The Chemistry of L-Ascorbic Acid Derivatives in the Asymmetric Synthesis of C2- and C3- Substituted Aldono-γ-lactones
A Dissertation by
Ayodele O. Olabisi
M. S., Wichita State University, 2004 B. S., Wichita State University, 1999
Submitted to the College of Liberal Arts and Sciences and the Faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy
August 2005 The Chemistry of L-Ascorbic Acid Derivatives in the Asymmetric Synthesis of C2- and C3- Substituted Aldono-γ-lactones
I have examined the final copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Chemistry.
______Professor Kandatege Wimalasena, Committee Chair
We have read this dissertation and recommend its acceptance:
______Professor William C. Groutas, Committee Member
______Professor Ram P. Singhal, Committee Member
______Professor Francis D’Souza, Committee Member
______Professor George R. Bousfield, Committee Member
Accepted for the College of Liberal Arts and Sciences
______Dr. William Bischoff, Dean
Accepted for the Graduate School
______Dr. Susan K. Kovar, Dean
ii DEDICATION
To My Parents
iii ACKNOWLEDMENTS
I wish to express my deepest and sincerest appreciation to my advisor, Dr
Kandatege Wimalasena for his positive guidance, enlightened mentoring and encouragement. His passion for the subject matter has greatly improved my knowledge and interest. My sincere appreciation extends to Dr. Shyamali Wimalasena and Dr.
Mathew Mahindaratne, who helped me with my initial research training and their assistance in the preparation of my manuscripts. I would also like to give my appreciation to my other committee members; Dr. William C. Groutas, Dr. Ram Singhal, Dr. Francis
D’Souza and Dr. George Bousfield for their significant recommendations.
I wish to express my heartfelt gratitude to my colleagues, Dr. Srimevan
Wanduragala, Dr. Mehul Bhakta, Dr. Rohan Perera and Samantha Ranaweera, for their invaluable friendship. It was a joy to work with them.
Finally, I acknowledge some of the many people without whom I could not have completed my education; my wife, Monica, and children, Angela, Dominique and Folade for their love, support and encouragement even at times of difficulty. My special gratitude deeply extends to my parents and sisters for their incomparable love, support and prayers.
This work was supported by a grant from the National Institutes of Health (NS 39423).
iv ABSTRACT
The antioxidant and redox properties of L-ascorbic acid are closely associated
with the electron rich 2, 3-enediol moiety of the molecule and therefore selective
functionalization of the 2- and 3-OH groups is essential for the detailed structure-activity
studies. Reactions of 5- and 6-OH protected ascorbic acid with electrophilic reagents
exclusively produce the corresponding 3-O-alkylated products under mild basic
conditions due to the high nucleophilicity of the C-3-OH. Based on the density functional
theory (B3LYP) electron density calculations, a novel and general method was devised
for the direct alkylation of the 2-OH group of ascorbic acid with complete regio- and
chemo-selectivity. A complete spectroscopic analysis of two complementary series of 2-
O-acetyl-3-O-alkyl and 2-O-alkyl-3-O-acetyl ascorbic acid derivatives was carried out to
define their spectroscopic characteristics and to resolve common inconsistencies in the
literature.
The asymmetric approach to the synthesis of natural products or other biologically active compounds is impeded by low abundance of natural sources as well as a limited number of efficient synthetic methods. Nevertheless, carbohydrate-based systems such as the aldono-1,4-lactones (also known as aldono-γ-lactones) which generate a host of chiral compounds have been particularly rewarding in this respect. This study shows a practical approach using 5,6-O-isopropylidene-L-ascorbic acid (ketal of L- ascorbic acid) as a single common starting material for facile asymmetric synthesis of protected, optically pure and functionalized aldono-1,4-lactones derivatives, valuable in the synthesis of derivatives of various pharmacologically active agents for structure-
v activity studies. The practicality of this new approach is demonstrated by the convenient
synthesis of a series of thermal Claisen-rearranged products of 5,6-O-isopropylidene-3-
O-allyl-L-ascorbic acid and 5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid as the
corresponding 5,6-O-isopropylidene-2-allyl-3-keto-L-galactono-γ-lactone and 5,6-O- isopropylidene-3-allyl-2-keto-L-galactono-γ-lactone respectively. The synthetic routes are economical, efficient, diastereospecific, and proceed in high yields.
vi TABLE OF CONTENTS
CHAPTER 1 ...... 1
INTRODUCTION ...... 1
CHAPTER 2 ...... 4
BACKGROUND AND SIGNIFICANCE...... 4
2.1 Discovery and History of L-Ascorbic Acid...... 4
2.2 Sources of L-Ascorbic Acid...... 10
2.3 Tissue Distribution of L-Ascorbic acid...... 12
2.4 Biosynthesis of L-Ascorbic Acid in Animals ...... 14
2.5 Biosynthesis of L-Ascorbic Acid in Plants...... 18
2.6 Commercial Scale Synthesis of L-Ascorbic acid...... 22
2.7 Biological Functions of L-Ascorbic Acid...... 26
2.7.1 L-Ascorbic Acid as an Enzyme Cofactor ...... 26
2.7.2 L-Ascorbic Acid in Electron Transport ...... 31
2.7.3 L-Ascorbic Acid as an Antioxidant in Biological Systems ...... 33
2.8 L-Ascorbic Acid Metabolic Enzymes...... 36
2.9 Degradation and Oxidation of L-Ascorbic Acid...... 37
2.10 Cellular Transport and Intestinal Absorption of L-Ascorbic Acid ...... 40
2.11 Molecular Structure of L-Ascorbic Acid ...... 41
2.12 Chemical and Physical Properties of L-Ascorbic Acid ...... 45
2.13 Synthetic Derivatives and Analogues of L-Ascorbic Acid...... 46
CHAPTER 3 ...... 53
vii RESEARCH OBJECTIVE ...... 53
CHAPTER 4 ...... 55
RESULTS AND DISCUSSION...... 55
4.1 Chemo- and Regio-Selective Alkylation of L-Ascorbic Acid...... 55
4.1.1 3-O-Alkylation of 5,6-O-Isopropylidene-L-Ascorbic Acid...... 56
4.1.2 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid...... 59
4.1.3 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid ...... 62
4.2 Acylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid...... 65
4.2.1 C3-O- to C2-O Rearrangements of 3-O-Acyl-L-Ascorbic Acid Derivatives
...... 67
4.3 NMR Spectroscopic Analyses of L-Ascorbic Acid and its Derivatives...... 70
4.3.1 NMR Spectroscopic Properties of 2-O- and 3-O-Substituted 5,6-O-
Isopropylidene-L-Ascorbic Acid ...... 71
4.4 The Sigmatropic Claisen Rearrangement of L-Ascorbic Acid Derivatives...... 75
4.4.1 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-
Isopropylidene-3-O-Allylic Derivatives of L-Ascorbic Acid...... 76
4.4.1.1 NMR Spectroscopic Analyses of Products from C3-O to C2
Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-3-O-
Allyl-L-Ascorbic Acid Derivatives ...... 77
4.4.2 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-
Isopropylidene-3-O-Cinnamyl-L-Ascorbic Acid Derivatives ...... 85
viii 4.4.2.1 NMR Spectroscopic Analyses of Products from C3-O to C2
Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-
Acetyl-3-O-Cinnamyl-L-Ascorbic Acid Derivative (10A) ...... 87
4.4.3 The C2-O to C3 Sigmatropic Claisen Rearrangement of 5,6-O-
Isopropylidene-2-O-Allyl-L-Ascorbic Acid Derivatives...... 90
4.4.3.1 NMR Spectroscopic Analyses of Products from C2-O to C3
Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-
Allyl-L-Ascorbic Acid Derivatives ...... 91
4.5 Comparative Analysis and Identificaton of Products of C3-O to C2 and C2-O to
C3 Claisen Rearrangement of L-Ascorbic Acid Derivatives ...... 102
4.6 Stereochemistry of Products of C3-O to C2 and C2-O to C3 Claisen
Rearrangement of L-Ascorbic Acid Derivatives...... 105
4.7 Chemo- and Diastereo-Selective Reduction of Claisen Rearranged Products (E
& F Series) of L-Ascorbic Acid Derivatives...... 110
CHAPTER 5 ...... 116
EXPERIMENTAL SECTION...... 116
LIST OF REFERENCES...... 144
APPENDIX...... 164
ix LIST OF TABLES
Table 1 Approximate Levels of L-Ascorbic Acid in Tissues ...... 13
Table 2 List of Enzymes Requiring L-Ascorbic Acid as a Cofactor or as a Modulator of
Activity (adapted from Ref. 118) ...... 28
Table 3 Physical Properties of L-Ascorbic Acid (adapted from Ref. 211 & 212)...... 47
Table 4 Products from 3-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid...... 58
Table 5 Products of 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)...... 61
Table 6 Products of 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic
Acid ...... 63
Table 7 Products of 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic
Acid ...... 64
Table 8 Products of 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid..... 65
Table 9 1H NMR (C-4-H) and 13C NMR (C-2 & C-3) Chemical Shifts (δ) of 2-O-Alkyl
and 3-O-Alkyl Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1) ...... 72
Table 10 1H NMR (C4-H) and 13C NMR (C2 & C3) Chemical Shifts (δ) of 2,3-O-
Disubstituted Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1) ...... 74
Table 11 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-
(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 79
Table 12 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-
(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 80
Table 13 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-
prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone ...... 81
x Table 14 13C-NMR Chemical shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-
(1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 82
Table 15 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-
(1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 83
Table 16 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-
(1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 84
Table 17 1H-NMR Chemical Shifts (δ) Claisen Rearranged 5,6-O-Isopropylidene-2-O-
Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 88
Table 18 13C-NMR Chemical Shifts (δ) Claisen Rearranged 5,6-O-Isopropylidene-2-O-
Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 89
Table 19 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-
prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone ...... 94
Table 20 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-
(1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 95
Table 21 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-
prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone ...... 96
Table 22 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-
(1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 97
Table 23 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-
methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 98
Table 24 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-
(1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 99
xi Table 25 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-
methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 100
Table 26 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3-
(1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone...... 101
Table 27 Comparison of Diastereoisomers of Allyl-L-Galactono-γ-Lactone ...... 103
xii LIST OF FIGURES
Figure 1 Depiction of L-Ascorbic Acid Biosynthesizing Abilities of Various Species of
Animals in Relation to their Phylogeny ...... 11
Figure 2 Cytochrome b561 in Trans-Membrane Electron Transport...... 32
Figure 3 Chemical Illustration of Radical Reactions in the Cell and Antioxidant Activities
(adapted from Ref. 129) ...... 35
Figure 4 L-Ascorbic Acid Redox System...... 36
Figure 5 Degradation of L-Ascorbic Acid (adapted from Ref. 35) ...... 39
Figure 6 L-Ascorbic Acid and its Diastereomers...... 44
Figure 7 Structural Forms of Dehydro-L-Ascorbic Acid ...... 45
Figure 8 Regioselective O-Alkylation of Ascorbic Acid...... 49
Figure 9 Potentials of L-Ascorbic Acid as a Chiral Synthon...... 52
Figure 10 Calculated Electrostatic Density Potential Diagrams of Monoanion Species of
1. Order of Electron Density: Blue < Green < Yellow < Red...... 57
Figure 11 Calculated Electrostatic Density Potential Diagrams of Dianion Species of 1.
Order of Electron Density: Blue < Green < Yellow < Red...... 60
Figure 12 Calculated Electrostatic Density Potential Diagrams of Neutral Species of 1.
Order of Electron Density: Blue < Green < Yellow < Red...... 70
Figure 13 Diastereoselective Reduction of C3-keto of E Series via Metal Chelation.... 112
Figure 14 Diastereoselective Reductive Amination Products of 1E, 5E and 1F (X, Y and
Z Respectively) ...... 115
xiii LIST OF SCHEMES
Scheme 1 Proposed Biosynthetic Pathway for L-Ascorbic Acid in Animals (adapted from
Ref. 118, 119 & 121)...... 15
Scheme 2 The Smirnoff-Wheeler Biosynthetic Pathway for L-Ascorbic Acid in Plants
(adapted from Ref. 118, 119 & 121) ...... 19
Scheme 3 The Reichstein Process for L-Ascorbic Acid Manufacture (adapted from Ref.
127) ...... 23
Scheme 4 Microbial-Engineered Pathway for L-Ascorbic Acid Manufacture (adapted
from Ref. 127)...... 25
Scheme 5 L-Ascorbic Acid (Exogenous Electron Donor) in DβM Enzymatic Reaction 30
Scheme 6 Four-Electron Reduction Process of Oxygen to Water...... 33
Scheme 7 3-O-Alkylation of 5,6-O-Isoprpylidene-L-Ascorbic Acid...... 58
Scheme 8 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1) ...... 61
Scheme 9 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid. 63
Scheme 10 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid. 64
Scheme 11 2,3-O-Disubstituted 5,6-O-Isopropylidene-L-Ascorbic Acid ...... 65
Scheme 12 Acylation of 5,6-O-Isopropylidene-L-Ascorbic Acid...... 67
Scheme 13 Irreversible Isomerization of 5,6-O-Isopropylidene-3-O-Acetyl-L-Ascorbic
Acid under Basic Conditions...... 68
Scheme 14 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3-
Keto-L-Galactono-γ-Lactone ...... 77
xiv Scheme 15 Direct Synthesis of 5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3-Keto-
L-Galactono-γ-Lactone from 1...... 86
Scheme 16 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1-
phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone...... 86
Scheme 17 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-prop-2-enyl)-2-
Keto-L-Galactono-γ-Lactone ...... 92
Scheme 18 C3-O (A) to C2 (E) Claisen Rearrangement Transition-State Geometry .... 107
Scheme 19 C2-O (C) to C3 (F) Claisen Rearrangement Transition-State Geometry .... 107
Scheme 20 The Reduction Products of 5, 6-O-Isopropylidene-2-Allyl-3-Keto-L-
Galactono-γ-Lactones (E) ...... 111
Scheme 21 The Reduction Product of 5,6-O-Isopropylidene-3-Allyl-2-Keto-L-Galactono-
γ-Lactone 2F ...... 112
xv CHAPTER 1
INTRODUCTION
Ascorbic acid is a versatile water soluble radical scavenger widely distributed in
aerobic organisms that plays a central role in the protection of cellular components
against oxidative damage by free radicals and oxidants that are involved in the
development and exacerbation of a multitude of chronic diseases such as cancer, heart
disease, brain dysfunction, aging, rheumatism, inflammation, stroke, emphysema, and
AIDS.1-17 & 217 It also plays a critical role as a physiological reductant for key enzymatic
transformations in catecholamine neurotransmitter, amidated-peptide hormone, and
collagen biosynthetic pathways. In addition, simple derivatives of L-ascorbic acid have
been shown to possess important pharmacological properties. For example, (a) 5,6-O-
modified ascorbic acid derivatives have been found to be effective anti-tumor agents for
various human cancers, and induce apoptosis in tumor cells;18-25 (b) C2 alkylated
derivatives have been shown to have immuno-stimulant activity;26-31 (c) C2-O and C3-O
alkylated derivatives are known to protect against peroxidation of lipids of the bio-
membrane.32-33 Recently, the chemistry of ascorbic acid has also been exploited to
develop strategies for central nervous system drug delivery.34 These antioxidant as well as redox and pharmacological benefits of L-ascorbic acid and its derivatives are closely associated with the electron rich C2,C3-enediol moiety of its five-membered lactone ring.35 Therefore, the selective modification of its C2- and C3-OH groups is essential for
detailed structure-activity studies of L-ascorbic acid. Consequently, our research group
1 was interested in studies13-17 involving various ascorbate derivatives as probes for
Dopamine-β-mono-oxygenase and Cytochrome b561, both of which use ascorbate as a
source of physiological reductant in catecholamine neurotransmitter biosynthesis.
In addition to well known physiological and pharmacological properties of L-
ascorbic acid and its derivatives,35 L-ascorbic acid has also been commonly used as an
inexpensive chiral synthon for the synthesis of a variety of natural products and
pharmacologically active agents.36-53 The common usage of the oxidatively cleaved C6-
C3 fragment of L-ascorbic acid as a chiral synthon36-48 and the selective alteration or
modification of its C2- and/or C3-OH functional groups provides a unique route to
different classes of aldono-1, 4-lactone derivatives which are important precursors for the
synthesis of modified sugars and non-carbohydrate natural compounds.54 One of the
widely used aldonolactones in the synthesis of natural products is D-gulono-γ-lactone
also known as D-gulono-1, 4-lactone and is easily obtained from L-gulono-γ-lactone by intramolecular Walden inversion.55 Gulono-1,4-lactone is a very versatile precursor for a
large number of pharmacologically active agents and natural products. For example, it is
used as a precursor in the synthesis of (a) rare sugars such as L-ribofuranose, which are
common starting materials for the synthesis of new nucleoside antibiotics such as novobiocin and anti-bacterial agents against Gram-positive bacteria;56-61 (b) pharmacological agents for the suppression of abnormal T-cell responses;62 (c) α- hydroxy-β-amino acid natural products that are known to display a broad range of biological activities which include antibiotic, antifungal, antitumor and potent aminopeptidase protease inhibitors;63-72 and (d) non-carbohydrate natural alkaloids
known for their antitumor activity.73 Furthermore, gulono-1,4-lactone also has
2 applications in polymer chemistry for the synthesis of potentially renewable, biomedical
polymeric materials which are biodegradable.74 Besides the usefulness of aldono-1, 4-
lactones as synthetic chemical precursors,75-76 both L-galactono-γ-lactone and L-gulono-
γ-lactone are also the key intermediate precursors of vitamin C biosynthesis in plants and animals, respectively.77-81
3 CHAPTER 2
BACKGROUND AND SIGNIFICANCE
2.1 Discovery and History of L-Ascorbic Acid
L-ascorbic acid is commonly referred to as vitamin C. The term “Vitamin C” is applied to substances that have anti-scorbutic activity and includes two compounds and their salts: L-ascorbic acid and its two-electron oxidized form, L-dehydroascorbic acid.
The major deficiency syndrome of vitamin C in animals is scurvy. Symptoms of scurvy include anorexia, anemia, arthralgia, bleeding gums, coiled hair, depression, dry eyes and mouth (Sjogren’s syndrome), ecchymosis, follicular hyperkeratosis, fatigue, frequent infections, impaired wound healing, inflamed gums, joint effusions, myalgia, muscle weakness, perifollicular hemorrhages, and petechiae. The disease’s later-stage conditions include patients exhibiting extreme exhaustion, kidney and pulmonary problems, as well as diarrhea, eventually leading to death. The necessity to take in raw animal flesh or fresh plant food in the diet to prevent scurvy disease was known from ancient times. Eber’s
Papyrus, an ancient Egyptian medical treatise in 1,500 BC, described scurvy as a disease characterized by spongy and bleeding gums and bleeding under the skin. Around 400 BC,
Hippocrates, a Greek physician known as the founder of medicine developed an Oath of
Medical Ethics for physicians to follow. This Oath known as the Hippocratic Oath is taken by physicians today as they begin their medical practice. He preached against one- sided nutrition and described how good a daily and healthy diet rich in foods that are
4 known today to contain great amounts of vitamin C could help prevent diseases such as
scurvy. In 1200 AD, the Crusaders were plagued with scurvy. From 1492 to 1600, world
exploration was threatened by scurvy. Ferdinand Magellan, a Portuguese sea captain
around 1520, lost 80% of his crew to scurvy (after he and his crew reached Cape
Virgennes on the southern tip of South America). Also, Vasco de Gama, a Portuguese conquistador famously known as Henry the navigator, was the first to sail across the
African coast on his way to India in 1492 and lost 100 of his 160 crew to scurvy. Scurvy was a severe threat to thousands of soldiers and sailors alike and many died of the disease
during military campaigns and lengthy ocean voyages, respectively, until in 1720, when
the physician J. G. H. Kramer found that fresh herbs and lemon cured the disease.82-111
In 1746, James Lind, a British naval surgeon on H.M.S. Salisbury, conducted a controlled test on 12 of his seamen suffering from the debilitating effects of scurvy and became the first person to give a scientific basis for the cause of scurvy. In 1753, James Lind published the results of his famous findings in a 400-page book, Treatise of the Scurvy, where for the first time, he established the benefit of citrus fruits in combating scurvy, and by 1795, the royal navy had mandated the use of lime juice or other citrus fruits as a scurvy preventative. In 1840, George Budd, a Londoner physicist, wrote that scurvy was due to the absence of an essential food factor that will be discovered in the near future by organic chemists. Up until 1907 scurvy was considered as a human-only disease as no other animal was known to be susceptible to it. However, in 1907, Alex Holst and
Theodore Frohlich, two Norwegian biochemists confirmed that guinea pigs were also susceptible to scurvy and later showed that laboratory monkeys were susceptible to scurvy as well. They also described the prevention of the illness by feeding fruits and
5 vegetables to patients. In 1908, the classic lectures of Sir Archibald Garrod on the
“Inborn Errors of Metabolism”, in which he showed that missing enzymes could cause
diseases such as scurvy, were ignored and neglected at a time when modern, widely
accepted biochemical and genetic concepts were unknown or unrecognized.82-111
In 1912, for the first time, the vitamin hypothesis was suggested by Polish-
American chemist Funk, part of which stated that scurvy was a deficiency disease caused by the lack of an unknown water-soluble substance called the anti-scorbutic factor. In
1920, Sir Jack Cecil Drummond, a Londoner, and the first Professor of Biochemistry in the University of London, suggested calling this substance as Vitamin C because man, guinea pigs, and certain monkeys unlike other mammals, cannot make their own ascorbic acid. This unknown water-soluble antiscorbutic substance was isolated from Ox adrenal cortex (and various plants) in 1928 by the Hungarian biochemist research team of Joseph
L. Svirbely and Albert Von Szent-Györgyi. In autumn of 1931, this reducing substance with the molecular formula C6H8O6, which he named hexuronic acid, was unequivocally
proven in experimentation as the powerful anti-scorbutic substance, and that the anti-
scorbutic activity of plant juices corresponded to their hexuronic acid content. About the
same time, the Americans Charles Glen King and William A.Waugh also reported
crystals obtained from lemon juice, which were actively anti-scorbutic and resembled
hexuronic acid. In 1932, Albert Von Szent-Györgyi and British chemist Sir Walter
Norman Haworth subsequently renamed hexuronic acid as Ascorbic acid. In 1933, the
main features of the constitution of ascorbic acid and its formula as a lactone of 2-keto-L-
gulonic acid, capable of reacting in various tautomeric forms, was first announced from
the University of Birmingham. At about the same time, the Polish Tadeus Reichstein, in
6 Switzerland, as well as Haworth’s group independently achieved the organic synthesis of vitamin C. The synthetic form of the vitamin was identical to the natural form and this made possible the cheap mass production of vitamin C. Three patent applications were filed in 1935 and the patents were granted in 1939 and 1940. Thus, the American biochemist and chemical engineer Dr. Irwin Stone obtained the first patents on an industrial application of ascorbic acid. Sir Walter Norman Haworth was awarded the
Noble Prize for chemistry largely for this contribution in 1937. Also, in 1937, Albert Von
Szent-Györgyi was awarded the Nobel Prize for the first isolation of vitamin C.82-111
In 1959, an American, J. J., Burns showed that the basic biochemical lesion in the few mammals susceptible to scurvy was primarily due to their inability to produce the active enzyme, L-gulonolactone oxidase which is the last of the four enzymes involved in the mammalian conversion of blood glucose to ascorbic acid, in the liver. According to
Dr. Hickey of Manchester University, humans carry a mutated and ineffective form of the enzyme. This was 51 years after Sir Archibald Garrod’s famous lecture pointed to the lack of an enzyme as the reason for scurvy. Up until 1965, it was assumed that all primates were unable to produce their own ascorbic acid and were as a result susceptible to scurvy. Then it was suggested by Dr. Irwin Stone that the whole order of primates should be examined for the presence of L-gulonolactone oxidase in their livers to determine in which primate ancestor of man this important enzyme system was lost. This challenge was picked up, tested and reported from Harvard University in 1966 by O.,
Eliott and 3 years later by the Yerkes Primate Research Center, wherein it was indicated that members of the suborder Anthropoidea showed an inactive form of the enzyme, L- gulonolactone oxidase, in their liver. As a result of these evolutionary studies, Dr. Irwin
7 Stone’s research on the genetics of scurvy had progressed to a point where it could then be said that scurvy was not a dietary disorder, but rather was a potentially lethal problem
in medical genetics that was due to an ineffective gene, which produces an inactive
enzyme. Therefore, present day humans still suffer from a mammalian inborn error of
carbohydrate metabolism indigenous to the liver. He produced four papers by 1966
describing a human birth defect existing in 100% of the population due to a defective
gene. The potentially fatal genetic liver enzyme disease, which makes it necessary for
man to obtain ascorbic acid from exogenous sources was named “Hypoascorbemia”, and
stated as the cause of scurvy.82-111 Stone profoundly believed in the distinctive healing
qualities of vitamin C and became convinced of its effectiveness when he and his wife
had an accident involving a head on collision with a drunk driver and used large doses of
vitamin C in their speedy and remarkable recovery. In 1968, the American and two-time
Nobel laureate Linus Pauling, who was introduced to vitamin C by Dr. Irwin Stone,
indicated that this evolutionary mutation may have had survival values at the time simply
because it freed the biochemical machinery (glucose-consumption) for other purposes
and conserved energy. Vitamin C is a hexose derivative, similar in structure to the six-
carbon sugar glucose. Pauling was initially skeptical of Dr. Irwin Stone’s ideas but was
intrigued by Stone’s theory regarding genetic deficiencies and genetic mutation. Pauling
later decided to follow Dr. Stone’s advice by taking 3 grams of vitamin C daily for 3
years. Pauling soon noted that his sense of wellness improved and he was not
experiencing the dreaded cold that plagued him for 40 years. He later described vitamin
C as an essential nutrient in the maintenance of a healthy immune system for humans
(optimum intake of about 2.3 to 9.5 grams per day) in early 1970. Linus Pauling
8 concluded that the intake of vitamin C could improve, as well as extend life expectancy, and therefore went forward to advocate its uses for various therapeutic uses for the remaining 30 years of his life.82-111
In the present day civilization, ascorbic acid is less known as the anti-scorbutic factor used for many centuries to cure the variety of clinical symptoms known as scurvy, as this pathological state in no longer very common. L-ascorbic acid is largely known as an antioxidant which efficiently scavenges toxic free radicals and other reactive oxygen species (ROS) formed in cell metabolism. ROS are associated with several forms of tissue oxidative damage by free radicals and oxidants that are involved in the development and exacerbation of a multitude of chronic diseases. A complete list of vitamin C uses can be found in the Clinical Guide to the Use of Vitamin C, edited by
Lendon H. Smith, M.D., Life Sciences Press, Tacoma, WA (1988). Some of these uses includes its benefit in combating (a) Allergic Rhinitis, (b) Alzheimer’s disease, (c)
Asthma, (d) Atherosclerosis, (e) Breast Cancer, (f) Burns, (g) Cataracts, (h) Cervical
Dysplasia, (i) Common-Cold, (j) Diabetes-Mellitus, (k) Eczema (l) Gallbladder disease
(m) Glaucoma, (n) Hypertension, (o) Hypercholesterolemia, (p) Macular-Degeneration,
(q) Myocardial-Infarction, (r) Obesity, (s) Osteoarthritis, (t) Pancreatitis, (u) Parkinson’s disease, (v) Photodermatitis, (w) Skin-Cancer, (x), Stroke, (y) Uveitis, and (z) Wounds.
