sign in sign up
<<
Home , Acetyl group, Alicyclic compound, Bioorganic chemistry, Chemical change, Meso compound

M. M. MELKONYAN

SELECTED LECTURES IN ORGANIC AND BIOORGANIC

Handbook

YEREVAN 2016 1

YEREVAN STATE MEDICAL UNIVERSITY AFTER M.HERATSI

M. M. MELKONYAN

SELECTED LECTURES IN ORGANIC AND BIOORGANIC CHEMISTRY

This handout is adopted by the Methodical Council of Foreign Students of the University

YEREVAN 2016 1

YEREVAN STATE MEDICAL UNIVERSITY AFTER M.HERATSI Department of General and Bioorganic Chemistry

MAGDA MHER MELKONYAN Professor, Head of Department of General and Bioorganic Chemistry

SELECTED LECTURES IN ORGANIC AND BIOORGANIC CHEMISTRY

The handbook ”SELECTED LECTURES IN ORGANIC AND BIOORGANIC CHEMISTRY” is intended to be studied by YSMU students of General medicine, Stomatological, Pharmacy facultes as well as those of Medical and Biological colleges. The present handbook includes all the chapters of Bioorganic Chemistry (in the frames of curriculum). Electronic and Steric constructions of organic compounds and the links between organic compounds structures and their biological activity is systematized in the textbook. The structure of the main compounds taking part in (static ), widely recognized commonly used drugs are given in the textbook as well as a special place is devoted to the discussion of chemical properties and main principles of reactive ability of organic compounds. The teaching material is illustrated by tables and pictures. Yerevan, YSMU, 2016, p. 224.

Ø.Ø. Ø»ÉùáÝÛ³Ý §úñ·³Ý³Ï³Ý ¨ λÝë³ûñ·³Ý³Ï³Ý ùÇÙdzÛÇ ¹³ë³ËáëáõÃÛáõÝ- Ý»ñÇ Ñ³í³ù³Íáõ¦: Ò»éݳñÏ Ý³Ë³ï»ëí³Í ºä´Ð ÁݹѳÝáõñ µÅßÏáõÃÛ³Ý ¨ ëïáÙ³ïáÉá·Ç³Ï³Ý ý³ÏáõÉï»ïÝ»ñÇ áõë³ÝáÕÝ»ñÇ, ÇÝãå»ë ݳ¨ Ï»Ýë³µ³Ý³Ï³Ý ¨ µÅßÏ³Ï³Ý Ã»ùáõÙáí áõëáõÙݳñ³ÝÝ»ñÇ, ùáÉ»çÝ»ñÇ áõë³ÝáÕÝ»ñÇ Ñ³Ù³ñ: ºñ¨³Ý, ºä´Ð, 2016, 224 ¿ç:

ISBN 99941- 40 -12 - 4 © Melkonyan M.M. 2

BIOORGANIC CHEMISTRY Introduction

According to the simplest definition, is the study of the compounds of . Perhaps the most remarkable feature of organic chemistry is that it is the chemistry of carbon and only a few other elements-chiefly, , , nitrogen, rare sulfur and phosphorus. have discovered or made well over ten million compounds composed of carbon and these three other elements. Organic compounds are every where around us—in our foods, flavors, and fragrances; in our medicines, toiletries, and cosmetics; in our plastics, films, fibers, and resins; in our paints and varnishes; in our glues and adhesives; and, of course, in our bodies and those of all living things. Organic chemistry has a long tradition of relating the properties of a substance to its molecular structure and, more than anything else, the relationship between how a substance behaves and the way its are connected. Extremely important are problems, concerning biological and physiological activity of organic compounds. Therefore this part of organic chemistry, which studies structure and function of compounds, which are compulsory and necessary for normal functioning of living bodies and especially and particularly human body, used to call bioorganic chemistry. The main goals of subject are: 1.To study the structure of all organic compounds from living sources, including biopolymers, such as , nucleic , polysaccharides and bioregulators, other metabolites and reveal connection between structure and function of these compounds. 2. Once a structure is known, to synthesize the compound in the laboratory and manufacture the compound if it is more economical, than to isolate from a natural sources. 3. To isolate, purify, identify and study the structure of active ingredients from remedies using in folk medicines and to synthesize the compound in the laboratory. 4. To synthesize the synthetic compounds with expected properties, much more efficient than analogues from natural sources. Unlike the course in where topics often appear unrelated, each new topic in organic chemistry builds on what has come before. At the beginning let us revise the nature of chemical bonds.

3

Formation of chemical bonds. According to Lewis' model, atoms bond together in such a way that each participating in a acquires a completed outer-shell electron configuration resembling that of the noble gas nearest it in the . Atoms acquire completed outer shells in two ways. 1. An atom may lose or gain enough electrons to acquire a completely filled outer shell. An atom that gains electrons becomes an anion (a negatively charged ), and an atom that loses electrons becomes a cation (a positively charged ion). A chemical bond between a positively charged ion and a negatively charged ion is called an ionic bond. 2. An atom may share electrons with one or more other atoms to complete its outer shell. A chemical bond formed by sharing electrons is called a . The main type of chemical bond in organic compounds is a covalent bond.

1. CLASSIFICATION OF ORGANIC COMPOUNDS The classes of are , , , and arenes. Alkanes are hydrocarbons in which all of the bonds are single bonds and are characterized by the molecular formula CnH2n+2. Functional groups are the structural units responsible for the characteristic reactions of a . The functional groups in an are its hydrogen substituents. Families of organic compounds are listed in Table 1. The simplest alkane is methane, CH4; ethane is C2H6, and propane is C3H8. Constitutional isomers are possible for alkanes with four or more . Thus there are two isomers of molecular formula C4H10. One of these has an unbranched carbon chain (СН3СН2СН2СН3) and is called n-butane; the other has a branched chain [(CH3)3CH)] and is called isobutane. n-Butane and isobutane are common names. Unbranched alkanes are sometimes called normal alkanes and are designated by the prefix n- in their common name . The prefixes n- and "iso" are joined by "neo" in the common names of the three isomeric C5H12 alkanes:

CH3CH2CH2CH2CH3 (CH3)2CHCH2CH3 (CH3)4C n-Pentane Isopentane Neopentane

A single alkane may have different names; a name may be a common name or it may be a systematic name developed by a well-defined set of rules. The system that is the most widely used in chemistry is IUPAC nomenclature. are alkanes in which a ring is present; they have the molecular formula CnH2n. The IUPAC rules for alkanes and cycloalkanes the rules for groups are given below. 4

Alkanes and cycloalkanes are essentially nonpolar and are insoluble in . The only forces of attraction between nonpolar are relatively weak induced dipole-induced dipole attractions. These forces are variously referred to as van der Waals attractions, London forces, or dispersion forces. Carbon combines with other atoms (e.g., C, H, N, O, S, ) to form structural units called functional groups-an atom or group of atoms within a molecule that shows a characteristic set of physical and chemical properties. Classification of organic compounds is based both on the structure of the chain and on the nature of functional groups existing in the compounds.

Table 1. Some important families of organic molecules Names of Common Family name Name Structures Functional formula ending Groups Contains only C-C and Alkane -ane C-H single bonds R-CH=CH-R -ene C = C –C  C– R-CC-R -yne Phenyl Arene none

-F, -Cl, -Br, -I (Hal) Halides R– Hal Alkyl Halide none –OH Hydroxyl R–OH , Phenol -ol –OR alkoxy R–OR none –SH Mercapto R–SH , Mercaptan Thiol –SR Alkylthio R–S—R, –NH R–NH 2 primary 2 >NH R –NH Amine -amine secondary 2 >N- R N tertiary 3 –CN Cyano R—C N >C=O Carbonyl -one

carboxyl

(carbonyl Carboxylic -ic (-CO2H) +hydroxyl) Carboxylic Acids -ate Ester Amide -amide

5

2. IUPAC NOMENCLATURE

2.1. IUPAC nomenclature of alkanes and cycloalkanes 2. 1. 1. Naming Alkanes (main rules). 1. Find the longest continuous chain of carbon atoms and assign a basis name to the compound corresponding to the IUPAC name of the unbranched alkane having the same number of carbons. The longest continuous chain in the alkane shown is six carbons.

This alkane is named as a derivative of hexane. 2. List the substituents attached to the longest continuous chain in alphabetical order. Use the prefixes di-,tri-, tetra-, etc., when the same substituent appears more than once. Ignore these prefixes when alphabetizing. The alkane bears two methyl groups and an . It is an ethyldimethylhexane.

3. Number the chain in the direction that giwes the lower locant to a substituent at the first point of difference. When numbering from left to right, the substituents appear at carbons 3,3, and 4. When numbering from right to left the locants are 3, 4, and 4. Therefore, number from left to right

Correct Incorrect

The correct name is 4-ethyl-3,3-dimethylhexane. 4. When two different numbering schemes give equivalent sets of locants, choose the direction that gives the lower locant to the group that appears first in the name. In the following example, the substituents are located at carbons 3 and 4 regardless of the direction in which the chain is numbered. 6

Correct Incorrect

Ethyl precedes methyl in the name; therefore 3-ethyl-4-methylhexane is correct.

2.1.2. Cycloalkanes 5. Count the number of carbons in the ring and assign a basis name to the corresponding to the IUPAC name of the unbranched alkane having the same number of carbons. The compound shown contains five carbons in its ring. It is named as a derivative of .

6. Name the alkyl group and append it as a prefix to the cycloalkane. No locant is needed if the compound is a monosubstituted cycloalkane. It is understood that the alkyl group is attached to C-1. The compound shown above is isopropylcyclopentane. Alternatively the alkyl group can be named according to the rules which be given below (later). The name becomes (1-methylethyl)cyclopentane. Parentheses are used to set off the name of the alkyl group as needed to avoid ambiquity. 7. When two or more different sutbstituents are present, list them in alphabetical order and number the ring in the direction that gives the lower number at the first point of difference. The compound shown is 1,1-diethyl-4-hexylcyclooctane.

8. Name the compound as a cycloalkyl-substituted alkane if the substituent has more carbons than the ring.

is pentylcyclopentane but is 1-cyclopentylhexane

7

2.1.3. Alkyl groups. An alkyl group can be thought of as the part of an alkane that remains when one hydrogen atom is removed to create an available bonding site. It's important to realize tht alkyl groups themselves are not compounds and that the "removal" of a hydrogen from an alkane is just a way of looking at things, not a . Alkyl groups are simply hypothetical part-structures that help us to name compounds. For example, removal of a hydrogen from methane gives the , -CH3, and removal of a hydrogen from ethane gives the ethyl group, -CH2CH3. These alkyl groups are named simply by replacing the -ane ending of the parent alkane with an -yl ending. Methane and ethane are special because each has only one "kind" of hydrogen. It doesn't matter which of the four methane is removed because all four are equivalent. Thus, there is only one possible methyl group. Similarly, it doesn't matter which of the six equivalent ethane hydrogens is removed, and only one ethyl group is possible. The situation is more complex for larger alkanes, which contain more than one structural kind of hydrogen. Propane, for example, has two different types of hydrogens. Removal of any one of the six hydrogens attached to an end carbon yields a straight-chain alkyl group called n-propyl (-CH2-CH2-CH3), whereas removal of one of the two hydrogens attached to the central carbon yields a branched-chain alkyl group called isopropyl –CH(CH3)2. (The "n" prefix in n-propyl stands for normal, meaning straight-chain.) Butane is even more complex. There are four 4-carbon (butyl) groups, named n-butyl, sec-butyl, isobutyl, and tert- butyl. The prefix sec- in sec-butyl stands for secondary, and the prefix tert- in tert-butyl stands for tertiary in reference to the number of other carbon atoms attached to the main alkyl carbon. There are four possible substitution patterns, called primary, secondary, tertiary, and quaternary. As indicated by the following structures, a primary (1°) carbon atom has one other carbon attached to it, a secondary (2°) carbon atom has two other carbons attached, a tertiary (3°) carbon atom has three other carbons attached, and a quaternary (4°) carbon atom has four other carbons attached.

8

The symbol R is used as a general abbreviation for any alkyl group. The group R may represent methyl, ethyl, propyl, or any of a vast number of other possibilities. Removal of a hydrogen atom from a primary carbon atom gives a straight-chain alkyl group, but removal from a secondary or tertiary carbon atom gives a branched alkyl group.

2.2. Nomenclature of complex compounds In earlier times, when relatively few pure organic chemicals were known, new compounds were named at the whim of their discoverer. Thus, is a crystalline substance first isolated from urine, and the barbiturates were named by their discoverer in honor of his friend Barbara. As more and more compounds became known, however, the need for a systematic method of naming compounds became apparent. An atom or group of atoms within a molecule that has characteristic chemical behavior, or the structural features that allow us to class compounds together are called functional groups. Functional groups are important for three reasons. First, they are the units by which we divide organic compounds into classes. Second, they are sites of characteristic chemical reactions; a particular functional group, in whatever compound it is found, undergoes the same types of chemical reactions. Third, functional groups serve as a basis for naming organic compounds. A given functional group undergoes the same reactions in every molecule it’s a part of.

2.2.1. IUPAC nomenclature of complex compounds. The basic rules for IUPAC system. The system of naming (nomenclature) now used is that devised by the International Union of Pure and Applied Chemistry, IUPAC. According to IUPAC system of nomenclature any given can be represented by one and only one molecular structure. In general, the IUPAC system of naming an organic compound consists of three parts. (a) Prefix(es) (b) Root word (c) Suffix(es)

The root word depends on the number of carbon atoms present in a suitable chain, called the parent chain, containing the functional group and as many of carbon - carbon multiple bonds(s) as possible. Appropriate suffix(es) is then added to the root word to denote the saturated or unsaturated character of the 9 parent chain and also the functional group present their in. Finally, prefix(es) is put to indicate the nature and position of the side chain or substituents.

The basic rules for IUPAC system are: 1. Longest chain rule The longest possible continuous chain of carbon atoms containing the functional group and carbon - carbon multiple bonds is first selected and the root word corresponding to it is noted.

Root word – Hex Root word — Hex

Root Words- No. of No. of Root Root word carbons carbons word 1 Meth- 6 Hex- 2 Eth- 7 Hept - 3 Prop- 8 Oct- 4 But- 9 Non- 5 Pent - 10 Dec -

2.Primary Suffix - A primary suffix is to be added to the root word to indicate saturation and unsaturation in the selected chain. The generic root word for any carbon chain is alk - (from alkane). Chain Type Suffix Generic Name 1. Saturated - ane Alk + ane 2. Unsaturated with one double bond - ene Alk + ene 3. Unsaturated with one triple bond - yne Alk + yne

4. Alkane - lH -yl. Alk + yl

10

3.Secondary Suffix - A secondary suffix is added to indicate the nature of functional group, if present in the compound. Some functional groups are given below : Secondary Functional Group Suffix Alcohol (-OH) -ol Aldehyde (-CHO) -al Ketone >C=Q - one (-COOH) - oic acid Ester (-COOR) - oate Amide (-CONH;) - amide Acid chloride (- COCl) - oyl chloride The terminal "e" of the primary suffix is replaced by secondary suffix.

No. of Root Primary Secondary IUPAC Formula carbons word suffix suffix Name HCHO 1 meth - - an -al Methanal CH3CH2OH 2 eth- - an -ol Ethanol CH3COOH 2 eth- - an - oic acid Ethanoic acid CH3CH2CH3 3 prop - - ane — Propane CH3CH=CH2 3 prop- - ene — Propene CH3-C≡CH 3 prop- - yne - Propyne CH3COCH2CH3 4 but- - an - one Butanone

Prefixes rule: Prefixes are to be added to the root word to represent the side chains and substituents.

4.Alphabetical arrangement of prefixes rule If there are more than one side chains/substituents, they should be prefixed in the alphabetical order. When two or more identical side chains/substituents are present, their location and number are represented by prefixing di (2), tri (3), tetra (4) etc. before the substituents/side chains.

3-ethyl-2-methyl hexane 2,3-dimethyl butane

5. Lowest sum rule The sum of the numbers used to indicate the position of substituents should 11 be minimum. The parent carbon chain is to be numbered from one end. The position of substituents and functional groups is indicated by the number of carbon atoms to which they are attached.

(Correct) (Incorrect)

3, 3, 5, trimethyl hexane 2, 4, 4, trimethyl hexane (I) - (Incorrect) (II)- (Correct)

In case of II, the sum of numbers of substituents is 2 + 4 + 4 = 10, which is less than in I, where the number of substituents is 3+3+5=11. In case the substituent on the parent chain is complex (containing more than 4 carbon atoms) it is named as substituted alkyl group whose carbon chain is numbered from the carbon carbon - atom attached to the main chain.

5- (1,2 dimethyl propyli) nonane

The position of a double bond (or triple bond) in alkenes (or alkynes) is indicated by prefixing the number of the carbon proceeding such a bond, the carbon chain being numbered from the end which assigns lower positional number to the double (or triple) bond.

2- ethyl-1 pentene 4,5-dimethyl-2– hexyne 2-pentene

6. Lowest number of functional group rule When a functional group is present in a compound, the lowest number must be given to the functional group, even if it violates the lowest sum rule. 12

3-hydroxy, 2, 2 dimethyl butane 3, 3-dimethyl butanol-2 (Incorrect) (Correct)

The parent chain in a substituted alcohol (I) would be numbered so as to give the lower number to the functional group -OH. 

(Correct) (Incorrect) (I)

The numbering of the parent chain in the alcohol II can be done in two ways:

(A) (B) (II)

In case B the functional group -OH gets the lower number 3, but in the case A it gets the higher number 4. However, the sum numbers used to indicate the position of the functional group and side chains in B is 4 + 4 + 3 = 11, and in case A it is 3 + 3 + 4 = 10. In accordance with this rule, the former system of numbering is preferred.

7. Numbering the carbon in terminal and non - terminal functional groups The carbon in chain terminating functional groups such as -COOH, -HO, -CONH2 , -CN etc. must always be given number 1. The carbon of non - terminal group (e.g. > C = 0) may or may not be assigned number 1. For example,

3-ethyl hexanoic acid 2-ethyl-3-methyl butanal

13

5-methyl hexanone-3

8. Numbering the carbon- carbon multiple bonds While numbering the parent chain containing carbon - carbon multiple bonds, the lower number of two carbons involved in the multiple bonds, is used to indicate the position of the multiple bond. Number 2, for example, is used to indicate the position of the double bond in the alkene, (III).

4-methyl hexene-2 (III)

9.Prefix for alicyclic compounds The word cycle is prefixed to the root word if the compound being named is an alicyclic compound. When alicyclic compounds contain side chains and/or substituents, they appear as secondary prefixes before the word cyclo.

10. Naming polyfunctional compounds When a compound contains two or more different functional groups, one of the functional groups is chosen as the principal functional group and the remaining functional groups (secondary functional groups) are treated as substituents in the IUPAC names. The following guidelines determine the choice of the principal and secondary functional group while giving IUPAC names to poly functional organic compounds. (a) When two or more than two functional groups are present in a compound, the principal functional group generally gets the following order of preference; Acids > Acid derivatives excluding > > Nitriles> >Ketones >Alcohols > > > > Alkenes > Alkynes. (b) While selecting the parent chain in a polyfunctional compound, it should be ensured that it includes the maximum number of functional groups present, including the principal functional group. (c) While numbering the parent chain in such cases the following is the decreasing order of preferences for giving the lowest numbers.

14

1, bromo-2-methyl butane 2methylhexane

2, 2, 4-trimethyl pentane 1,3-pentadiene

Table 2.1. The order of the substituents Functional Group Prefix Suffix (Ending) -(C)OOH1 - ic acid -COOH carboxy -SO3H sulfo -(C)  N - nitrile O // Al – C – H carbal (formil) -(C) = O oxo One - OH hydroxy Ol mercapto - SH thiol thio - NH2 amine amine 1(C) – this carbon atom are involved in the main chain Principal functional group > Double bond >Triple bond > Substituents

(d) If branching alkyl groups (side chains) contain multiple bonds and functional groups, the side chain is numbered separately so that the carbon atom in the side chain which is itself bonded to the parent chain is designated as 1. Characteristic groups which are used only as a prefix Family Functional group Prefix Halide -Br, -I, -F, -Cl fluoro, chloro, bromo, iodo Ether - OR alkoxy Sulfide - SR alkilthio Nitro derivates - NO2 nitro 15

3. ISOMERISM Various organic compounds represented by the same molecular formula are called isomers and this phenomenon is called isomerism. Types of Isomerism. Two common types of isomerism are: (1.) Structural isomerism. When two or more compounds possess the same molecular formula but different structural formulae, they are said to exhibit structural isomerism. (2) . The phenomenon exhibited by two or more compounds with the same molecular and structural formulae, but different spatial arrangements of atoms or groups is termed stereoisomerism.

3.1. Structural isomerism. Depending upon the nature of difference at structure, structural isomerism may be any of the following types: 3.1.(1) Chain or nuclear isomerism. Three isomers of pentane given below are chain isomers. CH3 H C–C–CH (i) H3C–CH2–CH2–CH2–CH3 (ii)H3C–CH2–CH–CH3 (iii) 3 3

CH3 CH3 n-pentane (straight chain) isopentane neopentane 3.1.(2) Position Isomerism. Isomerism caused by difference in position of the same substituent on the same chain is termed position isomerism: (i) CH3-CH2-CH2I and CH3-CHI-CH3 n-Propyl iodide isopropyl iodide or 1-iodopropane or 2-iodopropane Similarly (ii) CH2=CH-CH2-CH2 and CH3-CH=CH-CH3 1-Butene 2-Butene

3.1.(3) Functional isomerism. Monohydric are isomeric with ethers, the general formulae for both of these being CnH2n+2O. Thus ethanol, C2H5OH is isomeric with dimethyl ether, CH3-O-CH3 and propanol, С3Н7ОН with ethyl methyl ether, C2H5-O-CH3. This type of isomerism due to the presence of different functional groups is called functional isomerism and the isomers are termed as functional isomers. Some other example of functional isomers are: (i) C2H5CHO and CH3-CO-CH3 Propionaldehyde 16

both having the molecular formula C3H6O.

CH2

H C CH (ii) CH3-CH=CH2 and 2 2 Propene or trimethylene

both having the molecular formula C3H6. 3.1.(4) Metamerism. It is the type of isomerism exhibited by members of the same homologous series due to the difference in the nature of alkyl groups attached to the polyvalent atom of the functional group. For example, there are three isomeric ethers represented by the molecular formula C4H10O: C2H5-O-C2H5 CH3O-CH2СH2-СН3 СН3-O-CH(СН3)2 Diethyl ether Methyl n-propyl ether Methyl isopropyl ether 3.1.(5) Tautomerism. It is a dynamic isomerism and one isomer is constantly changing into the other and vice versa. It is termed tautomerism. Tautomerism is caused by the wandering of a labile hydrogen atom between two polyvalent atoms, carbon and oxygen in this case.

Fig. 3. 1.

Enols are compounds containing a double bond (C=C) as in alkenes and an OH group as in an alcohol. It is, therefore, known as keto-enol tautomerism.

3.2. . Stereoisomerism Stereochemistry refers to chemistry in three dimensions. Independently of each other, van't Hoff and Le Bel proposed that the four bonds to carbon were directed toward the corners of a tetrahedron so two compounds may be different because the arrangement of their atoms in space is different. Isomers that have the same constitution (the same molecular and structural formulae) but differ in the spatial arrangement of their atoms are called stereoisomers. Stereoisomers are divided into two groups: conformation and configuration isomers.

3.2.1.Conformations Different arrangements of atoms that can be converted into one another by rotation about single bonds are called conformations. The change from one to another involves rotation about the carbon-carbon bond, that there is free rotation about the carbon-carbon single bond. The cause of free rotation 17 about the carbon-carbon single bond is torsional . Let us discuss the conformations of ethane.

3.2.1.1. Conformations of acyclic compounds. Structure of ethane.

(a) (b) Fig. 3. 2. Ethane molecule. (a)Shape, (b)Carbon-carbon single bond:  bond

I II Eclipsed conformation Staggered conformation Fig. 3. 3. Actual structures of ethane: eclipsed and staggered conformations

There is an arrangement in which the hydrogens exactly oppose each other, and an arrangement in which the hydrogens are perfectly staggered, or an infinity of intermediate arrangements. All these are the actual structures of ethane which are formed due to rotation about the carbon-carbon single bond. Arrangement I is called the eclipsed conformation; arrangement II is called the staggered conformation (Fig. 3. 3.). The infinity of intermediate conformations are called skew conformations. It is used to draw these structures as so called Newman projections, named for M. S. Newman, of the Ohio State University, who first proposed their use. (Just sight along the carbon-carbon bond).

Fig. 3. 4. Newman`s projections: eclipsed and staggered conformation

Certain physical properties show that rotation is not quite free: there is an barrier of about 3 kcal/mol. The potential energy of the molecule is at a minimum for the staggered conformation, increases with rotation, and reaches a 18 maximum at the eclipsed conformation. Most ethane molecules, naturally, exist in the most stable, staggered conformation; or, any molecule spends most of its time in the most stable conformation. The 3-kcal barrier is not a very high one; even at room temperature the fraction of collisions with sufficient energy is large enough that a rapid interconversion between staggered arrangements occurs. The carbon- carbon single bond permits free rotation.

Fig. 3. 5. The potential energy of the molecule is at a minimum for the staggered conformation, increases with rotation, and reaches a maximum at the eclipsed conformation.

The barrier is considered to arise in some way from interaction among the electron clouds of the carbon-hydrogen bonds. The two sets of orbitals in ethane tend to be as far apart as possible-to be staggered. The energy required to rotate the ethane molecule about the carbon-carbon bond is called torsional energy. We speak of the relative instability of the eclipsed conformation-or any of the intermediate skew conformations-as being due to torsional str ain. As the hydrogens of ethane are replaced by other atoms or groups of atoms, other factors affecting the relative stability of conformations appear: van der Waals forces. Conformations of n-butane. Van der Waals repulsion. The n-butane molecule is similar to ethane, but with a methyl group replacing one hydrogen on each carbon. As with ethane, staggered conformations have lower torsional and hence are more stable than eclipsed conformations. But, due to the presence of the methyl groups, two new points are encountered here: first, there are several different staggered conformations; and second, a factor besides torsional strain comes into play to affect conformational stabilities. 19

There is the anti conformation, I, in which the methyl groups are as far apart as they can be (Fig. 3.6.).

I II III Fig. 3. 6. I-Anti conformation , II and III-Gauche conformations

There are two gauche conformations, II and III, in which the methyl groups are only 60° apart (Fig. 3.6.). Conformations II and III are mirror images of each other, and are of the same stability; nevertheless, they are different.

Fig. 3. 7. Potential energy changes during rotation about the C(2)-C(3) bond of n- butane.

The anti conformation, is more stable (by 0.8 kcal/mol) than the gauche. Both are free of torsional strain. But in a gauche conformation, the methyl groups are crowded together, and raise the van der Waals repulsion (or steric repulsion) between the methyl groups, and that the molecule is less stable because of van der Waals strain (or steric strain). The energy maximum reached when two methyl groups swing past each other (Fig. 3.7.).

3.2.1.2. Conforamation of cyclic aliphatic compounds. Baeyer strain theory In 1885 Adolf von Baeyer proposed a theory to account for certain aspects of the chemistry of cyclic compounds. According to Baeyer, when carbon is bonded to four other atoms, the angle between any pair of bonds is the 20 tetrahedral angle 109.5°. But the ring of cyclopropane is a triangle with three angles of 60°, and the ring of is a square with four angles of 90°. In cyclopropane or cyclobutane, therefore, one pair of bonds to each carbon cannot assume the tetrahedral angle, but must be compressed to 60° or 90° to fit the geometry of the ring. These deviations of bond angles from the "normal" tetrahedral value cause the molecules to be strained, and hence to be unstable compared with molecules in which the bond angles are tetrahedral. Cyclopropane and cyclobutane undergo ring-opening reactions since these relieve the strain and yield the more stable open-chain compounds. Because the deviation of the bond angles in cyclopropane (109.5° - 60° = 49.5°) is greater than in cyclobutane (109.5° - 90° = 19.5°), cyclopropane is more highly strained, more unstable, and more prone to undergo ring-opening reactions than is cyclobutane. The angles of a regular pentagon (108°) are very close to the tetrahedral angle (109.5°), and hence cyclopentane should be virtually free of angle strain. The angles of a regular hexagon (120°) are somewhat larger than the tetrahedral angle, and hence, Baeyer proposed (incorrectly), there should be a certain amount of strain in . Factors affecting stability of conformations. Any atom tends to have bond angles that match those of its bonding orbitals: tetrahedral (109.5°) for sp3 -hybridized carbon, for example. Any deviations from the "normal" bond angles are accompanied by angle strain. Any pair of tetrahedral carbons attached to each other tend to have their bonds staggered, like ethane, to take up a staggered conformation. Any deviations from the staggered arrangement are accompanied by torsional strain. Any two atoms (or groups) that are not bonded to each other can interact in several ways, depending on their size and polarity, and how closely they are brought together. These non-bonded interactions can be either repulsive or attractive, and the result can be either destabilization or stabilization of the conformation. Non-bonded atoms (or groups) that just touch each other - that is, that are about as far apart as the sum of their van der Waals radii-attract each other. If brought any closer together, they repel each other: such crowding together is accompanied by van der Waals strain (steric strain). Non-bonded atoms (or groups) tend to take positions that result in the most favorable dipole-dipole interactions: that is, positions that minimize dipole- dipole repulsions or maximize dipole-dipole attractions. (A particularly powerful attraction results from the special kind of dipole-dipole interaction called the ). All these factors, working together or opposing each other, determine the net stability of a conformation. 21

Conformations of cycloalkanes. Let us look more closely at the matter of puckered rings, starting with cyclohexane, make a model of the molecule and examine the conformations that are free of angle strain.

Chair conformation Boat conformation Twist-boat conformation Figure 3. 8. Conformations of cyclohexane that are free of angle strain.

First, there is the chair form (Fig. 3.8). If we sight along each of the carbon-carbon bonds in turn, we see in every case perfectly staggered bonds:

Staggered cyclohexane ethane Chair

The conformation is thus not only free of angle strain but free of torsional strain as well. The chair form is the most stable conformation of cyclohexane, and, indeed, of nearly every derivative of cyclohexane.Chair cyclohexane is: symmetrical, compact, and completely free of strain-angle, torsional, van der Waal`s strains. Every angle is the tetrahedral angle. About every carbon- carbon bond there is precise staggering. There is no crowding of hydrogen atoms. Indeed, the hydrogens closest together may well feel mild van der Waals attraction for each other: hydrogens on adjacent carbons, and certain hydrogens on alternate carbons-the three facing us and the three corresponding ones on the opposite face of the molecule. With an oxygen atom replacing one -similar chairs make up the most abundant building block of the organic world, D-glucose. Now, let us take chair cyclohexane and flip the "left" end of the molecule up (Fig. 3.9) to make the boat conformation (this involves only rotations about single bonds). Sighting along either of two carbon-carbon bonds, we see sets of exactly eclipsed bonds, and hence we expect considerable torsional strain: as much as in two ethane molecules. 22

Fig. 3. 9. Eclipsed cyclohexane ethane Boat

In addition, there is van der Waals strain due to crowding between the "flagpole" hydrogens, which lie only 1.83 A apart, considerably closer than the sum of their van der Waals radii (2.5 A). The boat conformation is a good deal less stable than the chair conformation. If chair cyclohexane is, conformationally speaking, the perfect specimen of a cycloalkane, planar cyclopentane (Fig. 3.10) must certainly be one of the poorest: there is exact bond eclipsing between every pair of carbons. To (partially) relieve this torsional strain, cyclopentane takes on a slightly puckered conformation, even, at the cost of a little angle strain. Evidence of many kinds strongly indicates that cyclobutane is not planar, but rapidly changes between equivalent, slightly folded conformations (Fig.3.10). Here, too, torsional strain is partially relieved at the cost of a little angle strain. Rings containing seven to twelve carbon atoms, too, are less stable than cyclohexane.

I II Fig. 3. 10. (I)-Planar cyelopentane: much torsional strain. The molecule is actually puckered, (II)-Cyclobutane: rapid transformation between equivalent non-planar "folded" conformations.

Equatorial and axial bonds in cyclohexane. Let us return to the model of the chair conformation of cyclohexane (Fig. 3.11). Although the cyclohexane ring is not flat, we can consider that the carbon atoms lie roughly in a plane. If we look at the molecule in this way, we see that the hydrogen atoms occupy two kinds of position: six hydrogens lie in the plane, while six hydrogens lie above or below the plane.

Fig. 3.11. Chair cyclohexane: equatorial and axial bonds 23

The bonds holding the hydrogens that are in the plane of the ring lie in a belt about the "equator" of the ring, and are called equatorial bonds. The bonds holding the hydrogen atoms that are above and below the plane are pointed along an axis perpendicular to the plane and are called axial bonds. In the chair conformation each carbon atom has one equatorial bond and one axial bond. Cyclohexane itself, in which only hydrogens are attached to the carbon atoms, is not only free of angle strain and torsional strain, but free of van der Waals strain as well. Hydrogens on adjacent carbons are the same distance apart (2.3 A) as in (staggered) ethane and, if anything, feel mild van der Waals attraction for each other. The three axial hydrogens on the same side of the molecule are thrown rather closely together, despite the fact that they are attached to alternate carbon atoms; as it happens, however, they are the same favorable distance apart (2.3 A) as the other hydrogens are. If a hydrogen is replaced by a larger atom or group, crowding occurs. The most severe crowding is among atoms held by the three axial bonds on the same side of the molecule; the resulting interaction is called 1,3-diaxial interaction. Except for hydrogen, a given atom or group has more room in an equatorial position than in an axial position. As a simple example of the importance of 1,3-diaxial interactions, let us consider methylcyclohexane. In estimating relative stabilities of various conformations of this compound, we must focus our attention on methyl, since it is the largest substituent on the ring and hence the one most subject to crowding. There are two possible chair conformations (Fig. 3.12), one with — CH3 in an equatorial position, the other with - CH3 in an axial position. We would expect the equatorial conformation to be the more stable, and it is, by about 1.8 kcal.

Equatorial- CH3 Axial- CH3 Fig. 3. 12. Chair conformations of methylcyclohexane.

Equatorial -CH3 Axial - CH3 Fig. 3. 13 1,3-Diaxial interaction in methylcyclohexane. 24

An axial - CH3 is more crowded than an equatorial - CH3. Most molecules (about 95% at room temperature) exist in the conformation with methyl in the uncrowded equatorial position. In an equatorial position, - CH3 points away from its nearest neighbours: the two hydrogens - one axial, and one equatorial - on the adjacent carbons. This is not true of - CH3 in an axial position, since it is held by a bond that is parallel to the bonds holding its nearest neighbours, the two axial hydrogens. In general, then, it has been found that (a) chair conformations are more stable than twist conformations, and (b) the most stable chair conformations are those in which the largest groups are in equatorial positions. As a prove for this could serve the predominant existence of the most abundant carbohydrate on the earth (D-glucose) in its -anomeric form.

3. 2. 2. Configurational isomerism Configurational isomerism includes two types of isomerism: (1) Optical Isomerism. The necessary structural feature of such type of compounds is the presence of asymmetric carbon atom. (2) Geometrical Isomerism. The isomerism due to difference in spatial arrangements of groups about the doubly bonded carbon atoms is called geometrical isomerism. Examining the structure of 2-Butene we find that its atoms can be arranged in two different ways. Conversion of them will involve rotation about the carbon-carbon double bond. The isomer in which the similar groups lie on the same side is called the cis isomers (Latin, cis = on same side) and the other in which the similar groups lie on the opposite sides is called the trans isomer (Latin trans = across). Due to these cis and trans isomers, geometrical isomerism is called cis-trans isomerism.

I II Fig. 3. 14. cis-2 Butene (b.p. 277 K) trans -2-Butene (b p. 274 K) Geometrical isomers.

Conversion of I into II will involve rotation about the carbon-carbon double bond which is a combination of one  bond and one  bond. To pass from structure I to II, the molecule must be twisted and the  bond is broken. 167.2 kJ of energy are required for breaking a  bond and an insignificant number of collisions possess this much energy. As a result of this hindered

25 rotation the positions of different groups attached to the two carbon atoms are fixed in space. Necessary condition for an olefin to show geometrical isomerism is that the two atoms or groups attached to each carbon atom joined by a double bond are different.

3.2.3. Molecular . The stereogenic center. . Symmetry of form abounds in classical solid geometry. A sphere, a cube, a cone, and a tetrahedron are all identical to, and can be superposed point for point on their mirror images. Mirror-image superposability also exists in many objects used every day. Cups and saucers, forks and spoons, chairs and beds, are all identical to their mirror images. Many other objects, however, cannot be superposed on their mirror images. Your left hand and your right hand, for example, are mirror images of each other but cannot be made to coincide point for point in three dimensions. In geometry, an object that is not superposable on its mirror image is said to be dissymmetric, where the prefix dis- signifies an opposite quality. In chemistry, the word that corresponds to dissymetric is chiral, as in a chiral molecule. Chiral is derived from the Greek word cheir, meaning "hand," in that it refers to the "handedness" of molecules. The opposite of chiral is achiral. A molecule that is superposable on its mirror image is achiral.

A B B Fig. 3.15. (a) Structures A and B are mirror-image representations of bromochlorofluoromethane (BrClFCH).

Structures A and B are mirror-image representations of bromochlorofluoromethane (BrClFCH). To test for superposability, let us reorient B by turning it 180° and compare A and B. The two do not match. A and B cannot be superposed upon each other. Therefore, bromochlorofluoromethane is a chiral molecule. The two mirror-image forms are enantiomers of one another. The two mirror-image forms are not superposable. So, molecules of the general type:

are chiral.

Van't Hoff pointed out that a molecule is asymmetric (without symmetry) 26 when four different groups are arranged in a tetrahedral fashion around one of its carbon atoms. The word describes a particular relationship between two objects. A chiral molecule can have one, and only one, enantiomer. Noting the presence of a stereogenic center in a given molecule is a simple, rapid way to determine that the molecule is chiral. For example, C-2 is a stereogenic centre in 2-butanol; it bears a hydrogen atom and methyl, ethyl, and hydroxyl groups as its four different substituents. By way of contrast, none of the carbon atoms bear four different groups in the achiral alcohol 2-propanol (Fig. 3.15).

Fig. 3.16. 2-Butanol 2-Propanol.

Molecules with stereogenic centers are very common, both as naturally occurring substances and as the products of . In the following examples, the stereogenic carbon is indicated by an asterisk (Fig.3.17.). (Carbons that are part of a double bond or a triple bond cannot be stereogenic centers.)

Fig. 3. 17. 4-Ethyl-4-methyloctane (a chiral alkane) Linalool

A carbon atom in a ring can be a stereogenic center if it bears two different substituents and the path traced around the ring from that carbon in one direction is different from that traced in the other. The carbon atom that bears the methyl group in 1,2-epoxypropane, for example, is a stereogenic center. The sequence of groups is CH2-O as one proceeds clockwise around the ring from that atom but is O-CH2 in the anticlockwise direction (Fig.3.18.). * CH3CH—CH2 O Fig. 3. 18. 1-2-Epoxypropane (product of epoxidation of propene)

Molecules with more than one stereogenic center may or may not be chiral. Symmetry in achiral structures. A molecule that has any element of symmetry, such as a plane of symmetry or a center of symmetry is superposable on its mirror image is achiral. 27

3.2.3.1 Properties of chiral molecules: optical activity The experimental facts that led van't Hoff and Le Bel to propose that molecules having the same constitution could differ in the arrangement of their atoms in space concerned a physical property called optical activity. Optical activity is measured by using an instrument called a . A polarimeter contains a device called a Nicol prism, which transmits only those light waves having their electric field components in the same plane. This is plane-polarized light.

Fig. 3. 19. Unpolarized The polarizer plane-polarized light from source light

A solution containing the substance being examined is then placed in the path of the beam of plane-polarized light. When the substance is achiral or contains equal amounts of enantiomers, the plane of polarization of the emergent beam is the same as that of the incident beam.

Fig. 3. 20. Plane-polarized Solution containing No change in plane of light. achiral substance or equal polarization quantities of enantiomers.

A substance which does not cause rotation of the plane of polarized light is said to be optically inactive. All achiral substances are optically inactive. A chiral substance is optically inactive if equal quantities of enantiomers are present. When the substance is chiral and one enantiomer is present in excess of the other, then the plane of polarization is rotated through some angle .

Fig. 3. 21. Plane-polarized Solution of a chiral substance Plane of polarization light in which more of one enantiomer has undergone than the other is present a rotation.

