Synthesis of Sugar Amino Acids via Base Hydrolysis of Sugar Aminonitriles

Transcription

1 Republic of Iraq Ministry of igher Education & Scientific Research University of Baghdad College of Science Department of Chemistry Synthesis of Sugar Amino Acids via Base ydrolysis of Sugar Aminonitriles A Thesis Submitted to the College of Science-University of Baghdad, in Partial Fulfillment of the Requirement for the Degree of Master in rganic Chemistry By mar Abdulateef Mohammed B.Sc. University of Baghdad 2000 Supervised By Prof. Dr. Yousif A. Al-Fattahi Prof. Suad Al-Araji

2 بسم االله الرحمن الرحيم و الل ه أ نز ل م ن الس م اء م اء ف ا ح ي ا ب ه الا ر ض ب ع د م و ت ه ا إ ن ف ي ذ ل ك لا ي ة ل ق و م ي س م ع ون {٦٥} و إ ن ل ك م ف ي الا ن ع ام ل ع ب ر ة ن س ق يك م م م ا ف ي ب ط و ن ه م ن ب ي ن ف ر ث و د م ل ب نا خ ال صا س ا ي غا ل لش ار ب ين { ٦٦ } و م ن ث م ر ات الن خ يل و الا ع ن اب ت ت خ ذ ون م ن ه س ك را و ر ز قا ح س نا إ ن ف ي ذ ل ك لا ي ة ل ق و م ي ع ق ل ون {٦٧} و أ و ح ى ر ب ك إ ل ى الن ح ل أ ن ات خ ذ ي م ن ال ج ب ال ب ي وتا و م ن الش ج ر و م م ا ي ع ر ش ون {٦٨} ث م ك ل ي م ن ك ل الث م ر ات ف اس ل ك ي س ب ل ر ب ك ذ ل لا ي خ ر ج م ن ب ط ون ه ا ش ر اب م خ ت ل ف أ ل و ان ه ف يه ش ف اء ل لن اس إ ن ف ي ذ ل ك لا ي ة ل ق و م ي ت ف ك ر ون {٦٩} صدق االله العظيم سورة النحل (الا يات ٦٥-٦٩)

3 Dedication To my Family With Love And Gratefulness MAR

4 Examination Committee We certify that we have read this thesis and as examining committee, examined the student in its contents and that in our opinion it is adequate for the partial fulfillment of the requirements for the degree of Master in organic chemistry, with (Excellent) standing. Signature: Name: Dr. Ahlam M. Al-Azzawi Title: Ass. Professor (Chairman) Date: / 7 / 2007 Signature: Name: Dr. Ilham M. Al-Rufai'i Title: Ass. Professor (mber) Date: / 7 / 2007 Signature: Name: Dr. Fatin Al-Qazzaz Title: Ass. Professor (mber) Date: / 7 / 2007 Signature: Name: Dr. Yousif A. Al-Fattahi Title: Professor (Supervisor) Date: / 7 / 2007 Signature: Name: Suad M. Al-Araji Title: Professor (Coadvisor) Date: / 7 / 2007 Approved by the College of Science Signature: Name: Dr. Khalid Shehab Al-Mukhtar Title: Professor Dean of the College of Science Date: / / 2007

5 Acknowledgements I d like first to express my deep appreciation to my supervisor Prof. Dr. Yousif A. Al-Fattahi for his suggestion of this research project and his guidance throughout the progress of the research work. Special thanks are due to the Chemistry Department, College of Science, University of Baghdad, especially to the coadvisor Prof. Suad Al-Araji, "ead of the Chemistry Department" for offering me all the requirements to facilitate the execution of this work, and to all members of staff who overwhelmed me with their kindness. My sincere thanks go to Miss. Muneera for her help in carrying out the FTIR measurements. My special affection is extended to Mrs. Thana'a Mahdi and to all my colleagues with whom I shared our common problems and ambitions. My gratefullness is to my friends Mustafa, Wathiq, Baha'a, Ali and Muqdad. Special gratitude is due to Mohammed Kadhim for typing this thesis. mar Al-Salihi 2007

6 Subject List of Contents Page - List of contents... I - List of Figures... III - List of Schemes. IV - List of Tables... V - Abbreviations... VI - Abstract..... VIII Chapter ne: Introduction 1.1. Introduction Structural Versatilities of Sugar Amino Acids Natural ccurrence of SAAs Synthesis of Amino Acids Synthesis of Sugar Amino Acids Synthesis of Sugar a-amino Acids (a-saas) Bucherer-Bergs Synthesis of a-saas Strecker Synthesis of a-saas Synthesis of Glycosyl-a-Amino Acids The Synthesis of Lower Classes of SAAs Aim of The Work...25 Chapter Two: Experimental 2.1. General Notes ,3:4,5-Di--isopropylidene-b-D-fructopyranose (89) ,3:4,5-Di--isopropylidene-b-D-arabino-hexosulo-2,6-pyranose (90) ,2:3,4-Di--isopropylidene-a-D-galactopyranose (91) ,2:3,4-Di--isopropylidene-a-D-galacto-hexodialdo-1,5-pyranose (92) General Amino-cyanation Procedure; Synthesis of Compounds (93-98) Benzylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,L-glycero-b- D-arabino-3-heptulopyranosononitrile (93) (sec-Butylamino)-2-deoxy-3,4:5,6-di--isopropylidene- D,Lglycero-b-D-arabino-3-heptulopyranosononitrile (94) Isobutylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,L-glycerob-D-arabino-3-heptulopyranosononitrile (95) Benzylamino-6-deoxy-1,2:3,4-di--isoproylidene-D,L-glycero-a- D-galacto-heptopyranurononitrile (96) (sec-Butylamino)-6-deoxy-1,2:3,4-di--isopropylidene-D,Lglycero-a-D-galacto-heptopyranurononitrile (97) Isobutylamino-6-deoxy-1,2:3,4-di--isopropylidene-D,L-glycero- 37 I

7 a-d-galacto-heptopyranurononitrile (98).. Subject Page ,4:5,6-Di--isopropylidene-D,L-glycero-b-D-arabino-3- heptulopyranosononitrile (90b) General Procedure for ydrolysis of a-aminonitriles (93-98); Synthesis of Compounds (99-104) Benzylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,L-glycero-b- D-arabino-3-heptulopyranosonic acid (99) (sec-Butylamino)-2-deoxy-3,4:5,6-di--isopropylidene- D,Lglycero-b-D-arabino-3-heptulopyranosonic acid (100) Isobutylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,L-glycerob-D-arabino-3-heptulopyranosonic acid (101) Benzylamino-6-deoxy-1,2:3,4-di--isopropylidene-D,L-glycero-a- D-galacto-heptopyranuronic acid (102) (sec-Butylamino)-6-deoxy-1,2:3,4-di--isopropylidene-D,Lglycero-a-D-galacto-heptopyranuronic acid (103) Isobutylamino-6-deoxy-1,2:3,4-di--isopropylidene-D,L-glyceroa-D-galacto-heptopyranuronic acid (104). 42 Chapter Three : Results and Discussion 3.1. Preface Synthesis of Compounds (89) and (91) xidation of Compounds (89) and (91) xidation with DMS-Ac xidation with Pyridinium chlorochromate (PCC) xidation with Pyridinium dichromate-acetic anhydride Amino-cyanation of Compounds (90) and (92) The Base ydrolysis of a-aminonitriles (93-98) Conclusions Suggested Future Work References. 84 II