Thus, this makes ascorbic acid ever more important than when scurvy was a major menace confronting human health. Furthermore, ascorbic acid is widely used in the food industry as a common additive to foods in order to improve the taste and as well as to restore the vitamin C loss due to processing and storage. It is used as a preservative to prevent oxidation or serve as a stabilizer in various food products and beverages. It is also
9 used in bread baking, brewing, wine making, and freezing of fruits. In addition, ascorbic
acid and some of its derivatives have important usage in industrial processes such as
polymerization reactions, photographic development and printing, and metal technology.
Most of these modern applications of ascorbic acid make use of the reducing properties
of the molecule.82-111
2.2 Sources of L-Ascorbic Acid
The main sources of L-ascorbic acid for humans are from plants and animals with
indigenous biosynthetic capabilities of producing L-ascorbic acid. The ubiquitousness
of L-ascorbic acid throughout the human body emphasizes its daily requirement and
vitality as a nutrient for healthy maintenance.112-114 Its biological half-life in humans
is 14-40 days after normal intake and a vitamin-C-free diet in a human develops
scurvy in about 3-4 months.115 It is required in the diet by only a few species of
animals (Figure 1): man, guinea pig, red-vented barbul, an Indian fruit-eating bat and
some related species of passeriform birds, and most but not all primates. Many
invertebrates and teleost fish are incapable of synthesizing vitamin C. L-Ascorbic
acid is also an essential nutrient for rainbow trout, carp, Coho salmon, and some
insects.
The vast majority of species of plants and animals are known to synthesize their own
vitamin C. A majority of vertebrates such as amphibians, reptiles, birds, and
mammals are able to synthesize L-ascorbic acid. Molecules similar to ascorbic acid
are made by some fungi but not by bacteria.
10 Figure 1 Depiction of L-Ascorbic Acid Biosynthesizing Abilities of Various Species of Animals in Relation to their Phylogeny
Adapted from Seib & Tolbert Am. Chem. Soc. Adv. Chem. Ser. 200; Washington, D. C., 1982 (Ref. 35)
All algal classes can synthesize vitamin C from glucose or other sugars. All higher
plant species can synthesize vitamin C and thus make it prevalent in the surrounding
food sources. For example, large concentrations of vitamin C are found in fruits such
as oranges, grapefruits, tangerines, lemons, limes, papaya, strawberries, and
11 cantaloupe. It is also found in the white linings of these fruits and other plants. Also,
many vegetables are known to pack in vitamin C and these include tomatoes,
broccoli, green and red bell peppers, raw lettuce and other leafy greens. A complete
listing of every food containing vitamin C according to the USDA food database is
available through The Vitamin C Foundation. 86
2.3 Tissue Distribution of L-Ascorbic acid
Tissue distribution of L-ascorbic acid (Table 1) offers a clue to its metabolic role
since its concentration in various tissues is found to be tightly controlled.114, 116-117 Its bio- availability status in the body is found to influence many metabolic systems such as iron and copper balance, fatty acid transport, hemostasis, endocrine function, control of blood pressure, collagen synthesis, peptide metabolism, the immune system, endothelial function, steroid metabolism and lipid metabolism.115 The biochemical mechanism of L-
ascorbic acid in each of the different systems appears to be related to its antioxidant
properties.35, 115-116 The plasma L-ascorbic acid concentration of a healthy person is 8-14
mg/L and it contributes around 10-15% of the total antioxidant strength of fasting
plasma.115 Some monocytes and adrenal cells such as the adrenal glands, corpus luteum,
pituitary, thymus and retina have L-ascorbic acid concentrations up to 100-fold that of the
plasma.115 The salivary gland, pancreas, leukocytes, kidney, thyroid, liver, small
intestinal mucosa, lymph glands, testicle, lung, spleen, ovary and the brain have in excess
of about 10-50 times that of the plasma L-ascorbic concentration.115
12 Table 1 Approximate Levels of L-Ascorbic Acid in Tissues
Human Rat Tissues (mg / 100mg tissue) (mg / 100mg tissue)
Adrenal glands 30-40 280-400 Brain 13-15 35 Eye lens 25-31 8-10 Heart muscle 5-15 5-10 Kidney 5-15 15-20 Liver 10-16 25-40 Lungs 7 20-40 Pancreas 10-16 - Pituitary gland 40-50 100-130 Plasma 0.4-1.0 1.6 Saliva 0.07-0.09 - Skeletal muscle 3-5 5 Spleen 10-15 40-50 Testes 3 25-30 Thymus 10-15 40 Thyroid 2 22
Adapted from Levine, M.; Mortia, K. In Vitamins and Hormones; Aurbach, G. D.; McCormick, D. B., Eds.; Vol. 42, academic Press Inc.: New York, NY., 1985, pp. 1-64.
The cardiac and smooth muscles, erythrocytes, and the skeletal muscle have
concentrations about 10 times that of plasma. The high level of L-ascorbic acid found in vital organs suggests that these regions have elevated anti-oxidation requirements and
thus serve to protect them against dietary deficiencies as well as the maintenance of their
structural integrity through collagen synthesis. Thus, they are enabled in performing their
specialized functions.
13 2.4 Biosynthesis of L-Ascorbic Acid in Animals
The biosynthesis of L-ascorbic acid in animals (Scheme 1) is integrated with the
glucuronic acid metabolic pathway. This metabolic pathway is involved in the metabolism of sugars under both normal and disease states and is regulated by the body’s
physiological functions.35, 115,118 It is an important pathway for major detoxification processes in the body and the activities of the synthesizing enzymes vary from species to
species.35, 115,118 The well-known evolutionary distribution of L-ascorbic acid
biosynthesis suggests that it started in the kidney of lower vertebrates such as amphibians
and reptiles, then transferred to the liver of mammals, and eventually lost in primates,
fruit bats and guinea pigs.35, 115,118 Even in vertebrates capable of synthesizing L-ascorbic
acid, this biosynthesis only takes place in a few cell types. For mammals, these cells are
the hepatocytes, whereas in reptiles, amphibians and egg-laying mammals, the
biosynthesis takes place in the kidney cells. However, in birds with the exception of the
passeriforms, which are incapable of L-ascorbic acid biosynthesis, this biosynthesis is
known to take place in the kidney, liver or both.35, 115,118 Most of the research on ascorbic
acid synthesis in animals have been carried out using rats.35, 115,118
14 Scheme 1 Proposed Biosynthetic Pathway for L-Ascorbic Acid in Animals (adapted from Ref. 118, 119 & 121)
ATP ADP
D-Glc D-Glc-6-P D-Glc-1-P UDP-D-Glc UDP-D-GlcUA 1 2 3 4
5
L-GulA D-GlcUA D-GlcUA-1-P 7 6 8
OH HO H HO H O HO O O O HO HO 9 H H HO OH L-GL Cyt Cox Cyt Cred L-Ascorbic acid
Catalytic Step Enzyme Substrate
D-Glucose 1 Hexokinase
D-Glucose-6-phosphate 2 Phosphoglucomutase
D-Glucose-1-phosphate 3 UDP-D-Glucose pyrophosphorylase
UDP-D-Glucose dehydrogenase (EC UDP-D-Glucose 4 1.1.1.22) D-Glucuronate-1-phosphate UDP-D-Glucuronic acid 5 uridylytransferase (EC 2.7.7.44)
6 D-Glucurono kinase (Hydrolase) UDP-D-Glucuronic acid-1-phosphate
D-Glucuronate reductase D-Glucuronic acid 7 (EC 1.1.1.19) L-Galacturonic acid 8 Aldonolactonase (EC 3.1.1.17)
L-Gulono-1,4-lactone dehydrogenase L-Gulono-1,4-lactone 9 (EC 1.1.3.8)
15 In 1960, the de novo biosynthesis of L-ascorbic acid in animals was established and
known for utilizing intermediates of the D-glucuronic acid (hexuronic acid) pathway.115,
118 In vivo, the hexose skeleton of L-ascorbic acid originated from D-glucose that is
mainly derived from the breakdown of glycogen.115, 118 This in vivo biosynthesis takes
place either in the liver or kidney, which are both glycogen-storing organs.115, 118
The deficiency in the biosynthesis of L-ascorbic acid found in some animals and humans has been localized to a lack of the terminal flavor-enzyme, L-gulono-1,4-lactone oxidase (GuLO, EC 1.1.3.8), which completely blocks the liver production of L-ascorbic acid in humans.35,115,118 This oxidizing enzyme is required in the last step of the
conversion of L-gulono-γ-lactone to 2-oxo-L-gulono-γ-lactone, which is a tautomer of L-
ascorbic acid that is spontaneously transformed into vitamin C. Although cloning and
chromosomal mapping studies have indicated that the gene encoding L-gulono-1,4-
lactone oxidase was found to be present in the human genome, nonetheless it is not
expressed due to the accumulation of a number of promoter defective mutations which
are without any selective pressure since it presumably ceased to function during evolution.35,115,118 This terminal enzyme, L-gulono-1,4-lactone oxidase, is found not to
be 100% specific for L-gulono-γ-lactone as substrate, but also known to catalyze the
oxidation of related aldono-lactones such as D-altrono-γ-lactone(16%), D-manono-γ- lactone (64%) and L-galactono-γ-lactone (70-90%), which is the direct precursor of L- ascorbic aid biosynthesis in plants.115,118 Studies with radioactive labeling techniques
have indicated that D-glucose is converted into L-ascorbic acid sequentially via D-
glucuronic acid, L-gulonic acid, L-gulono-γ-lactone and 2-keto-L-gulono-γ-lacone (2- oxo-L-gulono-γ-lactone) as intermediates.115, 118 Radiotracer studies with D-[6-14C]-
16 glucose, D-[2-14C]-glucose and D-[1-14C]-glucose indicated that the C1 carbonyl group
of L-ascorbic acid is derived from the oxidation of the C6 carbon rather than the C1 of D-
glucose115, 118and that this important reduction and oxidation conversion of the C1 and C6
respectively, takes place between D-glucuronic acid and L-gulonic acid, while the D-
glucose chain remains intact.115, 118 Consequently, L-ascorbic acid biosynthesis in animals
is known to follow a non-inversion type conversion of derivatives of D-glucose. Some prokaryotic organisms that contain the enzyme, L-gulono-γ-lactone dehydrogenase, which is able to synthesize L-ascorbic acid or one of its isomers have been isolated and characterized.115, 118 However, the chemical and physical properties of this enzyme are
entirely different from those of eukaryotic organisms. Both in vivo and in vitro studies
have established that L-ascorbic acid biosynthesis in animals is controlled by a direct
feedback mechanism and that the concentration of L-ascorbic acid in the cell culture
medium or in the blood helps to regulate the amount of L-ascorbic acid synthesized in the
liver or in hepatocytes of rat or mice.115, 118 For example, in hepatocytes, L-ascorbic acid
synthesis is stimulated by glucagon, dibutyryl, cyclic adenosine monophosphate (cAMP),
phenylephrine, vasopressin and okadaic acid.115, 118 The hepatic L-ascorbic acid
biosynthesis in mice has also been shown to be stimulated by enhanced
glycogenolysis.115, 118 In rats, uridine diphosphate (UDP) glucuronosyltransferase gene
expression is shown to be involved in the stimulation of L-ascorbic acid biosynthesis by
exposure to xenobiotic compounds such as 3,4-benzpyrene, 3-methylcholanthrene and
sodium Phenobarbital.115, 118 Xenobiotic compounds are known to induce biosynthesis of enzymes involved in the glucuronic acid pathway which is a part of the drug detoxification process in the body.115, 118 The rate of in vitro L-ascorbic acid biosynthesis
17 shows close correlation with the glucose release by hepatocytes.115, 118 In mice, the
injection of glucagon increases L-ascorbic acid concentrations in the liver and plasma
membrane.115, 118 On the other hand, the biosynthesis of L-ascorbic acid is impaired by the deficiency of vitamin A, vitamin E and biotin.
2.5 Biosynthesis of L-Ascorbic Acid in Plants
The biosynthesis of L-ascorbic acid in plants has not been clearly and easily
established when compared to its biosynthesis in animals. However, recent advances in
the understanding of L-ascorbic acid biosynthesis in plants have helped to resolve many
of the contradictions of the past decades. There is now a general consensus that the biosynthetic pathway, which proceeds via GDP-D-mannose and GDP-L-galactose35, 118-
121 as proposed by the Smirnoff group,120 represents the major L-ascorbic biosynthetic
pathway in plants (Scheme 2). This pathway is known today as the Smirnoff-Wheeler L-
ascorbic acid biosynthetic pathway. The first part of the pathway is also utilized for the
synthesis of cell wall polysaccharide precursors, while the later steps following GDP-L-
galactose are solely dedicated to plant biosynthesis of L-ascorbic acid. The earlier
observation on the conversion of L-galactono-γ-lactone to L-ascorbic acid also applied in
this case since, interestingly, this pathway also utilizes the same terminal enzyme L-
galactono-γ-lactone dehyrogenase, just as in the route originally proposed by Isherwood
et al.122
18 Scheme 2 The Smirnoff-Wheeler Biosynthetic Pathway for L-Ascorbic Acid in Plants (adapted from Ref. 118, 119 & 121)
ATP ADP D-Glc D-Glc-6-P D-Fru-6-P D-Man-6-P 1 2 3 4 PPi GTP GDP-L-Gal GDP-Man D-Man-1-P 6 5 GMP 7 OH NAD NADH HO O 8 H O L-Gal-1-P L-Gal HO 9 H Pi HO L-GL
HO H 10 Cyt C HO O O ox H Cyt Cred HO OH L-Asc
Catalytic Step Enzyme Substrate
D-Glucose 1 Hexokinase (E.C. 2.7.1.1)
Phosphoglucose isomerase (E.C. D-Glucose-6-phosphate 2 5.3.1.9) Phosphomannose isomerase (E.C. D-Fructose-6-phosphate 3 5.3.1.8) Phosphomannose mutase (E.C. D-Mannose-6-phosphate 4 5.4.2.8) GDP-Mannose pyrophoshorylase D-Mannose-1-phosphate 5 (E.C. 2.7.7.22) GDP-Mannose-3,5-epimerase GDP-D-Mannose 6 (E.C. 5.1.3.18) GDP-L-Galactose GDP-L-Galactose 7 pyrophosphatase L-Galactose-1-phosphate L-Galactose-1-phosphate 8 phosphatase L-Galactose 9 L-Galactose dehydrogenase
L-Galactono-1,4-lactone L-Galactono-1,4-lactone 10 dehydrogenase (E.C. 1.3.2.3)
19 The conversion of D-glucose to L-ascorbic acid in this pathway occurs without the earlier
proposed inversion of the hexose carbon skeleton, which suggested a site-specific
epimerization of the D-glucose carbon skeleton that causes the conversion from D to L
configuration. Therefore, this Smirnoff-Wheeler biosynthetic pathway for L-Ascorbic
Acid in Plants reconciles the crucial radio-labeling evidence from earlier works by
Loewus group.123-124 They partially purified a new L-galactose-dehydrogenase enzyme
from pea and Arabidopsis thaliana and established L-galactose as an effective precursor of L-ascorbic acid in vivo. This enzyme is known to catalyze the NAD-dependent
oxidation of the C1 of L-galactose to give L-galactono-γ-lactone with a Km of 0.3 mM for
L-galactose. This same enzyme was also able to slowly oxidize L-sorbosone to L-
ascorbic acid at a very low Km value, which may perhaps explain earlier literature reports.118 GDP-L-galactose, which is synthesized from the double epimerisation of
GDP-D-mannose, is incorporated as a minor component of certain cell wall
polysaccharides.118 The reaction is catalyzed by a poorly characterized enzyme isolated
from Chlorella pyrenoidosa and flax, which is known as GDP-D-mannose-3,5-
epimerase.118 Nonetheless, the enzyme responsible for converting GDP-L-galactose to L-
galactose remains unidentified in plants. On the other hand, it has been reported that
incubations with radio-labeled GDP-D-mannose in vitro resulted in the incorporation of
radio-labels into L-galactono-γ-lactone.118 Furthermore, additional genetic data in support
of this pathway is beginning to emerge from the characterization of the L-ascorbic-acid-
deficient Arabidopsis mutants.118 The locus of one of these mutants has recently been
shown to be D-mannose pyro-phosphorylase. Also, independent work on the anti-sense
inhibition of this enzyme in potato was reported to have produced plants with foliar L-
20 ascorbic acid levels of about 44% to 72% of wild type, and with a 30% to 50% reduction in their leaf cell wall mannose content.118 And upon transfer to soil, these plants
expressed developmental changes leading to early senescence.118 Therefore, the
importance of this new Smirnoff-Wheeler L-Ascorbic Acid biosynthetic pathway in
plants is that it integrates L-ascorbic acid biosynthesis into the pathways for central
carbohydrate metabolism and provides connections to protein glycosylation and
polysaccharide biosynthesis.118 Nevertheless, some important questions remained
unanswered such as the earlier reports on the in vivo conversion of uronic acid derivatives
for example, D-glucuronic acid, its lactone, D-glucuronolactone, and as D-galacturonic
acid methyl ester, which are found to be converted directly to L-ascorbic acid. These
conversions are found to occur without disruption of the carbon skeleton and with slight
redistribution of the radio-labels.118 The draw-back is that there are few available data on
the enzymes catalyzing these reactions and thus researchers are still uncertain of its significance in L-ascorbic acid biosynthesis. It is possible that L-ascorbic biosynthesis from these compounds may only be significant under certain cellular circumstances or in specific tissue types. However, what is known is that D-glucuronic acid and D- galacturonic acid are major components of plant non-cellulose type cell wall polysaccharides and their conversion to L-ascorbic acid might in part represent a mechanism to salvage carbon fragments arising from the breakdown of the cell walls, such as those that take place during growth, cell expansion, abscission, pollen grain maturation, fruit ripening and softening.118
21 2.6 Commercial Scale Synthesis of L-Ascorbic acid
At present, the bulk of commercially manufactured L-ascorbic acid is synthesized
via the seven-step Reichstein process (Scheme 3), which was developed soon after the
discovery of vitamin C by Albert Von Szent-Györgyi in 1928. The current world
production of L-ascorbic acid is estimated at 80,000 tons per year with a global market in
excess of US $600 million and with an annual growth rate of 3-4%.125-126 This enormous
demand for L-ascorbic acid is driven by its various uses in manufacturing, agricultural,
health and pharmaceutical industries. For example, (1) approximately 50% of the synthetic L-ascorbic acid is used in vitamin supplements and in pharmaceutical preparations such as in the making of ointments for the treatment of burns; (2) There is a rapidly growing market in cosmetic products which use L-ascorbic acid as an additive, due to its anti-oxidant properties and its potential to stimulate collagen production;35 (3)
This antioxidant properties are also exploited in food processing and beverage manufacturing, to protect against pigment discoloration and enzymatic browning. This helps to preserve flavor, aroma, and enhance or protect the nutrient content;35 (4) Farmers
frequently use L-ascorbic-acid-supplemented feeds to augment its bio-availability in
livestock for optimum growth and health.35
22 Scheme 3 The Reichstein Process for L-Ascorbic Acid Manufacture (adapted from Ref. 127)
D-Glucose 80-125 atm 2+ Ni 140-150 oC D-Sorbitol
Fermentation
L-Sorbose
CH3COCH3 Conc. H2SO4 Diacetone-L-Sorbose
Pd2+ 2-Keto-L-Gulonic Acid
HCl CH3OH
2-Keto-L-Gulonic Acid Methylester
o C2H5OH 100 C L-Ascorbic Acid
The Reichstein process uses D-glucose as the starting material and involves six chemical steps and one fermentation step for the oxidation of D-sorbitol to L-sorbose with an overall yield of 50%.125 The synthetic process is based on chemical methods and bears no relationship to the biochemical pathway used by L-ascorbic-acid-biosynthesizing organisms. In spite of its many years in development, the Reichstein process is still highly energy consuming and requires high temperatures and/or pressure for many of the steps. These and other economic factors have generated a substantial interest in the manufacturing of the Reichstein intermediates towards the synthesis of L-ascorbic acid in
23 a more economical and efficient manner. The more recent revelation of the plant biosynthesis pathway and genomic advancement have broadened new opportunities to explore recent innovations in technologies such as fermentation processes, cell-free bio- catalytic systems, biochemistry and recombinant DNA technology for a more efficient commercial synthesis of L-ascorbic acid via the Reichstein intermediates.127 Such methods involve the use of genetically engineered prokaryotes for the large scale synthesis of L-ascorbic acid. The two most commercially advanced methods are the oxidation of D-glucose to 2-keto-L-gulonate (2-KLG) via D-gluconate, 2-keto-D- gluconate and 2,5-diketo-D-gluconate [2,5-DKG pathway, (Scheme 4)] and the oxidation of D-sorbitol or L-sorbose to 2-keto-L-gulonate via the intermediate L-sorbosone
[sorbitol pathway, (Scheme 4)]. The first synthesis of L-ascorbic acid from a non- carbohydrate source was successfully attempted using enantiopure cis-1,2- dihydrocatechol. This precursor was obtained from microbial oxidation of chlorobenzene and converted via 3,5-O-benzylidene-L-gulonolactone into L-ascorbic acid.128 While this synthetic method may not yet be economically suitable for commercial scale synthesis of
L-ascorbic acid, however it offers a reaction sequence open to the preparation of labeled
L-ascorbic and its derivatives that could then be used in probing the in vivo functions of
L-ascorbic acid.
24 Scheme 4 Microbial-Engineered Pathway for L-Ascorbic Acid Manufacture (adapted from Ref. 127)
2,5-DiKeto-D-Gluconic Acid Pathway D-Sorbitol Pathway
COOH CHO CH2OH H OH H OH H OH HO H HO H HO H H OH H OH H OH H OH Glucose dehydrogenase H OH Hydrogenation H OH
CH2OH CH2OH CH2OH D-Gluconic acid D-Glucose D-Sorbitol
Gluconate dehydrogenase D-Sorbitol dehydrogenase
COOH CH2OH O O HO H HO H H OH H OH H OH HO H
CH2OH CH2OH
2-Keto-D-Gluconic acid L-Sorbose
2-Keto-D-gluconate dehydrogenase L-Sorbose dehydrogenase
COOH COOH CHO O O O HO H HO H HO H 2,5-DiKeto-D-gluconate- L-Sorbose- H OH dehydrogenase H OH dehydrogenase H OH O HO H HO H
CH2OH CH2OH CH2OH
2,5-Diketo-D-Gluconic acid 2-Keto-L-Gulonic acid L-Sorbosone
Esterification Lactonisation
C=O OH OH O H HO H
CH2OH
L-Ascorbic Acid
25 2.7 Biological Functions of L-Ascorbic Acid
There are three main types of biological activity distinctive to L-ascorbic acid in plants and animals. These are (1) its function as an enzyme co-factor; (2) as a direct physiological radical scavenger and finally; (3) as a donor/acceptor in electron transport in both plasma membrane and chloroplasts.35, 116, 118
2.7.1 L-Ascorbic Acid as an Enzyme Cofactor
L-ascorbic acid is involved in the modulation of a number of important enzymatic reactions such as in the metabolism of several amino acids which lead to the formation of hydroxyproline, hydroxylysine, norepinephrine, serotonin, carnitine and homogenistic acid. It has also been found to be essential for the normal functioning of the osteoblasts, fibroblasts, adrenal hormones and carnitine biosynthesis.35, 115, 118 Carnitine is a molecule present in the liver, heart and skeletal muscles, which is responsible for the transport of energy-rich activated long-chain fatty acids from the cytoplasm across the inner mitochondrial membrane to the matrix side, where they are catabolized to acetates.115, 118
Carnitine is synthesized from methionine and lysine by two hydroxylases through a series of reactions that require ferrous iron and L-ascorbic acid for optimum activity. Therefore, the deficiency of L-ascorbic acid is found to cause a decrease in both the rate of carnitine biosynthesis and the efficiency of carnitine renal re-absorption, and increase in the urinary carnitine excretion; these effects are linked to the buildup of triglycerides in blood, physical fatigue and lassitude in scurvy patients.115, 118 A list of enzymes that
26 requires L-ascorbic acid for optimal function is shown in Table 2. These enzymes are typically mono or di-oxygenases that contain transition metals such as iron or copper at their active sites and require L-ascorbic acid for optimum activity.35, 115, 118 The role of L- ascorbic acid in these enzymes is to maintain the transition metal ion centers in the reduced form, which is required for the optimum activity of the systems.35, 115, 118 For example, many of the symptoms of scurvy in animals, particularly those having to do with the connective tissues defects are traced back to the biochemical role of L-ascorbic acid as a cofactor for the two mixed-function oxidases, which are prolyl and lysyl hydroxylase enzymes involved in the formation of both hydroxyproline and hydroxylysine, which are two important components of collagens and the fibrous connective tissues in animals. Collagen is the principal components of tendons, ligaments, skin, bone, teeth, cartilage, heart valves, intervertebral disks, cornea, eye lens and the ground substances between cells. When collagen is synthesized, proline and lysine are post-translationally hydroxylated on the growing peptide chain.
Hydroxyproline and hydroxylysine are required for the formation of a stable extracellular matrix and cross-links in the fiber.