A substance which causes the plane of polarized light to undergo a rotation is said to be optically active. The angle of rotation  could be measured by polarimeter. Enantiomeric forms of a chiral molecule cause a rotation of the plane of 28 polarization in exactly equal amount but in opposite directions. Therefore a solution containing equal quantities of enantiomers exhibits no net rotation because all the tiny increments of clockwise rotation produced by molecules of one "handedness" are canceled by an equal number of increments of anticlockwise rotation produced by molecules of the opposite handedness. Mixtures containing equal quantities of enantiomers are called racemic mixtures. Racemic mixtures are optically inactive. Conversely, when one enantiomer is present in excess, a net rotation of the plane of polarization is observed. At the limit, where all the molecules are of the same handedness, we say the substance is optically pure. Rotation of the plane of polarized light in the clockwise sense is taken as positive (+), while rotation in the anticlockwise sense is taken as a negative (-) rotation. The classical terms for positive and negative rotations are dextrorotatory and levorotatory respectively. Dextro- and levo- are Latin prefixes, meaning "to the right" and "to the left," respectively. Formerly, the symbols d and l were used to distinguish between enantiomeric forms of a substance. Thus the dextrorotatory enantiomer of 2-butanol was called d-2- butanol, the levorotatory form l-2-butanol; and a of the two was referred to as dl-2-butanol. Current custom favors using algebraic signs instead, as in (+)-2-butanol, (-)-2-butanol, and (±)-2-butanol, respectively. Specific rotation is a physical property of a substance, just as melting point, boiling point, density, and solubility are. For example, the lactic acid obtained from milk is exclusively a single enantiomer. We cite its specific 25 rotation in the form []D = +3.8°. The temperature in degrees Celsius and the wavelength of light at which the measurement was made are indicated as superscripts and subscripts, respectively.

3.2.3.2. Displaying Molecular Shapes. Absolute and relative configuration The precise arrangement of substituents at a chiral center is its absolute configuration. Neither the sign nor the magnitude of rotation by itself provides any information concerning the absolute configuration of a substance. Thus, one of the following structures is (+)-2-butanol and the other is (-)-2-butanol, but in the absence of additional information we cannot tell which is which (Fig . 3.22.A).

? (+)-2-Butanol (-)-2-Butanol Fig . 3. 22. A B 29

While no absolute configuration was known for any substance before 1951, organic chemists had experimentally determined the configurations of thousands of compounds relative to one another (their relative configurations) through chemical interconversion. Since 1951, when the absolute configuration of a of (+)-tartaric acid was determined, the absolute configurations of all the compounds whose configurations had been related to (+)-tartaric acid stood revealed as well. Now for the pair of enantiomers mentioned above, their absolute configurations have been firmly established as shown (Fig . 3.22.B). Absolute configuration. Molecular shape is critical to the proper functioning of biological molecules. The tiniest difference in shape can cause two compounds to behave differently or to have different physiological effects in the body. It's therefore critical that chemists have techniques available both for determining molecular shapes with great precision and, for visualizing these shapes in useful and manageable ways. Three-dimensional shapes of molecules are determined by X-ray , a technique that allows us to "see" molecules in a crystal using X-ray waves. The molecular "picture" obtained by X-ray crystallography looks at first like a series of regularly spaced dark spots on a photographic film. After computerized manipulation of the data, however, recognizable molecules can be drawn. Relatively small molecules like are usually displayed on paper, but enormous biological molecules like immunoglobulins are best displayed on computer terminals where their structures can be enlarged, rotated, and otherwise manipulated for the best view.

3.2.3.3. Nomenclature for Chiral Molecules The discoveries of optical activity and enantiomeric structures made it important to develop suitable nomenclature for chiral molecules. Two systems are in common use today: the so-called D,L system and the (R,S) system. In the D,L system of nomenclature, isomers of glyceraldehyde are denoted as D- glyceraldehyde and L-glyceraldehyde, respectively (Fig. 3.23.). Absolute configurations of all other carbon-based molecules are referenced to D- and L- glyceraldehyde. When sufficient care is taken to avoid racemization of the amino acids during hydrolysis of proteins, it is found that all of the amino acids derived from natural proteins are of the L configuration. Amino acids of the D configuration are nonetheless found in nature, especially as components of certain peptide antibiotics, such as valinomycin, gramicidin, and actinomycin D, and in the cell walls of certain microorganisms.

30

Fig. 3.23. The assignment of L and D and (R) and (S) notation for glyceraldehyde.

In spite of its widespread acceptance, problems exist with the D,L system of nomenclature. For example, this system can be ambiguous for molecules with two or more chiral centers. To address such problems, the (R,S) system of nomenclature for chiral molecules was proposed in 1956. In this more versatile system, priorities are assigned to each of the groups attached to a chiral center on the basis of atomic number, atoms with higher atomic numbers having higher priorities. The newer (R,S) system of nomenclature is superior to the older D,L system. Nevertheless D,L –system continue be more favorable in the course of Bioorganic chemistry. Rules for Description of Chiral Centers in the (R,S) System* (* additional useful information) Naming a chiral center in the (R,S) system is accomplished by viewing the molecule from the chiral center to the atom with the lowest priority. If the other three atoms facing the viewer then decrease in priority in a clockwise direction, the center is said to have the (R) configuration (where R is from the Latin rectus meaning "right"). If the three atoms in question decrease in priority in a counterclockwise fashion, the chiral center is of the (S) configuration (where S is from the Latin sinistrus meaning "left"). If two of the atoms coordinated to a chiral center are identical, the atoms bound to these two are considered for priorities. For such purposes, the priorities of certain functional groups found in amino acids and related molecules are in the following order: SH > OH > NH2 > COOH > CHO > CH2OH > CH3 From this, it is clear that D-glyceraldehyde is (R)-glyceraldehyde, and L-alanine is (S)-alanine (see figure 3.23.). Interestingly, the -carbon configuration of all the L- amino acids except for is (S). Cysteine, by virtue of its thiol group, is in fact (-R)-cysteine. Fischer projection formulas. Stereochemistry is concerned with the three-dimensional arrangement of a molecule's atoms, and it is used to show stereochemistry with wedge-and-dash drawings and computer-generated ball- and-stick models. It is possible, however, to convey stereochemical information in an abbreviated form using a method devised by the German Emil Fischer. Fischer projections are always generated the same way: 31 the molecule is oriented so that the vertical bonds at the stereogenic center are directed away from you and the horizontal bonds point toward you. A projection of the bonds onto the page is a cross. The stereogenic carbon lies at the center of the cross but is not explicitly shown. It is customary to orient molecules with several carbons so that the carbon chain is vertical as shown for the Fischer projection of (R)-2-butanol.

(R)–2-Butanol or D-Butanol

Fischer projections offer an easy way to draw three-dimensional molecules on paper in two dimensions. The atoms are all projected onto one plane. Fig. 3.24. shows the projection idea for glyceraldehyde, for which the Fischer projections of the two enantiomers are

Fig. 3.24 D- glyceraldehyde L- glyceraldehyde

The Fischer rules for showing the array around a chiral center are as follows: 1 Write down or at least envision the carbon chain of the compound writ- ten vertically with the at the top. 2 Represent the chiral carbon(s) as the intersection of crossed lines, i.e.,

3 Substituents on the vertical lines are understood to be going back behind the plane of the paper. The chiral center is in the paper plane. 4. Substituents on the horizontal lines are understood to be coming forward out of the paper plane. Physical properties of enantiomers. The usual physical properties of density, melting point, and boiling point are identical within experimental error for both enantiomers of a chiral compound. Enantiomers can have striking differences, however, in properties that depend on the arrangement of atoms in space. Take, for example, the enantiomeric forms of carvone. (R)-(–)-carvone is the principal component of spearmint oil. Its enantiomer, (S)-(+)-carvone, is the principal 32 component of caraway seed oil. Each of two enantiomeric forms of carvone has its own characteristic odor.

CH3 CH3 O O

C C CH2 CH H3C H3C 2 Fig. 3.25 (R)-(-)-Carvone (S)-(+)-Carvone (from spearmint oil) (from caraway seed oil)

The reason for the difference in odor between (R)- and (S)-carvone results from their different behavior toward receptor sites in the nose. It is believed that volatile molecules occupy only those receptor sites that have the proper shape to accommodate them. These receptor sites are themselves chiral, so that one enantiomer may fit one kind of receptor site while the other enantiomer fits a different kind of receptor. One analogy that can be drawn is to hands and gloves. Your left hand and your right hand are enantiomers. You can place your left hand into a left glove but not into a right one. The receptor site (the glove) can accommodate one enantiomer of a chiral object (your hand) but not the other. The term chiral recognition has been coined to refer to the process whereby some chiral receptor or reagent interacts selectively with one of the enantiomers of a chiral molecule. Very high levels of chiral recognition are common in biological processes. (-)-Nicotine, for example, is much more toxic than (+)-nicotine, and (+)- adrenaline is more active in the constriction of blood vessels than (-)- adrenaline. (-)-Thyroxine is an amino acid of the thyroid gland, which speeds up metabolism and causes nervousness and loss of weight. Its enantiomer, (+)-thyroxine, exhibits none of these effects but is sometimes given to heart patients to lower their cholesterol levels.

Fig. 3.26. Nicotine Adrenaline Thyroxine

Stereochemistry in chemical reactions that produce chiral molecules. Many of the reactions can produce a chiral product from an achiral starting material. A large number of the reactions of alkenes, for example, fall into this 33 category. In the following group of examples, addition to their carbon-carbon double bonds converts alkenes to products that contain a stereogenic center (Fig. 3.27.).

Fig. 3. 27.

In these and related reactions, the chiral product is formed as a racemic mixture and is optically inactive. Remember, in order for a substance to be optically active, not only must it be chiral but one enantiomer must be present in excess of the other. It is a general principle that optically active products cannot be formed when optically inactive substrates react with optically inactive reagents. This principle holds irrespective of whether the addition is syn or anti, concerted or stepwise. No matter how many steps are involved in a reaction, if the reactants are achiral, formation of one enantiomer is just as likely as the other and a racemic mixture results. When a substrate is chiral but optically inactive because it is racemic, any products derived from its reactions with optically inactive reagents will be optically inactive. Optically inactive starting materials can give optically active products if they are treated with an optically active reagent or if the reaction is catalyzed by an optically active substance. The best examples of these phenomena are found in biochemical processes. Most biochemical reactions are catalyzed by . Enzymes are chiral and enantiomerically homogeneous; they provide an asymmetric environment in which chemical reaction can take place. Ordinarily, - catalyzed reactions occur with such a high level of stereoselectivity that one enantiomer of a substance is formed exclusively even when the substrate is achiral. The enzyme fumarase, for example, catalyzes the hydration of fumaric acid to malic acid in apples and other fruits. Only the S enantiomer of malic acid is formed in this reaction (Fig. 3. 28.). H COOH H H C=C + H2O ⇄ C=C HOOC H HOOC COOH Fig. 3.28. Fumaric acid (S)-(-)-Malic acid or L-malic acid 34

The reaction is a reversible one, and its stereochemical requirements are so pronounced that neither the cis isomer of fumaric acid (maleic acid) nor the R enantiomer of malic acid can serve as a substrate for the fumarase-catalyzed hydration-dehydration equilibrium. Achiral molecules with two stereogenic centers. For compounds with more than ONE chiral carbon, it sometimes turns out that there are fewer than the maximum number of stereoisomers. Tartaric acid offers an example of this phenomenon. Isomers III and IV are nonsuperimposable mirror images of one another; i.e., they are enantiomers (3.29). COOH COOH COOH COOH COOH COOH H – C – OH HO – C – H H – C – OH H – C – OH HO – C – H H – C – OH HO – C – H H – C – OH H – C – OH HO – C – H H – C – OH H – C – OH COOH COOH COOH COOH COOH COOH Fig.3.29. I II III IV

Isomers I and II appea to be enantiomers, but it happens that they are identical. If you rotate Fischer projection I 180° in the plane of the paper you will find that it superimposes exactly with II. If I=II, then there are only three stereoisomers for tartaric acid. A compound such as this unique isomer of tartaric acid is called a meso compound. Meso compounds are characterized by an internal reflection plane, that is, one-half of the molecule reflects the other. It is also true in meso compounds that each chiral carbon has the same set of four different substituents. For meso-tartaric acid this set is -H, -OH, — COOH, and –CHOH-COOH. Because the common carbohydrates that you will study all have differently substituted chiral carbons, you will not encounter meso compounds among them. That is, the 2n rule for the number of existing isomeric structures will apply. . Stereoisomers that are not related as an object and its mirror image are called diastereomers; diastereomers are stereoisomers that are not enantiomers. Thus, stereoisomer I is a of III and a diastereomer of IV. In order to convert a molecule with two stereogenic centers to its enantiomer, the configuration at both centers must be changed. Reversing the configuration at only one stereogenic center converts it to a diastereomeric structure. Enantiomers must have equal and opposite specific rotations. Diastereomeric substances can have different rotations, with respect to both sign and magnitude. Organic chemists use an informal nomenclature system based on Fischer projections to distinguish between diastereomers. When the carbon chain is vertical and like substituents are on the same side of the Fischer projection, 35

the molecule is described as the erythro diastereomer. When like substituents are on opposite sides of the Fischer projection, the molecule is described as the threo diastereomer. Thus, as seen in the Fischer projections of the stereoisomeric 2,3-dihydroxybutanoic acids, compounds I and II are erythro stereoisomers and III and IV are threo. COOH COOH COOH COOH COOH COOH H – C – OH HO – C – H H – C – OH H – C – OH HO – C – H H – C – OH HO – C – H H – C – OH H – C – OH HO – C – H H – C – OH H – C – OH COOH COOH COOH COOH COOH COOH I II III IV Fig.3. 30. erythro erythro threo threo

Because diastereomers are not mirror images of each other, they can have, and often do have, markedly different physical and chemical properties.

Fig. 3. 31. (2R,3R)-2,3-Butanediol (2.S,3S)-2,3-Butanediol meso-2,3-Butanediol

In the same way that a Fischer formula is a projection of the eclipsed conformation of meso-2,3-butanediol onto the page, the line drawn through its center is a projection of the plane of symmetry which is present in the eclipsed conformation

(a) (b) Fig. 3.32. (a) The eclipsed conformation of meso-2,3-butanediol has a plane of symmetry, (b) The anti conformation of meso-2,3-butanediol has a center of symmetry.

Molecules with multiple stereogenic centers. Many naturally occurring compounds contain several stereogenic centers. By an analysis similar to that 36 described for the case of two stereogenic centers, it can be shown that the maximum number of stereoisomers for a particular constitution is 2n, where n is equal to the number of stereogenic centers. When two or more of a molecule's stereogenic centers are equivalently substituted, meso forms are possible, and the number of stereoisomers is then less than 2n Thus, 2n represents the maximum number of stereoisomers for a constitutional formula containing n stereogenic centers. The best examples of substances with multiple stereogenic centers are the carbohydrates (Chapter 11). One class of carbohydrates, called hexoses, has the constitution

a hexose

Since there are four stereogenic centers and there is no possibility of meso forms, there are 24, or 16, stereoisomeric hexoses. All 16 are known, having been isolated either as natural products or as the products of chemical synthesis. Steroids represent another class of natural products with multiple stereogenic centers. One such compound is cholic acid, which can be obtained from bile. Cholic acid has 11 stereogenic centers, and so there are total (including cholic acid) of 211, or 2048, stereoisomers that have this constitution. Of these 2048 stereoisomers, how many of them are diastereomers of cholic acid? Only one of the stereoisomers is an enantiomer of cholic acid, while all the rest are diastereomers. Of the 2048 stereoisomers, one is cholic acid, one is its enantiomer, and the other 2046 are diastereomers of cholic acid. Only a small fraction of these compounds are known, and (+)- cholic acid is the only one ever isolated from natural sources. Eleven stereogenic centers may seem like a lot, but this number is nowhere close to a world record. Palytoxin, a very poisonous polyhydroxylated substance produced by a Tahitian marine organism, has 64 stereogenic centers. Even this number seems modest when we note that most proteins and nucleic acids have well over 100 stereogenic centers. If a molecule contains both stereogenic centers and double bonds, additional opportunities for stereoisomerism arise. For example, the configuration of the stereogenic center in 3-penten-2-ol may be either R or S, and that of the double bond may be either E or Z. Therefore, even though 3- penten-2-ol has only one stereogenic center, there are four stereoisomeric forms.

37

4. MUTUAL INFLUENCE OF ATOMS IN MOLECULES OF ORGANIC COMPOUNDS

4. 1 Conjugation as a factor of stabilization of organic compounds. Conjugation, mesomerism or resonance. It was found that no structural peculiarity could satisfactorily explain all the properties of certain compounds, e.g. , , especially their high stability. This led to the idea that such compounds, containing conjugated double bonds exist in a state which is some combination of two or more electronic structures. Relative stabilities of alkadienes. Electron delocalization in conjugated  –cojugation. The factor most responsible for the increased stability of conjugated double bonds is the greater delocalization of their  electrons compared with the  electrons of isolated double bonds (Fig. 4.1.).

Fig. 4.1. (a) Isolated double bonds (b) Conjugated double bonds

The  electrons of an isolated system occupy, in pairs, two noninteracting p orbitals. Each of these p orbitals encompasses two carbon 3 atoms (Fig. 4.1.). A sp hybridized carbon insulates the two pz orbitals from each other, preventing the exchange of electrons between them. In a conjugated diene, however, mutual overlap of the two pz orbitals, gives an orbital system in which each pz electron is delocalized over four carbon atoms. Delocalizing of electrons lowers their energy and gives a more stable molecule. Conjugate is a Latin verb meaning “to link or yoke together”. The simplest example of conjugate system is butadiene-1.3. CH2=CH–CH= CH2

Fig. 4.2. A conjugated diene- butadiene-1,3.

Such type of conjugation is called -conjugation (Fig.4.2) and is common also for the  unsaturated carbonyl compounds such as propenal 38 and propenoic acid. -conjugation is formed due to conjugation of double bonds between carbon- carbon and carbon and heteroatom as well. CH2 = CH – COH CH2 = CH – COOH Propenal propenoic acid

At 146 pm the C-2 — C-3 distance in 1,3-butadiene is relatively short for a carbon-carbon single bond (Fig.4.3). This is most reasonably seen as a hybridization effect. In ethane both carbons are sp3 hybridized and are separated by a distance of 153 pm. The carbon-carbon single bond in propene unites sp3 and sp2 hybridized carbons and is shorter than that of ethane. Both C-2 and C-3 are sp2 hybridized in 1,3-butadiene, and a decrease in bond distance between them is consistent with the tendency of carbon to attract electrons more strongly as its s character increases.

Figure 4.3.

A convenient way to represent benzene as a resonance hybrid of the two Kekule structures is by inscribing a circle inside a hexagon.

The circle reminds us of the delocalized nature of the electrons. It was first suggested by the British chemist Sir Robert Robinson as a convenient symbol for the aromatic sextet, the six delocalized n electrons. Ingold called the phenomenon mesomerism. Heisenberg from , supplied a theoretical background for mesomerism, it is called resonance. Arguments based on quantum mechanics shows that a resonating hybrid would be more stable than any single resonating structures i.e. the internal energy of a resonance hybrid is less than that calculated for any one of the resonating structures. The difference between the heat of formation of the actual compound i.e. the observed value and that of the resonating structure which has the lowest internal energy (obtaining by evaluation) is called the resonance energy. The resonance energy of a molecule is a property of the molecule in the ground state. Another property of the resonance hybrid which differs from that of any of the resonating structures is that of the bond length, i.e. the distance between atoms joined by a covalent bond. The normal length of the 39 carbonyl double bond =C=O in is about 1.22A, the value found in is 1.15A.For a given pair of atom, the length of single bond is greater than that of double bond, which, in turn, is greater than that of a triple bond. Resonance, therefore, account for the carbonyl bond in carbon dioxide not being single double or triple.

4.2. Conjugation in alkadienes and allylic systems. p, -conjugation Not all of the properties of alkenes can be understood by focusing exclusively on the functional group behaviour of the double bond. A double bond can affect the properties of a second functional unit to which it is directly attached. It can be a substituent, for example, or a positively charged carbon in an allylic carbocation, or a carbon that bears an unpaired electron in an allylic free radical; or it can be a substituent on a second double bond in a conjugated diene.

Allylic carbocation Allylic free radical Conjugated diene

Allylic carbocations, allylic free radicals, and conjugated dienes are all examples of conjugated systems. The . The group CH2=CHCH2— is known as allyl, which is both a common name and a permissible IUPAC name. It is most often encountered in functionally substituted derivatives, and the following compounds containing this group are much better known by their radicofunctional names than by their substitutive names: CH2=CHCH2OH CH2=CHCH2Cl CH2=CHCH2Br Allyl alcohol Allyl chloride Allyl bromide (2-propen-l-ol) (3-chloro-l-propene) (3-bromo-l-propene)

Allyl is derived from the botanical name for garlic (Album sativum). The adjective allylic denotes the structural unit C==C—C. The sp3 hybridized carbon of an allylic unit is called the allylic carbon, and an allylic substituent is that is attached to an allylic carbon. Conversely, the sp2 hybridized carbons of a carbon-carbon double bond are called vinylic carbons, and substituents attached to either one of them are referred to as vinylic substituents (Fig. 4.4.).

Fig. 4. 4. 40

Allylic is often used as a generic term to refer to a molecule which bears a functional group at an allylic position. Allylic carbocations. Allylic carbocations are carbocations that have a or substituted vinyl group as a substituent on their positively charged carbon. The allyl cation is the simplest allylic carbocation. Representative allylic carbocations

Allyl cation l-Methyl-2-butenyl cation 2-Cyclopentenyl cation

A substantial body of evidence indicates that allylic carbocations are more stable than simple alkyl cations. Structurally, the two carbocations differ in that the allylic carbocation has a vinyl substituent on its positively charged carbon in place of one of the methyl groups of tert-butyl cation.

tert-Butyl cation 1,1-Dimethylallyl cation (less stable) (more stable)

A vinyl group stabilizes a carbocation more than does a methyl group. Why? A vinyl group is an extremely effective electron-releasing substituent. A resonance interaction of the type shown permits its  electrons to be delocalized and disperses the positive charge. Because it is a resonance-stabilized species, this allylic carbocation is formed faster than tert-butyl cation. Allylic halides undergo ionization to form carbocations faster than do alkyl halides. Allylic free radicals (p –conjugation). Just as allyl cation is stabilized by electron delocalization, so is allyl radical (Fig. 3.5.):

or

Fig. 4.5. Allyl radical

Allyl radical is a in which a singly occupied 2p orbital overlaps with the  orbital of an adjacent double bond to give an extended  system. The  electrons are delocalized over all three carbons. The unpaired electron has an equal probability of being found at C-1 or C-3. Another example of p –conjugation is compounds having heteroatom with unshared pair of electrons attached to the carbon linked with double bond to the other neighbor atom (Fig. 4.6.). 41

C= C – X ; X= O, Hal, S

CH2 = CH CH2 = CH Z Cl CH2 = CH – O – CH = CH2 Fig. 4.6.

4.3. Arenes and . Benzene. The Huckel 4n+2 rule We have defined aromatic compounds as those that resemble benzene. The aromatic compounds are compounds whose molecular formulas would lead us to expect a high degree of unsaturation, and yet which are resistant to the addition reactions generally characteristic of unsaturated compounds. Aromatic compounds: 1. undergo electrophilic substitution reactions like those of benzene, 2. are resistant toward addition- evidence of unusual stability, 3. are cyclic-generally containing five-, six-, or seven-membered rings, 4. are found to have flat (or nearly flat) molecules, 5. a molecule that contains cyclic clouds of delocalized  electrons above and below the plane of the molecule; furthermore, the  clouds must contain a total of (4n + 2)  electrons. This requirement, called the Huckel rule, is based on quantum mechanics. Benzene has six n electrons, the aromatic sextet; six is, of course, a Huckel number, corres- ponding to n = 1. Arenes are hydrocarbons based on the benzene ring as a structural unit. Benzene, , and , for example, are arenes.

Benzene Toluene Naphthalene

A conjugated system of  electrons in arenes can have properties that are much different from those of open-chain polyenes. Arenes are also referred to as aromatic hydrocarbons. The word aromatic has nothing to do with odor but rather refers to a level of stability for arenes that is substantially greater than that expected on the basis of their formulation as conjugated trienes. For the particular degree of stability that characterizes an , delocalization alone is not enough. There must be a particular number of  electrons: 2, or 6, or 10, etc. This requirement, called the 4n+2 rule or Huckel rule, is based on quantum mechanics. The Huckel rule is strongly supported by the facts. Huckel's rule states: among planar, monocyclic, fully 42 conjugated polyenes, only those possessing (4n+2) electrons, where n is an integer (0,1,2, ….т) will have special aromatic stability. Besides benzene and its relatives (naphthalene, , ), we shall encounter a number of heterocyclic compounds that are clearly aromatic; these aromatic heterocycles are just the ones that can provide an aromatic sextet. Cyclobutadiene and cyclooctatetraene Structural studies confirm the absence of appreciable  electron delocalization in cyclooctatetraene. Cyclooctatetraene has four noninteracting double bonds. The evidence clearly indicates that cyclooctatetraene is not at all like benzene and is more appropriately considered to be a cyclic polyene.

Cyclobutadiene Cyclooctatetraene

Cyclobutadiene escaped chemical characterization for more than 100 years. Despite numerous attempts, all synthetic efforts met with failure. It became apparent not only that cyclobutadiene was not aromatic but that it was exceedingly unstable. Structural studies of cyclobutadiene and some of its derivatives indicate that it is best described as a diene with alternating single and double bonds and a rectangular, rather than a square, shape. All the available evidence shows that neither cyclooctatetraene nor cyclobutadiene are aromatic. Cyclic conjugation, while necessary for aromaticity, is not sufficient for it. These compounds didn`t meet the requirements of the Huckel rules. Only when the number of  electrons is 2,6,10, 14, and so on, can a closed-shell electron configuration be realized. Hückel's rule is now taken to apply to planar monocyclic completely conjugated systems generally, not just to neutral hydrocarbons. A planar monocyclic continuous system of orbitals possesses aromatic stability when it contains (4n + 2)  electrons. Aromatic include cyclopropenyl cation (two  electrons) and cyclooctatetraene dianion (ten electrons) also meet the Huckel rules requirements (Fig. 3.7.).

Cyclopropenyl Cyclooctatetraene cation dianion

Fig. 4.7.

43

Benzene reactivity. Under conditions in which bromine adds rapidly to alkenes and alkynes, benzene proved to be inert. When bromination was carried out in the presence of catalysts such as Fe(III) bromide, the reaction that took place was not addition but substitution!

The stability of benzene. Hydrogenation of benzene and other arenes is more difficult than hydrogenation of alkenes and alkynes. The more active catalysts are nickel, rhodium and platinum, and it is possible to hydrogenate arenes in the presence of these catalysts at room temperature and modest pressure. Benzene consumes three molar equivalents of hydrogen to give cyclohexane. The heat of hydrogenation of benzene is less than expected for a hypothetical 1,3,5-cyclohexatriene with noninteracting double bonds. This is the empirical resonance energy of benzene152 kJ/mol (36 kcal/mol). The picture portrayed in Figure 4.8. is a useful model of electron distribution in benzene and vividly depicts the delocalization of its  electrons. The resonance energy of benzene is quite large, 6 to 10 times the resonance energy of a conjugated triene. It is this very large increment of resonance energy that places benzene and related compounds in a separate category and accords to them the description aromatic.

Fig. 4. 8. (a) The 2p orbitals of benzene carbon atoms are suitably aligned for maximum a overlap, (b) Overlap of the 2p orbitals generates a  system encompassing the entire ring.

There are regions of high  electron density above and below the plane of the ring. All compounds that contain a benzene ring are aromatic, and substituted derivatives of benzene make up the largest class of aromatic compounds.

44

Table 4.1. Names of some common benzene derivatives (These common names are acceptable in IUPAC nomenclature).

Styrene Acetophenone Phenol Aniline

A class of compounds called polycyclic benzenoid aromatic hydrocarbons is composed of arenes which possess substantial resonance energies because they are collections of benzene rings fused together. Naphthalene, anthracene, and phenanthrene are the three simplest members of this class. They are all present in coal tar, a mixture of organic substances formed when coal is converted to coke by heating at high temperatures (about 1000 C) in the absence of air.

4.3.1. Physical properties of arenes They are nonpolar materials, insoluble in water, and less dense than water. In the absence of polar substituent groups, intermolecular forces are weak and limited to van der Waals attractions of the induced dipole-induced dipole type. Not long ago, and in spite of its flammability, benzene was widely used as a solvent. This use virtually disappeared once it was demonstrated that benzene is a carcinogen and statistical evidence revealed a greater than average incidence of leukemia among workers exposed to atmospheric levels of benzene as low as 1 ppm. Toluene has replaced benzene as an inexpensive organic solvent, because it has similar solvent properties but has not been determined to be carcinogenic in the cell systems and at the dose levels that benzene is.

4.3.2. Reactions of arenes A carbon atom that is directly attached to a benzene ring is called a benzylic carbon (analogous to the allylic carbon of C=C—C). A (C6H5—) is an even better conjugating substituent than a vinyl group 45

(C=C—), and benzylic carbocations and radicals are more highly stabilized than their allylic counterparts. The double bond of an alkenylbenzene is stabilized to about the same extent as that of a conjugated diene.

Side-chain oxidation of is important in certain metabolic processes. One way in which the body gets rid of foreign substances is by oxidation in the liver to compounds more easily excreted in the urine. Toluene, for example, is oxidized to benzoic acid by this process and is eliminated rather readily.

Toluene Benzoic acid

Benzene, with no alkyl side chain, undergoes a different reaction in the presence of these enzymes, which convert it to a substance capable of inducing mutations in DNA (deoxyribonucleic acid). This difference in chemical behavior seems to be responsible for the fact that benzene is carcinogenic while toluene is not.

4.4. Heterocyclic aromatic compounds Cyclic compounds that contain at least one atom other than carbon within their ring are called heterocyclic compounds, and those heterocyclic compounds which possess aromatic stability are called heterocyclic aromatic compounds. Some representative heterocyclic aromatic compounds are , pyrrole, furan, and thiophene. The structures and the IUPAC numbering system used in naming their derivatives are shown. In their stability and chemical behavior, all of these compounds resemble benzene more than they resemble alkenes.

Fig. 4. 9. Pyridine Pyrrole Furan Thiophene 46

Pyridine, pyrrole, and thiophene, like benzene, are present in coal tar. Furan is prepared from a substance called furfural obtained from corncobs. Heterocyclic aromatic compounds can be polycyclic as well. A benzene ring and a pyridine ring, for example, can share a common side in two different ways. One mode of fusion creates a compound called quinoline; the other gives isoquinoline.

Quinoline Isoquinoline

Fig. 4. 10. Indole Benzofuran Benzothiophene

Analogous compounds derived by fusion of a benzene ring to a pyrrole, furan, or thiophene nucleus are called indole, benzofuran, and benzothiophene. A large group of heterocyclic aromatic compounds are derived from pyrrole by replacing one of the ring carbons  to nitrogen by a second heteroatom. Compounds of this type are called azoles.

Imidazole Oxazole Thiazole

Cimetidine Firefly luciferin

*The most widely prescribed drug for the treatment of gastric ulcers has the generic name cimetidine and is a synthetic imidazole derivative. Firefly luciferin is a thiazole derivative which is the naturally occurring light-emitting substance present in fireflies. Firefly luciferin is an example of an azole that contains a benzene ring fused to the five-membered ring. Such structures are fairly common. Another example is benzimidazole, present as a structural unit in vitamin B12. Some compounds related to benzimidazole include purine and its amino-substituted derivative adenine, one of the so-called heterocyclic bases found in DNA and RNA). 47

Fig. 4. 11. Benzimidazole Purine Adenine

Hückel's rule applies to heterocyclic aromatic compounds in a manner similar to its application to hydrocarbons and ions. A single heteroatom can contribute either 0 or 2 of its lone-pair electrons as needed to the  system so as to satisfy the (4n + 2)  electron requirement. The lone pair in pyridine, for example, is associated entirely with nitrogen and is not delocalized into the aromatic  system as shown in Figure 4.12.(a). Pyridine is simply a benzene ring in which a nitrogen atom has replaced a CH group. The nitrogen is sp2 hybridized and the three double bonds of the ring contribute the necessary six  electrons to make pyridine a heterocyclic aromatic compound. The unshared electron pair of nitrogen occupies an sp2 orbital in the plane of the ring, not a p orbital aligned with the  system. In pyrrole, on the other hand, the unshared pair of nitrogen must be added to the four  electrons of the two double bonds in order to meet the six  electron require- ment. As shown in Figure 4.12.b, the nitrogen of pyrrole is sp2 hybridized and the pair of electrons occupies a p orbital where both electrons can participate in the aromatic  system. Pyridine and pyrrole are both weak bases, but pyridine is much more basic than pyrrole. When pyridine is protonated, its unshared pair is used to bond to a proton and, since the unshared pair is not involved in the  system, the aromatic character of the ring is little affected. When pyrrole acts as a , the two electrons used to form a bond to hydrogen must come from the  system and the aromaticity of the molecule is sacrificed on protonation.

(a) Pyridine

(b) Pyrrole ( c) Furan Fig. 4. 12. (a) Pyridine, (b) Pyrrole (c) Furan

The oxygen in furan has two unshared electron pairs (Figure 4.12). One pair is like the pair in pyrrole, occupying a p orbital and contributing two electrons to 48 complete the six - electron requirement for aromatic stabilization. The other electron pair in furan is an "extra" pair, not needed to satisfy the 4n + 2 rule for aromaticity, and occupies an sp2 hybridized orbital like the unshared pair in pyridine. The bonding in thiophene is similar to that of furan.

4.5. Inductive effect A covalent bond is a chemical bond formed by sharing electron pairs between two atoms whose difference in electronegativity is less than 1.9 . The covalent bond is more abundant type of bonds in organic compounds. Consider a carbon chain in which one terminal carbon atom is joined to chlorine atom. –C3 – C2 –C1 – Cl Chlorine has a greater electron affinity than carbon therefore the electron pair forming the covalent bond between the chlorine atom carbon will be displaced towards the chlorine atom to aquire a small negative charge and on carbon atom a small +ve charge. Since carbon atom one is +vely charged, it will attract towards itself the electron pair forming the covalent bond between first carbon atom and second carbon atom. This will cause carbon atom second to acquire a small +ve charge, but charge will be smaller than the first carbon atom’s, because chlorine's effect has been transmitted through the first carbon atom to second one. Similarly third carbon atom will acquire small +ve charge but less than first and second carbon atom. This type of electron displacement along a chain is known as the inductive effect, it is permanent and decreases as the distance from the source increases. The inductive effect may be represented in several ways. Inductive effect may be due to atoms or groups. The following is the order of decreasing inductive effects. For measurement of relative inductive effects hydrogen is chosen as reference in the molecule CR3-H as standard. If, when the H atom in this molecule is replaced by Z (ion, atom or group) the electron density in the CR3 part of the molecule is less in this part than in CR3-H, then Z is said to have a (-I) effect (electron attracting ). If the electron density in the CR3 part is greater than in CR3-H, the Z is said to have a (+I) effect (electron repelling).e.g.Br is (-I) and -C2H5 is (+I).

Fig. 4.13. Inductive effect: decreases with distance.

It is typical of inductive effects that they decrease rapidly with distance, and are seldom important when acting through more than four atoms. 49

4.6. The mesomeric effect The mesomeric effect is a permanent polarisation, a permanent displacement of electron pair, occurring in a system under the influence of substituents which are involved in the conjugation. This concerns compounds of following types and aromatic compounds: Z – C = C, e.g. Z = R2N ; Cl. Since there is no multiple bond in this molecule, the mesomeric effect is not possible. When the electronic displacement is away from the group the mesomeric (resonance) effect is said to be (+M) and when towards the group (-M). Inductive and mesomeric effects are permanently operating in the real molecule collectively they are known as the polarisation effect. If theoretically both effects could be observed, remember that almost in all cases mesomeric effect is predominant. Table 4.2. represents effects of substituents.

Table 4.2. Effects of substituents Electron Effects Net Effect Of Substituent Substituent Inductive Mesomeric In Conjugated Systems Alkyl (Methyl, +I - Electron Donor Ethyl) (–NH 2 -I +M +M >> -I -NHAlk1,NAlk2) –OH -I +M +M > -I Halogens -I +M -I > +M –NO2 -I -M Electron Acceptor –COOH -I -M Electron Acceptor –SO3H -I -M Electron Acceptor >C=O -I -M Electron Acceptor

5. ACIDS AND BASES Bronsted acids and bases In the Bronsted definition, an acid donates a proton and a base accepts a proton. The strengths of acids and bases are measured by the extent to which they lose or gain protons, respectively. In these reactions acids are converted to their conjugate bases and bases to their conjugate acids. Acid-base reactions go in the direction of forming the weaker acid and the 50 weaker base.To be called an acid, the species must be more acidic than water and be able to donate a proton to water. Some compounds, such as alcohols, are not acidic toward water, but have an H which is acidic enough to react with very strong bases or with Na.

5.1. Basicity (acidity) and structure The basicity of a species depends on the reactivity of the atom with the unshared pair of electrons, this atom being the basic site for accepting the H+. The more spread-out (dispersed, delocalized) is the electron density on the basic site, the less basic is the species. The acidity of a species can be determined from the basicity of its conjugate base, its stability.

 For bases of binary acids (HnX) of elements in the same group, the larger the basic site X, the more spread-out is the charge. Compared bases must have the same charge (degree of the delocalization of the negative charge depends on the size, polarize ability –SH, -OH).  For like-charged bases of binary acids in the same period, the more unshared pairs of electrons the basic site has, the more delocalized is the charge (the atom’s electronegativity ).  Delocalization can occur via the inductive effect (the radical’s effect), whereby an electronegative atom transmits its electron-withdrawing effect through a chain of  bonds. Electropositive groups are electron-donating and localize more electron density on the basic site. So in general, electron releasing radical increases basicity and decreases the acidity (positive inductive effect leads to the decrease of the partially positive charge on the atom in the acidic center and increase in the electron density) and electron withdrawing radical decreases basicity and increases the acidity (negative inductive effect leads to the increase in the partially positive charge on the atom in acidic center and decrease of the electron density). Lewis acids and bases A Lewis acid (electrophile) shares an electron pair furnished by a Lewis base (nucleophile) to form a covalent (coordinate) bond. The Lewis concept is especially useful in explaining the acidity of an aprotic acid (no available proton), such as BF3.

Lewis base Lewis acid

51

5.2. Classification of organic acids The degree of acidity is determined largely by the kind of atom that holds the hydrogen and in particular, by that atom’s ability to accommodate the electron pair left behind by the departing hydrogen ion. In other words, it depends on the stability of conjugate base formed from acid. The stability of conjugate base depends on the factors listed above. In general, almost all organic compounds could be referred to acids, because of presence of hydrogen in their structure. According to the nature of atom in the “acidic centre” organic acids are classified by convention as “S”, “O”, “N”, “C” acids. Within a given row of the Periodic Table, acidity increases as electronegativity increases: H-CH3 ( R) < H-NH2 (R) < H-OH(R) < H-F H-SH < H-Cl Within a given family (group), acidity increases as the size increases: H-F < H-Cl < H-Br < H-I H-OH < H-SH < H-SeH Sequence of relative acidity of some abundant organic compounds: R-SH > R-OH > R-NH2 > R-CH3 SONC (mnemonic rule)

Alcohols as acids and bases. Of the varied chemical properties of alcohols, there is one pair that underlies all the others; their acidity and basicity. These properties reside, of course, in the functional group of alcohols: the hydroxyl group, -OH. This group is like the hydroxyl group of water. Like water, alcohols are weak acids and weak bases-roughly, about as acidic and as basic as water. It is oxygen, with its unshared electron pairs, that makes an alcohol basic. Like water, alcohols are basic enough to accept a proton from strong acids like hydrogen chloride and hydrogen sulfate, and thus bring about complete dissociation of these acids. For example:

Alcohol Protonated alcohol StrongerBase Weaker base

In alcohols, hydrogen is bonded to the very electronegative element oxygen. The polarity of the O-H bond facilitates the departure of a proton (acidity); electronegative oxygen readily accommodates the negative charge of electrons left behind. The acidity of alcohols is shown by their reaction with active metals to liberate hydrogen gas. 52

The product is called alkoxide: ethoxide. With the possible exception of methanol, alcohols are weaker acids than water. When water is added to an alkoxide, there is obtained and the parent alcohol.

Stronger Stronger Weaker Weaker base acid base acid

he weaker acid, RO-H, is displaced from its salt by the stronger acid, HO- H. In other language, the stronger base, RO-, pulls the proton away from the weaker base, HO-; if RO- holds the proton more tightly than HO-, then RO-H must be a weaker acid than HO-H. Acidity of phenols. Phenols are converted into their salts by aqueous hydroxides, but not by aqueous bicarbonates. The salts are converted into the free phenols by aqueous mineral acids, carboxylic acids, or carbonic acid.

Stronger acid Weaker acid

Stronger acid Weaker acid

Phenols must therefore be considerably stronger acids than water, but considerably weaker acids than the carboxylic acids: most phenols have pKa values of about 10-10, whereas carboxylic acids have pKa, values of about 10-5. Although weaker than carboxylic acids, phenols are tremendously more acidic than alcohols, which have pKa, values in the neighborhood of 10-l6 to 10-18. It is due to differences in stabilities of reactants and products, or stability of conjugate bases. The phenoxide ion is much more stable than alkoxide because of conjugation, delocalization of the negative charge, unlike the alcoxide.

