows that radical hydrothiolation of alkenes and alkynes can occur in ionic liquids with at least as the same efficiency as in traditional solvents. The ionic liquids are compatible with the use of different radical initiation conditions, i.e. thermal decomposition of azo-initiators (80-100 °C), reaction of triethylborane with dioxygen (r.t.), and UV-photolysis at r.t. in the presence of either an azo-initiator (AIBN) or a photosensitizer (DMPA). The reaction products can be efficiently isolated from the ionic liquid by centrifuge- mediated extraction with diethyl ether, and the ionic liquid can be usually recycled up to 3 times without any significant change in yields and byproducts under any reaction conditions. Some results show that the ionic liquids, if compared with traditional solvents, seem to favor the hydrothiolation reaction, probably through stabilization of the transition state for hydrogen transfer from the starting thiol to the intermediate alkyl or vinyl radical. Although the results are merely preliminary and yields are not optimized, it seems that this protocol could be successfully applied to the synthesis of biologically interesting molecules through click-chemistry procedures carried out in ionic liquids.

2.4 Experimental Section General Remarks. IR spectra were recorded on a FT-IR Perkin Elmer

1 13 Spectrum RXI instrument in CHCl3 solutions. H- and C-NMR spectra were

1 recorded in CDCl3 solutions on a Varian Mercury Plus 400 MHz ( H: 400 MHz,

13 1 C: 100 MHz, internal ref. for H-NMR spectra, δ 7.26 ppm for CHCl3, internal ref.

13 for C-NMR spectra, δ 77.0 for CHCl3). In reporting spectral data, the following abbreviations were used: d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad singlet, br t = broad triplet. J values are reported in Hz. Electron spray ionization (ESI)mass spectra were recorded in acetonitrile solution with a Waters – Micromass ZQ4000 instrument. GC-MS analyses were performed using a ThermoFisher − Focus DSQ system. Column chromatography was carried out on ICN silica gel (63−200, 60 Å) by gradual elution with hexane/diethyl ether. Benzenethiol 2A, methyl thioglycolate 2B, L-cysteine ethyl ester hydrochloride,

63 L-cystine, 1-octene 1a, styrene 1b, p-chlorostyrene 1c, p-methoxystyrene 1d, methylenecyclohexane 1e, butyl vinyl ether 1f, vinyl acetate 1g, phenylacetylene 4a, 1- phenylpropyne 4b, 1-phenylbutyne 4c, 1-phenylpentyne 4d, 1- hexadecene, [bmim][PF6], [bmim][Tf2N], [bmim][BF4], DMPA, and AIBN were commercially available (AIBN was recrystallized at r.t. from chloroform/ methanol); VAZO® and triethylborane (1.0 M solution in hexane) were commercially available as well and were used as received. L-N-Fmoc-cysteine- tert- butyl ester17 and tetraacetyl O-allyl glucoside (1:1 α/β mixture)18 were prepared according to reported literature procedures.

General Procedure for the Addition of Thiols to Alkenes. For the reactions at 0-40 °C, a solution of alkene (1 mmol), thiol (1 mmol), and Et3B (100 μl) in [bmim][PF6] (1 mL) was kept under stirring for

® 0.5-1 h; the reactions at 80 °C were carried out by replacing Et3B with VAZO (10%) and by keeping the resulting solutions at 80 °C for 0.5-1 h; photolysis were performed by keeping the above stirred solutions at ca. 2.5 cm from the UV source (4 bulb-lamp, 15 W each, type 3, λ 310-400 nm) in the presence of either azo-bis-iso-butyronitrile (AIBN, 15%) or 2,2-dimethoxy- 2-phenylacetophenone (DMPA, 15%) as a photoinitiator. Workup was performed by adding diethyl ether (3 × 2 mL) and centrifuging the resulting mixture for 2 min at 2000 rpm; the ether layer was separated, the solvent evaporated and the residue analyzed by GC-MS/NMR. Addition products 3aA-eA, 3gA, 3aB, 3fB, and 3gB were identified by comparison with authentic samples19; spectral data of compound 3fA were as follows: 1H-NMR (400 MHz) δ 0.91 (3 H, t, J = 7.4), 1.32- 1.40 (4 H, m), 3.11 (2 H, t, J = 7.0), 3.44 (2 H, t, J = 6.7), 3.61 (2 H, t, J = 7.0), 7.17-7.20 (1 H, m), 7.27-7.31 (2 H, m), 7.35-7.38 (2 H, m); 13C-NMR (100 MHz) δ 13.9, 19.2 (CH2), 31.7 (CH2), 33.2 (CH2), 69.4 (CH2), 71.0 (CH2), 126.0, 128.9, 129.3, 129.8 (C); GC-MS m/z (rel. inten.) 210 (M+,34), 154 (36), 123 (77), 110 (86), 109 (44), 57 (100). Anal. Calcd. for C12H18OS: C, 68.52; H, 8.63. Found: C, 68.70; H, 8.67. Yields are reported in Table 1 and in the text.

64 General Procedure for the Addition of Thiols to Alkynes. For the reactions at r.t., a solution of alkyne (1 mmol), benzenethiol (1 mmol), and Et3B (100 μl) in the suitable ionic liquid (1 mL) was kept under stirring for 3 h; the reactions at 100 °C were carried out by replacing Et3B with VAZO® (10%) and by keeping the resulting solutions at 100 °C for 3 h. Workup was performed by adding diethyl ether (3 × 2 mL) and centrifuging the resulting mixture for 2 min at 2000 rpm; the ether layer was separated, the solvent evaporated and the residue analyzed by GC-MS/NMR. Addition products 5aA-dA were identified by comparison with authentic samples15a. Yields and E/Z ratios are reported in Table 2.

Reaction of L-N-Fmoc-Cysteine tert-Butyl Ester with 1-Hexadecene. A mixture of 1- hexadecene (0.9 mmol), aminoacid (0.3 mmol), and

DMPA (50%) in [bmim][PF6] (3 mL) was kept under stirring at ca. 2.5 cm from the UV source (4 bulb-lamp, 15 W each, type 3, λ 310-400 nm) for 5 h. Workup was performed by adding diethyl ether (3 × 2 mL) and centrifuging the resulting mixture for 2 min at 2000 rpm; the ether layer was separated, the solvent evaporated and the residue chromatographed to give adduct 6 (30% yield), which was identified by comparison with an authentic sample13.

Reaction of L-N-Fmoc-Cysteine tert-Butyl Ester with Tetraacetyl O-Allyl Glucoside. A mixture of tetraacetyl O-allyl glucoside (1:1 α/β mixture, 30 mg, 0.077 mmol), Fmoc-Cys-OtBu (31 mg, 0.077 mmol), and DMPA (2 mg, 7.70 μmol) in [bmim][PF6] (0.5 mL) was irradiated at room temperature for 1 h as described above. After usual workup, the resulting residue was eluted from a column of silica gel with 1:1 cyclohexane/ethyl acetate to give sulfide 7 (30 mg, 50%, ~ 1:1 anomeric mixture), slightly contaminated by the starting alkene; 1H NMR (selected data) δ 7.80 (2 H, d, J = 7.4, Ar), 7.64 (2 H, d, J = 7.4, Ar), 7.50-7.20 (4 H, m, Ar), 5.76 (0.5 H, d, J = 8.0, NH), 5.74 (0.5 H, d, J = 8.0, NH), 5.20 (0.5 H, d, J = 4.0, H-1α), 4.60 (0.5 H, d, J = 9.5, H-1β), 2.20-2.02(12 H, 8s, Me), 1.44 (4.5 H, s,

+ + t-Bu), 1.42 (4.5 H, s, t-Bu); ESI MS (787.87): 788.8 (M + H ), 805.5 (M + NH4 ).

65 Reaction of L-Cysteine Ethyl Ester Hydrochloride with Phenylacetylene. A solution of phenylacetylene (1 mmol), aminoacid (1 mmol), and

DMPA (5%) in [bmim][PF6] (1 mL) was kept under stirring at ca. 2.5 cm from the UV source (4 bulb-lamp, 15 W each, type 3, λ 310-400 nm) for 1h. Workup was performed by adding first an aqueous solution of sodium carbonate, then diethyl ether, and finally centrifuging the resulting mixture for 2 min at 2000 rpm; the ether layer was separated, the solvent evaporated and the residue chromatographed to give vinyl sulfide 8 (78% yield, 55:45 E/Z ratio): IR νmax 1188, 1598, 1735, 2934, 3011, 3305, 3376 cm−1; 1H-NMR (400 MHz) δ 1.26 (6 H, br t, J = 7.1, both isomers), 2.08 (4 H, br s, both isomers), 2.99-3.09 (2 H, m), 3.13-3.23 (2 H, m), 3.69-3.75 (2 H, m), 4.16 (2 H, q, J = 7.1), 4.18 (2 H, q, J = 7.1), 6.23 (1 H, d, J = 10.8, Z-isomer), 6.45 (1 H, d, J = 10.8, Z-isomer), 6.57 (1 H, d, J = 15.6, E-isomer), 6.68 (1 H, d, J = 15.6, E-isomer), 7.16-7.38 (8 H, m, both isomers), 7.44-7.48 (2 H, m, both isomers); 13C-NMR (100 MHz) δ 14.0, 37.9

(CH2), 40.7 (CH2), 54.1, 54.6, 61.2 (CH2), 61.3 (CH2), 123.9, 125.5, 126.3, 126.4, 126.7, 127.1, 128.1, 128.4, 129.5, 128.9, 136.4 (C), 136.5 (C), 173.3 (C), 173.5 (C); GC-MS m/z (rel. inten.) 251 (M+, 55), 178 (41), 150 (100), 149 (43), 135 (67), 134 (36), 116 (32), 115 (53), 102 (48), 91 (50), 74 (49). A control experiment performed in DMF for 1 h with the same reagents in the same amounts afforded, after basic aqueous workup, extraction with diethyl ether, and chromatography, vinyl sulfide 8 in 86% yield (55:45 E/Z ratio).

66 2.5 Bibliography 1 T. Lanza, M. Minozzi, A. Monesi, D. Nanni, P. Spagnolo and C. Chiappe, Curr. Org. Chem., 2009,13, pp. 1726 - 1732

2 a) Walton, J. C. Linking borane with N-heterocyclic carbenes: effective hydrogen-atom donors for radical reactions Angew. Chem. Int. Ed., 2009, 48, 1726; b) Bencivenni, G.; Lanza, T.; Leardini, R.; Minozzi, M.; Nanni, D.; Spagnolo, P.; Zanardi, G. Tin-free generation of alkyl radicals from alkyl 4- pentynyl sulfides via homolytic substitution at the sulfur atom Org. Lett., 2008, 10, 1127; c) Benati, L.; Bencivenni, G.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. Reaction of azides with dichloroindium hydride: very mild production of amines and pyrrolidin-2-imines through possible indium-aminyl radicals Org. Lett., 2006, 8, 2499; d) Healy, M. P.; Parsons, A. F.; Rawlinson, J. G. T. Consecutive approach to alkenes that combines radical addition of phosphorus hydrides with Horner−Wadsworth−Emmons-type reactions Org. Lett., 2005, 7, 1597; e) Benati, L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Strazzari, S.; Zanardi, G. A novel tin-free procedure for alkyl radical reactions Angew. Chem. Int. Ed., 2004, 43, 3598; f) Studer, A.; Amrein, S. Tin hydride substitutes in reductive radical chain reactions Synthesis, 2002, 835. See also: Togo, H., Advanced Free Radical Reactions for Organic Synthesis, Elsevier: Amsterdam, 2004, chp. 12, p. 247.

3a) Ionic Liquids in Synthesis, Peter Wasserscheid and Tom Welton Ed.s; Wiley-VCH: Weinheim, 2008; b) Pârvulesku, V. I.; Hardacre, C. Catalysis in ionic lLiquids Chem. Rev., 2007, 107, 2615; c) Harper, J. B.; Kobrak, M. N. Understanding organic processes in ionic liquids: achievements so far and challenges remaining Mini-Rev. Org. Chem., 2006, 3, 253; d) Chiappe, C.; Malvaldi, M.; Pomelli, C. S. Ionic liquids: solvation ability and polarity Pure Appl. Chem., 2009, 81, 767.

4 a) Carmichael, A. J.; Haddleton, D. M.; Bon, S. A. F.; Seddon, K. R. Copper(I) mediated living radical polymerisation in an ionic liquid Chem. Commun., 2000, 1237; b) Biedron, T.; Kubisa, P. Atom- transfer radical polymerization of acrylates in an ionic liquid Macromol. Rapid Commun., 2001, 22, 1237; c) Harrisson, S.; Mackenzie, S. R.; Haddleton, D. M. 16Unprecedented solvent-induced acceleration of free-radical propagation of methyl methacrylate in ionic liquids Chem. Commun., 2002, 2850; d) Biedron, T.; Kubisa, P. Atom transfer radical polymerization of acrylates in an ionic liquid: synthesis of block copolymers J. Polym. Sci. Part A, 2002, 40, 2799; e) Zhang, H.; Hong, K.; Jablonsky, M.; Mays, J. W. Statistical radical copolymerization of styrene and methyl methacrylate in a room temperature ionic liquid Chem. Commun., 2003, 1356; f) Thurecht, K. J.; Gooden, P. N.; Goel, S.; Tuck, C.; Licence, P.; Irvine, D. J. Free-radical polymerization in ionic liquids: the case for a protected radical Macromolecules, 2008, 41, 2814 and references therein. See also Ref. 4a, chp. 7, p. 619.

5 a) Bar, G.; Parsons, A. F.; Thomas, C. B. Manganese(III) acetate mediated radical reactions in the presence of an ionic liquid Chem. Commun., 2001, 1350; b) Yorimitsu, H.; Oshima, K. Triethylborane- induced radical reactions in ionic liquids Bull. Chem. Soc. Jpn., 2002, 75, 853; c) Bar, G.; Bini, F.; Parsons, A. F. CAN-Mediated oxidative free radical reactions in an ionic liquid Synth. Commun., 2003, 33, 213; d) Fukuyama, T.; Inouye, T.; Ryu, I. Atom transfer carbonylation using ionic liquids as reaction media J. Organomet. Chem., 2007, 692, 685. For other examples concerning the behavior of radical intermediates in ionic liquids, see: e) Grodkowski, J.; Neta, P. Reaction kinetics in the ionic liquid methyltributylammonium bis(trifluoromethylsulfonyl)imide. Pulse radiolysis study of •CF3 radical reactions J. Phys. Chem. A, 2002, 106, 5468; f) Zhao, D.; Wang, J.; Zhou, E. Oxidative desulfurization of diesel fuel using a Brønsted acid room temperature ionic liquid in the presence of H2O2 Green Chem., 2007, 9, 1219; g) Baj, S.; Chrobok, A. Autooxidation of hydrocarbons with oxygen in ionic liquids as solvents Int. J. Chem. Kin., 2008, 40, 287.

67 6 a) Griesbaum, K. Problems and possibilities of the free-radical addition of thiols to unsaturated compounds Angew. Chem. Int. Ed., 1970, 9, 273; b) Jarvis, B. B. Free-radical additions to dibenzotricyclo[3.3.0.02,8]-3,6-octadiene J. Org. Chem., 1970, 35, 924; c) Brown, H. C.; Kawakami, J. H.; Liu, K.-T. Additions to bicyclic olefins. V. Effect of 7,7- 17dimethyl substituents on the stereochemistry and rates of cyclic additions to norbornenes J. Am. Chem. Soc., 1973, 95, 2209.

7 a) Benati, L.; Bencivenni, G.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. Generation and cyclization of unsaturated carbamoyl radicals derived from S-4- pentynyl carbamothioates under tin-free conditions J. Org. Chem., 2006, 71, 3192; b) Benati, L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. Radical chain reaction of benzenethiol with pentynylthiol esters: production of aldehydes under stannane/silane-free conditions Synlett, 2004, 987; c) Benati, L.; Calestani, G.; Leardini, R.; Minozzi, M.; Nanni, D.; Spagnolo, P.; Strazzari, S. Generation and intramolecular reactivity of acyl radicals from alkynylthiol esters under reducing tin- free conditions Org. Lett., 2003, 5, 1313; d) Bachi, M. D.; Korshin, E. E.; Ploypradith, P.; Cumming, J. N.; Xie, S.; Shapiro, T. A.; Posner, G. H. Synthesis and in vitro antimalarial activity of sulfone endoperoxides Bioorg. Med. Chem. Lett., 1998, 8, 903; e) Bilokin, Y. V.; Melman, A.; Niddam, V.; Benhamu, B.; Bachi, M. D. Synthesis of thiabicyclic heterocycles through free radical cyclization of β- thioacrylates Tetrahedron, 2000, 56, 3425; f) Korshin, E. E.; Hoos, R.; Szpilman, A. M.; Konstantinovski, L.; Posner, G. H.; Bachi, M. D. An efficient synthesis of bridged-bicyclic peroxides structurally related to antimalarial yingzhaosu A based on radical co-oxygenation of thiols and monoterpenes Tetrahedron 2002, 58, 2449; g) Majumdar, K. C.; Debnath, P. Thiol- mediated radical cyclizations Tetrahedron, 2008, 64, 9799; h) Majumdar, K. C.; Taher, A. Wittig olefination and thiol mediated 7-endo-trig radical cyclization: novel synthesis of oxepin derivatives Tetrahedron Lett., 2009, 50, 228; i) Carta, P.; Puljic, N.; Robert, C.; Dhimane, A.-L.; Fensterbank, L.; Lacôte, E.; Malacria, M. Generation of phosphorus-centered radicals via homolytic substitution at sulfur Org. Lett., 2007, 9, 1061. See also ref. 3b.

8 For leading references, see: a) Dondoni, A. The emergence of thiol–ene coupling as a click process for materials and bioorganic chemistry Angew. Chem. Int. Ed., 2008, 47, 8995; b) Becer, C. R.; Hoogenboom, R.; Schubert, U. S. Click chemistry beyond metal-catalyzed 18cycloaddition Angew. Chem. Int. Ed., 2009, 48, 4900; c) Hoyle, C. E.; Lee, T. Y.; Roper, T. Thiol-enes: chemistry of the past with promise for the future J. Polym. Sci. Part A, 2004, 42, 5301, and references therein.

9 The Chemistry of the Thiol Group, Patai, S., Ed.; Wiley: New York, 1974.

10 a) Is treated before in Chapter 1 Thiol radical coupling, b) Kice, J. L. in Free Radicals, Jay K. Kochi, Ed.; Wiley: New York, 1973; Vol. II, p. 720.

11 A thorough discussion on this subject can be found in: Giese, B., Radicals in Organic Synthesis: Formation of Carbon-Carbon Bonds; Pergamon Press: Oxford, 1986; Chp. 2.

12 Morrow, T. I.; Maginn, E. J. Molecular dynamics study of the ionic liquid 1-n-butyl-3- methylimidazolium hexafluorophosphate J. Phys. Chem. B, 2002, 106, 12807; Jenner, G. Role of the medium in high pressure organic reactions. A review Mini-Rev. Org. Chem., 2004, 1, 9.

13 Triola, G.; Brunsveld, L.; Waldmann, H. Racemization-free synthesis of S-alkylated cysteines via thiol-ene reaction J. Org. Chem., 2008, 73, 3646.

14 Killops, K. L.; Campos, L. M.; Hawker, C. J. Robust, efficient, and orthogonal synthesis of dendrimers via thiol-ene “click” chemistry J. Am. Chem. Soc., 2008, 130, 5062.

68 15 a) Benati, L.; Montevecchi, P. C.; Spagnolo, P. Free-radical reactions of benzenethiol and diphenyl disulphide with alkynes. Chemical reactivity of intermediate 2-(phenylthio)vinyl radicals J. Chem. Soc., Perkin Trans. 1, 1991, 2103 and references therein. For scavenging of β-sulfanyl-substituted vinyl radicals, see also: b) Leardini, R.; Nanni, D.; Zanardi, G. Radical addition to isonitriles: a route to polyfunctionalized alkenes through a novel three-component radical cascade reaction J. Org. Chem., 2000, 65, 2763.

16 a) Jensen, K. J.; Brask, J. Carbohydrates in peptide and protein design Pept. Sci., 2005, 80, 747. For a recent example, see: b) Fauré, R.; Shiao, T. C.; Lagnoux, D.; Giguère, D.; Roy, R. 20En route to a carbohydrate-based vaccine against Burkholderia cepacia Org. Biomol. Chem.,2007, 5, 2704.

17 This compound was prepared from commercially available L-cystine according to: Mustapa, M. F. M.; Harris, R.; Bulic-Subanovic, N.; Elliott, S. L.; Bregant, S.; Groussier, M. F. A.; Mould, J.; Schultz, D.; Chubb, N. A. L.; Gaffney, P. R. J.; Driscoll, P. C.; Tabor, A. B. Synthesis of orthogonally protected lanthionines J. Org. Chem., 2003, 68, 8185.

18 Synthesis: Mandai, T.; Okumoto, H.; Oshitari, T.; Nakanishi, K.; Mikuni, K.; Hara, K.-J.; Hara, K.-Z. A practical synthetic method for α- and β-glycosyloxyacetic acids Heterocycles, 2000, 52, 129. Spectroscopic data for the β-anomer: Kishida, M.; Akita, H. Simple preparation of phenylpropenoid β- d-glucopyranoside congeners by Mizoroki–Heck type reaction using organoboron reagents Tetrahedron, 2005, 61, 10559. Spectroscopic data for the α-anomer: Roy, B.; Mukhopadhyay, B. Sulfuric acid immobilized on silica: an excellent catalyst for Fischer type glycosylation Tetrahedron Lett., 2007, 48, 3783.

19 3aA: Fernandez-Rodriguez, M. A.; Shen, Q.; Hartwig, J. F. A general and long-lived catalyst for the palladium-catalyzed coupling of aryl halides with thiols J. Am. Chem. Soc., 2006, 128, 2180; 3bA: Kumar, P.; Pandey, R. K.; Hegde, V. R. Anti-Markovnikov addition of thiols across double bonds catalyzed by H-Rho-zeolite Synlett, 1999, 12, 1921; 3cA: Ranu, B. C.; Mandal, T. Indium(I) iodide promoted cleavage of dialkyl/diaryl disulfides and subsequent anti-Markovnikov addition to styrenes: a new route to linear thioethers Tetrahedron Lett., 2006, 47, 6911; 3dA: Baciocchi, E.; Lanzalunga, O.; Pirozzi, B. Oxidations of benzyl and phenethyl phenyl sulfides. Implications for the mechanism of the microsomal and biomimetic oxidation of sulfides Tetrahedron, 1997, 53, 12287; 3eA: Mirafzal, G. A.; Liu, J.; Bauld, N. L. Hole transfer promoted hydrogenation: one-electron oxidation as a strategy for selective reduction of .pi.-bonds J. Am. Chem. Soc., 1993, 115, 6072; 3gA: Rosnati, V.; Di Vona, M. L.; Traldi, P. Neighboring-group participation. A quasi-SNi mechanism in the acetolysis and thioacetolysis of 1-(phenylthio)-2-[(p-tolylsulfonyl)oxy]ethane J. Org. Chem., 1989, 54, 761; 3aB: Jones, P. B.; Parrish, N. M.; Houston, T. A.; Stapon, A.; Bansal, N. P.; Dick, J. D.; Townsend, C. A. A new class of antituberculosis agents J. Med. Chem., 2000, 43, 3304; 3fB: Cadogan, J. I. G.; Sadler, I. H. Quantitative aspects of radical addition. Part IV. Rate-constant ratios for addition of trichloromethyl and thiyl radicals to olefins J. Chem. Soc. B, 1966, 1191; 3gB: Gouault, S.; Pommelet, J. C.; Lequeux, T. First synthesis of 4,4-difluoro-1,3-oxathiolan- 5-one Synlett, 2002, 6, 996.

69 70 CHAPTER 3 Thiol/Yne Coupling for Peptide Glycosidation1 3.1 Introduction Here, we report an explorative study on model substrates that aimed at getting more insight into the conditions affecting the coupling reaction outcome, particularly the ensuing mono- and bis-adduct ratio. Both classical organic solvents and water (or water/solvent mixtures) were taken into account for carrying out the reactions, since the aqueous medium would be crucial for applying Thiol-Yne Couplings as a ligation strategy, for instance, for peptide glycosylation, similarly to what has been done with TEC procedures.2 In addition, we are going to confirm that, compared to alkylacetylenes, C-C triple bonds linked to aromatic substrates (arylacetylenes) work as a better trap toward sulfanyl radicals:3 to the best of our knowledge, this enhanced behavior has been exploited neither in the domain of bioconjugation nor in that of material derivatization and could entail interesting consequences in both fields. Finally, some preliminary results will be reported showing that the data obtained from the explorative study could be successfully applied to some bioconjugation examples including glycosylation of the native form of glutathione and of an unmodified nonapeptide containing a cysteine residue.