27 Table 2 List of Enzymes Requiring L-Ascorbic Acid as a Cofactor or as a Modulator of Activity (adapted from Ref. 118)
Metal Ion Enzyme Physiological Role Enzymatic Activity Centre
1-Aminocyclopropane-1- Ethylene (plant hormone) Oxidation of 1-Aminocyclopropane to Iron carboxylate Oxidase biosynthesis ethylene and cyanoformic acid Cholesterol 7-alpha Cholesterol catabolism; bile acid Hydroxylation of Cholesterol - monooxygenase E.C. 1.14.13.17 synthesis (animals) Catechol-O-methyl transferase E.C. Adrenaline (epinephrine) inactivation Increased levels of adrenaline - 2.1.1.6 (animals) (epinephrine) Dopamine-β- monooxygenase Noradrenaline (norepinephrine) β-hydroxylation of dopamine Copper E.C. 1.14.17.1 synthesis Deacetoxycephalosporin C Antiobiotic metabolism (fungi) Penicillin N to deacetylcephalosporin Iron synthetase γ-Butyrobetaine-2-oxoglutarate-4- Hydroxylation of butyrobetaine to Carnitine biosynthesis Iron dioxygenase E.C. 1.14.11.1 carnitine Gibberellin-3-β-dioxygenase Gibberin (plant hormone) C oxidation decarboxylation and 20 Iron E.C. 1.14.11.15 biosynthesis activation of gibberellins Decarboxylation and hydroxylation of 4-Hydroxylphenylpyruvate Tyrosine metabolism 4-hydroxyphenyl pyruvic acid to Iron dioxygenase E.C. 1.13.11.27 homogenistic acid Lysine hydroxylase Collagen biosynthesis (animals) & Hydroxylation of Lysine Iron E.C. 1.14.11.4 Extensin biosynthesis (plants) Mitochondrial glycerol-3- NAD(P) H and ATP production; aid phosphate dehydrogenase Dehydrogenation of triose phosphate Iron in insulin release E.C. 1.1.99.5 Peptidyl glycine α-amidating Peptide amidation in peptide hormone C-terminal glycine amidation Copper monooxygenase E.C. 1.14.17.3 metabolism Procollagen proline 2-oxoglutarate- Procollagen biosynthesis (animals) Hydroxylation of proline (3- 3-dioxygenase Iron Extensin biosynthesis (plants) hydroxylating) E.C. 1.14.11.7 Proline hydroxylase Hydroxylation of proline (4- Procollagen synthesis (animals) Iron E.C. 1.14.11.2 hydroxylating) Pyrimidine deoxynucleoside 2’- Pyrimidine metabolism (fungi) Deoxyuridine to uridine Iron dioxygenase E.C. 1.14.11.3 Thioglucoside glucohydrolase Catabolism of glucosinolates (plants) Hydrolysis of S-glucosides - E.C. 3.2.3.1 Thymine dioxygenase Pyrimidine metabolism (fungi) 7-Hydroxylation of thymine Iron E.C. 1.14.11.6 Trimethyllysine 2-oxoglutarate Carnitine biosynthesis Hydroxylation of trimethyl lysine Iron dioxygenase E.C. 1.14.11.8 Zeaxanthin biosynthesis and the De-epoxidation of violaxanthin and Violaxanthin de-epoxidase - xanthophylls cycle (plants) antheroxanthin
28 L-ascorbic acid acts a physiological electron donor to the ferric and cupric ions at the metal centers of these enzymes, thus reducing them to their activated reduced states, which is essential for the reactions to proceed. Some collagens that are biosynthesized in the absence of L-ascorbic acid, such as what occurs in scurvy, are known to form abnormal fibers, resulting in skin lesions, blood-vessel fragility, etc.35, 115,118 In plants, the direct involvement of L-ascorbic acid in the biosynthesis of plant hydroxyproline-rich proteins118 has implications for cell expansion and cell division.118 High levels of hydroxyproline-rich glycoproteins such as the extensins found in the cell wall118 are developmentally regulated and are involved in the cross-linking of the cell wall in response to injury. Also, extensin genes are induced in response to wounding and pathogenic attacks.118 Furthermore, in the biosynthesis of a variety of neurotransmitters and hormones35, 115, 118 in animals, L-ascorbic acid is an important factor in many of the hydroxylation and decarboxylation processes involved in these metabolic pathways. For example, L-ascorbic acid is important for the initial hydroxylation step in the synthesis of serotonin, a neurotransmitter and vasoconstrictor, which is catalyzed by tryptophan hydroxylase. This step involves the hydroxylation and decarboxylation of tryptophan and
L-ascorbic acid is able to convert dihydrobiopterin (oxidized form) to tetrahydrobiopterin
(reduced form), which is the co-substrate for this hydroxylase enzyme.115, 118 Animals with deficiency in L-ascorbic acid are unable to catabolize tyrosine to fumaric and acetoacetic acid via homogenistic acid.115, 118 Also, tyrosine is metabolized in the presence of L-ascorbic acid to catecholamines by hydroxylation and decarboxylation to produce dopamine, norepinephrine, epinephrine, and adrenocrome. L-ascorbic acid is
29 directly involved as an electron donor to dopamine-β-monooxygenase (DβM) reaction for the conversion of dopamine to norepinephrine (Scheme 5).
Scheme 5 L-Ascorbic Acid (Exogenous Electron Donor) in DβM Enzymatic Reaction
Dopamine O2 H2O R-Norepinephrine
HO H NH2 2H + 2e- NH2 HO HO OH OH
II I DßM-E CU 2 + 2ASC DßM-E CU 2 + 2Semidehydro-ASC
2Semidehydro-ASC ASC + Dehydro-ASC
Catecholamine biosynthesis occurs in the adrenal glands and brain, both with relatively large amounts of L-ascorbic acid. L-ascorbic acid also protects catecholamines by direct chemical interactions and elimination of adrenochrome, a toxic product of catecholamine oxidation, which has been linked to certain mental diseases.115 There are complex interactions among catecholamines and their receptors with L-ascorbic acid to protect them from oxidative damage. Other enzymatic systems responsible for neurotransmitter and hormone synthesis, and dependent on the presence of oxygen and L-ascorbic acid, are the copper-containing peptidyl glycine amidating monooxygenases, which are found in the skin, atrium, adrenal and pituitary glands.35, 115-116, 118 The microsomal enzymatic system containing cytochrome P450-hydroxylases requires L-ascorbic acid for the hydroxylation reaction involved in the stepwise conversion of cholesterol to bile acid via
30 7α-hydroxycholesterol.35, 115, 118 In L-ascorbic acid deficient animals including humans, impaired cholesterol transformation to bile acids leads to cholesterol accumulation in the blood and liver, atherosclerotic changes in coronary arteries, and formation of cholesterol gallstones. Therefore, administration of L-ascorbic acid helps to lower the plasma chlolesterol concentration. L-ascorbic acid is also essential for the oxidation and decarboxylation of fatty acids in lipid metabolism. Animals with L-ascorbic acid- deficiency exhibit high levels of plasma triglycerides with a decrease in post-heparin plasma lipolytic activity and the half-life of plasma triglycerides increases, thereby causing triglyceride accumulation in the liver and arteries.35, 115, 118
2.7.2 L-Ascorbic Acid in Electron Transport
The biochemical and physiological functions of L-ascorbic acid primarily depend on its reducing properties and its role as an electron carrier.35, 115 L-ascorbic acid and its single-electron oxidized product, semidehydro-L-ascorbate functions as a cycling redox couple in various electron transport reactions and changes the activities of cytochromes, the electron membrane-protein carriers. Several ascorbate oxidoreductases have been identified and are involved in the electron transport reactions with a cytochrome b protein.35, 115 For instance, L-ascorbic acid is known as a major electron donor for a trans-
35, 115 membrane oxidoreductase of human erythrocytes. Cytochrome b561, an electron channel membrane-protein found in secretory and synaptic vesicles, catalyzes the trans- membrane electron transport.
31 Figure 2 Cytochrome b561 in Trans-Membrane Electron Transport
Adapted from Stewart & Klinman, Annu. Rev. Biochem., 1988, Vol. 57, 551-592
The transported electrons mediate equilibration of the L-ascorbate/semidehydro-L- ascorbate redox couple inside the secretory vesicles with those present in the cytoplasm.
The role of cytochrome b561 (Figure 2) is to regenerate L-ascorbic acid inside the vesicle for use by intravesicular monooxygenases such as dopamine β-monooxygenase and peptidylglycine α-amidating mooxygenase.35, 115, 118 The cytochrome is reduced by a single reducing equivalent donated by L-ascorbic acid in the cytosol and is oxidized by semidehydro-L-ascorbate in the granule matrix, thereby maintaining a redox equilibrium
32 between cytoplasmic and intravesicular pools of ascorbate and semidehydro-L- ascorbate.35,115-116,118
2.7.3 L-Ascorbic Acid as an Antioxidant in Biological Systems
Since oxygen is required for cell viability in both plant and animal systems, it is essential that a mechanism be available to control the reactive oxygen species (ROS) generated during cellular metabolism and from exogenous sources and environmental chemicals. L-ascorbic acid interacts enzymatically and non-enzymatically with ROS and their derivatives to neutralize their cellular damaging effects. Radical reactions are initiated by ROS mainly produced as side products from the mitochondria in animals and choloroplast in plants, where cellular energy is produced by the reaction of an oxygen molecule with 4 electrons and 4 protons resulting in the formation of water (Scheme 6).
These ROS such as superoxide and especially hydrogen peroxide undergo the so called
Fenton reaction in the presence of transition metal ions, especially Fe (II) to produce the hydroxyl radical, an extremely reactive radical.
Scheme 6 Four-Electron Reduction Process of Oxygen to Water
+ H O H +2H +2H+ O O O O H O O H e e 2e H O H - O2 O2 H2O2 2H2O
33 The hydroxyl radicals (HO.) undergo facile radical reactions with susceptible cellular components such as proteins, DNA, lipids and membrane lipids.129 For example, membrane lipids possess allylic hydrocarbon chains that can undergo facile reactions with hydroxyl radicals.129 The resulting carbon-centered radicals react with oxygen rapidly at a diffusion-controlled rate to form alkyl peroxy radicals (LOO.). This alkyl peroxy radicals abstract a hydrogen atom from lipids to generate LOOH.129 LOOH has a sufficient life-time to migrate and finally generate reactive radicals by reacting with metal ions to damage other cellular components in addition to the membrane. Therefore LOOH is capable of causing extensive tissue damage that may lead to cell death due to its radical effect called oxidative stress. L-ascorbic acid and glutathione, another water-soluble reducing agent, function together as antioxidants against oxidative stress and free radical damage in the body (Figure 3).129 Although, L-ascorbic acid cannot scavenge lipophilic radicals directly within the lipid compartment, it acts as a synergist with tocopherol for the reduction of lipid peroxide radicals. At the lipid-aqueous interphase, L-ascorbic acid interacts with the membrane-bound oxidized tocopherol radical to regenerate active reduced tocopherol for continued antioxidant functions.129 The biological importance of the antioxidant behavior of L-ascorbic acid is unlike other low-molecular-weight antioxidants (uric acid, carotenoids, flavonoids, α-tocopherol, etc.), in that it terminates the radical chain reactions and itself is transformed into non-toxic oxidized products, i.e., semidehydro-L-ascorbic acid radical and dehydro-L-ascorbic acid. Semidehydro-L- ascorbic acid radical, disproportinates back to L-ascorbic acid and dehydro-L-ascorbic acid (Figure 4).
34 Figure 3 Chemical Illustration of Radical Reactions in the Cell and Antioxidant Activities (adapted from Ref. 129)
Glucose Oxidation
NADP+ NADPH
GSSG Reductase
GSSG GSH
. GPX ASC ASC or DHASC
Membrane LOH LOOH . TOC TOC
Metal . . Ions LOOH LOO L + O2
Nucleic Acids Proteins Initiation . LH + X
Aldehydes Lipid Peroxidation
- H2O+ O2 H2O2 O2 Catalase, GSH PX SOD
2+ . Fe HO
Radical species in the cell (designated as X.) initiate radical chain reactions leading to oxidative stress. Thus, cell antioxidants (e.g. L-ascorbic acid, Asc.), antioxidant enzymes and glucose supplies reducing power to fight oxidative stress (adapted from Ref. 129).
35 Figure 4 L-Ascorbic Acid Redox System
HO OH HO OH O + - O - H , - e O H O +H+, + e- H O OH O O L-ascorbate (Asc) Semidehydro-L-ascorbic acid (SDA) or L-ascorbyl free radical
+ H+, + 2e- +e-
-H+, -2e- - e- HO OH
O O H
O O Dehyro-L-ascorbic acid (DA)
The non-enzymatic antioxidant activity of L-ascorbic acid provides reducing equivalents to a wide range of biological substrates to maintain their reduced and active forms. For example, L-ascorbic acid maintain the reduced form of folic acid which is needed in the many one-carbon transfer reactions, which are involved in the formation of a wide variety of biologically important bio-molecules.129
2.8 L-Ascorbic Acid Metabolic Enzymes
L-ascorbic acid is directly oxidized by two enzymes, ascorbate peroxidase and ascorbate oxidase. Ascorbate peroxidase is a hydrogen-peroxide-scavenging enzyme that functions to protect cells from hydrogen peroxide accumulation under normal and stressful conditions present in plants.35, 115-116,118 This enzyme is found both as membrane-
36 bound and soluble forms. In chloroplasts, it catalyzes the reduction of hydrogen peroxide, as an electron donor, to yield water and semidehydro-L-ascorbate radical as the primary product. Ascorbate oxidase is a member of the class of blue multicopper oxidases and catalyzes the oxidation of L-ascorbic acid to dehydro-L-ascorbic acid with the conversion of O2 to H2O2. This enzyme is associated with the rapidly growing regions in plants and has been found as protein bound to the cell wall and as soluble protein in the cytosol.35,
115-116,118 Semidehydro-L-ascorbate radical and dehydro-L-ascorbate, which are the two oxidized forms of L-ascorbic acid are respectively reduced by semidehydro-L-ascorbate reductase and dehydro-L-ascorbate reductase. Semidehydro-L-ascorbate reductase catalyzes the regeneration of L-ascorbic acid from semidehydro-L-ascorbic acid radical using nicotinamide–adenine dinucleotide phosphate (NADPH) as the electron donor. This enzyme scavenges toxic reactive oxygen species in plant tissues.35, 115-116,118 Dehydro-L- ascorbate reductase functions as a reducing agent for the regeneration of L-ascorbic acid from dehydro-L-ascorbic acid. It has been isolated from various plant and animal tissues.
Its ability to recycle L-ascorbic acid depends on the relative activity level of the enzymes and concentration of glutathione.35, 115-116,118
2.9 Degradation and Oxidation of L-Ascorbic Acid
L-ascorbic acid is metabolized in the liver, and to some extent in the kidneys. The principal pathway of L-ascorbic acid metabolism involves the direct loss of two electrons which produces dehydro-L-ascorbic acid.35, 115 The loss of one electron produces semidehydro-L-ascorbic acid radical, which can also undergo oxidation to reversibly
37 produce dehydro-L-ascorbic acid. Dehydro-L-ascorbic acid can irreversibly react with water to produce physiologically inactive 2,3-diketogulonic acid product. 2,3- diketogulonic acid is either cleaved to oxalic acid and threonic acid, or undergoes decarboxylation to produce carbon dioxide, xylose, and xylulose and eventually leads to the formation of L-xylonic acid and L-lyxonic acid. All these metabolites and L-ascorbic acid are excreted in the urine. The amount of each metabolite varies from species to species according to the amounts of L-ascorbic acid ingested. Some other metabolites besides those already mentioned above, such as 2-O-sulfate L-ascorbic acid and 2-O- methyl L-ascorbic acid have also been found in humans and rats.35, 115 A new metabolite,
2-O-β-glucuronide-L-ascorbic acid was recently identified in human urine and plasma.35,
115 The rate of chemical degradation of L-ascorbic acid depends on several factors among which are temperature, oxygen level, light, transition metals (e.g. copper & iron), and pH
(most stable at pH 4-6).35, 115 L-ascorbic acid undergoes slow two-electron autooxidation to dehydro-L-ascorbic acid depending on these factors mentioned above. In acidic solutions, degradation of L-ascorbic acid metabolites proceeds to form L-(+)-tartaric acid,
2-furfuraldehyde, 3-hydroxy-2-pyrone, and other furan derivatives, as well as some condensation products.35, 115 Dehydro-L-ascorbic acid nonenzymatically reacts with several amino acids to form brown-colored products, a reaction known to contribute to food spoilage.35, 115 L-Ascorbic acid content in foods significantly decreases during storage and the degradation process is greatly enhanced during cooking.
38 Figure 5 Degradation of L-Ascorbic Acid (adapted from Ref. 35)
Anaerobic HO OH HO OH Aerobic O O O -2H+ O H H +2H+ HO OH O O L-Ascorbic Acid Dehydro-L-Ascorbic Acid
Delactonization H2O
HO OH OH HO O H H2O O O CO2 CO 2 2,3-Diketogulonic Acid
Deoxypentose Xylosone
Furfural
± Amino Acid Reductones
Brown Pigments
Thermal decomposition of L-ascorbic acid is extensively studied because of its importance in food and beverage industries. Kurata and Sakurai131, 132 both studied the degradation process in acidic medium, which was found to progess through decarboxylation after the lactone ring opening and subsequent cyclization to give furfural
(Figure 5). Thermal degradation of L-ascorbic acid and dehydro-L-ascorbic acid also resulted in the formation of some furan derivatives.130, 133 A group of volatile furan-type compounds and reductones are detected by gas chromatography.115 Some of the browning
39 metabolite products have antioxidant activity, 35, 115 while others have destructive prooxidant effects, which include cytotoxicity, lipid peroxidation, mutagenesis, the adduct formation with proteins and nucleic acids.35, 115
2.10 Cellular Transport and Intestinal Absorption of L-Ascorbic Acid
L-ascorbic acid accumulates in human tissues as much as 50-fold compared to the plasma.134 L-ascorbic acid and its oxidized metabolite, dehydro-L-ascorbic acid are both transported and accumulated distinctly and neither competes with the other. L-ascorbic acid is transported by sodium-dependent carrier-mediated active transport. Dehydro-L- ascorbic acid transport and accumulation is at least 10-fold faster than L-ascorbic acid transport and is sodium-independent and biologically separable from its reduction to L- ascorbic acid. Studies have shown that dehydro-L-ascorbic acid, and not L-ascorbic acid, is preferentially transported intracellularly.35, 115 Once transported, dehydro-L-ascorbic acid is immediately reduced in the intracellular compartments to L-ascorbic acid. A number of detailed experimental criteria have been used to distinguish the two systems, which established that L-ascorbic acid and dehydro-L-ascorbic acid are transported into various cells such as the human neutrophils and fibroblasts by two distinct mechanisms.
This has also established L-ascorbic acid as preferentially available for intracellular utilization. Glucose Transporters I-V have been well characterized and are neither sodium-dependent nor have sites indicative of sodium dependency. Such sodium dependency is required for L-ascorbic acid but not for dehydro-L-ascorbic acid transport and accumulation. The activity of sodium-dependent uptake of radio-labeled L-ascorbic
40 acid is reported to be inhibited by excess unlabeled L-ascorbic acid and not glucose, thus implying that the putative L-ascorbic acid transporter is mediated by a different protein than that responsible for Glucose.115, 135 Intestinal absorption of L-ascorbic acid is achieved by this sodium-dependent transport system.115, 135 Its transport into the ileum is a carrier-mediated process at low mucosal concentrations of L-ascorbic acid. However, at high mucosal concentrations, the influx of L-ascorbic acid into the ileum is linearly dependent on its concentration and absorption occurs predominantly by simple diffusion.115, 135 Its gastro-intestinal absorption is inversely dependent on its dosage.115, 135
The amount of L-ascorbic acid absorbed decrease with the age of a person and L-ascorbic acid in large doses can cause intestinal discomfort and osmosis diarrhea.115, 135
2.11 Molecular Structure of L-Ascorbic Acid
One of the most difficult tasks facing earliest organic chemists was to work out a detailed structure of a compound from studying its reaction with a variety of known materials and then sequentially fitting different possible models to account for the chemistry. Therefore, it came as no surprise that L-ascorbic acid synthesis was first accomplished long before the correct structure was determined. Micheel and Kraft suggested that 2-(4,5-dihydo-3,4-dihydroxy-5-hydroxymethyl) furanyl-carboxylic acid as the constitutional formula of ascorbic acid, in 1933, after analyzing the chemical properties of the compound. This structure was later rejected after failing to account for the characteristic chemical and physical properties of ascorbic acid. One such property
41 was the mild oxidations of ascorbic acid which resulted in loss of its acidic properties.136,
137
On the contrary, the structure constituting the C2,C3-enediol lactone moiety suggested by Hirst received a worldwide recognition. In the same year, a detailed synthetic report was published, considering D-xylosone has a furanose structure, with the initial reaction involving the substitution of its hydroxyl group at the anomeric carbon by cyanide to give 2-oxo-1-cyano-1-deoxy-xylofuranose as the primary product. And successively upon its acid hydrolysis, the author suggested that the compound would lead to give the final product as D-ascorbic acid, having the structure similar to that earlier proposed by Micheel and Kraft. The physical and chemical properties of this final product were found to be very similar to the natural L-ascorbic acid, except for the differences in their specific optical rotations. Consequently, these findings were considered as an excellent model for the structure of L-ascorbic acid, and it helped to guide Haworth and coworkers using the same reaction sequence starting with L-xylosone to obtain L-ascorbic acid.106, 110 As a result of this brilliant work, they were able to isolate and characterize 1-imino-L-ascorbic acid as the final product, which upon hydrolysis eventually produced L-ascorbic acid as the final product. As a result of this outstanding work, they were able to give the precise structure of L-ascorbic acid that is used today.
The designation of the compound was changed from 2-oxo-L-threo-hexano-1,4-lactone-
2,3-enediol or vitamin C to L-ascorbic acid in 1965 by the IUPAC-IUB Commission on
Biochemical Nomenclature. The stereo-chemical assignments of ascorbic acid to the L- configuration was first established by the synthesis from L-xylose and later confirmed by
X-ray crystallography and neutron diffraction analysis.138-140
42 The L-ascorbic acid molecule has a molecular formula of C6H6O6 and includes two asymmetric carbon atoms, C4 and C5. Therefore, in addition to L-ascorbic acid itself, there are three other stereoisomers: L-isoascorbic acid, D-isoascorbic acid, and D- ascorbic acid. Of the four possible stereoisomeric forms of ascorbic acid, only the form identical to the natural vitamin C, that is the (+)-ascorbic acid (L-ascorbic acid), has the same anti-ascorbutic activity.141 However, all of the diastereomers show the same strong antioxidant properties. There is often the misconception that (+)-Ascorbic acid and (-)- isoascorbic acid, often labeled as L-ascorbic acid and D-erythrorbic acid, respectively, are enantiomers. These are not enantiomers, but rather are diastereomers as the structures are not mirror images. While L-ascorbic acid is utilized as a bioactive vitamin C nutrient, both L-ascorbic acid and D-erythorbic acid are commercially important as antioxidant preservatives, for instance in protecting the flavor profile of citrus soft drinks such as orange soda. D-erythorbic acid exhibits only 5% of the anti-ascorbutic activity compared to L-ascorbic acid.141 The structure of L-ascorbic acid and its three stereosiomers are shown in Figure 6. L-ascorbic acid is a dibasic acid with a C2,C3-enediol moiety built into a five-membered heterocyclic lactone ring. The ring is almost planar with a slight distortion of the lactone oxygen atoms out of the enediol plane. The molecule is stabilized by delocalization of the π electrons of the conjugated carbonyl with the enediol system. The chemical and physical properties of L-ascorbic acid are directly related to its structure. Dehydro-L-ascorbic acid, the first stable oxidation product of L-ascorbic acid is also present in biological tissues and retains physiological or vitamin C activity.
43 Figure 6 L-Ascorbic Acid and its Diastereomers.
HO HO (S) H (R) H HO HO O O (R) O (S) O H H HO OH HO OH
L-Ascorbic acid D-Ascorbic acid
HO (R) H HO HO (S) H O (R) O HO O O H (S) H HO OH HO OH
D-Arabo-ascorbic acid L-Arabo-ascorbic acid = D-Erythorbic acid = L-Erythorbic acid
The structure of dehyro-L-ascorbic acid was postulated as C2,C3-diketo-lactone, which has a side chain that forms a hydrated hemiketal (Figure 7, a). X-ray crystallography analysis was used to determine dehydro-L-ascorbic acid as a dimer (Figure 7, b). Nuclear magnetic resonance (NMR) studies have also indicated that dehyro-L-ascorbic acid in aqueous solution exists as a bicyclic hydrated monomer. Electrochemical studies have indicated that L-ascorbic acid and dehydro-L-ascorbic acid form a reversible redox couple. 35,115
44 Figure 7 Structural Forms of Dehydro-L-Ascorbic Acid
H O HO O OH H O O OO HO O O HO OH O OH OH O O H OH
(a) Dehydro-L-ascorbic acid (b) Dehydro-L-ascorbic acid hydrated Monomer hydrated Dimer (crystal from)
2.12 Chemical and Physical Properties of L-Ascorbic Acid
The role of L-ascorbic acid in biological systems stems from its basic functional structure. It is a five-membered lactone sugar acid and its C3 and C2 enolic hydroxyl groups can dissociate to form a dibasic acid. The 2,3-enediol moiety of L-ascorbic acid conjugated with its C1 carbonyl group, makes the proton on the C3 hydroxyl group significantly acidic (pK1 = 4.25: comparable to acetic acid with pKa = 4.8) in comparison to the proton on C2 hydroxyl group (pK2 = 11.79). These two acidic protons are the reason for the acidic properties of the molecule. The 2,3-enediol moiety enables L- ascorbic acid to donate one or two electrons (reducing equivalents) and form somewhat stable oxidized intermediate (semidehydro-L-ascorbic acid) and the final oxidized product (dehydro-L-ascorbic acid), a property from which, most if not all the chemical and biological functions of L-ascorbic acid are derived. The two hydroxyl groups at C5 and C6 are normal alcohol groups and thus react with aldehydes and ketones to give cyclic acetals and ketals, respectively. L-Ascorbic acid registers a positive value for optical rotation due to the two asymmetric centers at C4 and C5. The optical rotation is
45 not significantly affected by the acidity of the solution, but in contrast, it varies greatly with alkalinity, increasing over +160o in 2N NaOH solution.142 A number of physical properties of L-ascorbic acid are listed in Table 3.
2.13 Synthetic Derivatives and Analogues of L-Ascorbic Acid
It is more convenient to correlate the literature with respect to the reactivity of L- ascorbic acid with acylating and alkylating reagents under basic and acidic conditions, its ketal and acetal derivatization, and lastly with respect to the asymmetric chemistry of its oxidative cleavage and/or reduction towards producing chiral synthons. L-ascorbic acid has several reactive positions that are open to derivatization towards producing a number of compounds with interesting chemical and physical properties. There are many substituted derivatives at the C2, C3, C5 and C6 positions of L-ascorbic acid reported in the literature.35, 115 Electrophilic attack on L-ascorbic acid by acylating and alkylating reagents depends on the acidity (pKa) and steric constraints of the four hydroxyl groups at C2, C3, C5, and C6 of the molecule. The first ionization takes place at the most acidic proton which is the enolic C3-hydrogen (pK1 = 4.25). However, the delocalization of the negative charge in the monoascorbate anion causes susceptibility to alkylation at both the
C3-O and the C2 positions.