Phenol Phenoxide ion

The electron-attracting substituents increase the acidity of phenols, and electron-releasing substituents decrease acidity. Thus substituents affect acidity of phenols in the same way that they affect acidity of carboxylic acids. It is, of course, opposite to the way these groups affect basicity of amines. Electron- 53 attracting substituents tend to disperse the negative charge of the phenoxide ion, whereas electron-releasing substituents tend to intensify the charge. serve as antidote, effective compounds largely using for the detoxication of the heavy metal’s ions, preventing their poisonous action on the biological systems.

5.2.1. lonization of carboxylic acids. Acidity constant The relatively higher level of acidity of the carboxylic acids also could be explained by the resonance and delocalization of charge and equal disperse among all centers. In aqueous solution a carboxylic acid exists in equilibrium with the carboxylate anion and the hydrogen ion (actually, of course, the + hydronium ion, H3O ). As for any equilibrium, the concentrations of the components are related by the expression

Since the concentration of water, the solvent, remains essentially constant, we can combine it with Kaq to obtain the expression

in which Ka, equals Kaq H2O. This new constant, Ka is called the acidity constant. Every carboxylic acid has its characteristic Ka, which indicates how strong an acid is. Since the acidity constant is the ratio of ionized to un- ionized material, the larger the K, the greater the extent of the ionization (under a given set of conditions) and the stronger the acid. Unsubstituted aliphatic and aromatic acids have Ka values of about 10-4 to 10-5 (0.0001 to 0.00001) (Table 5.1.). This means that they are weakly acidic, with only a slight tendency to release protons. By the same token, carboxylate anions are moderately basic, with an appre- ciable tendency to combine with protons. They react with water to increase the concentration of hydroxide ions, a reaction often referred to as hydrolysis. As a result aqueous solutions of carboxylate salts are slightly alkaline:

The basicity of an aqueous solution of a carboxylate salt is due chiefly, of course, to the carboxylate anions, not to the comparatively few hydroxide ions they happen to generate.

54

The series of relative acidities and basicities are:

Certain substituted acids are much stronger or weaker than a typical acid like CH3COOH. As was mentioned above acidity is determined chiefly by the difference in stability between the acid and its anion. Structure of carboxylate ions.

Non-equivalent: Equivalent: resonance less important resonance more important

Both acid and anion are stabilized by resonance in a case of carboxylic acid, stabilization is far greater for the anion than for the acid. Equilibrium is shifted in the direction of increased ionization, and Ka, is increased. The acidity of a carboxylic acid is due to the powerful resonance stabilization of its anion. This stabilization and the resulting acidity are possible only because of the presence of the carbonyl group. According to the resonance theory, then, a carboxylate ion is a hybrid of two structures which, being of equal stability, contribute equally. Carbon is joined to each oxygen by a " one-and-a-half" bond. The negative charge is evenly distributed over both oxygen atoms.

That the anion is indeed a resonance hybrid is supported by the evidence of bond length.

5.2.2. Effect of substituents on acidity Any factor that stabilizes the anion more than it stabilizes the acid should increase the acidity; any factor that makes the anion less stable should decrease acidity. Electron-withdrawing substituents should disperse the negative charge, stabilize the anion, and thus increase acidity. Electron- releasing substituents should intensify the negative charge, destabilize the anion, and thus decrease acidity (Fig. 5.1). The Ka, values listed in Table are in agreement with this prediction. 55

G - withdraws electrons:, G -releases electrons; stabilizes anion destabilizes anion, strengthens acid weakens acid Fig. 5.1.

Table 5.1. Acidity constants of carboxylic acids

Looking first at the aliphatic acids, we see that the electron-withdrawing halogens strengthen acids: chloroacetic acid is 100 times as strong as , dichloroacetic acid is still stronger, and trichloroacetic acid is more than 10 000 times as strong as the unsubstituted acid. The other halogens exert similar effects. -Chlorobutyric acid is about as strong as chloroacetic acid. As the chlorine is moved away from the —COOH, however, its effect rapidly dwindles:  -chloro-butyric acid is only six times as strong as butyric acid, and chlorobutyric acid is only twice as strong. It is typical of inductive effects that they decrease rapidly with distance, and are seldom important when acting through more than four atoms:

Inductive effect: decreases with distance

The aromatic acids are similarly affected by substituents: -CH3, -OH, and – NH2 make benzoic acid weaker, and -Cl and -NO2 make benzoic acid stronger. Acid-weakening groups as the ones that activate the ring toward electrophilic substitution and acid-strengthening groups are the ones that deactivate toward electrophilic substitution. Furthermore, the groups that have the largest effects on reactivity-whether activating or deactivating-have the largest effects on acidity. Dicarboxylic acids are more acidic than consequent monocarboxylic acids for the same reason (Table 5.2.). 56

Table 5.2.

5.3. The basicity We handle basicity just as we handled acidity: compare the stabilities of amines with the stabilities of their ions; the more stable the ion relative to the amine from which it is formed, the more basic the amine. + - RNH2 + H2O ↔ RNH3 + OH [RNH ][OH-] K K [H O] 3 b eq 2 [RNH2] The basicity constant, Kb First of all, amines are more basic than alcohols, ethers, , thiols,etc., for the same reason that ammonia is more basic than water: nitrogen is less electronegative than oxygen, and can better accommodate the positive charge of the ion. N >O> S mnemonic rule - decrease of basicity.

An aliphatic amine is more basic than ammonia because the electron- releasing alkyl groups tend to disperse the positive charge of the substituted ammonium ion, and therefore stabilize it in a way that is not possible for the unsubstituted ammonium ion. From another point of view, an alkyl group pushes electrons toward nitrogen, and thus makes the fourth pair more available for sharing with an acid. The differences in basicity among primary, secondary, and tertiary aliphatic amines are due to a combination of solvation and polar factors. In the gaseous phase:

57

+I CH3 +I CH3 +I NH3 < CH3 NH2 < NH < +I N CH3 +I CH3 +I CH3 Increase of basicity ÑÇÙݳÛÝáõÃÛáõÝÁ ³×áõÙ ¿ + - Reaction of an amine with an acid yields an ammonium salt (RNH3 Cl ). The reaction is reversible, and ammonium salts can be reconverted to amines by treatment with OH-. Formation of an ammonium salt is often used to obtain a more water-soluble derivative of an amine. Reaction of tertiary amines with + - alkylhalides gives quaternary ammonium salts (R4N X ), which are unaffected by changes in the pH of a solution.

5.3.1. Aromatic amine`s basicity From another point of view, we can say that aniline is a weaker base than ammonia because the fourth pair of electrons is partly shared with the ring and is thus less available for sharing with a hydrogen ion. The tendency (through resonance) for the –NH2 group to release electrons to the aromatic ring makes the ring more reactive toward electrophilic attack; at the same time this tendency necessarily makes the amines less basic. An electron-releasing substituent like –CH3 increases the basicity of aniline, and an electron-withdrawing substituent like –NO2 decreases the basicity. A given substituent affects the basicity of an amine and the acidity of a carboxylic acid in opposite ways. Since basicity depends upon ability to accommodate a positive charge, and acidity depends upon ability to accommodate a negative charge. Acidic and basic functional groups are part of many biomolecules, which then exist as soluble ions at the pH of body fluids. The most common ionized groups in biomolecules are carboxylate ions, phosphate ions, and ammonium ions:

Carboxylate ion Phosphate ion Ammonium ion

Amino groups and nitrogen heterocycles are present in many biomolecules, including amino acids, nucleotides, and . The alkaloids, which include many poisons and many drugs, are a large family of nitrogen compounds found in . Many drugs are amines or nitrogen heterocycles. 58

6. THE MECHANISMS OF ORGANIC REACTIONS Classification of organic reactions is based on the following: 1. The nature of reagent (radical, ionic, synchronic) 2. The result of reaction (addition, substitution, elimination, oxidation, intramolecular rearrangement-isomerism) 3. The number of particles participating in the elementary step of reaction- (monomolecular, bimolecular, trimolecular) The nature of reagent (radical, ionic, synchronic) depends on the character of breaking a molecule into two particles. Thus of the two electrons making up the covalent bond, one goes to each fragment; such bond – breaking is called (radicals formation): A:B  A + B If two electrons of the covalent bond go to the same fragment, such bond– breaking is called ,(are formed anion + cation). A : B  A + :B According to the nature of particles which attack the reaction centres are known nucleophilic, electrophilic and radical reactions. Anion is called- nucleophile. Cation is called-electrophile. Taking into account all above mentioned the mechanisms of organic reactions are: Addition (A)- AR, AN, AE Substitution (S)- SR, SN, SE ; SN could be SN 1, SN2 Elimination (E)- E1, E2 Note: (subscripts concern reagents)

6.1. Oxidation-reduction in organic chemistry A useful generalization of the notion of oxidation number (also known as oxidation state) is given in the the Table 6.1. Table 6.1. Oxidation Number of Carbon in One-Carbon Compounds Structural Molecular Oxidation Compound formula formula Number Methane CH4 CH4 -4 Methanol CH3OH CH4O -2 Formaldehyde H2C=O CH2O 0

Formic acid CH2O2 +2

Carbonic acid H2CO3 +4

Carbon dioxide O=C=O CO2 +4 59

Oxidation of carbon corresponds to an increase in the number of bonds between carbon and oxygen and/or a decrease in the number of carbon-hydrogen bonds. Conversely, reduction corresponds to an increase in the number of carbon- hydrogen bonds and/or a decrease in the number of carbon-oxygen bonds. Oxidation of carbon occurs when a bond between carbon and an atom which is less electronegative than carbon is replaced by a bond to an atom that is more electronegative than carbon (O, N, S etc). The reverse process is reduction. Among the various classes of hydrocarbons, alkanes contain carbon in its most reduced state, and alkynes contain carbon in its most oxidized state.

Summarizing: The process of oxidation includes : 1) the loss of electrons, 2) the loss of protones (H+), 3) the loss of hydrogens (H), 3) the loss of hydride iones (H-), 4) the more polar bond formation between carbon and heteroatom. Reduction is the process opposite oxidation and is accompanied by the formation of new bonds with hydrogen, including the transfer of electrons to organic substrate.

6.1.1. Biological oxidation and reduction. Oxidation of alcohols. Alcohols can be oxidized, not only in the test tube, but in living organisms. Let us examine just one example of such an oxidation, the conversion of ethanol into .

Ethanol Acetaldehyde

Like almost all biological reactions, this one requires by an enzyme: in this case, alcohol dehydrogenase. The oxidizing agent is a very common one in biological systems, nicotinamide adenine dinucleotide, or NAD. It is a coenzyme, an organic molecule that works with an enzyme to cause a particular chemical change. Here, the enzyme brings together the ethanol and the coenzyme, and the coenzyme does the actual oxidizing. NAD is a much smaller molecule than the enzyme. Its structure is known, and so is the change in structure that takes place when it acts as an oxidizing agent. The mechanism of the oxidation process has been the subject of much study. For our present purpose we need only know that NAD oxidizes by abstracting a 60 hydrogen and a pair of electrons—a hydride ion, in effect—from the substrate. We shall represent the oxidized form of NAD as NAD+, and the reduced form as NADH.

NAD+ The oxidation of ethanol thus becomes:

Oxidized NAD Reduced NAD

Ethanol loses one of its -hydrogens with a pair of electrons, and then—or probably simultaneously—loses a proton from oxygen to give the aldehyde.

Like all catalysts, enzymes speed up reaction in both directions: under the proper conditions, alcohol dehydrogenase catalyzes the reduction of acetaldehyde to ethanol by NADH.

Reduced NAD Oxidized NAD

The reduction, of course, follows exactly the same path as the oxidation, but in the opposite direction. Acetaldehyde gains a hydride ion from NADH, and a proton from the solvent.

Relative oxidizeability of primary, secondary, tertiary alcohols. The oxidation of an alcohol involves the loss of one or more hydrogens - hydrogens) from the carbon bearing the —OH group. The kind of product that is formed depends upon how many of these -hydrogens the alcohol contains, 61 that is, upon whether the alcohol is primary, secondary, or tertiary. A primary alcohol contains two -hydrogens, and can either lose one of them to form an aldehyde, or both of them to form a carboxylic acid.

A 1° alcohol An aldehyde A 1° alcohol A carboxylic acid

Under the proper conditions, as we shall find, an aldehyde can itself be oxidized to a carboxylic acid. A secondary alcohol can lose its only - hydrogen to form a ketone.

no oxidation A 2° alcohol A ketone A 3° alcohol

Oxidation of primary alcohols to carboxylic acids is usually accomplished by use of potassium permanganate. Oxidation of alcohols to the aldehyde or ketone stage is usually accomplished by the use of Cr(VI). Oxidation of secondary alcohols to ketones is generally straightforward.

A 2° alcohol A ketone

A tertiary alcohol contains no -hydrogens and is not oxidized. (An acidic oxidizing agent can, however, dehydrate the alcohol to an alkene and then oxidize this.). Phenols are more easily oxidized than alcohols. The phenol oxidations that are of the most use are those involving derivatives of 1,2- benzenediol (pyrocatechol) and 1,4-benzenediol (hydroquinone). Oxidation of compounds of this type with silver oxide or with chromic acid yields conjugated dicarbonyl compounds called quinones.

6.1.2. Oxidation of alkenes. Hydroxylation. Formation of 1,2-diols Certain oxidizing agents convert alkenes into 1,2-diols: dihydroxy alcohol containing the two —OH groups on adjacent carbons. (They are also known as glycols.) The reaction amounts to addition of two hydroxyl groups to the double bond. 62

A 1,2-diol

Of the numerous oxidizing agents that bring about hydroxylation, two of the most commonly used are (a) cold alkaline potassium permanganate (KMnO4), and (b) peroxy acids, such as peroxyformic acid (HCO2OH). Hydroxylation with permanganate is carried out by stirring together at room temperature the alkene and the aqueous permanganate solution. For example:

Ethylene 1,2-Ethanediol

Hydroxylation of alkenes is the most important method for the synthesis of 1,2-diols. Epoxidation of alkenes. Three-membered oxygen-containing rings called or oxides of alkenes, are formed by the reaction of alkenes with sources of electrophilic oxygen. oxide and propylene oxide, for example, are the common names of two industrially important epoxides.

ethylene oxide Propylene oxide

Substitutive IUPAC nomenclature names epoxides as epoxy derivatives of alkanes. According to this system, ethylene oxide becomes epoxyethane, and propylene oxide becomes 1,2-epoxypropane. The prefix epoxy- always immediately precedes the alkane ending; it is not listed in alphabetical order in the manner of other substituents.

1,2-Epoxycyclohexane 2-Methyl-2,3-epoxybutane

Epoxides are the products of the reaction between an alkene and a peroxy acid. This process is known as epoxidation.

Alkene Peroxy acid Carboxylic acid

63

6.1.3. Epoxides in biological processes Many naturally occurring substances are epoxides. In most cases, epoxides are biosynthesized by the enzyme-catalyzed transfer of one of the oxygen atoms of an O2 molecule to an alkene. Since only one of the atoms of O2 is transferred to the substrate, the enzymes that catalyze such transfers are classified as monooxygenases. A biological reducing agent, usually the coenzyme NADH, is required as well.

A prominent example of such a reaction is the biological epoxidation of the polyene squalene, a biological precursor to cholesterol and the steroid , including testosterone, progesterone, estrone, and cortisone. The pathway from squalene 2,3-epoxide to these compounds will be discussed in Biochemistry.

6.1.4. Hydroxylation of the aromatic ring. L-Tyrosine formation One amino acid often serves as the biological precursor to another. L- Phenylalanine is classified as an essential amino acid, whereas its p-hydroxy derivative, L-tyrosine, is not. This is because mammals have the ability to convert L-phenylalanine to L-tyrosine by hydroxylation of the aromatic ring. An arene oxide is an intermediate.

L-Phenylalanine Arene oxide intermediate L-Tyrosine

Some individuals lack sufficient quantities of the enzymes necessary to convert L-phenylalanine to L-tyrosine. The L-phenylalanine that they obtain from their diet is therefore diverted along an alternative metabolic pathway that leads to formation of phenylpyruvic acid:

L-Phenylalanine Phenylpyruvic acid

Phenylpyruvic acid is produced in sufficient quantities to cause mental retardation in infants who are deficient in the enzymes necessary to convert L- phenylalanine to L-tyrosine. They are said to suffer from phenylketonuria, or PKU disease. PKU disease can be detected by a simple test routinely administered 64 to infants shortly after birth. It is an inborn error of metabolism and, while not presently correctable, can be controlled by restricting the dietary intake of L- phenylalanine. In practice this means avoiding foods such as meat that are rich in L-phenylalanine.

6.1.5. Oxidation of phenols. Quinones -Unsaturated ketones of a rather special kind are given the name of quinones: these are cyclic diketones of such a structure that they are converted by reduction into hydroquinones, phenols containing two –OH groups. Because they are highly conjugated, quinones are colored (p-benzoquinone, (for e. g. is yellow) and rather closely balanced, energetically, against the corresponding hydroquinones. The ready interconversion provides a convenient oxidation-reduction system.

Some quinones related to more complicated aromatic systems, such as coenzymes Q, have been isolated from biological sources (molds, fungi, higher plants). In many cases they seem to take part in oxidation-reduction cycles essential to the living organism, such as mitochondrial terminal oxidation chain. Quinones are colored; p-benzoquinone, for example, is yellow. Many occur naturally and have been used as dyes. The oxidation- reduction process that connects hydroquinone and benzoquinone involves two 1-electron transfers. The ready reversibility of this reaction is essential to the role that quinones play in cellular respiration, the process by which an organism utilizes molecular oxygen. Electrons are not transferred directly from the substrate molecule to oxygen but instead are transferred by way of an electron transport chain involving a succession of oxidation-reduction reactions. A key component of this electron transport chain is ubiquinone, or coenzyme Q:

Ubiquinone (coenzyme Q) 65

The name ubiquinone is a shortened form of ubiquitous quinone, a term coined to describe the observation that this substance can be found in all cells. The length of it side chain varies among different organisms; the most common form in vertebra has n = 10, while ubiquinones in which n = 6 to 9 are found in yeasts and plants. Another physiologically important quinone is vitamin K, was first identified as essential for the normal clotting of blood.

Vitamin K

6.2. Addition reactions (AE) The characteristic reaction of alkenes is addition to the double bond:

Alkene and electrophile Carbocation

Carbocation Nucleophile Product of electrophilic addition

The first step is rate-determining. It is the transfer of the pair of electrons of the alkene to the electrophile to form a high-energy intermediate, a carbocation. Following its formation, the carbocation undergoes rapid capture by some Lewis base present in the medium. The range of compounds represented as Y—E in this equation is quite large (H2, halogens, HX, etc), and addition reactions offer a wealth of opportunity for converting alkenes to a variety of other functional group types. When two atoms or groups add to the same face of a double bond, the process is referred to as syn addition.

syn addition of X—Y anti addition of X—Y

66

When atoms or groups add to opposite faces of the double bond, the process is referred to as anti addition. The terms syn and anti describe the stereochemical course of the addition reaction.

6.2.1. Hydrogenation of alkenes Hydrogenation is the addition of H2 to a multiple bond.

Ethylene Hydrogen Ethane

The uncatalyzed addition of hydrogen to an alkene, although exothermic, is a very slow process.

6.2.2. Addition of halogens to alkenes Halogens normally react with alkenes by electrophilic addition.

Alkene Halogen Vicinal dihalide

The products of these reactions are called vicinal dihalides (two like substituents attached to adjacent carbons are called vicinal, which means "neighboring.") The halogen is either chlorine (Cl2) or bromine (Br2), and addition takes place rapidly at room temperature and below.

4-Methyl-2-pentene Bromine 2,3-Dibromo-4-methylpentane(100%)

Bromine addition to alkenes is the basis of a qualitative test for alkenes. Solutions of bromine in carbon tetrachloride, like bromine itself, are reddish brown. When a solution of bromine in carbon tetrachloride is added dropwise to an alkene, reaction occurs practically instantaneously and the red color is discharged, giving a colorless solution.

Mechanism of halogen addition to alkenes. The generally accepted mechanism for bromine and chlorine additions to alkenes begins with electrophilic attack of the halogen on the  electrons of the double bond. Bromine and chlorine are not polar molecules, but they are moderately 67 electrophilic. Nucleophilic species, such as alkenes, interact with bromine and chlorine to break the weak halogen-. One halogen atom becomes bonded to the nucleophile; the other is lost as a halide anion. The overall reaction:

Ethylene Bromine 1,2-Dibromoethane

Ethylene Bromine 2-Bromoethyl cation Bromide ion (nucleophile) (electrophile) (leaving group)

A carbocation, which contains a source of electrons (the lone pairs on the bromine substituent) in close proximity to the positively charged carbon is close to form a cyclic bromonium ion.

The mechanism: Step 1: Reaction of ethylene and bromine to form a bromonium ion intermediate:

Ethylene Bromine Ethylenebromonium Bromide ion anion

Step 2: Nucleophilic attack of bromide anion on the bromonium ion:

Bromide Ethylenebromonium 1,2-Dibromoethane

68

6.2.3. Electrophilic addition of hydrogen halides to alkenes In a large number of addition reactions the attacking reagent, unlike H2 or Br2, is a polar molecule or one which is easily polarizable. Hydrogen halides, which are polarized H—X, are among the simplest examples of polar substances that add to alkenes:

Alkene Hydrogen halide Alkyl halide

A proton and a halogen add to the double bond of an alkene to yield an alkyl halide. The order of reactivity of the hydrogen halides reflects their ability to donate a proton. Hydrogen iodide is the strongest acid of the hydrogen halides and reacts with alkenes at the fastest rate. HF < HC1 < HBr < HI The mechanism. An alkene, can accept a proton from a hydrogen halide to form a carbocation. Normally this is the rate-determining step. The carbocations are the conjugate acids of alkenes.

Alkene Hydrogen halide Carbocation Anion (base) (acid) (conjugate acid) (conjugate base)

That carbocations, when generated in the presence of halide anions, react with them to form alkyl halides.

Carbocation Halide ion Alkyl halide (electrophile) (nucleophile)

It is called electrophilic addition because the reaction is triggered by the attack of an electrophile (an acid) on the  electrons of the carbon-carbon double bond. Alkenes are weak bases, and the site of their basicity is the component of the double bond.

69

6.2.3.1. of hydrogen halide addition: Markovnikov's rule In principle a hydrogen halide can add to an unsymmetrical alkene in either of two ways. In practice, addition is so highly regioselective as to be considered regiospecific.

Markovnikov's rule states that when an unsymmetrically substituted alkene reacts with a hydrogen halide, the hydrogen adds to the carbon that has the greater number of hydrogen substituents, and the halogen adds to the carbon having fewer hydrogen substituents. Markovnikov's rule, like Zaitsev's, is an empirical rule.

l-Butene Hydrogen bromide 2-Bromobutane (80%)

Mechanistic basis for Markovnikov's rule. In the reaction of a hydrogen halide HX with an unsymmetrically substituted alkene RCH=CH2 always is formed more substituted compound, because more substituted carbocation intermediate is more stable (alkyl groups are electron-releasing and have an stabilizing effect on the carbocation). Addition according to Markovnikov's rule:

Secondary Halide Observed carbocation ion product

The activation energy difference between a primary carbocation and a secondary carbocation is so great and their rates of formation are so different that essentially all the product is derived from the secondary carbocation. Markovnikov's rule holds because addition of a proton to the doubly bonded carbon that already has the greater number of hydrogen substituents produces the more stable carbocation intermediate:

70

Tertiary carbocation Secondary carbocation

The product of the reaction is derived from the more stable carbocation— in this case, it is a tertiary carbocation that is formed more rapidly than a secondary one. In general, alkyl substituents on the double bond increase the reactivity of alkenes toward electrophilic addition of hydrogen halides. Alkyl groups are electron-releasing, and the more electron-rich a double bond is the better it is able to donate its electrons to an electrophilic reagent. Hydrogen halides reacts with alkenes in accordance with Markovnikov's rule.

6.2.3.2. Acid-catalyzed hydration of alkenes The method by which alkenes may be converted to alcohols is through the addition of a molecule of water across the carbon-carbon double bond under conditions of acid catalysis.

Alkene Water Alcohol

This reaction is carried out in a dilute acid medium. Markovnikov's rule is followed: a proton adds to one carbon of the double bond and a hydroxyl group adds to the other.

2-Methyl-2-butene 2-Methyl-2-butanol (90%)

The general principles of electrophilic addition to alkenes, is that in the example cited, proton transfer to 2-methylpropene forms tert-butyl cation in the first step. This is followed in step 2 by reaction of the carbocation intermediate with a molecule of water acting as a nucleophile. 71

The overall reaction:

2-Methylpropene Water tert -Butyl alcohol

The mechanism: Step 1: Protonation of the carbon-carbon double bond in the direction that leads to the more stable carbocation:

2-Methylpropene Hydronium tret-Butyl cation Water ion

Step 2: Water acts as a nucleophile to capture tert-Butyl cation:

tert-Butyl cation Water tert-Butyloxonium ion

Step 3: Deprotonation of tert-Butyloxonium ion. Water acts as a Bronsted base:

The proposal that carbocation formation is rate-determining: the more stable the carbocation, the faster is its rate of formation.

6.3. Electrophilic aromatic substitution (SE). Reactions of arenes Characteristically, the reagents that react with the aromatic ring of benzene and derivatives are electrophiles. Electrophilic agents add to alkenes, but a different reaction takes place when electrophiles react with arenes. Substitution is observed instead of addition. Representing an arene by the general formula ArH, where Ar stands for an aryl group, the electrophilic portion of the reagent replaces one of the hydrogens on the ring.

Arene Electrophilic Product of reagent electrophilic aromatic substitution 72

We call this reaction electrophilic aromatic substitution. Electrophilic aromatic substitution is the principal method by which substituted derivatives of benzene are prepared industrially and in the laboratory. Mechanistic principles of electrophilic aromatic substitution The first step in the reaction of electrophilic reagents with benzene is similar to the first step of addition (look at AE). An electrophile accepts an electron pair from the  system of benzene to form a carbocation:

Benzene and electrophile Carbocation

A significant portion of the 125 to 150 kJ/mol (30 to 36 kcal/mol) of resonance energy of benzene is lost when it is converted to the cyclohexadienyl cation intermediate which is not aromatic. Once formed, the cyclohexadienyl cation rapidly suffers deprotonation, resulting in full restoration of the aromaticity of the ring and formation of the product of electrophilic aromatic substitution. Bromine reacts with benzene only in the presence of catalysts that enhance their electrophilicity.

6.3.1. Halogenation of benzene. Conversion of benzene to bromobenzene by electrophilic aromatic substitution Bromine is added to benzene in the presence of metallic .

Benzene Bromine Bromobenzene Hydrogen bromide

Bromine, while it is a good enough electrophile to react rapidly with alkenes, is insufficiently electrophilic to react at an appreciable rate with benzene. Iron is a catalyst which increases the electrophilic properties of bromine. The active catalyst is iron(III) bromide, formed by reaction of iron and bromine. Iron(lll) bromide (FeBr3) is also called ferric bromide.

Iron Bromine Iron (III) bromide

Iron(III) bromide is a weak Lewis acid. It reacts with bromine to form a Lewis acid-Lewis base complex.

Lewis base Lewis acid Lewis acid-Lewis base complex 73

Complexation of bromine with iron(III) bromide makes bromine more electrophilic, and it attacks benzene to give a cyclohexadienyl intermediate. In step 2, loss of a proton from the cyclohexadienyl cation is rapid and gives the product of electrophilic aromatic substitution. Some aromatic substrates are much more reactive than benzene and react rapidly with bromine even in the absence of a catalyst. Chlorination is carried out in a manner similar to bromination. Fluorination and iodination of benzene and other arenes are rarely used processes. Step 1: The bromine-iron(III) bromide complex is the active electrophile which attacks benzene. Two of the electrons of benzene are used to form a bond to bromine and give a cyclohexadienyl cation intermediate.

Benzene and bromine-iron (III) Cyclohexadienyl cation Tetrabromoferrate bromide complex ion intermediate ion

Step 2: Loss of a proton from the cyclohexadienyl cation yields bromobenzene.

Cyclohexadienyl Tetrabromoferrate ion Bromobenzene Hydrogen Iron(III) bromide сation intermediate bromide romide

Fluorine is so reactive an electrophile that its reaction with benzene is difficult to control. Iodination is both very slow and characterized by an unfavorable equilibrium constant.

6.3.2. Rate and orientation in electrophilic aromatic substitution of arenes Rate and orientation in electrophilic aromatic substitution of arenes that already bear at least one substituent. Consider the nitration of benzene, toluene, and (trifluoromethyl)benzene.

Toluene Benzene (Trifluoromethyl)benzene (most reactive) (least reactive)

74

Toluene is more reactive than benzene. It undergoes nitration some 20 to 25 times as fast as benzene. Because toluene is more reactive than benzene, we say that a methyl group activates the ring toward electrophilic aromatic substitution. (Trifluoromethyl)benzene is much less reactive than benzene. It undergoes nitration about 40,000 times more slowly than benzene. We say that a trifluoromethyl group deactivates the ring toward electrophilic aromatic substitution. Thus, the rate of electrophilic aromatic substitution depends markedly on a substituent already on the ring. Three products are possible from nitration of toluene: o-nitrotoluene, m-nitro- toluene, and p-nitrotoluene. All are formed, but not in equal amounts. The meta isomer is formed to only a very small extent (3 percent). Together, the ortho- and para-substituted isomers comprise 97 percent of the product mixture.

Toluene o-Nitrotoluene m-Nitrotoluene p-Nitrotoluene (63%) (3%) (34%)

Because substitution in toluene occurs primarily at positions ortho and para to methyl, we say that a methyl substituent is an ortho, para director. Nitration of (trifluoromethyl)benzene, on the other hand, yields almost exsively m- nitro(trifluoromethyl)benzene (91 percent). The ortho- and para-substituted isomers are minor components of the reaction mixture.

(Trifluoromethyl) o-Nitro(trifluoro- m-Nitro(trifluoro- p-Nitro(trifluoro benzene methyl)benzene methyl)benzene methyl)benzene (6%) (91%) (3%)

Because substitution in (trifluoromethyl)benzene occurs primarily at positions m to the substituent, we say that a trifluoromethyl group is a meta director.The regioselectivity of substitution, like the rate, is strongly affected by the substituent. A methyl group is a electron-releasing substituent and activates all of the ring carbons of toluene toward electrophilic attack. The ortho and para positions are activated more than the meta positions.The major influence of the methyl group is electronic. The most important factor is relative carbocation stability. All alkyl groups, not just methyl, are activating substituents and ortho, 75 para directors. This is because any alkyl group, be it methyl, ethyl, isopropyl, tert- butyl, or any other, stabilizes a carbocation site to which it is directly attached. The electrophilic aromatic substitution in (trifluoromethyl)benzene. Because of their high electronegativity the three fluorine atoms polarize the electron distribution in their  bonds to carbon, so that carbon bears a partial positive charge.

more stable than more stable than

Methyl group releases electrons Trifluoromethyl group stabilizes carbocation, withdraws electrons, destabilizes carbocation.

A trifluoromethyl group is a powerful electron-withdrawing substituent and destabilizes a carbocation site to which it is attached. Attack at the meta position leads to a more stable intermediate than attack at either the ortho or the para position, and so predominant meta substitution is observed in (trifluoromethyl)benzene. All the ring positions of (trifluoromethyl)benzene are deactivated as compared with benzene. The meta position is simply deactivated less than the ortho and para positions.

6.3.3. Substituent effects in electrophilic aromatic substitution Table 6.2 summarizes orientation and rate effects in electrophilic aromatic substitution reactions for a variety of frequently encountered substituents. It is arranged in order of decreasing activating power: 1.All activating substituents are ortho, para directors. 2.Halogen substituents are slightly deactivating but are ortho, para-directing. 3.Substituents more deactivating than halogen are meta directors. A group that makes the ring more reactive than benzene is called an activating group. A group that makes the ring less reactive than benzene is called a deactivating group. A group that causes attack to chiefly at positions ortho and para is called an ortho, para director, a group that causes attack to occur chiefly at positions meta to it is called a meta director. Classification of substituent groups. Determination of relative reactivity. As shown in table 6.2., nearly all groups fall into one of two classes: activating and ortho, para-directing, or deactivating and meta-directing. The halogens are in a class by themselves, being deactivating but ortho, para-directing. Just by knowing the effects summarized in these short lists, it is possible to predict fairly accurately the course of hundreds of aromatic substitution reactions. 76

Table 6.2. Effect of groups on electrophilic aromatic substitution Activating: Ortho,para directors Deactivating: Meta directors Strongly activating –NO2 + –NH2 (–NHR, –NR2) –N(CH3)3 –OH –CN –COOH (–COOR) Moderately activating –SO3H –OCH3 (–OC2H5, etc.) –CHO, –COR –NHCOCH3

Weakly activating Deactivating: Ortho,para directors –C6H5 –F, –Cl, –Br, –I –CH3 (–C2H5, etc.)

Table 6.3. The electronic effects of the substituents. The outcome of the electronic effects electronic effects of the substituent substituents on the conjugate inductive mesomeric and aromatic systems Alkyl (methyl -, ethyl – etc) +I - electronreleasing

-NH2 (-NHAlk1,-NAlk2) - I +M +M >>-I electronreleasing -OH -I +M +M >-I electronreleasing alkoxy- (methoxy-, ethoxy-, -I +M +M>-I electronreleasing etc) Halides -I +M -I >+M, electronwithdrawing

-NO2 -I -M electronwithdrawing -COOH -I -M electronwithdrawing

-SO3H -I -M electronwithdrawing >C=O -I -M electronwithdrawing *The seeming inconsistency between regioselectivity and rate that a halogen substituent can affect is explained by the stability of a cyclohexadienyl cation. First, halogens are electronegative and draw electrons away from the carbon to which they are bonded in the same way that a trifluoromethyl group does. However, like hydroxyl groups and amino groups, halogen substituents possess unshared electron pairs that can be donated to a positively charged carbon. This electron donation stabilizes the intermediates derived from ortho and from para attack. 77

Multiple substituent effects. When a benzene ring bears two or more substituents, both its reactivity and the site of further substitution can in most cases be predicted from the cumulative effects of its substituents.

6.4. Nucleophilic substitution (SN). Functional group transformation by nucleophilic substitution The net reaction is:

Nucleophilic substitution reactions of alkyl halides are related to elimination reactions in that the halogen acts as a leaving group on carbon, and is lost as an anion. The carbon-halogen bond of the alkyl halide is broken heterolytically; the pair of electrons in that bond are lost with the leaving group.

; The carbon-halogen bond in an alkyl halide is polarized and is cleaved on attack by a nucleophile so that the two electrons in the bond are retained by the halogen. The most frequently encountered nucleophiles in functional group transformations are anions, which are used as their lithium, sodium, or potassium salts. The anionic portion of the salt substitutes for the halogen of an alkyl halide. The metal cation portion becomes a lithium, sodium, or potassium halide salt.

Table 6.4. Representative Functional Group Transformations by Nucleophilic Substitution Reactions of Alkyl Halides  Alkoxide ion (RO-) The oxygen atom of a metal alkoxide acts as a nucleophile to replace the halogen of an alkyl halide. The product is an ether.

78

 Carboxylate ion (RC—O:-) An ester is formed when the negatively charged oxygen of a carboxylate replaces the halogen of an alkyl halide.

 Hydrogen sulfide ion (HS-). Use of hydrogen sulfide as a nucleophile permits the conversion of alkyl halides to compounds of the type RSH. These compounds are the sulfur analogs of alcohols and are known as thiols.

 Cyanide ion (CN-).The negatively charged carbon atom of cyanide ion is usually the site of its nucleophilic character. Use of cyanide ion as a nucleophile permits the extension of a carbon chain by carbon- carbon bond formation.

6.4.1. Relative reactivity of halide leaving groups. Among alkyl halides alkyl iodides undergo nucleophilic substitution at the fastest rate, alkyl fluorides at the slowest. Increasing rate of substitution by nucleophiles

Least reactive Most reactive

Alkyl iodides are several times more reactive than alkyl bromides and from 50 to 100 times more reactive than alkyl chlorides. Fluorine has the strongest bond to carbon and fluoride is the poorest leaving group. Leaving- group ability is also related to basicity. A strongly basic anion is usually a poorer leaving group than a weakly basic one. Fluoride is the most basic and the poorest leaving group among the halide anions, iodide the least basic and the best leaving group.

79

6.4.2. The bimolecular (SN2) mechanism of nucleophilic substitution The mechanisms by which nucleophilic substitution takes place have been the subject of much study. Methyl bromide reacts with sodium hydroxide to form methyl alcohol by a nucleophilic substitution reaction:

The rate of this reaction is directly proportional to the concentration of both methyl bromide and sodium hydroxide. It is first-order in each reactant, or second-order overall.

In the second-order reactions the rate-determining step is bimolecular, i.e., that both hydroxide ion and methyl bromide are involved at the transition state. SN2 mechanism is a concerted process, that is, a single-step reaction in which both the alkyl halide and the nucleophile are involved at the transition state. Cleavage of the bond between carbon and the leaving group is assisted by formation of a bond between carbon and the nucleophile. In effect, the nucleophile "pushes off" the leaving group from its point of attachment to carbon. For this reason, the SN2 mechanism is sometimes referred to as a direct displacement process. The SN2 mechanism for the hydrolysis of methyl bromide may be represented as

where carbon is partially bonded to both the incoming nucleophile and the departing leaving group at the transition state. Progress is made toward the transition state as the nucleophile begins to share a pair of its electrons with carbon and the halide ion leaves, taking with it the pair of electrons in its bond to carbon.

6.4.2.1. Stereochemistry of SN2 reactions Assuming that the transition state is bimolecular in reactions of primary and secondary alkyl halides with anionic nucleophiles, the two spatial arrangement of the nucleophile in relation to the leaving group possible. In the pathway shown in Figure 6.2.a, the nucleophile simply assumes the position occupied by the leaving group. It attacks the substrate at the same face from 80 which the leaving group departs. This is called "front-side displacement," or substitution with retention of configuration (Figure 6.2. a). In a second possibility, the nucleophile attacks the substrate from the side opposite the bond to the leaving group. This is called "backside displacement," or substitution with inversion of configuration (Figure 6.2. b).

(a) Nucleophilic substitution with retention of configuration

(b) Nucleophilic substitution in this case had occurred with inversion of configuration. Figure 6.2. (a,b)

Numerous experiments have demonstrated the generality of this observa- tion. Substitution reactions that proceed by the SN2 mechanism are stereospecific and proceed with inversion of configuration at the carbon that bears the leaving group.The hybridization of the carbon at which substitution occurs changes from sp3 in the alkyl halide starting material to sp2 in the transition state. The SN2 transition state is pentacoordinate; carbon is fully bonded to three substituents and partially bonded to both the leaving group and the incoming nucleophile. The bonds to the nucleophile and the leaving group are relatively long and weak at the transition state. Once past the transition state, the leaving group is expelled and carbon becomes tetracoordinate, its hybridization returning to sp3. Alkyl halides differ in reactivity according to the nature of their leaving group. The bond to the leaving group is partially broken in the SN2 transition state, and alkyl iodides react faster than other alkyl halides with nucleophilic reagents because they have the weakest carbon-halogen bonds.

6.4.2.2. Steric effects in SN2 reactions There are very large differences in reactivity among alkyl halides, which depend on the degree of substitution at the carbon that bears the leaving group. 81

The accepted explanation for the large rate difference among methyl, ethyl, iso-propyl, and tert-butyl bromides rests on the degree of steric hindrance each offers to nucleophilic attack. The nucleophile must approach the alkyl halide from the side opposite the bond to the leaving group, and, as illustrated in (Fig. 6.2.), this approach is hindered by alkyl substituents on the carbon that is being attacked. The three hydrogen substituents of methyl bromide offer little resistance to approach of the nucleophile, and a rapid reaction occurs. Replacing one of the hydrogens by a methyl group somewhat shields the carbon from attack by the nucleophile and causes ethyl bromide to be less reactive than methyl bromide. Replacing all three hydrogen substituents by methyl groups almost completely blocks back-side approach to the tertiary carbon of (CH3)3CBr and shuts down bimolecular nucleophilic substitution. In general, nucleophilic substitutions characterized by second-order kinetic behavior exhibit the following dependence of rate on substrate structure: Increasing rate of substitution by the SN2 mechanism R3CX < R2CHX < RCH2X < CH3X Tertiary Secondary Primary Methyl Least reactive Most reactive most crowded least crowded

Alkyl substituents at the carbon atom adjacent to the point of nucleophilic attack also decrease the rate of the SN2 reaction.

6.4.2.3. Nucleophiles and nucleophilicity The Lewis base that acts as the nucleophile in a nucleophilic substitution often is an anion. Neutral Lewis bases can also serve as nucleophiles. Solvolysis in water converts an alkyl halide to an alcohol.