3.2 Result and Discussion Our long, earlier interest in the radical reactions of thiols with alkynes4 led us to ascertain that, under thermal conditions (80-100 °C) and in hydrocarbon solvents (e.g., benzene or toluene), derived sulfanyl radicals usually lead to vinyl sulfide adducts only. Consistent with our original evidence, methyl thioglycolate 1 was presently found to react with equimolar amounts of 1-octyne 2 in toluene solution at 80 °C in the presence of AIBN as a radical initiator, affording vinyl sulfide 3 (62%) and disulfide 5 (28%) as the only identifiable reaction products (Table 1, entry 1). Nevertheless, the reaction outcomes alter to a considerable extent by changing conditions (i.e., solvent, temperature, concentration, and initiation method), as proved by a series of experiments (Tables 1-4) carried out between two thiols (methyl thioglycolate 1 and N-acetyl-L-cysteine methyl ester 10) and two alkynes (1-octyne 2 and phenylacetylene 6). The parallel behavior

71 of thiols with aromatic and aliphatic alkynes has never been addressed in the recently reported TYC studies, but in light of our previous investigations, we thought that this point should deserve adequate consideration. Different reaction solvents were chosen on the basis of their potential employment in bioconjugation procedures (water and 95:5 water/DMSO mixtures) or material derivatization (DMSO, DMF). All the reactions were carried out at room temperature(r.t.)5 for 1-2 h by irradiation with UV-lamp ( max 365 nm) using 2,2-dimethoxy-2-phenylacetophenone (DMPA, 10 mol %) as a radical initiator, that is, the conditions normally employed in this kind of radical couplings.6 Thus, the model reaction of thiol 1 with alkyne 2 (1 equiv) in toluene (0.5 M) solution afforded, under these conditions, both mono- (3) and bis-adduct (4) in comparable amounts (45:55 ratio, 76% overall yield), together with very minor Minozzi et al. JOCArticle 3 amounts (<5%) of disulfide 5 (Table similarly to what has been done with TEC procedures. In TABLE 1. Thiol-Yne Couplings of Methyl Thioglycolate 1 with a addition, we are going to confirm that, compared to alkyla- 1-Octyne 2 1, entry 2). cetylenes, C-C triple bonds linked to aromatic substrates Minozzi et al. 12 (arylacetylenes) work as a bettertraptowardsulfanylradicals: JOCArticle As far as the 3/4 ratio is concerned, 3 to the best of our knowledge,similarly thisto what enhanced has been behavior done with TEC has procedures. been In TABLE 1. Thiol-Yne Couplings of Methyl Thioglycolate 1 with exploited neither in theaddition, domain we areof bioconjugationgoing to confirm that, nor compared in that to alkyla- 1-Octyne 2a of material derivatizationcetylenes, and C- couldC triple entail bonds linked interesting to aromatic con- substrates almost identical results were (arylacetylenes) work as a bettertraptowardsulfanylradicals:12 sequences in both fields.to the best Finally, of our knowledge, some preliminary this enhanced behavior results has been will be reported showingexploited that neither the in data the domain obtained of bioconjugation from the nor in that obtained by substituting toluene with explorative study couldof material be successfully derivatization and applied could entail to someinteresting con- sequences in both fields. Finally, some preliminary results DMSO or DMF, albeit with lower bioconjugation exampleswill beincluding reported glycosylationshowing that the of data the obtained native from the form of glutathione andexplorative of an unmodified study could be nonapeptide successfully applied con- to some taining a cysteine residue.bioconjugation examples including glycosylation of the native overall yields. It would therefore form of glutathione and of an unmodified nonapeptide con- taining a cysteine residue. Results and Discussion seem that temperature plays a pivotal Results and Discussion Our long, earlier interest in the radical reactions of thiols 13 Our long, earlier interest in the radical reactions of thiols role in affecting the outcoming with alkynes led uswith to ascertain alkynes13 led that, us to under ascertain thermal that, under condi- thermal condi- tions (80-100 °C) andtions in (80 hydrocarbon-100 °C) and in solvents hydrocarbon (e.g., solvents ben- (e.g., ben- zene or toluene), derivedzene or sulfanyl toluene), derived radicals sulfanyl usually radicals lead usually to lead to sulfide-3/bis-sulfide-4 ratio (by vinyl sulfide adducts only.vinyl Consistent sulfide adducts with only. ourConsistent original with evidence,our original evidence, methyl thioglycolate 1 was presently found to react with entry 1/2 solvent (c [M]) 3/4 [%]b,c yield 3 4 [%]d b,c þ dcomparison of entries 1 and 2), as an methyl thioglycolateequimolar1 waspresently amounts of 1-octyne found2 in to toluene react solution with at 80 °Centry 11/2 1:1 Toluenesolvent (0.5) (ce[M]) 100/-3/4 [%] 62yield 3 4 [%] in the presence of AIBN as a radical initiator, affording vinyl f þ equimolar amounts of 1-octyne 2 in toluene solution at 80 °C 2 1:1 Toluene (0.5) e 45/55 76 sulfide 3 (62%) and disulfide 5 (28%) as the only identifiable 13 1:1 1:1 Toluene DMSO (0.02) (0.5) 80/20100/- 55 62 in the presence of AIBN as a radical initiator, affording vinyl f g expected result of a faster or slower back reaction products (Table 1, entry 1). Nevertheless, the reac- 24 1:1 1:1 Toluene H2O-DMSO (0.5) (0.5) 45/5545/55 90 76 sulfide 3 (62%) and disulfidetion outcomes5 (28%) alter to as a the considerable only identifiable extent by changing 35 1:1 1:1 DMSO H2O (0.5) (0.02) 11/89 80/20 85 55 6 1:1 [bmim][PF6]g 25/75 52 reaction products (Tableconditions 1, entry (i.e.,1). solvent, Nevertheless, temperature, the concentration, reac- and 47 1:1 2:1 H2 DMSOO-DMSO (0.5) (0.5) -/10045/55 75 90 fragmentation of the dithioalkyl radical g initiation method), as proved by a series of experiments 58 1:1 2:1 H2 HO2O (0.5)-DMSO (0.5) -/100 11/89 90 85 tion outcomes alter to a considerable extent by changing 9 2:1 H O (0.5) -/100 89 (Tables 1-4) carried out between two thiols (methyl thiogly- 6 1:1 [bmim][PF2 6] 25/75 52 conditions (i.e., solvent,colate temperature,1 and N-acetyl-L-cysteine concentration, methyl ester and10) and two 7aPhotoinduced 2:1 DMSO reactions (0.5) were carried out at- r.t./100 with a household 75 precursor of the bis-adduct. Interest- alkynes (1-octyne 2 and phenylacetylene 6). The parallel beha- UVA lamp apparatus at λmax 365 nm (seeg the Experimental Section for initiation method), as proved by a series of experiments 8 2:1 H2O-DMSO (0.5) -/100 90 vior of thiols with aromatic and aliphatic alkynes has never equipment setup). Reactions performed with 1 normally gave the (Tables 1-4) carried out between two thiols (methyl thiogly- 9corresponding 2:1 H disulfide2O (0.5)5 in <5% yield; only the-/100 reaction of entry 3 89 been addressed in the recently reported TYC studies, but in b ingly, unlike the aliphatic acetylene 2, a afforded 5 in ca. 10% yield. Values are relative percentages and were colate 1 and N-acetyl-lightL-cysteine of our previous methyl investigations, ester 10) we and thought two that this Photoinduceddetermined by GC reactions-MS and 1 wereH NMR carried analysis. outcAdduct at r.t.3 was with a 1.2:1 a household d alkynes (1-octyne 2 andpoint phenylacetylene should deserve adequate6). The parallelconsideration. beha- DifferentUVA lampmixture apparatus of E and Z isomers. at λmaxIsolated365 nm yield (see calculated the Experimental on the basis of the Section for e under the same conditions reaction solvents were chosen on the basis of their potentialequipmentstarting setup). alkyne. Reaction Reactions performed performed under thermal with conditions1 normally (80 °C) gave the vior of thiols with aromatic and aliphatic alkynes has never with AIBN as the radical initiator. fSimilar results were obtained in employment in bioconjugation procedures (water and 95:5corresponding disulfideg 5 in <5% yield; only the reaction of entry 3 DMSO or DMF. H2O/DMSO 95:5. been addressed in thewater/DMSO recently reported mixtures) TYCor material studies, derivatization but in (DMSO,afforded 5 in ca. 10% yield. bValues are relative percentages andphenylacetylene were 6 gave only vinyl sulfide DMF). All the reactions were carried out at room temperature 1 c light of our previous investigations, we thought that this determined14 by GC-MS and H NMR analysis. Adduct 3 was a 1.2:1 (r.t.) for 1-2 h by irradiationd with UV-lamp (λmax 365 nm) point should deserve adequate consideration. Different mixtureusing of E 2,2-dimethoxy-2-phenyland Z isomers. Isolatedacetophenone yield calculated (DMPA, 10 on mol the %) basis of the 7 (12) For comparisons between the reactivities of alkyl- and arylacety-starting alkyne. eReaction performed under thermal conditions7 (80 in 90% yield (Table 2, entry 1).°C) reaction solvents werelenes, chosen see refs 8d on above, the 13a, basis 13b, below, of their and (a) potential Ogawa, A.; Obayashi, R.; as a radical initiator, that is, thef conditions normally em- Ine, H.; Tsuboi, Y.; Sonoda, N.; Toshikazu, H. J. Org. Chem. 1998, 63, 881–with AIBNployed as in thethis kindradical of radical initiator. couplings.Similar9 Thus, results the were model obtained in employment in bioconjugation884. For another recent procedures example of radical (water monothiolation and 95:5 of arylacetylenes,DMSO or DMF. gH O/DMSO 95:5. water/DMSO mixtures)see: (b) or Beauchemin, material A.; Gareau, derivatization Y. Phosphorus, Sulfur, (DMSO, Silicon Relat. Elem. reactionDilution of of thiol21 with alkyne the 2 reaction mixture from (1 equiv) in toluene (0.5 M) 0.5 to 0.02 M led, with 1-octyne in 1998, 139, 187–192. It is worth emphasizing that a higher reactivity (kinetic solution afforded, under these conditions, both mono- (3) DMF). All the reactionsconstant were of more carried than one out order at of room magnitude temperature higher) of phenylacetylene with respect to an alkyl congener (1-propyne) has been also reported for 14and bis-adduct (4) in comparable amounts (45:55 ratio, 76% addition of alkyl (methyl) radicals, see: (c) Fischer, H.; Radom, L. Angew.DMSO, (r.t.) overallfor 1to yield),-2 preferential formation of mono-adduct h together by irradiation with very minor with amountsUV-lamp (<5%) (λmax of 365 nm) 3 (3/4 ratio ca. 4:1; Table 1, Chem., Int. Ed. 2001, 40, 1340–1371. usingdisulfide 2,2-dimethoxy-2-phenyl5 (Table 1, entry 2). acetophenone (DMPA, 10 mol %) (12) For comparisons between(13) (a) Benati, the reactivities L.; Montevecchi, of P.alkyl- C.; Spagnolo, and arylacety- P. J. Chem. Soc., Perkin As far as the 3/4 ratio is concerned, almost identical results lenes, see refs 8d above, 13a,Trans. 13b, 1 1991 below,,2103–2109.andreferencestherein.(b)Benati,L.;Montevecchi, and (a) Ogawa, A.; Obayashi, R.; as a radical initiator, that is, the conditions normally em- P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1992,1659–1664.(c)Benati,L.; were obtained by substituting toluene with DMSO9 or DMF, Ine, H.; Tsuboi, Y.; Sonoda,Capella, N.; Toshikazu, L.; Montevecchi, H. P. C.;J. Spagnolo,Org. Chem. P. J. Chem.1998 Soc.,, 63 Perkin, 881– Trans. 1 1995ployed, in this kind of radical couplings. Thus, the model 884. For another recent example1035–1038. of radical (d) Benati, monothiolation L.; Capella, L.; Montevecchi, of arylacetylenes, P. C.; Spagnolo, P. J. Org. albeit with lower overall yields. It would therefore seem that see: (b) Beauchemin, A.; Gareau,Chem. 1995 Y., 60Phosphorus,,7941–7946.(e)Montevecchi,P.C.;Navacchia,M.L. Sulfur, Silicon Relat. Elem. J. Org.reactiontemperature of thiol plays1 with a pivotal alkyne role in2 affecting(1 equiv) the in outcoming toluene (0.5 M) Chem. 1997, 62,5600–5607.(f)Montevecchi,P.C.;Navacchia,M.L.J. Org. sulfide-3/bis-sulfide-4 ratio (by comparison of entries 1 and 2), as 1998, 139, 187–192. It is worthChem. emphasizing1998, 63,537–542.(g)Montevecchi,P.C.;Navacchia,M.L.;Spagnolo,P. that a higher reactivity (kinetic solution afforded, under these conditions, both mono- (3) constant of more than oneTetrahedron order of1998 magnitude, 54,8207–8216.(h)Montevecchi,P.C.;Navacchia,M.L.; higher) of phenylacetylene an expected result of a faster or slower back fragmentation of with respect to an alkyl congenerSpagnolo, P. (1-propyne)Eur. J. Org. Chem. has1998 been,1219–1226.(i)Leardini,R.;Nanni,D.; also reported for and bis-adductthe dithioalkyl (4 radical) in comparable precursor of the amounts bis-adduct. (45:55 Interest- ratio,72 76% Zanardi, G. J. Org. Chem. 2000, 65,2763–2772.Werecentlyexploitedadditionof addition of alkyl (methyl)sulfanyl radicals, radicals see: to (c)alkynes Fischer, as a novel H.; tin-/metal-free Radom, method L. Angew. to generate assortedoverallingly, yield), unlike thetogether aliphatic acetylenewith very2, under minor the same amounts conditions (<5%) of Chem., Int. Ed. 2001, 40, 1340–1371.kinds of radicals, see: (j) Benati, L.; Calestani, G.; Leardini, R.; Minozzi, M.;disulfidephenylacetylene5 (Table 1,6 gave entry only 2). vinyl sulfide 7 in 90% yield (13) (a) Benati, L.; MontevNanni,ecchi, D.; Spagnolo, P. C.; Spagnolo, P.; Strazzari, S.P.Org.J. Chem. Lett. 2003 Soc.,, 5,1313–1316.(k)Benati, Perkin (Table 2, entry 1).15 L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. As far as the 3/4 ratio is concerned, almost identical results Trans. 1 1991,2103–2109.andreferencestherein.(b)Benati,L.;Montevecchi,Synlett 2004,987–990.(l)Benati,L.;Bencivenni,G.;Leardini,R.;Minozzi,M.; P. C.; Spagnolo, P. J. Chem. Soc.,Nanni, Perkin D.; Scialpi, Trans. R.; Spagnolo, 1 1992,1659–1664.(c)Benati,L.; P.; Zanardi, G. J. Org. Chem. 2006, 71,3192–were obtained(14) Under theseby substituting conditions, the reaction toluene vessel actually with warmed DMSO up to or DMF, Capella, L.; Montevecchi, P.3197. C.; Spagnolo, (m) Bencivenni, P. J. G.; Chem. Lanza, Soc., T.; Leardini, Perkin R.;Trans. Minozzi, 1 1995 M.;, Nanni, D.; 35-40 °C. Anyway, strictly identical results were obtained with reaction 1035–1038. (d) Benati, L.; Capella,Spagnolo, L.; P.; Zanardi,Montevecchi, G. Org. Lett. P. C.;2008 Spagnolo,, 10,1127–1130. P. J. Org. albeitmixtures with keptlower at 25 overall°C by simultaneous yields. air-cooling It would with compressed therefore air. seem that Chem. 1995, 60,7941–7946.(e)Montevecchi,P.C.;Navacchia,M.L.J. Org. temperature playsJ. a Org. pivotal Chem. Vol. role XXX in, affectingNo. XX, XXXX the outcomingC Chem. 1997, 62,5600–5607.(f)Montevecchi,P.C.;Navacchia,M.L.J. Org. sulfide-3/bis-sulfide-4 ratio (by comparison of entries 1 and 2), as Chem. 1998, 63,537–542.(g)Montevecchi,P.C.;Navacchia,M.L.;Spagnolo,P. Tetrahedron 1998, 54,8207–8216.(h)Montevecchi,P.C.;Navacchia,M.L.; an expected result of a faster or slower back fragmentation of Spagnolo, P. Eur. J. Org. Chem. 1998,1219–1226.(i)Leardini,R.;Nanni,D.; the dithioalkyl radical precursor of the bis-adduct. Interest- Zanardi, G. J. Org. Chem. 2000, 65,2763–2772.Werecentlyexploitedadditionof sulfanyl radicals to alkynes as a novel tin-/metal-free method to generate assorted ingly, unlike the aliphatic acetylene 2, under the same conditions kinds of radicals, see: (j) Benati, L.; Calestani, G.; Leardini, R.; Minozzi, M.; phenylacetylene 6 gave only vinyl sulfide 7 in 90% yield Nanni, D.; Spagnolo, P.; Strazzari, S. Org. Lett. 2003, 5,1313–1316.(k)Benati, 15 L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. (Table 2, entry 1). Synlett 2004,987–990.(l)Benati,L.;Bencivenni,G.;Leardini,R.;Minozzi,M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. J. Org. Chem. 2006, 71,3192– (14) Under these conditions, the reaction vessel actually warmed up to 3197. (m) Bencivenni, G.; Lanza, T.; Leardini, R.; Minozzi, M.; Nanni, D.; 35-40 °C. Anyway, strictly identical results were obtained with reaction Spagnolo, P.; Zanardi, G. Org. Lett. 2008, 10,1127–1130. mixtures kept at 25 °C by simultaneous air-cooling with compressed air.

J. Org. Chem. Vol. XXX, No. XX, XXXX C entry 3), whereas the reaction with alkyne 6 was not substantially affected, yielding again the mono adduct 7 (65%) as the only coupling product (Table 2, entry 2); both reactions also afforded disulfide 5 as a byproduct (ca. 10%). Since, in general, dilution was found to affect negatively the reaction outcome in terms of lower overall yields, formation of byproducts, and lower conversions, all the subsequent reactions were performed in 0.5 M solutions. When the reaction of 1 with 2 was carried out in water/DMSO 95:5, no change in product distribution was observed (Table 1, entry 4), whereas pure water8 strongly favored occurrence of bis-adduct 4 over the mono adduct 3 (3/4 ratio ca. 1:8; Table 1, entry 5). So strong seems this effect of water that with this solvent also phenyl acetylene 6 afforded small amounts (10%) of bis-adduct as a mixture of Article Minozzi et al. regioisomers 8 and 9 in a 2:1 JOC TABLE 2. Thiol-Yne Couplings of Methyl Thioglycolate 1 with SCHEME 2. Reaction Mechanism of Formation of Bis-Adducts ratio (Table 2, entry 4).The PhenylacetyleneJOCArticle 6a 4, 8, and 9 Minozzi et al. outcoming difference in the TABLE 2. Thiol-Yne Couplings of Methyl Thioglycolate 1 with SCHEME 2. Reaction Mechanism of Formation of Bis-Adducts Phenylacetylene 6a 4, 8, and 9 regiochemistry of the bis-sulfide adducts arising from octyne 2 and phenylacetylene 6 probably reflects the relative stability of the alkyl radicals R and à that could in principle arise from further addition of sulfanyl afforded small amounts (10%) of bis-adduct as a mixture radical to the respective double affordedof small regioisomers amounts (10%)8 and 9 ofin bis-adduct a 2:1 ratio as (Table a mixture 2, entry 4). of regioisomersThe outcoming8 and 9 in a difference 2:1 ratio (Table in the 2, regiochemistry entry 4). of the bis- The outcoming difference in the regiochemistry of the bis- sulfide adducts arising from octyne 2 and phenylacetylene 6 bond of vinyl sulfides 3 and 7 sulfide adducts arising from octyne 2 and phenylacetylene 6 probablyprobably reflects the reflects relative the stability relative of stability the alkyl radicalsof the alkylR radicals R (Scheme 2). With sulfide 3, the and β thatand couldβ that in could principle in arise principle from furtherarise from addition further of addition of sulfanylsulfanyl radical to radical the respective to the doublerespective bond double of vinyl bond sulfides of3 vinyl sulfides 3 entryentry 11/6 solventsolvent (c ([M])c [M]) 7/87/89 [%]9b[%],c yieldb,c yield7 8 7 9 [%]8 d 9 [%]d sulfanyl would strictly prefer to þ þ þ þþ þ and 7 (Schemeand 7 (Scheme 2). With sulfide 2). With3, the sulfide sulfanyl3, the would sulfanyl strictly would strictly 1 1:1 Toluene (0.5)e e 100/- 90 1 1:1 Toluene (0.5) 100/- 90 prefer toprefer form tothe form more the stable more radical stableβ,R=C radical6Hβ13,R=C, owing 6H13, owing 22 1:1 1:1 DMSO DMSO (0.02) (0.02) 100/ 100/- - 65 65 form the more stable radical β, R f to back-donationto back-donation stabilization stabilization provided by provided the attached by the attached 3 1:1 H2O-DMSO (0.5) f 100/- 87 3 1:1 H2O-DMSO (0.5) 100/g - 87 sulfur atom; with sulfide 7, the formation of the correspond- 4 1:1 H2O (0.5) 90/10 g 91 sulfur atom; with sulfide 7, the formation of the correspond- 4 1:1 H2O (0.5) 90/10g 91 = C6H13, owing to back-donation 5 1:1 [bmim][PF6] 90/10 g 58 ing radical β, R = Ph, would be discouraged to some extent g ing radical β, R = Ph, would be discouraged to some extent 56 1:1 2:1 DMSO [bmim][PF (0.5)6] 40/60 90/10 69 58 in favor of the resonance-stabilized benzylic radical R,R= f g g 67 2:1 2:1 H DMSO2O-DMSO (0.5) (0.5) 22/78 40/60 88 69 in favor of the resonance-stabilized benzylic radical R,R= f g g Ph (Scheme 2). stabilization provided by the 78 2:1 2:1 H H22OO (0.5)-DMSO (0.5) 5/9522/78 91 88 g It is worthPh (Scheme noting that 2). an analogous effect in favor of bis- 8aPhotoinduced 2:1 H2O reactions (0.5) were carried out 5/95 at r.t. with a household91 It is worth noting that an analogous effect in favor of bis- a hydrothiolation was observed by changing water with an attached sulfur atom; with UVAPhotoinduced lamp apparatus reactions at λmax 365 were nm carried (see the Experimental out at r.t. with Section a household for equipment setup). Reactions performed with 1 normally gave the ionic liquidhydrothiolation ([bmim][PF6]): was with observed this solvent, by 1-octyne changing gave water with an UVA lamp apparatus at λmax 365 nm (see the Experimental Section foradducts 4 and 3 in a 3:1 ratio (Table 1, entry 6) and phenyl- equipmentcorresponding setup). disulfide Reactions5 in <5% performed yield; only with the reaction1 normally of entry gave the ionic liquid ([bmim][PF6]): with this solvent, 1-octyne gave sulfide 7, the formation of the 2 afforded 5 in ca. 10% yield. bValues are relative percentages and were acetylene afforded again monoadduct 7 accompanied with corresponding disulfide 5 in1 <5% yield; onlyc the reaction of entry adducts 4 and 3 in a 3:1 ratio17 (Table 1, entry 6) and phenyl- determined by GC-MS and Hb NMR analysis. Adduct 7 was a 2:1 minor amounts of bis-adducts 8 and 9 (Table 2, entry 5). 2mixture afforded of Z5 andin ca.E isomers. 10% yield.dIsolatedValues yield are calculated relative on percentages the basis of the and were acetylene afforded again monoadduct 7 accompanied with corresponding radical β, R = Ph, 1 c Preferential occurrence of bis-sulfide 4 at the expense of vinyl determinedstarting alkyne. bye GCSimilar-MS results and wereH obtained NMR analysis. in DMSO orAdduct DMF. fH7 wasO/ a 2:1 17 d 2 minor amounts of bis-adducts 8 and 9 (Table 2, entry 5). mixtureDMSO 95:5.of Z andg8/9 E2:1.isomers. Isolated yield calculated on the basis of thesulfide 3 would possibly entail especially fast H-transfer from would be discouraged to some e ∼ f Preferential occurrence of bis-sulfide 4 at the expense of vinyl starting alkyne. Similar results were obtained in DMSO or DMF. H2O/thiol 1 to the intermediate dithioalkyl radical. Under these DMSO 95:5. g8/9 2:1. circumstances,sulfide 3 inwould fact, that possibly radical entail intermediate especially could fast H-transfer be from Dilution of the∼ reaction mixture from 0.5 to 0.02 M led, (seriously)thiol discouraged1 to the intermediate to suffer usual dithioalkylβ-elimination radical. of Under these with 1-octyne in DMSO, to preferential formation of mono- sulfanylcircumstances, radical yielding back in fact, vinyl that sulfide radical3 (see Schemeintermediate 1). could be adductDilution3 (3/ of4 ratio the reaction ca. 4:1; Table mixture 1, entry from 3), 0.5 whereas to 0.02 the M led,Thus, our(seriously) present findings discouraged with octyne to suffer2 in the usual above ionicβ-elimination of withreaction 1-octyne with alkyne in DMSO,6 was tonot preferential substantially formation affected, yield- of mono-liquid seemsulfanyl to substantiate radical yielding previous back chemical vinyl evidence sulfide 3 that(see Scheme 1). ing again the monoadduct 7 (65%) as the only coupling 6 adduct73 3 (3/4 ratio ca. 4:1; Table 1, entry 3), whereas thethiols inThus, ionic liquid our solvents present could findings act as verywith strong octyne H-donors.2 in the above ionic reactionproduct (Table with 2,alkyne entry 2);6 was both notreactions substantially also afforded affected, disulfide yield-Moreover, the corresponding findings achieved in pure 5 as a byproduct (ca. 10%). Since, in general, dilution was liquid seem to substantiate previous chemical evidence that ing again the monoadduct 7 (65%) as the only couplingwater first suggest that in such medium thiols should inter- 6 found to affect negatively the reaction outcome in terms of estinglythiols become in ionic even liquid stronger solvents H-donors. could Whether act as very the strong en- H-donors. productlower overall (Table yields, 2, entry formation 2); both of reactions byproducts, also and afforded lower con- disulfidehancedMoreover, H-donor properties the corresponding of thiol in ionic findings liquid achieved and, in pure 5versions,as a byproduct all the subsequent (ca. 10%). reactions Since, in were general, performed dilution in wasespecially,water water first would suggest result that from in some such solvent medium stabilization thiols should inter- found0.5 M tosolutions. affect negatively When the reaction the reaction of 1 with outcome2 was carried in terms ofof the H-transferestingly become transition even state, stronger as previously H-donors. suggested, Whether6 the en- lowerout in overall water/DMSO yields, 95:5, formation no change of byproducts, in product distribution and lower con-or wouldhanced just be aH-donor consequence properties of ‘neat’ reactions of thiol occurring in ionic liquid and, 18 versions,was observed all (Table the subsequent 1, entry 4), whereas reactions pure waterwere16 performedstrongly ininside organicespecially, droplets wateris would a debated result question from some that willsolvent be stabilization 0.5favored M solutions. occurrence When of bis-adduct the reaction4 over of the1 monoadductwith 2 was carried3 dealt withof thein future H-transfer studies. transition state, as previously suggested,6 Taking into account the overall results obtained with thiol out(3/4 inratio water/DMSO ca. 1:8; Table 95:5, 1, entry no 5). change So strong in product seems this distribution effect or would just be a consequence of ‘neat’ reactions occurring of water that with this solvent also phenylacetylene16 6 1 (Tables 1 and 2), we can infer that18 it is possible to modify was observed (Table 1, entry 4), whereas pure water stronglyproperlyinside the reaction organic outcome droplets by tuningis athe debated reaction question condi- that will be favored occurrence of bis-adduct 4 over the monoadduct 3tions. Startingdealt with from in equivalent future studies. amounts of thiol and alkyne, (15) All the vinyl sulfides were formed as mixtures of E- and Z-isomers: (3see/4 theratio Experimental ca. 1:8; Section Table for details. 1, entry Studies 5). on So the strong stereoselective seems formation this effect Taking into account the overall results obtained with thiol of the kinetic (Z)orthermodynamic(E)productarecurrentlyunderway. (17) Both1 (Tables in water and 1 and in the 2), ionic we liquid can the infer8/9 ratio that is ca. it 2:1. is possible to modify of(16) water Reactions that carried with out in this water solvent were heterogeneous also phenylacetylene mixtures, whereas 6 (18) For a recent discussion about organic synthesis “on water”, see: those carried out in water/DMSO 95:5 were normally homogeneous. Chanda,properly A.; Fokin, V. the V. Chem. reaction Rev. 2009 outcome, 109, 725–748. by tuning the reaction condi- tions. Starting from equivalent amounts of thiol and alkyne, D(15)J. All Org. the Chem. vinyl sulfidesVol. XXX were, No. formed XX, asXXXX mixtures of E- and Z-isomers: see the Experimental Section for details. Studies on the stereoselective formation of the kinetic (Z)orthermodynamic(E)productarecurrentlyunderway. (17) Both in water and in the ionic liquid the 8/9 ratio is ca. 2:1. (16) Reactions carried out in water were heterogeneous mixtures, whereas (18) For a recent discussion about organic synthesis “on water”, see: those carried out in water/DMSO 95:5 were normally homogeneous. Chanda, A.; Fokin, V. V. Chem. Rev. 2009, 109, 725–748.