8 Figure No. List of Figures Title Page No. 1-1 A fragment of polypeptide chain backbone illustrating the rigid 2 peptide bonds and the intervening N-C a and C a -C backbone linkages, which are free to rotate 1-2 The general structure of sugar amino acids (SAAs) The carbohydrate derivatives that investigated by Kuszmann et 14 al. [58] using Bucher-Bergs reaction 1-4 The a-saas synthesized by Koόš et al., structurally related to 16 alanine (50, 51), leucine (52, 53), and serine (54, 55) 1-5 Various synthesized sugar a-amino nitriles by using catalytic 18 Strecker reaction as reported by Postel et al. 1-6 Glycosyl-a-amino acids synthesized by Grison et al The first synthesized d-saa (72) by eyns and Paulsen [70], 21 and the first synthesized polyamide (74) of d-saa (73) by Fuchs and Lehmann 1-8 The somatostatin analog (85) prepared by Kessler et al. [74], and 24 the furanoid and pyranoid e-saas (86 and 87), and the cyclic peptide (88) prepared by verhand et al. 3-1 FTIR spectrum of compound (89) FTIR spectrum of compound (91) FTIR spectrum of compound (90) FTIR spectrum of compound (90a) FTIR spectrum of compound (92) FTIR spectrum of compound (90b) FTIR spectrum of compound (93) FTIR spectrum of compound (94) FTIR spectrum of compound (95) FTIR spectrum of compound (96) FTIR spectrum of compound (97) FTIR spectrum of compound (98) FTIR spectrum of compound (99) FTIR spectrum of compound (100) FTIR spectrum of compound (101) FTIR spectrum of compound (102) FTIR spectrum of compound (103) FTIR spectrum of compound (104) 79 III

9 List of Schemes Schemes No. Title 1-1 The general Strecker synthesis (A), and the Bucherer-Bergs Page No. 10 synthesis (B) of a-amino acids 1-2 The first synthesized pyranose a-saas by Yanagisawa et al The first synthesized furanose a-saa by Yanagisawa et al The multi-step synthetic path of (2S,4S)-4-amino-2-13 (hydroxymethyl) tetrahydrofuran-4-carboxylic acid (16), as reported by Yoshimura et al. 1-5 The mechanism of anomalous Bucherer reaction that 15 investigated by Kuszmann et al. 1-6 Catalytic Strecker-type reaction as reported by Postel et al The synthetic strategy of the glycosyl-a-amino acids as 20 reported by Grison et al. 1-8 The improved Kessler synthesis of d-saa The overall synthesis of N-protected b- and g-saas as 23 reported by Kessler et al. 3-1 The synthetic approach of the work The mechanism of PDC oxidation 57 IV

10 List of Tables Table No. Title 3-1 Comparison between the oxidation methods of compound Page No. 59 (89) 3-2 The Characteristic FTIR Absorption Bands of 71 Compounds (93-98) 3-3 The Characteristic FTIR Absorption Bands of 73 Compounds (99-104) 3-4 Some Physical Properties of The Synthesized Derivatives 80 V

11 Abbreviations Abbreviation Ac R a aq. Arg. Asp. as. atm. Bz Bn b Boc C cm dec. d DCC DCM DMS e equiv. FTIR Fmoc g GAA Gly g h (hr) ibu ipr Lit. m.p. mg aning Acetyl Alkyl Alpha Aqueous Arginine Aspartic acid Asymmetric Atmosphere Benzoyl Benzyl Beta tert-butyloxycarbonyl Centigrade Centimeter Decompostion Delta N,N-Dicylcohexylcarbodiimide Dichloromethane Dimethyl sulfoxide Epsilon Equivalent Fourier Transform Infrared 9-Fluorenylmethyloxycarbonyl Gamma Glyco amino acid Glycine Gram our Isobutyl Isopropyl Literature lting point thyl Milligram VI

12 Abbreviation aning ml Milliliter mm Millimeter mmol Millimole M Molar [] xidation % Percentage ph Phenyl PCC Pyridinium chlorochromate PDC Pyridinium dichromate R f Retardation factor r.t. Room temperature 2 Secondary sec Bu Secondary butyl SAA Sugar amino acid s. Symmetric TBABr Tetrabutylammonium bromide TF Tetrahydrofuran TEMP Free radical 2,2,6,6-tetramethyl-1-piperidinyl tert. Tertiary TEA Triethylamine TFA Trifluoroacetic acid TLC Thin-Layer Chromatography (v/v) Volume ratio VII

13 Abstract This work includes synthesis of new N-substituted sugar-a-amino acids, so that one of the monosaccharide carbon atoms represents the a- position of the a-amino acid. In order to obtain these derivatives; the known 2,3:4,5-di-isopropylidene-b-D-fructopyranose (89) and 1,2:3,4-di--isopropylidenea-D-galactopyranose (91) were prepared from D-fructose and D- galactose, respectively and used as starting materials. The oxidation of the primary alcohol (89) by using dimethyl sulfoxide/acetic anhydride, pyridinium chlorochromate or pyridinium dichromate/acetic anhydride afforded the corresponding aldehyde (90), but the latter method had the priority over the other two methods for the oxidation of (89) and also for the oxidation of the alcohol (91) to the corresponding aldehyde (92). Compounds (90) and (92) were subjected to a modified Streckertype reaction, whereby different primary aliphatic amines in common with the cyanide ion were introduced successfully, and new a- aminonitriles (93-98) were thus obtained in good yield. Thin-layer chromatography of the prepared a-aminonitriles, indicated that each derivative may exist in a mixture of two stereoisomers with different proportions. Base hydrolysis of the sugar-a-aminonitriles (93-98) by using (35%) aqueous sodium hydroxide solution, at reflux temperature gave the desired sugar-a-amino acids (99-104). FTIR spectroscopy was utilized for the characterization of the synthesized compounds. VIII

14 D-Fructose (, C 2 ) Acetone 2 S 4 r.t. C 2 (89) [] (1) Na, 2 -Et, reflux (2) resin ( + ) NR NC (93) : R = Bn (94) : R = sec. Bu (95) : R = ibu (1) aq. Na 2 S 2 5, RN 2 (2) NaCN C (90) NR C (99) : R = Bn (100): R = sec. Bu (101): R = ibu C 2 D-Galactose (, ) Acetone ZnCl 2, 2 S 4 (91) C 2 [] (1) Na, 2 -Et, reflux (2) resin ( + ) NC (96) : R = Bn (97) : R = sec-bu (98) : R = ibu NR (1) aq. Na 2 S 2 5, RN 2 (2) NaCN C (92) C NR (102) : R = Bn (103) : R = sec-bu (104) : R = ibu IX