46 Table 3 Physical Properties of L-Ascorbic Acid (adapted from Ref. 211 & 212)
Property Comments
Appearance White, odorless, crystalline solid with sharp acidic state
Formula / Molar mass C6H8O6 / 176.13 g/mol
Melting point 190-192oC
Density 1.65 g/cm3
pH ~3 (5 mg/ml); ~2 (50mg/ml)
pK1 4.17
pK2 11.57
Redox potential First stage: E1O + 0.166 V (pH 4)
Spectral properties Emax (1%, 1cm), 695 at 245nm (undissociated form) UV pH 2:
Spectral properties Emax (1%, 1cm), 940 at 265nm UV pH 6.4: (monodissociated form)
o o o [α]D at 25 C = +20.5 to +21.5 (C = 1 in Optical rotation water) o o [α]D at 23 C = +48 (C = 1 in methanol)
Solubility (g/ml)
Water 0.33
95% Ethanol 0.033
Propylene glycol 0.05
Glycerol (USP) 0.01
Fats and oil solvents: ether, chloroform, benzene, petroleum insoluble ether, etc.
47 The ambident (a chemical compound with two alternative and strongly interacting distinguishable reactive centers, to either of which a bond may be made in a reaction with the centers: the centers must be connected in such a way that reaction at either site stops or greatly retards subsequent attack at the second site) characteristic of the C3-O- monoanion to display nucleophilicity at both the C3-O and C2 was first reported by
Jackson and Jones,164 who were the first to report alkylation of sodium ascorbate with benzyl chloride to afford a mixture of C3-O and C2 benzylated products. Likewise, Poss and Belter obtained C2 allylated derivatives by treating potassium ascorbate with various allylic bromides in acetone as solvent.165 Also, dealkylation of 2-O-(E)-cinnamoyl-5,6-O- isopropylidene-3-O-methyl-L-ascorbic acid with lithium iodide and iodomethane in DME afforded the C2-alkylated isomer, where the C2 acted as a sink for the equilibrating mixture of dealkylated C3-O- and the C2.166 Furthermore, reaction of ascorbic acid with various Michael acceptors with α, β-unsaturated carbonyl compounds undergoing conjugated addition and 1,4-dialdehydes and 4-keto aldehydes giving aldol-derived products have all been extensively studied as means of generating C2-alkylated analogues.145-148 Therefore, reactions of C5- and C6-OH protected ascorbic acid with electrophilic reagents under mild basic conditions exclusively takes place at the C3-OH due to its high nucleophilicity.35 However, the C2-O-alkylated products could only be obtained after the protection of the C3-OH group with protecting groups (Figure 8) such as acetyl, MOM, benzyl, etc. On the other hand, in a strong basic conditions (pKa ≥ 12), alkylation of the di-anion of L-ascorbic acid occurs preferentially at the less stable C2-O position (pK2 11.79), allowing the direct and selective functionalization of this position among the other three hydroxyl groups. Under highly basic conditions, ionization of the
48 C4-hydrogen of L-ascorbic acid occurs to produce tri-anionic form of L-ascorbic acid. If a leaving group resides at the C5 position of L-ascorbic acid, elimination can occur via the ionization of the C4-hydrogen to produce a 4,5-dehydro-L-ascorbic acid derivative.
This has been observed in the case of 2,3,5,6-tetra-O-methyl-L-ascorbic acid, which on treatment with 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) or potassium hydride afforded the 5-deoxy-4,5-dehydro-2,3-di-O-methyl-L-ascorbic acid as a mixture of olefinic isomers.143
Figure 8 Regioselective O-Alkylation of Ascorbic Acid
H CCH H CCH HO OH 3 3 3 3 HO OH O O O O 1. Acetone,AcCl, O O conc. HCl, O O r.t. 4 h THF, r.t., 5 h O O O H 1 O H 2. R X, K2CO3, H H HO OH DMSO/THF, 6 h R1 + R1O OH 1 O R O OH or OH minor major BnBr, KHCO3, 1. DEAD, Ph3P,THF/DMF, o DMSO, 60 oC, 12 h -78 C 15 min. 2. BnOH, r.t., 2 h
HO OH HO OH HO OH O O O O O O R X, K CO , H 2 2 3 H H2-Pd/C H DMSO/THF AcOEt, r.t. 6 h BnO OR HO OR2 BnO OH 2
Adapted from Ref. 35,144,154-156,164-165, 217
In general, the selective C2-O and C3-O alkylation is difficult to achieve and requires protection of the C5-O and C6-O positions prior to alkylation in order to minimize their interference during reaction.217 However, Beifuss et al.144 has reported an efficient and regioselective approach to C2-O and /or C3-O alkylation without the C5- and C6-OH protection. Preferential and direct alkylation of C3-OH of the C5- and C6-OH
49 unprotected ascorbic acid under the Mitsunobu conditions has been recently reported.154-
156 Acid catalyzed esterification of L-ascorbic acid with an acylating reagent initially produces the C6-O-acylated derivative, and under a more vigorous conditions, eventually gives the 5,6-diester derivatives.35 Also, some C2-O-esters and C2 inorganic esters such as C2-O-phosphate and C2-O-sulfate have been synthesized.35 A detailed characterization of C2-O and C3-O-acetyl esters of 5,6-O-isopropylidine-L-ascorbic acid has been previously reported.163 Activated (α, β-unsaturated) aldehydes and ketone reagents have been used in a Michael addition reaction with L-ascorbic to protect the C2 and C3 positions.145-148 This process permits the selective modification of the primary and secondary alcohol groups on the products to produce a number of side-chain-oxidized derivatives.
The syntheses of 5,6-O-ketal or 5,6-O-acetal derivatives of L-ascorbic acid under acidic catalyzed conditions helps to maximize its solubility in organic solvent and also limit the interference of the 5,6-O-protected group in reactions involving the C2- and
C3-OH. For example, 5,6-O-isopropylidene and 5,6-O-benzylidene, which are ketal and acetal derivatives respectively are well-known and extensively used in various organic syntheses of L-ascorbic derivatives and analogues.217 In basic conditions, if both the C2- and C3-OH groups are protected, base-promoted alkylation or acylation takes place at the more sterically accessible primary hydroxyl group on C6 rather than C5 position.
Therefore, reactions at the C5 position occur only after derivatization of C2, C3, and C6 are completed. Since L-ascorbic acid possesses several chiral and pro-chiral centers, significant attention has been focused on the chemistry of this important molecules and its application in various asymmetric synthesis to obtain commercially unavailable and
50 highly functionalized chiral synthons (Figure 9). Some examples include a group of bicyclic alklidene-dimethyloxy butenolides, glycerol acetonides, threitols, erythritol and a series of hydroxyl-lactones, all synthesized using L-ascorbic acid as a starting material.36-
53 Compounds with quite similar structural features to L-ascorbic acid have been synthesized. For example, an analogue such as cyclopentenone has been synthesized. In this molecule, the C1-oxygen of the lactone ring is replaced with a carbon atom.149 Also, the total synthesis of 2-deoxy-L-ascorbic acid from methyl-3,4-O-isopropylidene-L- threonate has been reported.150, 151 Nitrogen analogues of L-ascorbic acid such as 2- amino-2-deoxy- and 2,3-diamino-2,3-deoxy-L-ascorbic acid are also known. Also, 6- halo-6-deoxy such as 6-fluoro-, 6-bromo-, 6-chloro- and 6-iodo-L-ascorbic analogues have been synthesized.152 The crystal structure of erythroascorbic acid has been reported.
This molecule contains a side chain with a methoxy group instead of an ethoxy group.153
51 Figure 9 Potential of L-Ascorbic Acid as a Chiral Synthon
HO OH O O H HO OH
H3C CH3 H3C CH3 H3C CH3 O O O O O O O O O O O O H H H HO OH O OH HO O
H C CH 3 3 H3C CH3 O O O O O O O O H H OH HO HO OH
H3C CH3 O O OH H H O
H3C CH3 H3C CH3 O O O O OH OH H H OH OH O
52 CHAPTER 3
RESEARCH OBJECTIVE
The acetyl functional group has been commonly used as a C3-O protecting group during the alkylation of the C2-OH of L-ascorbic acid. However, the high instability of the C3-O-acetyl derivatives and facile migration of acyl groups from C3-O to C2-O even under mild reaction conditions157-160 have led us161 and others162-163 to the misidentification and characterization of C2-O and C3-O substituted L-ascorbic acid derivatives. Although, the products of these reactions were once characterized as C3-O- acetyl-C2-O-alkyl derivatives, in most cases they were C2-O-acetyl-C3-O-alkyl derivatives which were predominantly formed due to the fast acetyl migration under typical reaction conditions.161 In order to resolve the discrepancies of the structure assignments of C2-O and C3-O substituted ascorbate derivatives, we sought to develop a specific and a direct method to alkylate the C2-O position of 5,6-O-isopropylidine-L- ascorbic acid, which to our knowledge had not been reported in the literatures. We used density functional theory (B3LYP) calculations to determine the electron density distributions and reactivities of the neutral, monoanion and dianion of L-ascorbic acid and found that electrophilic reactions with the monoanion and dianion of L-ascorbic acid should preferentially occur at the C3-O and C2-O positions, respectively. Based on these findings, we have devised a novel and general method for the direct alkylation of C2-O of
5,6-O-isopropylidene-L-ascorbic acid in good yields with complete regio- and chemo- selectivity with both activated and inactivated electrophiles. We have also carried out a complete spectroscopic analysis of two complementary series of C2-O-acetyl-C3-O-alkyl
53 and C2-O-alkyl-C3-O-acetyl ascorbic acid derivatives in order to clearly define the spectroscopic characteristics of these derivatives for future studies.
Our previous study161 showed that C2-O- and C3-O-allyl derivatives of 5,6-O- isopropylidene-L-ascorbic acid, which are cyclic enol ethers, undergo facile thermal
Claisen rearrangement providing an excellent, convenient and stereo controlled access to non-accessible C2- and C3-substituted L-galactono-γ-lactones. As a result, a direct and practical route to the synthesis of unknown C2- and C3- substituted gulono-1,4-lactone derivatives will be very valuable in the structure-activity studies of the various galactono-
γ-lactone-derived pharmacological agents for the improvement of their pharamacokinetics and thus their therapeutic values. Consequently, we extended our previous findings161 that thermal Claisen rearrangement of C2-O- and C3-O-allyl L- ascorbic acid derivatives easily provides a convenient entry to non-accessible C2- and
C3-substituted L-galactono-γ-lactones. More importantly, we show that the Claisen rearranged products could be stereoselectively reduced to produce a new series of C2- substituted L-gulono-γ-lactone derivatives, which are synthetically more demanding and could be used as chiral intermediates in the synthesis of a range of important natural products and pharmacologically active materials.54-81
54 CHAPTER 4
RESULTS AND DISCUSSION
4.1 Chemo- and Regio-Selective Alkylation of L-Ascorbic Acid
The alkylation and acylation of L-ascorbic acid under basic conditions is a function of the acidity (pKa) and steric environments of the four hydroxyl groups at the
C2, C3, C5 and C6 positions. These four hydroxyl groups show different reactivity toward electrophiles under basic reaction conditions. The presence of four hydroxyl functional groups makes L-ascorbic acid very hydrophilic and insoluble in organic solvents. Therefore, it is difficult to use L-ascorbic acid as a starting material for organic synthesis. The most synthetically useful and well-studied class of modified L-ascorbic acid is the 5, 6-O-isoproylidene-L-ascorbic acid derivatives (ketal of L-ascorbic acid).
These derivatives (5,6-O-ketal & 5,6-O-acetal) are significant in organic synthesis for protection of the 5,6-hydroxyl functions, which makes them more soluble in organic solvents and also limits the interference of the protected hydroxyl group from reactions involving the C2- and C3-enol hydroxyls. Consequently, all our syntheses began with
5,6-O-isopropylidene-L-ascorbic acid (1) as the starting material, which is cheaply and easily made from L-ascorbic and low-grade acetone (contaning small H2O%) under acid- catalyzed conditions with excellent yield.
55 4.1.1 3-O-Alkylation of 5,6-O-Isopropylidene-L-Ascorbic Acid
The C3-OH group of L-ascorbic acid is more reactive towards electrophiles under mild basic conditions in comparison to the C2-OH (Scheme 7). This is primarily due to the preferential deprotonation of C3-OH over C2-OH under mild basic conditions to produce the monoanion.35 The electron density distribution diagram of the monoanion of
1 (Figure 10) clearly shows that the negative charge of the monoanion is distributed between the C3-O─ and C1-carbonyl of the lactone ring with little electron density on C2-
OH. Therefore, the reactions of C5-OH- and C6-OH-protected ascorbic acid with various electrophilic reagents under mild basic conditions should predominantly occur at the C3-
OH position as experimentally observed with the synthesized compounds in Table 4. In addition, the electron density distribution diagram of the monoanion of 1 noticeably confirm that the electron density at the C2 of the monoanion is significantly higher than that of the C2-OH suggesting that the C2 position of the monoanion may also be susceptible to electrophilic reactions.
56 Figure 10 Calculated Electrostatic Density Potential Diagrams of Monoanion Species of 1. Order of Electron Density: Blue < Green < Yellow < Red
In agreement with the literature findings and the electron density distribution diagram
(Figure 10), C2-alkylated products were also observed as minor products in the alkylation of 1 under mild alkaline conditions. The electrophile isoprenyl bromide (4-bromo-2- methyl-2-butene) gave exclusively C2-alkylated product when reacted with 5,6-O- isopropylidene-L-ascorbate.191 The observed steady increment in the production of the
C2-alkylated product over the 3-O-alkylated product, when the electrophilic reagents changed from allyl, crotyl, benzyl, to cinnamyl bromide, clearly indicates the dependence of the transition state stability on the electrophile, which apparently plays a significant role in the product distribution (Table 4). Therefore, these empirical results show that the reactions of 1 with simple electrophiles under mild basic conditions can produce both C3-
O and C2 alkylated products.
57 Scheme 7 3-O-Alkylation of 5,6-O-Isoprpylidene-L-Ascorbic Acid
H C CH CH3 3 3 H3C CH3 H3C O O O O O O O O CH3I or RBr / K2CO3 O O O H + O H H HO OH DMSO:THF / r.t. / 4-6 h R O H R O O O H 1 A B
Table 4 Products from 3-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid
Products R Yielda (A:B ratio)b
1A CH3 91 (100:0)
2A CH2C6H5 86 (62:38)
3A CH2CH=CHCH3 72 (70:30)
4A CH2CH=CH2 80 (80:20)
5A CH2CH=CHC6H5 72 (35:65)
aAll the yields (%) are given for the chromatographically purified products. bCalculated based on the weights of the purified compound or by 1H-NMR signals for the nonresolvable mixtures.
58 4.1.2 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid
The C2-O-alkylated products of 1 could only be obtained after the protection of the C3-OH group with protecting groups such as acetyl, MOM, etc.144, 161, 167 because of the high nucleophilicity of C3-OH under mild basic condtions as previously mentioned.
In the literature, the acetyl group has been commonly used as a C3-O-protecting group in the alkylation of the C2-OH of ascorbic acid. However, the high instability of the C3-O- acetyl derivatives and facile migration of acyl groups from C3-O to C2-O position even under mild reaction conditions led to many misidentification157-163 and mischaracterization157-163 of C2,C3-O-disubstituted ascorbic acid derivatives. In all these cases, the products characterized as C3-O-acetyl-C2-O-alkyl derivatives of 1, were later confirmed as the C2-O-acetyl-C3-O-alkyl derivatives of 1. These thermodynamically favored C2-O-acetyl-C3-O-alkyl derivatives are predominantly formed due to the fast acetyl migration under the reaction conditions as previously mentioned.
Therefore, we sought to develop a specific and a direct method to alkylate the C2-O position of 5,6-O-isopropylidine-L-ascorbic acid (1), which has not been reported in the literature, to our knowledge. We used the results of density functional theory (B3LYP) calculations to determine the electron density distributions and thus the nucleophilicity of the dianion species of 1, towards electrophiles. Based on the molecular calculation results, we have devised a novel and general method for the direct alkylation of C2-OH of 1 in good yields with complete regio- and chemo-selectivity with both activated and unactivated electrophiles.
59 Figure 11 Calculated Electrostatic Density Potential Diagrams of Dianion Species of 1 Order of Electron Density: Blue < Green < Yellow < Red
Inspection of the electron density distribution of the dianion of 1 (Figure 11) shows that the negative charge of the C3-O is highly delocalized to the lactone carbonyl similar to that of the monoanion as previously discussed. However, the electron density of the C2-
O is highly localized in the dianion suggesting that electrophilic reagents should preferentially react with the C2-O rather than C3-O of the dianion of 1.
In excellent agreement, the dianion of 1 (Scheme 8), generated by reacting two equivalents of potassium tert-butoxide (t-BuOK) in DMSO/THF (3:2) at -10 oC, reacts with an equivalent amount of activated or unactivated electrophilic alkylating agents to exclusively produce the corresponding C2-O-alkylated products (Table 5) in good yields
(80-90%). Regardless of the nature of the electrophile used according to Scheme 8, no
60 detectable amounts of C3-O- and/or C2-substituted products were produced, under these experimental conditions. However, the addition of two equivalents of the electrophile cleanly produces the corresponding C2,C3-O-disubstituted derivatives.
Scheme 8 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)
H3C H3C CH3 CH3 CH3 O H3C O O O O O O O 2 equivs t-BuOK O O RBr O O H + - H -2H H DMSO:THF / HO OH - O o HO 1 O -10 C / 3 h O R C
Table 5 Products of 2-O-Alkylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid (1)
R Products Yield (%)
CH2CH=CH2 1C 80
CH3 2C 91
CH2CH=CHCH3 3C 72
CH2C6H5 4C 83
CH2CH=CHC6H5 5C 87
CH2(CH2)5CH3 6C 96
61 4.1.3 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid
2,3-O-disubstituted products of 5,6-O-isopropylidene-L-ascorbic acid (1) are known for their vast usefulness in synthetic chemistry and various biological and pharmaceutical application. The synthesis of most disubstituted products of 1, are simply achieved either by (a) the use of excess electrophilic reagents under the appropriate reaction conditions to directly produce 2,3-O-disubstituted products of 1; or (b) selectively alkylating C2-OH (Scheme 8) or C3-OH (Scheme 7) of 1 prior to the subsequent modification of these mono-substituted proudcts to generate the desired disubstituted products (Scheme 9-11) . Tables 6-8 show a summary of 2,3-O- disubstituted derivatives synthesized in the present study. The C2-O-alkylation of 5,6-O- isopropylidene-3-O-acetyl-L-ascorbic acid derivatives of 1 to produce 5,6-O- isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid products (Table 7) could only be achieved by this latter procedure. This is because the intra-molecular C3-O to C2-O acetyl migration is much faster than the alkylation of the C2-OH group of 5,6-O- isopropylidene-3-O-acetyl-L-ascorbic acid (1D), under the C2-O-alkylation conditions.
Therefore, C2-O-alkylation of 5,6-O-isopropylidene-3-O-acetyl-L-ascorbic acid produces the 5,6-O-isopropylidene-2-O-acetyl-3-O-alkyl-L-ascorbic acid products instead of the desired 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid derivatives.
Consequently, the synthesis of 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid derivatives must proceed by first synthesizing the 5,6-O-isopropylidene-2-O-alkyl-
L-ascorbic acid derivatives (Scheme 8), followed by the subsequent acylation (Scheme
10) to give the 5,6-O-isopropylidene-2-O-alkyl-3-O-acetyl-L-ascorbic acid derivatives.
62 Scheme 9 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid
H C 3 CH3 CH3 O H3C O O O O O O CH3COCl / Py O H H CH2Cl2 / RT / 2 h O RO OH RO O CH3 1A-5A 6A-10A
Table 6 Products of 2-O-Acetylation of 5,6-O-Isopropylidene-3-O-Alkylated-L-Ascorbic Acid
Starting R Product Yield (%) material
1A CH3 6A 90
2A CH2C6H5 7A 90
3A CH2CH=CHCH3 8A 78
4A CH2CH=CH2 9A 70
5A CH2CH=CHC6H5 10A 76
63 Scheme 10 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid
CH CH3 3 H C H3C O 3 O O O O O O O CH3COCl / Py H HO OR CH2Cl2 / RT / 2 h O OR O H3C 1C-5C 7C-11C
Table 7 Products of 3-O-Acetylation of 5,6-O-Isopropylidene-2-O-Alkylated-L-Ascorbic Acid
Starting R Product Yield (%) material
1C CH2CH=CH2 7C 70
2C CH3 8C 80
3C CH2CH=CHCH3 9C 84
4C CH2C6H5 10C 82
5C CH2CH=CHC6H5 11C 76
64 Scheme 11 2,3-O-Disubstituted 5,6-O-Isopropylidene-L-Ascorbic Acid
CH3 H3C CH H C 3 3 O O O O O O RBr / K CO H 2 3 O H O R1O OH DMSO:THF / RT / 3 h R O 1 OR2
Table 8 Products of 2,3-O-Disubstitution of 5,6-O-Isopropylidene-L-Ascorbic Acid
Starting R R Product Yield (%) material 1 2
4A CH2CH=CH2 CH3 11A 59
4A CH2CH=CH2 CH2CH=CH2 12A 61
2A CH2C6H5 CH2C6H5 13A 88
5C CH2CH=CHC6H5 CH2CH=CHC6H5 14A 75
1A CH3 CH2CH=CH2 12C 66
1A CH3 CH2CH=CHCH3 13C 65
1A CH3 CH2CH=CHC6H5 14C 71
2A CH2C6H5 CH2CH=CH2 15C 81
4.2 Acylation of 5, 6-O-Isopropylidene-L-Ascorbic Acid
Ascorbic acid has four hydroxyl groups that are susceptible to acylation.
However, by working with 1, the 5,6-O-isopropylidene derivative of L-ascorbic acid, the situation is simplified in that only the C2- and C3-hydroxyls remain accessible to acylation. The huge gap in the pKa of C2- and C3-OH groups synthetically favors a C3-
65 O-acyl product, which is predominantly formed over the pH range of 4-7 under acylation reaction condition.163 Therefore, many publications reported to have synthesized C3-O- acyl derivatives of L-ascorbic acid, which were supported by non-crystallographic structural evidence. In our acylation reactions, methylene chloride was used as the solvent, which also served as an indicator to determine the completion of reaction. Since the starting material (1) is only slightly soluble in methylene chloride, the reaction mixture, which begins as a heterogeneous mixture slowly changes from a cloudy mixture into a homogenous solution as the reaction progresses to completion. In order to avoid the formation of 2-O-acyl, and 2,3-O-di-acyl derivatives of 1, pyridine, a weak organic base with pKa close to 9168 was used to exclusively generate 3-O-acyl derivatives. When,
1 in methylene chloride was reacted with one equivalent of pyridine and one equivalent of acetyl chloride (acylating agent), a mixture of products was obtained in about 85% yield. TLC analysis of the crude mixuture shows three different products. 1H NMR analysis of the crude product shows three different signals for the C4 methine carbon proton doublet at 4.78, 4.84 and 5.15 ppm with calculated ratios of 21:49:30 respectively
(Scheme 12). Further purification and spectroscopic analysis of the crude mixture revealed the products as 2D, 1D and 3D respectively.
66 Scheme 12 Acylation of 5,6-O-Isopropylidene-L-Ascorbic Acid
H3C CH3 H C CH 3 3 H3C CH3 O O O O + O O CH + 3 O O O O H C O O 3 O CH3COCl / Py O O H H H O O O O O O H O H O H O CH O O 3 CH3 CH Cl / RT / 1 h H3C HO OH 2 2 H3C 2D 1 1D 3D
4.2.1 C3-O- to C2-O Rearrangements of 3-O-Acyl-L-Ascorbic Acid Derivatives
The favored formation of C2-O-acyl over C3-O-acyl has long been a serious problem confronting the acetylation of L-ascorbic acid over the years. The possible acyl migration (Scheme 13) was long associated with several inorganic esters of L-ascorbic acid such as the phosphate and sulfate derivatives of L-ascorbic acid. For example, some earlier reported C3-O-phosphate and C3-O-sulfate derivatives of L-ascorbic acid were later confirmed by crystallography to be the corresponding C2-O-esters derivatives.163
67 Scheme 13 Irreversible Isomerization of 5,6-O-Isopropylidene-3-O-Acetyl-L-Ascorbic Acid under Basic Conditions.
H C CH H3C CH3 3 3 O O O O O O O O 1D H H O O O O O H O - + H C H3C 3 B BH
H3C CH3 H3C CH3 O O O O O O O O 2D H O H O O O CH O - + 3 CH BH O 3 - + BH
It has not been possible to obtain a pure C3-O-acetyl ester of 1 under various reaction conditions without the contamination with some C2-O-ester and C2,C3-O-di-esters isomers. We observed that the ratio of the C3-O-acetyl over the C2-O-acetyl ester derivatives, even during work-up procedures with increasing ratio of the C2-O-acetyl ester over the C3-O-acetyl ester derivatives. The presence of proton donor contaminants such as methanol or water led to a reduced amount of the C3-O-acetyl derivative and an
68 increase in the C2-O-acetyl ester derivative. Also, the use of neutral form of 1, prolonged reaction time, and the use of inorganic bases such as K2CO3 and NaHCO3 in the acylation reaction led to an increased formation of C2-O-acetyl ester derivatives. The change in reaction condition from 25 oC to 0 oC did not substantially change the amount of C3-O- acetyl ester formation; however, this accelerated the overall formation of the C2-O-acetyl ester derivative. It is clearly shown from the molecular calculations result (Figure 12) that the C2-O atom of the neutral species of 1 has a slightly higher electron density than the
C3-O atom. These results are consistent with the literature findings that the C2-O is more nucleophilic than the C3-O atom in the neutral species of L-ascorbic acid and may have added to the increased formation of the C2-O-acetyl ester derivative.35, 158, 163 It is well known in the literature that the neighboring hydroxyl groups participate in the monophosphate isomerization observed in ribonucleoside-2’-phosphate, glycerol-2- phosphate, glycerol-1-phosphate and in some other β-hydroxy phosphate esters.169-186
Another possible factor influencing isomerization of C3-O-acyl esters of 1 is that the C2-
O and C3-O atoms are restricted to an eclipsed conformation as a result of the planar lactone ring and such restriction could enhance the intramolecular rearrangement by nucleophilic participation of the vicinal hydroxyl group.139, 187-190 There was no evidence for the formation of C2,C3-O-diesters from C3-O-acetyl-esters of 1, when stirred with
1.2 equivalents of base under nitrogen gas for over 12 h, which resulted in complete acetyl group migaration of the C3-O- to C2-O- position. Therefore, the findings that the increase in susceptibility of the C3-O-acetyl esters to rearrangement in the presence of protic impurities, prolonged reaction time and increase in temperature strongly suggests
69 an intra-molecular and not inter-molecular isomerization happening in the acetyl migration of L-ascorbic acid derivatives.163
Figure 12 Calculated Electrostatic Density Potential Diagrams of Neutral Species of 1 Order of Electron Density: Blue < Green < Yellow < Red
4.3 NMR Spectroscopic Analyses of L-Ascorbic Acid and its Derivatives
The detailed structural analysis of L-ascorbic acid and its derivatives by NMR spectroscopy is important in understanding their structural characteristics in relation to their biological and chemical properties. The diagnostic 1H- and 13C-NMR spectral signals of L-ascorbic acid has its most upfield resonance belonging to the C6, which is followed by the C5, C4, C2, C3 and the C1 respectively. Consequently, derivatives of L- ascorbic acid display distinctive characteristics in their 1H- and 13C-NMR chemical shifts
70 for the six carbon centers of L-ascorbic acid, which could be used to unequivocally identify the derivatives.