D J. Org. Chem. Vol. XXX, No. XX, XXXX extent in favor of the resonance-stabilized benzylic radical α, R = Ph (Scheme 2). JOCArticle Minozzi et al.

TABLE 2. Thiol-Yne Couplings of Methyl Thioglycolate 1 with SCHEME 2. Reaction Mechanism of Formation of Bis-Adducts Phenylacetylene 6a 4, 8, and 9

It is worth noting that an analogous effect afforded small amounts (10%) of bis-adduct in favor as a of bis-hydrothiolation mixture of regioisomers 8 and 9 in a 2:1 ratio (Table 2, entry 4). was observed by The changing water with an ionic outcoming difference in the regiochemistry liquid ([bmim][PF of the bis- 6]): with this sulfide adducts arising from octyne 2 and phenylacetylene 6 solvent, 1-octyne probably gave reflects adducts the4 relative and stability3 in a 3:1 ratio (Table 1, entry 6) and of the alkyl radicals R phenyl acetylene and β afforded that could again in principle mono arise adduct from further7 accompanied addition of with minor sulfanyl radical to the respective double bond of vinyl sulfides 3 entry 1/6 solvent (c [M]) 7/8 9 [%]b,c yield 7 8 9 [%]d þ þ þ and 7 (Scheme 2). With9 sulfide 3, the sulfanyl would strictly e amounts of bis-adducts 8 and 9 (Table 2, entry 5). Preferential occurrence of 1 1:1 Toluene (0.5) 100/- 90 prefer to form the more stable radical β,R=C6H13, owing 2 1:1 DMSO (0.02) 100/- 65 f bis-sulfide 4 at the expense of vinyl sulfide to back-donation stabilization provided3 would possibly entail especially fast by the attached 3 1:1 H2O-DMSO (0.5) 100/- 87 g sulfur atom; with sulfide 7, the formation of the correspond- 4 1:1 H2O (0.5) 90/10 91 g H-transfer from thiol 1 to the intermediate dithioalkyl radical. Under these 5 1:1 [bmim][PF6] 90/10 58 ing radical β, R = Ph, would be discouraged to some extent 6 2:1 DMSO (0.5) 40/60g 69 in favor of the resonance-stabilized benzylic radical R,R= f g circumstances, in fact, that radical intermediate could be (seriously) discouraged 7 2:1 H2O-DMSO (0.5) 22/78 88 g Ph (Scheme 2). 8 2:1 H2O (0.5) 5/95 91 It is worth noting that an analogous effect in favor of bis- a to suffer usual β-elimination of sulfanyl radical yielding back vinyl sulfide 3 (see Photoinduced reactions were carried out at r.t. with a household hydrothiolation was observed by changing water with an UVA lamp apparatus at λmax 365 nm (see the Experimental Section for equipment setup). Reactions performed with 1 normallyScheme 1). gave the Thus, ionic our liquid present ([bmim][PF findings with octyne 6]): with this solvent,2 1-octyne in the above ionic liquid gave corresponding disulfide 5 in <5% yield; only the reaction of entry adducts 4 and 3 in a 3:1 ratio (Table 1, entry 6) and phenyl- 2 afforded 5 in ca. 10% yield. bValues are relative percentagesseem and to were substantiate acetylene previous afforded again chemical monoadduct evidence 7 accompanied that thiols with in ionic liquid determined by GC-MS and 1H NMR analysis. cAdduct 7 was a 2:1 17 minor amounts of bis-adducts 8 and 109 (Table 2, entry 5). mixture of Z and E isomers. dIsolated yield calculated on thesolvents basis of the could act as very strong H-donors. Moreover, the corresponding e f Preferential occurrence of bis-sulfide 4 at the expense of vinyl starting alkyne. Similar results were obtained in DMSO or DMF. H2O/ DMSO 95:5. g8/9 2:1. findings achieved in pure water first suggest that in such medium thiols should sulfide 3 would possibly entail especially fast H-transfer from ∼ thiol 1 to the intermediate dithioalkyl radical. Under these interestingly become even stronger H-donors. circumstances, in fact, that radical intermediate Whether the could enhanced be H-donor Dilution of the reaction mixture from 0.5 to 0.02 M led, (seriously) discouraged to suffer usual β-elimination of with 1-octyne in DMSO, to preferential formationproperties of thiol in ionic liquid and, especially, water would result from some of mono- sulfanyl radical yielding back vinyl sulfide 3 (see Scheme 1). adduct 3 (3/4 ratio ca. 4:1; Table 1, entry 3), whereas the Thus, our present findings with octyne 2 in the above ionic 10 reaction with alkyne 6 was not substantially affected,solvent stabilization of the H-transfer transition state, as previously suggested, yield- liquid seem to substantiate previous chemical evidence that 6 ing again the monoadduct 7 (65%) as the onlyor coupling would just thiols be a in consequence ionic liquid solvents of could ‘neat’ act reactions as very strong occurring H-donors. inside organic product (Table 2, entry 2); both reactions also afforded disulfide Moreover, the corresponding findings achieved in pure 5 as a byproduct (ca. 10%). Since, in general, dilutiondroplets was11 is a debated question that will be dealt with in future studies.water first suggest that in such medium thiols should inter- found to affect negatively the reaction outcome in terms of estingly become even stronger H-donors. Whether the en- lower overall yields, formation of byproducts, and lowerTaking into account the overall results obtained with thiol con- hanced H-donor properties of thiol in ionic liquid and,1 (Tables 1 and versions, all the subsequent reactions were performed in especially, water would result from some solvent stabilization 2), we can infer that it is possible to modify properly the reaction outcome by 6 0.5 M solutions. When the reaction of 1 with 2 was carried of the H-transfer transition state, as previously suggested, out in water/DMSO 95:5, no change in product distribution or would just be a consequence of ‘neat’ reactions occurring tuning the reaction conditions. Starting 18 from equivalent amounts of thiol and was observed (Table 1, entry 4), whereas pure water16 strongly inside organic droplets is a debated question that will be favored occurrence of bis-adduct 4 over the monoadduct 3 dealt with in future studies. (3/4 ratio ca. 1:8; Table 1, entry 5). So strong seems this effect Taking into account the overall results obtained with thiol of water that with this solvent also phenylacetylene 6 1 (Tables 1 and 2), we can infer that it is possible to modify properly the reaction outcome74 by tuning the reaction condi- tions. Starting from equivalent amounts of thiol and alkyne, (15) All the vinyl sulfides were formed as mixtures of E- and Z-isomers: see the Experimental Section for details. Studies on the stereoselective formation of the kinetic (Z)orthermodynamic(E)productarecurrentlyunderway. (17) Both in water and in the ionic liquid the 8/9 ratio is ca. 2:1. (16) Reactions carried out in water were heterogeneous mixtures, whereas (18) For a recent discussion about organic synthesis “on water”, see: those carried out in water/DMSO 95:5 were normally homogeneous. Chanda, A.; Fokin, V. V. Chem. Rev. 2009, 109, 725–748.

D J. Org. Chem. Vol. XXX, No. XX, XXXX alkyne,with 1-octyne 2 (Table 1), formation of monoadduct 3 is favored at higher temperatures (AIBN-initiated reaction in toluene) or diluted mixtures (0.02 M in DMSO), whereas formation of bis-adduct 4 is strongly favored in an ionic liquid such as [bmim][PF6] and, particularly, in water. With phenyl- acetylene 6 (Table 2), the monoadduct 7 is always the exclusive product, with the exception of the small amounts of the regioisomeric bis-adducts 8 and 9 isolated in water or [bmim][PF6]. If total production of bis-sulfide is desired, this can be readily achieved by using a 2-fold excess of thiol reagent. Indeed, the photolytically initiated reaction of alkyne 2 in the presence of 2 equiv of thiol 1 was found to afford virtually quantitative amounts of bis-sulfide 4 irrespective of the solvent employed (DMSO, H2O/DMSO, or H2O) (Table 1, entries 7-9). Even in the presence of the same excess of thiol 1, the aromatic alkyne 6 behaved in a different fashion, since it could still afford significant amounts of monoadduct 7 both in DMSO and H2O/DMSO (Table 2, entries 6 and 7). In pure water, however, 6 succeeded in forming virtually exclusive amounts of the bis-sulfides 8 and 9, in line with the discovered effect of water solvent (Table 2, entry 8). As far as the cysteine thiol 10 is concerned (Tables 3 and 4), its reactions appear slower than those of the congener 1 as a possible result of a higher steric hindrance.12 This probably justifies the preferential formation of monoadduct 11,6d which is the major product both in DMSO, water/DMSO, and pure water (Table 3, entries 1-3). It is worth noting that highly selective production of the monoadduct 11 could be achieved by carrying out the reaction under diluted (0.02 M DMSO) conditions (35% yield of 11, with only 50% conversion; not reported) or using a 2-fold excess of the alkyne 2 (11/12 ratio ∼19:1; Table 3, entry 4). Conversely, the use of a 2-fold excess of cysteine 10 allowed highly selective occurrence of the bis-sulfide 12 (11/12 ratio >1:19 both in water/DMSO and water) in very good yield (>90%) (Table 3, entries 5 and 6). In parallel, the TYC of the same cysteine 10 with equimolar phenylacetylene 6 afforded the monoadduct 14 in a very selective fashion under all conditions employed (Table 4, entries 1-3). No evidence of formation of a bis-adduct was observed either with 2 equiv of thiol (entries 4 and 5).

75 Minozzi et al. JOCArticle Minozzi et al. with 1-octyne 2 (Table 1), formation of monoadduct 3 is TABLE 3. Thiol-Yne Couplings of N-Acetyl-L-cysteineJOCArticle Methyl Ester It is noteworthy that our favored at higher temperatures (AIBN-initiated reaction in 10 with 1-Octyne 2a with 1-octyne 2 (Table 1), formation of monoadduct 3 is TABLE 3. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl Ester toluene) or diluted mixtures (0.02 M in DMSO), whereas a explorative study with favored at higher temperatures (AIBN-initiated reaction in 10 with 1-Octyne 2 formation of bis-adduct 4 is strongly favored in an ionic liquid toluene) or diluted mixtures (0.02 M in DMSO), whereas octyne 2 and such as [bmim][PFformation6] of and, bis-adduct particularly,4 is strongly in water. favored With in an phenyl- ionic liquid acetylene 6 (Table 2), the monoadduct 7 is always the exclusive such as [bmim][PF6] and, particularly, in water. With phenyl- phenylacetylene 6 clearly product, withacetylene the6 exception(Table 2), the of monoadduct the small7 amountsis always the of exclusive the regioisomericproduct, bis-adducts with the exception8 and 9 ofisolated the small in amounts water or of the revealed a deeply different [bmim][PFregioisomeric6]. If total production bis-adducts of8 bis-sulfideand 9 isolated is desired, in water this or can be readily[bmim][PF achieved6]. If total by production using a 2-fold of bis-sulfide excess is of desired, thiol this behavior of these two reagent. Indeed,can be the readily photolytically achieved initiated by using reaction a 2-fold of excess alkyne of2 thiol in the presencereagent. of 2 Indeed, equiv ofthe thiol photolytically1 was found initiated to afford reaction virtually of alkyne 2 alkynes toward their radical in the presence of 2 equiv of thiol 1 was found to afford virtually quantitativequantitative amounts amountsof bis-sulfide of bis-sulfide4 irrespective4 irrespective of the of solvent the solvent employed (DMSO, H O/DMSO, or H O) (Table 1, entries coupling reactions with employed (DMSO,2 H2O/DMSO,2 or H2O) (Table 1, entries 7-9). Even7- in9). the Even presence in the presence of the same of the excess same excess of thiol of thiol1,the1,the aromatic alkynearomatic6 behaved alkyne 6 behaved in a different in a different fashion, fashion, since since it could it could thiols 1 , 10. I n d e e d , still affordstill significant afford significant amounts amounts of monoadduct of monoadduct7 both7 inboth DMSO in DMSO phenylacetylene 6 usually and H2O/DMSOand H2O/DMSO (Table 2, (Table entries 2, entries6 and 6 7). and In 7). pure In pure water, water, however, 6however,succeeded6 succeeded in forming in forming virtually virtually exclusive exclusive amounts amounts of of gave mono/bis-sulfide the bis-sulfidesthe bis-sulfides8 and 9,inlinewiththediscoveredeffectofwater8 and 9,inlinewiththediscoveredeffectofwater solvent (Table 2, entry 8). solvent (Table 2, entry 8). entry 10/2 solvent (c [M]) 11/12 [%]b,c yield 11 12 [%]d adducts in notably higher As far as the cysteine thiol 10 is concerned (Tables 3 and 4), entry 10/2 solvent (c [M]) 11/12 [%]b,c yield 11þ 12 [%]d As far asits the reactions cysteine appear thiol slower10 is concerned than those (Tables of the congener 3 and 4),1 as a 1 1:1 DMSO (0.5) 83/17 62 þ 19 1 1:1 DMSO (0.5)e 83/17 62 its reactionspossible appear result slower of a than higher those steric of hindrance. the congenerThis1 probablyas a 2 1:1 H2O-DMSO (0.5) 67/33 85 (overall) yields and, 19 e possible result of a higher steric hindrance. This probably9d 23 1:1 1:1 H H2O2O-DMSO (0.5) (0.5) 55/4567/33 82 85 justifies the preferential formation of monoadduct 11, which is 4 1:2 H O (0.5) 95/5 81 justifies the preferential formation of monoadduct 11,9d which is 3 1:1 H2O2 (0.5) 55/45 82 furthermore, showed a the major product both in DMSO, water/DMSO, and pure 5 2:1 H2O-DMSO (0.5) <5/>95 91 4 1:2 H2O (0.5)e 95/5 81 water (Table 3, entries 1-3). 6 2:1 H2O (0.5) <5/>95 92 the major product both in DMSO, water/DMSO, and pure 5 2:1 H2O-DMSO (0.5) <5/>95 91 It is worth noting that highly selective production of the a e distinct propensity to form water (Table 3, entries 1-3). 6Photoinduced 2:1 H2O reactions (0.5) were carried<5/>95 out at r.t. with a household 92 monoadduct 11 could be achieved by carrying out the UVA lamp apparatus at λmax 365 nm (see the Experimental Section for It is worth noting that highly selective production of the aPhotoinduced reactions were carried out at r.t. with a household reaction under diluted (0.02 M DMSO) conditions (35% equipment setup). Reactions performed with 10 normally gave the monosulfide rather than bis- monoadduct 11 could be achieved by carrying out the UVA lamp apparatus at λmax 365 nm (see the Experimental Section for yield of 11, with only 50% conversion; not reported) or using corresponding cystine 13 in <5% yield; only the reaction of entry 1 reaction under diluted (0.02 M DMSO) conditions (35% equipmentafforded 13 setup).in ca. Reactions20% yield. bValues performed are relative with percentages10 normally and gave were the a 2-fold excess of the alkyne 2 (11/12 ratio 19:1; Table 3, correspondingdetermined by cystine GC-MS13 andin <5%1H NMR yield; analysis. onlycAdduct the reaction11 was of a 1.5:1 entry 1 sulfide product. It is yield of 11, with only 50% conversion; not reported)∼ or using d entry 4). Conversely, the use of a 2-fold excess of cysteine 10 affordedmixture13 ofinZ ca.and 20%E isomers. yield. IsolatedbValues yield are relativecalculated percentages on the basis ofand the were a 2-fold excessallowed of highly the alkyne selective2 (11 occurrence/12 ratio of19:1; the bis-sulfide Table 3, 12 determinedstarting alkyne by GC (entries-MS and 1-31H and NMR 5-6) analysis. or the startingcAdduct thiol11 (entrywasa 4). 1.5:1 therefore plausible that the ∼ e d entry 4). Conversely,(11/12 ratio >1:19 the use both of a in 2-fold water/DMSO excess of and cysteine water) in10 very mixtureH2O/DMSO of Z and 95:5.E isomers. Isolated yield calculated on the basis of the allowed highlygood yield selective (>90%) occurrence (Table 3, entries of the 5 and bis-sulfide 6). In parallel,12 the starting alkyne (entries 1-3 and 5-6) or the starting thiol (entry 4). aromatic alkyne 6 could act e (11/12 ratioTYC >1:19 of the both same in cysteine water/DMSO10 with equimolar and water) phenylacetylene in very H2peptides.O/DMSO The 95:5. usefulness of this radical reaction in glycopep- good yield6 (>90%)afforded (Table the monoadduct 3, entries 514 andin a 6). very In parallel, selective thefashion as a much stronger trap for sulfur-centered radicals than the aliphatic congener tide chemistry has been recently validated by the synthesis of TYC of theunder same all cysteine conditions10 with employed equimolar (Table phenylacetylene 4, entries 1-3). No peptides.dually glycosylatedThe usefulness peptides of this by radical a two-step reaction strategy, in glycopep- which 6 affordedevidence the monoadduct of formation14 ofin a bis-adduct a very selective was observed fashion either 2tide. This point was actually substantiated by our additional finding that the usual involved chemistry first has the beenS-propargylation recently validated of a cysteine by the containing synthesis of with 2 equiv of thiol (entries 4 and 5). peptide, and then the photoinduced coupling with excess of under all conditions employed (Table 4, entries 1-3). No dually glycosylated10a peptides by a two-step strategy, which It is noteworthy that our explorative study with octyne 2 reaction glycosyl thiol.of cysteine Undoubtedly,10 with if peptide equimolar mono glycosyla- amounts of both alkynes 2 and 6 in evidence ofand formation phenylacetylene of a bis-adduct6 clearly revealed was observed a deeply either different involvedtion is first the target, the S-propargylation the direct TYC of asugar cysteine alkynes containing with with 2 equivbehavior of thiol of these(entries two 4 alkynes and 5). toward their radical coupling peptide,peptides and bearing then the a free photoinduced cysteine residue coupling appears with as a excess more of DMSO could 10a basically afford the phenylvinyl sulfide 14 at the expense of the It is noteworthyreactions with that thiols our explorative1, 10. Indeed, study phenylacetylene with octyne6 usually2 glycosylstraightforward thiol. Undoubtedly, strategy. Our investigation if peptide on mono this comple- glycosyla- and phenylacetylenegave mono/bis-sulfide6 clearly adducts revealed in anotably deeply higher different (overall) alkylvinyl one tionmentary is the approach target,11 thetook (11 direct advantage/14 ∼ 1:15, Scheme 3). TYC of the of information sugar alkynes gained with behavior ofyields these and, two furthermore, alkynes toward showedtheir a distinct radical propensity coupling to form peptidesfrom the bearing above explorative a free cysteine study and residue involved appears peptide portions as a more reactions withmonosulfide thiols 1, rather10. Indeed, than bis-sulfide phenylacetylene product.6 Itusually is therefore straightforwardof increasingAs anticipated, the ultimate goal of this study was to establish the potential complexity. strategy. Accordingly,Our investigation the coupling on this between comple- the peracetylated O-propargyl β-glycoside 15 with a single gave mono/bis-sulfideplausible that theadducts aromatic in notably alkyne 6 highercould act (overall) as a much mentary approach took advantage of the information gained role of photoinduced TYC as a click ligation tool for the direct monoglycosylation cysteine residue, that is, the N-Fmoc cysteine tert-butyl ester yields and,stronger furthermore, trap for showed sulfur-centered a distinct radicals propensity than theto form aliphatic from the above explorative study and involved peptide portions congener 2. This point was actually substantiated by our 16, was initially considered to establish optimal conditions monosulfide rather than bis-sulfide product. It is therefore of increasingfor the selective complexity. monohydrothiolation Accordingly, pathway the coupling of suchbetween more additional finding that the usual reaction of cysteine 10 with of unmodified peptides. The usefulness of this radical reaction in glycopeptide thecomplex peracetylated substratesO-propargyl (Table 5). β-glycoside 15 with a single plausible thatequimolar the aromatic amounts of alkyne both alkynes6 could2 and act6 asin DMSO a much could cysteineThe residue, photoinduced that is, TYC the (Nλ -Fmoc365 nm, cysteine DMPAtert 10-butyl mol %) ester stronger trapbasically for sulfur-centered afford the phenylvinyl radicals sulfide than14 theat the aliphatic expense of chemistry has been recently max validated by the synthesis of dually glycosylated congener 2. This point was actually substantiated by our 16,of was equimolar initially15 consideredand 16 proceeded to establish smoothly optimal under conditions homo- the alkylvinyl one 11 (11/14 1:15, Scheme 3). forgeneous the selective conditions monohydrothiolation with DMF (0.5 M) aspathway the solvent of such to give more additional findingAs anticipated, that the usual the ultimate reaction∼ goal of cysteineof this study10 with was to peptides by a two-step strategy, which involved first the S-propargylation of a complexexclusively substrates the monoadduct (Table 5).17 (25%) as a 1.5:1 mixture of equimolarestablish amounts the of potential both alkynes role of2 photoinducedand 6 in DMSO TYC could as a click TheE/Z photoinducedisomers (Table 5, TYC entry ( 1),λ albeit365 in nm, low DMPA yields. Complete 10 mol %) basically affordligation the tool phenylvinyl for the direct sulfide monoglycosylation14 at the expense of unmodified of cysteine containing peptide, and then the photoinduced coupling with excess of max of equimolarconversion of15 cysteineand 1616proceededwas conveniently smoothly achieved under by using homo- the alkylvinyl one 11 (11/14 1:15, Scheme 3). a 3-fold excess of13 sugar alkyne 15, thus, obtaining 17 in 88% (19) We cannot exclude∼ that polar factors may also play an additional role glycosyl geneous conditions thiol. with Undoubtedly, DMF (0.5 M) if as peptide the solvent mono to give glycosylation is the target, the As anticipated,in the overall the thiolation ultimate reaction, goal see: Escoubet, of this S.; Gastaldi,studywas S.; Vanthuyne, to isolated yield (entry 2). It is worth noting that, owing to the N.; Gil, G.; Siri, D.; Bertrand, M. P. J. Org. Chem. 2006, 71, 7288–7292. exclusivelyset of orthogonal the monoadduct protective groups,17 (25%) the glycosylas a 1.5:1 amino mixture acid of establish the potential role of photoinduced TYC as a click direct E/Z isomers TYC (Table of sugar 5, entry alkynes 1), albeit in with low yields. peptides Complete bearing a free cysteine residue ligation tool for the direct monoglycosylation of unmodified conversion of cysteineJ. Org.16 Chem.was conveniently Vol. XXX, No. achieved XX, XXXX by usingE a 3-fold excess of sugar alkyne 15, thus, obtaining 17 in 88% (19) We cannot exclude that polar factors may also play an additional role in the overall thiolation reaction, see: Escoubet, S.; Gastaldi, S.; Vanthuyne, isolated yield (entry 2). It is worth noting that, owing to the N.; Gil, G.; Siri, D.; Bertrand, M. P. J. Org. Chem. 2006, 71, 7288–7292. set of orthogonal protective groups, the glycosyl amino acid 76 J. Org. Chem. Vol. XXX, No. XX, XXXX E appears as a more straightforward strategy. Our investigation on this complementary approach took advantage of the information gained from the above explorative study and involved peptide portions of increasing complexity. Accordingly, the coupling between the peracetylated O-propargyl β-glycoside 15 with a single cysteine residue, that is, the N-Fmoc cysteine tert-butyl ester 16, was initially considered to establish optimal conditions for the selective monohydrothiolation pathway of such more complex substrates (Table 5). JOCArticle Minozzi et al. The photoinduced TYC TABLE 4. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl TABLE 5. Synthesis of Glycosyl Cysteine 17 and Bis-Armed Ester 10 with Phenylacetylene 6a ( max Cysteine 365 nm, 18a DMPA 10 mol %) of equimolar 15 JOCArticle Minozzi et al.