15 Chapter ne Introduction 1.1. Introduction It is being increasingly realized that, instead of creating abstract molecules in millions, it is better to design new molecules by emulating the basic principles followed by nature to build its vast repertoire of biomolecules [1]. The fundamental building blocks used by nature, like amino acids, sugars and nucleosides [2], can be amalgamated to produce nature-like, and yet unnatural, structural entities with multifunctional groups anchored on a single ensemble, based on which new molecules can be created [1]. Proteins are macromolecules consisting of one or more polypeptides, each polypeptide consists of a chain of amino acids linked together by peptide (amide) bonds. While the peptide bond has a rigid, planar structure, the other two bond types found in the polypeptide backbone (i.e. the N-C a bond and the C a -C bond, Figure, 1-1) are relatively free to rotate. The polypeptide backbone can thus be viewed as a series of planar plates which can rotate relative to one another. The angle of rotation around the N-C a bond is termed f (phi), while that around the C a -C bond is termed Y (psi). By convention, these angles are defined as being 180 when the polypeptide chain is in its fully extended; trans form. In principle, each bond can rotate to any value between -180 and owever, the degrees of rotation actually observed are restricted, depending upon the steric hindrance between atoms of the polypeptide backbone and those of amino acid side chains (the R- groups). These angles allowable around each C a in a polypeptide backbone obviously exerts a major influence upon the final threedimensional shape assumed by the polypeptide [3]. ۱

16 Chapter ne Introduction Planar (rigid) peptide bonds R 2 N C C N C C N C a a a R 1 R 3 N-C a bond free to rotate, angle of rotation = f C a -C bond free to rotate, angle of rotation = y Figure(1-1): A fragment of polypeptide chain backbone illustrating the rigid peptide bonds and the intervening N-C a and C a -C backbone linkages, which are free to rotate [3] Although many peptide sequences have been identified as potent bioactive agents, there are fundamental limitations associated with the development of peptides as therapeutics [4,5]. The inherent conformational flexibility of a small peptide makes it difficult to restrict short linear chains in any particular conformation that helps them to bind effectively to receptors, they exist in solution in large number of energetically equivalent conformations that can cause undesired effects by interacting with various receptors. In addition, there are a number of metabolic limitations restricting the use of peptides as therapeutics; poor permeability across membrane, the susceptibility of externally administered peptides to proteolytic degradation in the gastrointestinal ۲

17 Chapter ne Introduction tract, serum and other tissues. Furthermore, rapid clearance and in some cases poor solubility and a tendency to aggregate, all contribute to low oral availability of peptide based therapeuties [6,7]. Also, the lack of specific transport system restricts the efficient passage of peptides to the desired site of action [8]. For these reasons, there is a growing demand to design and synthesize new compounds that imitate or mimic the biological activity of peptides, and at the same time, to overcome the limitations inherent with their uses as therapeutics. A common approach, involves the design of conformationally rigid non-peptide scaffolds, which when inserted in the appropriate sites in the peptide, produce the specific secondary structure required for binding to the corresponding receptor, leading to the development of potent agonists/antagonists, and to increase the metabolic stabilities of this peptide towards proteolytic enzymes [5]. Such approach termed as "Peptidomimetics", refers to any compound designed to perform the function of a peptide. Generally, peptidomimetics are derived from a lead peptide sequence where structural modifications have been incorporated to improve binding affinity and/or metabolic resilience. These modifications involve changes to the peptide that will not occur naturally; such as altered backbones, and the incorporation of unnatural amino acids [4,9,10]. In this respect, the number of reports on the development of constrained non-peptide scaffolds in peptidomimetics is increasing exponentially [9-15], and yet very few peptide-based drugs have been developed [5]. Newer concepts are emerging where the natural building blocks, like amino acids, sugars and nuclosides, are amalgamated to produce nature-like, and yet unnatural, de novo structural entities with multifunctional groups fixed on a single ۳

18 Chapter ne Introduction ensemble [16-25]. ne such hybrid design is represented by a class of compounds called sugar amino acids [26,27]. A sugar amino acid (SAA) [or a glyco amino acid (GAA)] can be regarded as a hybrid between an amino acid and a carbohydrate. It consists of a carbohydrate core with at least one amino group and one carboxylic acid functionality directly attached to the carbohydrate, as depicted in Figure (1-2). 2 N n n` () n`` n C n` = 0, 1 ; n`` = 0-4 Figure (1-2): The general structure of sugar amino acids (SAAs). Structures with n > 1 (Figure 1-2), are generally understood to be SAAs as well although, the correct term would be glycosyl amino acids [1,26-28]. ٤

19 Chapter ne Introduction 1.2. Structural Versatilities of Sugar Amino Acids There are several advantages of sugar amino acids (SAAs) as building blocks in peptidomimetics, due mainly to the structural characteristics of the carbohydrate part of these compounds [1,5,26,27]. 1. The rigid furan and pyran rings of these molecules make them ideal candidates as non-peptide scaffolds in peptidomimetics where they can be easily incorporated by using their carboxyl and amino termini utilizing well-developed solid-phase or solution-phase peptide synthesis methods. 2. The presence of several chiral centers in carbohydrate molecules can give rise to a large number of possible isomers that can be used to create combinatorial libraries of sugar amino acid-based molecular frameworks predisposed to fold into architecturally beautiful ordered structures, which may also have interesting properties. 3. The protected/unprotected hydroxyl groups of sugar rings can also influence the hydrophobic/hydrophilic nature of such molecular assemblies. 4. The nonproteinogenic properties of SAAs will make compounds having them physiologically more stable. ٥

20 Chapter ne Introduction 1.3. Natural ccurrence of SAAs Although SAAs are generally regarded as unnatural amino acids, there are a number of naturally occurring compounds containing SAAs as a major constituents, for example, they belong to a family of natural compounds known as sialic acids; which are N- and - substituted derivatives of neuraminic acid (1) and muramic acid (2) as terminal units on cell wall glycoconjugates [29]. AcN C C (1) (2) N 2 ere, they take part in different recognition processes during bacterial and viral infections of cells, e.g. the initial binding of influenza virus to the cell surface [30]. Another example is lysomidase; a complex of three enzymes in a framework of repeating units of trisaccharide (3), the complex is capable of disrupting Gram positive bacterial cell walls [31]. NAc C (3) Ac NAc C NAc n ٦