Solvolysis in methyl alcohol converts an alkyl halide to an alkyl methyl ether:

82

In these and related solvolyses, nucleophilic substitution is the first step and is rate-determining. The proton transfer step that follows it is fast. Since, as we have seen, the nucleophile attacks the substrate in the rate-determining step of the SN2 mechanism, it follows that the rate at which substitution occurs may vary from nucleophile to nucleophile. Just as some alkyl halides are more reactive than others, some nucleophiles are more reactive than others. Nucleophilic strength, or nucleophilicity, is a measure of how fast a Lewis base displaces a leaving group from a suitable substrate. Neutral Lewis bases such as water, alcohols, and carboxylic acids are much weaker nucleophiles than their conjugate bases. When comparing species that have the same nucleophilic atom, a negatively charged nucleophile is more reactive than a neutral one.

Table 6. 5. Nucleophilicity of Some Common Nucleophiles Very good nucleophiles I-, HS-, RS------Good nucleophiles Br , HO , RO , CN , N3 - - - Fair nucleophiles NH3, Cl , F , RCO2 Weak nucleophiles H2O, ROH Very weak nucleophiles RCO2H

Compare reactivity of these species. As long as the nucleophilic atom is the same, the more basic the nucleophile, the more reactive it is. An alkoxide - ion (RO ) is more basic and more nucleophilic than a carboxylate ion (RCO2). The connection between basicity and nucleophilicity holds when comparing atoms in the same row of the periodic table. It does not hold when proceeding down a column in the periodic table. For example, I- is the least basic of the halide ions but is the most nucleophilic. F- is the most basic halide ion but the least nucleophilic. The factor that seems most responsible for the inverse relationship between basicity and nucleophilicity among the halide ions is the 83 degree to which they are solvated by hydrogen bonds. In general, smaller anions are more highly solvated by hydrogen bonding to solvents such as water and methanol than are larger ones. Among the halide anions, F- forms the strongest hydrogen bonds to water and alcohols, and I- the weakest. Thus, the nucleophilicity of F- is suppressed more than that of Cl-, Cl- more than Br-, and Br- more than I-. Similarly, HO- is smaller, more solvated, and less nucleophilic than HS-. Nucleophilicity is also related to polarizability. The partial bond between the nucleophile and the alkyl halide that characterizes the SN2 transition state is more fully developed at a longer distance when the nucleophile is very polarizable than when it is not. Among related atoms, polarizability increases with increasing size. Thus iodide is the most polarizable and most nucleophilic halide ion, fluoride the least.

6.4.3. The unimolecular (SN1) mechanism of nucleophilic substitution Recalling that tertiary alkyl halides are practically inert to substitution by the SN2 mechanism because of steric hindrance, but they undergo nucleophilic substitution SN1. Hughes and Ingold discovered that the hydrolysis of tert- butyl bromide occurs readily and is characterized by a first-order rate law:

The overall reaction: + - (CH3)3CBr + 2H2O  (CH3)3OH + H3O + Br tert-butyl bromide water tert-butyl alcohol hydronium ion bromide ion

Step 1: The alkyl halide dissociates to a carbocation and a halide ion.

Step 2: The carbocation formed in step 1 reacts rapidly with a water molecule. Water is a nucleophile. This step completes the nucleophilic substitution stage of the mechanism and yields an oxonium ion.

84

Step 3: This step is a fast acid-base reaction that follows the nucleophilic substitution. Water acts as a base to remove a proton from the oxonium ion to give the observed product of the reaction, tert-butyl alcohol.

The rate of hydrolysis depends only on the concentration of tert-butyl bromide. The first-order kinetics is interpreted as evidence for a unimolecular rate-determining step-a step that involves only the alkyl halide. The first step, a unimolecular dissociation of the alkyl halide to form a carbocation as the key intermediate, is rate-determining. The SN1 mechanism is an ionization mechanism. The nucleophile does not participate until after the rate-determining step has taken place. Carbocation stability and the rate of substitution by the SN1 mechanism Tertiary alkyl halides are good candidates for reaction by the SN1 mechanism because they are too sterically hindered to react by the SN2 mechanism and, since they form relatively stable carbocations. The relative rate order in SN1 reactions is exactly the opposite of that seen in SN2 reactions: SN1 reactivity: methyl < primary < secondary < tertiary SN2 reactivity: tertiary < secondary < primary < methyl The order of alkyl halide reactivity in SN1 reactions is in the same direction as the order of carbocation stability: the more stable the carbocation, the faster an alkyl halide reacts by the SN1 mechanism.

6.4.3.1. Stereochemistry of SN1reactions When the leaving group is attached to the stereogenic center of an optically active halide, ionization leads to a carbocation intermediate that is achiral. It is achiral because the three bonds to the positively charged carbon lie in the same plane, and this plane is a plane of symmetry for the carbocation. A symmetrical carbocation should react with a nucleophile at the same rate at either of its two faces. We expect the product of substitution by the SN1 mechanism to be formed as a racemic mixture and to be optically inactive. Normally, the product is formed with predominant, but not complete, inversion of configuration.

85

6.5. Elimination reactions. E1 and E2 These types of reactions are characteristic for alcohols and alkyl halides and lead to alkenes formation. For example, dehydration of 3-ethyl-3-pentanol ( elimination):

3-Ethyl-3-pentanol 3-Ethyl-2-pentene Water

6.5. 1. Regioselectivity in alcohol dehydration: the Zaitsev rule

2-Methyl-2-butanol 2-Methyl-1- butene 2-Methyl-2-butene (10%) (90%)

In its original form Zaitsev's rule stated that the alkene formed in greatest amount is the one that corresponds to removal of the hydrogen from the  carbon having the fewest hydrogen substituents ( Fig 6.3).

Fig 6.3.

Zaitsev's rule as applied to the acid-catalyzed dehydration of alcohols is now more often expressed in a different way: elimination reactions of alcohols yield the most highly substituted alkene as the major product. Since the most highly substituted alkene is also normally the most stable one, Zaitsev's rule is sometimes expressed in terms of a preference for predominant formation of the most stable alkene that could arise by  elimination.

2,3-Dimethyl-2-butanol 2,3-Dimethyl-1-butene 2,3-Dimethyl-2-butene (minor product) (major product) 86

In addition to being regioselective, alcohol dehydration reactions are stereoselective. A stereoselective reaction is one in which a single starting material can yield two or more stereoisomeric products, but gives one of them in greater amounts than any other. Alcohol dehydrations tend to produce the more stable stereoisomeric form of an alkene. Dehydration of 3-pentanol, for example, yields a mixture of trans-2-pentene and cis-2-pentene in which the more stable trans stereoisomer predominates.

3-Pentanol cis-2-Pentene (25%) trans-2-Pentene (75%) (minor product) (major product)

6.5. 2. The mechanism of acid-catalyzed dehydration of alcohols The relative reactivity of alcohols decreases in the order: tertiary > secondary > primary.

These means that carbocations are key intermediates in alcohol dehydration. The overall reaction:

tert-Butyl alcohol 2-Methylpropene Water

Step 1: Protonation of tert-butyl alcohol.

tert-Buty\ alcohol Hydronium ion tert-Butyloxonium ion Water

Step 2: Dissociation of tert-butyloxonium ion.

tert-Butyloxonium ion tert-Butyl cation Water

87

Step 3: Deprotonation of tert-butyl cation.

tert-Butyl cation Water 2-Methylpropene Hydronium ion

6.5. 3. Dehydrohalogenation of alkyl halides

l-Chlorooctadecane 1-Octadecene (86%)

The regioselectivity of dehydrohalogenation of alkyl halides follows the Zaitsev rule;  elimination predominates in the direction that leads to the more highly substituted alkene. The major alkene is normally the more stable one, which is formed by removing a proton from the  carbon that has the fewest hydrogen substituents.

2-Bromo-2- 2-Methyl-l-butene 2-Methyl-2-butene methylbutane (29%) (71%)

Mechanism of the dehydrohalogenation of alkyl halides: The E2 mechanism. The dehydrohalogenation reaction exhibits second-order kinetics; it is first-order in alkyl halide and first-order in base. The rate equation may be written Rate = k[alkyl halide][base] The rate of elimination depends on the halogen, the reactivity of alkyl halides increasing with decreasing strength of the carbon-halogen bond. Increasing rate of dehydrohalogenation RF < RC1

Fig. 6.4.

Since alkyl groups stabilize double bonds, they also stabilize a partially formed  bond in the transition state.

6.5.3.1. Anti elimination in E2 reactions: stereoelectronic effects. stereoselectivity

Syn periplanar Anti periplanar

Adjacent bonds are eclipsed when the H—C—C—X assembly is syn peri- planar, a transition state derived from this conformation is less stable than one that has an anti periplanar relationship between the proton and the leaving group. Effects that arise because one spatial arrangement of electrons (or orbitals or bonds) is more stable than another are called stereoelectronic effects. We say there is a stereoelectronic preference for the anti periplanar arrangement of proton and leaving group in E2 reactions. Alkyl halides such as sec-butyl bromide normally yield the more stable trans alkene as the major product on treatment with base.

2-Bromobutane 1-Butene cis-2-Butene trans-2-Butene

6.5.4. A different mechanism for alkyl halide elimination: the El mechanism The E2 mechanism is a concerted process in which the carbon-hydrogen and carbon-halogen bonds undergo cleavage in the same elementary step. One possibility is the two-step mechanism in which the carbon-halogen bond breaks first to give a carbocation intermediate (first, rate-determining step), followed by deprotonation of the carbocation in a second step. Because the 89 rate-determining step is unimolecular- it involves only the alkyl halide and not the base - this mechanism is known by the symbol El. It exhibits first-order kinetics. Rate = k[alkyl halide]

The mechanism: Step 1: Alkyl halide dissociates by heterolytic cleavage of carbon-halogen bond. (Ionization step, like in SN1):

2-Bromo-2-methylbutane 1, 1 -Dimethylpropyl cation bromide ion

Step 2: Ethanol acts as a base to remove a proton from the carbocation to give the alkene products. (Deprotonation step):

Ethanol 1, 1-Dimethylpropyil cation Ethyloxonium ion 2-Methyl-l-butene

Ethanol 1, 1-Dimethylpropyl cation Ethyloxonium ion 2-Methyl-2-butene

Increasing rate of elimination by the E1 mechanism RCH2X < R2CHX < R3CX Primary alkyl halide: slowest rate of El elimination Tertiary alkyl halide: fastest rate of El elimination

Alkyl iodides have the weakest carbon-halogen bond and are the most reactive; alkyl fluorides have the strongest carbon-halogen bond and are the least reactive. The best examples of E1 eliminations are those carried out in the absence of added base. At even modest concentrations of strong base, elimination by the E2 mechanism is faster than El elimination.

90

7. THE CARBONYL COMPOUNDS

Several families of compounds that are widespread in organic and biological chemistry those that contain a carbonyl group. Every carbonyl group consists of a carbon and oxygen atom connected by a double bond. The carbonyl group containing compounds used to classify into to large groups: the carbonyl compounds (aldehydes and ketones) and carboxylic acids and their derivatives.

A carbonyl group Aldehyde Ketone R= H, Alk, Ar R, R''= Alk, Ar R= H, Alk, Ar

Aldehydes and ketones are the two most simple types of carbonyl compounds. In aldehydes the carbonyl group has one R group and one H atom on the carbonyl carbon atom, and in ketones there are two R groups on the carbonyl carbon. Aldehydes and ketones are the simplest of the families of carbonyl compounds, which differ according to what substituents are bonded to the carbonyl-group carbon atom. Because oxygen attracts electrons more strongly than carbon, carbonyl groups are strongly polarized, with a partial positive charge on carbon and a partial negative charge on oxygen (Fig.7.1.). As shown in following sections, this polarity of the carbonyl group helps to explain how many of its reactions take place.

120° angles in a planar triangle Fig.7.1.

Another property common to all carbonyl groups is planarity. The carbonyl carbon atom is surrounded by three regions of electron density, and the bond angles between the three substituents on carbon are 120° or close to it. It's useful to divide carbonyl compounds into two groups based on their chemical properties. In one group are aldehydes and ketones, which have similar properties because their carbonyl groups are bonded to atoms that don't attract electrons strongly-carbon and hydrogen. In the second group are 91 carboxylic acids, esters, anhydrides, and . The carbonyl-group carbon in these compounds is bonded to an oxygen or nitrogen atom, which does attract electrons strongly.

Table 7.1. Some Kinds of Carbonyl Compounds

7.1. Reactions that lead to aldehydes and ketones  Oxidation of alcohols: O [O] [O] R CH C R C O R CH2OH R C H O aldehyde ketone

In the biological systems oxidation takes place in enzymatic way (NAD and FAD-dehydrogenases and oxidases are involved ).  Hydrolysis of gem- dihalides: O H2O R – CCl2 – R R – C – R' + 2HCl O H2O R – CH Cl2 R – C + 2HCl H  Reducing of some derivatives of carboxylic acids, more often acyl chlorides (Rosenmund reduction). 92

O Pd O R – C + H2 R – C + HCl Cl H  Hydrolysis of alkynes (Kucherov`s reaction, in the presence of Hg2+salts): Hg2+ O HC CH + H2O CH3 – C H O Hg2+ CH3 – C CH + H2O CH3 – C CH3

7.2. Reactions of aldehydes and ketones Reactivity of carbonyl compounds depends on the nature of reactive centers in the static conditions (Fig. 7.1.). In general reaction centers could be expressed schematically:

Fig. 7.2.

According to the existing centers, carbonyl compounds undergo following types of reactions: 1. Nucleophilic addition 2. Nucleophilic addition-elimination (condensation). Reactions with amines and amines derivatives. Enolization. Base - catalyzed enolization. Enolate anion formation. 4. Aldol addition and condensation. 5. -Halogenation of aldehydes and ketones 6. Oxidation of aldehydes.

7.2.1. Nucleophilic addition (AN). Principles of nucleophilic addition to carbonyl groups Hydration of aldehydes and ketones. The hydration of carbonyl group is a reversible reaction. The position of equilibrium depends strongly on the nature of the carbonyl group and is influenced by a combination of electronic and steric effects.

93

Electronic effects: As the starting material becomes more stable due to electronic effects of substituents (+I) the smaller will be its equilibrium constant for hydration and vice versa: formaldehyde > acetaldehyde > acetone (one alkyl substituent) (two alkyl substituents)

Aldehydes generally undergo nucleophilic addition more readily than ketones due to a combination of electronic and steric effects. Steric effects: Geminal diol product is more crowded than the starting aldehydes and ketones. Increased crowding can be better tolerated when the substituents are hydrogen than when they are alkyl groups. The degree of hydration of aldehydes and ketones depends on the structure of substrate. Thus, more than 99,9 % of the formic aldehyde and about 50% of acetaldehyde are hydrated in the water solution, but acetone practically isn’t hydrated. Resulting product of hydration, as a rule, is unstable, therefore it is impossible to separate from solution by distillation. Aldehydes generally undergo nucleophilic addition more readily than ketones due to a combination of electronic and steric effects. Trichloroacetic aldehyde is completely hydrated because of a combination of electronic and steric effects. The presence of electro acceptor trichloromethane group (CCl3-) has a significant stabilizing effect, that is why this compound could be dehydrated only in the presence of strong acid, such as H2SO4.

Trichloroacetic aldehyde is used in medicine as sedative and soporific medicine. Acid-catalyzed nucleophilic addition. If acid is present, hydrogen ion becomes attached to carbonyl oxygen (Fig. 7.2.). Protonation of carbonyl oxygen makes carbonyl carbon more susceptible to nucleophilic attack; then, addition will be favored by high acidity. Thus nucleophilic addition to aldehydes and ketones can be catalyzed by acids (sometimes, by Lewis acids)

Fig. 7.3. Undergoes nucleophilic attack more readily

94

7.2.1.1. Addition of alcohols. formation Alcohols add to the carbonyl group of aldehydes in the presence of anhydrous acids to yield :

There is good evidence that in alcoholic solution an aldehyde exists in equilibrium with a compound called a hemiacetal:

A hemiacetal

In the presence of acid the hemiacetal, acting as an alcohol, reacts with more of the solvent alcohol to form the acetal, an ether:

Hemiacetal Acetal (An alcohol) (An ether)

The reaction involves the formation (step 1) of the ion I, which then combines (step 2) with a molecule of alcohol to yield the protonated acetal.

Step 1.

step 2

Acetal

7.2.1.2.Cyclic hemiacetals Nucleophilic addition of an alcohol to an aldehyde leads to hemiacetal, and when the hydroxyl and aldehyde functions are part of the same molecule, a cyclic hemiacetal is formed. Cyclic hemiacetal formation is most favorable when the ring that results is five-membered or six-membered.

95

 ⇄

The hemiacetal formation is abundant in the biological systems. Carbohydrates, aldoses, such as glucose et al. mostly exist in form of cyclic hemiacetals. Aldoses have two types of functional group, an aldehyde group and the hydroxyl group, which are capable of reacting with each other.

 ⇄

7.2.1.3. Addition of cyanide. Cyanohydrin formation The elements of HCN add to the carbonyl group of aldehydes and ketones to yield compounds known as cyanohydrins:

The reaction is often carried out by adding mineral acid to a mixture of the carbonyl compound and aqueous sodium cyanide. Addition appears to involve nucleophilic attack on carbonyl carbon by the strongly basic cyanide ion; subsequently (or possibly simultaneously) oxygen accepts a hydrogen ion to form the cyanohydrin product:

Cyanohydrin

96

Cyanohydrins are nitriles and their principal use is based on the fact that, like other nitriles, they undergo hydrolysis producing -hydroxy acids or unsaturated acids.

7.2.2. Addition of derivatives of ammonia.Condensation reactions

The products contain a carbon-nitrogen double bond resulting from elimination of a molecule of water from the initial addition products. This mechanism of interaction is a background of transamination reactions in living systems (chapter 10.3.2.). Some of these reagents and their products are:

Semicarbazone

Like ammonia, these derivatives of ammonia are basic, and therefore react with acids to form salts. and formation are used for separation and identification of aldehydes and ketones from reaction mixture. These derivatives are solid compounds with precise melting points.

7.2.3. Oxidation of Aldehydes and ketones In the presence of alkali aldehydes undergo oxidation by heavy metal ions, especially silver and copper. In a result of reactions ions are reduced. These reactions used chiefly for detection of aldehydes.

97

The one of them Tollens' test, or so called “Silver mirror” reaction is familiar to everyone.

Colorless solution Silver mirror

C'annizzaro reaction is a special sort of oxidation and reduction. In the presence of concentrated alkali, aldehydes containing no - hydrogens undergo self-oxidation-and-reduction to yield a mixture of an alcohol and a salt of a carboxylic acid. Under these conditions an aldehyde containing -hydrogens would undergo aldol condensation faster.

An aldehyde with no Acid Alcohol -hydrogens salt

Formaldehyde Formate ion Methanol

7.2.4. Reduction. Reduction to alcohols Aldehydes can be reduced to primary alcohols, and ketones to secondary alcohols, either by catalytic hydrogenation or by use of chemical reducing agents like lithium aluminum hydride, LiAlH4.

This time the nucleophile is hydrogen transferred with a pair of electrons - as a hydride ion, H:- —from the metal to carbonyl carbon.

98

7.2.5. Haloform reaction is the characteristic test for detecting the presence of – COCH3 group. Acetaldehyde and methyl ketones react rapidly with halogens including iodine in alkaline solution and undergo substitution reaction called haloform reaction. A topical antiseptic iodoform, called also triiodomethane with specific odor is formed.

or

7.2.6. Aldol condensation Under the influence of dilute base, two molecules of an aldehyde or a ketone may combine to form a -hydroxy aldehyde or  -hydroxy ketone. This reaction is called the aldol condensation.

Acetaldehyde  -Hydroxybutyraldehyde 2 moles (3-Hydroxybutanal)

If the aldehyde or ketone does not contain an -hydrogen, a simple aldol condensation cannot take place. (In concentrated base, however, such aldehydes may undergo the Cannizzaro reaction.) The general mechanism for the base-catalyzed condensation: step 1. Hydroxide ion abstracts a hydrogen ion from the -carbon of the aldehyde to form carbanion I (nucleophilic reagent), which attacks carbonyl carbon of the other molecule of aldehyde.

Carbanion I

99 step 2. Carbanion I attacks carbonyl carbon to form ion II.

step 3. Ion II (an alkoxide) abstracts a hydrogen ion from water to form the -hydroxy aldehyde III, regenerating hydroxide ion.

II III

The purpose of hydroxide ion is thus to produce the carbanion I, which is the actual nucleophilic reagent. The carbonyl group plays two roles in the aldol condensation. It not only provides the unsaturated linkage at which addition (step 2) occurs, but also makes the -hydrogens acidic enough for carbanion formation (step 1) to take place.

7.2.6.1. Dehydration of aldol products (crotonic condensation) The -hydroxy aldehydes and  -hydroxy ketones obtained from aldol condensations are very easily dehydrated; the major products have the carbon- carbon double bond between the - and  -carbon atoms. For example:

Aldol 2-Butenal 3-hydroxybutanol

Both the ease and the orientation of elimination are related to the fact that the alkene obtained is a particularly stable one, since the carbon-carbon double bond is conjugated with the carbon-oxygen double bond of the carbonyl group.

7.3. Use of aldol condensation in synthesis Catalytic hydrogenation of   -unsaturated aldehydes and ketones yields saturated alcohols, addition of hydrogen occurring both at carbon-carbon and at carbon-oxygen double bonds. It is for the purpose of ultimately preparing saturated alcohols that the aldol condensation is often carried out. For example, n-butyl alcohol is prepared on an industrial scale in this way: 100

Acetaldehyde Aldol 2-Butenal

n-Butyl alcohol

7.3.1. Aldol Condensations in the Biological World Carbonyl condensations are among the most widely used reactions in the biological world for the assembly of new carbon-carbon bonds in such important biomolecules as fatty acids, cholesterol, steroid hormones, terpenes, carbohydrates, hydroxyacids. One source of carbon atoms for the synthesis of these biomolecules is acetyl-CoA, a of acetic acid and the thiol group of coenzyme A.

Acetyl-CoA Acetyl-CoA Acetoacetyl-CoA Coenzyme A

Condensation of Acetyl-CoA with Oxaloacetate to Form Citrate The first reaction of the TCA (tricarboxylic acids cycle) is catalyzed by citrate synthase and involves a carbanion formed at the methyl group of acetyl-CoA that undergoes aldol condensation with the carbonyl carbon atom of the oxaloacetate:

Acetyl-CoA Oxaloacetate Citrate

This reaction is practically irreversible and has a  G° of -7.7 kcal/mol (-32.2 KJ/mol). Formation of citrate is the committed step of the cycle and is regulated by allosteric effectors. Another example of aldol condensation reaction is Fructose 1,6- bisphosphate formation. It is rate determining step of gluconeogenesis (synthesis of glucose de novo) which is reversible process of decay during glycolysis and will be discussed in details during course of Biochemistry.

101

8. CARBOXYLIC ACIDS AND THEIR DERIVATIVES

Carboxylic acids are classified according to the quantity of fuctional groups (mono-, di, tri etc), structure of carbon-carbon chain (branched, unbranched), types of bonds (saturated, unsaturated) (Table 8.1.).

Ester Anhydride (RCOOH or RCO2H) (RCOOR ' or RCO2R ') (RCO2COR)

Salt Thioether Amide

Substituted amide Acyl hydrazide Acyl halide

8. 1. Sources of carboxylic acids Many carboxylic acids were first isolated from natural sources and were given names indicative of their origin. Formic acid (Lalia formica, "ant") was obtained by distilling ants. Since ancient times acetic acid (Latin acetum, "vinegar") has been known to be present in wine that has turned sour. Butyric acid (Latin butyrum, "butter") contributes to the odor of rancid butter, and lactic acid (Latin lac "milk") has been isolated from sour milk. In most cases the large-scale preparation of carboxylic acids relies on chemical synthesis. 8.1.1. Preparation of carboxylic acids Preparation of carboxylic acids by oxidation of different compounds.  Oxidation of petroleum-derived starting materials. Several catalytic processes of preparation of carboxylic acids have been developed. Two of these involve oxidation of petroleum-derived starting materials:

102

Table 8. 1. Carboxylic acids Common Name Melting Number of “C” Structure And IUPAC Name point t°C Monocarboxylic acids Formic acid HCOOH C - 8 Methanoic acid 1 Acetic acid CH COOH C 3 - 2 Ethanoic acid 2 Propionic acid CH CH COOH C 3 2 16 propanoic acid 3 Butyric acid CH (CH ) COOH C 3 2 2 31.5 Butanoic acid 4 Valeric acid CH (CH ) COOH C 3 2 3 44 Pentanoic acid 5 Caproic acid CH (CH ) COOH C 3 2 4 54 Hexanoic acid 6 Phenylacetyc acid C8 C6H5CH2COOH 64 Benzoic acid C7 C6H5COOH 70 Saturated Dicarboxylic Acids Oxalic or Ethandioic acid C2 HOOC- COOH 189 Malonic or Propanedioic acid C3 HOOC- CH2- COOH 135 Succinic or Butanedioic acid C4 HOOC- (CH2)2- COOH 185 Glutaric or Pentanedioic acid C5 HOOC- (CH2)3- COOH 98 Unsaturated Carboxylic Acids Acrylic acid, C3 CH2=CHCOOH 14 Propenoic acid 2-butenoic acid (trans), C4 CH3CH =CHCOOH 52 Crotonic acid Unsaturated Dicarboxylic Acids H H Maleic acid, C3 C=C 130 cis-butendioic acid HOOC COOH H COOH Fumaric acid, C4 C=C 287 trans-butendioic acid HOOC H C8 COOH 208 Phthalic acid COOH tere-Phthalic acid C8 HOOC COOH 300

103

 Primary alcohols may be oxidized either to an aldehyde or to carboxylic acid: H O [O] [O] O CH3 – CH – O – H CH3 – C CH3 – C H OH  Primary aldehydes may be oxidized to carboxylic acid:

8.1.2. Hydrolysis of acid derivatives  Acid chlorides. Low-molecular-weight acid chlorides react very rapidly with water to form carboxylic acids and HX. Higher-molecular-weight acid halides are less soluble and consequently react less rapidly with water.

 Acid Anhydrides. Anhydrides are generally less reactive than acid chlorides. However, the lower-molecular-weight anhydrides also react readily with water to form two molecules of carboxylic acid. Because acid anhydrides (RCO2OCR) react rapidly with water to regenerate acids, they are not found in plants and animals.

Their reactivity, however, makes anhydrides useful in the synthesis of other carboxylic acid derivatives. The R—C=O portion of an anhydride combines with an alcohol to give an ester or with an amine to give an amide, while the remainder of the anhydride adds hydrogen to give an acid:

 Esters. Esters are hydrolyzed only very slowly, even in boiling water. Esters are fairly stable in neutral aqueous media but are cleaved while heated with water in the presence of strong acids or bases. 104

Ester Water Carboxylic Alcohol acid

Hydrolysis of esters in dilute aqueous acid is also an equilibrium reaction and proceeds by the same mechanism as esterification, except in reverse. The role of the acid catalyst is to protonate the carbonyl oxygen. In doing so, it increases the electrophilic character of the carbonyl carbon toward attack by water to form a tetrahedral carbonyl addition intermediate. Collapse of this intermediate gives the carboxylic acid and an alcohol. In this reaction, acid is a catalyst; it is consumed in the first step but is regenerated at the end of the reaction.  Amides. Amides require considerably more vigorous conditions for hydrolysis in both acid and base than esters. Amides undergo hydrolysis in hot aqueous acid to give a carboxylic acid and an ammonium ion. One mol of acid is required per mol of amide. + H (H2O) + R–CONH2 R–COOH + NH4 carboxylic acid

 Synthesis of carboxylic acids by the preparation and hydrolysis of nitriles. Primary and secondary alkyl halides may be converted to the next higher carboxylic acid by a two-step synthetic sequence involving the preparation and hydrolysis of nitriles. Nitriles, also known as alkyl cyanides, are prepared by nucleophilic substitution.

Alkyl halide Cyanide ion Nitrile Halide ion (alkyl cyanide)

The cyano group of a nitrile is hydrolyzed in aqueous acid to a carboxyl group and ammonium ion. Net reaction is:

Nitrile Water Carboxylic Ammonium acid

Mechanism of hydrolysis of a cyano group. In hydrolysis of a cyano group in aqueous acid, protonation of the nitrogen atom gives a cation that reacts with water to give an imidic acid (the enol of an amide). Keto-enol 105 tautomerism of the imidic acid gives an amide. The amide is then hydrolyzed to a carboxylic acid and ammonium ion.

An imidic acid An amide (enol of an amide)

The reaction conditions required for acid-catalyzed hydrolysis of a cyano group are typically more vigorous than those required for hydrolysis of an amide, and in the presence of excess water, a cyano group is hydrolyzed first to an amide and then to a carboxylic acid.

8.1.3. The malonic synthesis of monosubstituted and disubstituted acids. The malonic ester synthesis The malonic ester synthesis is useful for the preparation of monosubstituted and disubstituted acetic acids of the following types.

Step 1. The -hydrogens of diethyl malonate are more acidic than ethanol (pKa 15.9), and, therefore, diethyl malonate is converted completely to its anion by sodium ethoxide or other alkali metal alkoxide.

Diethyl malonate Sodium ethoxide Sodium salt of Ethanol diethyl malonate

Step 2. The anion of diethyl malonate is a nucleophile and reacts by an SN2 pathway with methyl and primary alkyl halides, -haloketones, and haloesters. In the following example, the anion of diethyl malonate is alkylated with bromoalkane.

106

Step 3,4 Hydrolysis of the alkylated malonic ester in aqueous NaOH followed by acidification with aqueous HCl gives a -dicarboxylic acid.

Step 5. Heating the -dicarboxylic acid slightly above its melting point brings about decarboxylation to give monocarboxylic acid.

A disubstituted acetic acid can be prepared by interrupting the previous sequence after Step 2, treating the monosubstituted diethyl malonate with a second equivalent of base, carrying out a second alkylation, and then proceeding with Steps 3 through 5.

8.2. Reactions of carboxylic acids and their derivatives. Characteristic properties. Reaction centers The most apparent chemical property of carboxylic acids, their acidity, has already been examined in the chapter of Acids and Bases.

n – basic center

O. electrophile center R–CH C O H OH –acidic center

H CH – acidic center

The most common reaction theme of acid halides, anhydrides, esters, and amides is addition of a nucleophile to the carbonyl carbon to form a tetrahedral carbonyl addition intermediate. In this sense, the reaction of these functional groups is similar to nucleophilic addition to the carbonyl groups in aldehydes and ketones. This reaction can also be catalyzed by acid, in which case protonation of the carbonyl oxygen precedes the attack of the nucleophile. For functional derivatives of carboxylic acids, the fate of the tetrahedral carbonyl addition intermediate is quite different; the intermediate collapses to expel the leaving group Y and regenerate the carbonyl group. The result of this addition-elimination sequence is nucleophilic acyl substitution.

107

8.2.1. Nucleophilic acyl substitution.

addition intermediate Substitution product

The major difference between the nucleophilic addition to the carbonyl groups in aldehydes and ketones and acids is that aldehydes and ketones do not have a group that can leave as a relatively stable anion. Aldehydes and ketones undergo only nucleophilic acyl addition. The carboxylic acid derivatives do have a group, Y, that can leave as a relatively stable anion; they undergo nucleophilic acyl substitution. Neutral molecules, such as water, alcohols, ammonia, and amines, may also serve as nucleophiles and leaving groups in the acid-catalyzed version of this reaction. The leaving group here as an anion. An important point about leaving groups is: the weaker the base, the better the leaving group. Relative reactivities of carboxyl derivatives toward nucleophilic acyl substitution. Interconversion of functional derivatives. The weakest base in the series, and the best leaving group, is halide ion; acid halides are the most reactive toward nucleophilic acyl substitution. The strongest base, and the poorest leaving group, is amide ion; amides are the least reactive toward nucleophilic acyl substitution. Acid halides and acid anhydrides are so reactive that they are not found in nature. Esters and amides, however, are universally present.

Reactivity toward nucleophilic acyl substitution

Increasing leaving group

Increasing basicity

Each derivative has certain characteristic reactions of its own.

108

Thioesters are most effective acylating agents because of the same reasons: the-SR groups are more easy leaving groups compare with (to) the alcoxide ion. In addition, many reactions of carboxyl derivatives occur by acid catalysis. In these reactions, the carbonyl group is protonated in the first step, which increases its electronegativity. Then the leaving group is protonated in a later step to decrease its basicity and make it a better leaving group. Treatment of a carboxylic acid with thionyl chloride (the acid chloride of sulfurous acid) converts it to the more reactive acid chloride. Carboxylic acids are about as reactive as esters under acidic conditions, but are converted to the unreactive carboxylates under basic conditions. Acid chlorides are the most reactive toward nucleophilic acyl substitution and that amides are the least reactive. Another useful way to think about the relative reactivities of these four functional derivatives of carboxylic acids is following: any compound (functional group) at left hand (in row) could be prepared from those at right hand by treatment. An acid chloride, for example, can be converted to an acid anhydride, an ester, an amide, or a carboxylic acid. Acid anhydrides, esters, and amides, however, do not react with chloride ion to give acid chlorides. * Acetyl coenzyme A The form in which is used in most of its important biochemical reactions is acetyl coenzyme A. Acetyl coenzyme A is a thioester. Its formation from pyruvate involves several steps and is summarized in the overall equation:

Pyruvic Coenzyme A Acetyl Carbon Proton acid coenzyme A dioxide

All the individual steps are catalyzed by enzymes. NAD+ is required as an oxidizing agent, and coenzyme A is the acetyl group acceptor. Coenzyme A is a thiol; its chain terminates in a sulfhydryl (— SH) group. of the sulfhydryl group of coenzyme A gives acetyl coenzyme A. Because sulfur does not donate electrons to an attached carbonyl group as well as oxygen does, compounds of the type are better acyl transfer agents than is Both properties are apparent in the properties of acetyl coenzyme A. In some of its reactions acetyl coenzyme A acts as an acetyl transfer agent, whereas in others the  carbon atom of the acetyl group is the reactive site. In vivo reactions (reactions in living systems) are enzyme-catalyzed and occur at rates that are far greater than when the same transformations are carried out in vitro ("in glass") in the absence of enzymes. These transformations are essential fundamental processes of organic chemistry. 109

8.3. Preparation of functional derivatives of carboxylic acids  A more reactive derivative may be converted to a less reactive derivative by treatment with an appropriate reagent.  Acyl chlorides readily available and are normally prepared from carboxylic acids by the reaction with thionyl chloride or other non-metal

halides (PCl3, PCl5, SOCl2):

Carboxylic acid Thionyl Acyl chloride Sulfur Hydrogen chloride dioxide chloride

 Amides are available by heating of ammonium salts of carboxylic acids. O t  O R – C R – C + H2O ONH4 NH2 Preparation of carboxylic acids anhydrides a) The customary method for the laboratory synthesis of acid anhydrides is the reaction of acyl chlorides with carboxylic acids or their salts

b)Are assessable by dehydration of carboxylic acids in the presence of P4O10. This compound is known as” phosphorous pentoxide”, because it was once thought to have molecular formula P2O5. Phosphorous pentoxide is the anhydride of phosphoric acid and is used in a number of reactions that require a dehydrating agent.

 Acid catalised esterification reaction. Acid-catalyzed esterification of carboxylic acids is one of the fundamental reactions of organic chemistry. The net reaction is: O H+ O R – C + H – O – R' R – C +H2O OH O – R'

Mechanism of esterification. Protonation of the carbonyl oxygen activates the carbonyl group toward nucleophilic addition. Addition of alcohol gives a tetrahedral intermediate (shown in the box), which has the capacity to revert to starting materials or to undergo dehydration to yield an ester. Esters can be 110 hydrolyzed to carboxylic acids and alcohols under conditions of acid catalysis.

8.4. Chemical properties of functional derivatives of carboxylic acids 8.4.1. Saponification Hydrolysis of an ester in aqueous NaOH or KOH to an alcohol and the sodium or potassium salt of a carboxylic acid is known as saponification (after the Latin word sapo, "soap"). The product of saponification is a carboxylate anion rather than a free carboxylic acid.

Although the carbonyl carbon of an ester is not strongly electrophilic, hydroxide ion is a good nucleophile and adds to the carbonyl carbon to form a tetrahedral carbonyl addition intermediate, which in turn collapses to give a carboxylic acid and an alkoxide ion. The carboxylic acid reacts with the alkoxide ion or other base present to form a carboxylic acid anion.

8.4.2. Halogenation at carbon atom of carboxylic acids. An acid is treated with chlorine or bromine in the presence of a catalytic quantity of phosphorus or a phosphorus trihalide:

Carboxylic acid Halogen -Halo acid

8.4.3.Decarboxylation The loss of a molecule of carbon dioxide from a carboxylic acid is known as a decarboxylation reaction.

Carboxylic acid Alkane Carbon dioxide 111

Decarboxylation of simple carboxylic acids takes place with great difficulty and is rarely encountered. Decarboxylation is of limited importance for aromatic acids, and highly important for certain substituted aliphatic acids: malonic acids and -keto acids.

Decarboxylation of malonic acid and related compounds Compounds that do undergo ready thermal decarboxylation include those related to malonic acid. On being heated above its melting point, malonic acid is converted to acetic acid and carbon dioxide.

Malonic acid Acetic acid Carbon dioxide (propanedioic acid) (ethanoic acid)

It is important to recognize that only one carboxyl group is lost in this process. The second carboxyl group is not cleaved under these reaction conditions. The thermal decarboxylation of malonic acid derivatives is the last step in a multistep synthesis of carboxylic acids known as the malonic ester synthesis. Compounds that have substituents other than hydroxyl groups at  position undergo an analogous decarboxylation. The compounds most frequently encountered in this reaction are  -keto acids, that is, carboxylic acids in which the  carbon is a carbonyl function. Decarboxylation of - keto acids leads to ketones.

8.4.4. Reduction of esters Like many organic compounds, esters can be reduced in two ways: (a) by catalytic hydrogenation using molecular hydrogen, or (b) by chemical reduction. In either case, the ester is cleaved to yield (in addition to the alcohol or phenol from which it was derived) a primary alcohol corresponding to the acid portion of the ester.

112

Chemical reduction is carried out by use of sodium metal and alcohol, or more usually by use of lithium aluminum hydride. For example:

8.5. Dicarboxylic acids Dicarboxylic acids have been prepared from dihalides by the following way:

1,3-Dibromopropane 1,5-Pentanedinitrile 1,5-Pentanedioic acid

Dicarboxylic acids are characterized by separate ionization constants, designated K1, and K2, respectively, for each ionization step. First representative of dicarboxylic acid, oxalic acid is poisonous and occurs naturally in a number of plants including sorrel and begonia.

oxalic acid hydrogen oxalate (monoanion), pK1= 1.2

hydrogen oxalate oxalate (monoanion) (dianion) pK2 = 4.3

The first ionization constant of dicarboxylic acids is larger than Ka for monocarboxylic analogs. There are two potential sites for ionization rather than one. One carboxyl group acts as an electron-withdrawing group to facilitate dissociation of the other. This is particularly noticeable when the two carboxyl groups are separated by only a few bonds. Oxalic and malonic acid, for example, are several orders of magnitude stronger than simple alkyl derivatives of acetic acid (see 5.2.2.). Heptanedioic acid, in which the carboxyl groups are well separated from each other, is only slightly stronger than acetic acid. HO2CCO2H HO2CCH2CO2 HO2C(CH2)5CO2H Oxalic acid Malonic Heptanedioic acid -2 -3 -5 K1, 6.5x10 K1 1.4x10 K1 3.1x10 (pK1= 1.2) (p K1= 2.8) (p K1= 4.3)

113

1,1-Dicarboxylic acids and -keto acids are subject to ready thermal decarboxylation by a mechanism in which a  carbonyl group assists the departure of carbon dioxide. The distinguish feature of succinic (CH2CO2H)2 and glutaric CH2(CH2CO2H)2 acids, is that by heating they undergo dehydration by converting into cyclic anhydrides: O O CH – C – OH – C 2 t CH2

CH2 – C – OH - H2O O CH – C O 2 O succinic acid anhydride of succinic acid O

CH2 – C – OH CH2 – C = O t H2 C H2C O - H2O CH2 – C – OH CH2 – C = O O glutaric acid anhydride of glutaric acid

CH2(CO2C2H5)2 – diethylmalonic ester is used as a precursor in the synthesis of carboxylic acids and barbituric acid, which is in turn in the presence of base serves as a substrate for synthesis of many sedative medicines, such as barbital (veronal) and pheno barbital (luminal).

barbital (veronal) pheno barbital (luminal)

8.6. Unsaturated carboxylic acids The main representatives are: CH2=CH–COOH CH3–CH=CH–COOH acrylic acid crotonic acid propenoic acid butenoic acid

Unsaturated dicarboxylic acids (maleic acid, fumaric acid)

114

maleic acid anhydride of maleic acid

8.7. Carbonic acid and its derivatives

The simplest dicarboxylic acid, carbonic acid, is not even classified as an organic compound. Because many minerals are carbonate salts, nineteenth-century chemists placed carbonates, bicarbonates, and carbon dioxide in the inorganic realm. Nevertheless, the essential features of carbonic acid and its salts are easily understood on the basis of our knowledge of carboxylic acids. Carbonic acid is formed when carbon dioxide reacts with water. Hydration of carbon dioxide is far from complete, however. Almost all the carbon dioxide that is dissolved in water exists as carbon dioxide; only 0.3 percent of it is converted to carbonic acid. Carbonic acid is a weak acid and ionizes to a small extent to bicarbonate ion.