4.3.1 NMR Spectroscopic Properties of 2-O- and 3-O-Substituted 5,6-O- Isopropylidene-L-Ascorbic Acid
The C2- and C3-OH group substitution of the 5,6-O-isopropylidene-L-ascorbic acid (1) causes a diagnostic chemical shift of its indigenous carbon and hydrogen that are useful in the characteristic identification of the various derivatives. The data presented in
Table 9 demonstrate that C2-O- and C3-O-monosubstituted derivatives of 1 display characteristic 13C-NMR chemical shifts for their C2 and C3 carbon signals and the 1H-
NMR chemical shifts for their C4-H that could be used to unequivocally identify the C2-
O- and C3-O-monosubstituted derivatives. The standard 13C chemical shifts of C2 and C3 of ascorbic acid and 1 are 118.8 ppm, 120.5 ppm, 156.3 ppm, and 158.4 ppm respectively.35, 161 The 13C-NMR signals of C2 and C3 are in the ranges of 119-120 ppm and 148-150 ppm for C3-O-alkylated derivatives and 121-123 ppm and 156-158 ppm for
C2-O-alkylated derivatives, respectively (Table 9), and are in good agreement with the previously reported literature values.144, 154-156, 167 Substitution at the C3-OH (3-O- alkylation) causes an upfield shift (8.5 to 10.2 ppm) of 13C signals of C3 (1A-4A) with respect to 1 depending on the nature of the alkyl substituent. On the other hand, the effects of the C2-O-substitution (1C-6C) on the 13C signals of C-2 are considerably smaller and in the range of 0.3 to 2.6 ppm (upfield). The large chemical shift difference in the 13C signals of C3 in the C3-O-substitued derivatives (1A-4A) in comparison to the
C2 in the C2-O-substititued derivatives (1C-6C) must be due to the lack of efficient delocalization of the C3-O electron density into the C1-carbonyl in C3-O-substituted
71 derivatives (1A-4A) in comparison to C2-O-substituted derivatives (1C-6C). This effect
is also clearly visible in 1H–NMR chemical shifts of C4-H (Table 9), where C3-O-
substitution (1A-4A) caused an upfield shift of the C4-H in comparison to the C2-O-
substituted derivatives (1C-6C).
Table 9 1H NMR (C-4-H) and 13C NMR (C-2 & C-3) Chemical Shifts (δ) of 2-O-Alkyl and 3-O-Alkyl Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1)
CH3 H3C O O O O 4 1 3 2 R1O OR2
13C 13C 13C 13C 1H Derivatives R1 R2 δ (2-C) ∆(2-C)a δ(3-C) ∆(3-C)a δ(4-C-H)
1 H H 120.5 - 158.4 - 4.91
1A CH3 H 119.5 -1.0 149.9 -8.5 4.53
2A CH2C6H5 H 119.5 -1.0 148.6 -9.8 4.57
3A CH2CH=CHCH3 H 119.1 -1.4 148.6 -9.8 4.55
4A CH2CH=CH2 H 119.2 -1.3 148.2 -10.2 4.58
1C H CH2CH=CH2 121.4 +0.9 156.4 -2.0 4.72
2C H CH3 123.1 +2.6 155.8 -2.6 4.71
3C H CH2CH=CHCH3 120.8 +0.3 157.9 -0.5 4.69
4C H CH2C6H5 121.1 +0.6 157.5 -0.9 4.60
5C H CH2CH=CHC6H5 121.2 +0.7 157.1 -1.3 4.64
6C H CH2(CH2)5CH3 121.8 +1.3 156.6 -1.8 4.71 a 13 The difference in C chemical shifts of C2 and C3 (∆(C-2) and ∆(C3)) were calculated by subtracting the chemical shifts of various derivatives from the corresponding values of the compound
72 In the C2,C3-O-disubstituted series (2,3-O-dialkylation), the C2 and C3 showed characteristic shifts of 13C signals (Table 10) with respect to 1 that could be used to distinguish the 2-O-alkyl-3-O-acetyl derivatives (7C-11C) from the 2-O-acetyl-3-O-alkyl derivatives (6A-10A). The 13C signals of C3 of 2-O-alkyl-3-O-acetyl derivatives (7C-
11C) showed a large upfield shift in the range of 16.1 to 13.8 ppm with respect to 1. On the other hand, 13C signals of C2 of these derivatives showed large downfield shifts (in the range of 9.6 to 14.3 ppm) with respect to 1. As discussed above for C3-O-alkylated derivatives (1A-4A), these large 13C shifts of the C3 signals of C3-O-acetylated derivatives (7C-11C) must be due to the significant perturbation of the native electronic structure of 1 by the electron withdrawing C3-O-acetate group. The inhibition of the delocalization of C3-O electron to the C1-carbonyl group by the C3-O-acetate group leads to an increase of the electron density at C3 causing 13C signal to shift upfield, and a decrease of electron density at C2 causing 13C signal to shift significantly downfield. A significant downfield shift of the 1H-NMR signal (Table 10) of C4-H of 7C-11C, in comparison to 1, further confirms the significant perturbation of the native electronic structure of 1 by the electron withdrawing nature of the C3-O-acetyl group. In 2-O- acetyl-3-O-alkyl derivatives (6A-10A), the effects were more localized to the C2 as expected. The 13C signals of the C2 of these derivatives shifted upfield in comparison to
1, which is in sharp contrast to the effects of C3-O-acetate substitution, suggesting that the direct electron withdrawing effect of the C2-O-acetate group primarily determines the
13C chemical shift of the C2 of these derivatives.
73 Table 10 1H NMR (C4-H) and 13C NMR (C2 & C3) Chemical Shifts (δ) of 2,3-O- Disubstituted Derivatives of 5,6-O-Isopropylidene-L-Ascorbic Acid (1)
CH3 H3C O O O O 4 1 3 2 R1O OR2
13C 13C 13C 13C 1H Derivatives R1 R2 δ (2-C) ∆(2C)a δ(3-C) ∆(3-C)a δ(4-C)H 1 H H 120.5 - 158.4 - 4.91
6A CH2-CH=CH2 O=C-CH3 114.6 -5.9 159.5 +1.1 4.69
7A CH3 O=C-CH3 114.5 -6.0 160.7 +2.3 4.67
8A CH2-CH=CHCH3 O=C-CH3 114.4 -6.1 159.7 +1.3 4.66
9A CH2-C6H5 O=C-CH3 114.8 -5.7 159.8 +1.4 4.71
10A CH2-CH=CH-C6H5 O=C-CH3 114.7 -5.8 159.7 +1.3 4.70
11A* 123.0 +2.5 155.1 -3.3 4.53 CH2CH=CH2 CH3 12A 121.5 +1.0 155.7 -2.7 4.55 CH2CH=CH2 CH2CH=CH2 13A 121.2 +0.7 156.4 -2.0 4.53 CH2C6H5 CH2C6H5 14A* 121.3 +0.8 156.1 -2.3 4.55 CH2CH=CHC6H5 CH2CH=CHC6H5
7C O=C-CH3 CH2-CH=CH2 130.1 +9.6 143.8 -14.6 5.18
8C O=C-CH3 CH3 131.2 +10.7 142.3 -16.1 5.15
9C O=C-CH3 CH2-CH=CHCH3 132.2 +11.7 143.8 -14.6 5.18
10C O=C-CH3 CH2-C6H5 130.1 +9.6 144.6 -13.8 5.15
11C O=C-CH3 CH2-CH=CH-C6H5 134.8 +14.3 144.6 -13.8 5.17
12C* 121.5 +1.0 157.1 -1.3 4.52 CH3 CH2CH=CH2 13C* 121.4 +0.9 157.3 -1.1 4.51 CH3 CH2CH=CHCH3 14C* 121.4 +0.9 157.4 -1.1 4.51 CH3 CH2CH=CHC6H5 15C* 121.4 +0.9 155.9 -2.5 4.55 CH2C6H5 CH2CH=CH2 a 13 The difference in C chemical shifts of C2 and C3 (∆(C2) and ∆(C3)) were calculated by subtracting the chemical shifts of various derivatives from the corresponding values of the compound *Data obtained from Mahindaratne, M. P. D. Thesis Ref. 191
74 The C4-H of C2-O-acetyl derivatives (6A-10A), showed a small but significant upfield shift, again demonstrating the electron withdrawing inductive effect of the C2-O-acetate group. Furthermore, in C2,C3-O-dialkyl derivatives (11A-14A & 12C-15C), the 13C signals of C3 showed moderate upfield shifts in the range of 1.1 to 2.7 ppm with respect to 1. In contrast, 13C signals of C2 of these derivatives (11A-14A & 13C-15C) showed modest downfield shifts in the range of 0.7 to 2.5 ppm with respect to 1. As discussed above for C3-O-alkylated derivatives (1A-4A), these upfield 13C shifts of C3 of C2,C3-
O-dialkyl derivatives (11A-14A & 13C-15C) must be due to the significant perturbation of the native electronic structure of 1 by the C3-O-alkyl group. The inhibition of the delocalization of C3-O electron to the C1-carbonyl by the C3-O-alkyl group leads to the increase of the electron density at C3 causing 13C signal to shift upfield, and a decrease of electron density at C2 causing 13C signal to shift downfield moderately. This effect is also clearly visible in 1H–NMR chemical shifts of C4-H, where C3-O-substitution of the
C2,C3-O-dialkyl derivatives (11A-14A & 13C-15C) caused an upfield shift of the C4-H in comparison to the free non-substituted ascorbate 1 and thus further confirms the considerable perturbation of the native electronic structure of 1 by the inherent nature of the substitution group.
4.4 The Sigmatropic Claisen Rearrangement of L-Ascorbic Acid Derivatives
Selective modification of the C2- and C3-OH groups of L-ascorbic acid provides a unique route to different ascorbate derivatives with great potentials as chiral synthons.
Therefore, in order to further explore the versatility of L-ascorbic acid, our group had
75 previously exploited the possibility of using L-ascorbic acid derivatives as Claisen substrates for the synthesis of new C2- and C3-substituted aldonolactone (L-galactono-γ- lactone) derivatives. Therefore, the sigmatropic Claisen rearrangement of the allyl vinyl ether’s moiety of a series of 5,6-O-isopropylidene-3-O-allyl-L-ascorbic acid (A, Scheme
14-16) and 5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid derivatives (C, Scheme 17) to their corresponding 5,6-O-isopropylidene-2-allyl-3-keto-L-galactono-γ-lactone (E, Table
11-18) and 5,6-O-isopropylidene-3-allyl-2-keto-L-galactono-γ-lactone (F, Table 19-26) respectively were synthesized and fully characterized for comparative purposes.
4.4.1 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-
Isopropylidene-3-O-Allylic Derivatives of L-Ascorbic Acid
5,6-O-isopropylidene-3-O-allyl-L-ascorbic acid derivatives progressed through a facile
Claisen rearrangement (100% conversion) according to Scheme 14 to give products with a β-keto-ester functional group known as 5,6-O-isopropylidene-2-allyl-3-keto-L- galactono-γ-lactone. This aldono-γ-lactone products are less polar and less-UV-active than their corresponding starting materials. 1H- and 13C-NMR analysis of the crude products revealed a C2-allylated diastereomeric excess with a minor diastereomeric product. These products were very visible in an iodine bath and were easily separated by careful silica gel chromatography with ethyl acetate and hexane in high purity for characterization.
76 Scheme 14 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-(1-prop-2-enyl)-3- Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 H3C O 7 7 O 14 O O O 1 O OR2 5 4 2 6 5 O O 3 6 13 4 1 (a) 12 H reflux / toluene H R1 3 O 2 14 6 h 11 10 O OR2 10 12 13 11 R 1 A E
Reactant R1 R2 Product(b)
4A H H 1E
9A H COCH3 2E
11A H CH3 3E
12A H CH2CH=CH2 4E
3A CH3 H 5E
8A CH3 COCH3 6E
(a)100% conversion of A to E was obtained in 6 h. (b) 1H-NMR analysis of crude reaction mixtures indicated that the product is a mixture of two diastereomers with > 90% of the major.
4.4.1.1 NMR Spectroscopic Analyses of Products from C3-O to C2 Sigmatropic
Claisen Rearrangement of 5,6-O-Isopropylidene-3-O-Allyl-L-Ascorbic Acid
Derivatives
The comparative 1H-NMR signals (E: Table 11, 13 & 15) of the Claisen rearranged products of a series of 5,6-O-isopropylidene-3-O-allylic derivatives of L-ascorbic acid and their corresponding starting materials (A: Scheme 14) showed characteristic chemical
77 shift patterns that could be used to identify the products. For instance, there is a downfield shift of the C10 proton signals of the starting materials (A) by a range of 0.25 to 0.58 ppm in forming the corresponding products (E). The newly formed C2-C12 bond on the products resulted in an upfield chemical shift of the C11-H, C12-H and C13-H proton signals of the starting materials by about 0.05 to 0.32 ppm, 2.55 to 2.93 ppm and
0.54 to 2.77 ppm respectively. The C3-β-ketone-lactone moiety on the products showed in general the deshielding of the product’s C4-H and C5-H proton signals when compared to their corresponding starting materials.
The comparative 13C NMR signals of the starting material and their corresponding C2- allylated products (E: Table 12, 14, & 16) revealed distinctive chemical shift patterns that could be used in the identification of the compounds. For example, C10 carbon signals of the products showed a downfield chemical shift ranging from 45.5 to 50.6 ppm when compared to the corresponding starting materials (A). The newly created C2-C12 bond resulted in a large upfield chemical shift of the C12 carbon signals of the products by about 77.8 to 85.1 ppm when compared to their starting materials. Furthermore, C3 and
C2 carbon signals of the products appeared in the ranges of 200.9-206.1 ppm (~ 41.2 to
57.3 ppm downfield) and 73.8-80.4 ppm (~ 37.1 to 44.7 ppm upfield) respectively, when compared to the corresponding starting materials. These significant chemical shift patterns of the C2 and C3 carbon signals reflect the disappearance of the conjugated enone moiety in the products.
78 Table 11 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H C CH 3 3 9 CH3 7 H3C O O O 7 O O 13 O 1 OR 6 5 O O 5 4 2 6 3 4 1 H H 12 3 2 13 O 11 10 O OR 12 10 11 A E
Proton # R = H R = CH3 A E A E ∆ (δ H)a ∆ (δ H)a (4A) (1E) (11A) (3E) δ (4-H) 4.58 4.66 -0.08 4.53 4.54 -0.01
δ (5-H) 4.28 4.54 -0.26 4.30 4.65 -0.35
δ (6-H’) 4.04 4.08 -0.04 4.04 4.06 -0.02
δ (6-H”) 4.15 4.19 -0.04 4.14 4.17 -0.03
δ (8-CH3) 1.37 1.35 +0.02 1.36 1.32 +0.04
δ (9-CH3) 1.40 1.41 -0.01 1.40 1.38 +0.02
δ (10-H’) 4.97 5.26 -0.29 4.93 5.20 -0.27
δ (10-H”) 4.97 5.28 -0.31 4.93 5.25 -0.32
δ (11-H) 6.01 5.69 +0.32 5.98 5.73 +0.25
δ (12-H’) 5.31 2.66 +2.65 5.33 2.63 +2.70
δ (12-H”) 5.41 2.66 +2.75 5.40 2.63 +2.77
δ (13-H) - - - 3.85 3.34 +0.51
aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
79 Table 12 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 H C O 7 3 7 O O O O 13 O 1 OR 6 O 5 4 2 5 O 6 3 4 1 H H 12 3 2 13 O 11 10 O OR 10 12 11 A E
Carbon # R = H R = CH3 A E A E ∆ (δ C)a ∆ (δ C)a (4A) (1E) (11A) (3E) δ (1-C) 171.0 172.6 -1.60 168.7 171.2 -2.50
δ (2-C) 119.2 74.5 +44.7 123.0 80.4 +42.6
δ (3-C) 148.2 205.5 -57.3 155.1 206.1 -51.0
δ (4-C) 75.6 81.5 -5.90 74.6 81.5 -6.90
δ (5-C) 74.3 72.0 +2.30 74.0 74.3 -0.30
δ (6-C) 65.3 64.8 +0.50 65.3 64.9 +0.40
δ (7-C) 110.3 111.3 -1.00 110.3 110.9 -0.60
δ (8-C) 25.5 25.3 +0.20 25.5 25.5 0.00
δ (9-C) 25.9 25.5 +0.40 25.8 25.6 +0.20
δ (10-C) 72.3 122.9 -50.6 72.2 122.1 -49.9
δ (11-C) 132.2 127.6 +4.60 131.8 127.8 +4.00
δ (12-C) 119.1 39.8 +79.0 118.8 41.0 +77.8
δ (13-C) - - - 59.9 55.8 +4.10
aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
80 Table 13 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 7 H3C O O O 7 O O 13 O 1 OR 6 5 O O 5 4 2 6 3 4 1 H H 12 3 2 13 O 11 10 O OR 12 10 11 A E
R = COCH Proton # 3 R = CH CH=CH 2 2 A E A E ∆ (δ H)a ∆ (δ H)a (9A) (2E) (12A) (4E) δ (4-H) 4.69 4.87 -0.18 4.55 4.54 +0.01
δ (5-H) 4.38 4.53 -0.15 4.30 4.64 -0.34
δ (6-H’) 4.08 4.10 -0.02 4.04 4.06 -0.02
δ (6-H”) 4.16 4.20 -0.04 4.14 4.17 -0.03
δ (8-CH3) 1.37 1.37 0.00 1.36 1.33 +0.03
δ (9-CH3) 1.41 1.42 -0.01 1.39 1.39 0.00
δ (10-H’) 4.81 5.19 -0.38 4.94 5.19 -0.25
δ (10-H”) 4.81 5.26 -0.45 4.94 5.20 -0.26
δ (11-H) 5.95 5.90 +0.05 5.99 5.74 +0.25
δ (12-H’) 5.35 2.80 +2.55 5.35 2.67 +2.68
δ (12-H”) 5.40 2.80 +2.60 5.39 2.67 +2.72 δ (13-H) 2.27 2.16 +0.11 4.62, 3.98, +0.64, 5.27, 5.25, +0.02, 5.31, 5.30, +0.01, 5.98 5.89 +0.09 aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
81 Table 14 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 H3C O 7 7 O O O O 13 O 1 OR 6 O 5 4 2 5 O 6 3 4 1 H H 12 3 2 13 O 11 10 O OR 10 12 11 A E
R = COCH3 Carbon # R = CH2CH=CH2 A E A E ∆ (δ C)a ∆ (δ C)a (9A) (2E) (12A) (4E) δ (1-C) 167.6 170.9 -3.30 168.9 171.4 -2.50
δ (2-C) 114.6 73.8 +40.8 121.5 79.9 +41.6
δ (3-C) 159.5 201.0 -41.5 155.7 206.1 -50.4
δ (4-C) 75.3 82.1 -6.80 74.7 81.5 -6.80
δ (5-C) 73.7 73.7 0.00 74.0 74.2 -0.20
δ (6-C) 65.2 65.2 0.00 65.3 64.9 +0.40
δ (7-C) 110.6 110.8 -0.20 110.3 110.9 -0.60
δ (8-C) 25.5 25.5 0.00 25.6 25.4 +0.20
δ (9-C) 25.8 25.8 0.00 25.9 25.7 +0.20
δ (10-C) 72.5 120.8 -48.3 72.5 118.0 -45.5
δ (11-C) 131.0 128.3 +2.70 132.9 127.8 +5.10
δ (12-C) 119.6 34.5 +85.1 119.2 41.2 +78.0 δ (13-C) 20.3, 19.2, +1.10, 72.3, 69.2, +3.10, 166.8 169.7 -2.90 118.9, 122.2, -3.30, 131.9 133.2 -1.30
aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
82 Table 15 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 H3C O 7 7 O O O O 14 O 1 OR 6 5 4 2 5 O O 3 1 6 13 4 12 H CH3 H 3 2 14 O 11 O OR 10 12 10 11 A E H C 3 13
Proton # R = H R = COCH3 A E A E ∆ (δ H)a ∆ (δ H)a (3A) (5E) (8A) (6E) δ (4-H) 4.55 4.60 -0.05 4.66 4.51 +0.15
δ (5-H) 4.26 4.53 -0.27 4.36 4.51 -0.15
δ (6-H’) 4.02 4.07 -0.05 4.07 4.13 -0.06
δ (6-H’’) 4.13 4.18 -0.05 4.15 4.13 +0.02
δ (8-CH3) 1.37 1.34 +0.03 1.36 1.38 -0.02
δ (9-CH3) 1.40 1.40 0.00 1.40 1.48 -0.08
δ (10-H’) 4.89 5.26 -0.37 4.73 5.28 -0.55
δ (10-H’’) 4.89 5.28 -0.39 4.73 5.31 -0.58
δ (11-H) 5.90 5.75 +0.15 5.87 5.71 +0.16
δ (12-H’) 5.68 2.75 +2.93 5.62 2.90 +2.72
δ (13-H) 1.75 1.18 +0.57 1.76 1.22 +0.54
δ (14-H) - - - 2.27 2.16 +0.11
aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
83 Table 16 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- (1-methyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 8 H3C CH3 9 CH3 H3C O 7 7 O O O O 14 O 1 OR 6 5 4 2 5 O O 3 1 6 13 4 12 H CH3 H 3 2 14 O 11 O OR 10 12 10 11 A E H C 3 13
Carbon # R = H R = COCH3 A B A B ∆ (δ C)a ∆ (δ C)a (3A) (5E) (8A) (6E) δ (1-C) 171.8 172.8 -1.00 167.6 170.3 -2.70
δ (2-C) 119.1 74.4 +44.7 114.4 77.3 +37.1
δ (3-C) 148.6 205.7 -57.1 159.7 200.9 -41.2
δ (4-C) 75.7 81.8 -6.10 75.3 84.8 -9.50
δ (5-C) 74.4 74.3 +0.10 73.7 75.1 -1.40
δ (6-C) 65.3 64.8 +0.50 65.2 65.2 0.00
δ (7-C) 110.3 111.0 -0.70 110.5 110.0 +0.50
δ (8-C) 25.6 25.3 +0.30 25.5 25.3 +0.20
δ (9-C) 25.9 25.4 +0.50 25.7 26.7 -1.00
δ (10-C) 72.4 120.0 -47.6 72.6 120.6 -48.0
δ (11-C) 132.6 134.3 -1.70 133.2 132.9 +0.30
δ (12-C) 125.2 44.3 +80.9 124.0 41.4 +82.6
δ (13-C) 17.8 12.5 +5.30 17.7 12.3 +5.40 δ (14-C) - - - 167.0, 169.3, -2.30, 20.2 19.2 +1.00
aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
84 4.4.2 The C3-O to C2 Sigmatropic Claisen Rearrangement of 5,6-O-Isopropylidene-
3-O-Cinnamyl-L-Ascorbic Acid Derivatives
5,6-O-isopropylidene-2-O-acetyl-3-O-cinnamyl-L-ascorbic acid derivative (10A) was rearranged according to Scheme 16 to give 5,6-O-isopropylidene-2-O-acetyl-2-(1- phenyl-1-prop-2-enyl)-3-keto-L-galactono-γ-lactone, which is a C2-allylated aldono-γ- lactone derivative. Interestingly, when 5,6-O-isopropylidene-L-ascorbic acid (1) was treated with 2 molar equivalents of potassium tert-butoxide in DMSO/THF solvent system for 3 hours (Scheme 15), the reaction exclusively produced a C2-allylated aldono-
γ-lactone derivative, 5,6-O-isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3-keto-L- galactono-γ-lactone (7E). This reaction is unique to using cinnamyl chloride as the electrophilic reagent. For instance, when cinnamyl bromide was used under the same reaction condition, the product of this reaction was 5,6-O-isopropylidene-2-O-cinnamyl-
L-ascorbic acid (5A), a C3-O-allylated compound in place of the C2-allylated compound
(7E). The crude product of 10A was easily separated by careful silical gel chromatography followed by recrystallization to give a diastereomeric excess (9E) and a minor diastereomeric product (10E).
85 Scheme 15 Direct Synthesis of 5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-3- Keto-L-Galactono-γ-Lactone from 1
9 9 H3C 8 9 8 H3C 8 CH3 CH O 7 H3C CH3 O 3 7 6 O O 7 O 6 O 5 5 O H 4 6 5 O O 2Eq t-Buok, DMSO/THF O (Ac)2O /DMAP / Et 3N 1 O 1 H 4 3 4 1 2 O 3 H 2 2 O O 3 o Cinnamyl Chloride, 3 h O CH2Cl2, -79 C 13 HO OH (quant.) C6H5 12 O 12 OR CH3 13C6H5 11 10 11 10 1 7E 8E
Scheme 16 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-2-O-Acetyl-2-(1- phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 8 9 H C CH H3C 8 3 3 CH O 3 O 7 O 7 6 O 6 5 O O 5 4 1 O H 2 O (a) H 4 3 1 reflux / toluene 3 2 O O 10 O O 6h O 12 CH3 11 13 12 13 C6H5 O CH3 C6H5 11 10
10A E
Reactant E: Product (Major) E: Product (Minor) 10A 9E(b) 10E(b) (a)100% conversion of A to E was obtained in 6 h. (b)1H-NMR analysis of crude reaction mixtures indicated that the product is a mixtures of two diastereomers with > 75% of the major.