21 Chapter ne Introduction In addition, there are a number of examples of SAA repeating units in some bacterial polysaccharide constituents, such as 2,3-diacetamido- 2,3-dideoxy-D-glucuronic acid (4) in Ps.aeruginosa type 6 [32], and 5- actamido-3,5,7,9-tetradeoxy-7-[(r)-3-hydroxybutyramido]-l-glycero-lmanno-nonulosnic acid (5) in S.boydii type 7 and Ps.aeruginosa 10a [33]. C NAc N C 1 2 C 2 C 2 NAc NAc (4) (5) Moreover, the -specific polysaccharide of L.pneumophila serogroups 2-14 consists of a homopolymer of 5-acetamidino-7- acetamido-3,5,7,9-tetradeoxy-d-glycero-d-talo-nonulosonic acid (6) [34]. 9 R 8 7 AcN N C 2 N 2 R = or Ac (6) n the other hand, SAAs play an important role as constituents in certain nucleoside antibiotics, like gougerotin (7) and aspiculamycin (8), both are anti-mycoplasma, acaridical and antibacterial antibiotics [35,36] ; where the basic structural framework is a pyranoside sugar b-amino acid. ۷

22 Chapter ne Introduction N 2 N b N (7) a N N N 2 N N 2 N b N a N (8) N N 2 N N The antifungal antibiotics, ezomycin A 1 (9) and A 2 (10), also contain SAA constituents [37]. N 2 N 2 S C 2 N C N (9) N 2 N C N N C 2 N N 2 N C (10) N N 2 N In amipurimycin (11) [38,39], and miharamycin (12) [40], the 6-deoxy- 6-aminoheptopyranuronic acid moieties are basically sugar a-amino acids. N 2 N a C (11) N N N N N 2 N N 2 N N() N 2 a C (12) N N N N N 2 ۸

23 Chapter ne Introduction Nucleoside antifungal agent, polyoxin J (13) [41,42], and herbicide hydantocidin (14) [43,44] also contain sugar a-amino acids, the latter having an anomeric a-amino acid moiety. 2 N N 2 N C (13) The glycopeptide antibiotics, A40926a-b (15) owe their bioactivity to the N-acylated pyranoside sugar amino acids [45]. a N N a (14) N N R 2 C N N Cl N N Cl N N N R 1 R 1 = a-d-mannopyranoside C C R 2a = N, R 2b = N (15) The 4-amino-2-(hydroxymethyl)tetrahydrofuran-4-carboxylic acid (16), was isolated from diabetic urine [46], and its configuration was determined to be (2S, 4S) [47]. 2 C N 2 (16) C ۹

24 Chapter ne Introduction 1.4. Synthesis of Amino Acids ne of the oldest and reliable routes towards a-amino acids is the treatment of a carbonyl compound (aldehyde or ketone) with ammonia and then with hydrogen cyanide to form the a-amino nitrile; the so-called Strecker synthesis [48]. The a-amino acid, is then obtained by the acid or base hydrolysis of the a-amino nitrile. The latter may also be obtained by reacting the carbonyl compound, in one step, with ammonium chloride and sodium cyanide, (Scheme 1.1,A). Also, the Bucherer-Bergs synthesis [49] provides hydantoin; the direct precursor of a-amino acids. That is, when a carbonyl derivative is allowed to react with two moles of potassium cyanide and four moles of ammonium carbonate in 50% aqueous alcoholic warm solution, the hydantoin may usually be isolated on cooling. The a-amino acid will then be obtained by base or acid hydrolysis of the hydantoin. (Scheme 1.1,B). N 3 (A) N 2 CN a acrbonyl compound N 4 Cl KCN, 2 CN acid or base N 2 2, an a-amino nitrile C N 2 an a-amino acid (B) KCN, (N 4 ) 2 C 3 50% aq. Et o C C N N C a hydantoin acid or base 2, Scheme (1-1): The general Strecker synthesis (A), and the Bucherer-Bergs synthesis (B) of a-amino acids ۱۰

25 Chapter ne Introduction 1.5. Synthesis of Sugar Amino Acids SAAs are classified on the basis of position of the amino group compared to the carboxylic acid, with Greek letters to state this position. In this way, the common nomenclature of SAAs reflects normal amino acids, (i.e.; a-, b-, g-, d-, e-saas,..etc.) [28]. Whereas normal amino acids are generally synthesized by using common approaches (1.4), the synthesis of SAAs on the other hand, required some variations on such approaches, or in some cases special pathways [50,51], according to the nature of the selected sugar molecule, as a starting material, and the desired end product (the SAA) Synthesis of Sugar a-amino Acids (a-saas) ne class of SAAs, a-saas, is of special interest, since they mimic many of naturally occurring compounds, (1.3), and they also became an attractive goal in the area of peptodomimetic studies [26] for designing new powerful drugs, as well as, new model compounds for specific enzymetic studies [52,53]. The first attempt of synthesizing a-saa was reported in 1969, when Yanagisawa et al. [54] obtained eight stereoisomers of methyl (+)- and (-)-3-amino-3-C-carboxy-3-deoxypentopyranoside (25, 26) from the catalytic hydrogenation of the optically active a-nitro esters (21, 22), and subsequent base hydrolysis of the a-amino esters (23, 24). Periodate oxidative cleavage of methyl b-l-arabinopyranoside (17) and methyl b- D-xylopyranoside (18) provided the diglycoaldehydes (19 and 20) respectively, which were condensed with ethyl nitroacetate afforded (21, 22), as outlined in Scheme 1.2. ۱۱

26 Chapter ne Introduction (17) (18) i C C R1 R 2 (19): R 1 =, R 2 = (20): R 1 =, R 2 = ii R 1 R 2 2 N CEt (21): R 1 =, R 2 = (22): R 1 =, R 2 = iii R 1 R 2 2 N CEt (23): R 1 =, R 2 = (24): R 1 =, R 2 = 2 N R 1 CEt R 2 iv (25): R 1 =, R 2 = (26): R 1 =, R 2 = Scheme (1-2): The first synthesized pyranose a-saas by Yanagisawa et al. [54] Reagents and conditions: i) I 4, 2, 20 C, 20h; ii)ethyl nitroacetate, KAc, K 2 C 3 ; iii) Raney Ni T-4, atm., r.t., 4h; iv) Ba() 2.8 2, reflux, (1N) 2 S Bucherer-Bergs Synthesis of a-saas Yanagisawa and co-workers [55] utilized a modified Bucherer- Bergs reaction [56] for synthesizing new nucleoside derivatives having an a-amino acid structure in their carbohydrate moiety, as a part of a research project for improvement the activity of the antibiotic puromycin. The 3-ulose derivative (28), obtained from the oxidation of 5--benzoyl- 1,2--isopropylidene-a-D-xylofuranose (27), was allowed to react with Bucherer-Bergs reagents under a carbon dioxide atmosphere in an autoclave afforded the spirohydantoin (29), which upon base hydrolysis provided the a-saa (30). (Scheme 1.3). ۱۲