TABLE 4. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl TABLE 5. Synthesisand of Glycosyl16 Cysteine proceeded 17 and Bis-Armed Ester 10 with Phenylacetylene 6a Cysteine 18a smoothly under homogeneous conditions with DMF (0.5 M) as the solvent to give exclusively the monoadduct 17 (25%) as a 1.5:1 mixture of E/Z

b,c entry 10/6 10/6 solvent (c [M])solvent (c [M]) yield 14 [%]b,c yield 14 [%] isomers (Table 5, entry 1), 1 1:1 DMSO (0.5) 60 1 1:1 DMSOd (0.5) 60 2 1:1 H2O-DMSO (0.5) 78d albeit in low yields. Complete 32 1:1 1:1 H2O (0.5) H2O-DMSO (0.5) 88 78 43 2:1 1:1 DMSO (0.5) H2O (0.5) 86 88 d conversion of cysteine 16 was 54 2:1 2:1 H2O-DMSO DMSO (0.5) (0.5)91 86 aPhotoinduced reactions were carried out at r.t. with a householdd 5 2:1 H2O-DMSO (0.5) 91 UVA lamp apparatus at λmax 365 nm (see the Experimental Section for conveniently achieved by using a 3- equipmentaPhotoinduced setup). Reactions reactions performed were with carried10 normally out gave at r.t. the with a household corresponding cystine 13 in <5% yield; only the reaction of entry 1 UVA lamp apparatusb at λ 365 nm (see the Experimental Section for afforded 13 in ca. 20% yield. Adductmax14 was a 1:1 mixture of Z and E fold excess of sugar alkyne 15, thus, b equipmentisomers. cIsolated setup). yield calculated Reactions on the performed basis∼ of the starting with alkyne.10 normallyentry gave15/16 the solvent (c [M]) time (h) yield 17/18 [%] d c H2O/DMSO 95:5. 1 1:1 DMF (0.5) 1 25 /- corresponding cystine 13 in <5% yield; only the reactionobtaining of entry 117 in 88% isolated yield c b 2 3:1 DMF (0.5) 1 88 /- d afforded 13 in ca. 20% yield. Adduct 14 was a 1:1 mixture3 of Z and 1:1E H2O (0.5) 2 -/- b c ∼ 4 1:1 H O-PhMeentry (0.01)e 15/1626solventc/81f (c [M]) time (h) yield 17/18 [%] isomers.SCHEME 3.IsolatedCompetitive yield Reaction calculated of N-Acetyl- on theL-cysteine basis of the starting(entry alkyne. 2). It 2 is worth noting that, d 5 1:2 H O-PhMe (0.01)e 2 /85f MethylH O/DMSO Ester 10 with 95:5. 1-Octyne 2 and Phenylacetylene 6 2 - c 2 a 1 1:1 DMF (0.5) 1 25 /- (1:1:1 Ratio) Photoinduced reactions were2 carried out 3:1 at r.t. with DMF a household (0.5) 1 88c/- owing to the set of orthogonal protective groups, UVA lamp apparatus the glycosyl at λmax 365 nm (seeamino the Experimental acid 17 Section for b d equipment setup). Isolated yield3 calculated 1:1 on the basis H of2O the (0.5) starting 2 -/- alkyne (entries 1, 3-5) or the starting thiol (entry 2). c14Adduct 17 was a e c f appeared to be a suitable substrate for a co-translational 4d approach 1:1 H2O-PhMe to (0.01) 26/81 SCHEME 3. Competitive Reaction of N-Acetyl-L-cysteine1.5:1 mixture of E and Z isomers. Cysteine 16 stuck on reaction vessel e f e 5 1:2 H O-PhMe (0.01) 2 /85 Methyl Ester 10 with 1-Octyne 2 and Phenylacetylenewalls. 6 Toluene (10% v/v) was used to disperse reagents and2 catalyst in - glycopeptides through N-Fmoc-based peptide synthesis.water. fd.r. 1:1. a (1:1:1 Ratio) ∼ Photoinduced reactions were carried out at r.t. with a household UVA lamp apparatus at λmax 365 nm (see the Experimental Section for From a mechanistic point of view, possible formation of the bis-adduct conditions in the model couplingsequipment of 1-octyne setup). b (TableIsolated18 1, entry yield calculated on the basis of the starting c 5 and Table 3, entry 3), thealkyne photoinduced (entries coupling 1, 3- (5)λmax or365 the nm, starting thiol (entry 2). Adduct 17 was a seemed to be strongly inhibited by steric factors DMPA 10 in agreement with the results mol %) of sugar1.5:1 alkyne mixture15 ofandE cysteineand Z isomers.16 was dCysteine 16 stuck on reaction vessel initially performed in Hwalls.2O (0.5eToluene M). Unfortunately, (10% v/v) was these used to disperse reagents and catalyst in reported above and previous observations on photoinduced TYC of bulky thiols.conditions did not producewater. any results,fd.r. very1:1. likely because of the ‘sticky’ nature of 16, which resulted∼ in agglomeration and 6d Nevertheless, due to the relevance of ‘bis-armed’ hence precluded amino the intimate acids contact of type between18 the in reaction 17 appeared to be a suitable substrate for a co-translational partners (entry 3). On the other hand, when equimolar 15, 16, 20 conditions in the model couplings of 1-octyne (Table 1, entry peptide chemistry,approach to glycopeptides15 through the double hydrothiolation of sugar alkyne N-Fmoc-based peptide and the sensitizer DMPA (10 mol15 %) with cysteine were previously dissolved synthesis. with minimal toluene, the5 subsequent and Table addition 3, entry of H2 3),O (0.01 the M) photoinduced coupling (λmax 365 nm, From a mechanistic point of view, possible formation of resulted in the formationDMPA of organic 10 droplets mol which %) dispersed of sugar alkyne 15 and cysteine 16 was the bis-adduct 18 seemed to be strongly inhibited by steric under vigorous magnetic stirring. Irradiation of that mixture factors in agreement with the results reported above and for 2 h afforded, after concentrationinitially performed and column inchroma- H2O (0.5 M). Unfortunately, these previous observations on photoinduced TYC of bulky thiols.9d tography, the bis-glycosylatedconditions cysteine did derivative not produce18 as the any results, very likely because of Nevertheless, due to the relevance of ‘bis-armed’ amino acids main product (81%, d.r. 1:1) along with small amounts of 21 of type 18 in peptide chemistry, the double hydrothiolation77 the monoadduct 17 (6%;the∼ entry ‘sticky’ 4). The selective nature formation of 16, which of resulted in agglomeration and of sugar alkyne 15 with cysteine 16 was actively pursued. the bis-adduct 18 washence finally achieved precluded by simply the using intimate 2 contact between the reaction Thus, bearing in mind the beneficial effect of heterogeneous equiv of cysteine 16 underpartners the same (entry conditions 3). On (entry the 5). other hand, when equimolar 15, 16, 17 appeared to be a suitable substrate for a co-translationalAlthough a detailed analysis of this reaction outcome goes (20) McGarvey,20 G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett. and the sensitizer DMPA (10 mol %) were previously dissolved approach2002, 4, 3591–3594. to glycopeptides through N-Fmoc-basedbeyond peptide the object of this research, it can be speculated that synthesis.(21) For leading references on the synthesis of bis-amino acids, see: (a) Li, reactants concentrationwith into organic minimal droplets toluene, by means the subsequent of addition of H2O (0.01 M) C.; Tang, J.; Xie, J. Tetrahedron 2009, 65, 7935–7941. For an interesting application, see: (b) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem. hydrophobic interactions is responsible for the observed rate From a mechanistic point of view, possible formation of resulted in the formation18,22 of organic droplets which dispersed Res. 2008, 41, 1387–1398. acceleration of the double hydrothiolation reaction. the bis-adduct 18 seemed to be strongly inhibited by steric under vigorous magnetic stirring. Irradiation of that mixture FfactorsJ. Org. inChem. agreement Vol. XXX, No. with XX, XXXX the results reported above and for 2 h afforded, after concentration and column chroma- previous observations on photoinduced TYC of bulky thiols.9d tography, the bis-glycosylated cysteine derivative 18 as the Nevertheless, due to the relevance of ‘bis-armed’ amino acids main product (81%, d.r. 1:1) along with small amounts of 21 of type 18 in peptide chemistry, the double hydrothiolation the monoadduct 17 (6%;∼ entry 4). The selective formation of of sugar alkyne 15 with cysteine 16 was actively pursued. the bis-adduct 18 was finally achieved by simply using 2 Thus, bearing in mind the beneficial effect of heterogeneous equiv of cysteine 16 under the same conditions (entry 5). Although a detailed analysis of this reaction outcome goes (20) McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett. 2002, 4, 3591–3594. beyond the object of this research, it can be speculated that (21) For leading references on the synthesis of bis-amino acids, see: (a) Li, reactants concentration into organic droplets by means of C.; Tang, J.; Xie, J. Tetrahedron 2009, 65, 7935–7941. For an interesting application, see: (b) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem. hydrophobic interactions is responsible for the observed rate 18,22 Res. 2008, 41, 1387–1398. acceleration of the double hydrothiolation reaction.

F J. Org. Chem. Vol. XXX, No. XX, XXXX JOCArticle Minozzi et al.

TABLE 4. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl TABLE 5. Synthesis of Glycosyl Cysteine 17 and Bis-Armed Ester 10 with Phenylacetylene 6a Cysteine 18a

16 was actively entry pursued. 10/6 Thus, bearing solvent (c [M]) in mind the yield 14 beneficial [%]b,c effect of 1 1:1 DMSO (0.5) 60 heterogeneous conditions in the model couplings of 1-octyne (Table d 1, entry 5 2 1:1 H2O-DMSO (0.5) 78 3 1:1 H2O (0.5) 88 and Table 3,entry 3),the photoinduced coupling( max 365nm, DMPA 10 mol %) 4 2:1 DMSO (0.5) 86 d 5 2:1 H2O-DMSO (0.5) 91 of sugar alkyne 15aPhotoinduced and cysteine reactions16 were was carried initially out at r.t. performed with a household in H2O (0.5 M). UVA lamp apparatus at λmax 365 nm (see the Experimental Section for Unfortunately, these conditions did not produce any results, very likely because equipment setup). Reactions performed with 10 normally gave the corresponding cystine 13 in <5% yield; only the reaction of entry 1 b of the ‘sticky’ afforded nature 13 of in ca.16 20%, yield. which Adduct resulted 14 was a in 1:1 mixture agglomeration of Z and E and hence b isomers. cIsolated yield calculated on the basis∼ of the starting alkyne. entry 15/16 solvent (c [M]) time (h) yield 17/18 [%] d c precluded the intimate contact between the reaction partners (entry 3).H2O/DMSO 95:5. 1 1:1 DMF (0.5) 1 25 /- 2 3:1 DMF (0.5) 1 88c/- d 3 1:1 H2O (0.5) 2 -/- 4 1:1 H O-PhMe (0.01)e 26c/81f SCHEME 3. Competitive Reaction of N-Acetyl-L-cysteine 2 5 1:2 H O-PhMe (0.01)e 2 /85f Methyl Ester 10 with 1-Octyne 2 and Phenylacetylene 6 2 - a (1:1:1 Ratio) Photoinduced reactions were carried out at r.t. with a household UVA lamp apparatus at λmax 365 nm (see the Experimental Section for equipment setup). bIsolated yield calculated on the basis of the starting alkyne (entries 1, 3-5) or the starting thiol (entry 2). cAdduct 17 was a 1.5:1 mixture of E and Z isomers. dCysteine 16 stuck on reaction vessel walls. eToluene (10% v/v) was used to disperse reagents and catalyst in water. fd.r. 1:1. ∼

conditions in the model couplings of 1-octyne (Table 1, entry 5 and Table 3, entry 3), the photoinduced coupling (λmax 365 nm, DMPA 10 mol %) of sugar alkyne 15 and cysteine 16 was initially performed in H2O (0.5 M). Unfortunately, these conditions did not produce any results, very likely because of the ‘sticky’ nature of 16, which resulted in agglomeration and hence precluded the intimate contact between the reaction On the other hand, 17 appeared when equimolar to be a suitable substrate15, 16 for, and the sensitizer DMPA (10 a co-translational partners (entry 3). On the other hand, when equimolar 15, 16, approach20 to glycopeptides through N-Fmoc-based peptide and the sensitizer DMPA (10 mol %) were previously dissolved mol %) were previously dissolved with minimal toluene, the subsequent synthesis. with minimal toluene, the subsequent addition of H2O (0.01 M) From a mechanistic point of view, possible formation of resulted in the formation of organic droplets which dispersed addition of H2O (0.01 M) resulted in the formation of organic droplets which the bis-adduct 18 seemed to be strongly inhibited by steric under vigorous magnetic stirring. Irradiation of that mixture dispersed under vigorous magnetic stirring. factors in agreement with the results Irradiation of that mixture reported above and for for 2 h 2 h afforded, after concentration and column chroma- previous observations on photoinduced TYC of bulky thiols.9d tography, the bis-glycosylated cysteine derivative 18 as the afforded, after concentration and column chromatography, Nevertheless, due to the relevance of ‘bis-armed’ amino the bis-glycosylated acids main product (81%, d.r. 1:1) along with small amounts of 21 of type 18 in peptide chemistry, the double hydrothiolation the monoadduct 17 (6%;∼ entry 4). The selective formation of cysteine derivative of sugar18 as alkyne the main 15 with product cysteine 16 (81%, was actively d.r. ∼1:1) pursued. along with small the bis-adduct 18 was finally achieved by simply using 2 Thus, bearing in mind the beneficial effect of heterogeneous amounts of the monoadduct 17 (6%; entry 4). The selective formation of the bis-equiv of cysteine 16 under the same conditions (entry 5). Although a detailed analysis of this reaction outcome goes (20) McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett. adduct 18 was finally achieved by simply using 2 equiv of cysteine 2002, 4, 3591–3594. 16 under the beyond the object of this research, it can be speculated that (21) For leading references on the synthesis of bis-amino acids, see: (a) Li, reactants concentration into organic droplets by means of same conditions (entry 5). Although a detailed analysis of this reaction outcome C.; Tang, J.; Xie, J. Tetrahedron 2009, 65, 7935–7941. For an interesting application, see: (b) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem. hydrophobic interactions is responsible for the observed rate 18,22 goes beyond the Res. object 2008, 41, of 1387–1398. this research, it can be speculated that acceleration reactants of the double hydrothiolation reaction. F J. Org. Chem. Vol. XXX, No. XX, XXXX concentration into organic droplets by means of hydrophobic interactions is responsible for the observed rate acceleration of the double hydrothiolation reaction.16,11

78 Minozzi et al. JOCArticle

SCHEME 4. Synthesis of Orthogonally Protected Bis-Armed SCHEME 5. Optimized Conditions for the Complete Cysteine 20 Glycosylation of Glutathione 22 by Sugar Alkynes 21 and 24

The discovery of a reaction window for both mono- and The discovery double-hydrothiolation of a reaction of sugarwindow alkyne for 15 prompted both mono- us to and double- hydrothiolation investigate of sugar the alkyne sequential15 hydrothiolation prompted us of to 15 investigate the using the two sequential different cysteine derivatives 16 and 19 (Scheme 4). Indeed, hydrothiolation we of thought15 using the two different cysteine derivatives this proof-of-principle experiment might be of 16 and 19 interest to establish the potential of photoinduced TYC/ (Scheme 4). Indeed, TEC sequences we thought this in dual-labeling proof-of-principle experiment investigations.11 Thus, the vinyl might be of thioether intermediate 17 was first prepared by TYC of alkyne interest to establish the potential of photoinduced TYC/ TEC sequences in dual-15 with cysteine 16 under optimized conditions (Table 5, entry2), of a mixture of glutathione 22, DMPA (10 mol %), and sugar alkyne 24 (1.1 equiv) in H O-MeOH 3:1 (v/v) (0.1 M)23 labeling investigations.and then subjected17 Thus, to the the photoinduced vinyl thioether TEC (λmax intermediate 365 nm, 2 h, 17 was first 2 DMPA 10 mol %) with cysteine 19 (2 equiv) in diluted water/ resulted in quantitative formation of the glycoconjugate 25 as 1 prepared by TYC toluene of alkyne (10:1 v/v)15 (0.01 with cysteine M) to give the16 target under bis-adduct optimized 20 conditions judged by HNMRandLC-MS analyses of the crude reaction (d.r. 1:1) in 66% overall yield (Scheme 4). Noteworthy, mixture (see Supporting Information). These optimal coupling (Table 5, entry 2), and then subjected to the photoinduced TEC ( max 365 nm, 2 the orthogonal∼ protection of the bis-amino acid 20 allows conditions did not involve any significant excess of either for differential peptide chain elongation via Boc- and Fmoc- reagents as required by a true ‘click’ reaction, and then allowed h, DMPA 10 mol %) with cysteine based peptide synthesis.2119 (2 equiv) in diluted water/ toluene (10:1 for a simple, rapid purification process of 25.Thiscompound The investigation of ‘click’ equimolar glycosylation of was readily isolated by short-column chromatography with v/v) (0.01 M) to cysteine-containing give the target peptides bis-adduct via photoinduced20 (d.r. ∼1:1) in 66% overall yield TYC was Sephadex LH20 in 82% yield as a 6:1 mixture of E/Z isomers (Scheme 5). With the pure glycopeptide 25 in hand, the (Scheme 4). Noteworthy, the next step in the our orthogonal program. To this protection aim, the readily of the available bis-amino acid 20 glycosyl alkyne 21 and the natural tripeptide glutathione 22 (γ-L- stability of the vinyl thioether linkage in aqueous medium was 1 allows for differential Glu-L-Cys-Gly, peptide GSH) inchain its native elongation form were considered via Boc- suitable and Fmoc- next based investigated by recording HNMRspectraofaD2O substrates for testing the efficiency of this approach (Scheme 5). solution of 25 (0.03 M) over the time. Rewardingly, no degrada- peptide synthesis.After15 some experimentation, full conversion of GSH 22 could be tion occurred during a week, as it was also confirmed by a final achieved under irradiation (λmax 365 nm, DMPA 10 mol %, 1 h) LC-MS analysis of the same solution. 23 The investigation in a 19:1 H of ‘click’ 2O-MeOH equimolar mixture when glycosylation of using at least a 5-fold cysteine-containing Paralleling the previous study on peptide glycosylation via 24 excess of sugar alkyne 21,asitwasestablishedbyLC-MS photoinduced thiol-ene coupling, the efficacy of the opti- peptides via photoinduced TYC was analyses (see Supporting Information). the next step in our This rather disappoint- program. To this aim, mized thiol-yne coupling was evaluated in aqueous solu- the readily available glycosyl ing, even though successful, alkyne result21 and the natural tripeptide glutathione prompted us to synthesize an tions at physiological pH with the higher synthetic nonapeptide aromatic counterpart of the alkyne 21 (Scheme 5) in view of our TALNCNDSL 26,25 which displays a single cysteine residue as 22 (-L- Glu-L-Cys-Gly, previous discovery GSH) that in its an aromatic native alkyne form should were be considered more well(Scheme suitable 6).Hence, thecouplingof 24 with 26 was optimized effective than an aliphatic one in photoinduced TYC (Scheme 3). by adding a solution of DMPA (50 mol %) and 24 (5 equiv) in substrates for testing Accordingly, the the efficiency unknown ethynylbenzyl of this approach β-D-glucopyranoside (Scheme 5). After DMSO some (5% final volume content) to a solution of 26 in 20 mM 24 was obtained by quantitative hydroxyl groups deprotection phosphate buffer (pH 7.4) and maintaining irradiation (λmax experimentation, full conversion of GSH (NaOMe/MeOH) of the corresponding22 could be achieved under irradiation peracetylated deriva- 365 nm) for 1 h. Under these conditions, peptide 26 was tive, which in turn was prepared, under nonoptimized conditions18 quantitatively converted into the glycoconjugate 27,whichwas (max365nm, DMPA 10mol%, 1h) in a 19:1 H2O-MeOH mixture when using at (45% yield), by BF3 3 OEt2-promoted glycosylation of β-D- obtained in 68% isolated yield (Sephadex LH20) as a 2:1 least a 5-fold excess of sugar alkyne glucose pentaacetate with21 ethyn, as it was established by LC-MS analyses ylbenzyl alcohol (Scheme S1, mixture of E/Z isomers. The selective attachment of 24 to the Supporting Information). Gratifyingly, irradiation for 1 h sulfhydryl group of 26 was duly confirmed by LC-MS/MS

(22) Pirrung, M. C.; Das Sarma, K.; Wang, J. J. Org. Chem. 2008, 73, (24) Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.—Eur. J. 2009, 8723–8730. 15, 11444–11449. (23) Homogeneous conditions were required to achieve good conversions (25) This compound was kindly provided as trifluoroacetic salt by of GSH 22. 79 UFPeptides S.r.L. (University of Ferrara).

J. Org. Chem. Vol. XXX, No. XX, XXXX G (see article). This rather disappointing, even though successful, result prompted us to synthesize an aromatic counterpart of the alkyne 21 (Scheme 5) in view of our previous discovery that an aromatic alkyne should be more effective than an aliphatic one in photoinduced TYC (Scheme 3). Accordingly, the unknown ethynylbenzyl β-D-glucopyranoside 24 was obtained by quantitative hydroxyl groups deprotection (NaOMe/MeOH) of the corresponding peracetylated derivative, which in turn was prepared, under nonoptimized conditions (45%

yield), by BF3 OEt2-promoted glycosylation of β-D- glucose pentaacetate with ethynylbenzyl alcohol (Scheme S1). Gratifyingly, irradiation for 1 h of a mixture

of glutathione 22, DMPA (10 mol %), and sugar alkyne 24 (1.1 equiv) in H2O- MeOH 3:1 (v/v) (0.1 M)18 resulted in quantitative formation of the glycoconjugate 25 as judged by 1H NMR and LC-MS analyses of the crude reaction mixture (see article). Minozzi et al. JOCArticle

SCHEME 4. Synthesis of Orthogonally Protected Bis-Armed SCHEME 5. Optimized Conditions for the Complete Cysteine 20 Glycosylation of Glutathione 22 by Sugar Alkynes 21 and 24

The discovery of a reaction window for both mono- and double-hydrothiolation of sugar alkyne 15 prompted us to investigate the sequential hydrothiolation of 15 using the two different cysteine derivatives 16 and 19 (Scheme 4). Indeed, we thought this proof-of-principle experiment might be of interest to establish the potential of photoinduced TYC/ TEC sequences in dual-labeling investigations.11 Thus, the vinyl thioether intermediate 17 was first prepared by TYC of alkyne 15 with cysteine 16 under optimized conditions (Table 5,These optimal coupling conditions did not involve any significant excess of entry2), of a mixture of glutathione 22, DMPA (10 mol %), and sugar 23 and then subjected to the photoinduced TEC (λmax 365 nm, 2 h, alkyne 24 (1.1 equiv) in H2O-MeOH 3:1 (v/v) (0.1 M) DMPA 10 mol %) with cysteine 19 (2 equiv) ineither diluted reagents water/ resulted as required in quantitative by a formation true ‘click’ of the reaction, glycoconjugate and 25 then as allowed for a 1 toluene (10:1 v/v) (0.01 M) to give the target bis-adduct 20 judged by HNMRandLC-MS analyses of the crude reaction (d.r. 1:1) in 66% overall yield (Scheme 4).simple, rapid purification process of Noteworthy, mixture (see Supporting Information).25. This compound was readily isolated by These optimal coupling the orthogonal∼ protection of the bis-amino acid 20 allows conditions did not involve any significant excess of either for differential peptide chain elongation via Boc-short-column and Fmoc- chromatography reagents as required by with a true ‘clicSephadex k’ reaction, LH20 and then in allowed 82% yield as a 6:1 based peptide synthesis.21 for a simple, rapid purification process of 25.Thiscompound The investigation of ‘click’ equimolar glycosylation of was readily isolated by short-column chromatography with cysteine-containing peptides via photoinduced TYC was Sephadex LH20 in 82% yield as a 6:1 mixture of E/Z isomers the next step in our program. To this aim, the readily available (Scheme 5). With the pure glycopeptide 25 in hand, the glycosyl alkyne 21 and the natural tripeptide glutathione 22 (γ-L- stability of the vinyl thioether linkage in aqueous medium was 80 1 Glu-L-Cys-Gly, GSH) in its native form were considered suitable next investigated by recording HNMRspectraofaD2O substrates for testing the efficiency of this approach (Scheme 5). solution of 25 (0.03 M) over the time. Rewardingly, no degrada- After some experimentation, full conversion of GSH 22 could be tion occurred during a week, as it was also confirmed by a final achieved under irradiation (λmax 365 nm, DMPA 10 mol %, 1 h) LC-MS analysis of the same solution. 23 in a 19:1 H2O-MeOH mixture when using at least a 5-fold Paralleling the previous study on peptide glycosylation via 24 excess of sugar alkyne 21,asitwasestablishedbyLC-MS photoinduced thiol-ene coupling, the efficacy of the opti- analyses (see Supporting Information). This rather disappoint- mized thiol-yne coupling was evaluated in aqueous solu- ing, even though successful, result prompted us to synthesize an tions at physiological pH with the higher synthetic nonapeptide aromatic counterpart of the alkyne 21 (Scheme 5) in view of our TALNCNDSL 26,25 which displays a single cysteine residue as previous discovery that an aromatic alkyne should be more well(Scheme 6).Hence, thecouplingof 24 with 26 was optimized effective than an aliphatic one in photoinduced TYC (Scheme 3). by adding a solution of DMPA (50 mol %) and 24 (5 equiv) in Accordingly, the unknown ethynylbenzyl β-D-glucopyranoside DMSO (5% final volume content) to a solution of 26 in 20 mM 24 was obtained by quantitative hydroxyl groups deprotection phosphate buffer (pH 7.4) and maintaining irradiation (λmax (NaOMe/MeOH) of the corresponding peracetylated deriva- 365 nm) for 1 h. Under these conditions, peptide 26 was tive, which in turn was prepared, under nonoptimized conditions quantitatively converted into the glycoconjugate 27,whichwas (45% yield), by BF3 3 OEt2-promoted glycosylation of β-D- obtained in 68% isolated yield (Sephadex LH20) as a 2:1 glucose pentaacetate with ethynylbenzyl alcohol (Scheme S1, mixture of E/Z isomers. The selective attachment of 24 to the Supporting Information). Gratifyingly, irradiation for 1 h sulfhydryl group of 26 was duly confirmed by LC-MS/MS

(22) Pirrung, M. C.; Das Sarma, K.; Wang, J. J. Org. Chem. 2008, 73, (24) Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.—Eur. J. 2009, 8723–8730. 15, 11444–11449. (23) Homogeneous conditions were required to achieve good conversions (25) This compound was kindly provided as trifluoroacetic salt by of GSH 22. UFPeptides S.r.L. (University of Ferrara).