Carbon Water Carbonic Bicarbonate dioxide acid ion

The systematic name for bicarbonate ion is hydrogen carbonate. Thus, the systematic name for sodium bicarbonate (NaHCO3) is sodium hydrogen carbonate. The equilibrium constant for the overall reaction is related to an apparent equilibrium constant K1 for carbonic acid ionization by the expression

Carbonic anhydrase is an enzyme that catalyzes the hydration of carbon dioxide to bicarbonate. The uncatalyzed hydration of carbon dioxide is too slow to be effective in transporting carbon dioxide from the tissues to the lungs, and so mammals have developed catalysts to speed this process. The activity of carbonic anhydrase is remarkable; it has been estimated that one molecule of this enzyme can catalyze the hydration of 3.6 x 107 molecules of carbon dioxide per minute.

115

As with other dicarboxylic acids, the second ionization constant of carbonic acid is far smaller than the first.

Bicarbonate ion Carbonate ion

-11 The value of K2 is 5.6 x 10 (pKa 10.2). Bicarbonate is a weaker acid than carboxylic acids but a stronger acid than water and alcohols. 8.7.1.Functional derivatives of carbonic acid Much of the chemistry of the functional derivatives of carbonic acid is already quite familiar to us through our study of carboxylic acids. The first step in dealing with one of these compounds is to recognize just how it is related to the parent acid. Since carbonic acid is , each of its derivatives, too, contains two functional groups; these groups can be the same or different. For example:

Carbonic acid Phosgene Urea Ethyl carbonate Acid (Carbonyl chloride) (Carbamide) Ester Acid chloride Amide

Ethyl chlorocarbonate Cyanamide Urethane Carbamic acid Acid chloride-ester Amide-nitrile (Ethyl ) Ester-amide

We use these functional relationships to carbonic acid simply for convenience. In general, a derivative of carbonic acid containing an—OH group is unstable, and decomposes to carbon dioxide. For example:

116

Most derivatives of carbonic acid are made from one of three industrially available compounds: phosgene, urea, or cyanamide. Phosgene, COCI2, a highly poisonous gas, is manufactured by the reaction between and chlorine.

Phosgene It undergoes the usual reactions of an acid chloride.

Urea, H2NCONH2, is excreted in the urine as the chief nitrogen-containing end product of metabolism. It is synthesized on a large scale for use as a fertilizer and as a raw material in the manufacture of urea-formaldehyde plastics and of drugs.

Urea undergoes hydrolysis in the presence of' acids, bases, or the enzyme urease.

117

Urea reacts with nitrous acid to yield carbon dioxide and nitrogen; this is a useful way to destroy excess nitrous acid in diazotizations.

Urea is converted by hypohalites into nitrogen and carbonate.

Treatment of urea with acid chlorides or anhydrides yields ureides.

Acetylurea A ureid importance are the cyclic ureides formed by reaction with malonic esters; these are known as barbiturates and are important hypnotics (sleep- producers). For example:

Urea Ethyl malonate Barbituric acid (Malonylurea)

The qualitative and quantitative determination of urea. In the biuret test the sample is treated with an alkaline copper sulfate reagent that produces a violet color due to presence of two peptide bonds. The biuret is a product of heating of urea:

Biuret

Biuret Violet complex

Derivatives of guanidine. The guanidinium group of arginine is transferred to to form guanidinoacetate, N of which forms creatine, precursor of creatine phosphate, a high energy phosphate–storage compound found in muscle. 118

Arginine Guanidinoacetate Creatine

9. POLYFUNCTIONAL AND HETEROFUNCTIONAL COMPOUNDS

9.1. Polyfunctional compounds. Polyfunctional compounds are compounds containing two and more of the same functions. According to the function they are classified as polyalcohols (OH-containing-ethylene glycol, glycerin, pyrocatechine, resorcine, hydroquinone, inositol etc.), di- and polyamines (NH2 containing putrescine, cadaverine), dicarboxylic and tricarboxylic acids, most of which have important role in the metabolic transformations in living systems due their physiological and biochemical effects. CH2OH–CH2OH CH2OH–CHOH–CH2OH 1,2- ethanediol 1,2,3- propanetriol (ethylene glycol) (glycerol)

Vicinal diols are diols that have their hydroxyl groups on adjacent carbons.The most commonly encountered diol is 1,2-ethane diol or ethylene glycol. Ethylene glycol is a poisonous liquid, which is used as an antifreeze in cars. Glycerol is the structural component of many biologically active compounds such as triacylglycerols, phospholipids. Industrially it is used for preparation of ointments and for other purposes. The acidic properties of polyatomic alcohols are more expressed due to the negative inductive effects of neighboring hydroxyl groups. Chemical properties of polyatomic alcohols are similar to the simple alcohols. At the same time more than one functional group could be involved in the reaction. One of the simplest methods of identification of presence of diol fragments is based on the reaction with some metal hydroxides with complex (chelate) formation in the presence of a base. Particularly widely used copper (II) hydroxide with bright blue colored complex formation.

119

2 - CH O CH2OH 2 OCH2 + 2 + Cu(OH)2 + 2NaOH Cu 2Na -4H2O CH O CH2OH 2 OCH2

Depending on the conditions at which reaction is carried out, dehydration of ethylene glycol could lead to intramolecular or intermolecular dehydration. Intramolecular dehydration leads to ethylene oxide formation. The result of intermolecular dehydration is dioxan, which is poisonous compound, but very good solvent. O CH2OH CH2OH t  H2 SO4 2 H2C CH2 2 -H2O - 2 H2O CH OH , 2 O CH2OH O Ethylene glycol Ethylene oxide dioxan Glycerol eliminates water by heating and is converted into -unsaturated aldehyde-acrolein.

CH2OH CH2 O CH – OH t  C -2H2O CH2 = CH – C H H CH2OH CH — O

Some derivatives of glycerol, especially esters, have many applications in medicine. Trinitro glycerol (1% solution in ethanol) is used as vasodilatator, glycerol phosphates as well as phospholipids have regenerative action in so called membrane diseases. Interaction of glycerol and phosphoric acid leads to the formation of mixture of and glycerol phosphates.

- glycerol phosphate - glycerol phosphate

The main representatives of cyclic diols, pyrocatechine, resorcine, hydroquinone reveal significant, but different biological effects. OH OH OH OH OH OH pyrocatechine resorcine hydroquinone 120

Pyrocatechine is a structural part of epinephrine and norepinephrine (adrenaline and noradrenaline), the hormones and neuromediators, compounds with highly expressed physiological activity. Hydroquinone is a structural part of coenzymes Q, the participants of mitochondria’s terminal oxidation chain. The phosphoric esters of six member cyclic alcohol inositol serve as intracellular secondary messengers, regulators. OH OH 2 3 HO H 1 H H HO 4 H 5 H 6 OH OH H ÙÇá-ÇÝá½ÇïáÉ mioinozitol

Acyclic (malonic, succinic, oxalic, glutaric, maleic, fumaric acids) and cyclic dicarboxylic acids (phthalic acid, terephthalic acid) also belong to the polyfunctional compounds. Rotting of amino acids is accompanied with diamines or ptomaines formation, which possessed toxic properties. H2N–CH2–CH2–CH2–CH2–NH2 H2N–CH2–CH2–CH2–CH2–CH2–NH2 1, 2-diaminobutane 1, 5 -diaminepentane putrescine cadaverine

9.2. Heterofunctional compounds. Classification of heterofunctional compounds Compounds containing two and more different functions in the same molecule are called heterofunctional compounds. Depending on the nature of functions, compounds containing only two different functions are classified as is shown in Table 9.1:

Table 9. 1. Classes of heterofunctional compounds Functional groups Classes – OH, -NH2 amine alcoholes – OH, -COOH hydroxyl acids

oxo acids, keto acids

–NH2, – COOH amino acids carbohydrates – OH

121

These compounds give all the reactions characteristic for present functions (functional groups), simultaneously they possess some distinguished chemical properties. All of represented functions have electronegative effect in aliphatic compounds and because of that increase reactivity of each other. For example, 2- hydroxypropanoic acid (pKa = 3.08), is a stronger acid than propanoic acid (pKa = 4.87) because of the electron-withdrawing inductive effect of the hydroxyl oxygen. Depending on the location of functional groups there are  - isomers. The chemical activity also depends on that how close functions to each other are: with increase of distance between them activity decreases.

9.2.1. Amino alcohols. Ethanolamine, choline, Preparation, biological role. In the laboratory synthesis one of the most well known amino alcohol, ethanolamine, is carried out in two ways:

Ethylene oxide ethanolamine Ethylene

In the living tissues synthesis of colamine takes place presumably by decarboxylation of amino acid serine

serine ethanolamine

The main derivative of ethanolamine- choline, could be synthesized by methylation both in the cells and in the laboratory. The laboratory synthesis take place in the following ways: ( CH 3 ) 3 N, H 2O H 2C – CH 2 O + HO – CH 2 – CH 2 – N ( CH 3 ) 3 OH HOCH 2 CH 2 NH 2 AgOH,3CH I 3

The main biological function of ethanolamine and choline is that these compounds are structural components of phospholipids. Besides, choline is a precursor of neuromediator acetylcholine. The formation of acetylcholine from choline in the biological systems takes place by choline interaction with acetyl- CoA: 122

choline acetylcholine

Some naturally occurring amines mediate the transmission of nerve impulses and are referred to as neurotransmitters. Two examples are: OH OH

CH – CH2 – NH2 CH – CH2 – NH – CH3

OH OH OH OH norepinephrine epinephrine (noradrenaline) (adrenaline )

Some cyclic aminoalcohols and their derivatives are used as medicines (drugs):

p-aminophenol phenetidide p-acetamidophenol phenacetin

p-acetamidophenol (paracetamol) and phenacetin are an analgesic and antipyretic medicines and in combination with other components are wellknown as tylenol, coldrex, teraflu, etc.

9.2.2. Hydroxyl acids The general formula of hydroxides of monocarboxylic acids is: R– CHOH – (CH2) n – COOH R = H, Alk, Ar, n = 0, 1, 2, ... Structural (etc), geometrical and optical isomerism are observed in hydroxyl acids. Main representatives are given in Table 9.2.

123

Table 9.2. Main representatives of hydroxy acids

glycolic acid 2 – hydroxyethanoic acid lactic acid 2 – hydroxypropanoic acid

 - hydroxybutyric acid  - hydroxybutyric acid 3 – hydroxybutanoic acid 4 - hydroxybutanoic acid

9.2.2. 1. Preparation of hydroxyl acids  By nucleophilic substitution of acyl halids in reaction with bases:

Monochloracetic acid Glycolic acid

 By nucleophilic addition of HCN to carbonyl compounds and further hydrolysis of formed cyanohydrins:

 - hydroxy valeric acid

This method is used only for preparation of  -hydroxyacids.  By hydration of  unsaturated acids only -hydroxy acids are formed:

- hydroxy propionic acid propenoic acid 3- hydroxy propanoic acid 124

 By oxidation of primary hydroxyl groups of diols,

etane diol hydroxyethanoic acid ethylene glycol glycolic acid

By reducing of ketoacids:

lactic acid

9.2.2.2. Chemical properties of hydroxyl acids A hydroxy acid is both alcohol and acid. Chemical properties are determined by the existing functional groups: hydroxyl acids give all reactions which are characteristic for both –OH and -COOH functional groups: etherification, oxidation, substitution. At the same time they possess new properties, which are determined by the presence and mutual influence of different groups. In such compounds reaction of nuclephylic substitution could take place not only between different molecules.  Hydroxy acids, i.e., compounds that contain both a hydroxyl and a carboxylic acid function within the same molecule, have the capacity to form cyclic esters called lactones. This intramolecular esterification reaction takes place spontaneously and is particularly favored when the formed ring is five- or six-membered. Lactones, a five-membered cyclic esters are referred to - lactones because they arise from  -hydroxy carboxylic acids. Their six- membered analogues are known as -lactones.

4-Hydroxybutanoic acid  - butyrolactone

Treatment with base (actually hydrolysis of an ester) rapidly opens the lactone ring to give the open-chain salt. The characteristic reaction of -hydroxyacids is cyclic esters (lactides) formation due to intermolecular dehydration For example, lactic acids by heating are formed lactide. 125

O O C – OH H – O C – O t H3 C – CH HC – CH3 H3 C – CH HC – CH3 - 2H2O O – H HO – C O – C O O lactic acids lactide

-Hydroxyacids undergo intramolecular dehydration, forming  -unsaturated carboxylic acid. For example:

- hydroxy butyric acid 2- butenoic acid 3-butanoic acid

These specific reactions are used for identification of structural isomers of hydroxyacids. Many derivatives of cyclic hydroxyacids are used as medicines:

p-aminosalicylic acid acetylsalicylic acid .

9.2.3. Hydroxy and oxo-derivatives of di- and tricarboxylic acids The main representatives are malic acid (2-hydroxybutanedioic acid), citric acid (2-hydroxy-1,2,3-tricarboxypropane, see Topic 7.2.6), 2,3-dihydroxy- butanedioic acids. The most important derivatives are participants of tricarboxylic acid cycle (Krebs cycle).

Fumaric acid malic acid oxaloacetic acid (trans-butendioic acid) (hydroxy butendioic acid) oxobutendioic acid

126

9.2.4. Ketoacids The general structural formula is:

R – C – ( CH2) n – COOH R = H, Alk, Ar, n = 0, 1, 2, ... O The most abundant representatives are:

glyoxalic acid 2- oxobutanedioic acid or oxaloacetic acid

2-oxopropanoic or pyruvic acid 2- oxopentanedioic acid or - ketoglutaric acid

3- oxobutanoic acid or

9.2.4.1. Preparation of ketoacids  The main way of preparation of ketoacids is oxidation of hydroxy acids:

hydroxy acid keto acid

Lactic acid Pyruvic acid  Pyruvic acid or 2-oxopropanoic acid is the important metabolite of glucose aerobe oxidation and also is formed by alanine transamination:

Alanine Pyruvic acid

 Pyruvic acid could be prepared in the reaction of with potassium cyanide (KCN):

acetyl chloride nitrile of acetic acid pyruvic acid

127

Decarboxylation. Most carboxylic acids are quite resistant to moderate heat without decarboxylalion. Exceptions are carboxylic acids that have an oxo group  to the carboxyl group. This type of carboxylic acid undergoes decarboxylation quite readily on mild heating. For example, when 3-oxobutanoic acid is heated moderately, it undergoes decarboxylation to give acetone and carbon dioxide. Decarboxylation on mild heating is a unique property of -oxocarboxylic (- ketoacids) and is not observed with other classes of ketoacids. Oxaloacetic acid, one of the main substrates of TCA (Krebs) cycle, is formed by transamination of aspartic acid and carboxilation of pyruvic acid. Acetoacetic acid (-oxobutyric acid) which is formed by oxidation of - hydroxybutyric acid, is unstable and even at the room temperature undergo decarboxilation with acetone formation. [O] H3 C – CH – CH2 – COOH H3 C – C – CH2 – COOH H3 C – C – CH3 CO2 OH O O - hydroxybutyric acid -ketobutyric acid acetone

* and diabetes mellitus The keto-enol tautomerism is observed in such compounds and enol is converted into the keto form due to movable hydrogen and vice versa.

R – C – CH – COOH R – C = CH – COOH O H OH keto enol

3-Oxobutanoic acid (acetoacetic acid) and its reduction product, 3-hydroxybutanoic acid are synthesized in the liver from acetyl-CoA, a product of the metabolism of fatty acids and certain amino acids. 3-Hydroxybutanoic acid , 3-oxobutanoic acid and acetone are known collectively as ketone bodies. The concentration of ketone bodies in the blood of healthy, well-fed humans is approximately 0.01 mM/L. However, in persons suffering from starvation or diabetes mellitus, the concentration of ketone bodies may increase to as much as 500 times normal. Under these conditions, the concentration of acetoacetic acid increases to the point where it undergoes spontaneous decarboxylation to form acetone and carbon dioxide. Acetone is not metabolized by humans and is excreted through the kidneys and the lungs. The odor of acetone is responsible for the characteristic "sweet smell" of the breath of severely diabetic patients. Acetoacetate, -hydroxybutyrate, and acetone are commonly known within the health sciences as ketone bodies, in spite of the fact that one of them is not a ketone at all. They are products of human metabolism and are always present in blood plasma. Most tissues, with the notable exception of the brain, have the enzyme systems necessary to use them as energy sources. Synthesis of ketone bodies occurs by the enzyme-catalyzed reactions. Reaction (4) is spontaneous and uncatalyzed. 128

Application of acids in medicine A strongly acid solution can damage the skin as seriously as a flame can. Nevertheless, as administered by physicians such solutions have found the place in the treatment of a variety of skin conditions. And weakly acidic solutions used in skin treatments fall at the borderline of the distinction between prescription drugs and cosmetics. Trichloracetic acid, a strong acid, is used for chemical peeling of the skin (a treatment for eczema or psoriasis), for removal acne scars, and sometimes for removing wrinkles. The depth of removal (and the length of the healing period) varies with the strength of the acid and how long it is left on the skin. As the result of healing, old skin is replaced by a new, smoother skin surface. A number of naturally occurring - hydroxyl acids, which are acetic-acid-like weak acids, provide less aggressive skin treatments (Glycolic acid, Lactic acid, Salicylic acid). Glycolic acid is used by physicians for a "mini" peel that can be done quickly and from which the skin returns to a normal appearance in just a few hours. In higher concentrations, glycolic acid is used for "spot" removal of precancerous lesions or unsightly brown, thickened skin (keratoses). The alpha-hydroxyls have a further promotional advantage because they are all "nature's own" chemicals. Lactic acid, which at 12% concentration is a prescription drug, is present in many over-the-counter lotions and creams at lower concentration.

129

10. AMINO ACIDS

So far more than 300 amino acids were separated and identified from different natural sources such as animal tissues, plants, yeasts, microbes and so on. They vary in large scale in their structures, physiological activity and biological roles. Most of them serve as a regulators of different metabolic processes, such as - amino butyric acid (GABA), others are found as a structural components of bioactive compounds such as coenzymes (-alanine in CoASH, p-amino benzoic acid in the tetrahydrofolic acid, FH4). Proteins are the most abundant class of organic compounds in the healthy, lean human body, constituting more than half of its cellular dry weight. Proteins are the indispensable agents of biological function, and amino acids are the building blocks of proteins. Proteins are polymers of amino acids and have molecular weights ranging from approximately 10,000 to more than one million. Biochemical functions of proteins include catalysis, transport, contraction, protection, structure, and metabolic regulation. The stunning diversity of the thousands of proteins found in nature arises from the intrinsic properties of only 20 commonly occurring amino acids. These features include (1) the capacity to polymerize, (2) novel acid-base properties, (3) varied structure and chemical functionality in the amino acid side chains, and (4) chirality. Amino acids are the monomeric units, or building blocks, of proteins joined by a specific type of covalent linkage. The properties of proteins depend on the characteristic sequence of component amino acids, each of which has distinctive side chains. Amino acid polymerization requires elimination of a water molecule as the carboxyl group of one amino acid reacts with the amino group of another amino acid to form a covalent amide bond. The repetition of this process with many amino acids yields a polymer, known as a polypeptide. The amide bonds linking amino acids to each other are known as peptide bonds. Each amino acid unit within the polypeptide is referred to as a residue. The sequence of amino acids in a protein is dictated by the sequence of nucleotides in a segment of the DNA in the chromosomes.

10.1. L--Amino Acids: Structure Almost all of the naturally occurring amino acids in proteins are L-- amino acids. There are principal 20 amino acids in proteins. The structure of a single typical amino acid is shown in (Fig. 10.1). Central to this structure is the tetrahedral alpha () carbon (Ca), which is covalently linked to both the amino group and the carboxyl group. Also bonded to this -carbon is a 130 hydrogen and a variable side chain, so-called R group, that gives each amino acid its identity.

Fig. 10.1

Proline is an exception because it has a cyclic structure and contains a secondary amine group (called an imino group) instead of a primary amine group (called an amino group). In neutral solution (pH 7), the carboxyl group exists as —COO- and the + amino group as -NH3 . Because the resulting amino acid contains one positive and one negative charge, it is a neutral molecule called a zwitterion. Amino acids are also chiral molecules. With four different groups attached to it, the - carbon is said to be asymmetric. The two possible configurations for the- carbon constitute nonidentical mirror image isomers or enantiomers. Several amino acids, including isoleucine, threonine, hydroxyproline, and hydroxylysine, have two asymmetric carbons. The isomer obtained from digests of natural proteins is arbitrarily designated L-isoleucine. Except for glycine (R = H), the amino acids have at least one asymmetrical carbon atom (the -carbon). The absolute configuration of the four groups attached to the -carbon is conventionally compared to the configuration of L-glyceraldehyde (Fig. 10.2). The D and L designations specify absolute configuration and not the dextro (right) or levo (left) direction of rotation of plane-polarized light by the asymmetrical carbon center. In organic chemistry, the assignment of absolute configurations of an asymmetrical center is made presumably by the R and S classification of isomers. But we prefer to use D and L designations for this class of compounds.

D-Glyceraldehyde L-Glyceraldehyde D-Alanine L-Alanine Fischer perspective formulas Fig. 10.2. Different representations of the configurational stereoisomers of glyceraldehyde and alanine. 131

10.2. Classifications of common amino acids. There are several ways to classify the common amino acids. The modern classification of Amino acids is based on the chemical properties of the R- groups of amino acids. All the amino acids except proline have both free - amino and free -carboxyl groups. There are the three-letter and one-letter codes used to represent the amino acids (Table 10.1).

Table 10.1. The structures and names of 23 amino acids that have been found in proteins. Certain of these (marked e) are the essential amino acids.

132

133

The most useful of these classifications is based on the polarity of the side chains at pH 7.0: (1) Nonpolar or hydrophobic amino acids. Hydrophilic amino acids in turn are divided into: (2) Neutral (uncharged) but polar amino acids, (3) Acidic amino acids (which have a net negative charge at pH 7.0), (4) Basic amino acids (which have a net positive charge at neutral pH). This classification system is very important for predicting protein properties. Within each class, R-groups differ in size, shape, and other properties. The structure of each amino acid is given according to this classification with the R-group outlined. Ionizable structures are drawn as they would exist at pH 7.0. The eight essential amino acids (Table10.2) are those which humans cannot synthesize and which must be supplied in the diet. The remaining amino acids are synthesized in the body by various biochemical pathways. (a) Nonpolar Amino Acids

Alanine Valine Leucine

Isoleucine Proline Phenylalanine

Methionine Tryptophan

(b) Polar, negatively charged (hydrophilic, acids in the protonated state)

Aspartate Glutamate (c) Polar, positively charged (hydrophilic, basic in the protonated state)

Histidine Lysine 134

Arginine

(d) Polar, neutral (hydrophilic)

Serine Threonine Tyrosine

Asparagine Glutamine

Cysteine Glycine

According to the biological significance amino acids are divided into 2 groups: essential and non-essential (Table 10.2.).

Table 10.2. Amino acid abbreviations and nutritional property Designation Nutritional Amino acids Abbreviation letter property* Alanine Ala A non -essential Arginine Arg R conditionally essential Asparagine Asn N non -essential Aspartic acid Asp D non -essential Cysteine Cys С non -essential Glutamic acid Glu E non -essential Glutamine Gin Q conditionally essential Glycine Gly G non -essential Histidine His H conditionally essential Isoleucine Ile I essential Leucine Leu L essential Lysine Lys К essential Methionine Met M essential Phenylalanine Phe F essential Proline Pro P non -essential Serine Ser S non -essential Threonine Thr Т essential Tryptophan Trp W essential Tyrosine Tyr Y non -essential Valine Val V essential 135

* The eight essential amino acids are not synthesized in the body and have to be supplied in the diet. The conditionally essential amino acids, although synthesized in the body, may require supplementation during certain physiological conditions such as pregnancy. The non-essential amino acids can be synthesized from various metabolites.

10.2.1.Nonpolar or hydrophobic amino acids Alanine. The side chain of alanine is a hydrophobic methyl group, — CH3. Other amino acids may be considered to be chemical derivatives of alanine, with substituents on the -carbon. Alanine and glutamate provide links between amino acid and carbohydrate metabolism. Valine, Leucine, and Isoleucine. These branched-chain aliphatic amino acids contain bulky nonpolar R-groups and participate in hydrophobic interactions. All three are essential amino acids. A defect in their catabolism leads to maple syrup urine disease. Isoleucine has asymmetrical centers at both the - and -carbons and four stereoisomers, only one of which occurs in protein. The bulky side chains tend to associate in the interior of water-soluble globular proteins. Thus, the hydrophobic amino acid residues stabilize the three-dimensional structure of the polymer. Phenylalanine. A planar hydrophobic phenyl ring is part of the bulky R-group of phenylalanine. It is an essential amino acid whose metabolic conversion to tyrosine is defective in phenylketonuria. Phenylalanine, tyrosine, and tryptophan are the only -amino acids that contain aromatic groups and consequently are the only residues that absorb ultraviolet (UV) light. Tryptophan and tyrosine absorb significantly more energy than phenylalanine at 280 nm, the wavelength generally used to measure the concentration of protein in a solution. Tryptophan. A bicyclic nitrogenous aromatic ring system (known as an indole ring) is attached to the -carbon of alanine to form the R-group of tryptophan. Tryptophan is a precursor of serotonin, melatonin, nicotinamide, and many naturally occurring medicinal compounds derived from plants. It is an essential amino acid. The indole group absorbs UV light at 280 nm - a property that is useful for spectrophotometric measurement of protein concentration. Tryptophan and tyrosine both show fluorescence; however, tryptophan absorbs more intensely. Methionine. This essential amino acid contains an R-group with a methyl group attached to sulfur. Methionine serves as donor of a methyl group in many transmethylation reactions, e.g., in the synthesis of epinephrine, creatine, and melatonin. Almost all of the sulfur-containing compounds of the body are derived from methionine. 136

Proline. Proline contains a secondary amine group, called an imine, instead of a primary amine group and is called an imino acid. Since the three- carbon R-group of proline is fused to the -nitrogen group, this compound has a rotationally constrained rigid-ring structure. As a result, prolyl residues in a polypeptide introduce restrictions on the folding of chains. In collagen, the principal protein of human connective tissue, certain prolyl residues are hydroxylated (Figure 10.4). The hydroxylation occurs during protein synthesis and requires ascorbic acid (vitamin C) as a . Deficiency of vitamin С causes formation of defective collagen and scurvy.

10.2.2. Acidic Amino Acids Aspartic Acid. The -carboxylic acid group of aspartic acid has a pK' of 3.86 and is ionized at pH 7.0 (the anionic form is called aspartate). The anionic carboxylate groups tend to occur on the surface of water-soluble proteins, where they interact with water. Such surface interactions stabilize protein folding. Glutamic Acid. Glutamate is the primary excitatory in the brain. In some proteins, the -carbon of glutamic acid contains an additional carboxyl group. Residues of  -carboxyglutamic acid bear two negative charges and can strongly bind calcium ions.  -Carboxylation of glutamic acid residues is a posttranslational modification and requires vitamin К as a cofactor.  - Carboxyglutamate residues are present in a number of blood coagulation proteins, osteocalcin, a protein present in the bone (Fig .10.4).

10.2.3. Basic Amino Acids Lysine. Lysine is an essential amino acid. The lysyl side chain forms ionic bonds with negatively charged groups of acidic amino acids. The  -NH2 groups of lysyl residues are covalently linked to biotin (a vitamin), lipoic acid, and retinal, a derivative of vitamin A and a constituent of visual pigment. In collagen and in some glycoproteins, C5-carbons of some lysyl residues are hydroxylated, and moieties are attached at these sites. In elastin and collagen, some  -carbons of lysyl residues are oxidized to reactive aldehyde (-CHO) groups, with elimination of NH3. These aldehyde groups then react with other -NH2 groups to form covalent cross-links between polypeptides, thereby providing tensile strength and insolubility to protein fibers. Arginine. The positively charged guanidinium group attached to the - carbon of arginine is stabilized by resonance between the two NH2 groups and has a pK' value of 12.48. Arginine is utilized in the synthesis of creatine and it 137 participates in the urea cycle. The nitrogen of the guanidino group of arginine is converted to (NO) by nitric oxide synthase. NO is unstable, highly reactive, and has a life span of only a few seconds. However, NO affects many biological activities, including vasodilation, inflammation, and neurotransmission. Histidine. The pK' value of histidyl residues in protein varies depending on the nature of the neighboring residues. The imidazolium-imidazole buffering pair has a major role in acid-base regulation (e.g., hemoglobin). The imidazole group functions as a nucleophile, or a general base, in the active sites of many enzymes and may bind metal ions. Histidine is nonessential in adults but is essential in the diet of infants and individuals with uremia (a kidney disorder). Decarboxylation of histidine to yield histamine occurs in mast cells present in loose connective tissue and around blood vessels, basophiles of blood, and enterochromaffin-like (ECL) cells. The many specific reactions of histamine are determined by the type of receptor present in the target cells. N N N N CH2 CH – COOH HN CH2 CH2 – CH2 – NH2 CH – COOH Cú 2 HN CH2 – CH2 – NH2 HN Cú2 NH2 HN NH2 hÇëï³ÙÇÝ hÇëïǹÇÝ histidine histaminehÇëï³ÙÇÝ hÇëïǹÇÝ Fig.10.3.

10.2.4. Neutral (polar, uncharged) amino acids Glycine. Glycine , the smallest and the simplest amino acid, has only a single hydrogen for an R group, and this hydrogen is not a good hydrogen bond former. Glycine's solubility properties are mainly influenced by its polar amino and carboxyl groups, and thus glycine is best considered a member of the polar, uncharged group. It is the only -amino acid that is not optically active. The small R-group provides a minimum of steric hindrance to rotation about bonds; therefore, glycine fits into crowded regions of many peptide chains. Collagen, a rotationally restricted fibrous protein, has glycyl residues in about every third position in its polypeptide chains. Glycine is used for the of many nonprotein compounds, such as porphyrins and purines. Glycine and taurine are conjugated with bile acids, products derived from cholesterol, before they are excreted into the biliary system. Conjugated bile acids are amphipathic and are important in absorption. Glycine also is a neurotransmitter; it is inhibitory in the spinal cord and excitatory in the cerebral cortex and other regions of the forebrain.

138

Serine. The primary alcohol group of serine can form esters with phosphoric acid and glycosides with . The phosphorylation and dephosphorylation processes regulate the biochemical activity of many proteins. Active centers of some enzymes contain seryl hydroxyl. Threonine. This essential amino acid has a second asymmetrical carbon atom in the side chain and therefore can have four isomers, only one of which, L-threonine, occurs in proteins. The hydroxyl group, as in the case of serine, participates in reactions with phosphoric acid and with sugar residues. Cysteine. The weakly acidic (pK' = 8.33) sulfhydryl group (-SH) of cysteine is essentially undissociated at physiological pH. Free -SH groups are essential for the function of many enzymes and structural proteins. Heavy metal ions, e.g., Pb2+ and Hg2+, inactivate these proteins by combining with their -SH groups. Two cysteinyl -SH groups can be oxidized to form cystine. A covalent bond of cystine can join two parts of a single polypeptide chain or two different polypeptide chains through cross-linking of cysteine residues. These -S-S- bonds are essential both for the folding of polypeptide chains and for the association of polypeptides in proteins that have more than one chain, e.g., insulin and immunoglobulins. Tyrosine. The phenolic hydroxyl group of this aromatic amino acid has a weakly acidic pK' of about 10 and therefore is unionized at physiological pH. In some enzymes, the hydrogen of the phenolic hydroxyl group can participate in hydrogen bond formation with oxygen and nitrogen atoms. The phenolic hydroxyl group of tyrosine residues in protein can be sulfated (e.g., in gastrin and cholecystokinin) or phosphorylated by a reaction catalyzed by the tyrosine-specific protein kinase that is a product of some oncogenes. Tyro- sine kinase activity also resides in a family of cell surface receptors that includes receptors for such anabolic polypeptides as insulin, epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor type 1. Tyrosine accumulates in tissues and blood in tyrosinosis and tyrosinemia, which are due to inherited defects in catabolism of this amino acid. Tyrosine is the biosynthetic precursor of thyroxine, catecholamines and melanin. Tyrosine and its biosynthetic precursor, phenylalanine, both absorb UV light. Asparagine. The R-group of this amide derivative of aspartic acid has no acidic or basic properties but is polar and participates in hydrogen bond formation. It is hydrolyzed to aspartic acid and ammonia by the enzyme asparaginase. In glycoproteins, the carbohydrate side chain is often linked through the amide group of asparagine. Glutamine. This amide of glutamic acid has properties similar to those of asparagine. The -amido nitrogen, derived from ammonia, can be used in the 139 synthesis of purine and pyrimidine nucleotides, converted to urea in the liver, or released as NH3 in the kidney tubular epithelial cells. The last reaction, catalyzed by the enzyme glutaminase, functions in acid-base regulation by neutralizing H+ ions in the urine. Glutamine is the most abundant amino acid in the body. It composes more than 60% of the free amino acid pool in skeletal muscle. It is metabolized in both liver and gut tissues. Glutamine, along with alanine, are significant precursors of glucose production during fasting. Glutamine is enriched in enteral and parenteral nutrition to promote growth of tissues; it also enhances immune functions in patients recovering from surgical procedures. Thus, glutamine may be classified as a conditionally essential amino acid during severe trauma and illness.

10.2.5. Uncommon Amino Acids Several amino acids occur only rarely in proteins. These include hydroxylysine and hydroxyproline, which are found mainly in the collagen and gelatin proteins, and thyroxine and 3,3',5-triiodothyronine, iodinated amino acids that are found only in thyroglobulin, a protein produced by the thyroid gland. (Thyroxine and 3,3',5-triiodothyronine are produced by iodination of tyrosine residues in thyroglobulin in the thyroid gland. Degradation of thyroglobulin releases these two iodinated amino acids, which act as hormones to regulate growth and development.). -carboxyglutamic acid present in certain blood-clotting proteins.

 -Carboxy glutamate Fig .10.4. The structures of several amino acids that are less common but nevertheless found in certain proteins.

10.2.6. Unusual Amino Acids. Several L-amino acids have physiological functions as free amino acids rather than as constituents of proteins. Certain amino acids and their derivatives, although not found in proteins, nonetheless are biochemically important. -Aminobutyric acid, or GABA, is produced by 140 the decarboxylation of glutamic acid and is a potent neurotransmitter. Histamine, which is synthesized by decarboxylation of histidine, and serotonin, which is derived from tryptophan, similarly function as neurotransmitters and regulators. -Alanine is found in nature in the peptides carnosine and anserine and is a component of pantothenic acid (a vitamin), which is a part of coenzyme A. Epinephrine (also known as adrenaline), derived from tyrosine, is an important . Penicillamine is a constituent of the penicillin antibiotics. Ornithine, betaine, homocysteine, and homoserine are important metabolic intermediates. Citrulline is the immediate precursor of arginine.

-Alanine Homocysteine Homoserine Cystine

Ornithine Citrulline Serotonin Epinephrine Histamine

Fig.10.5. The structures of some amino acids that are not normally found in pro- teins but that perform other important biological functions.

Epinephrine, histamine, and serotonin, although not amino acids, are derived from and closely related to amino acids.

10.3. Amino Acids formation in living systems. 10.3.1. Protein Hydrolysis Hydrolysis is effected by the action of acids, alkalis or enzymes. In protein hydrolysis, the reverse of protein formation, peptide bonds are 141 hydrolyzed to yield amino acids. of proteins in the diet involves nothing more than hydrolyzing peptide bonds. For example,

Alanine Glycine Cysteine Aspartic Acid Fig. 10.6.

Although a chemist in the laboratory might choose to hydrolyze a protein by heating it with a solution of hydrochloric acid, most digestion of proteins in the body takes place in the stomach and small intestine, where the process is catalyzed by enzymes. Once formed, individual amino acids are absorbed through the wall of the intestine and transported in the bloodstream to wherever they are needed.

10.3.2. Transamination Transamination is an enzymatic process carried out with transaminases. The coenzyme of transaminases is pyridoxal phosphate, derivative of vitamin B6. O

HO– – CH2– O– P– OH =R OH H3С– N pyridoxal phosphate coenzyme of transaminases Fig.10.7.

The net reaction is: OO OO CH 3 – CH – COOH + H C– CH2 – CH2 – C– C H O NH 2 alanine - ketoglutaric acid

CH 3 – C – COOH OO 2 2 OO + H C– CH– CH – CH – C H O NH 2 pyruvic acid glutamic acid 142

Mechanism of transamination.

1st step. Transfer of amine-group on the coenzyme, pyridoxal phosphate with pyridoxamine phosphate formation.

alanine

Schiff base I Schiff base II

pyruvic acid pyridoxamine phosphate

2nd step. Reaction between some -ketoglutaric acid and pyridoxamine phosphate part of enzyme with amino acid formation.

- ketoglutaric acid pyridoxamine phosphate

Schiff base III Schiff base IV

glutamic acid pyridoxal phosphate

10.3.3. Reducing amination of ketoacids: + NH3,NADH+H HOOC–CH2–CH2–C–COOH HOOC–CH2–CH2–CH–COOH H2O,NAD O NH2 - ketoglutaric acid glutamic acid 143

10.3.4. Amination of  - unsaturated acids: NH3 R–CH=CH–COOH R–CH–CH2–COOH NH2 unsaturated acid amino acid

10.4. Preparation In Laboratory 10.4.1.Amination of haloacids:

O O O HOH H CHCN N NHNH3 3 R –R C– –C H– H R –R C– –CC–C N N

10.4.2.By cyanhydrin synthesis: H H ÑǹÑǹñûùëÇÝÇïñÇÉ ñû ù ëÇÝÇï ñÇÉ O OH NHNH2 2 2H O HCN NH3 2H2 2O R – C – H R – C–C N R –R C– –CC–C N N R –CR –CH–COOH H –C O O H -N H-NH3 3 NH2 H H H NH2 ÑǹñûùëÇÝÇïñÇÉ NH2-hydroxynitrile 2H O R – C–C N 2 R –CH–COOH 10.5. Electrolyte-NH3 and acid-base properties of amino acids AminoH acids are weak polyproticNH2 acids, ampholytes, i.e., they contain both acidic and basic groups. Free amino acids can never occur as neutral nonionic molecules: instead, they exist as neutral zwitterions that contain both positively and negatively charged groups,which are electrically neutral and so do not migrate in an electric field. In acidic solution (below pH 2.0), the predominant species of an amino acid is positively charged and migrates toward the cathode. The ionizable groups are not strongly dissociating ones, and the degree of dissociation thus depends on the pH of the medium.

pH 1 pH 7 pH 13 Net charge +1 Net charge 0 Net charge -1 Cationic form Zwitterion (neutral) Anionic form

Fig. 10.8. The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain. The cationic form is the low pH form, and the of the cationic species with base yields the zwitterion and finally the anionic form.

144

The isoelectric point (pI) of an amino acid is the pH at which the molecule has an average net charge of zero and therefore does not migrate in an electric field. The pI is calculated by averaging the pK' values for the two functional groups that react as the zwitterion becomes alternately a monovalent cation or a monovalent anion (pI = ½( pK1+ pK2). At physiological pH, monoaminomonocarboxylic amino acids, e.g., glycine and alanine, exist as zwitterions. All the amino acids contain at least two dissociable hydrogens. At low pH, both the amino and carboxyl groups of glycine are protonated and the molecule has a net positive charge. If the pH is increased, the carboxyl group is the first to dissociate, yielding the neutral zwitterionic species Gly° (Fig. 10.9.). Further increase in pH eventually results in dissociation of the amino group to yield the negatively charged glycinate. The side chains of several of the amino acids also contain dissociable groups. Thus, aspartic and glutamic acids contain an additional carboxyl function, and lysine possesses an aliphatic amino function. Histidine contains an ionizable imidazolium proton, and arginine carries a guanidinium function. -carboxyl group of aspartic acid and the -carboxyl side chain of glutamic acid exhibit pKa values intermediate to the -COOH on the one hand and typical aliphatic carboxyl groups on the other hand.

Fig. 10.9. Titration profile of glycine, a monoaminomonocarboxylic acid. In basic solution (above pH 9.7), the predominant species is negatively charged and migrates toward the anode. 145

In a similar fashion, the -amino group of lysine exhibits a pK3 that is higher than the -amino group but similar to that for a typical aliphatic amino group ( Table 10.3.).