27 Chapter ne Introduction Bz 2 C i Bz 2 C ii Bz 2 C o N o N iii Bz 2 C 2 N C (27) (28) (29) (30) Scheme (1-3): The first synthesized furanose a-saa by Yanagisawa et al. [55] Reagents and conditions: i) DMS, DCC, pyridine, CF 3 C, dry benzene, r.t., 18hr.; ii) KCN, (N 4 ) 2 C 3,, C 2 50 atm., autoclave, 60 C, 10h; iii)ba() 2.8 2, 2, 125 C, 12hr. Similar approach was followed by Yoshimura et al. [57] to synthesize the natural product (16) [46] from (2S)-benzyloxymethyl-4-oxotetrahydrofuran (31), the latter was prepared starting from D-ribose in multi-step synthesis (Scheme 1.4). D-Ribose Bn 2 C (31) 2 C (32) N N 2 C 2 N C (16) Scheme (1-4): The multi-step synthetic path of (2S,4S)-4-amino-2-(hydroxymethyl) tetrahydrofuran-4-carboxylic acid (16), as reported by Yoshimura et al. [57] While Bucherer-Bergs reaction provides a suitable way for the introduction of an a-amino acid function, there are some obstacles restrict its use in some carbohydrate derivatives. Kuszmann and co-workers [58], investigated this reaction on a series of protected aldehyde and keto derivatives of carbohydrates (33-39), Figure (1-3). ۱۳

28 Chapter ne Introduction C C (33) (34) (35) (36) C C C C (37) (38) (39) N N (40) Figure (1-3): The carbohydrate derivatives that investigated by Kuszmann et al. [58] using Bucher-Bergs reaction. They found that, when the normal conditions were applied [KCN, (N 4 ) 2 C 3 in 50% aq. Et at 50 ], the keto derivative (33); named 3- ulose, reacted smoothly affording mainly the spiro-hydantoin derivative (40), whereas the other derivatives (34-39) having a free aldehydo group attached to an acetal ring in an a-position proceeded an anomalous Bucherer reaction afforeded undesired hydatoin derivatives via an elimination reaction. They explained such anomalous reaction in the following mechanism. Aldehydes (41) scheme (1-5), substituted in the a- position by an oxygen atom incorporated into an acetal ring can react with cyanide to give an equilibrium mixture of the diastereomeric cyanohydrins (42 and 43), because of the strong electron-withdrawing properties of the geminal hydroxyl and cyano groups, -1 can be ۱٤

29 Chapter ne Introduction removed even under very weakly basic conditions of the Bucherer reaction leading simultaneously to trans elimination of -2 and yielding the unsaturated derivatives (44 and 45), respectively, from which the -isopropylidene group is lost and the corresponding unsaturated hydantoins with E (46) and Z configuration (47) are formed, as depicted in Scheme 1-5. R (41) C R CN N - + CN + + N R R (42) (44) (46) CN CN N N R R R (43) (45) (47) Scheme (1-5): The mechanism of anomalous Bucherer reaction that investigated by Kuszmann et al. [58] ۱٥

30 Chapter ne Introduction Recently, Koόš et al. [59-62] managed to convert the dialdo derivative (48), which obeys the anomalous Bucherer reaction [58], into the keto derivatives (49), by the Grignard reaction with the former and subsequent oxidation of the resulting alcohols. R R C RMgX dry ether reflux, 1hr. C pyridinium dichromate Ac 2, C 2 Cl 2 reflux, 2hr. C (48) 2 o alcohols (49) By utilizing the suitable Grignard reagent, they managed to synthesize a number of a-saas structurally related to some natural a- amino acids through the usual Bucherer-Bergs reaction on (49), (Figure 1-4). 2 N C 2 N C 2 C( ) 2 C 2 N C (50) (51) (52) C C 2 C( ) 2 2 N C 2 C C 2 2 N C 2 N (53) (54) (55) Figure (1-4): The a-saas synthesized by Koόš et al., structurally related to alanine (50, 51), leucine (52, 53), and serine (54, 55). ۱٦

31 Chapter ne Introduction Strecker Synthesis of a-saas The direct introduction of the a-amino nitrile function into the carbonyl derivatives of carbohydrates using the classical Strecker conditions [N 4 Cl, KCN, 2 ], is unprofitable, and only the corresponding cyanohydrins (58 and 59) were obtained, form the 3-ulose (56) [63,64] and the dialdo (57) [65] derivatives, respectively. Bn CN C Bn CN (56) (57) (58) (59) Instead, various catalytic Strecker-type reactions have been used for this purpose. The most earlier reports in this respect, is that of Postel and his associates [66,67], when they used titanium (IV) isopropoxide as a mild Lewis acid catalyst for the one-pot a-aminocyantion procedure of some protected keto derivatives of carbohydrates (Scheme 1-6). (33) a N b NC 2 N (60) Scheme (1-6): Catalytic Strecker-type reaction as reported by Postel et al. [66] Reagents and conditions: a)ti(ipr) 4 (1.2 equiv.), N 3 -, r.t., 5hr., then b) 3 SiCN (1 equiv.), r.t., 12hr., 80%. ۱۷

32 Chapter ne Introduction It is noteworthy that the procedure was also successfully applied for introducing different alkyl- and aryl- amines as well as amino acids [52,68] (Figure 1-5). R` NC N 2 Tr NC R`C N R NC R`C N R (61) R` = Bn (70%) (62) R` = Bz (82%) (63) R = (80%) (64) R` = (47%) (65) R = (53%) (66) R` = (23%) Figure (1-5): Various synthesized sugar a-amino nitriles by using catalytic Strecker reaction as reported by Postel et al. [52,68] Synthesis of Glycosyl-a-Amino Acids Recently, Grison et al. [69] have synthesized a number of glycosyla-amino acids (Figure 1-6, 67-71), in which the a-c of the amino acid moiety is connected to C-6 of a pyranose (67), or to the C-5 of pentoses (68-71), as a part of a study concerning the synthesis of more stable C- glycopeptide analogs to improve the metabolic stability of such potential drugs. ۱۸

33 Chapter ne Introduction ph N C C 2 (67) ph N C C 2 (68) ph N C C 2 (69) ph N C C 2 ph N C 2 C (70) (71) Figure (1-6): Glycosyl-a-amino acids synthesized by Grison et al. [69] The overall synthetic strategy of these glycosyl amino acids involved four distinct steps from dialdoses : 1) a diastereoselective Darzens reaction between the potassium anion derived from isopropyl dichloroacetate and a suitable protected dialdose (a), 2) the one-pot transformation of the so-obtained isopropyl glycosyl-a-chloroglycidic ester (b) with MgI 2, then NaS 3, into an isopropyl glycosyl-a-keto ester (c), 3)the reductive amination of (c) with (S)- a-methylbenzylamine and a hydrogenating reagent, afforded the a-amino ester (d), 4) the C-selective deprotection afforded the a-amino acid salt (e), as outlined in Scheme 1-7. ۱۹