J. Org. Chem. Vol. XXX, No. XX, XXXX G mixture of E/Z isomers (Scheme 5). With the pure glycopeptide 25 in hand, the stability of the vinyl thioether linkage in aqueous medium was next investigated

1 by recording H NMR spectra of a D2O solution of 25 (0.03 M) over the time. Rewardingly, no degradation occurred during a week, as it was also confirmed by a final LC-MS analysis of the same solution. JOCArticle Minozzi et al.

SCHEME 6. Preparation of Glycopeptide 27 of a cysteine-containing nonapeptide under physiological con- ditions. Aromatic alkynes have hence become a new, attractive tag for bioconjugation studies based on the TYC ligation strategy.

Experimental Section Photoinduced reactions were carried out in a glass vial (diameter, 1 cm; wall thickness, 0.65 mm), sealed with a natural rubber septum, located 2.5 cm away from the UVA lamp (irradiation on sample: 365 nm, 1.04 W/m2). General Procedure for the Thiol/Yne Radical Couplings Re- ported in Tables 1-4. A mixture of alkyne (0.50 mmol), thiol (0.50 mmol unless otherwise stated), 2,2-dimethoxy-2-phenyla- cetophenone (13 mg, 0.05 mmol), and the stated solvent (1 mL unless otherwise stated) was irradiated at room temperature for 1-2 h under magnetic stirring. The crude mixtures of reactions performed in DMSO, H2O/DMSO, or DMF were diluted with H O (5 mL) and extracted with AcOEt (3 5 mL). The com- 2 Â bined organic phases were washed several times with H2O, dried (Na2SO4), and concentrated. The crude mixtures of the reac- tions carried out in [bmim][PF ] were extracted with AcOEt (3 6 Â 5 mL). The combined organic phases were dried (Na2SO4) and concentrated. For the reactions performed in toluene, the solvent was simply evaporated off. The resulting residues were eluted from a column of silica gel with the suitable elution system. Yields are reported in Tables 1-4. (E/Z)-Methyl 2-(Oct-1-enylthio)acetate (3). Column chroma- tography with 5:1 hexanes-AcOEt afforded 3 as a 1.2:1 mixture Paralleling analyses the previous of both ( study E)and( on Z peptide )isomersofglycopeptide glycosylation via photoinduced 27 of E and Z isomers. GC-MS: Z-3, r.t. 8.18, m/z 216 (Mþ, 25), 145 (59), 143 (39), 109 (59), 85 (47), 71 (100), 55 (44); E-3, r.t. (Supporting19 Information). Inevitably, the use of a 5-fold excess thiol-ene coupling, the efficacy of the optimized thiol-yne coupling 8.23, m was /z 216 (Mþ, 39), 145 (74), 143 (40), 109 (50), 85 (42), 71 of sugar alkyne 24 was required to drive the coupling to com- (100), 55 (37). 1H NMR (400 MHz): δ = 6.02-5.94 (m, 1 H, evaluated in aqueous pletion (LC solutions -MS analysis). at physiological This result, however, pH with appears the quite higher synthetic H-10), 5.77 (ddd, 0.55 H, J2 ,3 a = 7.0 Hz, J2 ,3 b = 7.1 Hz, J1 ,2 = satisfactory if compared to that obtained in the parallel TEC 0 0 0 0 0 0 20 14.9 Hz, H-20(E)), 5.65 (ddd, 0.45 H, J2 ,3 a = 7.3 Hz, J2 ,3 b = nonapeptide TALNCNDSL study.24 In that26 occasion,, which displays a single cysteine residue as well even a 30-fold excess of a peracetylated 0 0 0 0 7.4 Hz, J10,20 = 9.5 Hz, H-20(Z)), 3.74 (s, 3 H, OMe), 3.35 (s, 2 H, allyl C-glycoside was required to achieve complete conversion of 2H-2), 2.17-2.04 (m, 2 H, 2H-30), 1.42-1.20 and 0.92-0.80 (Scheme 6). Hence, the coupling of 24 with 26 was optimized by adding a 13 the same peptide 26.Itshouldbenoted,however,thatadetailed (2 m, 11 H, 2H-40, 2H-50, 2H-60, 2H-70, 3H-80). C NMR (100 solution of DMPA comparison (50 mol between %) the and two24 methodologies (5 equiv) isin complicated DMSO (5% at final MHz): volume δ = 170.3, 134.3, 131.9, 122.7, 120.7, 52.5, 52.4, 35.1, this stage by too many variables,andtherefore,itwillbethe 35.0, 33.0, 31.9, 31.7, 31.6, 29.7, 29.3, 29.0, 28.9, 28.7, 22.7, 22.6, content) to a solution of object of a dedicated26 in 20 mM phosphate buffer (pH 7.4) and maintaining study. 14.1, 14.0. ESI MS (216): 239 (M Naþ). HRMS (ESI/Q-TOF): þ calcd m/z for C H NaO S[M Na]þ, 239.1082; found, irradiation ( max 365 nm) for 1 h. Under these conditions, peptide 26 was 11 20 2 þ Conclusions 239.1087. quantitatively converted into the glycoconjugate 27, which was obtained in 68% (R/S)-Methyl 2-[(1-[(2-Methoxy-2-oxoethyl)sulfanyl]methyl- Our explorative study showed that the radical alkanethiol/ heptyl)sulfanyl]acetate (4). Column chromatography with 5:1 hex- isolated yield (Sephadex terminal alkyne LH20) as coupling a reactions 2:1 mixture of E/Z are strongly affected isomers. by The selective anes-AcOEt afforded 4 as a yellow oil. GC-MS: r.t. 11.52, m/z 322 the adopted experimental conditions, including thiol/alkyne (Mþ, <1%), 263 (7), 216 (40), 203 (47), 146 (19), 143 (73), 110 (26), 1 attachment of molar24 to the sulfhydryl group of ratio, temperature and, above26 all, was solvent. duly confirmed by LC-MS/ A proper 107 (29), 97 (35), 87 (27), 69 (46), 55 (100). H NMR (600 MHz): choice of the reaction conditions can hence favor highly selective δ = 3.71 (s, 3 H, OMe), 3.70 (s, 3 H, OMe), 3.32 and 3.21 (2 d, 2 H, J =14.5Hz,CH CO), 3.25 and 3.21 (2 d, 2 H, J =14.7Hz, occurrence of either mono- or bis-sulfide coupling product. This 2 CH2CO), 3.00-2.91 (m, 2 H, H-1a, H-2), 2.81-2.75 (m, 1 H, H-1b), study also showed that an aromatic alkyne, besides being much 1.80-1.70, 1.50-1.40, 1.38-1.10 (3 m, 10 H, 2H-3, 2H-4, 2H-5, 2H- more reactive than an aliphatic81 congener, has a notably en- 6, 2H-7), and 0.85 (t, 3 H, J = 7.0, 3H-8). 13C NMR (150 MHz): hanced propensity to form the monocoupling product exclusive δ = 171.2, 171.0, 51.5, 51.4, 44.8 (C-2), 37.1 (C-1), 32.7, 32.2, 31.5, of the double-coupling one. Our present observations, which are 30.6, 28.0, 25.5, 21.5, 13.0. ESI MS (322): 345 (M Naþ). HRMS þ unprecedented in the reported studies of TYC reactions in the (ESI/Q-TOF): calcd m/z for C14H26NaO4S2 [M Na]þ,345.1170; fields of bioconjugation and/or material derivatization, may found, 345.1173. þ possibly pave the way to new important applications of (R/S)-Methyl 2-(2-[(2-Methoxy-2-oxoethyl)sulfanyl]-1-phenyl- the TYC strategy in those fields. The findings were preliminarily ethylsulfanyl)acetate (8). Column chromatography with 5:1 exploited in successful glycosylation of cysteine derivatives as hexanes-AcOEt afforded 8 as a yellow oil. GC-MS: r.t. 12.05, m/z 314 (Mþ, <1), 208 (55), 195 (100), 177 (18), 149 (38), 135 (29), 121 well as in the production of a bis-armed cysteine through dual (97), 115 (20), 104 (27), 91 (25), 77 (18). 1H NMR (400 MHz): δ = labeling of an alkynyl sugar in a TYC/TEC sequence. Further, 7.36-7.24 (m, 5 H, Ph), 4.26 (t, 1 H, J1,2 = 7.8 Hz, H-1), 3.71 (s, 3 H, the appealing behavior of aromatic alkynes in TYC was reward- OMe), 3.67 (s, 3 H, OMe), 3.15 (d, 2 H, 2H-2), 3.14 and 3.08 (2 d, ingly applied to glycosylation of the native form of GSH just 2H,J =14.7Hz,CH2CO), 3.13 and 2.98 (2 d, 2 H, J =15.1Hz, 13 using virtually equimolar amounts of a sugar bearing an aryla- CH2CO). C NMR (100 MHz): δ = 170.4 (2C), 139.4, 129.1, cetylene moiety and, more importantly, to a similar glycosylation 128.5, 128.2, 128.0, 127.8, 52.2 (2C), 49.3, 37.7, 33.4, 32.6. ESI MS

H J. Org. Chem. Vol. XXX, No. XX, XXXX MS analyses of both (E) and (Z) isomers of glycopeptide 27. Inevitably, the use of a 5-fold excess of sugar alkyne 24 was required to drive the coupling to completion (LC-MS analysis). This result, however, appears quite satisfactory if compared to that obtained in the parallel TEC study.19 In that occasion, even a 30-fold excess of a peracetylated allyl C-glycoside was required to achieve complete conversion of the same peptide 26. It should be noted, however, that a detailed comparison between the two methodologies is complicated at this stage by too many variables, and therefore, it will be the object of a dedicated study.

3.3 Conclusions Our explorative study showed that the radical alkanethiol/ terminal alkyne coupling reactions are strongly affected by the adopted experimental conditions, including thiol/alkyne molar ratio, temperature and, above all, solvent. A proper choice of the reaction conditions can hence favor highly selective occurrence of either mono- or bis-sulfide coupling product. This study also showed that an aromatic alkyne, besides being much more reactive than an aliphatic congener, has a notably enhanced propensity to form the monocoupling product exclusive of the double-coupling one. Our present observations, which are unprecedented in the reported studies of TYC reactions in the fields of bioconjugation and/or material derivatization, may possibly pave the way to new important applications of the TYC strategy in those fields. The findings were preliminarily exploited in successful glycosylation of cysteine derivatives as well as in the production of a bis-armed cysteine through dual labeling of an alkynyl sugar in a TYC/TEC sequence. Further, the appealing behavior of aromatic alkynes in TYC was rewardingly applied to glycosylation of the native form of GSH just using virtually equimolar amounts of a sugar bearing an aryla- cetylene moiety and, more importantly, to a similar glycosylation of a cysteine-containing nonapeptide under physiological conditions. Aromatic alkynes have hence become a new, attractive tag for bioconjugation studies based on the TYC ligation strategy.

82 3.4 Experimental Section Photoinduced reactions were carried out in a glass vial (diameter, 1 cm; wall thickness, 0.65 mm), sealed with a natural rubber septum, located 2.5 cm away from the UVA lamp (irradiation on sample: 365 nm, 1.04 W/m2). General Procedure for the Thiol/Yne Radical Couplings Reported in Tables 1-4. A mixture of alkyne (0.50 mmol), thiol (0.50 mmol unless otherwise stated), 2,2-dimethoxy-2-phenylacetophenone (13 mg, 0.05 mmol), and the stated solvent (1 mL unless otherwise stated) was irradiated at room temperature for 1-2 h under magnetic stirring. The crude mixtures of reactions performed in

DMSO, H2O/DMSO, or DMF were diluted with H2O (5 mL) and extracted with AcOEt (3 x 5 mL). The combined organic phases were washed several times with

H2O, dried (Na2SO4), and concentrated. The crude mixtures of the reactions carried out in [bmim][PF6] were extracted with AcOEt (3 x 5 mL). The combined organic phases were dried (Na2SO4) and concentrated. For the reactions performed in toluene, the solvent was simply evaporated off. The resulting residues were eluted from a column of silica gel with the suitable elution system. Yields are reported in Tables 1-4.

(E/Z) Methyl 2-(oct-1-enylthio)acetate 3. as a 1.2:1 mixture of E and Z isomers. GC-MS: Z-5, r.t. 8.18, m/z 216 (M+, 25), 145 (59), 143 (39), 109 (59), 85 (47), 71 (100), 55 (44); E-5, r.t. 8.23, m/z 216 (M+, 39), 145 (74), 143 (40), 109 (50), 85 (42), 71 (100), 55 (37). 1H NMR

(400 MHz): δ = 6.02-5.94 (m, 1 H, H-1’), 5.77 (ddd, 0.55 H, J2’,3’a = 7.0 Hz, J2’,3’b =

7.1 Hz, J1’,2’ = 14.9 Hz, H-2’(E)), 3.65 (ddd, 0.45 H, J2’,3’a = 7.3 Hz, J2’,3’b = 7.4 Hz, J1’,2’ = 9.5 Hz, H-2’(Z)), 3.74 (s, 3 H, OMe), 3.35 (s, 2 H, 2H-2), 2.17-2.04 (m, 2 H, 2H-3’), 1.42-1.20 and 0.92-0.80 (2 m, 11 H, 2H-4’, 2H-5’, 2H-6’, 2H-7’, 3H-8’). 13C NMR (400 MHz): δ = 170.3, 134.3, 131.9, 122.7, 120.7, 52.5, 52.4, 35.1, 35.0, 33.0, 31.9, 31.7, 31.6, 29.7, 29.3, 29.0, 28.9, 28.7, 22.7, 22.6, 14.1, 14.0. ESI MS (216):

+ + + 239 (M Na). HRMS (ESI/Q-TOF): calcd m/z for C11H20NaO2S [M+Na] : 239.1082; found: 239.1087.

83 (R/S) Methyl 2-[(1-[(2-methoxy-2-oxoethyl)sulfanyl]methylheptyl)sulfanyl] acetate 4. GC-MS: r.t. 11.52, m/z 322 (M+, <1%), 263 (7), 216 (40), 203 (47), 146 (19), 143 (73), 110 (26), 107 (29), 97 (35), 87 (27), 69 (46), 55 (100). 1H NMR (600 MHz): δ = 3.71 (s, 3 H, OMe), 3.70 (s, 3 H, OMe), 3.32 and 3.21 (2 d, 2 H, J = 14.5

Hz, CH2CO), 3.25 and 3.21 (2 d, 2 H, J = 14.7 Hz, CH2CO), 3.00-2.91 (m, 2 H, H-1a, H-2), 2.81-2.75 (m, 1 H, H-1b), 1.80-1.70, 1.50-1.40, 1.38-1.10 (3 m, 10 H, 2H-3, 2H-4, 2H-5, 2H-6, 2H-7), and 0.85 (t, 3 H, J = 7.0, 3H-8). 13C NMR (600 MHz): δ = 171.2, 171.0, 51.5, 51.4, 44.8 (C-2), 37.1 (C-1), 32.7, 32.2, 31.5, 30.6, 28.0, 25.5, 21.5, 13.0. ESI MS (322): 345 (M + Na+). HRMS (ESI/Q-TOF): calcd m/z for

+ C14H26NaO4S2 [M+Na] : 345.1170; found: 345.1173.

(R/S) Methyl 2-(2-[(2-methoxy-2-oxoethyl)sulfanyl]-1phenylethylsulfanyl) acetate 8. GC-MS: r.t. 12.05, m/z 314 (M+, <1), 208 (55), 195 (100), 177 (18), 149 (38), 135 (29), 121 (97), 115 (20), 104 (27), 91 (25), 77 (18). 1H NMR (400 MHz): δ = 7.36-7.24 (m, 5 H, Ph), 4.26 (t, 1 H, J1,2 = 7.8 Hz, H-1), 3.71 (s, 3 H, OMe), 3.67 (s, 3 H, OMe), 3.15 (d, 2 H, 2H-2), 3.14 and 3.08 (2 d, 2 H, J = 14.7 Hz,

13 CH2CO), 3.13 and 2.98 (2 d, 2 H, J = 15.1 Hz, CH2CO). C NMR (400 MHz): δ = 170.4 (2C), 139.4, 129.1, 128.5, 128.2, 128.0, 127.8, 52.2 (2C), 49.3, 37.7, 33.4, 32.6. ESI MS (314): 337 (M + Na+). HRMS (ESI/Q-TOF): calcd m/z for

+ C14H18NaO4S2 [M+Na] : 337.0544; found: 337.0548.

Methyl 2-(1-[(2-methoxy-2-oxoethyl)sulfanyl]-2-phenylethylsulfanyl)acetate 9. GC-MS: r.t. 11.08, m/z 314 (M+, <1), 241 (27), 223 (50), 209 (26), 177 (48), 149 (100), 135 (50), 115 (38), 103 (14), 91 (51). 1H NMR (400 MHz): δ =

7.35-7.22 (m, 5 H, Ph), 4.38 (t, 1 H, J1,2 = 7.1 Hz, H-1), 3.70 (s, 6 H, OMe), 3.45 and

13 3.22 (2 d, 4 H, J = 15.2 Hz, 2 CH2CO), 3.13 (d, 2 H, 2H-2). C NMR (400 MHz): δ = 170.6 (2C), 137.5, 129.3 (2C), 128.4 (2C), 127.0, 53.8, 52.4 (2C), 42.2, 32.4 (2C).

+ ESI MS (314): 337 (M + Na ). HRMS (ESI/Q-TOF): calcd m/z for C14H18NaO4S2 [M +Na]+: 337.0544; found: 337.0549.

84 (2R,E/Z) Methyl 2-acetamido-3-(oct-1-enylthio)propanoate 11. as a 1.5:1 mixture of Z and E isomers. GC-MS: Z-11, r.t. 9.41, m/z 287 (M+, 1), 228 (22), 177 (8), 144 (16), 111 (13), 87 (26), 81 (11), 69 (38), 55 (32), 43 (100); E-11, r.t. 9.56, m/z 287 (M+, 2), 228 (18), 177 (5), 144 (14), 111 (13), 87 (14), 81 (15), 69 (33), 55 (34), 43 (100). 1H NMR: δ = 6.33 (bd, 1 H, J = 7.0 Hz,

NH), 5.90-5.70 (m, 1.4 H, H-1’(E), H-1’(Z), H-2’(E)), 5.58 (ddd, 0.6 H, J2’,3a’ = 7.2

Hz, J2’,3’b = 7.3 Hz, J1’,2’ = 9.3 Hz, H-2’(Z), 4.91-4.83 (m, 1 H, H-2), 3.75 (s, 3 H, OMe), 3.23-3.04 (m, 2 H, 2H-3), 2.17-2.00 (m, 2 H, 2H-3’), 1.42-1.20 and 0.98-0.84 (2 m, 11 H, 2H-4’, 2H-5’, 2-H6’, 2H-7’, 3-H8’). 13C NMR (selected data): δ = 170.8, 169.7, 134.2, 131.4, 123.8, 121.5, 52.6, 52.5, 52.1, 36.0, 35.3, 33.1, 31.6, 31.5, 29.0, 28.9, 28.8, 28.7, 23.1, 22.6, 22.5, 14.0. ESI MS (287): 310 (M + Na+). HRMS (ESI/Q-

+ TOF): calcd m/z for C14H25NNaO3S [M+Na] : 310.1453; found: 310.1457.

Dimethyl (4R,11R)-7-hexyl-2,13-dioxo-6,9-dithia-3,12-diazatetradecane-4,11- dicarboxylate 12. as a 1:1 mixture of C2 diastereoisomers. GC-MS: r.t. ~25 (very broad peak), m/z 320 (M+ − N-Ac-Cys, 2%), 288 (18), 287 (13), 246 (14), 232 (17), 228 (28), 176 (34), 144 (9), 43 (100). 1H NMR (400 MHz): δ = 6.64 (t, 1 H, J = 7.2 Hz, NH), 6.54 (t, 1 H, J = 7.9 Hz, NH), 4.89-4.79 (m, 2 H, H-2’, H-2”), 3.78 (s, 6H, OMe), 3.10-2.91 and 2.84-2.65 (2 m, 7 H, 2H-1, H-2, 2H-3’, 2H-3”), 2.07 and 2.06 (2 s, 6 H, C(O)Me), 1.78-1.60, 1.52-1.40, and 1.39-1.20 (3 m, 10 H, 2H-3, 2H-4, 2H-5, 2H-6, 2H-7), 0.88(t, 3 H, J = 6.7 Hz, 3H-8). 13C NMR (400 MHz, selected data): δ = 171.3, 171.2 (2C), 170.0, 169.9, 52.7, 52.6, 52.3, 52.1, 52.0, 46.0, 38.7 (2C), 35.1, 35.0, 34.0, 33.9, 32.9, 32.7, 31.6, 29.0, 26.7, 26.6, 23.0, 22.5, 14.0. ESI MS (464):

+ + 487 (M + Na ). HRMS (ESI/Q-TOF): calcd m/z for C20H36N2NaO6S2 [M+Na]: 487.1912; found: 487.1921.

(2R,E/Z) Methyl 2-acetamido-3-(styrylthio)propanoate 14. as a ~1:1 mixture of E and Z isomers (reported NMR spectra are for a 4:1 Z/ E mixture). GC-MS: Z-14, r.t. 10.27, m/z 279 (M+, 14), 220 (45), 161 (23), 144 (63), 134 (43), 116 (28), 115 (54), 98 (72), 91 (46), 84 (17), 77 (23), 43 (100); E-14, r.t. 10.52, m/z 279 (M+, 14), 220 (49), 161 (25), 144 (62), 134 (47), 116 (28), 115 (61), 98 (74), 91 (51), 84 (17), 77 (25), 43 (100). 1H NMR (400 MHz): δ

= 7.46-7.16 (m, 5 H, Ph), 6.64 and 6.56 (2 d, 1 H, J1’,2’ = 15.6 Hz, H-1’(E), H-2’(E),

85 6.42 and 6.12 (2 d, 1 H, J1’,2’ = 10.7 Hz, H-1’(Z), H-2’(Z), 6.38 (d, 1 H, J = 7.1 Hz, NH), 4.97-4.88 (m, 1 H, H-2), 3.77 (s, 1.5 H, OMe), 3.72 (s, 1.5 H, OMe), 3.38-3.21 (m, 2 H, 2H-3), 2.03 (s, 3 H, C(O)Me), 2.00 (s, 3 H, C(O)Me). 13C NMR (400 MHz, selected data): δ = 171.0 (E), 170.7 (Z), 170.5 (E), 169.8 (Z), 136.3, 129.4, 128.6, 128.5, 128.1, 127.3, 126.9, 126.5, 126.2, 125.5, 123.7, 60.3, 52.6, 52.4, 52.2, 37.7, 35.0, 22.9, 20.9, 14.0. ESI MS (279): 302 (M + Na+). HRMS (ESI/Q-TOF): calcd m/

+ z for C14H17NNaO3S [M+Na] : 302.0827; found: 302.0830.

(2R,E/Z) Ethyl 2-amino-3-(oct-1-enylthio)propanoate 14. as a 2:1 mixture of Z and E isomers. GC-MS: Z, r.t. 8.43, m/z 259 (M+, 5), 186 (85), 158 (18), 143 (21), 126 (10), 116 (22), 110 (36), 102 (75), 87 (71), 81 (29), 74 (62), 67 (33), 55 (45), 43 (100); E, r.t. 8.53, m/z 259 (M+, 2), 186 (58), 158 (13), 143 (15), 126 (9), 116 (19), 110 (29), 102 (61), 87 (45), 81 (37), 74 (55), 67 (32), 55 (51), 43 (100). 1H NMR (400 MHz): δ = 5.92-5.86 (m, 1 H, H-1’(E),

H-1’(Z), 5.76 (ddd, 0.33 H, J2’,3a’ = 7.0 Hz, J2’,3b’= 7.2 Hz, J1’,2’ = 15.0 Hz, H-2’(E)),

5.58 (ddd, 0.66 H, J2’,3a’ = 6.9 Hz, J2’,3b’= 7.0 Hz, J1’,2’ = 9.2 Hz, H-2’(Z)), 4.19 (q, 2 H, J

= 7.0 Hz, OCH2CH3), 3.68-3.64 (m, 1 H, H-2), 3.10-3.00 (m, 1 H, H-3a(E), H-3a(Z)),

2.93 (dd, 0.66 H, J2,3b = 7.1 Hz, J3a,3b = 13.8 Hz, H-3b(Z)), 2.85 (dd, 0.33 H, J2,3b =

7.5 Hz, J3a,3b = 13.9 Hz, H-3b(E)), 2.18-2.00 (m, 2 H, 2H-3’), 1.40-1.24 (m, 11 H, 2-

13 H4’, 2H-5’, 2H-6’, 2H-7’, OCH2CH3), 0.92-0.84 (m, 3 H, 3H-8’). C NMR (400 MHz, selected data): δ = 173.6, 133.7, 131.2, 123.9, 121.6, 61.2, 54.7, 54.2, 38.9, 38.2, 33.0, 31.6 (2C), 29.0, 28.8, 28.8, 28.7, 22.5, 22.4, 14.1, 13.9. ESI MS (259): 282 (M

+ + + Na ). HRMS (ESI/Q-TOF): calcd m/z for C13H25NNaO2S [M+Na] : 282.1504; found: 282.1497.