That is, at a pH of 6.9-7.4, the-carboxyl group (pK' 2.4) is dissociated to yield a negatively charged carboxylate ion (-COO-), while the -amino group + (pK' 9.7) is protonated to yield an ammonium group (-NH3 ). The buffering capacities of weak acids and weak bases are maximal at their pK values. Thus, monoaminomonocarboxylic acids exhibit their greatest buffering capacities in the two pH ranges near their two pK' values, namely, pH 2.3 and pH 9.7. The pK' value of the -carboxyl group is considerably lower than that of a comparable aliphatic acid, e.g., acetic acid (pK' 4.6). This stronger acidity is due to electron withdrawal by the positively charged ammonium ion and the consequent increased tendency of a carboxyl hydrogen to dissociate. The titration profile of glycine hydrochloride is nearly identical to the profiles of all other monoaminomonocarboxylic amino acids with nonionizable R-groups (Ala, Val, Leu, Ile, Phe, Ser, Thr, Gln, Asn, Met, and Pro). Neither these amino acids nor the-amino or -carboxyl groups of other amino acids (which have similar pK' values) have significant buffering capacity in the neutral (physiological) pH range. The only amino acids with R-groups that 146

have buffering capacity in the physiological pH range are histidine (imidazole; pK' 6.0) and cysteine (sulfhydryl; pK' 8.3). The pK' values for R- groups vary with the ionic environment.

Table 10.3.

Amino pK'1 pK'2 pK'3 pI Acid (-COOH) Alanine 2.34 9.69 (-NH +) 6.0 3

Aspartic + 2.09 3.86 (-COOH) 9.82 (-NH3 ) acid

Glutamic + 2.19 4.25 (-COOH) 9.67 (-NH3 ) acid Arginine 12.48 2.17 9.04 (-NH +) 3 (Guanidinium)

Histidine 6.00 + 1.82 9.17 (NH3 ) (Imidazolium) + + Lysine 2.18 8.95 (-NH3 ) 10.53 (-NH3 )

Cysteine + 1.71 8.33 (-SH) 10.78 (-NH3 )

+ 10.07 Tyrosine 2.20 9.11 (-NH3 ) (Phenol OH) + 13.6 Serine 2.21 9.15 (-NH3 ) (Alcohol OH) *The pK' values for functional groups in proteins may vary significantly from the values for free amino acids. pK'and pI Values of Selected Free Amino Acids at 25°C* 6.00

10.6. Reactions of Amino Acids There are three reasons to consider these reactivities. 1.) Proteins can be chemically modified in very specific ways by taking advantage of the chemical reactivity of certain amino acid side chains. 2.) The detection and quantification of amino acids and proteins often depend on reactions that are specific to one or more amino acids and that result in color, radioactivity, or some other quantity that can be easily measured. 3.) Finally and most importantly, the biological functions of proteins depend on the behavior and reactivity of specific R groups. The carboxyl groups of amino acids undergo all the simple reactions common to this functional group. Reaction with ammonia and primary amines yields 147 unsubstituted and substituted amides, respectively. Esters and acid chlorides are also readily formed. Esterification proceeds in the presence of the appropriate alcohol and a strong acid. Free amino groups may react with aldehydes to form Schiff bases and can be acylated with acid anhydrides and acid halides.

10.6.1. Carboxyl and Amino Group Reactions The -carboxyl and -amino groups of all amino acids exhibit similar chemical reactivity (salts and chelates formation, Fig. 10.10.).

Fig. 10.10.

 Amino acids can join via peptide bonds The crucial feature of amino acids that allows them to polymerize to form peptides and proteins is the existence of their two identifying chemical groups: + - the amino ( -NH3 ) and carboxyl (-COO ) groups.

10.6.2. Deamination Deamination is one of the important metabolic transformations of nitrogen-containing compounds in living systems. There are several ways of deamination.  Intramolecular deamination is specific for -aminoacids.

amino acid unsaturated acid

aspartic acid fumaric acid

 Oxidative deamination is an enzymatic process and one of the main ways of nitrogen metabolism in the living tissues. Extremely important is reaction of desamination of glutamic acid, catalyzed by glutamate dehydrogenase, coenzyme NAD:

148

+ H2O,NAD HOOC–CH2–CH2–CH–COOH HOOC–CH2–CH2–C–COOH NH ,NADH+H+ 3 O NH2 glutamic acid - ketoglutaric acid

 The side chains, however, exhibit specific chemical reactivities, depending on the nature of the functional groups (dehydration-deamination).

treonine en-aminoacid

imino acid ketoacid

10.6.3. Amino acids decarboxilation The process is important in the body in the formation of histamine from histidine and in the production of other amines from amino acids.

HúúC– CH2 – CH2 – CH – COOH HúúC– CH 2 – CH2 – CH 2 – NH2 Cú 2  - aminobutyric acid NH 2 glutamic acid

N N CH 2 CH – COOH CH2 CH2 NH2 HN Cú 2 HN – – NH 2 histidine histamine

10.6.4. Distinguish features of , - amino acids - amino acids undergo cyclic dipeptides formation O O C – OH H – N – H C – NH t H 3  C – CH HC – CH3 H 3 C – CH HC – CH3 - 2H 2 O H – N – H HO – C HN – C O O cyclic dipeptide (diketopiperazine)

149

 , - amino acids undergo lactam formation

H 2 C – CH2 H 2 C – CH2 H 2 C CH 2 t H 2 C CH2 HN HHO C - H 2O HN – C O O cyclic amide (lactam) -amino acids undergo intramolecular desamination with - unsaturated acids formation. This is a reverse reaction of amino acid formation from unsaturated acids ( see. 10.6.2.).

10.6.5. Physiologically important chemical reactions of amino acids The reactions of amino acids with carbon dioxide, metal ions and glucose are of great physiological importance. In tissue capillaries, CO2combines with free -amino groups of hemoglobin to form carbaminohemoglobin; in pulmonary capillaries, this reaction is reversed to release CO2 into the alveoli. This mode of transport is limited to only about 10% of the carbon dioxide transported in the blood. Metal ions can form complexes with amino acids. Metal ions that function in enzymatic or structural biochemical systems include those of iron, calcium, copper, zinc, magnesium, cobalt, manganese, molybdenum, nickel, and chromium, forming chelates. Metal ions can also react with amino acid functional groups to abolish the biological activity of proteins. Heavy metal ions that form highly insoluble sulfides (e.g., HgS, PbS, CuS, Ag2S) characteristically react with sulfhydryl groups of cysteinyl residues. If the reactive -SH group is involved in biological activity of the protein, the displacement of the hydrogen and the addition of a large metal atom to the S atom usually cause a major change in protein structure and loss of function. Hence, heavy metals are often poisons. In contrast, amino acid residues in proteins may undergo nonenzymatic chemical reactions that may or may not alter biological activity. An example of this type of reaction is the formation of glycated proteins. The N-terminal group of the  -chains of hemoglobin (amino groups of proteins) combine with carbonyl groups of sugars (glucose) to form labile aldimines (Schiff bases), which are isomerized to yield stabile ketoamine (fructosamine) products. The degree of glycation achieved in a protein is determined by the concentration of sugar in the environment of the protein,particularly in high quantity are produced with prolonged 150 hyperglycemia and undergo progressive nonenzymatic reactions involving dehydration, condensation, and cyclization. These compounds are collectively known as advanced glycosylation end products and are involved in the chronic complications of diabetes mellitus (cataracts and nephropathy).

10.7. Qualitative and quantitative detection of aminoacids. Universal and specific reactions. The detection and quantification of amino acids and proteins often depend on reactions that are specific to one or more amino acids and that result in color, radioactivity, or some other quantity that can be easily measured. 10.7.1.Universal reaction.The Ninhydrin Reaction -Amino acids can be readily detected and quantified by reaction with ninhy- drin. Ninhydrin, or triketohydrindene hydrate, is a strong oxidizing agent and causes the oxidative deamination of the -amino function. The products of the reaction are the resulting aldehyde, ammonia, carbon dioxide, and hydrindantin, a reduced derivative of ninhydrin. The ammonia produced in this way can react with the hydrindantin and another molecule of ninhydrin to yield a purple product (Ruhemann's Purple) that can be quantified spectrophotometrically at 570 nm. The appearance of CO2 can also be monitored. Indeed, CO2 evolution is diagnostic of the presence of an -amino acid. -Imino acids, such as proline and hydroxyproline, give bright yellow ninhydrin products with absorption maxima at 440 nm, allowing these to be distinguished from the -amino acids. O O O O C C OH C RCH 2 C + NH 2 CHCOOH C = N – C + OH CO2 C C C R H2O O O OH ninhydrin amino acid

Because amino acids are one of the components of human skin secretions, the ninhydrin reaction was once used extensively by law enforcement and forensic personnel for fingerprint detection. (Fingerprints as old as 15 years can be successfully identified using the ninhydrin reaction.) More sensitive fluorescent reagents are now used routinely for this purpose.

10.7.2. Specific reactions. Xanthoproteic reaction (yellow protein reaction) The addition of concentrated nitric acid to protein solutions generally causes the formation of a white 151

precipitate which turns yellow upon heating, the color becoming orange when the solution is made alkaline ( due to dissociation of OH group). The Xanthoproteic reaction is due to nitration of the phenyl rings present in tyrosine and phenylalanine to give yellow nitro substitution products. HNO3 HO CH2 – CH – COOH HO HNO3 CH2 – CH – COOH HO CH2 – CH – COOH HO CH2 – CH – COOH NH2 NH2 NO2 NH2 NH2 NO2 NaOH + NaOHNa O CH2 – CH – COO Na Na O CH2 – CH – COO Na+ NH2 NO2 NH2  Unoxidized sulfurNO test2 for detection cysteine amino acid. CH2 – SH CH2 – OH

CH – NH2 + 2NaOH CH – NH2 + Na2S + H2O COOH COOH (CH3COO)2Pb + 2NaOH Pb(ONa)2 + 2CH3COOH Na2S + Pb(ONa)2 + 2H2O PbS + 4NaOH Black lead sulfide

10.7.3. Quantitative Determination  Van Slyke reaction for determination -amino acids: Reaction of amino acids with nitrous acid to form the corresponding hydroxy acids, with the liberation of nitrogen gas:

R – CH – COOH + HNO2 R – CH – COOH + N2 + H2 O

NH2 OH Van Slyke has utilized the reaction for the estimation of free amino groups in amino acids, peptides, and proteins.  Sørensen’s reaction. Reaction of amino acids with formaldehyde. Sorensen’s formal titration. The carboxyl group of -amino acids cannot be accurately titrated in water solution with alkali, because it reacts with basic amino group to form zwitterions. Sorensen observed that if amino acid solutions are treated with a large excess of neutralized formaldehyde solution, the mixture becomes acid and can be titrated with standard alkali. The amount of alkali required for this titration was found to correspond to the complete titration of the carboxyl group of the amino acid.

152

H H RR – – CH CH – –COOH CO O H + O + O = C = C R – R CH – CH – COOH – CO O H H H NHNH2 2 NHNH – CH – CH2 OH2 OH HH R –R CH – CH – COOH – COOH + O + O = C= C R R – –CH CH – –COOH COOH R R – –CH CH – COOH – CO O H - H2 O H H - H 2 O NN = =CH CH2 2 NHNH2 2 NHNH – –CH CH2 OH2 OH

10.7.4. R –RSpecific CH – CH – COOH– COOHReactions of Amino Acid side Chains - H-2A HO 2number O of reactio ns of amino acids have become important in recent years N N= CH= CH2 2 because they are essential to the degradation, sequencing, and chemical syn- thesis of peptides and proteins. In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. There are numerous other reactions involving specialized reagents specific for particular side chain functional groups (see 10.8.1.).

10.8. Peptides. Proteins. Classification

Amino Acids Can Join via Peptide Bonds The crucial feature of amino acids that allows them to polymerize to form peptides and proteins is the existence of their two identifying chemical groups: + - the amino (-NH3 ) and carboxyl (-COO ) groups. The amino and carboxyl groups of amino acids can react in a head-to-tail fashion, eliminating a water molecule and forming a covalent amide linkage, which, in the case of peptides and proteins, is typically referred to as a peptide bond. Peptide is the name assigned to short polymers of amino acids. Peptides are classified by the number of amino acid units in the chain. Each unit is called an amino acid residue, the word residue denoting what is left after the release of H2O when an amino acid forms a peptide link upon joining the peptide chain (Fig. 10.11.). In the name of peptide -yl ending on amino acid residue indicates that the carboxyl group of an amino acid is linked to amino group (e.g., in a peptide bond)of another amino acid (is not free). O O O -2H2 O H2 N – CH – C– OH+ H2 N – CH – C– OH+ H2 N – CH – C – OH

R R 2 R 1 3 peptibe bond

153

O O O

H 2 N – CH – C – NH – CH – C – NH – CH – C – úH

R 3 R 1 R 2 + Fig. 10.11. The -COOH and -NH3 groups of two amino acids can react with the resulting loss of a water molecule to form a covalent amide bond. Dipeptides have two amino acid residues, tripeptides have three, tetrapeptides four, and so on. After about 12 residues, this terminology becomes cumbersome, so peptide chains of more than 12 and less than about 20 amino acid residues are usually referred to as oligopeptides, and, when the chain exceeds several dozen amino acids in length, the term polypeptide is used. The distinctions in this terminology are not precise. The terms polypeptide and protein are used interchangeably in discussing single polypeptide chains. Proteins having only one polypeptide chain are monomeric proteins. Proteins composed of more than one polypeptide chain are multimeric proteins. Multimeric proteins may contain only one kind of polypeptide, in which case they are homomultimeric, or they may be composed of several different kinds of polypeptide chains, in which instance they are heteromultimeric. Greek letters and subscripts are used to denote the polypeptide composition of multimeric proteins. Thus, an 2-type protein is a dimer of identical polypeptide subunits, or a homodimer. Hemoglobin consists of four polypeptides of two different kinds; it is an 22 hetero-multimer. Chemically, proteins are unbranched polymers of amino acids linked head to tail, from carboxyl group to amino group, through formation of covalent peptide bonds, a type of amide linkage. The peptide "backbone" of a protein consists of the repeated sequence —N—С—С—, where the N represents the amide nitrogen, the Ca is the -carbon atom of an amino acid in the polymer chain, and the final С is the carbonyl carbon of the amino acid, which in turn is linked to the amide N of the next amino acid down the line. The carbonyl oxygen and the amide hydrogen are trans to each other. This conformation is favored energetically because it results in less steric hindrance between nonbonded atoms in neighboring amino acids. Because the -carbon atom of the amino acid is a chiral center, the polypeptide chain is inherently asymmetric. The peptide bond has partial double bond character.

10.8.1.Amino Acid Analysis of Proteins. Acid Hydrolysis of Proteins The unique characteristic of each protein is the distinctive sequence of amino acid residues in its polypeptide chain(s) is the amino acid sequence 154 of proteins that is encoded by the nucleotide sequence of DNA. By convention, the amino acid sequence is read from the N-terminal end of the polypeptide chain through to the С-terminal end. Determination of primary structure is one of the most important goals of modern biochemistry. Peptide bonds of proteins are hydrolyzed by either strong acid or strong base. Because acid hydrolysis proceeds without racemization and with less destruction of certain amino acids (Ser, Thr, Arg, and Cys) than alkaline treatment, it is the method of choice in analysis of the amino acid composition of proteins and polypeptides. Typically, samples of a protein are hydrolyzed with 6 N HCl at 110°C for 24, 48, and 72 hr in sealed glass vials. Tryptophan is destroyed by acid and must be estimated by other means to determine its contribution to the total amino acid composition. Peptide bonds involving hydrophobic residues such as valine and isoleucine are only slowly hydrolyzed in acid. The hydrolysate is dissolved in a small volume of an acidic buffer to obtain the protonated form of the amino acid and then chromatographed on a cation exchange resin column. The intensity of binding of each type of amino acid to the resin depends upon the number of positive charges on the molecules, the nature and size of the R-groups, and the pK' values of the functional groups of the amino acids. Basic amino acids (lysine, histidine and arginine) have more than one positive charge and therefore bind more tightly than the neutral amino acids. Acidic amino acids (glutamic and aspartic acid) bind less tightly. Amino acids are weakly attracted by hydrophobic and van der Waals interactions through their side chains.

10.8.2. Amino acid sequence determination Separation and quantitation of amino acids by ion exchange or by reversed-phase high-pressure liquid chromatography (HPLC). In ion exchange chromatography, the amino acids are separated and then quantified following reaction with ninhydrin. In HPLC, the amino acids are converted to phenylthiohydantoin (PTH) derivatives via reaction with Edman's reagent prior to chromatography (precolumn derivatization). The process of separation and quantitation of amino acids has been automated. In newer procedures, the complete analysis can be performed in about I hour and permit detection of as little as 1-2 nmol of an amino acid. Amino acid separation and quantitation can also be accomplished by reverse- phase high-pressure liquid chromatography of amino acid derivatives—a rapid and sensitive procedure. Determination of the amino acid sequence of a protein involves the following steps: 155

1. Identification of the N- and C-terminal amino acid residues, 2. Cleavage of any disulfide bonds present, 3. Limited cleavage of the peptide into overlapping smaller fragments, 4. Purification of the fragments, and 5. Their stepwise cleavage into individual amino acid residues.

10.8.2.1. Identification of the N-Terminal residue Determination of the N-terminal residue is carried out by labeling the free unprotonated -amino groups. Three alternative labeling reagents are used: 2,4-dinitrofluorobenzene (DNFB; Sanger's reagent), dansyl chloride (l- dimethylaminonaphthalene-5-sulfonyl chloride), and phenylisothiocyanate (PITC; Edman's reagent). DNFB and dansyl chloride react with free amino groups under basic conditions. The labeled peptide is hydrolyzed with acid to yield the labeled N-terminal residue and other free amino acids (Fig.10.12.). The 2,4-dinitrophenyl amino acid derivatives (DNP-amino acids) have a yellow color and are separable by chromatographic methods and identifiable by comparison with reference DNP-amino acids. DNFB reacts with the - amino groups of lysyl residues to yield -DNP-lysine after hydrolysis. N- Terminal lysine produces di(DNP)-lysine, whereas an internal lysine produces a derivative with only one dinitrophenyl group ( -DNP-lysine).

Fig. 10.12. Determination of N-terminal amino acid residues by use of 2,4-dinitrofluorobenzene (Sanger's reagent).

156

Treatment of a peptide with dansyl chloride followed by hydrolysis yields a dansyl derivative of the N-terminal amino acid and other unlabeled amino acids. The dansyl amino acid is separated and identified by chromatographic methods. The dansyl procedure is about 100 times more sensitive than the DNFB method because the dansyl amino acids are highly fluorescent and therefore detectable in minute quantities. In the Edman procedure, PITC reacts under basic conditions with the free-amino group to form a phenylthiocarbamoyl peptide (Fig.10.13.). Treatment with anhydrous acid yields the labeled terminal amino residue plus the remainder of the peptide. In this process, the terminal amino acid is cyclized to the corresponding phenylthiohydantoin derivative (PTH-amino acid), which can be identified by , reverse-phase high- pressure liquid chromatography, thin-layer chromatography, or as the free amino acid after hydrolysis.

Phenylisothiocyanate (PITC) Peptide 

→

a b Fig. 10.13. a) Phenylthiohydantoin derivative of N-terminal amino acid (PTH-amino acid) b) Remaining peptide

After removal of the N-terminal amino acid, the remainder of the peptide remains intact and a new N-terminal amino acid is available for removal by the next reaction cycle. A significant advantage of the Edman procedure is that on removal of the N-terminal residue, the remaining peptide is left intact and its N-terminal remaining peptide group is available for another cycle of the procedure. This procedure can thus be used in a stepwise manner to estab- lish the sequence of amino acids in a peptide starting from the N-terminal.

157

All methods of separation and analysis are fully automated in instruments called amino acid analyzers. Analysis of the amino acid composition of a 30-kD protein by these methods requires less than 1 hour and only 6 g (0.2 nmol) of the protein. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or С- terminal end. 10.8.3. Secondary Structure of Proteins The spatial relationship of amino acids near each other along one or more polypeptide chains is referred to as secondary structure. Three well-defined, orderly secondary structures are known. Two, the -helix and the -sheet, are held in place by hydrogen bonds between backbone peptide groups. The third is a different type of helix found in collagen, a structural protein.

10.8.3.1. The -Helix -Helix a common secondary protein structure in which a protein chain wraps into a coil stabilized by hydrogen bonds between peptide groups in the backbone. Although the strength of each individual hydrogen bond is low (about 5 kcal/mol), the large number in the helix results in an extremely stable secondary structure. Wool, hair, fingernails, and feathers are made of a strong, fibrous protein known as-keratin that is composed almost completely of -helixes, with three -helixes twisted together into small fibrils, which in turn are twisted into larger and larger bundles.

Figure 10.14. Secondary structures. 158

10.8.3.2. The -Sheet In the -sheet structure, polypeptide chains are held in place by hydrogen bonds between every pair of peptide groups along their backbones. Natural silk is almost entirely composed of -sheets formed by hydrogen bonding be- tween different protein chains. When the polypeptide chains of protein molecules bend and fold in order to assume a more compact three-dimensional shape, a tertiary (3°) level of struc- ture is generated. It is by virtue of their tertiary structure that proteins adopt a globular shape. A globular conformation gives the lowest surface-to-volume ratio, minimizing interaction of the protein with the surrounding environment.

10.8.4. Protein Conformation. Protein Shape. Tertiary structure The overall three-dimensional architecture of a protein is generally referred to as its conformation. Of the great number of theoretical conformations a given protein might adopt, only a very few are favored energetically under physiological conditions. Proteins can be assigned to one of three global classes on the basis of shape and solubility: fibrous, globular, or membrane. Fibrous proteins tend to have relatively simple, regular linear structures. These proteins often serve structural roles in cells. Typically, they are insoluble in water or in dilute salt solutions. In contrast, globular proteins are roughly spherical in shape. The polypeptide chain is compactly folded so that hydrophobic amino acid side chains are in the interior of the molecule and the hydrophilic side chains are on the outside exposed to the solvent, water. Consequently, globular proteins are usually very soluble in aqueous solutions. Most soluble proteins of the cell, such as the cytosolic enzymes, are globular in shape. Membrane proteins are found in association with the various membrane systems of cells. For interaction with the nonpolar phase within membranes, membrane proteins have hydrophobic amino acid side chains oriented outward. As such, membrane proteins are insoluble in aqueous solutions but can be solubilized in solutions of detergents. Membrane proteins characteristically have fewer hydrophilic amino acids than cytosolic proteins.

10.8.4.1. Shape-Determining Interactions in Proteins Covalent Bonding: Disulfide Bridges. Thiols, RSH, react with mild oxidizing agents to yield , RS—SR. Covalent bonding between the R groups of cysteine residues is the only one that is covalent. There are also four kinds of noncovalent interactions: hydrogen bonding along the backbone, hydrogen bonding between R groups, ionic attraction 159 between R groups, and hydrophobic interaction between R groups. The shape of a protein depends solely on the primary structure because the amino acid sequence alone determines where the following kinds of interaction will occur. If the thiol groups are on the side chains of two cysteine amino acid residues, then disulfide bond formation links the residues together (Fig. 10.15).

Fig.10.15. Cysteine (Cys)

Insulin is a biomolecule classified as a hormone because it is secreted by the endocrine system and carries a chemical message to molecules elsewhere in the body. Like numerous other chemical messengers, it is a relatively small polypeptide.The structure and function of insulin are of intense interest because of its role in glucose metabolism and the need for supplementary insulin by individuals with one form of diabetes. Two polypeptide chains (21 and 30) linked together by disulfide bridges in two places. One of the chains also has a loop caused by a third disulfide bridge (Fig.10.16.).

Fig.10.16.

Noncovalent Interactions  Hydrogen bonds along the backbone.  Hydrogen bonds between R groups. Wherever amino acid side chains contain appropriate functional groups, hydrogen bonds between them can connect different parts of a protein chain, sometimes close together and sometimes far distant from each other.

160

 Ionic attractions between R groups. Where there are ionized acidic and basic side chains, their attraction to each other produces what are sometimes known as salt bridges. An acidic glutamate and a basic lysine side chain have formed a salt bridge in the middle of the protein. salt bridge C = O - C = O O + C – CH 2 CH 2 C = ú H 3 NCH 2 CH 2 CH 2 CH 2 CH

NH NH

Giutamic acid residue Lysine residue

 Hydrophobic interactions among R groups. Nonpolar hydrocarbons side chains tend to cluster together in the same way that oil molecules cluster on the surface of water.

hydrophobic interactions

The intermolecular attractions among the water molecules are so strong that the groups cannot come between them. By being excluded from water, the hydrophobic R groups have created a pocket in the protein chain.

10.8.5. Quaternary protein structure Quaternary Structure: The way in which two or more protein chains aggregate to form large, ordered structures. Many proteins consist of two or more interacting polypeptide chains of characteristic tertiary structure, each of which is commonly referred to as a subunit of the protein. Hemoglobin, the oxygen carrier in red blood cells (erythrocytes), consists of four polypeptide chains held together primarily by hydrophobic interactions. Each chain 161 contains one heme unit and is very similar in composition and tertiary structure to myoglobin.

Fig.10.16. a b Whereas the primary structure of a protein is determined by the covalently linked amino acid residues in the polypeptide backbone, secondary and higher orders of structure are determined principally by noncovalent forces such as hydrogen bonds and ionic, van der Waals, and hydrophobic interactions. It is important to emphasize that all the information necessary for a protein molecule to achieve its intricate architecture is contained within its 1° structure, that is, within the amino acid sequence of its polypeptide chain (s).

10.8.6. Protein denaturation Denaturation is the loss of secondary, tertiary, or quaternary protein structure due to disruption of disulfide bonds or noncovalent interactions that leaves peptide bonds intact. Such a disruption in shape without affecting the protein's primary structure is known as denaturation. When denaturation of a globular protein occurs, for example, the structure unfolds from a well- defined globular shape to a randomly looped chain, Fig.10.16. (a, b). Denaturation is accompanied by changes in physical, chemical, and biological properties. Solubility is often decreased by denaturation, as occurs when egg white is cooked and the albumins coagulate into an insoluble white mass. Enzymes lose their catalytic activity and other proteins are no longer able to carry out their biological functions when their shapes are altered by denaturation. The agents that cause denaturation include heat, mechanical agitation, detergents, organic solvents, extremely acidic or basic pH, inorganic salts and oxidizing agents: • Heat. The weak side-chain attractions in globular proteins are easily! disrupted by heating, in many cases only to temperatures above 50°C. Cooking meat converts some of the insoluble collagen into soluble gelatin, which can be used in glue and gell and for thickening sauces. • Mechanical agitation. The most familiar example of denaturation by agitation is the foam produced by beating egg whites. Denaturation of proteins at the surface of the air bubbles stiffens the protein and causes the bubbles to be held in place.

162

• Detergents. Even very low concentrations of detergents can cause denaturation by disrupting the association of hydrophobic side chains. • Organic compounds. Polar solvents such as acetone and ethanol interfere with hydrogen bonding by competing for hydrogen-bonding sites. The disinfectant action of ethanol, for example, results from its ability to denature bacterial protein. • pH change. Excess H+ or OH- ions react with the basic or acidic side chains in amino acids and disrupt salt bridges. One familiar example of denaturation by pH change is the protein coagulation that occurs when milk turns sour. • Inorganic salts. Ions can disturb salt bridges, and heavy metal ions such as those of lead, mercury, and silver react with -SH groups.Many heavy metals (such as mercury and lead) are poisons because they denature and inactivate crucial enzymes by such reactions. Most denaturation is irreversible. Hard-boiled eggs don't soften when their temperature is lowered. Many cases are known, however, in which unfolded proteins spontaneously undergo renaturation—a return to their natural state. Renaturation is accompanied by recovery of biological activity, indicating that the protein has completely returned to its stable secondary and tertiary structure. By refolding into their active shapes, proteins demonstrate that all the information needed to determine these shapes is present in the primary structure.

11. CARBOHYDRATES

Carbohydrates are defined as polyhydroxyaldehydes and polyhydroxy- ketones or materials which can be broken down into structures with those functional-group classifications. Carbohydrates are widely distributed in plants and animals, where they fulfill both structural and metabolic roles. Several examples of main functions are: 1 energic- of the tissues of mammals (glucose, starch, glycogen etc) ; 2-structural-cellulose- plants cell`s wall, 3-receptor, in certain complex ; 4-in combination with protein in glycoproteins and proteoglycans. Carbohydrates are classified as follows: 1.monosaccharides are those carbohydrates that cannot be hydrolysed into simpler carbohydrates; 2- disaccharides yield two molecules of monosaccharides when hydrolysed; 3. oligosaccharides yield two to ten molecules of monosaccharide units on hydrolyses; 4. polysaccharides yield more than ten molecules of monosaccharide units on hydrolyses. Monosaccharides are also called simple 163 sugars. Glucose, or blood sugar, and starch (corn starch, potato starch) are common examples of carbohydrates. Carbohydrates are also called sugars or saccharides.

164

11. 1. The nomenclature and structures of the monosaccharides. 11.1.1. Classification: aldoses versus ketoses There is a systematic nomenclature for carbohydrates, but the parent or base names are all common names. For example, note the names of the following:

Fig 11.1. Glucose mannose fructose

The suffix -ose denotes a simple sugar or monosaccharide, but there is no system based on structure for the parent names; glucose, mannose, and fructose are historical common names. One classification rests on the occurence of an aldehyde or ketone group in the sugar and chain length. If an aldehyde group is present, the sugar is an aldose. The presence of a ketone group leads to the classification of a ketose. Thus glucose and mannose are aldoses, and fructose is a ketose. Glucose, mannose, and fructose are all hexoses, that is, six carbon sugars. Similarly pentoses are five- sugars. Both classifications—functional (aldehyde vs. ketone) and chain length – can be reflected in one name. Thus, glucose and mannose are both aldohexoses and fructose is a ketohexose.

11.1.2. Stereoisomerism Biological systems require specific stereoisomers in their reactions and will not function if the wrong isomer is supplied. Glucose's molecular formula was determined as C6H12O6 in the nineteenth century, and early in this century it was known that this compound was an aldehyde and also had five alcohol (OH) groups. But there are 16 stereoisomers corresponding to this aldohexose structure because there are four chiral carbons (starred on structure) (Fig11.1). The maximum number of stereoisomers that can exist is 2n where: n equals the number of chiral carbons. Figure 11.2.depicts the 8 isomers of D-aldohexoses. Among these 8 different three-dimensional arrangements, glucose is by far the most prevalent stereoisomer found in nature, and it is of premier importance in carbohydrate metabolism. Only isomers very similar to glucose in structure, 165 such as mannose or galactose, can function metabolically, and these do so often by first undergoing isomerization to the glucose structure. Because there are four chiral carbons in the aldohexose structure, there are 16 stereoisomers, that is, eight pairs of enantiomers. The carbohydrate isomers are divided into two major categories depending on the arrangement of groups around the chiral carbon which bears the highest number in the carbon chain, i.e., is farthest away from the carbonyl group. The two categories are called the D- family and the L-family. Each enantiomer is classified as D or L depending on the orientation of —OH at the 5 carbon.

A sugar isomer is assigned to the D-family if the —OH substituent on the highest-numbered chiral carbon appears on the right in the Fischer projection. D is used because dexter is the Latin word for "right." This structure for glucose is designated as D-glucose because of the position of—OH on the carbon in position 5. The orientation of the —OH groups on the other chiral carbons in no way influences the assignment. Alternatively, a sugar is of the L-family if the —OH substituent on the highest-numbered chiral carbon appears on the left in the Fischer projection. L is used because laevus is the Latin word for "left." In this case, of course, there is also a correspondence to the English word. The configurations of H and OH groups on the asymmetric carbon atoms of D- and L-glyceric aldehyde are identical with the configurations of these groups on asymmetric carbon 5 of D- and L-glucose, respectively. All the D-aldose sugars may be considered derivatives of D- glyceric aldehyde, and all the L-aldose sugars derivatives of L-glyceric aldehyde. Most biologically active carbohydrates belong to the D-family (Fig 11.2). We readily metabolize D-glucose, D-mannose, D-fructose, and D- galactose. L-sugars usually cannot be used by the body.

166

Configurations of The Aldoses. D-Series

Fig 11.2.

11.1.3. Ring structures and tautomeric forms of the sugars Intramolecular hemiacetals and hemiketals, anomers. D-glucose and the other monosaccharides exist as cyclic hemiacetals (or hemiketals) fashion in nature because of the ability of the carbonyl group to react with an alcohol group intramolecularly. Haworth projections. If we attempt to write the hemiacetal forms of glucose in a Fischer projection, they would look like a and b in the Fig.11.3.:

-D-glucopyranose -D-glucopyranose , -D-glucopyranose Fig. 11.3. (a) (b) (c) 167

These structures (a and b) are clearly not good representations of a six- membered ring. Consequently, Haworth devised a system for transposing substituents of the Fischer projection onto a more reasonably shaped ring, in a correctly oriented fashion (c) The squiggle (d) means that OH may point out either up or down (Fig. 11.3). (For convenience and clarity, ring hydrogens usually are not shown.). The system can be summarized by three rules: 1. The hemiacetal ring is represented and numbered as it is shown in Fig.11.3. 2. The shading of the ring indicates that it should be viewed as perpendicu- lar to the paper plane. The carbons in positions 1 and 4 are in the plane, the carbons at positions 2 and 3 come toward you, and the carbon at position 5 and the oxygen are behind the plane (a). 3. For all D-sugars (the only ones we'll consider), the carbon at position 6 is attached above the plane of the ring (b). 4. Groups on the right in the Fischer projection are written down below the ring plane. Groups on the left are written up. The H's on carbons at positions 2, 3, and 4 have been omitted to avoid clutter in the representation. Ring closure produces a new chiral center at C-1, which was previously achiral. Thus for every open-chain form there are two cyclic stereoisomers. Consequently, all of the aldohexoses form hemiacetals through the reaction of the aldehyde group with the alcohol group at C-5, the six-membered ring so formed includes five carbons (at positions 1 through 5) and one oxygen (the O at C-5) and is called a pyranose ring. Aldopentoses form five-membered rings; e.g., ribose closes as shown:

ribose

fructose

The hexose fructose also forms a five-membered ring: Five-membered ring sugars with four carbons and one oxygen in the ring are called furanoses. Biologically active monosaccharides are aldohexopyranoses, aldopentofuranoses, and the one ketohexofuranose, fructose. 168

pyranose ring furanose ring

The pyranose and furanose ring structures of the sugars proposed by Haworth more accurately represent their actual configurations than do the older projection structures used by Fischer and other early workers. It is customary, in writing pyranose and furanose rings to leave out the C atoms and write them as follows:

-D-glucopyranose D-glucose aldehyde form D-glucopyranose (+ 19°) only trace present ( +112°)

-D-fructofuranose -D-fructofuranose Fig. 11.4.

C-1 isomers, anomers (for aldoses). Ring closure can be effected in two ways leading to two isomers. If the OH at C-5 attacks C-1 when the carbonyl oxygen is pointing downward, the new OH at C-1 will point downward. If, on the other hand, the OH at C-5 attacks C-1 when the carbonyl oxygen is pointing upward, the new OH at C-1 will point upward. By convention, the one with —OH down is designated  and the one with —OH up is designated . When open-chain monosaccharides close, they form two anomers, the - anomer and the  -anomer (Fig. 11.4.). The carbon atom giving rise to the  and  forms is called the "anomeric carbon atom".In the D-series of the aldohexopyranoses the terminal primary alcoholic group, —CH2OH, projects above the plane of the ring, while in the L-series it lies below the ring.

11.1.4. Mutarotation In the solid state, glucose is a white crystalline solid. One might have a sample of pure -D-glucose (mp = 146°C and = +112°), a sample of pure -D-glucose (mp = 150°C and optical rotation = +19°), or a mixture of the two with intermediate properties. In any case, the solid would look a lot like table sugar. 169

Because hemiacetal formation is reversible, in solution the glucose ring can open up; i.e., the hemiacetal group is reconverted to the aldehyde and alcohole groups from which it originally formed. When the ring has opened up, free rotation around carbon-carbon bonds (which is restricted in the ring) becomes possible. This rotation provides a route for the conversion of the - form to the -form or the reverse process. Pure -D-glucose displays an optical rotation of +112° if this is measured the moment the sample solution is prepared. With time, the numerical value of the rotation decreases and eventually reaches a lower limit of +52.7°. Pure -D-glucose displays an optical rotation of +19° in a freshly prepared sample. As the solution sits, this value increases and eventually reaches an upper limit of +52.7°. Reaching the value of 52.7° in each case indicates that equilibrium has been attained. The value reflects the equilibrium concentrations for the two anomers of 36% - D-glucose and 64%  -D-glucose. That is, given that the rotation of pure - glucose is +112° and pure  -glucose +19°, it is possible to calculate that the equilibrium percentages of - and  -glucose must be 36 and 64 percent respectively to yield a rotation of +52.7° for the mixture of anomers. The optical rotation of a freshly prepared solution of glucose finally becomes constant. This change in the rotation of sugar solutions upon standing is a general property of reducing sugars, with the exception of some ketoses, and is called "mutarotation." The prefix muta- means "change."

(36%)

(64%) Fig. 11.5. Glucose exists in isomeric forms which in solution change into the same equilibrium mixture regardless of which form is dissolved 170

It was showen that glucose exists in isomeric forms which in solution change into the same equilibrium mixture regardless of which form is dissolved: In glucose solutions approximately two thirds of the sugar exists as the - form at equilibrium.

11.1.5. Conformations It appears that the common configurations of the pyranose rings are chair forms such as A and B (Fig.11.6). In solutions of the free sugars, where the ring form is in equilibrium with the aldehyde form, there is a mixture of the various ring types in equilibrium.

A B Fig. 11.6. Chair type of pyranose ring Boat type of pyranose ring

But it is obvious that prevalent forms in solutions for D-glucose are glucopyranoses (64%), because the hemiacetal hydrogen in these isomers has equatorial location and the whole structure is more stable than glucopyranose (Fig.11.7.).

-Dglucopyranose -Dglucopyranose (36%) Chair types (64%) Fig.11.7.

Blood sugar concentration is approximately 70 to 110 mg/100 mL 2 to 4 h after eating. As you will see in the course of biochemistry, carbohydrate metabolism is largely the metabolism of glucose. Fructose, which is also called fruit sugar, is unique among the hexoses. It is the only ketohexose we've seen; it exists in the furanose ring. Its optical rotation is levorotatory, for this reason it is also known as levulose. All other monosaccharides exhibit dextrorotation. Fructose is the sweetest sugar. It is nearly twice as sweet as sucrose or table sugar, and it is three times as sweet as glucose. Galactose is sometimes called brain sugar because it is a component of polymeric glycoproteins (carbohydrate-protein combinations) in brain and nerve tissue. The major dietary sources of galactose are milk products because 171 galactose is part of the disaccharide lactose, or milk sugar. Galactose is metabolized in the body by first being converted to glucose. Mannose is the least frequently encountered aldohexose, it occurs mostly in nature as the monomer unit of polysaccharide called mannans, components of some berry plants. Mannose is metabolized by conversion into fructose.

11.1.6. Chemical reactions (properties) 11.1.6. 1. Reactions of sugars due to hydroxyl groups The hydroxyl groups of sugars have the properties of ordinary alcoholic groups but, because of the number present, may give rise to derivatives that are impossible with monohydroxy alcohols.  Hemiacetal hydroxyl group. Formation of glycosides. Glycosides are compounds formed from condensation between hydroxyl group of anomeric carbon of monosaccharide, or monosaccharide residue, and a second compound that may, or may not (in the case of aglycone) be another monosaccharide. The general reaction of preparation may be represented as follows:

 form of sugar  form of sugar  glycoside glycoside Hemiacetal structures Acetal structures Fig.11.8.