34 Chapter ne Introduction C R (a) CCl 2 CiPr Et 2, iprk/ipr 0 o C ph N (d) CiPr MgI 2, Et 2 35 o C Cl R (b) CiPr 2 or NaBN C ipr, 20 o 2 C R ph N C 2 R CiPr R CiPr I ph NaS 3 2 N 2 (c) (2 equiv.) Molecular sieves ipr, 20 o C, 24h CiPr R (i) Li, /TF 1/2, reflux, 2.5h (ii) K, /TF 1/3, reflux, 4h ph (e) N C 2 R C - K + R =,,,, Scheme (1-7): The synthetic strategy of the glycosyl-a-amino acids as reported by Grison et al. [69] The Synthesis of Lower Classes of SAAs The first report on a synthetic SAA is that of eyns and Paulsen [70], when they reported in 1959 the synthesis of d-saa (72, Figure 1-7) as a research project exploring the composition of a bacterial cell wall. In a close project, Fuchs and Lehmann [71] synthesized the d-saa; 5-amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-heptonic acid (73) as a potential monomer for the preparation of bacterial polysaccharide analogues. ۲۰

35 Chapter ne Introduction C a (72) d b g N 2 N 2 C 2 (73) C N C 2 C N n (74) C 2 C Figure (1-7): The first synthesized d-saa (72) by eyns and Paulsen [70], and the first synthesized polyamide (74) of d-saa (73) by Fuchs and Lehmann [71] In 2002, Kessler's group published an improved method for the synthesis of a d-saa [72] (82, Scheme 1.8). Employing a g(cn) 2 melt on 2,3,4,6-tetra--acetyl-β-D-glucopyranosyl bromide (75) furnished the cyano-derived glucopyranoside (76) in 81% yield. The latter was easily converted to (77) by means of reduction with LiAl 4. Chemoselective N- Boc protection gave (78). Regioselective monomethoxytrityl (MMtr) protection on the primary hydroxyl group followed by benzylation of the remaining secondary hydroxyl groups afforeded (79). The fully protected compound was submitted to acidic treatment by the chemoselective N- Fmoc protection. The resulting alcohol (80) was subsequently oxidized to give the desired target (82). ۲۱

36 Chapter ne Introduction Ac Br i Ac CN ii N 2 Ac Ac Ac Ac Ac Ac (75) (76) (77) a b g d NFmoc vii R NR 2 iii Bn Bn R 1 R 1 Bn (82) iv v vi R 1 (78) (78) R = R 1 =, R 2 = Boc (79) R = MMtr, R 1 =, R 2 = Boc (80) R = MMtr, R 1 = Bn, R 2 = Boc (81) R =, R 1 = Bn, R 2 = Fmoc Scheme (1-8): The improved Kessler synthesis of d-saa. Reagents and conditions: i) g(cn) 2 melt, 85 C, 1h, 81%; ii) LiAl 4, TF, 0 C, 4h; iii)boc 2, TF, 2, r.t., 15h, 81%; iv)mmtrcl, TEA, DMAP, DMF, r.t., 15h, 78%; v) BnBr, 18-crown-6, K, TF, r.t., 15h, 63%; vi) a.tfa, DCM, 2, r.t., 1h; b.fmoccl, NaC 3, TF, 2, r.t., 2h; vii)temp, KBr, TBABr, NaCl, DCM, NaCl, NaC 3, 2, r.t., 75min, 96% The same group [73] reported the development of two new sugar amino acids- a β-saa (83) and a g-saa (84), as detailed in Scheme 1.9. ۲۲

37 Chapter ne Introduction 1. Tf 2, Py, -10 o C C 2 Cl 2 2. NaN 3, DMF Cat. Bu 4 NCl N 3 77% Ac 3h, 65 o C quantitative N 3 a b FmocN (83) Pd/C, 2, FmocCl, p 8-9 TF/ 70% N 3 1. NaI 4,, 10 o C, 5h 2. KMn 4, 50% Ac, 90% b a g FmocN TEMP C 2 Cl 2 / 2 60% FmocN 1. Pd/C, 2, 2. FmocCl, p 8-9 / 2 90% (84) Scheme (1-9): The overall synthesis of N-protected b- and g-saas as reported by Kessler et al. [73] The β-saa (83) was used by Kessler to prepare the somatostatin analog (85) that showed anti-proliferative and apoptotic activity against both multidrug-resistant and drug-sensitive hepatoma carcinoma cells [74] (Figure 1-8). n the other hand, verhand's group [75] developed furanoid (86) and pyranoid (87) e-saas, the former was used to prepare the cyclic tertrapeptide (88) which showed a high inhibition activity due to its highly binding affinity to some integrin receptors [76] (Figure 1-8). ۲۳

38 Chapter ne Introduction 2 N Tr N N N N N N 2 N e d g (86) b a C (85) Arg-Gly-Asp N 2 N e d g b a C (88) (87) Figure (1-8): The somatostatin analog (85) prepared by Kessler et al. [74], and the furanoid and pyranoid e-saas (86 and 87), and the cyclic peptide (88) prepared by verhand et al. [75,76] ۲٤

39 Chapter ne Introduction 1.6. Aim of The Work The use of sugar amino acids as building blocks in peptidomimetic studies is increasing remarkably, and yet very few peptide-based therapeutics have been developed. In addition, most of natural and synthetic glycopeptides are incorporating N- or - linkage between the sugar residue and the peptide backbone; such linkages are subjected to both enzymatic and chemical degradation. In this regards, this work aimed to synthesize more stable C- gylco amino acids that may be used in peptidomimetic studies as conformationally constrained building blocks for drug design, and to mimic some of naturally occurring glycopeptide antibiotics. ۲٥

40 Chapter Two Experimental 2.1. General Notes 1. lting points were recorded by using Gallenkamp capillary melting point apparatus and were uncorrected. 2. Infrared spectra were recorded on a Shimadzu FTIR-8400S spectrophotometer, as KBr discs or thin flims. 3. Thin layer chromatography (TLC) was performed on aluminum plates precoated with 0.25 mm layer of silica-gel 60 supplied by Fluka and spots were detected with iodine vapour. 4. Column chromatography was carried out with silica-gel 60 (Fluka). 5. Solutions were usually evaporated under reduced pressure by using rotary evaporator. 6. Solvents and liquid reagents were purified and dried in the usual manner before being used. 7. All chemicals used were supplied by rck, Fluka, BD chemicals, Thomas Baker and Riedel-De aen AG. ۲٦

41 Chapter Two Experimental ,3:4,5-Di--isopropylidene-b-D-fructopyranose (89) [77] C 2 (89) Concentrated sulfuric acid (8.75 ml) was added, dropwise, with stirring, to an ice-cold anhydrous acetone (175 ml), and to this cooled solution, was added; dry, finely powdered D-fructose (9 g, 50 mmol) portionwise, with stirring. The resulting suspension was stirred magnetically at room temperature, with the exclusion of moisture, until all of the sugar had dissolved (15-20 min). the pale-yellow solution was kept at room temperature for an additional 80 min, and then cooled in ice. An ice-cold solution of sodium hydroxide (27.5 g) in water (125 ml) was gradually added, with stirring, and the resulting inorganic salts were removed by suction filteration and washed with acetone. The filtrate and washings were combined and evaporated under reduced pressure, and the residual aqueous suspension was extracted with dichloromethane (3 25 ml). The extracts were combined, washed with water (2 15 ml), dried (anhydrous Na 2 S 4 ), filtered, and evaporated to a pale-yellow solid (11 g, 85%), of crude (89), m.p ne recrystallization, by dissolving in boiling ether (5 ml/g), cooling, and adding pentane (5 ml/g), gave 7.5 g (58%) of compound (89), as white rosettes of needles, m.p , lit. [77,78] m.p. 97. Additional crop (1.2 g) of compound (89), was obtained from the mother liquor, when the latter was kept overnight in a ۲۷