86 (2R,E/Z)-tert-Butyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(3’-(2”,3”, 4”,6”-tetra-O-acetyl-β-D-glucopyranosyl)oxyprop-1-enylthio)propanoate 17. A mixture of propargyl glycoside 15 (300 mg, 0.78 mmol), cysteine derivative 16 (103 mg, 0.26 mmol), 2,2-dimethoxy-2-phenyl-acetophenone (7 mg, 0.026 mmol), and DMF (1.5 mL) was irradiated at room temperature for 1 h under magnetic stirring, and then concentrated. The resulting residue was eluted from a column of silica gel with (i-Pr)2O and then with 9:1 (i-Pr)2O / AcOEt to give 17 (179 mg, 88%) as 1.5:1 mixture of E and Z isomers. 1H NMR

(DMSO-d6, 120 °C, mixture of E and Z isomers): δ = 7.86 (d, 2 H, J = 7.4 Hz, Ar), 7.68 (d, 2 H, J = 7.4 Hz, Ar), 7.46-7.39 and 7.38-7.30 (2m, 4 H, Ar), 7.14 (bs, 1 H,

NH), 6.36 (ddd, 0.6 H, J1’,3a’ = 0.5 Hz, J1’,3b’ = 0.6 Hz, J1’,2’ = 15.0 Hz, H-1’(E)), 6.32

(ddd, 0.4 H, J1’,3a’ = 0.5 Hz, J1’,3b’ = 0.6 Hz, J1’,2’ = 9.5 Hz, H-1’(Z)), 5.75-5.60 (m, 1 H,

H-2’), 5.28-5.15 (m, 1 H, H-3”), 4.94 (dd, 0.4 H, J3”,4” = 9.0 Hz, J4”,5” = 9.5 Hz,

H-4”(Z)), 4.92 (dd, 0.6 H, J3”,4” = 9.0 Hz, J4”,5” = 9.5 Hz, H-4”(Z)), 4.85-4.70 (m, 1 H,

H-1”, H-2”), 4.40-4.32 (m, 2 H, FmocCH2), 4.30-4.06 and 4.00-3.88 (2 m, 7 H, FmocCH, H-2, 2 H-3’, H-5”, 2 H-6”), 3.20-2.80 (m, 2 H, 2 H-3), 2.05-1.92 (8 s, 12 H,

13 CH3), 1.45 (s, 9 H, t-Bu). C NMR (mixture of E and Z isomers and conformers, selected data): δ = 170.3, 169.3, 115.6, 143.8, 141.3,128.5, 127.7, 125.1, 123.7, 120.0, 99.2, 83.2, 72.8, 71.7, 713, 69.3, 68.4, 67.2, 65.5, 61.9, 54.8, 54.1, 47.1, 37.0, 35.2, 27.9, 20.7. ESI MS (785.27): 803.6 (M + NH4+). HRMS (ESI/Q-TOF):

+ calcd m/z for C39H51N2O14S [M+NH4] : 803.3050; found: 803.3061.

(2R,2’R/S,2”R)-tert-Butyl 2-((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(1’- (2”-((9H-fluoren-9-yl)methoxy)carbonylamino)-3”-tert-butoxy-3”- oxopropylthio)-3’-(2”’,3”’,4”’,6”’-tetra-O-acetyl-β-D-glucopyranosyl) oxypropan-2’-ylthio)propanoate 18 To a mixture of cysteine derivative 16 (155 mg, 0.39 mmol), propargyl glycoside 15 (75 mg, 0.20 mmol), and 2,2-dimethoxy-2-phenyl-acetophenone

(10 mg, 0.039 mmol) in toluene (2 mL) was added H2O (19 mL). The resulting dispersion was irradiated at room temperature for 2 h under vigorous magnetic stirring, then concentrated, and eluted from a column of silica gel with (i-Pr)2O and then with 9:1 (i-Pr)2O /AcOEt to give 18 (206 mg, 85%) as a ~1:1 mixture of

1 C2’-diastereoisomers. H NMR (DMSO-d6, 120 °C, mixture of diastereoisomers): δ = 7.86 (d, 4 H, J = 7.4 Hz, Ar), 7.68 (d, 4 H, J = 7.4 Hz, Ar), 7.45-7.36 and

87 7.35-7.28 (2 m, 8 H, Ar), 7.06 (bs, 2 H, NH), 5.22 (dd, 0.5 H, J3”’,4”’ = 9.0 Hz, J2”’,3”’ =

9.5 Hz, H-3”’(diast. I)), 5.20 (dd, 0.5 H, J3”’,4”’ = 9.0 Hz, J2”’,3”’ = 9.5 Hz, H-3”’(diast.

II)), 4.93 (dd, 0.5 H, J4”’,5”’ = 9.0 Hz, H-4”’(diast. I)), 4.92 (dd, 0.5 H, J4”’,5”’ = 9.0 Hz, H-4”’(diast. II)), 4.86-4.74 (m, 2 H, H-1”’, H-2”’), 4.44-3.89 (m, 12 H, 2 FmocCH, 2

FmocCH2, H-2, H-2”, H-3’a, H-5”’, 2 H-6”’), 3.82 (dd, 0.5 H, J2’,3’b = 5.0 Hz, J3’a,3’b =

10.5 Hz, H-3’b (diast. I), 3.70 (dd, 0.5 H, J2’,3’b = 5.5 Hz, J3’a,3’b = 10.5 Hz, H-3’b

(diast. II), 3.20-2.70 (m, 5 H, 2 H-3, 2 H-1”, H-2’), 2.02-1.92 (8 s, 12 H, CH3), 1.46-1.42 (4 s, 18 H, t-Bu). 13C NMR (mixture of diastereoisomers and conformers, selected data): δ = 170.6, 169.4, 155.8, 143.8, 141.3, 127.7, 127.0, 125.1, 120.0, 101.2, 100.8, 83.0, 82.9, 72.7, 72.6, 71.8, 71.0, 68.3, 67.2, 61.8, 54.6,

+ 53.4, 47.0, 34.4, 28.0, 20.7, 20.6. ESI MS (1184.42): 1203.1 (M + NH4 ). HRMS

+ (ESI/Q-TOF): calcd m/z for C61H76N3O18S2 [M+NH4] : 1202.4560; found: 1202.4551.

(2R,2’R/S,2”R)-Methyl 2-((tert-butoxycarbonylamino)-3-(1’-(2”-((9H- fluoren-9-yl)methoxy)carbonylamino)-3”-tert-butoxy-3”-oxopropylthio)-3’-(2”’, 3”’,4”’,6”’-tetra-O-acetyl-β-D-glucopyranosyl)oxypropan-2’-ylthio)propanoate 20. To a mixture of alkene 17 (150 mg, 0.19 mmol), cysteine derivative 19 (90 mg, 0.38 mmol), 2,2-dimethoxy-2-phenyl-acetophenone (10 mg, 0.038 mmol) in toluene (2.0 mL) was added H2O (19 mL). The resulting dispersion was irradiated at room temperature for 2 h under magnetic stirring, and then concentrated. The resulting residue was eluted from a column of silica gel with

8:1 (i-Pr)2O /AcOEt to give 20 (146 mg, 75%) as a ~1:1 mixture of C2’-

1 diastereoisomers. H NMR (DMSO-d6, 120 °C, mixture of diastereoisomers): δ = 7.86 (d, 2 H, J = 7.4 Hz, Ar), 7.70 (d, 2 H, J = 7.4 Hz, Ar), 7.45-7.38 and 7.37-7.30

(2 m, 4 H, Ar), 7.14 (bs, 1 H, NH), 6.60 (bs, 1 H, NH), 5.35 (dd, 0.5 H, J3”’,4”’ = 9.0

Hz, J2”’,3”’ = 9.5 Hz, H-3”’(diast. I)), 5.90 (d, 0.5 H, J1”’,2”’ = 7.5 Hz, H-1”’ (diast. I),

5.24 (dd, 0.5 H, J3”’,4”’ = 9.0 Hz, J2”’,3”’ = 9.5 Hz, H-3”’(diast. II)), 5.00-4.75 (m, 2.5 H, H-2”’, H-4”’, H-1”’ (diast. II), 4.40-4.32, 4.28-4.06, 4.00-3.90, and 3.80-3.64 (4 m,

13 H, FmocCH, FmocCH2, H-2, H-2”, 2 H-3’, H-5”’, 2 H-6”’, OCH3), 3.10-2.80 (m, 5

H, H-3, H-1”), 2.05-1.94 (8 s, 12 H, CH3), 1.46 (s, 9 H, t-Bu), 1.43 and 1.42 (2 s, 9 H, t-Bu). 13C NMR (mixture of diastereoisomers and conformers, selected data): δ = 170.7, 169.4, 155.8, 155.2, 143.8, 141.3, 127.7, 127.1, 125.2, 120.0, 101.2,

88 100.8, 91.7, 83.0, 72.8, 72.6, 71.9, 71.0, 68.3, 68.2, 67.2, 61.8, 61.4, 60.4, 54.7, 53.4, 52.6, 47.0, 46.8, 45.8, 40.3, 35.9. 29.7, 28.3, 28.0, 21.0, 20.9. ESI MS

+ (1020.36): 1038.9 (M + NH4 ). HRMS (ESI/Q-TOF): calcd m/z for C48H68N3O18S2

+ [M+NH4] : 1038.3934; found: 1038.3922.

Glycopeptide 23. A mixture of sugar alkyne 21 (272 mg, 1.25 mmol), reduced glutathione 22 (77 mg, 0.25 mmol), 2,2- dimethoxy-2-phenyl-acetophenone (6 mg, 0.025 mmol), MeOH (0.1 mL), and H2O (1.9 mL) was irradiated at room temperature for 1 h under magnetic stirring, and then concentrated. The resulting white solid residue was suspended in MeOH (2 mL) and triturated with portions of MeOH (32 mL) which were pipetted off. The residue left after trituration was eluted from a column of Sephadex LH20 with 3:1 H2O-MeOH to give 23 (102 mg, 78%) as a 8:1 mixture of (E)- and (Z)-isomers (reported NMR spectra are for a 2:1 E/Z mixture). The occurrence of the E-isomer as the major compound was confirmed by analysis of the vinyl protons region of the 1H NMR spectrum of the crude

1 reaction mixture. H NMR (300 MHz, D2O, (E/Z)-isomers): δ = 6.25 (d, 0.66 H, J = 15.0 Hz, CH=CH (E)), 6.19 (d, 0.33 H, J = 10.5 Hz, CH=CH (Z)), 5.70-5.60 (m, 1 H,

CH=CH), 4.48-4.40 (m, 1 H, H-5), 4.30 (d, 0.66 H, J1’’’,2’’’= 8.0 Hz, H-1’’’(E)), 4.28 (d,

0.33 H, J1’’’,2’’’= 8.5 Hz, H-1’’’(Z)), 4.22-4.00 (m, 2 H, 2 H-3’’), 3.80-3.50 (m, 6 H, 2 H-2, H-10, H-5’’’ 2 H-6’’’), 3.34-3.04 (m, 4 H, H-1’a, H-2’’’, H-3’’’, H-4’’’), 2.94-2.82 (m, 1 H, H-10 b), 2.40-2.30 (m, 2 H, 2 H-8), 2.02-1.94 (m, 2 H, 2 H-9).13C NMR (75 MHz, D2O, (E/Z)-isomers; selected data): δ = 174.7, 174.6, 173.8, 173.6, 172.1, 172.0, 129.7, 129.0, 128.3, 124.8, 124.4, 101.1, 100.7, 75.9, 75.8, 75.7, 72.9, 69.5, 69.4, 65.6, 60.6, 53.7, 52.9, 41.7 31.1, 25.9. ESI MS (525): 526 (M + H+). HRMS (ESI/Q-TOF): calcd m/z for C19H32N3O12S [M + H]+, 526.1707; found, 526.1701. The LC- MS analysis of the reaction mixture was performed to establish the full conversion of glutathione and confirm the E/Z ratio of 23 (see Figure S1)

Glycopeptide 25 A mixture of sugar alkyne 24 (79 mg, 0.27 mmol), reduced glutathione 22 (75 mg, 0.24 mmol), 2,2- dimethoxy-2-phenyl-acetophenone (6 mg, 0.024

89 mmol), and MeOH (0.5 mL), and H2O (1.5 mL) was irradiated at room temperature for 1 h under magnetic stirring, and then concentrated. The resulting residue was eluted from a column of Sephadex LH20 with 1:1 H2O- MeOH to give 25 (120 mg, 82%) as a 6:1 mixture of (E)- and (Z)-isomers. The occurrence of the E-isomer as the major compound was confirmed by analysis of the vinyl protons region of the 1H NMR spectrum of the crude reaction mixture.

1 H NMR (400 MHz, D2O, (E)-isomer): δ = 7.40-7.20 (m, 4, H, Ar), 6.72 and 6.54 (2 d, 2 H, J = 15.6 Hz, CH=CH), 4.74 and 4.57 (2 d, 2 H, J = 11.7 Hz, 2 H-2’), 4.56-4.50

(m, 1 H, H-5), 4.36 (d, 1 H, J1”’,2”’ = 8.0 Hz, H-1”’), 3.77 (dd, 1 H, J5”’,6”’a = 2.0 Hz, J6”’a,

6”’b = 12.5 Hz, H-6”’a), 3.74 (s, 2 H, 2H-2), 3.58 (dd, 1 H, J5”’,6”’b = 5.5 Hz, H-6”’b), 3.57 (t, 1 H, J = 7.0 Hz, H-10), 3.40-3.10 (m, 5 H, H-2”’, H-3”’, H-4”’, H-5”’, H-1’a),

3.03 (dd, 1 H, J1’b,5 = 8.0 Hz, J1’a,1’b = 14.5 Hz, H-1’b), 2.36-2.28 (m, 2 H, 2 H-8),

2.05-1.90 (m, 2 H, 2 H-9). 1H NMR (400 MHz, D2O, (E)-isomer; selected data): δ =

6.49 and 6.22 (2 d, 2 H, J = 10.8 Hz, CH=CH), 4.37 (d, 1 H, J1”’,2”’ = 8.0 Hz, H-1”’).

13 C NMR (D2O, (E/Z)-isomers; selected data): δ = 174.9, 173.8, 172.4, 116.7, 135.9, 129.5, 129.0, 127.3, 126.0, 124.3, 101.3, 76.1, 76.0, 73.3, 71.3, 69.9, 61.0,

+ 54.0, 53.7, 41.8, 33.8, 31.4, 26.1. ESI MS (601.19): 619.9 (M + NH4 ). HRMS (ESI/

+ Q-TOF): calcd m/z for C25H39N4O12S [M+NH4] : 619.2280; found: 619.2275. The LC-MS analysis of the reaction mixture was performed to establish the full conversion of glutathione and evaluate the E/Z ratio of 25 (see Figure S2).The LC MS/MS analysis of 25 was also performed to confirm the selective glycosylation of the cysteine residue of glutathione.

Glycopeptide 25 A solution of sugar alkyne 24 (13.8 mg, 0.047 mmol) and DMPA (1.2 mg, 4.71 mol) in DMSO (0.25 mL) was added to a solution of peptide 26 (10.0 mg, 9.41 mol) in 20 mM phosphate buffer (pH 7.4, 5.0 mL). The resulting solution was irradiated at room temperature for 1 h under magnetic stirring, and then lyophilized. The resulting residue was eluted from a column of Sephadex LH20 with H2O to give 27 (8.0 mg, 68%) as a 2:1 mixture of (E)- and (Z)-isomers but slightly contaminated by excess sugar alkyne (reported 1H NMR spectrum is for a ∼1:2 E/Z mixture). The occurrence of the E-isomer as the major compound was confirmed by analysis of the vinyl protons region of the 1H NMR spectrum of

90 1 the crude reaction mixture. H NMR (400 MHz, D2O, (E/Z)- isomers, selected data): δ = 6.64 and 6.58 (2 d, 066 H, J = 13.5 Hz, CH=CH(E)), 6.44 and 6.165 (2 d, 1.32 H, J = 9.5 Hz, CH=CH- (Z)). The LC-MS analysis of the reaction mixture was performed to establish the full conversion of peptide 26 and confirm the E/Z ratio of 27 (see Figure S4). The LC MS/MS analyses of peptide 26 and glycopeptide 27 were performed to confirm the selective glycosylation of the cysteine residue of 26

91 3.5 Bibliography 1 M. Minozzi, A. Monesi, D. Nanni, P. Spagnolo, N. Marchetti and A. Massi, J. Org. Chem., 2011, 76, 450-459.

2 van Dijk, M.; Rijkers, D. T. S.; Liskamp, R. M. J.; van Nostrum, C. F.; Hennink, W. E. Bioconjugate Chem. 2009, 20, 2001–2016. 3 a) Ogawa, A.; Obayashi, R.; Ine, H.; Tsuboi, Y.; Sonoda, N.; Toshikazu, H. J. Org. Chem. 1998, 63, 881– 884. For another recent example of radical monothiolation of arylacetylenes, see: b) Beauchemin, A.; Gareau, Y. Phosphorus, Sulfur, Silicon Relat. Elem. 1998, 139, 187–192. It is worth emphasizing that a higher reactivity (kinetic constant of more than one order of magnitude higher) of phenylacetylene with respect to an alkyl congener (1-propyne) has been also reported for addition of alkyl (methyl) radicals, see: c) Fischer, H.; Radom, L. Angew. Chem., Int. Ed. 2001, 40, 1340–1371. 4 a) Benati, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1991, 2103–2109. and references therein. b) Benati, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1992, 1659–1664. c) Benati, L.; Capella, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1995, 1035–1038. d) Benati, L.; Capella, L.; Montevecchi, P. C.; Spagnolo, P. J. Org. Chem. 1995, 60, 7941–7946. e) Montevecchi, P. C.; Navacchia, M. L. J. Org. Chem. 1997, 62, 5600–5607. f) Montevecchi, P. C.; Navacchia, M. L. J. Org. Chem. 1998, 63, 537–542. g) Montevecchi, P. C.; Navacchia, M. L.; Spagnolo, P. Tetrahedron 1998, 54, 8207–8216. h) Montevecchi, P. C.; Navacchia, M. L.; Spagnolo, P. Eur. J. Org. Chem. 1998, 1219–1226. i) Leardini, R.; Nanni, D.; Zanardi, G. J. Org. Chem. 2000, 65, 2763–2772. We recently exploited addition of sulfanyl radicals to alkynes as a novel tin-/metal-free method to generate assorted kinds of radicals, see: j) Benati, L.; Calestani, G.; Leardini, R.; Minozzi, M.; Nanni, D.; Spagnolo, P.; Strazzari, S. Org. Lett. 2003, 5, 1313–1316. k) Benati, L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. Synlett 2004, 987–990. l) Benati, L.; Bencivenni, G.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. J. Org. Chem. 2006, 71, 3192– 3197. m) Bencivenni, G.; Lanza, T.; Leardini, R.; Minozzi, M.; Nanni, D.; Spagnolo, P.; Zanardi, G. Org. Lett. 2008, 10, 1127–1130. 5 Under these conditions, the reaction vessel actually warmed up to 35-40 C. Anyway, strictly identical results were obtained with reaction mixtures kept at 25 °C by simultaneous air-cooling with compressed air. 6 a) Fairbanks, B. D.; Scott, T. F.; Kloxin, C. J.; Anseth, K. S.; Bowman, C. N. Macromolecules 2009, 42, 211–217. b) Chen, G.; Kumar, J.; Gregory, A.; Stenzel, M. H. Chem. Commun. 2009, 6291–6293. c) Chan, J. W.; Hoyle, C. E.; Lowe, A. B. J. Am. Chem. Soc. 2009, 131, 5751–5753. d) Hensarling, R. M.; Doughty, V. A.; Chan, J. W.; Patton, D. L. J. Am. Chem. Soc. 2009, 131, 14673–14675. e) Konkolewicz, D.; Gray-Weale, A.; Perrier, S. J. Am. Chem. Soc. 2009, 131, 18075–18077. f) Lowe, A. B.; Hoyleb, C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20, 4745–4750. g) Fairbanks, B. D.; Sims, E. A.; Anseth, K. S.; Bowman, C. N. Macromolecules 2010, 43, 4113–4119. h) Chan, J. W.; Shin, J.; Hoyle, C. E.; Bowman, C. N.; Lowe, A. B. Macro- molecules 2010, 43, 4937–4942. i) Hoogenboom, R. Angew. Chem., Int. Ed. 2010, 49, 3415–3417. 7 All the vinyl sulfides were formed as mixtures of E- and Z-isomers: see the Experimental Section for details. Studies on the stereoselective formation of the kinetic (Z) or thermodynamic (E) product are currently underway. 8 Reactions carried out in water were heterogeneous mixtures, whereas those carried out in water/DMSO 95:5 were normally homogeneous.

9 Both in water and in the ionic liquid the 8/9 ratio is ca. 2:1. 10 a) Chapter before, b) Lanza, T.; Minozzi, M.; Monesi, A.; Nanni, D.; Spagnolo, P.; Chiappe, C. Curr. Org. Chem. 2009, 13, 1726–1732.

92 11For a recent discussion about organic synthesis “on water”, see: Chanda, A.; Fokin, V. V. Chem. Rev. 2009, 109, 725–748. 12 We cannot exclude that polar factors may also play an additional role in the overall thiolation reaction, see: Escoubet, S.; Gastaldi, S.; Vanthuyne, N.; Gil, G.; Siri, D.; Bertrand, M. P. J. Org. Chem. 2006, 71, 7288–7292. 13 Lo Conte, M.; Pacifico, S.; Chambery, A.; Marra, A.; Dondoni, A. J. Org. Chem. 201075, 4644–4647. 14 McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett. 2002, 4, 3591–3594. 15 For leading references on the synthesis of bis-amino acids, see: (a) Li, C.; Tang, J.; Xie, J. Tetrahedron 2009, 65, 7935–7941. For an interesting application, see: (b) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem. Res. 2008, 41, 1387–1398. 16 Pirrung, M. C.; Das Sarma, K.; Wang, J. J. Org. Chem. 2008, 73, 8723–8730. 17 For an outstanding example, see: van Kasteren, S. I.; Kramer, H. B.; Jensen, H. H.; Campbell, S. J.; Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C.; Davis, B. G. Nature 2007, 446, 1105–1109. 18 Homogeneous conditions were required to achieve good conversions of GSH 22 19 Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.—Eur. J. 2009, 15, 11444–11449. 20 This compound was kindly provided as trifluoroacetic salt by UFPeptides S.r.L. (University of Ferrara).

93 94 CHAPTER 4 Theorical approach for radical additions Computational Methods 4.1 Theoretical approach This chapter is focused on the computational methods adopted in this thesis. All calculations presented here have been performed within the density functional theory (DFT) formulation of the quantum mechanics. The reason is that DFT is one of the most computationally efficient first principle methods to calculate atomic structures and electronic states in a wide variety of materials. Furthermore, it allows for dynamics, thus offering a versatile tool for performing finite-temperature simulations. More recently, DFT-based dynamics has been combined with methods suitable to sample chemical reaction paths and applied successfully to complicated chemical, catalytic and biochemical reactions. This chapter is organized as follows. Theoretical and practical aspects of DFT calculations are described in sections X.X and X.X, respectively. The basics of the first principles (DFT-based) dynamics are presented in section X.X. Finally, in the latter section, the free energy sampling technique, known as Blue Moon Ensemble, used to inspect chemical reaction pathways and to work out free energy barriers is briefly discussed in view of its applications.

4.1.1 Density Functional Theory The density functional theory (DFT) was originally proposed in the early 60s by Hohenberg and Kohn (1964)1, and Kohn and Sham (1965)2 with important contributions also from the group of Pople3 (Pople et al. 1981, Pople et al. 1989, Johnson et al. 1993). Its importance in the advancement of computational quantum chemistry and related fields was internationally acknowledged by the Nobel Prize in Chemistry in 1998 awarded jointly to Walter Kohn and John A. Pople. The DFT is a formulation of the many-body quantum mechanics in terms of an electron density distribution, ρ(x), which describes the ground state of a general system composed of interacting electrons and classical nuclei at given positions {RI}. Several excellent books and review articles have been published on the fundamentals of DFT (Parr and Yang4, Marx and Hutter5). For this reason, I limit the discussion to the basic details necessary

95 to what has been done in this thesis and refer the reader to the rich literature, part of which is cited in the references listed at the end of this chapter. The first step in DFT consists in giving an explicit form for the electron density distribution; in a way similar to the hypotheses introduced in the HF approach, also here single-particle wavefunctions i(x) are used to express the many-body mathematical function ρ (x). The major difference and, at the same time, dramatic simplification, is the fact that not even the specific analytic form of the complex function ψi(x) matters, but only its square modulus, so that the electron

( 1 ) density readsThis expression is clearly a single Slater determinant constructed from the single-particle wavefunctions representing all the Nocc occupied orbitals. The coefficients fi are the integer occupation numbers, and they are equal to 1 in the case in which the spin is explicitly considered (spin- unrestricted) or equal to 2 if the spin is neglected and energy levels are considered as doubly-occupied (spin-restricted). Furthermore, the wavefunctions ψi(x) are subject to the orthonormality constraint.