86 4.4.2.1 NMR Spectroscopic Analyses of Products from C3-O to C2 Sigmatropic
Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-Acetyl-3-O-Cinnamyl-L-
Ascorbic Acid Derivative (10A)
The 1H-NMR (Table 17) identifiable features of 5,6-O-isopropylidene-2-O-acetyl-3-O- cinnamyl-L-ascorbic acid derivatives (10A) after sigmatropic Claisen rearrangement showed distinctive chemical shift patterns that could be used in the identification of the products. For example, there is a downfield shift of the C10 proton signal by a range of
0.33 to 0.41 ppm in the rearranged products (9E & 10E) when compared to the starting material (10A). Also, the C12-H, C13-H and C14-H proton signals of the products shifted upfield by about 2.24 to 2.31 ppm, 0.04 to 0.08 ppm and 0.14 to 0.15 ppm respectively when compared to the starting material. The newly formed C2-C12 bond on the products resulted in an upfield chemical shift of the C11-H proton signals by about 0.28 to 0.37 ppm than the starting material. In general, the C3-β-ketone-lactone moiety on the products (E) resulted in the deshielding of the C4-H and C5-H proton signals when compared to the starting materials (A). However, the C4-H of 10E and C5-H of 9E showed a shielding of their proton signals by 1.00 ppm and 1.43 ppm respectively than the starting compound (10A).
87 Table 17 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2-O- Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9
9 8 H3C 8 H3C CH3 CH3 O 7 O 7 O 6 O 5 6 5 O O O 4 1 H 4 1 H 2 3 2 O 3 O 10 O OAC 13 12 12 C6H5 OAC 11 13 C6H5 11 10 A E Proton # A E ∆ (δ H)a E ∆ (δ H)a (10A) (9E) (10E)
δ (4-H) 4.70 4.75 -0.05 3.70 +1.00
δ (5-H) 4.40 2.97 +1.43 4.42 -0.02
δ (6-H’) 4.09 3.82 +0.27 4.05 +0.04
δ (6-H”) 4.17 3.97 +0.20 4.05 +0.12
δ (8-CH3) 1.36 1.27 +0.09 1.32 +0.04
δ (9-CH3) 1.41 1.45 -0.04 1.34 +0.07
δ (10-H’) 4.97 5.17 -0.20 5.31 -0.34
δ (10-H”) 4.97 5.30 -0.33 5.38 -0.41
δ (11-H) 6.71 6.43 +0.28 6.34 +0.37
δ (12-H) 6.29 4.05 +2.24 3.98 +2.31 δ (13-H) 7.30 7.24 +0.06 7.22 +0.08 7.40 7.36 +0.04 7.35 +0.05
aThe difference in 1H chemical shifts of A and E [∆ (δ H)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
88 Table 18 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-2- O-Acetyl-2-(1-phenyl-1-prop-2-enyl)-3-Keto-L-Galactono-γ-Lactone
9 9 8 H C H C CH 3 8 3 3 CH3 O 7 7 O O 6 O 5 6 5 O O 1 O 4 H 4 H 2 1 3 3 2 O 10 O OAC O 13 12 C H 12 OAC 11 6 5 13 C H 6 5 11 10 A E Carbon # A E ∆ (δ C)a E ∆ (δ C)a (10A) (9E) (10E)
δ (1-C) 167.6 169.3 -1.70 169.1 -1.50
δ (2-C) 114.7 76.0 +38.7 76.7 +38.0
δ (3-C) 159.7 201.5 -41.8 201.8 -42.1
δ (4-C) 75.3 83.9 -8.60 85.1 -9.80
δ (5-C) 73.6 72.7 +0.90 75.0 -1.40
δ (6-C) 65.2 64.9 +0.30 65.1 +0.10
δ (7-C) 110.6 110.2 +0.40 109.8 +0.80
δ (8-C) 25.5 25.4 +0.10 25.3 +0.20
δ (9-C) 25.7 26.4 -0.70 26.6 -0.90
δ (10-C) 72.6 119.9 -47.0 121.0 -48.4
δ (11-C) 135.5 134.9 +0.60 133.8 +1.70
δ (12-C) 121.6 51.3 +70.0 52.9 +68.7 δ (13-C) 126.7, 128.5 -1.80 128.8 -2.10 128.6, 129.3 -0.70 129.1 -0.50 128.7, 129.8 -1.10 129.3 -0.60 129.3 132.5 -3.20 131.1 -1.80
aThe difference in 13C chemical shifts of A and E [∆ (δ C)] were calculated by subtracting the chemical shifts of A from the corresponding values of the Claisen compound E
89 The differences in the C4-H and C5-H chemical shifts of 9E and 10E (Table 17) are possibly due to the nature of the combined steric influence of the bulky substituents at their C2, C4 and C12 positions. The comparative 13C NMR signals of C3-O (A: 10A) to
C2 (E: 9E & 10E) sigmatropic Claisen rearrangement (Table 18) revealed distinctive chemical shift patterns that are useful in the identification of products. Notable examples are the C10 (~ 47.3 to 48.4 ppm downfield), C12 (~ 68.7 to 70.0 ppm upfied), C2 (~ 38.0 to 38.7 ppm upfield) and C3 (~ 41.8 to 42.1 ppm downfield) carbon signals of the products when they were compared to the starting material. These distinctive chemical shift patterns are diagnostic of the disappearance of the conjugated enone moiety of the starting material (A) and the formation of products (E) without the enone moiety and thus the reason for their poor UV-activity. The C3-carbonyl signals of the β-keto-ester group
(E, 200.9 to 205.7 ppm) and their lactone C1-carbonyl (E, 170.3-172.8 ppm) are in the same range of typical β-keto-γ-lactone carbonyl NMR signals, which usually appear around 200 ppm and 170 ppm respectively. 208-210,215-216
4.4.3 The C2-O to C3 Sigmatropic Claisen Rearrangement of 5,6-O-
Isopropylidene-2-O-Allyl-L-Ascorbic Acid Derivatives
5,6-O-isopropylidene-2-O-allyl-L-ascorbic acid derivatives were subjected to sigmatropic Claisen rearrangement according to Scheme 17 to give products with an α- keto-ester functional group known as 5,6-O-isopropylidene-3-allyl-2-keto-L-galactono-γ- lactone. This compound is a C3-substituted aldono-γ-lactone derivative and the 1H- and
13C-NMR analysis of the crude products revealed a diastereomeric excess (>95%). The
90 products are very visible in iodine and are very sensitive to hydrolysis in silica gel chromatography even under very careful conditions.
4.4.3.1 NMR Spectroscopic Analyses of Products from C2-O to C3 Sigmatropic
Claisen Rearrangement of 5,6-O-Isopropylidene-2-O-Allyl-L-Ascorbic Acid
Derivatives
The comparative 1H-NMR signals (Table 19, 21, 23 & 25) of the series of 5,6-O- isopropylidene-2-O-allylic derivatives of L-ascorbic acid (C) after sigmatropic Claisen rearrangement to produce their corresponding products (F), showed disntintive chemical shift patterns that could be used to identify this series of compounds. For example, the
C10 methylene proton signals of the products showed a downfield chemical shift of about
0.45 to 0.77 ppm when compared to the corresponding starting materials (C). Also, as a result of the newly created C3-C12 bond on the products (F), their C11-H, C12-H and
C13-H proton signals showed an upfield chemical shift in the ranges of 0.01 to 0.29 ppm, 2.40 to 2.96 ppm, and 0.07 to 0.64 ppm respectively, when compared to their corresponding starting materials. The C2-α-ketone-ester moiety on the products showed in general the deshielding of the C4-H and C5-H proton signals when compared to their corresponding starting materials. However, 3F and 6F both showed a moderate shielding of their C4-H and C5-H proton signals, perhaps due to the combined steric and electronic influence of the bulky substituents at C4 and the acetate group at C3 position.
91 Scheme 17 Synthesis of Claisen Rearranged 5,6-O-Isopropylidene-3-(1-prop-2-enyl)-2- Keto-L-Galactono-γ-Lactone
8 9 8 9 H C CH CH3 3 3 H3C 7 7 O O O O O O 1 5 4 6 5 O O 6 2 1 3 4 (a) H O 3 2 reflux / toluene H 14 13 12 R2O R1 14 O R2O 11 10 10 11 C 12 F
13 R1
Reactant R1 R2 Product
1C H H 1F
7C H COCH3 2F
12C H CH3 3F
15C H CH2C6H5 4F
3C CH3 H 5F
9C CH3 COCH3 6F
13C CH3 CH3 7F
(a)100% conversion of C to F was obtained in 24 h. 1H-NMR analysis of the crude reaction mixtures indicated that only a single diastereomer was detectable
The comparative 13C NMR signals of C2-O (C) to C3 (F) sigmatropic Claisen rearrangement (Table 20, 22, 24 & 26) showed distinguishable chemical shift patterns that are useful for the identification of the series of compounds. For instances, the C10 carobn signals of the products revealed a downfield chemical shift ranging from 46.3 to
92 50.6 ppm when compared to the corresponding starting materials. Also, the newly formed
C3-C12 bond on the products resulted in a large shielding (80.0 to 84.0 ppm) of the non- oxygen bearing C12 carbon signals when compared to the starting materials.
Furthermore, the C2 and C3 carbon signals of the products appear in the ranges of 186.3 to 195.3 ppm (~ 55.0 to 74.5 ppm downfield) and 77.2 to 79.3 ppm (~ 65.0 to 79.2 ppm upfield) respectively, when compared to the corresponding starting materials. These significant chemical shift patterns reflect the disappearance of the conjugated enone moiety in the products and these carbon signals are within the same range of typical α- keto-γ-lactone NMR signals.208-210,215-216
93 Table 19 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 9 CH3 H3C CH3 H3C 7 7 O O O O O O 1 5 4 6 5 O 6 2 O 3 4 1 H 3 2 O H 13 RO 12 13RO O 11 10 10 11 C 12 F
Proton # R = H R = CH3 C F C F ∆ (δ H)a ∆ (δ H)a (1C) (1F) (12C) (3F) δ (4-H) 4.72 4.69 +0.03 4.52 4.58 -0.06
δ (5-H) 4.43 4.53 -0.10 4.28 4.49 -0.21
δ (6-H’) 4.02 4.09 -0.07 4.03 3.99 +0.04
δ (6-H”) 4.16 4.16 0.00 4.13 4.14 -0.01
δ (8-CH3) 1.38 1.29 +0.09 1.36 1.33 +0.03
δ (9-CH3) 1.43 1.32 +0.11 1.39 1.33 +0.03
δ (10-H’) 4.62 5.24 -0.62 4.61 5.26 0.65
δ (10-H”) 4.62 5.30 -0.68 4.61 5.31 -0.70
δ (11-H) 5.98 5.81 +0.17 6.00 5.75 +0.25
δ (12-H’) 5.28 2.53 +2.75 5.28 2.51 +2.77
δ (12-H”) 5.36 2.53 +2.83 5.36 2.66 +2.70
δ (13-H) - - - 4.15 3.70 +0.45
aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
94 Table 20 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 9 CH3 H3C CH3 H3C 7 7 O O O O O O 1 5 4 6 5 O 6 2 O 3 4 1 H 3 2 O H 13 RO 12 13RO O 11 10 10 11 C 12 F
Carbon # R = H R = CH3 C F C F ∆ (δ C)a ∆ (δ C)a (1C) (1F) (12C) (3F) δ (1-C) 168.8 159.4 +9.40 169.9 159.9 +10.0
δ (2-C) 121.4 193.9 -72.5 121.5 192.2 -70.7
δ (3-C) 156.4 77.2 +79.2 157.1 79.3 +77.8
δ (4-C) 73.9 80.8 -6.90 74.5 79.5 -5.00
δ (5-C) 73.8 73.1 +0.70 73.8 72.5 +1.30
δ (6-C) 64.9 64.4 +0.50 64.2 65.0 -0.80
δ (7-C) 110.6 111.3 -0.70 110.3 111.3 -1.00
δ (8-C) 25.2 24.7 +0.50 25.5 24.7 +0.80
δ (9-C) 25.7 24.8 +0.90 25.8 25.2 +0.60
δ (10-C) 72.1 121.9 -49.80 72.7 121.7 -49.0
δ (11-C) 133.0 129.0 +4.00 132.8 129.1 +3.70
δ (12-C) 119.6 39.6 +80.0 119.3 35.0 +84.0
δ (13-C) - - - 59.6 52.8 +6.80
aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
95 Table 21 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 9 CH3 H3C CH3 H3C 7 7 O O O O O O 1 5 4 6 5 O 6 2 O 3 4 1 H 3 2 O H 13 RO 12 13RO O 11 10 10 11 C 12 F
Proton # R = COCH3 R = CH2C6H5 C F C F ∆ (δ H)a ∆ (δ H)a (7C) (2F) (15C) (4F) δ (4-H) 5.18 4.94 +0.24 4.55 4.63 -0.08
δ (5-H) 4.29 4.28 +0.01 4.30 4.55 -0.25
δ (6-H’) 4.02 4.04 -0.02 4.03 4.00 +0.03
δ (6-H”) 4.15 4.14 +0.01 4.11 4.11 0.00
δ (8-CH3) 1.36 1.32 +0.04 1.36 1.34 +0.02
δ (9-CH3) 1.38 1.34 +0.04 1.38 1.35 +0.03
δ (10-H’) 4.78 5.23 -0.45 4.54 5.30 -0.76
δ (10-H”) 4.78 5.23 -0.45 4.54 5.31 -0.77
δ (11-H) 5.97 5.68 +0.29 5.94 5.82 +0.12
δ (12-H’) 5.28 2.76 +2.52 5.26 2.57 +2.69
δ (12-H”) 5.37 2.97 +2.40 5.34 2.75 +2.59 δ (13-H) - - - 5.48 4.90 +0.58 5.48 5.25 +0.23 7.37 7.30 +0.07
aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
96 Table 22 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 9 CH3 H3C CH3 H3C 7 7 O O O O O O 1 5 4 6 5 O 6 2 O 3 4 1 H 3 2 O H 13 RO 12 13RO O 11 10 10 11 C 12 F
Carbon # R = COCH3 R = CH2C6H5 C F C F ∆ (δ C)a ∆ (δ C)a (7C) (2F) (15C) (4F) δ (1-C) 166.4 158.2 +8.20 168.8 159.8 +9.00
δ (2-C) 130.1 186.3 -56.0 121.4 192.2 -70.8
δ (3-C) 143.8 78.4 +65.0 155.9 79.3 +76.6
δ (4-C) 74.5 81.1 -6.60 74.7 79.4 -4.70
δ (5-C) 72.8 72.6 +0.20 73.9 72.7 +1.20
δ (6-C) 65.1 65.1 0.00 65.2 64.9 +0.30
δ (7-C) 110.5 111.1 -0.60 110.3 111.2 -0.90
δ (8-C) 25.3 25.0 +0.30 25.6 24.9 +0.70
δ (9-C) 25.6 25.2 +0.40 25.8 25.4 +0.40
δ (10-C) 71.3 121.9 -50.6 72.5 121.9 -49.4
δ (11-C) 132.4 128.1 +4.30 132.8 137.5 -4.70
δ (12-C) 118.9 36.9 +82.0 119.3 36.3 +83.0 δ (13-C) 20.6 20.0 +0.60 73.5, 67.1, +6.40, 127.7, 127.5, +0.20, 128.5, 127.9, +0.60, 128.7, 128.4, +0.30, 129.1 129.2 -0.10 aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
97 Table 23 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 H C CH 9 3 3 CH3 H3C O 7 O 7 O O O O 6 5 O O 1 4 1 5 4 6 2 H 3 2 H 3 O 13 14 12 14 RO O RO CH3 10 11 11 10
12 C CH 13 3 F Proton # R = H C F ∆ (δ H)a (3C) (5F) δ (4-H) 4.69 4.75 -0.06
δ (5-H) 4.39 4.52 -0.13
δ (6-H’) 4.05 4.08 -0.03
δ (6-H’’) 4.17 4.18 -0.01
δ (8-CH3) 1.37 1.29 +0.08
δ (9-CH3) 1.41 1.32 +0.10
δ (10-H’) 4.49 5.18 -0.69
δ (10-H’’) 4.49 5.18 -0.69
δ (11-H) 5.80 5.79 +0.01
δ (12-H’) 5.64 2.71 +2.93
δ (13-H) 1.71 1.15 +0.56 δ (14-H) - - -
aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
98 Table 24 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 H3C CH3 9 CH3 H3C O 7 O 7 O O O O 6 5 O O 1 4 1 5 4 6 2 H 3 2 H 3 O 13 14 12 14RO O RO CH3 10 11 11 10
12 C CH 13 3 F Carbon # R = H C F ∆ (δ C)a (3C) (5F) δ (1-C) 170.1 159.5 +10.6
δ (2-C) 120.8 195.3 -74.5
δ (3-C) 157.9 78.9 +79.0
δ (4-C) 74.6 80.0 -5.40
δ (5-C) 73.9 73.4 +0.50
δ (6-C) 65.0 64.4 +0.60
δ (7-C) 110.4 111.2 -0.80
δ (8-C) 25.3 24.8 +0.50
δ (9-C) 25.7 24.9 +0.80
δ (10-C) 72.3 118.6 -46.3
δ (11-C) 132.6 135.3 -2.70
δ (12-C) 125.8 43.8 +82.0
δ (13-C) 17.8 13.2 +4.60
δ (14-C) - - -
aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
99 Table 25 1H-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 H C CH 9 3 3 CH3 H3C O 7 O 7 O O O O 6 5 O O 1 4 1 5 4 6 2 H 3 2 H 3 O 13 14 12 14 RO O RO CH3 10 11 11 10
12 C CH 13 3 F
Proton # R = COCH3 R = CH3 C F C F ∆ (δ H)a ∆ (δ H)a (9C) (6F) (13C) (7F) δ (4-H) 5.18 4.83 +0.35 4.51 4.63 -0.12
δ (5-H) 4.28 4.30 -0.02 4.27 4.45 -0.18
δ (6-H’) 4.01 4.04 -0.03 4.03 3.98 +0.05
δ (6-H’’) 4.14 4.13 +0.01 4.13 4.14 -0.01
δ (8-CH3) 1.36 1.33 +0.03 1.36 1.33 +0.03
δ (9-CH3) 1.38 1.35 +0.03 1.39 1.33 +0.06
δ (10-H’) 4.70 5.22 -0.50 4.52 5.14 -0.62
δ (10-H’’) 4.70 5.22 -0.52 4.52 5.17 -0.65
δ (11-H) 5.83 5.73 +0.10 5.82 5.69 +0.13
δ (12-H’) 5.64 3.17 +2.47 5.66 2.70 +2.96
δ (13-H) 1.73 1.10 +0.63 1.74 1.10 +0.64
δ (14-H) 2.31 2.16 +0.15 4.14 3.73 +0.41
aThe difference in 1H chemical shifts of C and F [∆ (δ H)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
100 Table 26 13C-NMR Chemical Shifts (δ) of Claisen Rearranged 5,6-O-Isopropylidene-3- (1-methyl-1-prop-2-enyl)-2-Keto-L-Galactono-γ-Lactone
8 9 8 H C CH 9 3 3 CH3 H3C O 7 O 7 O O O O 6 5 O O 1 4 1 5 4 6 2 H 3 2 H 3 O 13 14 12 14 RO O RO CH3 10 11 11 10
12 C CH 13 3 F
Carbon # R = COCH3 R = CH3 C F C F ∆ (δ C)a ∆ (δ C)a (9C) (6F) (13C) (7F) δ (1-C) 166.5 158.0 +8.50 169.1 159.8 +9.30
δ (2-C) 132.2 187.5 -55.0 121.4 192.6 -71.2
δ (3-C) 143.8 77.3 +66.0 157.3 78.5 +78.8
δ (4-C) 74.4 79.7 -5.30 74.5 81.1 -6.60
δ (5-C) 72.9 73.4 -0.50 73.9 72.9 +1.00
δ (6-C) 65.0 65.5 -0.50 65.2 65.2 0.00
δ (7-C) 110.5 111.0 -0.50 110.3 111.2 -0.90
δ (8-C) 25.3 25.2 +0.10 25.6 24.9 +0.70
δ (9-C) 25.6 25.5 +0.10 25.8 25.4 +0.40
δ (10-C) 71.4 119.7 -48.3 72.2 119.3 -47.1
δ (11-C) 130.1 134.7 -4.60 132.4 135.9 -3.50
δ (12-C) 125.4 42.1 +83.3 125.8 42.3 +83.5
δ (13-C) 17.7 12.8 +4.90 17.8 13.6 +4.20 δ (14-C) 20.5 19.9 +0.60 59.6 54.6 +5.00 166.3 170.9 -4.60
aThe difference in 13C chemical shifts of C and F [∆ (δ C)] were calculated by subtracting the chemical shifts of C from the corresponding values of the Claisen compound F
101 4.5 Comparative Analysis and Identificaton of Products of C3-O to C2 and C2-O to C3 Claisen Rearrangement of L-Ascorbic Acid Derivatives
The Claisen rearrangement of C3-O-allyl ascorbate derivatives listed in Table 11-
18 quantitatively produced the C2-allylated products in about 6 h in boiling toluene. The
1H-NMR analysis of the crude reaction mixtures revealed that both possible diastereomers were produced with more than 75% diastereomeric excess. In contrast to the smooth rearrangement of C3-O-allyl ascorbate derivatives under relatively mild conditions (Scheme 14-16), the rearrangement of their C2-O-allyl counterparts (Scheme
17) was found to be much slower and required much more drastic reaction conditions.
For example, C2-O-allyl ascorbate derivatives typically require about 12 h to 24 h refluxing in toluene for the complete conversion in comparison to 6 h for corresponding
C3-O-allyl ascorbate derivatives. In addition, only a single diastereomer was detected by
1H-NMR analysis of the crude rearranged reaction mixtures of C2-O-allyl derivatives
(Table 19-26). Sterically more hindered C2-O-allyl ascorbate derivatives such as 11C &
14C do not undergo any significant rearrangement, even in boiling xylene or styrene for
72 h.
102 Table 27 Comparison of Diastereoisomers of Allyl-L-Galactono-γ-Lactone
9 8 9 8 9 8 H C CH H C 3 3 8 3 CH3 H3C CH3 9 CH3 7 7 O 7 O O H3C O O O O 7 O 14 6 6 O O O 1 5 O 6 5 O 5 Reflux / toluene O 4 OR O O 4 1 2 2 4 1 Reflux / toluene 4 1 5 13 13 6 3 H H 2 H 2 R1 3 2 R 3 3 14 1 10 H 12 10 OR2 11 R2O O 12 O O O 14 OR2 11 12 14 12 11 10 11 10 13 A E R1 F R1 C 13
Carbon # R1 = H & R2 = H R1 = H & R2 = COCH3 A E ∆a C F ∆a A E ∆a C F ∆a
(4A) (1E) (δ C) (1C) (1F) (δ C) (9A) (2E) (δ C) (7C) (2F) (δ C)
δ (1-C) 171.0 172.6 -1.60 168.8 159.4 +9.40 167.6 170.9 -3.30 166.4 158.2 +8.20 δ (2-C) 119.2 74.5 +44.7 121.4 193.9 -72.5 114.6 73.8 +40.8 130.1 186.3 -56.0 δ (3-C) 148.2 205.5 -57.3 156.4 77.2 +79.2 159.5 201.0 -41.5 143.8 78.4 +65.0 δ (4-C) 75.6 81.5 -5.90 73.9 80.8 -6.90 75.3 82.1 -6.80 74.5 81.1 -6.60 δ (5-C) 74.3 72.0 +2.30 73.8 73.1 +0.70 73.7 73.7 0.00 72.8 72.6 +0.20 δ (6-C) 65.3 64.8 +0.50 64.9 64.4 +0.50 65.2 65.2 0.00 65.1 65.1 0.00 δ (10-C) 72.3 122.9 -50.6 72.1 121.9 -49.8 72.5 120.8 -48.3 71.3 121.9 -50.6 δ (11-C) 132.2 127.6 +4.60 133.0 129.0 +4.00 131.0 128.3 +2.70 132.4 128.1 +4.30 δ (12-C) 119.1 39.8 +79.0 119.6 39.6 +80.0 119.6 34.5 +85.1 118.9 36.9 +82.0 δ (13-C) ------20.6 20.0 +0.60 ------20.3 19.2 +1.10 - - - δ (14-C) ------166.8 169.7 -2.90 - - -
Carbon # R1 = H & R2 = CH3 R1 = CH3 & R2 = COCH3 A E ∆a C F ∆a A E ∆a C F ∆a
(11A) (3E) (δ C) (12C) (3F) (δ C) (8A) (6E) (δ C) (9C) (6F) (δ C)
δ (1-C) 171.8 172.8 -1.00 170.1 159.5 +10.6 167.6 170.3 -2.70 166.5 158.0 +8.50 δ (2-C) 119.1 74.4 +44.7 120.8 195.3 -74.5 114.4 77.3 +37.1 132.2 187.5 -55.0 δ (3-C) 148.6 205.7 -57.1 157.9 78.9 +79.0 159.7 200.9 -41.2 143.8 77.3 +66.0 δ (4-C) 75.7 81.8 -6.10 74.6 80.0 -5.40 75.3 84.8 -9.50 74.4 79.7 -5.30 δ (5-C) 74.4 74.3 +0.10 73.9 73.4 +0.50 73.7 75.1 -1.40 72.9 73.4 -0.50 δ (6-C) 65.3 64.8 +0.50 65.0 64.4 +0.60 65.2 65.2 0.00 65.0 65.5 -0.50 δ (10-C) 72.4 120.0 -47.6 72.3 118.6 -46.3 72.6 120.6 -48.0 71.4 119.7 -48.3 δ (11-C) 132.6 134.3 -1.70 132.6 135.3 -2.70 133.2 132.9 +0.30 130.1 134.7 -4.60 δ (12-C) 125.2 44.3 +80.9 125.8 43.8 +82.0 124.0 41.4 +82.6 125.4 42.1 +83.3 δ (13-C) 17.8 12.5 +5.30 17.8 13.2 +4.60 17.7 12.3 +5.40 17.7 12.8 +4.90 ------20.2 19.2 +1.00 20.5 19.9 +0.60 δ (14-C) ------167.0 169.3 -2.30 166.3 170.9 -4.60 aThe difference in 13C-NMR chemical shifts (∆) was calculated by subtracting the chemical shifts of various derivatives (E & F) from their corresponding starting materials (A & C).
103 The relative unfavorability for the rearrangement of C2-O-allyl in comparison to C3-O- allyl derivatives could be due to a combination of steric and electronic effects. First, the steric constraints on the transition state for the C2-O to C3 allyl migration (Scheme 14-
16) is more pronounced relative to that of the C3-O to C2 migration (Scheme 17), due to the presence of a bulky 1,2-O-isopropylidene-1,2-ethanodiol moiety at the C4 of the molecule. Second, the relatively high lability of C3-O-allylic ether linkage compared to that of the C2-O-allylic ether linkage due to the direct interaction of the C3-O with the conjugated enone moiety could also facilitate the rearrangement to produce the thermodynamically more stable C2-allylated products. Therefore, the facile rearrangement of the C3-O-allyl derivatives in comparison to the non-catalyzed conventional Claisen rearrangement which requires high temperatures (150-240 oC) suggests that the thermodynamic lability of C3-O-allyic ether bond facilitates an efficient rearrangement to the C2 allylated products. This notion is further supported by the observation that the diallyl ascorbate derivative such as 12A exclusively rearranged to produce the C2-allylated product, 4E, and not the potential, corresponding C3-allylated product.