42 Chapter Two Experimental freezer. verall yield (8.7 g, 67%). R f (benzene-methanol; 24:1) 0.24, FTIR (KBr disc); 3300 cm -1 (-) ,3:4,5-Di--isopropylidene-b-D-arabino-hexosulo-2,6- pyranose (90) [79] C (90) Three methods of oxidation were examined to obtain compound (90). thod A; xidation with Dimethyl sulfoxide-acetic anhydride [79] Compound 89 (2.5 g, 9.6 mmol) was dissolved in a mixture of dry dimethyl sulfoxide (6.7 ml, 94 mmol) and acetic anhydride (8.3 ml, 89 mmol). The resulting colorless solution was kept at room temperature, with the exclusion of moisture, for four days. The light-yellow solution was diluted with 50 ml of chloroform, washed successively with water (4 25 ml), saturated aqueous Na 2 C 3 solution (4 25 ml), and water (2 25 ml). It was then dried (anhydrous Na 2 S 4 ), filtered, and evaporated to a yellow syrup (2 g, 81%). TLC (benzene-methanol; 24:1), showed that the product consisted of two components with R f s 0.42 and 0.7. Column chromatography (benzene-methanol; 24:1 as eluent) afforded (90); R f 0.42 as a pale-yellow syrup, yield 0.35 g (14%), FTIR (film) 2737 cm -1 (aldehyde C-) and 1751 cm -1 (C=). The ۲۸

43 Chapter Two Experimental component with R f 0.7 was isolated as a pale-yellow syrup, yield 1.1 g (36%) that identified to be (90a). C 2 C 2 S (90a) thod B; xidation with Pyridinium chlorochromate (PCC). (i) Preparation of Pyridinium chlorochromate [80] Anhydrous chromium trioxide Cr 3 (10 g, 100 mmol) was rapidly weighed into a 100-ml beaker, and 18.4 ml of 6 M hydrochloric acid solution (110 mmol) was added with stirring. The resulting dark-red solution was cooled to 0 C, and 7.9 g (8 ml, 100 mmol) of redistilled pyridine was added to it, dropwise and with stirring; at such a rate that the temperature of the mixture did not exceed 10 C. After completion of the addition, the mixture was recooled to 0 C, and the resulting yelloworange solid was collected, with suction, on a sintered-glass funnel, drained well, and dried over fresh silica gel pellets in a vacuum desiccator for at least 48 hours. Yield; 16.8 g (78.4%). (ii) xidation Procedure PCC (9 g, 41.7 mmol) was suspended in anhydrous dichloromethane (50 ml) in a dry, 100-ml round-bottomed flask, and to this suspension was added a solution of compound 89 (3.5 g, 13.5 mmol) in anhydrous dichloromethane (ca. 5 ml) with magnetic stirring. The flask ۲۹

44 Chapter Two Experimental was fitted with a dry double-surface condenser, protected by a CaCl 2 guard-tube, and the mixture was refluxed on a water-bath (bath temperature; 50 ), with vigorous stirring, until all of the starting material had consumed (2-2.5 hour); as monitered by TLC. The black reaction mixture was diluted with about 50 ml of ethyl acetate, and about 2 hours thereafter, the precipitated chromium salts were filtered off, and washed with a little ethyl acetate. The deep-brown filtrate was passed through a Celite pad, and the filter cake was washed with ethyl acetate. The filtrate and washings were combined, dried (anhydrous Na 2 S 4 ), and evaporated under reduced pressure to afford the product as a faint-yellow syrup; 1.2 g (35%), that was homogeneous by TLC (benzene-methanol; 24:1). R f 0.43, FTIR (film) 1751 cm -1 (C=), 2738 cm -1 (aldehyde C-). thod C; xidation with Pyridinium dichromate-acetic anhydride (i) Preparation of Pyridinium dichromate (PDC) [82] Redistilled pyridine; 7.9 g (8 ml, 100 mmol) was added dropwise (from a dropping funnel) to a cooled solution of anhydrous chromium trioxide; Cr 3 (10 g, 100 mmol) in water (10 ml) with occasional stirring, at such a rate that the temperature maintained below 25 C. After completion of the addition, the dark reaction mixture was diluted with acetone (40 ml), and stored overnight in a freezer. The resulting brightorange crystals were collected, with suction, on a sintered-glass funnel; washed with acetone (3 5 ml), drained well, dried in an oven (70 C) for 1-2 hours, and kept in a desiccator overnight; yield 13.5 g (72%), m.p C, lit. [82] C. ۳۰

45 Chapter Two Experimental (ii) xidation Procedure [81,83] PDC (7 g, 18.6 mmol) and about 2 g of molecular sieves powder Type 3A, were suspended in anhydrous dichloromethane (30 ml) in a dry, 100-ml round-bottomed flask, and acetic anhydride (9.4 ml, 100 mmol) was rapidly added with stirring. The flask was stoppered, and immersed in an ice-bath. After about 10 min., a solution of compound 89 (8 g, 30.8 mmol) in anhydrous dichloromethane (15 ml) was added portionwise, and with stirring. Upon completion of the addition, the reaction flask was fitted with a dry, double-surface condenser protected by a CaCl 2 guardtube, and the mixture was then refluxed, with vigorous magnetic stirring, on a water bath (bath temperature; 50 C). 1.5 hour thereafter, the oxidation was complete (as determined by TLC). The black reaction mixture was then diluted with ethyl acetate (ca. 50 ml), and kept at room temperature for 3 hours, and then filtered to remove the precipitated chromium salts. The dark filtrate was passed through a column of silica gel 60 (5 20 cm), equilibrated and eluted with ethyl acetate. The colorless eluate was concentrated under reduced pressure, and toluene (2 10 ml) was evaporated from the residue to remove traces of pyridine and acetic acid. This was afford compound (90) as a faint-yellow syrup, 5.6 g (71%). R f 0.42 (benzene-methanol; 24:1), FTIR (film) 1751 cm -1 (C=) and 2737 cm -1 (aldehyde C-) ,2:3,4-Di--isopropylidene-a-D-galactopyranose (91) [84] C 2 (91) ۳۱