( 2 )

as in any quantum mechanics approach. The Kohn-Sham (KS) DFT total energy of the system in its ground state is then written as

( 3 )

In Eq. (3) the first three terms on the right-hand side (Ek, EH, Exc) describe all the electron-electron interactions, the fourth term (EeI) refers to the electron- nucleus interaction and the fifth one (EII) the nucleus-nucleus interaction. (exactly as sketched in Fig. 4. Let’s revise briefly the explicit form and the meaning of each one of these terms. Ek is the Schrödinger-like kinetic energy expressed in terms of the single-particle wavefunctions ψi(x) as

96 ( 4 ) and it is completely diagonal both in the index i and in the argument x of the wavefunctions, as in a non-interacting system of Nocc electrons. We remark, in passing, that this expression for the kinetic energy does not depend on the density (x) but directly on the wavefunctions. The second term, EH, is the Hartree energy already encountered in the HF approach, it accounts for the Coulomb electrostatic interaction between two charge distributions and is written as

( 5 )

This is generally computed via the associate Poisson equation solved for the Hartree potential

( 6 )

The exchange interaction and the electron correlations due to many-body effects are represented by the term Exc[ρ], whose exact analytical expression is unfortunately unknown; this represents in a sense a limit of the DFT. There are good approximations derived from the homogeneous electron gas limit for the

6 exchange interaction . Namely, the pure exchange part of the functional, Ex[ρ] can be written in the so-called local density approximation (LDA) as

( 7 )

This is the simplest possible form and the name comes from the fact that an interacting but homogeneous electron distribution is assumed, in which the (unknown) density is given by the local density ρ(x) at a specific point x in the inhomogeneous system. Similarly, the LDA version of the correlation energy, originally proposed by Kohn and Sham2 and successively improved by Perdew and Zunger (1981)7, reads

97 ( 8 ) where the explicit analytic form of the function εc(ρ(x)) comes from a parameterization of the results of random phase approximation calculations. Due to the insufficiency of a simple LDA approximation in the treatment of many real systems, such as proteins and nucleic acids, non-local approximations including the gradient of the density, ρ (x) are often adopted and the exchange-correlation functional becomes

( 9 )

In practical applications, however, the gradient enters only with its modulus | ρ (x)|, thus adding only a modest computational cost to DFT calculations. These generalized gradient corrections (GGA) are indeed a bit arbitrary, in the sense that they do not represent a regular perturbation expansion as, for instance, in the case of MP2 corrections. Nonetheless, they are generally based on solid physical and mathematical argumentations and anyhow their accuracy can be assessed a posteriori by test calculations and comparisons with both exact results and experiments3c. As far as the exchange term is concerned, the original work of Becke6 represents a milestone in the field and it is still regarded as a standard. It was subsequently refined and improved by the same author8 with remarkable success in a wealth of applications. Also correlations, have received special attention from several research groups, sometimes including also the exchange part into a single functional9. Among all the possible functional forms for the exchange and correlation functional, the ones that have been most widely used in the applications of DFT DFT for chemistry and aqueous solution is the Becke exchange in combination with the Lee, Yang and Parr correlation, termed BLYP6,9e in the literature. This is also the choice done in the simulations treated in this chapter. Indeed, as far as it has been checked over the years, BLYP provides a better performance in terms of geometrical parameters and relative energies for a wide variety of organic and inorganic molecules10, two of the main ingredients in proteins and nucleic acids. In general, the explicit form adopted to describe the exchange and correlation

98 interactions represents a delicate step in the set-up of the simulation framework and must be carefully tested and benchmarked. As a word of warning, let us also stress the fact that no one of the present versions of Exc included van der Waals interactions. In the cases in which they are dominant, such as, for instance, in the base pair stacking of DNA and RNA, special corrections ad hoc must be included11, although they are not entirely (or not at all) self-consistent with the DFT formulation. The analytic form of the electrostatic interaction between the two sets of variables, electrons and nuclei, is simply given by the Coulomb attraction between a point-like charge at nuclear positions RI and the electron density distribution,

( 10 )

th where ZI is the charge of the I nucleus. However, for most of the applications that will be discussed in the following paragraphs, this expression turns out to be computationally expensive. In fact, in a large protein or nucleic acid system there are two different length scales that come into play: a small one for the core electrons, characterized by rapidly varying wavefunctions, especially in the region very close to the nucleus, and a longer one for the valence electrons that form chemical bonds and vary more smoothly. Clearly, in an all-electron calculation, the first one would dominate and add a computational workload that would make impractical dynamical simulations, and often even static optimizations, of large biomolecules. Provided that the model system is small enough and long dynamical quantum simulations are not required, it is certainly possible to use and all-electron DFT approach. However, one can observe that core electrons are generally inert and do not participate to chemical bonds. This crucial observation led to the use of pseudopotentials. Namely, core electrons are eliminated and a potential describing the core-valence interaction is built by fitting to the all-electron solutions of the Schrödinger or Dirac equation for the single atom of the chemical species considered12. Alternatively, one can consider the inner electron wavefunctions close to the nuclei as frozen orbitals and

99 describe them with appropriate angular momentum projectors13 . In a pseudopotential (PP) approach, the electron-nucleus interaction is rewritten as

( 11 )

The elimination of the core electron from the calculation allows one to get rid of the short length scale problem. The computational cost can be further reduced, provided that a separable form is chosen for the pseudopotential14 such as

( 12 )

where r = x – RI and on the right-hand side we have separated the PP into a local term Vloc(r), depending only on the position r, and a non-local (NL) part represented by a sum over all the orbital angular momenta l,m. The functions φlm

(r)= fl(r)Ylm(Ω) are eigenfunctions of the atomic Hamiltonian in which the core- valence interaction has been replaced by the PP, fl(r) indicates the radial part of the solution, whereas the angular dependence is represented by the spherical harmonics Ylm(Ω). Several analytical forms for the PPs have been reported over the years15 and widely used in applications ranging from gas-phase molecules, to solid state system and soft matter. In general they are norm-conserving. The meaning of this definition is that the square norm of the wavefunction is preserved in the pseudopotential construction. In fact, a general PP (radial) wavefunction φPP(r) is nodeless and matches the all-electron one φAE(r) only beyond a certain distance r0 from the nucleus, called cut-off radius,

( 13 )

Nonetheless, the norm of the wavefunction is conserved in the sense that

( 14 )

This rather stringent condition can be released as shown by Vanderbilt (1990)13a, leading to the so-called ultra-soft pseudopotentials. On the other

100 hand, an augmented charge has to be added in order to conserve the total charge density as obtained upon integration of the square modulus wavefunction. As a final observation, let us point out that in some cases the presence of a core electron charge density distribution can be properly accounted for via the introduction in the pseudopotential of a non-linear core correction16. Finally, the fifth and last term in right-hand side of Eq. (3) is simply the Coulomb interaction between two classical nuclei I and J and is written as where

ZI and ZJ must be intended as the net valence charge only in a PP approach.

( 15 )

The total energy Etot of the ground state of such a system of interacting electrons and nuclei can then obtained by minimizing the KS functional with respect to the single-particle orbitals ψi(x),

( 16 ) which, in practice, means solving the KS Schrödinger-like equations given by the variational derivative of the KS functional with respect to the single-particle wavefunctions ψi(x),

KS !E ["] KS * ! fiH #i (x) = fi$i#i (x) ( 17 ) !#i

A question that at this point is still unanswered is “what is ψi(x) ?”. In a way similar to what has been done in the case of HF, also in DFT approaches we need to select a proper basis set on which orbitals can be expanded. One possible choice is to adopt a Slater or Gaussian set of functions, as successfully done in many quantum chemistry approaches. An alternative choice, and rather popular in the physics community, is represented by plane waves (PW). In this expansion, the single-particle wavefunctions ψi(x) become:

( 18 )

101 where the Hilbert space spanned by the plane waves exp(iGx) is truncated at a suitable cut-off generally expressed as an energy and measured in Rydberg, Ecut = (Gmax)2/2. The advantage of plane waves is that they do not depend on the atomic positions RI, the accuracy can be systematically (variationally) improved by increasing the cut-off and they form a complete orthonormal basis-set. The first property is useful in the calculation of the forces required for instance in dynamics, since Pulay forces are exactly zero, and the Hellmann-Feynman theorem17,

( 19 )

Furthermore, the calculations of gradients ( ) or

Laplace operators ( ), such as the electronic kinetic energy, is reduced to simple products in the Fourier space; the use of the fast Fourier transform (FFT) makes the calculation straightforward and distributable in parallel processing18. As a word of warning, since often radicals are involved in the reactions studied here, we checked all the results with self-interaction corrections.

4.1.2 Car-Parinello Method Most of the quantum chemical applications are static calculations of the electronic structure performed either on stable experimental configurations or on stationary points obtained via geometry optimization. These are indeed instructive and rich of information. Nonetheless, finite temperature and entropy effects are two of the dominant features in chemical reactions, particularly in solution, and the role of the explicit solvent is far from negligible. In this respect, the so called first principles molecular dynamics (FPMD) has represented a huge step forward in quantum simulations of condensed phases in general and, more recently, in biological systems. In practice, the interactions among atoms, instead of being described by an analytic function of the atomic coordinates RI, is directly computed from the total energy Etot, which is simultaneously a function

102 of the electron wavefunctions and of the atomic coordinates in the sense specified at the beginning of this paragraph. The interactions are supposed to be electrostatic, i.e. dependent just on the charges and positions of the particles and not on their velocities; in particular, the Born-Oppenheimer (BO) approximation, at a given instant t in time, consists in an optimization of the electronic structure at the corresponding (fixed) nuclear positions RI(t). Then the nuclear forces are computed as gradients of the total energy with respect to the ionic position and the variables RI(t) updated to RI(t+δt) = R’I(t’). The set of equations that one has to solve iteratively reads

( 20 )

( 21 )

and imply the recalculation of the electronic ground state after each displacement of the atoms. The BO approximation assumes that electrons stay “frozen” on their ground state, whereas nuclei move and for this reason it is referred to as “adiabatic approximation”. An alternative to this scheme has represented a real breakthrough in first principles dynamical simulations: the Lagrangean-based method proposed by Car and Parrinello in 198519. Two major problems arise in FPMD: On one hand one has to integrate the equations of motion for the nuclear positions, which represent the long time scale part to the problem. On the other hand, one has to propagate dynamically the smooth time-evolving (ground state) electronic subsystem. The Car-Parrinello molecular dynamics (CPMD) is able to satisfy this second requirement in a numerically stable way and makes an acceptable compromise for the time step length of the nuclear motion. The formulation is an extension of a classical molecular dynamics Lagrangean in which the electronic degrees of freedom are added to the system, along with any other dynamical

20 21 variable qα(t), such as a thermostat or a barostat .

103 2 CP 1 ! 2 3 ! 1 2 tot L = !M I RI +!µ " d x !i (x) + !!" q!! # E [!,{RI }, q! ] 2 I i 2 !

d 3x *(x) (x) +!!ij ( ! !i ! j !!ij ) ( 22 ) ij

The first three terms in the right-hand side of Eq. (21) are the kinetic energies of the nuclei, of the electrons and of the additional dynamical variables, the fourth one is the total energy, in practice the DFT functional in the applications that will be discussed, and the last addendum is the orthonormality constraint for the wavefunctions. The kinetic energy for the electronic degrees of freedom is the novelty of the CPMD approach. The fictitious mass µ assigned to the orbitals ψi(x) is the parameter that controls the speed of the updating of the wavefunctions with respect to the nuclear positions and, for this reason, it determines degree of adiabaticity of the two subsystems, electrons and nuclei. A rigorous mathematical proof has been given22, showing that the CPMD trajectory {RCP(t)} stay close to the BO one {RBO(t)} and the upper bound is given by |RCP (t)- RBO(t)| < C µ1/2, where C is a positive constant. This is nothing else but a strategy to update on-the-fly the wavefunctions when ions undergo a displacement. The related Euler-Lagrange equations of motion read.

( 23 )

( 24 )

( 25 )

Let us observe that it is straightforward to give a Hamiltonian, instead of a Lagrangean, formulation of the CPMD method, via a simple Legendre transform. Despite the fact that the method dates back to more than a quarter of century,

104 applications to proteins and nucleic acids became possible only in the late 90s, when large systems could be computationally afforded.

4.1.3 Blue Moon One of the earliest techniques for sampling rare events and related free energy landscapes, also in the sense of the historical development, was the Blue Moon22. For all the details, we refer the reader to the cited publications. Just to summarize the essential points, let us recall that in a quantum dynamical simulation, the method rely on the identification of a reaction coordinate, or order parameter, ξ = ξ(RI) of a given subset of atomic coordinates (Lagrangean variables) RI able to track the chemical reaction on which one wants to focus. The simplest example is represented by the distance between two atoms that are expected to form or break a chemical bond as in all the cases studied here. This analytical function is added to, e.g., a Car-Parrinello Lagrangean as a holonomic constraint,

( 27 )

where λk is a Lagrange multiplier and ξk is a given assigned value of ξ(RI) that one wants to sample. For each sampled value, a constrained dynamics is performed and then the average constraint force fξ computed as the time average

( 28 )

turning out to be the gradient of the free energy F along ξ. The free energy profile of the reaction is then computed as

( 29 )

where ξ0 is the initial value of the reaction coordinate and ξf the final one.

105 4.2 Introduction of our problem The main target of the present set of simulations has been the radical reaction. The reactions were realized in laboratory in several usual and unusual environments, namely toluene, benzene, ionic liquids or even simple water23. Minozzi et al. JOCArticle The general reaction mechanism is shown Scheme 1 required in order to obtain complete conversions, a problem SCHEME 1. Mechanisms of TYC and TEC Radical Chains due to the reversibility of the attack of sulfur-centered radicals to C-C double bonds.7 In those cases, additional workup is necessary to get rid of the excess of alkene/thiol and one of the main advantages of click-reactions is therefore lost. In prin- ciple, a possible way of overcoming this drawback could be to perform the reactions in the presence of alkynes instead of alkenes, that is, to carry out Thiol-Yne Couplings (TYCs). Indeed, although reported as a slower process, sulfanyl radical addition to C-Ctriplebondsisknowntobevirtuallyirrevers- ible, at least with alkanesulfanyl radicals.8 This breakthrough has actually been developed in a group of very recent papers where the authors exploited this idea in the field of polymer chemistry.9 It should, however, be emphasized that those authors aimed at creating very highly functionalized materials (polymers and dendrimers) by means of a one-pot reaction and are neverthelessScheme 1. attained Mechanisms of TYC and TEC Radical Chains through an ionic addition of thiols under very mild conditions. Hence, they drove the reaction to to suitable electron-deficient alkynes. full completion by letting the initially formed vinyl sulfide Scheme 1 shows the radical chainreactionsthatcanoperate adducts to react with additional thiol through a consecutiveIn the first understep, TYC on which conditions. we Addition focused, of a is sulfanyl the thio radical-radical to the addiction to the thiol-ene coupling. On the other hand, the issue of the selective terminal carbon of the alkyne C-Ctriplebondgivesa formation of the primary vinyl sulfide TYC productselectron could-rich be systemβ-sulfanylvinyl named TYC; radical thatthis readily step abstractsproceeds hydrogen with the from formation of a β- crucial in the field of bioconjugation, where molecular complex- the thiol to regenerate the chain-carrier, sulfur-centered radical ity might be a secondary concern with respectsulfanylvinyl to finding a radicalintermediate (see Figure. with concomitant 1). The specific formation thio of the-radical vinyl sulfide selected is the methyl reliable, ‘clickable’ way of derivatization of valuable biological monoadduct (TYC chain). Under certain circumstances, the substrates. As far as we know, only two papersthioglycolate, have so far themonoadduct smallest molecule can undergo used further during addition the of anothersynthesis sulfa- work. For electron appeared focusing somewhat on this matter: the more recent nyl radical to give an R,β-disulfanyl-disubstituted alkyl radical one10a deals with dual glycosylation of peptides,rich thus, molecules, concen- attentionthat can subsequently has been abstractpaid to hydrogen terminal from alkyne, the thiol in toparticular 1-octine; give the final bis-sulfide bis-adduct (TEC chain). Unlike the trating again on bis-addition coupling products,the whereas approach the is equivalent to alkene TEC, characterized by mono and bis other one10b actually focuses on vinyl sulfide adducts, which primary vinyl radical, the successive alkyl-counterpart oc- curs in a reversible fashion: this allows for properly tuning (7) (a) Benati, L.; Montevecchi, P. C.; Spagnolo, P. J.adduction Chem. Soc., Perkin duringreaction the different conditions in synthesis. order to favor The formation β-sulfanylvinyl of the bis- formed readily Trans. 1 1991, 2103–2109. The reversibility of the addition of sulfanyl adduct (TYC-TEC sequence) or to stop the reaction at the radicals to C-C double bonds precludes, for example, theabstracts radical addition hydrogen from thiols in solution, to regenerate the chain-carrier (R1-S⋅), of disulfides to C-C double bonds, which is inefficient unless either by vinyl sulfide stage (TYC). employing alkynes instead of alkenes (see refs 7a above and 13a below) or in In our opinion, the latter outcome is that deserving the presence of a very good radical trap such as a diselenide,sulfur see: (b)-centered Ogawa, radicalthorough intermediate, attention. Indeed, with the simultaneous (virtually) irreversible formation oc- of vinyl sulfide A.; Tanaka, H.; Yokoyama, H.; Obayashi, R.; Yokoyama, K.; Sonoda, N. J. Org. Chem. 1992, 57,111-115 or a very efficient H-donor,mono see: (c) Taniguchi,adduct (TYCcurrence chain of). vinyl sulfide adduct from the radical TYC T.; Fujii, T.; Idota, A.; Ishibashi, H. Org. Lett. 2009, 11,3298–3301. process could be exploited in a more ‘clickable’ reaction than (8) (a) Heiba, E. I.; Dessau, R. M. J. Org. Chem. 1967, 32, 3837–3840. (b) Montevecchi, P. C.; Navacchia, M. L. J. Org. Chem. 1997, 62,The second reaction step (TEC) could not be studied within the limits of the 5600–5607. the TEC one, since it could provide easier access to assorted (c) Melandri, D.; Montevecchi, P. C.; Navacchia, M. L. Tetrahedron 1999, sulfur-tethered products employing equivalent amounts of 55, 12227–12236. For a kinetic study on the addition of benzenesulfanyl starting materials. This latter point could be of extreme radicals to alkynes, see: (d) Ito, O.; Omori, R.; Matsuda,present thesis. In such a step, M. J. Am. Chem. the mono adduct can undergo further addition of Soc. 1982, 104, 3934-3937. where that addition has been suggested to be importance in the field of bioconjugation, where the isola- slightly reversible. It is worth noting that addition of sulfur-centeredanother sulfanyl radical to give an α,β-disulfanyl-disubstituted alkyl radical that radicals tion of target molecules from complex reaction mixtures con- to alkynes is not always necessarily an irreversible process, provided that the 10a radicals possess additional stabilization with respect to sulfanyls: sulfonyl taining excess substrates is often a difficult task. Further- radicals, for example, have been reported to add reversiblycan subsequently abstract hydrogen to C-C triple more, synthesis and activity from of bioconjugates the thiol to containing give thethe final bis-sulfide bonds, see: (e) Rosenstein, I, J. In Radicals in Organic Synthesis; Renaud, P., vinyl sulfide linkage are highly worthy of being explored and, Sibi, M. P., Eds.: Wiley-VCH: Weinheim, Germany, 2001; Vol. 1, Chapter 1.4, pp 50-71. (f) Bertrand, M. P.; Ferreri, C. In Radicalsbis-adduct in Organic (TEC to chain, date, only Scheme a unique example 1). Our dealing studies, with this issueinstead, has been were pursued by Synthesis; Renaud, P., Sibi, M. P., Eds.: Wiley-VCH: Weinheim, Germany, reported.10b Finally, the feasibility of selective syntheses of Vol. 2, Chapter 5.5, pp 485-504. (g) Chatgilialoglu, C.;focusing on the crucial intermediates Vinil-E and Vinil-Z. Ferreri, C. In The Chemistry of Triple-Bonded Functional Groups;Patai,S.,Ed.;Wiley:Chichester, monoadducts can open the door to TYC-TEC sequences lead- U.K., 1994; Vol. 2, pp 917-944. (h) Chatgilialoglu, C.; Bertrand, M. P.; ing to nonsymmetric bis-functionalizations, hence, paving Ferreri, C. In S-Centered Radicals; Alfassi, Z. B., Ed.; Wiley: Chichester, the way to very attractive dual-labeling investigations.11 U.K., 1999; pp 311-354. (9) (a) Fairbanks, B. D.; Scott, T. F.; Kloxin, C. J.; Anseth, K. S.; Here, we report an explorative study on model substrates Bowman, C. N. Macromolecules 2009, 42, 211–217. (b) Chen, G.; Kumar, that aimed at getting more insight into the conditions affect- J.; Gregory, A.; Stenzel, M. H. Chem. Commun. 2009, 6291–6293. (c) Chan, J. W.; Hoyle, C. E.; Lowe, A. B. J. Am. Chem. Soc. 2009, 131, 5751–5753. ing the coupling reaction outcome, particularly the ensuing (d) Hensarling, R. M.; Doughty, V. A.; Chan, J. W.; Patton, D. L. J. Am. mono- and bis-adduct ratio. Both classical organic solvents Chem. Soc. 2009, 131, 14673–14675. (e) Konkolewicz, D.; Gray-Weale, A.; and water (or water/solvent mixtures) were taken into Perrier, S. J. Am. Chem. Soc. 2009, 131,18075–18077.(f)Lowe,A.B.;Hoyleb, C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20,4745–4750.(g)Fairbanks,B.D.; account for carrying out the reactions, since the aqueous Sims, E. A.; Anseth, K. S.; Bowman, C. N. Macromolecules 2010, 43,4113–4119. medium would be crucial for106 applying Thiol-Yne Couplings (h) Chan, J. W.; Shin, J.; Hoyle, C. E.; Bowman, C. N.; Lowe, A. B. Macro- molecules 2010, 43,4937–4942.(i)Hoogenboom,R.Angew. Chem., Int. Ed. 2010, as a ligation strategy, for instance, for peptide glycosylation, 49,3415–3417. (10) (a) Lo Conte, M.; Pacifico, S.; Chambery, A.; Marra, A.; Dondoni, (11) For an outstanding example, see: van Kasteren, S. I.; Kramer, H. B.; A. J. Org. Chem. 201075,4644–4647.(b)Shiu,H.-Y.;Chan,T.-C.;Ho,C.-M.; Jensen, H. H.; Campbell, S. J.; Kirkpatrick, J.; Oldham, N. J.; Anthony, Liu, Y.; Wong, M.-K.; Che, C.-M. Chem.—Eur. J. 2009, 15,3839–3850. D. C.; Davis, B. G. Nature 2007, 446, 1105–1109.

B J. Org. Chem. Vol. XXX, No. XX, XXXX 4.3 Computational Section First-principles dynamical simulations have been performed with the CPMD package version 3.15.124. The simulated systems were always placed in cubic or orthorhombic supercells (of lateral sizes in the range of 10-20 Å) on which periodic boundary conditions were applied whenever the solvent was present. In the cases in which reactions in vacuum were studied, these periodic boundary conditions were released and the system treated as isolated. On the other hand, the solvated conditions have been reproduced by periodic boundary conditions anb surrounding the solute molecule (or molecules) with a number of water molecules sufficient to reproduce at least 2-3 hydration shells, keeping the density of the solvent water to the experimental liquid density (0.99 g/cm3). Valence electrons are expanded in a plane-wave basis set with an energy cut-off of 70 Ry. All the calculations presented here, since they involve radicals or, in general, unpaired electrons have been performed in the spin-unrestricted local spin density approximation (LSD), thus allowing for a correct treatment of the open-shell cases. The core-valence interaction was expressed in terms of Trouiller-Martins norm conserving pseudopotentials for C, O and S, whereas a von Barth-Car norm conserving pseudo- potential was used for H atoms. The Kleinman–Bylander factorization was used for the nonlocal part and the BLYP gradient corrections mentioned at the beginning of this chapter were used for the exchange-correlation functional. The integration time step was set to 3.0 a.u. (0.12 fs) for vacuum system, and 4.0 a.u in case of solution (0,096 fs). This choice is driven by the necessity to keep into account the fast H stretching modes, which are a bit slowed down in solution, due to the presence of the hydrogen bond network of water, hence allowing for a slightly larger time step. A fictitious electron mass of 380-400 a.u. ensured good control of the constants of motion during the dynamics. The simulations have been performed for total times ranging from 0.5 ps for Vinyl to more than 5.0 picoseconds for the inspection of reaction pathways within the Blue Moon ensemble approach. The temperature was set to 300 K (i.e. 26-27 °C) and controlled with a Nosé-Hoover thermostat25 after an initial equilibration phase where a velocity

107 rescaling algorithm was used to thermalize the system. An example is reported for the case of the β-sulfanylvinyls, where simulations have been performed on the E and Z configurations, which are experimentally known to co-exist and between which our solute can easily switch during its exploration of the phase space. Has also explicated local spin density approximation (LSD) to split the calculation for all valence electrons present in the calculation and not consider the valence orbital like close shell. In all the constrained dynamics simulations, done within the Blue Moon scheme23, the reaction coordinate was chosen to be the distance between the atoms involved in the reactions. Specifically: (i) for thio-radical addictions the reaction coordinate adopted was the distance of the S atom of the thio-radical molecule (methyl-thioglycolate, R-S⋅) from the terminal carbon of the alkyne (C). (ii) Analogously for the H transfer, although some intrinsic difficulties imposed a slight variation; namely, in an initial stage, the distance between the S atom of thiol-molecule (R-SH) and the radical carbon center of β-sulfanylvinyl (C⋅) was used as a constraint, just to approach the two reacting molecules preventing artificial de-hydrogenation which could be induced by constraints on H. In fact, the bond between S and H is rather weak, and risk could break (artificially) apart if an uncareful constraint, corresponding to a pulling force, is applied. Only in a second stage, when the molecules were eventually separated by about 3.0 Å we switched to the H-C distance constraint. All molecules have been constructed and sketched with the ChemBioDraw version 12.0.3 or MarvinSketch version 5.3.8 in 2D, and subsequently transformed in 3D system by MarvinSpace or MarvinView version 5.3.8. The 3D coordinate has been transported in CPMD input file. These structure were used as initial configurations and both wavefunctions and geometries were initially fully optimized before each dynamical run. Data were visualized and analyzed using the VMD software26, gnuplot27 and XmGrace

108 4.4 Result and Discussion Reaction conditions: no solvent Thiyl-radical addiction The calculations discussed here focus on the addition of methylthiyl-radical to 1-octyne (Figure 1, a). After an initial unconstrained CPMD dynamics, needed to equilibrate the system, the free energy profile was computed with the Blue Moon ensemble approach, using as a reaction coordinate (RC) the distance dS-C = |R(S)- R(C)| show in Figure 1a. For each fixed constraint value, simulations were run for about 1–1.5 ps and thirty points; each value was characterized by different RC values, ranging from 7.14 Å to 1.85 Å. At the end of the constrained dynamics simulations, all constraint on the final reaction product were released and a free dynamics was performed to confirm the stability of the bond formed. On the reactants side, the simulation started with a planar angle configuration between

Cα and S⋅, as shown in Figure 1a. After few femtoseconds we observed the thiol radical moving in a position in which the two reactants form on average a 90° angle, a position compatible with the generally accepted reaction pathway always assumed as a starting point in all former ab-initio calculations.28 This is an important result that suggests that our reactants seem to rearrange spontaneously in such way as to allow for the studied reaction to occur without imposing any ad-hoc geometry which could bias the whole simulation.