A comparative 13C-NMR spectra analysis of C3-O to C2 and C2-O to C3 (Table
27) rearranged products show some interesting and characteristic features which could be used in their unequivocal identification. For example, the C3 and C2 carbonyl signals of two series of rearranged products, E and F, appear in the ranges of 205.7–200.9 and
186.3–195.3 respectively. The significant upfield shift of the 13C-NMR signals of C2 carbonyls of F series must be due to the electronic effects of the adjacent C1 lactone- carbonyl in comparison to that of the isolated C3 carbonyl group of E series. Similarly,
104 the C1 13C-NMR carbonyl signals of the two series were also quite distinguishable and appear in the ranges of 170.3–172.8 for E and 158–159.4 for F, again due to the electronic effects of the C2 carbonyl of F in comparison to that of the C3 carbonyl of
E.215-216 Interestingly, C4 13C-NMR signals of the two series of rearranged products were not significantly different. However, C4 13C-NMR signals of both E and F series show a significant downfield shift (∆δ in the range of 5.3–9.5) in comparison to the corresponding starting materials (A and C). This most likely reflects the deshielding effects of the disappearance of the conjugated enone moiety of the starting materials due to the rearrangement. Therefore, the comparative analysis of the 13C-NMR characteristics of the starting materials and their products could conveniently be used to distinguish between the two series of Claisen rearranged products (E and F).
4.6 Stereochemistry of Products of C3-O to C2 and C2-O to C3 Claisen
Rearrangement of L-Ascorbic Acid Derivatives
The intrinsic stereochemistry at C4 and C5 positions of all the starting materials
(A & C series), rearranged products (E & F series) and their derivatives are fixed, since
L-ascorbic acid is used in all cases. The stereochemistry of the C2 of the Claisen- rearranged products (E) was assigned by their NMR spectroscopic characteristics and confirmed by X-ray crystallographic studies. 191 For example, we unequivocally established that the least-bulky C3-O-allyl Claisen substrate, 4A (A series), yielded a major diastereomer (~90%) which is C2 allylated from the bottom face of the lactone ring
(A to E rearrangement). Therefore, we believe the preferential migration of the allylic
105 functionality from the bottom face of the lactone ring must be primarily due to the steric constraints imposed by the bulky C4 side chain (1,2-O-isopropylidene-1,2-ethanediol moiety) on the top face of the molecule. Based on these arguments, we also concluded that C2-O-allyl (C series) Claisen rearrangements must exclusively occur from the bottom face of the molecule, since the steric constraints of the C4 bulky substituent must even be more pronounced for the C2-O to C3 migrations (C to F rearrangement) in comparison to C3-O to C2 migration (A to E rearrangement). The importance of the C4 bulky side chain group (1,2-O-isopropylidene-1,2-ethanediol moiety) in the control of the new stereogenic center was more profound in the C2-O to C3 Claisen rearrangement
(Scheme 17). For example, C2-O-crotyl (3C, 9C, & 13C) Claisen substrates rearranges to give two additional chiral centers at their C3 and C12 positions (5F, 6F & 7F) and therefore could have four isomers. However, since we detected only one major product
(~98%) from these Claisen substrates, it is safe to conclude that the Claisen rearrangement went through the usual chairlike transition state conformation to mainly produce products having the migrated C2-O-allylic group (crotyl) in opposite position
(trans) to the C4 bulky side chain group, especially given that they are brought much more closer to each other than in the C3-O to C2 migrated products (Scheme 14-16).
However, 5,6-O-isopropylidene-2-O-trans-cinnamyl-3-O-acetyl-L-asorbic acid (11C) and
5,6-O-isopropylidene-2-O-trans-cinnamyl-3-O-methyl-L-asorbic acid (14C) were not susceptible to sigmatropic rearrangement even under vigorous conditions (refluxing in xylene and styrene for over 72 h).
106 Scheme 18 C3-O (A) to C2 (E) Claisen Rearrangement Transition-State Geometry
C H 3CCH3 4 CH 3 H 3C O H Minor O 14 O O H R1 O C1 Pathway O (S) 1 OR 2 4 2 O 1 O (R) (S) 4 = O 3 H O H (S) C 13 H H 12 3 2 H O R1 R1 OR 2 11 threo R2O Boat (A) 10CH 2 C 4 C 1 (7E & 8E) H 3CCH3 C 4 10 14 OR H 2C O 2 H H H O O Major CH 3 H C O E,Z O = C 1 Pathway 3 12C C 11 O O 14 4 1 O 1 OR O O (S) OR 2 13 H H 3 2 2 4 2 R Schemes 15-17 (R) (S) 1 R 3 H O R1 H 1 H OR H (R) C 13 2 O 12 R1 erythro Chair 11
CH 2 10 (E)
Scheme 19 C2-O (C) to C3 (F) Claisen Rearrangement Transition-State Geometry
H 3C CH (C) 3 9 O (S) O C 4 C 1 H C 8 3 (R) CH 3 C 1 O 7 14 10 O H 4 1 O H 14 3 2 R2O OCH 6 O H Minor 2 5 R1 R O C4 Pathway 2 (R) H H 4 O (S) O 12 1 O Z,E 11 CC O = 13 C O R 1 12 11 H 13 H R 3 H 1 R1 2 H R O OR 2 10 CH 2 threo Boat 2
9 9 8 H C 8 C1 H3C 3 CH 3 CH 3 O 7 7 H H O O O Major (S) 6 C 4 Pathway 6 5 5 (R) O O = O 4 1 O H 4 14 H 1 O 3 2 3 R O R2O 2 2 H Schemes 19-20 R2O (R) R 1 O R H (R) O erythro 1 Chair C 13 R 1 12 11 H 10 CH 2 (F)
107 This is in sharp contrast to 5,6-O-isopropylidene-2-O-acetyl-3-O-trans-cinnamyl-L- asorbic acid (10A), which was susceptible to facile Claisen rearrangement even under very mild conditions (at room temperature, 100% conversion was obtained within 3 weeks) to yield two diastereomeric products with a ratio similar to that obtained under reflux in toluene for 6 h (Scheme 16). Since the rearrangement of C2-O-trans-crotyl (3C,
9C & 13C), C3-O-trans-crotyl (3A & 8A) and C3-O-trans-cinnamyl (10A) derivatives generate an additional chiral center at their C12 positions, we have thoroughly investigated these reactions in order to establish the stereochemistry of the C12 site on these derivatives. The factors affecting the stereochemical outcome of the products of thermal Claisen rearrangement have been extensively studied and established that the stereochemistry of the newly generated stereogenic centers is controlled by the steric and electronic features of the system in the transition state.192-204 The restriction imposed by the orbital symmetry rules on the highly ordered cyclic transition state allows excellent prediction of the stereochemical result.203 One of the well established strategies that has been developed to control the stereoselectivity of Claisen rearrangement, is the intraannular stereo-selection (i.e., the stereogenic elements accounting for selectivity are incorporated into the cyclic structure of the transition state) by achiral auxiliary (i.e., e.g.
CH3 of crotyl derivatives and C6H5 of cinnamyl derivatives) intrinsic to the stereochemistry of the vinylic double bond. Therefore, this makes it possible to determine the stereoselectivty of the rearrangement process regardless of the optical purity of the product’s newly created chiral centers, in which case, the E/Z selectivity rule of the new double bond is considered. The Claisen rearrangement progresses preferentially through a chair-like transition state in order to minimize the steric interactions of various
108 substituents as illustrated in Schemes 18 & 19. Thus, the relative stereochemistry
(erythro/threo) at the newly generated allylic stereo-centers is controlled by the relative geometry of the allylic double bond of the Claisen substrate.203 Therefore, as shown in
Schemes 18 & 19, (Z,Z) and (E,E) Claisen substrates produce threo products, while (E,Z) and (Z,E) Claisen substrates produce erythro as the major products. Based on these arguments, and since the allylic functionality migrates from the bottom face of the lactone ring during the C3-O to C2 and C2-O to C3 rearrangements as argued above, we conclude that the major isomer of the Claisen-rearranged products of trans-crotyl (3A,
8A, 3C, 9C & 13C) and trans-cinnamyl (5A) derivatives must have R stereochemistry at their C12 as shown in Schemes 18 & 19. In cyclic systems such as the five-membered lactone ring, conformational constraints can take priority over the intrinsic preference for the chair-like transition state in both Cope and Claisen rearrangements and consequently lead to a partial involvement if not the dominance of a boat-like transition state structure,
204 which might explain why 10A produced 9E (S stereochemistry at C12) and 10E (R stereochemistry at C12) as major and minor diastereomeric isomers respectively (Scheme
18). 1H- and 13C-NMR of the C2-O acetylation product of 7E revealed this product, 8E as the same minor diastereomeric compound 10E, that was obtained as a minor product from 10A Clasien rearrangement. The NMR signal patterns, the selective 2D-NMR spectra and X-ray structures helped to unequivocally identify all the Claisen rearranged products (E & F Series).
109 4.7 Chemo- and Diastereo-Selective Reduction of Claisen Rearranged Products (E &
F Series) of L-Ascorbic Acid Derivatives
In order to further explore the chemistry and synthetic utility of the Claisen- rearranged products E and F, we carried out the selective reduction of their C3- and C2- keto groups by conventional methods. The reduction of the major isomers of the rearranged products of the E series (Scheme 20) with sodium borohydride in ethanol proceeds with high chemo- and diastereo-selectivity in producing a ~7:1 diastereomeric mixtures of alcohol products, G Series (diastereomeric excess), in almost quantitative yield. The diastereomeric mixture was easily separated by normal phase silica gel column chromatography using ethyl acetate/hexane solvent system. Subsequent acetylation of G under the reaction conditions of Scheme 20 gave the corresponding acetates (H Series) in quantitative yield. In contrast to the E series, the attempts made to reduce the rearranged
C2-keto products (F series) gave uncharacterizable complex reaction mixtures under the same conditions. However, under carefully controlled conditions, we were able to obtain a clean reduced product I from 2F in 60% yield (Scheme 21) after careful silica gel chromatography with ethyl acetate/hexane solvent system.
The structures of the diastereomeric excess of sodium borohydride reduced products (G) and their acetates (H) were unequivocally identified by their 1H- and 13C-
NMR characteristics. The stereochemistry of reduction by NaBH4, which is a nucleophile, although with relatively small steric demand, delivered the hydride from the less hindered direction perhaps due to the bulkier C4 substituent (1,2-O-isopropylidene-
110 1,2-ethanediol moiety on the top face of the molecule) generating cis alcohol diastereomers (G), which were identified by NOESY-NMR studies.
Scheme 20 The Reduction Products of 5, 6-O-Isopropylidene-2-Allyl-3-Keto-L-
Galactono-γ-Lactones (E)
CH3 CH3 H C O H C O 3 O 3 O O O O 1 O 1 4 OH* OAc* NaBH4, EtOH 5 2 (Ac)2O /DMAP / Et3N 5 4 2 6 3 6 3 E H H H R o H R HO CH2Cl2, -79 C AcO (quant.) G H
(E) (G) (H) R
1E 1G 1H H
4E 4G 4H H
*H = CH2CH=CH2 *Ac = CH2CH=CH2
5E 5G 5H CH3
7E 7G 7H C6H5
It is understood that the involvement of sodium ions from NaBH4 is not required for reduction to take place, 205-207 however; due to the inherent steric constraints of E, sodium metal ions may well have played a crucial role in the stereochemical course of the reduction to produce G. An elegant but ponderable explanation for the stereochemical results (Scheme 20) is that reduction of starting materials (E) may possibly proceed via a
111 well-known chelation205-207 in which the sodium ion is coordinated by the β-carbonyl oxygen atom (C3-O) and the oxygen atom of the α-hydroxy group of the E series (OR or
OHI) as depicted in Figure 13, thus, favoring and strengthening the diastereoselective formation of products (G). Subsequent acetylation of these compounds (G) according to
Scheme 20 gave the corresponding acetate products (H) in quantitative yield.
Scheme 21 The Reduction Product of 5,6-O-Isopropylidene-3-Allyl-2-Keto-L- Galactono-γ-Lactone 2F
O H3C O O 1 H H3C 2 NaBH4, EtOH 4 3 2F O 5 OH 6 H O
OCH3 I
Figure 13 Diastereoselective Reduction of C3-keto of E Series via Metal Chelation
O H OH H'O Na OH'
2 O C 10 - 3 H C10 OH' C C 10 4 C4 C4
The nucleophile, H-, approaches C3-keto of the much more conformationally rigid chelated intermediate along the allylic side group trajectory, which is much smaller than the C4 (1,2-O-isopropylidene-1,2-ethanediol moiety on the top face of the molecule) side group.
112 An unambiguous structural identification of reduced products G or I, and their corresponding acetylated product H was established by their NMR spectroscopic characteristics such as 1H-1H, 13C-1H and NOESY correlation studies. For example, upon reduction, the C4-H doublets at 4.54-4.66 ppm in the starting materials (E, Scheme 20) were shifted to doublet of doublets at 4.44-4.48 ppm for resultant cis-hydroxyl derivatives (G, Scheme 20) or at 4.08-4.43 ppm for their corresponding acetates (H,
Scheme 20) as expected. In addition, a new 1H-NMR signals appeared in the range of
4.15-4.25 ppm for all G derivatives, and at 5.51-6.11 ppm for their corresponding acetates H, due to the newly-generated C3-H of G and H upon reduction (Scheme 20).
Furthermore, the characteristic 13C-NMR β-carbonyl carbon singlets of starting materials
E in the range of 200.9-206.1 ppm had disappeared upon reduction and new C3 13C- methine carbon signal (doublet) appeared in the range of 71.9-74.7 ppm for alcohols (G,
Scheme 20) and 72.2-73.0 ppm for corresponding acetates (H, Scheme 20). In contrast, the reduction of the C2-carbonyl of C2-O to C3 rearranged product, 2F (I, Scheme 21), resulted in one significant change in 1H-NMR and two visible changes in 13C-NMR of the product. First, the appearance of a new 1H-NMR singlet at 4.09 ppm corresponding to the newly-generated C2-H. Second, the C2-carbonyl carbon 13C-NMR singlet of the starting material at 186.3 ppm is converted to a doublet at 71.8 ppm and there is a significant 11 ppm downfield shift of C1 carbon signal (from 158.2 ppm to 169.4 ppm) of the product
(I). These changes in the 1 H- and 13C-NMR characteristic, further confirms the reduction of the C2-carbonyl of 2F to produce I (Scheme 21).
The stereochemistries of these compounds were confirmed by NOESY correlation studies. For instance, an intense qualitative NOESY correlation between the newly
113 generated C3-H and the C4-H of all the products listed in Scheme 20 (G & H Series) strongly suggests that the configuration of these protons are cis to each other and identical to the overall configuration of L-gluono-γ-lactones. However, since the C2-O- to C3-allylated rearranged product 2F and its reduced counterpart, I, showed no significant qualitative NOESY correlation among the C4-H, C2-H, or C3-allylic- substituent hydrogens (C12-H2), the C2-stereochemistry of the product I could not be assigned with certainty. All the 1H- and 13C-NMR spectra of the sodium borohydride reduced products of G or I, and corresponding acetylated products H series were in good agreement with reported literature spectra for similar compounds.208-210
These studies have shown the importance of C2- and C3-allylated derivatives of
L-ascorbic acid and the potential for their applications in synthetic organic and pharmaceutical chemistry. Also, it shows a new synthetic route to other interesting compounds by possible derivatization of these L-ascorbic-acid-derived L-galactono-γ- lactones (E and F series) through the reductive-amination of their keto groups. This could potentially produce a series of biologically-active amines. We are currently working on the chemo- and diastereo-selective reductive-amination of E and F series of compounds and are currently working on developing a general method for synthesis and also the complete structural characterization of all derivatives. A list of some compounds already synthesized but yet to be fully characterized is listed in Figure 14.
114 Figure 14 Diastereoselective Reductive Amination Products of 1E, 5E and 1F (X, Y and Z Respectively)
H3C CH H3C H3C 3 CH3 O CH3 O O O O O O O H O O O H H H2N O H2N H H H HO HO N OH CH3 H X Y Z
115 CHAPTER 5
EXPERIMENTAL SECTION
General: All reagents and chemicals were obtained from various commercial sources at the highest purity available and used without further purification. Chromatographic separations were carried out using Davisil grade 1740 type 60A (200-424mesh, Fisher) silica gel and the reaction products were eluted with a mixture of ethyl acetate and n- hexane with varying ratios depending on the nature of the compound. The TLC analyses were performed on pre-coated silica gel GF plates (250µm, Analtech) and the products were observed under UV light and/or by exposure to iodine vapor. All solvents used were dried with appropriate drying agents and freshly distilled. NMR Spectra were recorded on a Varian XL-300-NMR spectrometer operating at 300 MHz for 1H and at 75.4 MHz for
13C or on a Varian UNITY INOVA 400-NMR spectrometer operating at 400 MHz for 1H and at 100.6 MHz for 13C. All chemical shifts are reported on the δ (ppm) scale relative
1 13 to TMS (0.00 ppm) for H-NMR and to CDCl3 (77.0 ppm) for C-NMR. The elemental analyses were carried out at Desert Analytical, Tucson, Arizona. The exact mass FAB experiments were performed at the University of Kansas, Mass Spectrometry laboratory,
Lawrence, Kansas.
Computational Modeling: The initial calculations were carried with AM1 semi- empirical method using Winmopac v2 to obtain a reasonable initial model to serve as a foundation for the subsequent ab initio calculations. All the ab initio calculations were carried out by the density function B3LYP method and basis set 6-31G* using Gaussian
116 98 programs.213 The electrostatic potential diagrams of the electronic density were obtained from these calculations. The electron density distributions of the molecules are plotted as the electrostatic potential with the standard color-coding using the standard built in features of the Gaussian 98 programs213 (order of electron density: blue < green < yellow < red).
5,6-O-Isopropylidene-L-ascorbic Acid (1). This was synthesized in 82% yield according to the procedure of Jung et al., 36 mp 204-206 oC [Lit.33 201-203 oC]: 1H-NMR
(400 MHz, D2O) δ 1.37 (6H, s), 4.17 (1H, dd, J = 9.1, 5.0 Hz), 4.31 (1H, dd, J = 9.1, 7.3
Hz), 4.59 (1H, ddd, J = 7.3, 5.0, 2.4 Hz), 4.91 (1H, d, J = 2.4 Hz); 13C-NMR (100 MHz,
D2O): δ 26.70, 27.51, 67.75, 75.65, 78.54, 113.46, 120.54, 158.37, 176.08.
5,6-O-Isopropylidene-3-O-methyl-L-ascorbic Acid (1A). This compound was synthesized according to the procedure of Wimalasena and Mahindaratne.161 A mixture of 1 (1 g, 4.63 mmol) and 1.2 equiv of K2CO3 (0.77 g, 5.56 mmol) in DMSO/THF (9:8) were stirred for 20 min at room temperature. Then 1.2 equiv of methyl iodide (0.79 g,
5.56 mmol) in the same solvent was added dropwise and the mixture was vigorously stirred for 4-6 h at room temperature. The reaction mixture was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was thoroughly washed with water and dried over anhydrous Na2SO4 and the solvents were removed under reduced pressure. The product was purified by conventional silica gel column chromatography using 4:1 n-hexane:ethyl acetate to give 91% yield as a viscous oil: 1H-NMR (300 MHz,
CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 4.02 (1H, dd, J = 8.5, 6.6 Hz), 4.13 (1H, dd, J = 8.5,
117 6.7 Hz), 4.18 (3H, s), 4.23 (1H, dt, J = 6.7, 3.8 Hz), 4.53 (1H, d, J = 3.8 Hz); 13C-NMR
(75 MHz, CDCl3): δ 25.2, 25.6, 59.4, 65.0, 73.9, 75.3, 110.0, 119.5, 149.9, 171.2.
5,6-O-Isopropylidene-3-O-benzyl-L-ascorbic Acid (2A). This was synthesized from
1 and benzyl bromide in 86% yield as a semisolid using the same procedure as for 1A:
1 H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.02 (1H, dd, J = 8.6, 6.7 Hz),
4.10 (1H, dd, J = 8.6, 6.7 Hz), 4.26 (1H, dt, J = 6.7, 3.8 Hz), 4.57 (1H, d, J = 3.8 Hz),
13 5.52 (2H, m), 7.35-7.42 (5H, m); C-NMR (75 MHz, CDCl3): 25.5, 25.9, 65.3, 73.5,
74.2, 75.7, 110.3, 119.5, 128.4, 128.6, 128.7, 135.7, 148.6, 171.1.
5,6-O-Isopropylidene-3-O-trans-crotyl-L-ascorbic Acid (3A). This was synthesized from 1 and trans-crotyl bromide in 72% yield as a light yellow oil using the same
1 procedure as for 1A: H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 1.75 (3H, dq, J = 6.5, 1.5 Hz), 4.02 (1H, dd, J = 8.6, 6.7 Hz), 4.13 (1H, dd, J = 8.6, 6.7 Hz), 4.26
(1H, dt, J = 6.7, 3.9 Hz), 4.55 (1H, d, J = 3.9 Hz), 4.89 (2H, m), 5.68 (1H, dtq, J = 15.3,
13 6.6, 1.5 Hz), 5.90 (1H, dtq, J = 15.3, 6.5, 1.5 Hz); C-NMR (75 MHz, CDCl3): δ 17.8,
25.6, 25.9, 65.3, 72.4, 74.4, 75.7, 110.3, 119.1, 125.2, 132.6, 148.6, 171.8.
5,6-O-Isopropylidene-3-O-allyl-L-ascorbic Acid (4A). This was synthesized from 1 and allyl bromide in 80% yield as a light transparent oil using the same procedure as for
1 1A: H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.40 (3H, s), 4.04 (1H, dd, J = 8.6, 6.7
Hz), 4.15 (1H, dd, J = 8.6, 6.7 Hz), 4.28 (1H, dt, J = 6.6, 3.8 Hz), 4.58 (1H, d, J = 3.8
Hz), 4.97 (2H, d, J = 5.7 Hz), 5.31 (1H, dq, J = 10.4, 1.4 Hz), 5.41 (1H, dq, J = 17.2, 1.4
13 Hz), 6.01 (1H, ddt, J = 17.2, 10.4, 5.7 Hz); C-NMR (75 MHz, CDCl3): δ 25.5, 25.9,
65.3, 72.3, 74.3, 75.6, 110.3, 119.1, 119.2, 132.2, 148.2, 171.0.
118 5,6-O-Isopropylidene-3-O-trans-cinnamyl-L-ascorbic Acid (5A). This was synthesized from 1 and cinnamyl chloride in 72% yield as light yellowish oil using the same procedure as for 1A. However, this compound undergoes very fast and facile
Claisen rearrangement to its C2-allylated rearranged product (7E). Therefore, 7E was characterized by NMR spectroscopy.
5,6-O-Isopropylidene-2-O-acetyl-3-O-methyl-L-ascorbic Acid (6A). This compound was synthesized according to the procedure of Wimalasena and Mahindaratne.161 1A (1 g,
4.34 mmol) and 1.4 equiv of pyridine (0.481 g, 6.1 mmol) in CH2Cl2 was stirred for 20 min at room temperature and 1.2 equiv of acetyl chloride (0.409 g, 5.21 mmol) was added drop-wise. The mixture was vigorously stirred until the solution became homogenous and was further stirred for 2 h at room temperature. The reaction mixture was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was thoroughly washed with water and dried over anhydrous Na2SO4 and the solvents were removed under reduced pressure. The product was isolated and purified with conventional silica gel column chromatography using 7:1 n-hexane:ethyl acetate to give
1 90% yield as a transparent oil: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s),
2.31 (3H, s), 4.00 (3H, s), 4.02 (1H, dd, J = 8.5, 6.6 Hz), 4.15 (1H, dd, J = 8.5, 6.6 Hz),
13 4.29 (1H, dt, J = 6.6, 2.4 Hz), 5.14 (1H, d, J = 2.4 Hz); C-NMR (75 MHz, CDCl3): δ
20.5, 25.3, 25.6, 58.4, 65.1, 72.9, 74.4, 110.6, 131.3, 142.4, 166.3, 166.4.
5,6-O-Isopropylidene-2-O-acetyl-3-O-benzyl-L-ascorbic Acid (7A). This was synthesized from 2A and acetyl chloride in 90% yield as a light yellow oil using the same
1 procedure as for 6A: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 2.22 (3H, s), 4.07 (1H, dd, J = 8.6, 6.7 Hz), 4.14 (1H, dd, J = 8.6, 6.7 Hz), 4.38 (1H, dt, J = 6.7, 2.9
119 Hz), 4.71 (1H, d, J = 2.9 Hz), 5.31 (1H, d, J = 11.5 Hz), 5.37 (1H, d, J = 11.5 Hz), 7.30-
13 7.50 (5H, m); C-NMR (75 MHz, CDCl3): δ 20.1, 25.5, 25.7, 65.1, 73.6, 73.9, 75.2,
110.5, 114.8, 127.5, 128.8, 129.0, 134.5, 159.8, 166.8, 167.5.
5,6-O-Isopropylidene-2-O-acetyl-3-O-trans-crotyl-L-ascorbic Acid (8A). This was synthesized from 3A and acetyl chloride in 78% as a light yellow semisolid using the
1 same procedure as for 6A: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.40 (3H, s), 1.76
(3H, dq, J = 6.5, 1.6 Hz), 2.27 (3H, s), 4.07 (1H, dd, J = 8.6, 6.6 Hz), 4.15 (1H, dd, J =
8.6, 6.6 Hz), 4.36 (1H, dt, J = 6.6, 3.1 Hz), 4.66 (1H, d, J = 3.1 Hz), 4.73 (2H, m), 5.62
(1H, dtq, J = 15.4, 6.5, 1.6 Hz), 5.87 (1H, dtq, J = 15.4, 6.5, 1.6 Hz); 13C-NMR (75 MHz,
CDCl3): δ 17.7, 20.2, 25.5, 25.7, 65.2, 72.6, 73.7, 75.3, 110.5, 114.4, 124.0, 133.2, 159.7,
167.0, 167.6.