46 Chapter Two Experimental Freshly fused, and powdered anhydrous zinc chloride (9.5 g, 70 mmol) was rapidly weighed into a dry 250-ml Erlenmeyer flask, and (100 ml) of dry acetone was added, the flask was stoppered, and the suspension was stirred magnetically at room temperature until the chloride had dissolved (a small amount of undissolved zinc hydroxide remained in suspension). Concentrated sulfuric acid (0.32 ml) was rapidly added dropwise, and with stirring, from a pipet in such a way that no acid touched the inside of the flask s neck, and to the resulting colorless solution was added finely powdered, anhydrous D-galactose (9 g, 50 mmol), with stirring. The flask was stoppered, and the suspension was stirred vigorously for four hours. A suspension of 16 g of anhydrous sodium carbonate in 28 ml of water was added in portions (cautiously, but as fast as possible), and the mixture was stirred (at first cautiously, and then vigorously) for about one hour. The suspension was then filtered with suction, and the inorganic salts were washed several times with acetone. The filtrate and washings were evaporated under reduced pressure (bath temperature; 40 C); whereupon the desired acetal separated as an oily upper layer. The mixture was extracted with ether (3 25 ml), and the extracts were dried (anhydrous Na 2 S 4 ), filtered, and evaporated (rotary evaporator, bath temperature; 30 C), to remove the ether. The bath temperature was then rasied to 100 C, to remove condensation products of the acetone, and this was left 6.5 g (57%) of compound (91) as a pale-yellow syrup. R f 0.21 (benzene-methanol; 24:1), FTIR (film) 3487 cm -1 (-). ۳۲

47 Chapter Two Experimental ,2:3,4-Di--isopropylidene-a-D-galacto-hexodialdo-1,5- pyranose (92) [85] C (92) Compound 91 (6 g, 23 mmol) was oxidized with PDC (5.3 g, 14 mmol) and acetic anhydride (7 ml, 75 mmol), following the detailed procedure that was given for the oxidation of compound (89); [thod C, (ii)]. After 2.5 hour reflux, compound (92) was isolated as a yellow, thick syrup (3.8 g, 64%), that was homogeneous by TLC (benzene-methanol; 24:1). R f 0.36, FTIR (film); 1743 cm -1 (C=), 2739 cm -1 (aldehyde C-) General Amino-cyanation Procedure; Synthesis of Compounds (93-98) A solution of the aldehyde (0.3 g, 1.16 mmol) in just sufficient ethanol was added to a solution of sodium metabisulfite (0.6 g, 3 mmol) in water (1 ml), and the mixture was stirred vigorously for 30 min at C ; during which time a slurry of the bisulfite adduct was formed. A solution of the amine (1.4 mmol) in ethanol (1 ml) was added, and the stirring was continued for min at C. The reaction vessel was then immersed in an ice bath inside the hood, and 0.1 g (2 mmol) of sodium cyanide was added. The vessel was tighted, and stirring was ۳۳

48 Chapter Two Experimental continued, at room temperature, until TLC (ether-petroleum ether; 3:1) indicated the completion of the reaction. The reaction mixture was diluted with chloroform-water (10 ml each), and the aqueous layer was extracted with chloroform (3 10 ml). The combined chloroform solutions were washed successively with brine (2 10 ml), and water (10 ml), dried (MgS 4 ), filtered, and evaporated to afford the mixture of epimeric a-aminonitriles. The following a-aminonitriles were synthesized by using the selected amines; Benzylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,Lglycero-b-D-arabino-3-heptulopyranosononitrile (93) NC NC 2 C 6 5 (93) Starting from the aldehyde (90), benzylamine 0.15 ml (0.15 g, 1.4 mmol) was used, and the reaction completed during 18 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitriles (93) was isolated as a yellow syrup. Yield g (66%). R f 0.86 and 0.81 (ether-petroleum ether; 3:1), FTIR(film) 3320 cm -1 (N-), 2245 cm -1 (C N). ۳٤

49 Chapter Two Experimental (sec-Butylamino)-2-deoxy-3,4:5,6-di--isopropylidene- D,Lglycero-b-D-arabino-3-heptulopyranosononitrile (94) NC NC C 2 5 (94) Starting from the aldehyde (90), sec-butylamine 0.14 ml (0.1 g, 1.4 mmol) was used, and the reaction completed during 24 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitriles (94) was isolated as a pale-yellow syrup by column chromatogyaphy (etherpetroleum ether; 5:1, as eluent). Yield 0.24 g (61%). R f 0.5 and 0.43 (ether-petroleum ether; 3:1), FTIR(film) 3400 cm -1 (N-), 2250 cm -1 (C N) Isobutylamino-2-deoxy-3,4:5,6-di--isopropylidene-D,Lglycero-b-D-arabino-3-heptulopyranosononitrile (95) (95) NC NC 2 C Starting from the aldehyde (90), isobutylamine 0.14 ml (0.1 g, 1.4 mmol) was used, and the reaction completed during 24 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitiles (95) ۳٥

50 Chapter Two Experimental was isolated as a pale-yellow syrup, by column chromatography (etherpetroleum ether; 5:1, as eluent). Yield 0.23 g (58%). R f 0.66 and 0.63 (ether-petroleum ether; 3:1), FTIR (film): 3410 cm -1 (N-), 2237 cm -1 (C N) Benzylamino-6-deoxy-1,2:3,4-di--isoproylidene-D,Lglycero-a-D-galacto-heptopyranurononitrile (96) NC NC 2 C 6 5 (96) Starting from the aldehyde (92), benzylamine 0.15 ml (0.15 g, 1.4 mmol) was used, and the reaction completed during 16 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitriles (96) was isolated as a yellow, thick syrup. Yield 0.28 g (64%). R f 0.71 and 0.68 (ether-petroleum ether; 3:1), FTIR (film): 3332 cm -1 (N-), 2230 cm -1 (C N) (sec-Butylamino)-6-deoxy-1,2:3,4-di--isopropylidene-D,Lglycero-a-D-galacto-heptopyranurononitrile (97) NC NC C 2 5 (97) ۳٦

51 Chapter Two Experimental Starting from the aldehyde (92), sec-butylamine 0.14 ml (0.1 g, 1.4 mmol) was used, and the reaction completed during 20 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitriles (97) was isolated, as a yellow syrup, by column chromatography (etherpetroleum ether; 5:1, as eluent). Yield 0.25 g (63%). R f 0.48 and 0.4 (ether-petroleum ether; 3:1), FTIR (film): 3395 cm -1 (N-), 2238 cm -1 (C N) Isobutylamino-6-deoxy-1,2:3,4-di--isopropylidene-D,Lglycero-a-D-galacto-heptopyranurononitrile (98) NC NC 2 C (98) Starting from the aldehyde (92), isobutylamine 0.14 ml (0.1 g, 1.4 mmol) was used, and the reaction completed during 20 hours after the addition of sodium cyanide. The mixture of epimeric a-aminonitriles (98) was isolated, as a yellow syrup, by column chromatography (etherpetroleum ether; 5:1, as eluent). Yield g (60%). R f 0.6 and 0.54 (ether-petroleum ether; 3:1), FTIR (film): 3390 cm -1 (N-), 2252 cm -1 (C N). ۳۷