The pyranose glycosides are called "pyranosides," whereas the furanose glycosides are "furanosides." The derivatives of each sugar are named according to the name of the sugar; that is, the derivatives of glucose are glucosides, of mannose mannosides, of galactose galactosides, and of arabinose, arabinosides, etc. In general, the ring forms of the simple sugars react with alcohols in the presence of hydrogen chloride as catalyst to form the glycosides. Only the hydroxyl on carbon 1 (glycosidic hydroxyl) of the sugar reacts under these conditions.

ethyl  -D-galactopyranoside ethyl -D-galactopyranoside 172

Generally a mixture of the - and -glycosides is obtained, and when the reaction is carried out at elevated temperatures the glycosides contain the pyranose ring. Reaction at room or lower temperatures also may produce glycosides with the furanose ring. CH OH 2 CH2OH CH2OHCH2OH CHO2OHO CHO2OHO CH3OH,CH 3HClOH, ( HClãáñ )(ãáñ) O CH2COH H3OH,HCl(anhydrous) CHO 2OH OH OH CH3OH, HCl (ãáñ) OH OH O O HO HOOH OH OH CH3OH, HCl (ãáñHO) HOOH OCH3OCH3 OH HO OH OHOH HO OHOCH3 HO OH HOOH OHOCH OHD - ·ÉÛáõÏáåÇñ³Ýá½ OH 3 -D-glucopyranoD - ·ÉÛáõÏáåÇñ³Ýá½seOH Ù»ÃÇÉ Ù»ÃÇÉ -  methyl D-  - ·ÉÛáõÏá-D- - -·ÉÛáõÏá-D-glucopyranose D - ·ÉÛáõÏáåÇñ³Ýá½ Ù»ÃÇÉ - D - OH·ÉÛáõÏá- D - ·ÉÛáõÏáåÇñ³Ýá½ åÇñ³Ýá½Ç¹åÇñ³Ýá½Ç¹ åÇñ³Ýá½Ç¹Ù»ÃÇÉ - D - ·ÉÛáõÏá- CH2OH åÇñ³Ýá½Ç¹CH OH CH2OH CH2OH 2 CH2OH CH2OH OCH2OH CH OHO O OH CH3OH, HCl ( ãáñ) O 2 OCH3 OH CH3OH, HCl ( ãáñ) OCH3 O OH O CHOH OH, HCl ( ãáñ) OOCHOOCH3 OH OH 3 CH3OH, HCl ( ãáñ) OH 3 HOOH OH HO OH HO HOOHOH HO HO OH HO HO OH OH DOH - ·ÉÛáõÏáåÇñ³Ýá½OH OHOHOH D - ·ÉÛáõÏáåÇñ³Ýá½D - ·ÉÛáõÏáåÇñ³Ýá½ Ù»ÃÇÉ - D - ·ÉÛáõÏá- -D-glucopyranoseD - ·ÉÛáõÏáåÇñ³Ýá½ Ù»ÃÇÉ Ù»ÃÇÉ åÇñ³Ýá½Ç¹ Ù»ÃÇÉ methyl- -  D- D - -·ÉÛáõÏá- D·ÉÛáõÏá- --D ·ÉÛáõÏá--glucopyranose åÇñ³Ýá½Ç¹åÇñ³Ýá½Ç¹ åÇñ³Ýá½Ç¹ The glycosides do not reduce alkaline copper solutions, because the sugar (hemiacetyl) group is combined. For the same reason, they are resistant to the action of alkali. They may be hydrolyzed to the constituent reducing sugars by boiling with dilute mineral acids. -Glycosides are hydrolyzed by maltase, an enzyme from yeast, while -glycosides are hydrolyzed by the enzyme emulsin, from bitter almonds. Enzyme hydrolysis thus affords a method of distinguishing between the two forms. Many glycosides occur in the roots, bark, and fruit, and frequently the leaves of various plants. Glycosides are usually well-crystallized, colorless, bitter solids, soluble in water and alcohol. A number of the natural glycosides are important in medicine or otherwise. The group attached to the sugar in a glycoside is often referred to as the "aglucone" or "aglycone".

11.1.6.2. Preparation of ethers When methyl a- or -glucoside or the free sugar is treated with alkali and dimethyl sulfate or methyl iodide under proper conditions, all the free hydroxyl groups are methylated to form pentamethylated glucose, which is a mixture of the a and  forms when the free sugar is methylated:

173

or methyl -D-glucopyranoside methyl tetra-O-methyl--D-glucopyranoside The second step is hydrolyze of glycosidic bond with ether formation:

a b Fig 11. 9. a) Methyl tetra-O-methyl-D-glucopyranoside; b) 2,3,4,6-tetra-O-methyl--D-glucopyranose (ether)

The glycosides of sugars may be completely methylated by treatment with methyl iodide in the presence of alkali, or methyl iodide and silver oxide. In this process a methyl group replaces the hydrogen of each hydroxyl group. The rigorous systematic nomenclature of sugar ethers such as the methylated sugars designates all of the alkyl groups except the glycoside alkyl (anomeric alkyl) as "-O-," indicating that the alkyl groups are attached to oxygen and not to carbon. Thus the fully methylated product of glucose shown is methyl tetra-O-methyl-D-glucopyranoside. The methylated sugars are much more stable toward the action of reagents in general than the free sugars are. Only the glycoside methyl group (on carbon 1) is removed by ordinary acid hydrolysis. When pentamethyl glucose is boiled with dilute mineral acid, the glycoside methyl group is hydrolyzed off, and tetramethyl- O-glucose is formed (Fig. 11.9.). It is obvious that a glycoside such as methyl -D-glucoside may have either the pyranose or the furanose structure, and in order to differentiate between such substances, they are referred to as "pyranosides" or "furanosides," respectively:

174

methyl -D-glucopyranoside methyl  -D-glucofuranoside

11.1.6.3. Formation of esters The hydroxyl groups of the sugars may be esterified to give esters such as the sugar , propionates, stearates, and benzoates. This is generally accomplished by treating the sugar with the appropriate acid anhydride or acyl chloride under the proper conditions.

a b c Fig. 11.10. a) D-glucopyranose; b) 1,2,3,4,6-penta-O-acetyl--D-glucopyranose or pentaacetylglucose; c) 1,2,3,4,6-penta-O-acetyl--D-glucopyranose

For example, when glucose is treated with , pentaacetyl glucose or glucose pentaacetate is formed. By varying the conditions the  or  form may be obtained as the chief product. In the case of sugar esters rigid systematic nomenclature requires that the acyl groups be designated "-O-acyl" as in "penta-O-acetyl--D-glucopyranose." The acetyl group on the first carbon is readily hydrolyzed off, leaving tetra-acetyl glucose with a free sugar group. All the acetyl groups may be removed by mild alkaline hydrolysis to re-form the sugar. The sugar acetates and other esters are used especially in the preparation of other sugar derivatives. They are generally insoluble in water and soluble in organic solvents.  Biologically active esters of monosaccharides. The sugar phosphates are outstanding in biological importance. The breakdown or metabolism of glucose and other sugars by animal tissues and by yeast and other microorganisms involves a succession of the phosphates of sugars and sugar derivatives. 175

Nucleoproteins of cell nuclei also contain sugar phosphates in combination. Sugar phosphates have been found as intermediate products in the carbohydrate metabolism of plants. Many of these substances have been isolated and identified, and a number have been synthesized. The structures of phosphates of glucose and fructose which are of biological importance are given below:

- D-glucopyranosyl-1 –phosphate -D-glucopyranosyl-6-phosphate glucose-1 -phosphate glucose-6-phosphate

D-fructofuranosyl-6-phosphate D-fructofuranosyl-l,6-diphosphate fructose-6-phosphate fructose diphosphate

It will be observed that, whereas glucose phosphates have pyranose rings, the phosphates of fructose are of the furanose type.

11.1.6.4.Reactions of monosaccharides characteristic of the aldehyde and ketone groups Reaction with hydrazines to form and osazones, with hydrogen cyanide to form cyanhydrins, with hydroxylamine to form (see 7.). Reduction to form sugar alcohols. Both aldoses and ketoses may be reduced to the corresponding polyhydroxy alcohols. This may be accomplished with sodium amalgam or, better, electrolytically or by hydrogen under high pressure in the presence of a catalyst. The alcohols formed from glucose, mannose, and fructose are:

176

Mannose → D-mannitol D-sorbitol ← glucose

glycerol erythritol ribitol dulcitol Fig. 11.11. The formulas of sugar alcohols

Each aldose yields the corresponding alcohol upon reduction, while a ketose forms two alcohols because of the appearance of a new asymmetric carbon atom in the process. Reduction of galactose gives dulcitol (11.11.) Each of these sugar alcohols is formed by reduction of both the D and L- corresponding sugar, and the alcohols are not designated by either the "D" or the "L" prefix. They are optically inactive, though erythritol, ribitol, and dulcitol contain asymmetric carbon atoms. This is due to the fact that the molecules are symmetrical and are internally compensated relative to polarized light just as meso-tartaric acid is. All the sugar alcohols discussed previously are natural products. As a class, the sugar alcohols are well-crystallized compounds, soluble in water and alcohol, and they have a sweet taste.  Oxidation to produce sugar acids. When oxidized under the proper condi- tions, the aldoses may form monobasic aldonic acids, or dibasic saccharic acids, or monobasic uronic acids containing the aldehyde group. 177

aldonic acids alduronic acids aldaric acids

 Aldonic acids. Oxidation of an aldose with bromine water converts the alde- hyde group to a carboxyl group and thereby forms the corresponding acid. Bromine reacts with water to form hypobromous acid, HOBr, which acts as the oxidizing agent. Oxidation of glucose leads to gluconic acid formation.

D-glucose D-gluconic acid

Similarly, mannose, galactose and arabinose give mannonic, galactonic, and arabonic acids, respectively. Other aldoses form the corresponding aldonic acids. Ketoses are not readily oxidized by bromine water. Calcium gluconate is often administered as a source of calcium. Solutions of it are given intravenously to raise the blood calcium.  Saccharic or aldaric acids. Oxidation of aldoses with nitric acid under the proper conditions converts both aldehyde and primary alcohol groups to carboxyl, forming dibasic sugar acids, the saccharic or aldaric acids. The more recent designation of the dibasic sugar acids as aldaric acids. According to the aldaric acid system, the name of the acid from a sugar is the name of the sugar with the ending "-aric" replacing "-ose"—for example, "glucaric," "xylaric," "arabaric," "threaric," "hex-aric" (from ahexose), "pentaric" (from a pentose), etc.

D-glucosaccharic acid D-mannosaccharic acid D-galactosaccharic acid D-glucaric acid D-mannaric acid D-galactaric acid 178

The acid salts of the aldaric acids are often used in the identification of sugars because of their low solubility in water. Galactaric (galactosaccharic) or mucic acid, produced by oxidation of galactose, is relatively insoluble in water and well crystallized, and its formation is used as a test for galactose in both the free and combined states. The aldaric acids have been of much value in proving the configurations of the aldose sugars.  Uronic acids. When an aldose is oxidized in such a way that the primary alcohol group is converted to carboxyl without oxidation of the aldehyde group, a uronic acid is formed. In doing this in the laboratory, the aldehyde or sugar group is protected by conversion to a glycoside.

or Fig. 11. 12.

Phosphate group may be removed by careful acid hydrolysis to yield D- glucuronic acid. D-Glucuronic acid occurs combined in materials. It is also a constituent of the chondroitin and mucoitin sulfuric acids of glycoproteins. Glucuronic acid is formed in the animal body in the process of detoxifying substances such as camphor, and benzoic acid, the glucuronic acid compounds of these substances being excreted in the urine. D-Galacturonic acid is widely distributed as a constituent of pectins and many plant gums and mucilages.

11.1.6.5. Interconvertion of monosaccharides, action of alkalies upon sugars D-Glucose and D-mannose differ only in the orientation of the —OH group at C-2, so they are epimers. The difference between D-glucose and D- fructose is the reversal of the —OH and the carbonyl functional groups at C-1 and C-2. D glucose, D-mannose, and D-fructose are identical from C-3 through C-6: 179

D-glucose D-mannose D-fructose Fig. 11.13.a

Monosaccharides, both aldoses and ketoses, and compound carbohydrates containing a free sugar group tautomerize and form the enol salt in alkaline solution.The enol forms of the sugars are enediols, because two hydroxyl groups are attached to the double-bonded carbon system. It will be noted that glucose, mannose, and fructose form the same enediol and by acidification the enediol tautomerizes into all three sugars as follows:

D-mannose 1-2 enediol form D-glucose D-fructose Fig. 11.13.b

11.1.6.6. Action of acids upon carbohydrates Polysaccharides and the compound carbohydrates in general are hydrolyzed into their constituent monosaccharides by boiling with dilute (0.5-1.0 M) mineral acids, such as hydrochloric or sulfuric. In general the monosaccharides are relatively stable to these hot dilute acids, though the ketoses are appreciably decomposed by prolonged action. When the concentration of acid is increased to several normalities, the monosaccharide molecules are decomposed. Pentoses yield the cyclic aldehyde furfural, as illustrated by the reaction for ribose and glucose: 180

D-ribose furfural hydroxyl methylfurfural Fig. 11.14.

This reaction is used for the quantitative determination of pentoses and compound carbohydrates containing pentoses (pentosans, etc.). Twelve percent hydrochloric acid has been found the most satisfactory acid for decomposition. Furfural forms, with phloroglucinol, a relatively insoluble compound, furfural-phloroglucide, which may be used in estimating the furfural formed in the reaction as a measure of the pentose present. The furfural may also be determined by use of its color reaction with aniline acetate or by a titrimetric reaction. Hexoses are decomposed by hot strong acid to give hydroxymethylfurfural, which decomposes into levulinic acid and other products.

11.1.7. Other Sugar Derivatives of Biological Importance  Amino sugars. Amino groups may be substituted for various hydroxyl groups of sugars to give amino sugars. Although many of these substances have been synthesized, relatively few are natural products, among which the best known are 2-amino-2-deoxy-D-glucose, or glucosamine, and 2- amino-2-deoxy-D-galactose, or galactosamine. These substances have the following formulas:

181

or

both  and  anomers

2-amino-2-deoxy-D-glucose 2-amino-2-deoxy-D-galactose or glucosamine or chitosamine galactosamine or chondrosamine

Fig. 11.15.a -D-Glucuronic acid N-Acetyl-D-glucosamine

Glucosamine and galactosamine exist in ring forms similar to those of glucose and galactose. They give chemical reactions characteristic of the sugars. The amino sugars occur combined as N-acetyl derivatives (amino groups acetylated) in a number of important biological substances (Fig. 11.15.a).

Sialic acids. The sialic acids represent a group of naturally occurring substances widely distributed in tissues, particularly in mucins and blood group substances. They are components of complex lipids and carbohydrates (mucopolysaccharides of mucoproteins). The sialic acids are acetyl derivatives of a 9-carbon 3-deoxy-5-amino sugar acid called "neuraminic acid," in which the amino group is acetylated, and in some cases a hydroxyl group also is acetylated. Neuraminic acids may be considered to be derived from an amino sugar (D-mannosamine) and pyruvic acid by an aldol condensation.

182

Neuraminic acid Sialic acid Fig. 11.15.b

The sialic acids present in mucopolysaccharides are linked with a sugar by a glycosidic bond at carbon 2 of the sialic acid. Deoxy sugars. Deoxy sugars represent sugars in which the oxygen of a hydroxyl group has been removed, leaving the hydrogen. Several of the deoxy sugars have been synthesized, and others are natural products. Ribose and 2-deoxyribose are the most important aldopentoses because they occur in genetic materials RNA (ribonucleic acid) and DNA (oxyribonucleic acid). The prefix 2-deoxy- means "without oxygen at position 2."

2-deoxy-D-ribose 6-deoxy-l-galactose (L-fucose)

Ascorbic acid or vitamin C. This is a very interesting sugar derivative found widespread in plant and animal tissues and is especially abundant in citrus juices, Hungarian paprika, and green walnuts. Its presence in the diet is essential for the prevention of scurvy in man and some animals. It is referred to as the antiscorbutic vitamin and vitamin C. The structure of ascorbic acid was first proved in Haworth's laboratory. It was found to be an enediol of the lactone of L-gulonic acid. Ascorbic acid is synthesized commercially in large amounts from L-sorbose. Ascorbic acid is a fairly strong organic acid (pK = 183

4.21), and it owes its acidic property to the enolic hydroxyl groups. It is stable in the crystalline state but is readily oxidized in aqueous solution by oxygen and other oxidizing agents because of its enediol structure. The first product of oxidation is dehydroascorbic acid:

L-ascorbic acid L-dehydroascorbic acid

Applied chemistry. Reducing properties of monosaccharides. Detection of glucose in the urine. All monosaccharides are classified as reducing sugars. The term reducing in this case comes from the oxidation-reduction reaction that occurs when an aldehyde (or -hydroxyketone) is oxidized In Tollens' or Benedict's reagent. In Tollens' or Benedict's solution a monosaccharide such as glucose opens to the free aldehyde form, the aldehyde group is oxidized, and the oxidizing agent is reduced. The monosaccharide is thereby acting as a reducing agent. These tests, especially Benedict's test, are used clinically to detect the presence of glucose in the urine, a condition known as glucosuria. Clinitest tablets are solid Benedict's reagent. A positive test is a change in color from blue to red. Tollens' test

Glucose oxidized glucose Benedict's test

Glucose oxidized glucose 184

The presence of glucose in urine is a symptom of hyperglycemia (high blood sugar) and diabetes mellitis. Benedict's and Tollens' tests are not specific for glucose, but rather show a positive result for all monosaccharides, some disaccharides, and other organic substances capable of being readily oxidized.

11.2. Compound carbohydrates The compound carbohydrates are derivatives of the monosaccharides in which the monosaccharides are joined together through acetal (glycoside) linkages. The molecular complexity of the compound sugars varies from those made up of two monosaccharide units to those containing hundreds or thousands of these units, such as the starches and glycogen. The simpler compound carbohydrates containing only a few monosaccharide units are crystalline substances with a sweet taste, form true solutions in water, and give the characteristic sugar reactions if a free sugar group is present in the molecule. These carbohydrates are called "oligosaccharides" because they are composed of only a few (oligos) monosaccharide units. The more complex compound carbohydrates, such as celluloses, starches, glycogen, and dextrins, are composed of many monosaccharide units, and most of them do not crystallize but are amorphous solids. The complex compound carbohydrates are called "polysaccharides" because their structures contain many (poly) monosaccharide units. All the compound carbohydrates are hydrolyzed by hot dilute mineral acids into their constituent monosaccharides. Alkalies, however, do not hydrolyze them. Many of them are hydrolyzed by specific glycosidase enzymes. For example, sucrose is hydrolyzed by sucrase, or invertase; lactose by lactase; maltose by maltase; cellulose by cellulase; and the starches, dextrins, and glycogens by the amylases or diastases. All the compound carbohydrates are optically active as a result of the optical activity of their constituent monosaccharides. In general, the specific rotations of the polysaccharides are much higher than those of the monosaccharides. The compound carbohydrates represent an exceedingly important group of substances biochemically. Sucrose, lactose, maltose, starches, and dextrins consti- tute the bulk of man's carbohydrate food. Glycogen is the form in which reserve carbohydrate is stored in the liver and muscles and is the primary carbohydrate involved in supplying energy for muscle contraction. The celluloses make up the larger proportion of the woody and fibrous structures of plants and are used directly and as derivatives for many purposes. The celluloses are by far the most abundant of all organic compounds in nature. 185

11.2.1. Oligosaccharides. Disaccharides. Reducing and nonreducing sugars The oligosaccharides are composed of the disaccharides, trisaccharides, tetrasaccharides and so on, so designated to indicate the number of monosaccharide units involved in their structures. Those which contain free sugar (hemiacetal) groups exist in the a and  forms, just as the monosaccharides do. When two monosaccharides are joined into a disaccharide, the linkage is always through the anomeric carbon of one of them (C-1 for hexosehemiacetals or C-2 for hexosehemiketals). Any —OH group of the other monosaccharide could potentially react and thereby link up with the anomeric carbon. Most often, however, it is the —OH at C-4 that reacts. The following tabulation gives the better-known disaccharides with their component monosaccharides (Table 11.1.). Those possessing a free sugar group, which consequently are reducing sugars and give the other characteristic sugar reactions, are indicated as reducing sugars. Sucrose, lactose, and maltose are the most important disaccharides. Recognizing a sugar as reducing or nonreducing is based on determining the presence or absence of a hemiacetal (or hemiketal) group. Free aldehyde or hemiacetal (hemiketal) group present = reducing sugar. Free aldehyde or hemiacetal (hemiketal) group absent = nonreducmg sugar.

Table 11. 1. Disaccharides Constituent C12H22O11 Monosaccharides I. Reducing Sugars Maltose Glucose, glucose Lactose Glucose, gaiactose Cellobiose Glucose, glucose II. Nonreducing Sugars Sucrose Glucose, fructose Trehalose Glucose, glucose

All sugars with hemiacetal functional groups are reducing sugars because they can undergo the reactions discussed above. Thus, the disaccharides, maltose, lactose, and cellobiose are reducing sugars. 186

maltose cellobiose

lactose sucrose Fig. 11.16.

Sucrose is a nonreducing sugar. Notice that there is no hemiacetal group. This is because the glucose and fructose units are joined through their anomeric positions, and both anomeric carbons are involved in acetal linkages (Fig. 11.16.). Acetal groups are stable in basic solution. Therefore, under the conditions of the Tollens' or Benedicts test the acetal linkage will not revert back to the free aldehyde form and there is no oxidation-reduction reaction.

11.2.1.1. Formation of di- and polysaccharides A buildup of monomer units into dimers (disaccharides) and polymers (polysaccharides) is possible because of the ability of sugar structures to form acetals as well as hemiacetals. Similarly, after the open-chain glucose has closed through the reaction of the aldehyde group with one internal alcohol group, the second reaction can occur if an external alcohol group is provided. If instead of a simple alcohol providing —OH, another monosaccharide molecule provides —OH, then a disaccharide forms.

187

That is, the two monosaccharides are joined together

The process of linking up monomer units through the anomeric carbon can continue so that trisaccharides, tetrasaccharides, pentasaccharides, etc., and polysaccharides form. Different monosaccharide units can link together, for example, glucose and galactose in lactose. Polysaccharides can be broken down by a hydrolysis process which is just the reverse of acetal formation. This hydrolysis process is carbohydrate digestion. Linkages between monosaccharides are always described by numbers which denote the ring carbons joined by the linkage. Thus, the link shown above is a 1,4-linkage. These links or bonds between monomer units could be called acetalic. More often chemists refer to them as glycosidic linkages. Differently oriented linkages produce different isomers with different properties. Two glucose units joined by -1,4 bond constitute the disaccharide maltose, -l,4-glycosidic linkage between glucose units characterizes cellobiose. Maltose, or malt sugar, is not naturally prevalent. It is encountered most often as an intermediate product in the digestive breakdown of starch. It is a reducing sugar and capable of mutarotation; i.e., the —OH at C-1 in the right-hand ring can be or . Lactose or milk sugar, is a disaccharide composed of glucose and galactose. Galactose provides the anomeric carbon of the glycosidic linkage in a - orientation. Glucose is hooked on through C-4. It is a reducing sugar. Lactose intolerance in adults is an unpleasant, though not life threatening condition that is prevalent in all populations other than those of Northern European descent. In fact, it has been suggested that the absence of this condition in adults rather than its presence is the deviation from the usual. Apparently, lactase, the enzyme that allows lactose digestion by infants, either disappears or is inactivated in adulthood; the explanation is not yet known. Because lactose remains in the intestines rather than being absorbed, it raises the osmolality there, which draws in excess water. in the intestine also ferment the lactose to produce lactic acid, carbon dioxide, and hydrogen. The result is bloating, cramps, flatulence, and diarrhea. The condition is treated with a lactose-free diet, which can extend to limitations on taking many medications and artificial sweeteners in which lactose is used as an inactive ingredient.

188

Lactose, often found in the urine of lactating women, or arabinose, from a diet containing large amounts of fruit or fruit juices, or vitamin C in urine from excess dietary intake of C, might give a positive Benedict's test which could be mistaken for an indication of glucosuria. To avoid this misdiagnosis, a specific enzyme test is employed. The enzyme glucose oxidase catalyzes only the oxidation of the monosaccharide glucose; a color change accompanies this oxidation of glucose. Sucrose is the sugar we encounter daily as table sugar. It is a nonreducing sugar because the constituent monomers, glucose and fructose, are both linked through their anomeric carbons. The glycosidic linkage in sucrose is 1,2 and is  with respect to the glucose ring and  with respect to the fructose ring. Sucrose—plain table sugar—is probably the most common highly purified organic chemical used in the world. Although it's found in many plants, sugar beets (20% by weight) and sugarcane (15% by weight) are the most common sources of sucrose. Hydrolysis of sucrose yields one molecule of glucose and one molecule of fructose. The 50:50 mixture of glucose and fructose that results, often referred to as invert sugar, is commonly used as a food additive because it's sweeter than sucrose. Sucrose differs from maltose and lactose in that it has no hemiacetal group because a 1,2 link joins both anomeric carbon atoms. The absence of a hemiacetal group means that sucrose is not a reducing sugar. Sucrose is the only common disaccharide that is not a reducing sugar.

or Fig. 11.17.

Sucrose is obtained from sugar cane or from sugar beets. Invert sugar is the name given to the 50:50 mixture of glucose and fructose obtained when the disaccharide sucrose is cleaved by hydrolysis.

189

The name arises because of the change or inversion of sign of optical rotation that occurs. Sucrose is dextrorotatory; the mixture of monosaccharides is levorotatory. Invert sugar is sweeter than table sugar because of the free fructose units. Honey is mostly invert sugar, as are many "liquid sugars," i.e., solutions of the crystalline solid sugars.

11.2.2. Polysaccharides 11.2.2.1. Homopolysaccharides Whereas polysaccharides could be made up of any monosaccharides, all important and abundant polysaccharides are made up of glucose monomer units. The differences between the polymers lie in the nature of the glycosidic linkages. All polysaccharides are nonreducing sugars. It is true that polysaccharides usually have a terminal hemiacetal carbon, however, polysaccharides are only sparingly soluble, and the one unit with a hemiacetal group is only one out of thousands. Starch, plant nutrient material, is actually a mixture of two polysaccha- rides, amylose and amylopectin. Amylose (Fig.11.8.) is characterized by - l,4-glucosidic linkages betwen glucose monomers, which lead to a linear chain of glucose units. Amylopectin, on the other hand, is a branched polymer because it contains 1,6- as well as -l,4-glucosidic links. The amylose gave a blue iodine reaction indicating the presence of amylose. Partial hydrolysis products of intermediate size (dextrins) are obtained. Glycogen is sometimes called animal starch because like plant starch material it is characterized by -glucosidic links. It has a branched structure similar to that of amylopectin; however, glycogen molecules are more highly branched with shorter branching chains. Also, in general, glycogen molecules have a greater number of glucose units than amylopectin. The particular number of glucose units in the polymer depends on the species producing the glycogen or amylopectin. The term animal starch for glycogen comes from the fact that it is made by animals. Blood glucose monomers are assembled into glycogen polymers for storage in muscles and in the liver. Biochemically glycogen is one of the most important substances in the body. Liver glycogen is broken down to glucose and passed into the blood stream for use by the tissues (Fig.11.9.). Muscle glycogen is a source of energy for muscle contractions.

190

(a)

Fig.11.18. (a) Amylose. -l,4-Glucosidic linkages produce a linear array of monomer units, (b) Amylopectin. There are -l,6-Glucosidic linkages as well as a-1,4-linkages. The structure is described as branched.

Fig.11.19. Hydrolysis of glycogen

Digestive enzymes in human beings allow for the hydrolysis of a- glucosidic linkages.

191

Cellulose is the most abundant polysaccharide. It is the structural material in plants. Wood, cotton, and paper are all mostly cellulose. The glycosidic linkages between glucose units in cellulose are -l,4-glucosidic linkages. This type of linkage produces a linear polymer. Typically the chains are 2000 to 13,000 glucose units long (Fig.11.10.). These long chains remain straight in nature and line up in parallel arrays held together by hydrogen bonding between chains. This gives the fibrous quality to cellulose materials. Humans do not possess the appropriate enzyme for the hydrolysis of - glucosidic linkages. Because of this, polysaccharides such as cellulose, which has -linkages between glucose units, are not digestible. We can digest plant starch (amylose and amylopectin), but not the cellulose which makes up more than 50 percent of the in plants. Cellulose does not give a characteristic reaction with iodine.

Fig.11.20. Cellulose: -l,4-Glucosidic linkages produce a linear array of monomer units

The average molecular weight of native cellulose is about 570,000. Undigested cellulosic fiber from vegetables and whole-grain cereals makes up most of the bulk of feces. Cows unlike humans have microorganisms in their stomachs, which can hydrolyze -glucosidic linkages, allowing the cow to digest cellulose-containing grass. The undigested plant food in our diet, of which cellulose forms a large proportion, is called dietary fiber. Some studies suggest that adequate dietary fiber intake is necessary to reduce the risk of diseases of the large intestine, heart disease, diabetes, and obesity. One theory is that fiber may dilute the concentration of chemical carcinogens in the intestine and thereby lower their cancer-causing potential. Fiber may also aid in the excretion of cholesterol and other fats into the feces and thereby possibly result in a lower incidence of heart disease

11.2.2.2. Heteropolysaccharides. Mucopolysaccharides The substances referred to as "mucopolysaccharides" often are composed of amino sugar and uronic acid units as the principal components, though some are chiefly made up of amino sugar and monosaccharide units without the presence of uronic acid (see 11.1.7.). The hexosamine present is generally acetylated. Glucosamine occurs widely in nature as a constituent of

192 mucopolysaccharides and mucoproteins, such as hyaluronic acid, heparin, and blood group substances. It is the chief organic component of the cell wall of fungi and of the shells of crustaceae (lobsters, crabs, etc.), where it occurs as . Chitin is made up of many molecules of N-acetylated glucosamine joined in a polysaccharide type of linkage and because of its relation to chitin, glucosamine is often called "chitosamine." Galactosamine occurs as the N-acetylated form in a group of complex sulfated mucopolysaccharides present in chondroproteins found in cartilage, adult bone, cornea, skin, tendons, and heart valves. Because of its presence in chondroitin, galactosamine is also called "chondrosamine." The mucopolysaccharides are essential components of tissues, where they are generally present, at least in part, combined with protein as mucoproteins or mucoids. The mucopolysaccharides such as hyaluronic acid, heparin, and the chondroitin sulfates, which are acidic in character, are called the acid mucopolysaccharides.

Fig. 11.21. Chitin

Cartilage and tendons contain polymers composed of varying combinations of disaccharides that include -D-glucuronic acid, N-acetyl-D- glucosamine, or galactose derivatives with sulfate groups. Sulfate groups, also appear in a repeating unit of heparin. Hyaluronic acid. Hyaluronic acid was isolated from vitreous humor and synovial fluid, skin, umbilical cord, hemolytic streptococci, and other sources. Hyaluronic acid, the unbranched or nearly unbranched chain polymer of N-acetylated hyalobiuronic acid units is integral part of the gel- like ground substance of connective and other tissues:

Fig.11.22. Repeating units in hyaluronic acid structure 193

Hyaluronic acid in tissues acts as a cementing substance and contributes to tissue barriers which permit metabolites to pass through but resist penetration by bacteria and other infective agents. Heparin. Heparin -heparin) is a blood anticoagulant present in liver (from which it was originally isolated), lung, thymus, spleen, and blood. Heparin is a polymer of D-glucuronic acid and D-glucosamine. The amino groups and some of the hydroxyl groups are combined with sulfuric acid. The molecular weight of heparin appears to be in the range 17,000 to 20,000. It is strongly acidic, due to sulfuric acid groups, and readily forms salts. The barium salt is used in its isolation. The highly charged heparin binds strongly to a blood clotting factor and acts as an anticoagulant. It is used clinically to prevent clotting after surgery or serious injury.

Fig.11.23. Repeating units in heparin

Also, a coating of heparin is applied to any surface that will come into contact with blood and at which clotting should be prevented, such as the interiors of containers used for transportation and storage of blood. Chondroitin sulfates. The chondroitin sulfates are among the principal mucopolysaccharides in the ground substance of mammalian tissues and cartilage, and occur combined with proteins. Three chondroitin sulfates have been isolated and designated A, B, and C.

Fig.11.24. Repeating units in chondroitin sulfate A

The structure of chondroitin sulfate C is the same as that of chondroitin sulfate A except that the sulfate group is at position 6 of the galactosamine group instead of at position 4. 194

11.3. Glycoproteins Glycoproteins contain carbohydrates bonded to proteins, either through C—N glycosidic bonds or through C—O glycosidic bonds (Fig.11.15.):

Fig.11.25.

Glycoproteins have important functions at cell surfaces, where the protein portion of the molecule is within the cell membrane and the hydrophilic carbohydrate portion extends into the surrounding fluid. Glycoproteins provide communication between a cell and its surroundings. For example, they are responsible for the familiar A,B,O system of typing blood (Fig.11.15.); they also identify pathogens that should be destroyed. The relative proportions of protein and carbohydrate in glycoproteins varies widely, from less than 1% carbohydrate in collagen to more than 80% in the blood group substances. The penicillins, a large family of antibiotics that share the structure, act by interfering with the synthesis of glycoproteins. Bacterial cells, unlike those of higher organisms, are enveloped by a protective coating (a cell wall). In the final stages of cell wall synthesis, strands of glycoproteins known as peptidoglycans are cross-linked together to form the final three-dimensional web. The crucial cross-linking step involves enzyme-catalyzed reaction of a D-Ala residue at the end of one strand with a Gly residue at the end of a neighboring strand (Fig.11.16.)

a) Penicillin G: b)Cell wall cross-linking in bacteria Fig 11.26.

Other penicillins have other acyl side chains. Penicillin's three-dimensional shape is evidently similar enough in that of the Ala-Ala end of the 195

peptidoglycan side chain that it is able to fit into the active site of the transpeptidase enzyme. Once in the active site penicillin binds irreversibly by covalent bond formation between the enzyme and the carbonyl group of the -lactam ring. With bacterial cell wall synthesis thus halted, the cell contents leak out through the weakened wall and the cell dies. Since the cells of higher organisms have no cell walls, penicillin is completely specific for bacteria and is nontoxic to all other organisms.

11.3.1.Cel1 Surface carbohydrates and blood type. Blood-group polysaccharides The so-called blood-group polysaccharides are present in erythrocytes, saliva, gastric mucin, cystic fluids, and other body secretions. When combined with proteins, they constitute the A, B, O (H), Rh, and other antigens of the erythrocytes and differentiate the blood groups or types. When red cells containing a specific type polysaccharide antigen are mixed with specific antibodies of serum, agglutination of the cells takes place: the erythrocytes of type A blood are agglutinated by antibodies (isoagglutinins) found in serum of type B or O blood; group A serum agglutinates red cells of blood types B and AB; etc. Thus, by working out the agglutination characteristics of cells and sera, it is possible to determine whether or not a given blood is of the proper type for transfusion into a particular patient so that agglutination and disastrous results will not follow. Agglutination indicates that the recipient's immune system has recognized foreign cells in the body and has formed antibodies to them. Cells of types A, B, and O each have on their surfaces characteristic oligosaccharides bound to proteins or lipids in the cell membrane. . The three blood group determinants are known. These are called antigenic determinants. Cells of type AB have both A and B determinants. The determinants can provoke an immune response that results in the production of antibodies. The marker for blood group O is a trisaccharide whose constituent sugars are common to all three types, whereas the markers for blood groups A and B have one additional sugar unit (Fig.11.17).

196

Blood groups:

O A B Fig.11.27. Cell surface

The results of test of blood types determination represented in table 11.2. Table 11.2. Blood types and agglutination

12. LIPIDS

Lipids, in contrast, to other cannot be defined structurally. The class called lipids contains a "mixed bag" of compounds with a variety of functional groups and structural features which are not soluble in water; they are hydrophobic. All lipids have extensive hydrocarbon parts in their structures. Lipids are soluble in ether, chloroform, and benzene, that is, in nonpolar solvents. Many lipids have ester functional groups and are therefore susceptible to saponification, a reaction of esters. The lipid class can be divided into saponifiable lipids (esters) and nonsaponifiable lipids (Fig. 12.1). The ester, or saponifiable, class is further divided into simple or compound lipids based on the complexity of the ester and the products of their hydrolysis or saponification. Simple lipids yield only alcohols and salts of carboxylic acids upon saponification; compound lipids yield other components, such as phosphoric acid and amines. 197

Saponifiable lipids

Simple lipids Compound lipids

Waxes, Fats, Glycolipids Glycerophospho esters oils Sphingolipids lipids

Fig. 12.1

Lipids have in common extensive hydrocarbon parts that are hydrophobic. Many also have polar, hydrophilic regions.

12.1. Saponifiable lipids 12.1.1. Simple lipids Simple lipids are esters of alcohols and carboxylic acids. The simplest of these are the waxes, which are mixtures of esters of long-chain, carboxylic acids called fatty acids and long-chain alcohols. The composition of wax can be represented by the formula:

or Long chain Ester Long chain from acid linkage from alcohol

If R and R' are sufficiently long (about 15 carbons), the ester will acquire the physical properties associated with waxes- malleable solids which melt at only moderately elevated temperatures. Natural waxes are found in insects and whales and on the surfaces of almost all plants. They are protective, water-resistant coatings. The ester waxes are very important cosmetically and medicinally because they have been designated safe for external application and can even be ingested in small amounts. For example, carnauba wax is used to polish candies and pills. Fatty acids are carboxylic acids (RCOOH) in which R is a long hydrocarbon chain. Table 12.1. represents some common fatty acids.

198

Table 12.1. Common Fatty Acids* Num- ber of Melting Name Formula double point °C bonds Saturated fatty acids 4 0 Butyric CH 3 (CH 2)2COOH -8 6 0 Caproic CH 3 (CH 2)4COOH -3 8 0 Caprylic CH 3 (CH 2)6COOH 17 10 0 Capric CH 3 (CH 2)8COOH 32 12 0 Lauric CH 3 (CH 2)10COOH 44 14 0 Myristic CH 3 (CH 2)12COOH 54 16 0 Palmitic CH 3 (CH 2)14COOH 63 18 0 Stearic CH 3 (CH 2)16COOH 70 20 0 Arachidic CH 3 (CH 2)18COOH 75 22 0 Behenic CH 3 (CH 2)20COOH 80 24 0 Lignoceric CH 3 (CH 2)22COOH 84 26 0 Cerotic CH 3 (CH 2)24COOH 88 Uusaturaled fatty acids 16 1 Palmitoleic CH 3 (CH 2)5CH=CH(CH2)7COOH -1 18 1 Oleic CH 3 (CH 2)7CH= CH(CH2)7COOH 14 18 2 Linoleic CH 3 (CH 2)4CH=CHCH2CH=CH(CH2)7COOH -5 CH CH CH=CHCH CH=CHCH CH= 18 3 Linolenic 3 2 2 2 -11 =CH(CH2)7COOH CH (CH ) CH=CHCH CH=CHCH CH= 20 4 Arachidonic 3 2 4 2 2 -50 =CHCH2CH=CH(CH2)3COOH

Table 12.1. lists several fatty acids. Linoleic and linolenic acids are essential and called vitamin F. Fatty acids contain even numbers of carbons; the cause is the way in which they are synthesized biologically. Fatty acids are unbranched, may be saturated, i.e., contain only single bonds between carbons, or they may contain one or more carbon-carbon double bonds, in which case they are unsaturated. The arrangement around the double bonds in unsaturaled fatty acids is almost always the cis arrangement. This contributes to lower melting points than for a saturated fatty acid or an unsaturated acid with a trans double bond. The cis arrangement produces a molecular shape that is more difficult to pack together in the solid state, and thus the dispersion forces between the hydrocarbon chains are weaker and as a consequence the melting point is lower. Butyric acid is included among long-chain fatty acids because of its occurrence in butter and its importance in fatty acid metabolism. 199

 Triacylglycerols. The most prevalent simple lipid compounds are triesters of the triol, glycerol and fatty acids.

Fatty acids +Glycerol → Triacylglycerol

Acyl groups Triesters are called variously triglycerides, triacylglycerols, fats or oils.

Fig. 12.2

Esterification reaction proceeds exactly as we have seen before: water is boxed out, and the acid and alcohol fragment are joined together. Because glycerol has three alcohol groups, it can react with one, two, and three acids to form mono-, di-, or trimesters. These could be called mono- ,di- or triglycerides, or mono-, di-, or triacylglycerols. The triesters are the most common and are the ones that will concern us (Fig. 12.2).

monoacylglycerol diacylglycerol triacylglycerol

The term triglyceride, though in common use, is unsatisfactory from an organic nomenclature point of view. Because all triacylglycerols contain the glycerol backbone, differences between the compounds must arise from the different acyl groups that may be attached (Fig. 12.2).

200

Properties of fats and oils. In terms of physical properties, fats are solids at room temperature, i.e., they have high melting points, and oils are liquids at room temperature. These physical properties are dictated by the structure of the acyl groups (fatty acids). Saturated acyl chains produce the higher melting points of fats. Triacylglycerols in animals tend to have salurated acyl chains and are thus fats, whereas the triacylglycerols in plants typically have unsaturated acyl groups and are usually oils. Polyunsaturated cooking oils from plant sources contain multiple double bonds (polyunsaturated fatty acids). Fats and oils that we encounter in everyday life are mixtures of triacylglycerols.

12.1.2. Chemical properties of triacylglycerols 12.1.2. 1. The addition reactions  The saturation reaction

Unsaturated oil with low melting point

Saturated fat with higher melting point Figure 12.3

This saturation reaction is important in the margarine industry (Fig. 12.3). Hydrogen gas is bubbled through liquid vegetable oils until the desired semisolid buttery consistency is achieved. Oils are generally not totally saturated to make butter substitutes. Total saturation would produce too hard a margarine, a brittle butter. The addition reaction of iodine -iodine number. The addition reaction is also used analytically to determine the degree of unsaturation of oils. In this case, the addition reaction of iodine is observed.

201

The presence of double bonds in a compound can be verified by adding of drops of an iodine solution to the compound. If double bonds are present and an addition reaction occurs, the iodine loses its color.