109 (b)

(c) (a) ds-c

(d)

Figure 1. Localization of Spin in the key point of simulation show in Figure 2

In such a process the thiol radical aligns onto the plane of the C-C triple bond with formation of the β-sulfanylvinyl radical, whose formation requires an activation energy of 10.5 kcal/mole; Figure 1d shows the whole reaction pathway. During the simulation the interaction between the two reaction centers (Figure 2, upper panel) is positive (> 0 au), that is the forces between the two atoms are repulsive, until the transition state is reached; afterwards, the forces becomes attractive (< 0 au) to form the stable sulfanylvinyl radical. The constraint force returns repulsive when the distance between the atoms further decreases. The lower panel of Figure 2 shows, the free total energy involved in the reaction, with the maximum indicating the transition state. Figure 1, in which the cyan surfaces represent spin density distribution, clearly show that the transition state can be attained through two different approaches, i. e. with the sulfanyl radical coming closer to the C-C triple bond on either sides with respect to the hydrocarbon chain of the alkyne, hence forming Z- and E-β-sulfanylvinyl radicals (structures b and c). It is worth noting that, both in the transition states and in the final intermediate, the spin distribution is correctly predicted to be on the internal carbon of the C-C unsaturated bond.

110 (c) (b)

(d) (a)

Figure 2..thiol-radical addition path way. Is also represented the localization of Spin in the key point of simulation. Initial state C-S distance 7.0 Å, Transition state, between 2.0 and

Calculations afforded the thermodynamically less stable Z-vinyl radical (Structure d) but this point is of no importance as far as the stereochemical outcome of the reaction is concerned, since, as previously reported, E- and Z- vinyl radicals are known to interconvert very rapidly into each other. β-sulfanylvinyls (E and Z) have been simulated for 0.4-0.7 ps, to observe the interchangeability between the two structures. Figure 3 shows the temperature of the Vinyl interconversion system as a function of the simulation step for the (NVT) calculation using a Nosé-Hoover thermostat for temperature control. In the initial stage, a velocity-rescaling algorithm was used to attain thermal equilibrium (after red line), and then a canonical thermostat of the type indicated above was adopted. Figure 4 shows the corresponding energies calculated step by step during the simulation.

111 400

300

200

Temperature (°K) 100

0

0 20000 40000 60000 80000 100000 Step Figure 3. Simulation with Nosé-Hoover thermostat. After an initial rescaling

Car-Parrinello-like simulations critically depend on the ability to control the drift of the electronic wave functions away from the instantaneous Born- Oppenheimer (BO) ground state and, to ensure a good degree of adiabaticity of the CPMD method during the simulation, a very good control of the electron fictitious kinetic energy must be ensured. Figure 4 shows in red the EKS Kohn- Sham energy, equivalent to the potential energy in classical MD; in green the EHAM energy of the total CP-Hamiltonian, that must be conserved during the simulation; in blue the total classical energy (ECLAS), i. e. the difference between the EHAM and the electron kinetic energy (EKINC).

-129.2

-129.22

-129.24

-129.26

-129.28 Energy ( a. u. )

-129.3

-129.32

0 20000 40000 60000 80000 100000

Step Figure 4. Vinyl E simulation, Red EKS, blue ECLAS, green EHAM

Figures 3 and 4 report comparable results, showing that the two vinyl structures are able to interconvert into each other at 300 K, giving Z/E lifetime

112 ratios of about 2/1 and 4/1 starting from the E- and Z-configuration, respectively (Figure 5).

β-sulfanylvinyl Z

β-sulfanylvinyl E

Figure 5. β-sulfanylvinyl E and β-sulfanylvinyl Z.

Hydrogen transfer: vinyl sulfide mono adduct formation The simulation in total took more than 3.0 ps. By accosting the β- sulfanylvinyl radical (Figure 6, a), and the thiol molecule (Figure 6, b) we expected, at a proper distance, the hydrogen thiol to be transferred to the β- sulfanylvinyl radical, giving the final vinyl sulfide mono adduct. We performed twenty-one calculations of different RC values, from 5.00 Å - to 2.40 Å, for the distance between the thiol and the vinyl radical, in particular between the thiol sulfur atom and the vinyl carbon radical.29 Heuster and Kalvoda30 proposed, for hydrogen transfer between C and O, a C-O distance of less than 2.8 Å. Houk et al.31 found for the analogous transition-state geometry a C-O distance of 2.48 Å. Sulfur has larger size and a more diffused electron cloud than carbon or oxygen atoms; indeed Jørgensen et al.32 calculated for C-S H- transfer a C–H∙∙∙S distance in the transition state close to 3.0 Å with a C–H∙∙∙S angle close to 130°. Subsequently Himo33 confirmed those data, and proposed for hydrogen atom transfer from cysteine to glycyl radical a C-S distance close to 3.0 Å.

113 0

-0.005

-0.01

-0.015 Constraint Force (au) -0.02 a -0.025

8 2.5 3 3.5 4 4.5 b

6

4 F (Kcal/mol)

2

0 2.5 3 3.5 4 4.5 C-S distance (!)

Figure 6. Hydrogen transfer. Constrained simulations between C-S

As shown in Figure 6, our first simulation did not give the expected results, since, instead of hydrogen transfer, the formation of a new bond between carbon and sulfur occurred. Therefore, starting from the geometry calculated for a C-S distance of 3.5 Å (circled point in Figure 6), a parameter in line with Himo and Jørgensen, we initially restarted the simulation with a double constraint involving both the C-S and the C-H bonds. After few femtoseconds the former constraint was eliminated and the simulation carried out with the latter only. Simulation was performed for twenty points more, with the C⋅-H distance ranging from 2.70 Å to 1.30 Å. At nearly 1.70 Å, a transition state was observed, and the final formation of the C-H bond was observed at 1.50 Å (Figure 7, Ts and Fs). The thiol molecule is shown to approach the vinyl radical with a C=C-H angle of about 90° to 120°. In the transition state the C･-S distance during hydrogen transfer was found to be 3.07 Å, in line with previous results.32,33 In the vacuum, the activation energy for hydrogen transfer from methyl thioglycolate to 1-octyne was calculated as ∼4,7 kcal/mol.

114 1.0

0.8

0.6 Ts

0.4

0.2

Constraint force (au) 0.0

1.4 1.6 1.8 2 2.2 2.4 2.6 2.0

0.0

-2.0 Fs -4.0

-6.0

F (kcal/mol) -8.0 1.4 1.6 1.8 2.0 2.2 2.4 2.6 C-H distance ( Å )

Figure 7.. Hydrogen transfer. Constrained simulations between C-H, after initial constraint C-S

To conclude this section it is worth pointing out that, during the simulations shown above, all molecules/intermediates were completely free to move in the space, with the only exceptions of the constraints explained above. That means that vinyl radicals were free to interconvert between their E- and Z- configurations. After hydrogen transfer, calculations predicted formation of the Z-vinyl sulfide, that is the expected kinetic product.

Reaction conditions: solvent (water) There are different types of effects induced by the solvent. Solvent molecules can be part of the reaction, like for instance in hydrolysis reactions. In this case, the only way to model the situation is, of course, to include the parts that are needed in the quantum calculation. The same applies to short-range solvent effects, where, for instance, strong hydrogen bonds are known to form or break during the course of the reaction. Other methods use the dielectric constant to emulate the solvent. CPMD calculations fill the calculation box with molecules of the solvent taken into consideration. Therefore we initially built a simulation box characterized by similar dimensions with respect to the previous simulations, but filled with so many water molecules as to obtain the normal water density

115 value of 0,99 g/cm3 at RT. After an initial free dynamics of 1.0 -2.0 ps, we needed to eliminate some water molecules to ensure a constant pressure value of the reaction vessel.

Thiyl-radical addiction Simulations of about 1–1.5 ps were performed for twenty points of different RC values. Figure 8 (lower panel) shows the free energy of the reactants (red line) and the total energy (black line) for the solvated system; the upper panel reports the interaction force between the two reaction centers. We can observe that, close to 4.0 Å, the total energy decreases of few kilocalories/moles in correspondences with a calculated structure where there is a proton sharing between the thiyl radical and the terminal carbon of the alkyne Figure 8 a. Although more constrained by the solvent molecules with respect to the vacuum, the thiyl radical was observed to be able to move rapidly in a position orthogonal to the carbon-carbon triple bond, analogously to what predicted in the vacuum. Also in this case preferential formation of the Z-vinyl radical was observed. The transition state was located between 2.2 Å and 2.5 Å (Figure 8 b) and the activation barrier for formation of the β-sulfanylvinyl radical (Figure x c) was calculated as 6.8 kcal/mol (17.0 kcal/mol for the total energy).

116 b)

c) a)

Figure 8.Thiol-radical addition path way in solution. a) Proton sharing, b)Transition state c) Final state.

Hence, the solvent effect in lowering the activation energy for addition of the thiyl radical to the alkyne can be estimated as approximatively 4.0 kcal/ mole. As far as E/Z interconversion is concerned, the results in water are comparable with those obtained in the vacuum. This means that vinyl radicals, although more constrained by the solvent, are able to flip and interconvert very rapidly at 300 K: Z/E lifetime ratios were about of about 1/1 and 2/1 starting from the E- and Z-configuration, respectively (Figure 9). This result would suggest that water could influence the carbon chain mobility in such a way as to favour vinyl radical interconversion, although this is a preliminary result that has to be supported by further, more sound experimental data. This result is however in line with the data obtained for additions of thiyl radicals to alkynes carried out in water (Chapter 4, Table 3).

117 β-sulfanylvinyl Z β-sulfanylvinyl E

Figure 9. β-sulfanylvinyl E and β-sulfanylvinyl Z solvated in water.

Hydrogen transfer: vinyl sulfide mono adduct formation After an initial unconstrained simulations of 0.5 ps, the thiol was observed to perform a spontaneous hydrogen transfer to the vinyl radical. Figure 10 shows the transition state structure (b) and the activation energy can be evaluated as lower than 2.5 kcal/mole at 300 K. This can only be an evaluation, since that value is comparable to the the error bar of the method. However it can be inferred that hydrogen transfer from a thiol to a vinyl radical seems to be a quasi-spontaneous process at room temperature in water.

0

-0.001

-0.002 Constraint force (au)

-0.003 b)

3.4 3.5 3.6 3.7 3.8 3.9 1.00 c) 0.80

0.60

0.40 F (Kcal/mol) 0.20 a) 0.00

3.4 3.5 3.6 3.7 3.8 3.9 C-S distance (Å)

Figure 10.Hydrogen transfer. Constrained simulations between C-S

118 Mulliken population Analysis Performing Mulliken charge density analyses during the simulations, allows for studying charge evolution along the reaction pathway. Graphic 1 shows the trends obtained by analyzing thiyl-radical addition in the vacuum and in water and shows the main atoms involved in the reaction (Figure 11) and their charge evolution (Table 1). The only significant modification of the charge distribution moving from the reactants to the product through the transition state was observed for the sulfur atom and, unexpectedly, for the sp3 carbon atom linked to the C-C triple bond. In the transition state the sulfur atom shows a propensity for assuming a partial negative charge and this result is in line with the polar characteristics of many transition states involved in radical reactions. Of course, the presence of water as the solvent can have a very beneficial effect by stabilizing charge separation in the transition state. The development of the positive charge on the carbon atom is instead much more puzzling and currently we don’t have any explanation for this unprecedented datum. Graphic 2 shows the analogous trends obtained for hydrogen transfer from the thiol moiety to the vinyl radical. Data are summarized in Table 2 and the involved atoms are labelled in Figure 12. A first, notable difference between the two reaction conditions (vacuum and water) is a strong charge separation in the reactants atoms (IS) before hydrogen transfer in the vacuum; this situation rapidly evolves towards a more uniform charge distribution even before the transition state is reached. In water it seems the solvent effect could decrease the local charge throughout the whole system, hence decreasing Coulomb interactions. Under these conditions, the only important trend is that of the carbon atom that is the terminus of hydrogen transfer: this atom increases its positive charge by approaching the transition state, as requested by polar effects in hydrogen atom transfers, and then becomes more and more negative by coming closer to the final state, as a result of conjugation with the sulfur atom. At the same time, the thiol sulfur lowers its positive charge in approaching the transition state, although the effect is much smaller than expected. The fact that in water the electron distribution of the initial state is much closer to that of the

119 transition state with respect to the vacuum conditions could suggest that the system would not require such an important rearrangement to be transformed into the products as in the vacuum: this hypothesis may well give an explanation for the calculated very low activation barrier for hydrogen transfer in aqueous solution.

Dec_Mulliken A B C D E F Vacuum

Initial State -0,279 -0,050 -0,239 -0,417 0,475 0,131

TS_2_41 -0,039 0,034 -0,172 -0,408 0,452 0,063

Final State -0,279 0,023 -0,295 -0,407 0,470 0,210 Water

Initial State -0,283 -0,021 -0,236 -0,413 0,475 0,121

TS 0,273 0,022 -0,218 -0,372 0,448 -0,050

Final State -0,287 0,063 -0,328 -0,417 0,478 0,297

Table1. Mulliken spin densities on different atoms take in consideration.

F D B E A C

Figure 11.Atoms take in consideration in Table 1

Vacuum Water 0,500 0,500 0,375 0,375 0,250 0,250 0,125 0,125 0 0 -0,125 -0,125 -0,250 -0,250 -0,375 -0,375 -0,500 -0,500 IS TS FS IS TS FS A B C D E F Graphic 1. Mulliken spin densities of Table 1

120 Quench_Mulliken A B C D E F

Vacuum Initial State -0,830 0,498 -0,884 0,884 0,394 0,110 TS_150 -0,247 0,059 -0,325 0,266 0,159 0,098 TS_160 -0,293 0,151 -0,311 0,220 0,070 0,040 TS_170 -0,290 0,162 -0,343 0,275 0,037 0,042 Final State -0,264 -0,132 -0,251 0,244 0,222 0,187 Water Initial State -0,301 0,127 -0,342 0,267 0,076 0,096 TS_G_74501 -0,275 0,174 -0,272 0,194 0,050 0,080 TS_G_75401 -0,293 0,115 -0,277 0,265 0,096 0,034 TS_G_75741 -0,275 -0,084 -0,273 0,301 0,149 0,178 Final State -0,236 -0,123 -0,270 0,271 0,155 0,190

Table2. Mulliken spin densities on different atoms take in consideration for Hydrogen Transfer

E F

C D A B

Figure 12.Atoms take in consideration in Table 2 Vacuum Water 0,900 0,400 0,675 0,300 0,450 0,200 0,225 0,100 0 0 -0,225 -0,100 -0,450 -0,200 -0,675 -0,300 -0,900 -0,400 IS TS_160 FS IS TS_G_75401 FS

A B C D E F A B C D E F

Graphic 2. Mulliken spin densities of Table 2

121 4.5 Conclusion CPMD calculations were carried out in order to get some more insight into the mechanism of the thiol/yne coupling (TYC), particularly the solvent effect. The model reaction, i.e. addition of methyl thioglycolate to 1-octyne, was considered in its two steps: i) addition of the corresponding sulfanyl radical to the alkyne, followed by ii) hydrogen atom transfer from the starting thioglycolate to the resulting β-sulfanyl-substituted vinyl radical. Simulations gave, for the first step, an activation barrier in the vacuum of 10.5 kcal/mol, with the two possible final intermediates, the E- and Z-vinyl radicals, rapidly interconverting at 300 K, as expected on the basis of previous results. The activation energy of the second step was calculated as 4.7 kcal/mol, suggesting that the rate-determining step of the whole TYC reaction is addition of the sulfanyl radical to the alkyne. In water the corresponding barriers were found to be 6.8 kcal/mol and ca. 2 kcal/mol, respectively, with the latter being an estimated value since the results of calculations were inside the error bar of the method. Neverthelss, this data suggest that both sulfanyl radical addition to the alkyne and, above all, hydrogen transfer to the resulting vinyl radical are significantly faster in water with respect to vacuum. This could be the result of charge stabilization by the water molecules, which, in the case of the hydrogen transfer step, seems to prevent a major reorganization for the system to pass through the transition state. In the addition step, on the contrary, the very similar charge distributions in reactants and products together with the significant charge development in some of the atoms directly involved in the reaction suggests formation in water of a transition state with a major charge separation: this is a situation that is commonly believed to accelerate many radical reactions, especially, like in our case, in the presence of heteroatoms that can tolerate that charge distribution. A faster addition step in water may well justify the better results obtained in that environment for TYC reactions but also a faster hydrogen transfer step may account for the preferred production of reduction products (vinyl sulfides) when the intermediate vinyl radicals may evolve by different routes.

122 It is however worth pointing out that, before saying that, with respect to vacuum, the effect of water in accelerating our reactions resides in its polar character or its ability to give raise to hydrogen bondings (and not, for instance, simply to entropic and/or micellar effects), it will be crucial to carry out analogous calculations in the presence of another non-polar, aprotic solvent such as benzene. Those calculations are currently underway.

123 4.6 Bibliography 1 P. Hohenberg and W. Kohn, Physical Review 1964, 136, B864. 2 W. Kohn and L. J. Sham, Physical Review 1965, 140, A1133. 3 a) D. J. DeFrees, B. A. Levi, S. K. Pollack, W. J. Hehre, J. S. Binkley and J. A. Pople, Journal of the American Chemical Society 1979, 101, 4085-4089. b) Raghavachari, K., G. W. Trucks, M. Head-Gordon, and J. A. Pople, 1989, Chem. Phys. Lett. 157, 479. c) Johnson B G, Gill P M W and Pople J A 1993,J. Chem. Phys. 98 5612–26. 4 R. G. Y. Parr, W., Density-functional theory of atoms and molecules, New York: Oxford University Press., New York, 1988, p. 333. 5 H. J. Marx D., Modern Methods and Algorithms of Quantum Chemistry,J. Grotendorst (Ed.), John von Neumann Institute for Computing, Ju ̈lich, NIC Series 2000, 1, 301-449. 6 A. D. Becke, Physical Review A 1988, 38, 3098. 7 J. P. Perdew and A. Zunger, Physical Review B 1981, 23, 5048. 8 a) A.D Becke. J. Chem. Phys., 98 (1993), p. 5648; b) A.D. Becke, J. Chem. Phys. 98, 1372 (1992). 9 a) J. P. Perdew, Phys. Rev. B 33, 8822 (1986); b) J.P. Perdew and Y. Wang, Phys. Rev. B 45, 13244 (1992);c) J.P. Perdew, K. Burke and M. Ernzerhof, Phys. Rev. Lett. 77, 3865 (1996); 78, 1396 (1997); d) S.J. Vosko, L. Wilk and M. Nusair, Can. J. Phys. 58, 1200 (1980); e) C. Lee, W. Yang and R. G. Parr, Phys. Rev. B 37, 785 (1988);f) F.A.Hamprecht, A.J.Cohen, D.J.Tozer and N.C. Handy, J. Chem. Phys. 109, 6264 (1998) g) A. J. Cohen and N. C. Handy, J. Chem. Phys. 117, 1470 (2002). H) X. Xu and W. A. Goddard III, Proc. Natl. Acad. Sci. USA, 101, 2673 (2004). 10 F. A. Hamprecht, A. J. Cohen, D. J. Tozer, and N. C. Handy, J. Chem. Phys. 109, 6264 (1998). 11 a) T. Schwabe and S. Grimme, Phys. Chem. Chem. Phys. 9, 3397 (2007). b) P. L. Silvestrelli, Phys. Rev. Lett. 100, 053002 (2008). 12 a) D. Vanderbilt. Phys. Rev. B, 41:7892, 1990.; b) P. E. Blochl. Phys. Rev. B, 50:17953, 1994. 13 L. Kleinman and D. M. Bylander, Physical Review Letters 1982, 48, 1425-1428. 14 a) G. B. Bachelet, D. R. Hamann and M. Schlter, Physical Review B 1982, 26, 4199-4228. b) R. Stumpf X. Gonze and M. Scheffler. Analysis of separable potentials. Phys. Rev. B, 44:8503, 1991. c) N. Troullier and J. L. Martins, Phys. Rev. B 43, 1993 (1991). 15 S. G. Louie, S. Froyen, and M. L. Cohen, Phys. Rev. B 26, 1738 (1982). 16 R. P. Feynman, Physical Review 1939, 56, 340-343. 17 W.H. Press, S.A. Teukolsky, W.T. Vetterling & B.P. Flannery (1992) 18 R. Car and M. Parrinello, Physical Review Letters 1985, 55, 2471. 19 a) S. NosÈ, J. Chem. Phys. 1984, 81, 511. b) W. G. Hoover, Physical Review A 1985, 31, 1695. 20 a) H. Andersen, J. Chem. Phys. 1980, 72, 2384. b) M. Parrinello and A. Rahman, Physical Review Letters 1980, 45, 1196.

124 21 F. A. a. S. Bornemann, Num. Math., 78 (3). 1998, 359-376. 22 G. Ciccotti and J. P. Ryckaert, Computer Physics Reports 4, 346-392. 23 a) Lanza, T.; Minozzi, M.; Monesi, A.; Nanni, D.; Spagnolo, P.;Chiappe, C. Curr. Org. Chem. 2009, 13, 1726–1732. b) M. Minozzi, A. Monesi, D. Nanni, P. Spagnolo, N. Marchetti and A. Massi, J. Org. Chem., 2011, 76, 450-459 24 CPMD Consortium (2010) CPMD Consortium page.www.cpmd.org 25 Martyna J, Klein ML, Tuckerman M (1992) Nosé–Hoover chains: the canonical ensemble via continuous dynamics. J Chem Phys 97:2635–2643 26 Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38 27 http://www.gnuplot.info/ 28 a) T. L. Jacobs,G. E. Illingworth Jr., The Addition of Thiyl Radicals to Allenic Hydrocarbons',The Journal of Organic Chemistry 1963 28 (10), 2692-2698, b) T. Kawamura, M. Ushio, T. Fujimoto, T. Yonezawa, Electron Spin Resonance Study on Intermediate Free Radicals in the Addition of Thiols to Unsaturated Compounds, Journal of the American Chemical Society / 93:4, February 24, 1971, 909 c) Y. A. Borisova, A. K. Dyusengalievb, G. A. Orazovab, and T. P. Serikovb, Quantum-Chemical Study of the Photochemical Addition Reaction of Dialkyl Disulfides with Olefins, PETROLEUM CHEMISTRY, Vol. 49, No. 2009 29 A. F. Shestakov, T. G. Denisova, E. T. Denisov,à and N. S. Emel ́yanova, Reactions of intramolecular hydrogen abstraction, Russ.Chem.Bull., Int.Ed., Vol. 51, No. 4, April, 2002., 30 Heusler, K.; Kalvoda, J. Angew. Chem., Int. Ed. Engl. 1964, 3, 525-596 31 Houk K. N., Tucker J. A., Dorigo A. E., Quantitative Modeling of Proximity Effects on Organic Reactivity, Acc. Chem. Res., Vol. 23, No. 4, 1990 32 Ma, L., Jørgensen, A.-M. M., Sørensen, G. O., Ulstrup,J. & Led, J. J. J. Am. Chem. Soc. 122, 9473–9485 (2000). 33 F. Himo, C–C bond formation and cleavage in radical enzymes, a theoretical perspective, Biochimica et Biophysica Acta 1707 (2005) 24–33

125 126 List of Publications • Spagnolo, P., Nanni, D., Chiappe, C., Minozzi, M., Lanza, T., Monesi, A., “Radical Additions of Thiols to Alkenes and Alkynes in Ionic Liquids“ Curr. Org. Chem., 2009, 13, pp. 1726 - 1732

• T. Lanza, M. Minozzi, A. Monesi, D. Nanni, P. Spagnolo, G. Zanardi, Improved Radical Approach to N-Unsubstituted Indol-2-one and Dihydro-2-quinolinone Compounds Bearing Spirocyclic Cyclohexanone/Cyclohexadienone Rings, Adv. Synth. Catal. 2010, 352, 2275 – 2280

• M. Minozzi, A. Monesi, D. Nanni, P. Spagnolo, N. Marchetti and A. Massi, An Insight into the Radical Thiol/Yne Coupling: The Emergence of Arylalkyne-Tagged Sugars for the Direct Photoinduced Glycosylation of Cysteine-Containing Peptides, J. Org. Chem., 2011, 76, 450-459.

127 128