Table 12.2. Fatty Acid Composition, by Percent, and Iodine Numbers of Common Fats and Oils Fatty Acid Vegetable oils Animal fats Number of Number of Coco Cotton Sun Beef Human carbons in double Olive Butter nut seed flower tallow fat acid chain bonds Saturated < 8 0 6 8 0 8 1 10 0 7 3 12 0 48 3 14 0 17 21 Trace 4 3 11 3 16 0 9 2 9 3 29 29 25 18 0 2 Trace 2 1 19 11 8 20 0 Trace 22 0 Trace Unsaturated 16 1 4 18 1 6 33 83 34 46 28 46 18 2 3 44 6 58 3 3 14 18 3 1 22 1 1 20 Unsat. 1 1 22 Unsat 1 Iodine 10 100 83 128 42 32 68 number

That is, elemental iodine is deep purple (pink in dilute solution), but the addition product is colorless. The test can be used quantitatively. If the concentration of the iodine solution is known and the volume added to a known volume of oil is measured, then one can calculate the number of double bonds in the oil. The iodine is added to the oil in a dropwise fashion until just a very faint color persists. At that point, all double bonds have added 202 iodine, no more iodine can react, and any iodine put into the solution will retain its color. The iodine number of an oil or fat is defined as the number of grams of iodine absorbed per 100 g of fat or oil. Compounds with high iodine numbers are very unsaturated. Low iodine numbers correspond to more saturated materials. Animal fats typically have iodine numbers less than 70; for example, for human fat it is 68. On the other hand, the more unsaturated vegetable oils have iodine values that are usually more than 100. Sunflower oil's iodine number is 128.

12.1.2.2. Hydrolysis of simple lipids The hydrolysis of a simple lipid is the hydrolysis of an ester. There are three main types of hydrolysis: acidic, alkaline and enzymatic. Beeswax, if hydrolyzed, would yield a long-chain acid and a long-chain alcohol:

Myricyl palmitate Palmitic acid (principal ester in beeswax) The hydrolysis of a triacylglycerol requires 3 mol of water per mole of fat or oil (Fig. 12.3.):

Three aciyl Glycerol Three fatty acids Glycerol fragments fragment Fat or oil

Fig. 12.3. Behenyl caproyl lauryl glycerol 203

As usual, the ester is cleaved between the carbonyl carbon and the oxygen, and the OH of water goes to the carbonyl carbon. The products of the hydrolysis of a fat or oil are always glycerol and three fatty acids. Rancidity. Unwanted hydrolysis reactions lead to spoilage, called rancidity, in fats and oils. For example, under moist air conditions the triacylglycerols in butter can hydrolyze to form butyric and caproic acids, which have rancid odors. Microorganisms present in the air furnish enzymes which speed this reaction. Oxygen in air reacts with the double bonds and cleaves long chains into shorter-chain fatty acids with rancid odors. To avoid unwanted oxidation, the food industry adds antioxidants and prevent the reaction of oxygen with double bonds in polyunsaturated oils. Saponification. Alkaline hydrolysis of fats and oils or of any ester is called saponification. Salts of long-chain fatty acids are soaps. The products of the saponification of a fat or oil are always glycerol and three soaps, i.e.; salts of fatty acids.

Glycerol Three soaps

12.2. Compound lipids Compound lipids are important structural components of the body. They form the lipid bilayer of cell membranes.

12.2.1. Phospholipids Phospholipids are saponifiable compound lipids which contain a phosphate group.

12.2.1.1. Glycerophospholipids The crucial representative is phosphatidic acid:

phosphatidic acid 204

The most prevalent of the phospholipids are the phosphoglycerides, which can be represented as derivatives of phosphatidic acid.

Glycerol Phosphoric Choline Lecithin (phosphatidylcholine) Acid Phosphoglyceride Fig. 12.4.

Upon hydrolysis (Fig. 12.4.) phosphoglycerides yield glycerol, two fatty acids, phosphoric acid, and an amino alcohol (serine, ethanolamine, choline):

Serine ethanolamine choline

When the esterifying amino alcohol is choline, the phosphoglyceride formed is identified as a lecithin. Another common amino alcohol component of phosphoglycerides is ethanolamine, these phospholipids are called cephalins. Some cephalins incorporate the amino acid serine, rather than ethanolamine.

mio-inositol phosphatidylinositol

The special group of phosphoglycerides is plasmalogenes. At the first position glycerol is etherified by unsaturated alcohols instead of fatty acid.

Plasmalogene 205

At physiological pH (~7.4), phosphoric acid groups are usually ionized. Consequently, nearby amine nitrogens would be protonated. At physiological pH, correct structural representations of the lecithins and cephalins would be

Phosphatidyl choline (a lecithin) Phosphatidyl ethanolamine (a cephalin)

The ionic nature of the phosphate and amino portion of the molecule is essential to the structure of the lipid bilayer of cell (Fig. 12.5. ).

Hydrophobic head

Fig. 12.5. Hydrophilic head

12.2.1.2. Sphingolipids Compounds, containing sphingosine are called sphingolipids and are divided into two groups: sphingophospholipids or sphingomyelins and glycolipids. The common part of theses compounds is ceramide.

ceramide

Sphingophospholipids. Sphingophospholipids, which are also found in cell membranes, are saponifiable compound lipids characterized by the presence of sphingosine as their backbone, rather than glycerol. In one type of sphingolipid, called sphingomyelins, the amino group of sphingosine is bonded to a fatty acid by an amide linkage, and the primary alcohol group of sphingosine is esterified with phosphoric acid. 206

Sphingosine Sphingomyeline

Fig. 12.6. Sphingomyeline

The phosphoric acid residue also forms a second ester linkage with an amino alcohol. At physiological pH the phosphate is ionized as shown and the sphingomyelins, like the phosphoglycerides, have a hydrophilic head and hydrophobic tail (Fig. 12.6.). Like the phosphoglycerides, sphingomyelins are prevalent in cell membranes, especially in the myelin sheath around certain nerve cells.

12.2.2. Glycolipids Cerebrosides are the simplest glycolipids; the carbohydrate component is a glucose or galactose ring. Gangliosides are more complex; oligosaccharide chains of up to seven units make up the carbohydrate component

Fig. 12.7. Galactocerebroside 207

Cerebrosides, which incorporate galactose, occur in the cell membranes of the brain. Gangliosides are found at cell surfaces in neural tissue and are often parts of the receptor sites for neurotransmitters Several genetic diseases involve the inability of some individuals to break down sphingolipids (12.8.). The result is their accumulation in tissues, especially brain tissue, which leads to swelling and disastrous physiological effects (in Niemann-Pick, Tay- Sachs disease) presumably mental retardation and eventually death.

Figure 12.8. The fluid mosaic model of membrane structure.

Proteins and lipids can float laterally through the sea of phospholipids which make up the cell membrane. Movement through or across the membrane from the outside to the inside of the cell (or vice versa) is carefully controlled by the proteins in or on the membrane. For example, the proteins on the surface are the receptor sites for neurotransmitters and antigens and hormones, the body's chemical messengers.

12.3. Nonsaponifiable lipids 12.3.1. Prostaglandins Prostaglandins are formed from 20-carbon fatty acid, arachidonic acid:

Arachidonic acid

Because these lipids do not contain an ester linkage, they are nonsaponifible lipids. Prostaglandins were originally isolated from prostate glands, from which 208 they were named, but they are now known to appear in most cells. They are synthesized in cell membranes from phosphoglycerides which have an arachidonic acyl residue at position 2 at the glycerol backbone. The reaction sequence begins with the hydrolysis of the arachidonic ester linkage. The conversion of arachidonic acid to various prostaglandins and related compounds called thromboxanes and leukotrienes is called the arachidonic acid cascade.

arachidonic acid prostanoic acid 5,8,11,14- eicosatetraenic thromboxane A2(TXA2)

leukotriene A4(LTA4) prostaglandin E2(PGE2)

12.3.2. Steroids These compounds have no ester linkages (i.e., they are nonsaponifiable) and no fatty acid residues in their structures. Steroids are characterized by a tetracyclic ring structure with the four rings labeled A, B, C, D and the carbons numbered as shown below;

cyclo pentane phenantrene cyclo pentane perhydrophenantrene

Many steroids have methyl groups at the junction of rings A and B (carbon 10) and rings C and D (carbon 13) and a side chain at position 17. Cholesterol 209 is the most abundant of the steroids and probably the most important because it has its own cellular functions and also serves as the raw material for the synthesis of other steroids. Cholesterol has the -ol ending because it is an alcohol, or more precisely a sterol. Cholesterol, which is found dissolved in dietary fats and oils, is also synthesized in the liver from acetyl coenzyme A. Cholesterol is found in all cell membranes and is essential for proper cell function. Many other steroids, including many hormones (adrenal corticoids, sex hormones), are made by the body from cholesterol. Testosterone regulates the development of male reproductive organs and masculine characteristics. One of the estrogens which regulate female characteristics is estradiol. Progesterone prepares the uterus wall to accept a fertilized egg and maintain pregnancy. Sunlight converts the steroid to vitamin D3, which is needed for healthy bones and teeth. Note. Nowadays one of the most important processes responsible for the development of many pathological states and deseases is lipid peroxidation processes. Lipid peroxidation (LPO) is responsible for rebuilding of the membranes structural components, particularly fatty acids composition of phospholipids. This process takes place in all cells with low intensity which is necessary to destroy the existing fatty acid and substitute by new ones. But in several cases, for example in stress conditions, by the action of physical, such as nuclear radiation, chemical, mechanical, biological agents etc, this process could be activated, the intensity of peroxidation arises and leads to significant disorders of the membrane structure. The main dangerous intermediates .) which could cause activation of LPO are hydroxyl radical (OH , peroxy .) . .) radical ( ROO , superoxide radical ( O 2), nitric oxide radical ( NO , peroxynitrite (ONOO-). Most of them are formed in the presence of Fe3+.

210

13. BIOLOGICALLY ACTIVE HETEROCYCLIC COMPOUNDS

In the biological world heterocyclic compounds are everywhere. A heterocyclic compound is one that contains a ring made up of more than one kind of atom: in addition to carbon, most commonly nitrogen, oxygen, or sulfur. For example:

O N O H ethylene ethylene tetrahydrofuran oxide imine

Figure 13.1.

Heterocyclic aromatic compounds can be polycyclic as well.

Quinoline Isoquinoline Indole Benzofuran Benzothiophene

Carbohydrates are heterocyclic; so are chlorophyll and heme. A numerous biologically active compounds are derivatives of heterocycles, which used to classify according to the size, nature of heteroatom(s), their number, and the nature of bonds. More often one can encounter with three-, four- five-, six- membered rings, main of which are aromatic (Fig.13.1). Most of biologically active compounds content aromatic heterocycles. In the numbering of ring positions, hetero atoms are generally given the lowest possible numbers.

211

13.1. Heterocyclic aromatic compounds Cyclic compounds that contain at least one atom other than carbon within their ring and possess aromatic stability are called heterocyclic aromatic compounds. Some representative heterocyclic aromatic compounds are pyridine, pyrrole, furan, and thiophene. In their stability and chemical behavior, all of these compounds resemble benzene (see Chapter 4.3.).

13.1.1.Derivatives of pyrrole The most important derivatives of five-membered one nitrogen containing heterocycle pyrrole are proline (amino acid); indole (polycyclic, structural part of amino acid tryptophan), tetrapyrrolic compounds, such as porphyrins, protoporphyrins, hem. Heme consists of protoporphyrin IX and an iron atom. Protoporphyrin, a highly conjugated system of double bonds, is composed of four 5-membered heterocyclic rings (pyrroles) joined together to form a tetrapyrrole macrocycle, porphin and of methyl, vinyl, and propionate side chains. The specific isomeric arrangement of side chains shown is protoporphyrin IX. Coordination of an atom of ferrous iron (Fe2+) by the four pyrrole nitrogen atoms yields heme (Fig.13.2.).

Porphin (C20H14N4) Protoporphyrin IX Heme (Fe-protoporphyrin IX) Fig. 13.2.

13.1.2. Pyridine and its derivative Among the most important and most interesting heterocycles is the only representative of the six-membered one nitrogen containing aromatic compound, pyridine, and the ones that possess aromatic properties. The chemical properties of pyridine are those we would expect on the basis of its structure. The ring undergoes the electrophilic substitution, typical of aromatic rings. Pyridine is one of the best organic solvents, but reveals neurotoxic effect. 212

The main derivatives of pyridine possessing biological importance are the nicotinic acid or niacin (3-Pyridinecarboxylic acid, a vitamin Anti-pellagra factor) and its amide; vitamin B6 (pyridoxal, pyridoxamine, pyridoxol). The amide of nicotinic acid is a constituent part of coenzymes NAD and NADP (see Chapter 6.1.1.). The isonicotinic acid (4- pyridinecarboxylic acid isomer) has been used in the form of its hydrazide in the treatment of tuberculosis; cardiamine, as a cardiotrop drug (Fig. 13.3.).

pyridoxal pyridoxamine pyridoxol Vitamin B6

Nicotinic Nicotinamide Isonicotinic Isonicotinic Cardiamine acid acid acid hydrazid Niacin (Isoniacin) (Isoniazid)

Pyridine piperidine promedol Figure 13.3.

The derivatives of completely reduced pyridine serve as medicines. One of the largely used preparations is promedol.

13.1.3. Pyrimidine ring (1,3 diazine) and purine ring system Pyrimidines are six-membered heterocyclic aromatic rings containing two nitrogen atoms. In the pyrimidine ring and purine ring system, by convention, atoms are numbered as indicated in Fig. 13.4. The purine ring structure is represented by the combination of a pyrimidine ring with a five-membered imidazole ring to yield a fused ring system. Both are relatively insoluble in water, as might be expected from their pronounced aromatic character.

213

Pyrimidine is a constituent part of many physiologically active compounds such as vitamin B1, vitamin B2, Folic acid, nucleosides, nucleotides, nucleic acids, etc.

The pyrimidine The purine Vitamin B1

Fig. 13.4.

13.1.3.1.Nitrogenous Bases. Common Pyrimidines and Purines The bases of nucleotides and nucleic acids are derivatives of either pyrimidine or purine. The common naturally occurring pyrimidines are cytosine, uracil, and thymine (5-methyluracil). Cytosine and thymine are the pyrimidines typically found in DNA, whereas cytosine and uracil are common in RNA. Various pyrimidine derivatives, such as dihydrouracil, are present as minor constituents in certain RNA molecules. Adenine (6-amino purine) and guanine (2-amino-6-oxypurine), the two common purines, are found in both DNA and RNA (Fig. 13.5.b). Other nat- urally occurring purine derivatives include hypoxanthine, xanthine and uric acid (13.6.). Hypoxanthine and xanthine are found only rarely as constituents of nucleic acids. Uric acid, the most oxidized state for a purine derivative, is never found in nucleic acids and is a main end product of purine metabolism.

Cytosine Uracil Thymine (2-oxy-4-amino (2-oxy-4-oxy (2-oxy-4-oxy pyrimidine) pyrimidine) 5-methyl pyrimidine)

Lactam Lactim Keto form Enol form The keto/enol tautomerism of uracil. The tautomerism of the guanine. Fig 13.5.a 214

The nitrogen bases could exist in different tautomeric forms. The common pyrimidine bases-cytosine, uracil, and thymine and purine base guanine are represented in the tautomeric forms predominant at pH 7 in Fig 13.5.a.

Adenine (6-amino purine) Guanine (2-amino-6-oxy purine) Fig 13.5.b

Other naturally occurring purine derivatives-hypoxanthine, xanthine and uric acid (Fig 13.6.).

Hypoxanthine Xanthine Uric acid Fig 13.6.

Hydrogen bonding between purine and pyrimidine bases is fundamental to the biological functions of nucleic acids, as in the formation of the double helix structure of DNA. The important functional groups participating in H- bond formation are the amino groups of cytosine, adenine, and guanine; the ring nitrogens at position 3 of pyrimidines and position 1 of purines; and the strongly electronegative oxygen atoms attached at position 4 of uracil and thymine, position 2 of cytosine, and position 6 of guanine.

Fig. 13.7.

Another property of pyrimidines and purines is their strong absorbance of ultraviolet (UV) light, which is also a consequence of the aromaticity of their

215 heterocyclic ring structures. This property is particularly useful in quantitative and qualitative analysis of nucleotides and nucleic acids.

13.2. Nucleosides. The pentoses of nucleotides and nucleic acids RNA contains the pentose D-ribose, while 2-deoxy-D-ribose is found in DNA: D-ribofuranose for RNA and 2-deoxy-D-ribofuranose for DNA. When these ribofuranoses are found in nucleotides, their atoms are numbered as 1', 2', 3', and so on to distinguish them from the ring atoms of the nitrogenous bases. The seemingly minor difference of a hydroxyl group at the 2'-position has far-reaching effects on the secondary structures available to RNA and DNA, as well as their relative susceptibilities to chemical and enzymatic hydrolysis.

Furanose form of D-Ribose Furanose form of -2- Deoxyribose

Glycosidic bonds link nitrogenous bases and sugars to form nucleosides.

Cytidine Uridine Fig. 13.8.a.-N1-glycosidic bond in pyrimidine ribonucleosides

216

Adenosine Guanosine Inosine, Hypoxanthine an uncommon nucleoside Fig. 13.8.b.  -N9 glycosidic bond in purine ribonucleosides

Nomenclature of nucleosides. The naming of nuclesides based on the following rule: 1. For pyrimidine nucleosides: Root + idine = uridine, tymidine, cytidine 2. For purine nucleosides: Root + osine = Adenosine, guanosine For the nuclesides, containing desoxyribose, the small “d” must be added in the front of the name: d-cytidine, d-adenosine, d-guanosine. Nucleosides are more water-soluble than free bases. Nucleosides are much more water-soluble than the free bases because of the hydrophilicity of the sugar moiety. Like glycosides (see 11.1.6. 1.), nucleosides are relatively stable in alkali. Pyrimidine nucleosides are also resistant to acid hydrolysis, but purine nucleosides are easily hydrolyzed in acid to yield the free base and pentose. Adenosine: A Nucleoside with Physiological Activity. (human biochemistry). For the most part, nucleosides have no biological role other than to serve as component parts of nucleotides. Adenosine is an exception. In mammals, adenosine functions as an "local hormone." This nucleoside circulates in the bloodstream, acting locally on specific cells to influence such diverse physiological phenomena as blood vessel dilation, smooth muscle contraction, neuronal discharge, neurotransmitter release, metabolism of fat; adenosine acts in regulating heartbeat-slows the heart rate. In addition, adenosine is implicated in sleep regulation. During periods of extended wakefulness, extracellular adenosine levels rise as a result of metabolic activity in the brain, and this increase promotes sleepiness. During sleep, adenosine levels fall. Caffeine promotes wakefulness by blocking the interaction of extracellular adenosine with its neuronal receptors.

Caffeine

217

13.3. Nucleotides Nucleotides are nucleoside phosphates. A nucleotide results when phosphoric acid is esterified to a sugar —OH group of a nucleoside (Fig. 13.9.). The nucleoside ribose ring has three —OH groups available for esterification, at C-2', C-3', and C-5' (although 2'-deoxyribose has only two). The vast majority of monomeric nucleotides in the cell are ribonucleotides having 5'-phosphate groups. Fig. 13.9. shows the structures of the common four ribonucleotides, whose formal names are adenosine 5'-monophosphate, guanosine 5'-monophosphate, cytidine 5'-monophosphate, and uridine 5'- monophosphate. These compounds are more often referred to by their abbre- viations: 5'-AMP, 5'-GMP, 5'-CMP, and 5'-UMP, or even more simply as AMP, GMP, CMP, and UMP. Nucleoside 3'-phosphates and nucleoside 2'- phosphates (3'-NMP and 2'-NMP, where N is a generic designation for "nucleoside") do not occur naturally, but are biochemically important as products of polynucleotide or nucleic acid hydrolysis. Because the pKa value for the first dissociation of a proton from the phosphoric acid moiety is 1.0 or less, the nucleotides have acidic properties.

Adenosine Adenosine Guanosine 5'-monophosphate 3'-monophosphate 5'- monophosphate (or AMP or adenylic acid) (or GMP or guanylic acid)

Cytidine 5'-monophosphate Uridine 5'-monophosphate (or CMP or cytidylic acid) (or UMP or uridylic acid) Fig. 13.9.

218

This acidity is implicit in the other names by which these substances are known—adenylic acid, guanylic acid, cytidylic acid, and uridylic acid. The pKa value for the second dissociation, is about 6.0, so at neutral pH or above, the net charge on a nucleoside monophosphate is -2. Nucleic acids, which are polymers of nucleoside monophosphates, derive their name from the acidity of these phosphate groups.

13.4. Cyclic nucleotides Nucleoside monophosphates in which the phosphoric acid is esterified to two of the available ribose hydroxyl groups (Fig. 13.10.) are found in all cells. Forming two such ester linkages with one phosphate results in a cyclic struc- ture. 3',5'-cyclic AMP, often abbreviated cAMP, and its guanine analog 3',5'- cyclic GMP, or cGMP, are important regulators of cellular metabolism.

3',5'-CyclicAMP,or cAMP 3',5'-cyclic GMP, or cGMP Fig. 13.10.

13.5. Nucleoside diphosphates and triphosphates Additional phosphate groups can be linked to the phosphoryl group of a nucleotide through the formation of phosphoric anhydride linkages. Addition of a second phosphate to AMP creates adenosine 5'-diphosphate, or ADP, and adding a third yields adenosine 5'-triphosphate, or ATP. The respective phosphate groups are designated by the Greek letters  and , starting with the -phosphate as the one linked directly to the pentose (Fig. 13.11.). The abbreviations GTP, CTP, and UTP represent the other corresponding nucleoside 5'-triphosphates. Like the nucleoside 5'-monophosphates, the nucleoside 5'-diphosphates and 5'-triphosphates all occur in the free state in the cell, as do their deoxyribonucleoside phosphate counterparts, represented as dAMP, dADP, and dATP; dGMP, dGDP, and dGTP; dCMP, dCDP, and dCTP; dUMP, dUDP, and dUTP; and dTMP, dTDP, and dTTP.

219

ADP (adenosine 5'-diphosphate) ATP (adenosine 5'-triphosphate) Fig. 13.11.

NDPs and NTPs Are Polyprotic Acids.Nucleoside 5'-diphosphates (NDPs) and nucleoside 5'-triphosphates (NTPs) are relatively strong polyprotic acids, in that they dissociate three and four protons, respectively, from their phosphoric acid groups. The resulting phosphate anions on NDPs and NTPs form stable complexes with divalent cations such as Mg2+ and Ca2+. Because Mg2+ is present at high concentrations (5 to 10 mM) intracellularly, NDPs and NTPs occur primarily as Mg2+ complexes in the cell (Fig. 13.12.). The phosphoric anhydride linkages in NDPs and NTPs are readily hydrolyzed by acid, liberating inorganic phosphate (often symbolized as Pi) and the corresponding NMP. A diagnostic test for NDPs and NTPs is quantitative liberation of P, upon treatment with 1N HCl at 100°C for 7 min.

Fig. 13.12. In a result of hydrolysis, depending on the pH, are formed different products: pH >7 pH=4 H3PO4 + adenosine  AMP  adenosine + H3PO4  pH =1 D – ribose + adenine + H3PO4

Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy Nucleoside 5'-triphosphates are indispensable agents in metabolism because the phosphoric anhydride bonds they possess are a prime source of 220 chemical energy to do biological work. GTP is the major energy source for protein synthesis, CTP is an essential metabolite in phospholipid synthesis, and UTP forms activated intermediates with sugars that go on to serve as substrates in the biosynthesis of complex carbohydrates and polysaccharides. The evolution of metabolism has led to the dedication of one of these four NTPs to each of the major branches of metabolism. The four NTPs and their dNTP counterparts are the substrates for the synthesis of the most great class of biomolecules-the nucleic acids.

13.6. Nucleic acids Nucleic acids are polynucleotides. Nucleic acids are linear polymers of nucleotides linked 3' to 5' by phosphodiester bridges (Fig. 13. 12). They are formed as 5'-nucleoside monophosphates are successively added to the 3'-OH group of the preceding nucleotide. Polymers of ribonucleotides are named ribonucleic acid, or RNA. Deoxyribonucleotide polymers are called deoxyribonucleic acid, or DNA. Because C-l' and C-4' in deoxyribonucleotides are involved in furanose ring formation and because there is no 2'-OH, only the 3'- and 5'-hydroxyl groups are available for internucleotide phosphodiester bonds. In the case of DNA, a polynucleotide chain may contain hundreds of millions of nucleotide units. Any structural representation of such molecules would be cumbersome at best, even for a short oligonucleotide stretch. The chains of RNA and DNA can be visualized as running from 5' to 3' along the atoms of one furanose and thence across the phosphodiester bridge to the furanose of the next nucleotide in line. This backbone can be portrayed by the symbol of a horizontal line representing the furanose and a slash representing the phosphodiester link, as shown in Fig. 13.13. The diagonal slash runs from the middle of a furanose line to the end of an adjacent one to indicate the 3'- (middle) to 5'- (bottom) carbons of neighboring furanoses joined by the phosphodiester bridge.

Fig. 13.13. Segment of unwound double helix illustrating the antiparallel orientation of the complementary strands. 221

The base attached to each furanose is indicated by a one-letter designation: A, C, G, or U (or T). The convention in all notations of nucleic acid structure is to read the polynucleotide chain/row the 5'-end of the polymer to the 3'-end. Note that this reading direction actually passes through each phosphodiester from 3' to 5'. Base Sequence. The only significant variation that commonly occurs in the chemical structure of nucleic acids is the nature of the base at each nucleotide position. These bases are not part of the sugar-phosphate backbone but instead serve as distinctive side chains, much like the R groups of amino acids along a polypeptide backbone. They give the polymer its unique identity. A simple notation of these structures is merely to list the order of bases in the polynucleotide using single capital letters-A, G, C, and U (or T). The presence of 3'- or 5'-phosphate termini, however, must still be specified, as in GACGUAp for a 3'-PO4 terminus. To distinguish between RNA and DNA sequences, DNA sequences are typically preceded by a lowercase "d" to denote deoxy, as in d-GACGTA.

Fig. 13.14. Ribonucleic acid Deoxyribonucleic acid

Classes of Nucleic Acids. The two major classes of nucleic acids are DNA and RNA. DNA has only one biological role, but it is the more central one. The information to make all the functional macromolecules of the cell (even 222

DNA itself) is preserved in DNA and accessed through of the information into RNA copies. Coincident with its singular purpose, there is only a single DNA molecule (or "chromosome") in simple life forms such as viruses or bacteria. Eukaryotic cells have many chromosomes, and DNA is found principally in two copies in the diploid chromosomes of the nucleus, but it also occurs in mitochondria and in chloroplasts, where it encodes some of the proteins and RNAs unique to these organelles. In contrast, RNA occurs in multiple copies and various forms. Cells contain up to eight times as much RNA as DNA. RNA has a number of important biological functions, and on this basis, RNA molecules are categorized into several major types: messenger RNA, ribosomal RNA, and transfer RNA. Eukaryotic cells contain an additional type, small nuclear RNA (snRNA). DNA. The DNA isolated from different cells and viruses characteristically consists of two polynucleotide strands wound together to form a long, slender, helical molecule, the DNA double helix, identified by J.Watson and F. Crick in 1953. The strands run in opposite directions; that is, they are antiparallel and are held together in the double helical structure through interchain hydrogen bonds (Fig. 13.15.). These H bonds pair the bases of nucleotides in one chain to complementary bases in the other, a phenomenon called base pairing.

Fig. 13.15. Segment of Watson and Crick's Double Helix

Chargaff's Rules. Chargaff noted that certain pairs of bases, namely, adenine and thymine, and guanine and cytosine, are always found in a 1:1 ratio and that the number of pyrimidine residues always equals the number of purine residues. These findings are known as Chargaff's rules: [A] = [T]; [C] = [G]; [pyrimidines] = [purines]. The DNA is a complementary double helix.. Two strands of deoxyribonucleic acid are held together by hydrogen 223 bonds formed between unique base pairs, always consisting of a purine in one strand and a pyrimidine in the other. Base pairing is very specific: if the purine is adenine, the pyrimidine must be thymine. Similarly, guanine pairs only with cytosine (Fig. 13.7.). So the sequence of bases in one strand has a complementary relationship to the sequence of bases in the other strand. That is, the information contained in the sequence of one strand is conserved in the sequence of the other. Therefore, separation of the two strands and faithful replication of each, leads to two progeny molecules identical in every respect to the parental double helix. Elucidation of the double helical structure of DNA represented one of the most significant events in the history of science. This discovery more than any other marked the beginning of molecular .

224

REFERENCES

1. R.T. Morrison and R.N. Boyd. 2. J.I. Kroschwitz, M. Winokur. Chemitry: General,Organic, Biological. Second edition. 1990. 3. R.H. Garret, Ch. M. Grisham. Biochemistry. Second edition. 1999. 4. W.H. Brown. Organic Chemistry. Second edition. 1998. 5. N.V. Bhagavan. Medical Biochemistry. Fourth edition. 2001. 6. J. McMurry, M.E. Castellion. Fundamentals of General, Organic and Biological Chemistry. Second edition. 1996. 7. F.A. Carey. Organic Chemistry. Second edition. 1992. 8. E.S. West, W.R. Todd, H.S. Mason, J.T. Van Bruggen. Text Book of Biochemistry. Fourth edition. 1966.

225

CONTENT

BIOORGANIC CHEMISTRY ...... 3 Introduction ...... 3 1. CLASSIFICATION OF ORGANIC COMPOUNDS ...... 4 2. IUPAC NOMENCLATURE ...... 6 2.1. IUPAC nomenclature of alkanes and cycloalkanes ...... 6 2.1.1. Naming Alkanes (main rules)...... 6 2.1.2. Cycloalkanes ...... 7 2.1.3. Alkyl groups ...... 8 2.2. Nomenclature of complex compounds ...... 9 2.2.1. IUPAC nomenclature of complex compounds. The basic rules for IUPAC system...... 9

3. ISOMERISM ...... 16 3.1. Structural isomerism...... 16 3.2. Stereochemistry. Stereoisomerism...... 17 3.2.1.Conformations...... 17 3.2.1.1. Conformations of acyclic compounds. Structure of ethane...... 18 3.2.1.2. Conforamation of cyclic aliphatic compounds. Baeyer strain theory ...... 20 3.2.2. Configurational isomerism ...... 25 3.2.3. Molecular chirality. The stereogenic center. Enantiomers...... 26 3.2.3.1 Properties of chiral molecules: optical activity...... 28 3.2.3.2. Displaying Molecular Shapes. Absolute and relative configuration...... 29 3.2.3.3. Nomenclature for Chiral Molecules...... 30

4. MUTUAL INFLUENCE OF ATOMS IN MOLECULES OF ORGANIC COMPOUNDS ...... 38 4. 1 Conjugation as a factor of stabilization of organic compounds...... 38 226

4.2. Conjugation in alkadienes and allylic systems. p, -conjugation...... 40 4.3. Arenes and aromaticity. Benzene. The Huckel 4n + 2 rule ...... 42 4.3.1. Physical properties of arenes ...... 45 4.3.2. Reactions of arenes ...... 45 4.4. Heterocyclic aromatic compounds ...... 46 4.5. Inductive effect ...... 49 4.6. The mesomeric effect...... 50

5. ACIDS AND BASES ...... 50 5.1. Basicity (acidity) and structure ...... 51 5.2. Classification of organic acids...... 52 5.2.1. lonization of carboxylic acids. Acidity constant ...... 54 5.2.2. Effect of substituents on acidity...... 55 5.3. The basicity...... 57 5.3.1. Aromatic amine`s basicity ...... 58

6. THE MECHANISMS OF ORGANIC REACTIONS ...... 59 6.1. Oxidation-reduction in organic chemistry...... 59 6.1.1. Biological oxidation and reduction. Oxidation of alcohols...... 60 6.1.2. Oxidation of alkenes. Hydroxylation. Formation of 1,2-diols...... 62 6.1.3. Epoxides in biological processes ...... 64 6.1.4. Hydroxylation of the aromatic ring. L-Tyrosine formation ...... 64 6.1.5. Oxidation of phenols. Quinones ...... 65 6.2. Addition reactions (AE) ...... 66 6.2.1. Hydrogenation of alkenes ...... 67 6.2.2. Addition of halogens to alkenes ...... 67 6.2.3. Electrophilic addition of hydrogen halides to alkenes ...... 69 6.2.3.1. Regioselectivity of hydrogen halide addition: Markovnikov's rule ...... 70 227

6.2.3.2. Acid-catalyzed hydration of alkenes ...... 71 6.3. Electrophilic aromatic substitution (SE). Reactions of arenes ...... 72 6.3.1. Halogenation of benzene. Conversion of benzene to bromobenzene by electrophilic aromatic substitution ...... 73 6.3.2. Rate and orientation in electrophilic aromatic substitution of arenes ...... 74 6.3.3. Substituent effects in electrophilic aromatic substitution ...... 76 6.4. Nucleophilic substitution (SN). Functional group transformation by nucleophilic substitution...... 78 6.4.1. Relative reactivity of halide leaving groups...... 79 6.4.2. The bimolecular (SN2) mechanism of nucleophilic substitution ...... 80 6.4.2.1. Stereochemistry of SN2 reactions ...... 80 6.4.2.2. Steric effects in SN2 reactions ...... 81 6.4.2.3. Nucleophiles and nucleophilicity ...... 82 6.4.3. The unimolecular (SN1) mechanism of nucleophilic substitution ...... 84 6.4.3.1. Stereochemistry of SN1reactions ...... 85 6.5. Elimination reactions. E1 and E2 ...... 86 6.5.1. Regioselectivity in alcohol dehydration: the Zaitsev rule ...... 86 6.5.2. The mechanism of acid-catalyzed dehydration of alcohols ...... 87 6.5.3. Dehydrohalogenation of alkyl halides ...... 88 6.5.3.1. Anti elimination in E2 reactions: stereoelectronic effects. Stereoselectivity ...... 89 6.5.4. A different mechanism for alkyl halide elimination: the El mechanism ...... 89

7. THE CARBONYL COMPOUNDS ...... 91 7.1. Reactions that lead to aldehydes and ketones ...... 92 7.2. Reactions of aldehydes and ketones ...... 93 7.2.1. Nucleophilic addition (AN). Principles of nucleophilic addition to carbonyl groups ...... 93

228

7.2.1.1. Addition of alcohols. Acetal formation...... 95 7.2.1.2. Cyclic hemiacetals ...... 95 7.2.1.3. Addition of cyanide. Cyanohydrin formation ...... 96 7.2.2. Addition of derivatives of ammonia. Condensation reactions ...... 97 7.2.3. Oxidation of Aldehydes and ketones ...... 97 7.2.4. Reduction. Reduction to alcohols ...... 98 7.2.5. Haloform reaction ...... 99 7.2.6. Aldol condensation ...... 99 7.2.6.1. Dehydration of aldol products (crotonic condensation) ...... 100 7.3. Use of aldol condensation in synthesis ...... 100 7.3.1. Aldol Condensations in the Biological World ...... 101

8. CARBOXYLIC ACIDS AND THEIR DERIVATIVES ...... 102 8. 1. Sources of carboxylic acids ...... 102 8.1.1. Preparation of carboxylic acids ...... 102 8.1.2. Hydrolysis of acid derivatives ...... 104 8.1.3. The malonic synthesis of monosubstituted and disubstituted acids. The malonic ester synthesis ...... 106 8.2. Reactions of carboxylic acids and their derivatives. Characteristic properties. Reaction centers ...... 107 8.2.1. Nucleophilic acyl substitution. Acylation ...... 108 8.3. Preparation of functional derivatives of carboxylic acids ...... 110 8.4. Chemical properties of functional derivatives of carboxylic acids ...... 111 8.4.1. Saponification ...... 111 8.4.2. Halogenation ...... 111 8.4.3.Decarboxylation ...... 111 8.4.4. Reduction of esters ...... 112 8.5. Dicarboxylic acids ...... 113 8.6. Unsaturated carboxylic acids ...... 114 8.7. Carbonic acid and its derivatives ...... 115 8.7.1.Functional derivatives of carbonic acid ...... 116 229

9. POLYFUNCTIONAL AND HETEROFUNCTIONAL COMPOUNDS ...... 119 9.1. Polyfunctional compounds...... 119 9.2. Heterofunctional compounds. Classification of heterofunctional compounds...... 121 9.2.1. Amino alcohols. Ethanolamine, choline, acetylcholine. Preparation, biological role...... 122 9.2.2. Hydroxyl acids ...... 123 9.2.2.1. Preparation of hydroxyl acids...... 124 9.2.2.2. Chemical properties of hydroxy acids ...... 125 9.2.3. Hydroxy and oxo-derivatives of di- and tricarboxylic acids ...... 126 9.2.4. Ketoacids ...... 127 9.2.4.1. Preparation of ketoacids ...... 127

10. AMINO ACIDS ...... 130 10.1. L--Amino Acids: Structure ...... 130 10.2. Classifications of common amino acids...... 132 10.2.1.Nonpolar or hydrophobic amino acids ...... 136 10.2.2. Acidic Amino Acids ...... 137 10.2.3. Basic Amino Acids ...... 137 10.2.4. Neutral (polar, uncharged) amino acids ...... 138 10.2.5. Uncommon Amino Acids ...... 140 10.2.6. Unusual Amino Acids...... 140 10.3. Amino Acids formation in living systems...... 141 10.3.1. Protein Hydrolysis ...... 141 10.3.2. Transamination ...... 142 10.4. Preparation In Laboratory ...... 144 10.5. Electrolyte and acid-base properties of amino acids ...... 144 10.6. Reactions of Amino Acids ...... 147 10.6.1. Carboxyl and Amino Group Reactions ...... 148 10.6.2. Deamination ...... 148 10.6.3. Amino acids decarboxilation ...... 149 10.6.4. Distinguish features of - amino acids ...... 149

230

10.6.5. Physiologically important chemical reactions of amino acids ...... 150 10.7. Qualitative and quantitative detection of aminoacids. Universal and specific reactions...... 151 10.7.1.Universal reaction.The Ninhydrin Reaction ...... 151 10.7.2. Specific reactions...... 151 10.7.3. Quantitative Determination ...... 152 10.7.4. Specific Reactions of Amino Acid side Chains ...... 153 10.8. Peptides. Proteins. Classification ...... 153 10.8.1.Amino Acid Analysis of Proteins. Acid Hydrolysis of Proteins ...... 154 10.8.2. Amino acid sequence determination ...... 155 10.8.2.1. Identification of the N-Terminal residue ...... 156 10.8.3. Secondary Structure of Proteins...... 158 10.8.3.1. The -Helix ...... 158 10.8.3.2. The -Sheet ...... 158 10.8.4. Protein Conformation. Protein Shape ...... 159 10.8.4.1. Shape-Determining Interactions in Proteins ...... 159 10.8.5. Quaternary protein structure ...... 161 10.8.6. Protein denaturation ...... 162

11. CARBOHYDRATES ...... 163 11.1. The nomenclature and structures of the monosaccharides...... 164 11.1.1. Classification: aldoses versus ketoses ...... 164 11.1.2. Stereoisomerism ...... 164 11.1.3. Ring structures and tautomeric forms of the sugars ...... 166 11.1.4. Mutarotation...... 168 11.1.5. Conformations...... 170 11.1.6. Chemical reactions (properties) ...... 171 11.1.6.1. Reactions of sugars due to hydroxyl groups ...... 171 11.1.6.2. Preparation of ethers ...... 172 11.1.6.3. Formation of esters ...... 174 11.1.6.4.Reactions of monosaccharides characteristic of the aldehyde and ketone groups ...... 175

231

11.1.6.5. Interconvertion of monosaccharides, action of alkalies upon sugars ...... 178 11.1.6.6. Action of acids upon carbohydrates ...... 179 11.1.7. Other Sugar Derivatives of Biological Importance ...... 180 11.2. Compound carbohydrates ...... 184 11.2.1. Oligosaccharides. Disaccharides. Reducing and nonreducing sugars ...... 185 11.2.1.1. Formation of di- and polysaccharides ...... 186 11.2.2. Polysaccharides ...... 189 11.2.2.1. Homopolysaccharides ...... 189 11.2.2.2. Heteropolysaccharides. Mucopolysaccharides ...... 191 11.3. Glycoproteins ...... 194 11.3.1.Cel1 Surface carbohydrates and blood type. Blood-group polysaccharides ...... 195

12. LIPIDS ...... 196 12.1. Saponifiable lipids ...... 197 12.1.1. Simple lipids ...... 197 12.1.2. Chemical properties of triacylglycerols ...... 200 12.1.2.1. The addition reactions ...... 200 12.1.2.2. Hydrolysis of simple lipids ...... 202 12.2. Compound lipids ...... 203 12.2.1. Phospholipids ...... 203 12.2.1.1. Glycerophospholipids ...... 203 12.2.1.2. Sphingolipids ...... 205 12.2.2. Glycolipids ...... 206 12.3. Nonsaponifiable lipids ...... 207 12.3.1. Prostaglandins ...... 207 12.3.2. Steroids ...... 208 13. BIOLOGICALLY ACTIVE HETEROCYCLIC COMPOUNDS ...... 210 13.1. Heterocyclic aromatic compounds ...... 211 13.1.1.Derivatives of pyrrole ...... 211 13.1.2. Pyridine and its derivative ...... 211 13.1.3. Pyrimidine ring (1,3 diazine) and purine ring system ...... 212 232

13.1.3.1.Nitrogenous Bases. Common Pyrimidines and Purines ...... 213 13.2. Nucleosides. The pentoses of nucleotides and nucleic acids ...... 215 13.3. Nucleotides ...... 217 13.4. Cyclic nucleotides ...... 218 13.5. Nucleoside diphosphates and triphosphates ...... 218 13.6. Nucleic acids ...... 220 References ...... 224

233