5,6-O-Isopropylidene-2-O-acetyl-3-O-allyl-L-ascorbic Acid (9A). This was synthesized from 4A and acetyl chloride in 70% yield as a colorless semisolid using the
1 same procedure as for 6A: H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.41 (3H, s), 2.27
(3H, s), 4.08 (1H, dd, J = 8.6, 6.6 Hz), 4.16 (1H, dd, J = 8.6 , 6.6 Hz), 4.38 (1H, dt, J =
6.6, 3.0 Hz), 4.69 (1H, d, J = 3.0 Hz), 4.81 (2H, m), 5.35 (1H, dq, J =10.6, 1.6 Hz), 5.40
(1H, dq, J = 17.7, 1.6 Hz), 5.95 (1H, ddt, J = 17.7, 10.6, 5.5 Hz); 13C-NMR (75 MHz,
CDCl3): δ 20.3, 25.5, 25.8, 65.2, 72.5, 73.7, 75.3, 110.6, 114.6, 119.6, 131.0, 159.5,
166.8, 167.6.
5,6-O-Isopropylidene-2-O-acetyl-3-O-trans-cinnamyl-L-ascorbic Acid (10A). A crude mixture of 5,6-O-isopropylidene-2-O-acetyl-L-ascorbic acid, 2D (1 g, 3.87 mmol) and 1.2 equiv of K2CO3 (0.64 g, 4.65 mmol) in DMSO/THF (9:8) were stirred for 20 min at room temperature. Then 1.2 equiv of cinnamyl bromide (0.92 g, 4.65 mmol) in the
120 same solvent was added dropwise and the mixture was vigorously stirred for 4-6 h at room temperature. The reaction mixture was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was thoroughly washed with water and dried over anhydrous Na2SO4 and the solvents were removed under reduced pressure. The product was purified by conventional silica gel column chromatography using 7:1 n-hexane:ethyl
1 acetate to give 76% yield as colorless crystals: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.41 (3H, s), 2.28 (3H, s), 4.09 (1H, dd., J = 8.6, 6.6 Hz), 4.17 (1H, dd., J = 8.6, 6.6
Hz), 4.40 (1H, d t, J = 6.6, 2.9 Hz), 4.70 (1H, d, J = 2.9 Hz), 4.90-5.03 (2H, m), 6.29 (1H, dt, J = 15.9, 6.3 Hz), 6.71 (1H, d, J = 15.9 Hz), 7.23-7.36 (3H, m), 7.37-7.42 (2H, m);
13 C-NMR (75 MHz, CDCl3): δ 20.2, 25.5, 25.7, 65.2, 72.6, 73.6, 75.3, 110.6, 114.7,
121.6, 126.7, 128.6, 128.7, 129.3, 135.5, 159.7, 166.9, 167.6.
5,6-O-Isopropylidene-3-O-allyl-2-O-methyl-L-ascorbic Acid (11A). This was prepared from 4A and methyl iodide (Scheme 11) in 59% yield as a semi-solid: 1H-NMR
(300 MHz, CDCl3) δ 1.36 (3H, s), 1.40 (3H, s), 3.85 (3H, s), 4.04 (1H, dd, J = 8.5, 6.6
Hz), 4.14 (1H, dd, J = 8.5, 6.7 Hz), 4.30 (1H, dt, J = 6.7, 3.3 Hz), 4.53 (1H, d, J = 3.3
Hz), 4.93 (2H, dt, J = 5.6, 1.4 Hz), 5.33 (1H, dq, J = 10.5, 1.3 Hz), 5.40 (1H, dq, J = 17.2,
13 1.5 Hz), 5.98 (1H, ddt, J = 17.2, 10.5, 5.6 Hz); C-NMR (75 MHz, CDCl3): δ 25.5, 25.8,
59.9, 65.3, 72.2, 74.0, 74.6, 110.3, 118.8, 123.0, 131.8, 155.1, 168.7.
5,6-O-Isopropylidene-2,3-O-diallyl-L-ascorbic Acid (12A). This was prepared from
4A and allyl bromide (Scheme 11) in 61% yield as a light yellow oil: 1H-NMR (300
MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.04 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (1H, dd, J
= 8.5, 6.7 Hz), 4.30 (1H, dt, J = 6.7, 3.2 Hz), 4.55 (1H, d, J = 3.2 Hz), 4.62 (2H, m), 4.94
(2H, dt, J = 5.6, 1.4 Hz), 5.27 (1H, dq, J = 10.8, 1.5 Hz), 5.31 (1H, dq, J = 10.5, 1.3 Hz),
121 5.35 (1H, dq, J = 17.2, 1.5 Hz), 5.39 (1H, dq, J = 17.3, 1.5 Hz), 5.98 (1H, ddt, J = 17.3,
13 10.5, 5.6 Hz), 5.99 (1H, ddt, J = 17.2, 10.3, 6.1 Hz); C-NMR (75 MHz, CDCl3): δ 25.6,
25.9, 65.3, 72.3, 72.5, 74.0, 74.7, 110.3, 118.9, 119.2, 121.5, 131.9, 132.9, 155.7, 168.9.
5,6-O-Isopropylidene-2,3-O-dibenzyl-L-ascorbic Acid (13A). This was prepared from 2A and benzyl bromide (Scheme 11) in 88% yield as a light transparent semi-solid:
1 H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.41 (3H, s), 4.00 (1H, dd, J = 8.5, 6.8 Hz),
4.07 (1H, dd, J = 8.5, 6.8 Hz), 4.27 (1H, dt, J = 6.8, 3.2 Hz), 4.53 (1H, d, J = 3.1 Hz),
5.07 (1H, d, J = 11.3 Hz), 5.11 (1H, d, J = 11.7 Hz), 5.14 (1H, d, J = 11.3 Hz), 5.20 (1H,
13 d, J = 11.7 Hz), 7.15-7.25 (2H, m), 7.30-7.50 (8H, m); C-NMR (75 MHz, CDCl3): δ
25.7, 25.9, 65.3, 73.5, 73.7, 74.0, 74.6, 110.2, 121.2, 127.7, 128.6, 129.1, 135.4, 136.0,
156.4, 168.9.
5,6-O-Isopropylidene-2,3-O-dicinnamyl-L-ascorbic Acid (14A). This was prepared from 5C and cinnamyl bromide (Scheme 11) in 75 % yield as a light transparent semi-
1 solid: H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.36 (3H, s), 4.05 (1H, dd, J = 8.5, 6.8
Hz), 4.13 (1H, dd, J = 8.5, 6.8 Hz), 4.33 (1H, dt, J = 6.8, 3.0 Hz), 4.55 (1H, d, J = 3.0
Hz), 4.82 (2H, m), 5.13 (2H, m), 6.32 (1H, dt, J = 15.9, 6.2 Hz), 6.36 (1H, dt, J = 15.9,
6.6 Hz), 6.68 (1H, d, J = 15.9 Hz), 6.69 (1H, d, J = 15.9 Hz), 6.36 (1H, dt, J = 15.8, 6.6
Hz), 6.68 (1H, d, J = 15.9 Hz), 6.69 (1H, d, J = 15.9 Hz), 7.27-7.38 (10H, m); 13C-NMR
(75 MHz, CDCl3): δ 25.6, 25.8, 65.3, 72.1, 72.3, 73.9, 74.7, 110.4, 121.3, 122.8, 123.8,
126.7, 128.1, 128.3, 128.6, 128.7, 134.7, 135.0, 135.8, 136.2, 156.1, 169.0.
5,6-O-Isopropylidene-3-Keto-2-benzyl-L-galactono-γ-lactone (2B). This was isolated as a minor product from a reaction mixture of 1 and benzyl bromide (Scheme 7)
1 as a colorless semi-solid: H-NMR (300 MHz, CDCl3) δ 1.31 (3H, s), 1.37 (3H, s), 1.65-
122 1.95 (1H, br s), 3.25 (1H, d, J = 12.6 Hz), 3.27 (1H, d, J = 12.6 Hz), 3.91 (1H, dd, J =
8.7, 7.0 Hz), 3.92 (1H, d, J = 2.0 Hz), 4.06 (1H, dd, J = 8.7, 7.0 Hz), 4.42 (1H, dt, J = 7.0,
2.0 Hz), 6.02 (1H, dt, J = 15.9, 7.8 Hz), 7.12-7.16 (2H, m), 7.26-7.35 (3H, m).
5,6-O-Isopropylidene-3-Keto-2-(trans-1-methyl-1-prop-2-enyl)-L-galactono-γ- lactone (3B). This was isolated as a minor product from a reaction mixture of 1 and
1 trans-crotyl bromide (Scheme 7): H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.41 (3H, s), 1.61-1.79 (3H, m), 2.60 (2H, dt, J = 7.5, 1.1 Hz), 3.00-3.40 (1H, br s), 4.08 (1H, dd, J
= 8.7, 7.0 Hz), 4.19 (1H, dd, J = 8.7, 7.0 Hz), 4.53 (1H, dt, J = 7.0, 2.0 Hz), 4.62 (1H, d, J
= 2.0 Hz), 5.29 (1H, dtq, J = 15.2, 7.6, 1.7 Hz), 5.69 (1H, dtq, J = 15.2, 6.5, 1.2 Hz); 13C-
NMR (75 MHz, CDCl3): δ 18.1, 25.3, 25.5, 38.9, 64.9, 72.2, 74.5, 81.4, 111.2, 119.7,
134.4, 172.8, 205.8.
5,6-O-Isopropylidene-3-Keto-2-(trans-3-phenyl-1-prop-2-enyl)-L-galactono-γ- lactone (5B). This was isolated as a major product from a reaction mixture of 1 and trans-cinnamyl bromide (Scheme 7) as a yellowish viscous oil: 1H-NMR (300 MHz,
CDCl3) δ 1.32 (3H, s), 1.41 (3H, s), 2.83 (2H, d, J = 7.8 Hz), 4.06 (1H, dd, J = 8.7, 7.0
Hz), 4.16 (1H, dd, J = 8.7, 7.0 Hz), 4.52 (1H, dt, J = 7.0, 2.0 Hz), 4.62 (1H, d, J = 2.0
Hz), 6.02 (1H, dt, J = 15.7, 7.8 Hz), 6.54 (1H, d, J = 15.8 Hz), 7.20-7.40 (5H, m); 13C-
NMR (75 MHz, CDCl3): δ 25.3, 25.5, 39.2, 64.8, 72.2, 74.5, 81.5, 111.2, 118.1, 126.5,
128.2, 128.6, 136.0, 137.5, 172.8, 205.8.
5,6-O-Isopropylidene-2-O-allyl-L-ascorbic Acid (1C). A solution of 2 equivalents of potassium tert-butoxide (t-BuOK) (1.04 g, 9.26 mmol) in dry DMSO/THF (3:2) was added dropwise to a solution of 1 (1 g, 4.63 mmol) in the same solvent at -10 oC under nitrogen to produce a bright yellow solution with an orange tint. The stirring of the
123 mixture was continued for about 2 min after which, 1.1 equivalents of allyl bromide (0.62 g, 5.09 mmol) in the same solvent was added dropwise over a period of 3 min with stirring continued for an additional 5 min at -10 oC. The cooling bath was removed, and the reddish orange solution was stirred for 3 h at room temperature. The reaction mixture was quenched with a cold solution of 0.25 M HCl (20 mL) and extracted with ethyl acetate (3 X 100 mL). The organic layer was dried over Na2SO4 and the solvents were removed under reduced pressure. The product was purified by conventional silica gel column chromatography using 3:1 n-hexane:ethyl acetate to give 80% yield as a white
214 1 solid: H-NMR (300 MHz, CDCl3) δ 1.38 (3H, s), 1.43 (3H, s), 4.02 (1H, dd, J = 9.0,
6.8 Hz), 4.16 (1H, dd, J = 9.0, 6.8 Hz), 4.43 (1H, dt, J = 6.8, 3.6 Hz), 4.62 (2H, dt, J =
6.3, 1.2 Hz), 4.72 (1H, d, J = 3.6 Hz), 5.28 (1H, dq, J = 10.2, 2.0 Hz), 5.36 (1H, dq, J =
13 17.3, 2.0 Hz), 5.98 (1H, ddt, J = 17.3, 10.2, 6.3 Hz); C-NMR (75 MHz, CDCl3): δ 25.2,
25.7, 64.9, 72.1, 73.8, 73.9, 110.6, 119.6, 121.4, 133.0, 156.4, 168.8. Anal. Calcd for
C12H16O6: C, 56.24; H, 6.29; O, 37.47. Found: C, 56.31; H, 6.21; O, 37.48.
5,6-O-Isopropylidene-2-O-methyl-L-ascorbic Acid (2C). This was synthesized from
1 and methyl iodide in 91% yield as a semisolid using the same procedure as for 1C: 1H-
NMR (300 MHz, CDCl3) δ 1.40 (3H, s), 1.45 (3H, s), 3.87 (3H, s), 4.07 (1H, dd, J = 9.0,
6.8 Hz), 4.19 (1H, dd, J = 9.0, 6.8 Hz), 4.46 (1H, dt, J = 6.8, 3.3 Hz), 4.71 (1H, d, J = 3.3
13 Hz); C-NMR (75 MHz, CDCl3); δ 25.4, 25.8, 59.6, 65.1, 73.96, 74.2, 111.0, 123.1,
155.8, 169.3. Anal. Calcd for C10H14O6: C, 52.17; H, 6.13; O, 41.70. Found: C, 52.43; H,
6.34; O, 41.23.
5,6-O-Isopropylidene-2-O-trans-crotyl-L-ascorbic Acid (3C). This was synthesized from 1 and trans-crotyl bromide in 72% yield as a light yellow oil using the same
124 1 procedure as for 1C: H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.41 (3H, s), 1.71 (3H, dq, J = 6.9, 1.7 Hz), 4.05 (1H, dd, J = 8.5, 6.8 Hz), 4.17 (1H, dd, J = 8.5, 6.8 Hz), 4.39
(1H, dt, J = 6.8, 3.6 Hz), 4.49 (2H, dt, J = 6.6, 1.0 Hz), 4.69 (1H, d, J = 3.6 Hz), 5.64
(1H, dtq, J = 15.7, 6.9, 1.6 Hz), 5.797 (1H, dtq, J = 15.7, 6.9, 1.6 Hz); 13C-NMR (75
MHz, CDCl3): δ 17.8, 25.3, 25.7, 64.99, 72.3, 73.9, 74.6, 110.4, 120.8, 125.8, 132.6,
157.9, 170.1. HRMS (FAB +) m/z exact mass calcd for C13H19O6 (M + 1) 271.1180, found m/z 271.1182.
5,6-O-Isopropylidene-2-O-benzyl-L-ascorbic Acid (4C). This was synthesized from
1 and benzyl bromide in 83% yield as a semisolid using the same procedure as for 1C:
1 H-NMR (400 MHz, CDCl3) δ 1.34 (3H, s), 1.37 (3H, s), 3.86 (1H, dd, J = 8.2 , 6.7 Hz),
4.04 (1H, dd, J = 8.2, 6.7 Hz), 4.31 (1H, dt, J = 6.7, 3.6 Hz), 4.60 (1H, d, J = 3.6 Hz),
13 5.11 (2H, two d), 7.31-7.41 (5H, m); C-NMR (100 MHz, CDCl3): δ 25.3, 25.8, 64.9,
73.3, 73.9, 74.2, 110.4, 121.1, 128.4, 128.5, 128.7, 136.4, 157.5, 169.3. Anal. Calcd for
C16H18O6: C, 62.74; H, 5.92; O, 31.34. Found: C, 62.95; H, 5.80; O, 31.25.
5,6-O-Isopropylidne-2-O-trans-cinnamyl-L-ascorbic Acid (5C). This was synthesized from 1 and trans-cinnamyl bromide in 87% yield as a semisolid using the
1 same procedure as for 1C: H-NMR (300 MHz, CDCl3) δ 1.30 (3H, s), 1.35 (3H, s), 3.99
(1H, dd, J = 8.8, 6.7 Hz), 4.09 (1H, dd, J = 8.8, 6.7 Hz), 4.36 (1H, dt, J = 6.7, 3.6 Hz),
4.64 (1H, d, J = 3.6 Hz), 4.74 (2H, d, J = 6.6 Hz), 6.32 (1H, dt, J = 13.8, 6.6 Hz), 6.36
13 (1H, d, J =13.8 Hz), 7.213-7.375 (5H, m), 8.667 (1H, s); C-NMR (75 MHz, CDCl3): δ
25.13, 25.63, 64.85, 72.00, 73.73, 74.13, 110.59, 121.25, 123.77, 126.69, 128.15, 128.59,
135.09, 136.06, 157.11, 169.50. Anal. Calcd for C18H20O6: C, 65.05; H, 6.07; O, 28.88.
Found: C, 65.34; H, 5.75; O, 28.91.
125 5,6-O-Isopropylidene-2-O-heptyl-L-ascorbic Acid (6C). This was synthesized from 1 and 1-bromoheptane in 96% yield as a semisolid using the same procedure as for 1C: 1H-
NMR (400 MHz, CDCl3) δ 0.88 (3H, t), 1.28-1.35 (8H, m), 1.38 (3H, s), 1.42 (3H, s),
1.66 (2H, quin, J = 7.0 Hz), 4.03-4.10 (2H, m), 4.18 (2H, dd, J = 8.8, 7.0 Hz), 4.43 (1H, dt, J = 6.6, 3.4 Hz), 4.71 (1H, d, J = 3.4 Hz), 8.79 (1H, s); 13C-NMR (100 MHz, CDCl3):
δ 13.7, 22.51, 25.18, 25.46, 25.72, 29.00, 29.57, 31.69, 64.99, 72.04, 73.82, 74.47,
110.50, 121.78, 156.58, 170.01. HRMS (FAB +) m/z exact mass calcd for C16H27O6 (M +
1) 315.1810, found m/z 315.1808.
5,6-O-Isopropylidene-3-O-acetyl-2-O-allyl-L-ascorbic Acid (7C). A mixture of 5,6-
O-isopropylidene-2-O-allyl-L-ascorbic acid, 1C (1 g, 3.90 mmol) and 1.4 equivalents of pyridine (0.43 g, 5.46 mmol) in CH2Cl2 stirred for 20 min at room temperature. Then, 1.2 equivalents of acetyl chloride (0.37 g, 4.68 mmol) was added dropwise under nitrogen.
The mixture was vigorously stirred until the solution became homogenous and was further stirred for 2 h at room temperature. The reaction mixture was diluted with water
(4-fold) and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and the solvents were removed under reduced pressure. The product was purified by conventional silica gel column chromatography using 7:1 n-hexane:ethyl acetate to give
214 1 in 70% yield a light yellow oil: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.38 (3H, s), 2.31 (3H, s), 4.02 (1H, dd, J = 8.3, 6.6 Hz), 4.15 (1H, dd, J = 8.3, 6.6 Hz), 4.29 (1H, dt, J = 6.6, 2.5 Hz), 4.78 (2H, tt, J = 5.1, 1.5 Hz), 5.18 (1H, d, J = 2.5 Hz), 5.28 (1H, dq, J
= 10.5, 2.1 Hz), 5.37 (1H, dq, J = 17.3, 2.1 Hz), 5.97 (1H, ddt, J = 17.3, 10.5, 5.1 Hz);
13 C-NMR (75 MHz, CDCl3): δ 20.6, 25.3, 25.6, 65.1, 71.3, 72.8, 74.5, 110.5, 118.9,
126 130.1, 132.4, 143.8, 166.3, 166.4. HRMS (FAB +) m/z exact mass calcd for C14H19O7 (M
+ 1) 299.1130, found m/z 299.1131.
5,6-O-Isopropylidene-3-O-acetyl-2-O-methyl-L-ascorbic Acid (8C). This was synthesized from 2C and acetyl chloride in 80% yield as a transparent viscous oil using
1 the same procedure as for 7C: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s),
2.32 (3H, s), 4.00 (3H, s), 4.04 (1H, dd, J = 9.0, 6.9 Hz), 4.16 (1H, dd, J = 9.0, 6.9 Hz),
13 4.30 (1H, dt, J = 6.9, 2.3 Hz), 5.15 (1H, d, J = 2.3 Hz); C-NMR (75 MHz, CDCl3): δ
20.5, 25.3, 25.6, 58.4, 65.1, 72.8, 74.4, 110.5, 131.2, 142.3, 166.2, 166.4. HRMS (FAB
+) m/z exact mass calcd for C12H17O7 (M + 1) 273.0970, found m/z 273.0974.
5,6-O-Isopropylidene-3-O-acetyl-2-O-trans-crotyl-L-ascorbic Acid (9C). This was synthesized from 3C and acetyl chloride in 84% yield as a light yellow oil using the same
1 procedure as for 7C: H-NMR (400 MHz, CDCl3) δ 1.36 (3H, s), 1.38 (3H, s), 1.73 (3H, dq, J = 6.4, 1.8 Hz), 2.31 (3H, s), 4.01 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (1H, dd, J = 8.5, 6.6
Hz), 4.28 (1H, dt, J = 6.6, 2.4 Hz), 4.70 (2H, dt, J = 6.4, 1.7 Hz), 5.18 (1H, d, J = 2.4 Hz),
5.64 (1H, dtq, J = 16.8, 6.4, 1.7 Hz), 5.83 (1H, dtq, J = 16.8, 6.4, 1.7 Hz); 13C-NMR (100
MHz, CDCl3); δ 17.7, 20.5, 25.3, 25.6, 65.0, 71.4, 72.9, 74.4, 110.5, 125.4, 130.1, 132.2,
143.8, 166.3, 166.5. Anal. Calcd for C15H20O7: C, 57.69; H, 6.45; O, 35.86. Found: C,
57.74; H, 6.54; O, 35.72.
5,6-O-Isopropylidene-3-O-acetyl-2-O-benzyl-L-ascorbic Acid (10C). This was synthesized from 4C and acetyl chloride in 82% yield as a light yellow oil using the same
1 procedure as for 7C: H-NMR (400 MHz, CDCl3) δ 1.33 (3H, s), 1.34 (3H, s), 2.21 (3H, s), 3.96 (1H, dd, J = 8.4, 6.8 Hz), 4.11 (1H, dd, J = 8.4, 6.8 Hz), 4.25 (1H, dt, J = 6.8, 2.4
Hz), 5.15 (1H, d, J = 2.4 Hz), 5.28 (1H, d, J = 11.6 Hz), 5.33 (1H, d, J = 11.6 Hz) 7.31-
127 13 7.39 (5H, m); C-NMR (100 MHz, CDCl3): δ 20.6, 25.3, 25.6, 65.0, 72.5, 72.8, 74.40,
110.5, 127.8, 128.4, 128.5, 130.1, 135.9, 144.6, 166.1, 166.5. HRMS (FAB +) m/z exact mass calcd for C18H21O7 (M + 1) 349.1290, found m/z 349.1298.
5,6-O-Isopropylidene-3-O-acetyl-2-O-cinnamyl-L-ascorbic Acid (11C). This was synthesized from 5C and acetyl chloride in 76% yield as viscous oil using the same
1 procedure as for 7C: H-NMR (300 MHz, CDCl3) δ 1.30 (3H, s), 1.32 (3H, s), 2.30 (3H, s), 4.02 (1H, dd, J = 8.4, 6.6 Hz), 4.13 (1H, dd, J = 8.4, 6.6 Hz), 4.29 (1H, dt, J = 6.6, 2.4
Hz), 4.89-5.00 (2H, m), 5.17 (1H, d, J = 2.4 Hz), 6.33 (1H, dt, J = 13.6, 6.5 Hz), 6.08
13 (1H, d, J = 13.6 Hz), 7.19- 7.41 (5H, m); C-NMR (75 MHz, CDCl3): δ 25.3, 25.4, 25.6,
65.1, 71.3, 72.9, 74.5, 110.6, 123.4, 126.7, 128.3, 128.6, 128.8, 134.8, 136.0, 144.6,
166.3, 166.6. HRMS (FAB +) m/z exact mass calcd for C20H23O7 (M + 1) 375.1440, found m/z 375.1444.
5,6-O-Isopropylidene-2-O-allyl-3-O-methyl-L-ascorbic Acid (12C). This was prepared from 1A and allyl bromide (Scheme 11) in 66 % yield as a yellowish viscous
1 oil: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 4.03 (1H, dd, J = 8.6, 6.6
Hz), 4.13 (1H, dd, J = 8.6, 6.6 Hz), 4.15 (3H, s), 4.28 (1H, dt, J = 6.6, 3.1 Hz), 4.52 (1H, d, J = 3.1 Hz), 4.60 (1H, ddt, J = 12.3, 6.2, 1.2 Hz), 4.62 (1H, ddt, J = 12.3, 6.3, 1.1 Hz),
5.28 (1H, dq, J = 10.3, 1.5 Hz), 5.36 (1H, dq, J = 17.2, 1.5 Hz), 6.00 (1H, ddt, J = 17.2,
13 10.3, 6.2 Hz); C-NMR (75 MHz, CDCl3): δ 25.5, 25.8, 59.6, 64.2, 72.7, 73.8, 74.5,
110.3, 119.3, 121.5, 132.8, 157.1, 169.9.
5,6-O-Isopropylidene-2-O-trans-crotyl-3-O-methyl-L-ascorbic Acid (13C). This was prepared from 1A and trans-crotyl bromide (Scheme 11) in 65 % yield as yellowish
1 viscous oil: H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.39 (3H, s), 1.74 (3H, dq, J =
128 6.4, 1.3 Hz), 4.03 (1H, dd, J = 8.5, 6.6 Hz), 4.13 (1H, dd, J = 8.5, 6.6 Hz), 4.14 (3H, s),
4.27 (1H, dt, J = 6.6, 3.1 Hz), 4.51 (1H, d, J = 3.1 Hz), 4.52 (2H, m), 5.66 (1H, dtq, J =
13 15.3, 6.7, 1.5 Hz), 5.82 (1H, dtq, J = 15.3, 6.4, 1.5 Hz); C-NMR (75 MHz, CDCl3): δ
17.8, 25.6, 25.8, 59.6, 65.2, 72.6, 73.9, 74.5, 110.3, 121.4, 125.8, 132.4, 157.3, 169.1.
5,6-O-Isopropylidene-2-O-trans-cinnamyl-3-O-methyl-L-ascorbic Acid (14C). This was prepared from 1A and trans-cinnamyl bromide (Scheme 11) in 71 % yield as
1 colorless solid: H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.36 (3H, s), 4.03 (1H, dd, J
= 8.5, 6.7 Hz), 4.12 (1H, dd, J = 8.5, 6.7 Hz), 4.16 (3H, s), 4.27 (1H, dt, J = 6.7, 3.0 Hz),
4.51 (1H, d, J = 3.0 Hz), 4.74 (1H, ddd, J = 12.1, 6.8, 1.2 Hz), 4.81 (1H, ddd, J = 12.1,
6.6, 1.2 Hz), 6.36 (1H, dt, J = 15.9, 6.6 Hz), 6.68 (1H, d, J = 15.9 Hz), 7.23-7.36 (3H, m),
13 7.37-7.42 (2H, m); C-NMR (75 MHz, CDCl3): δ 25.5, 25.8, 59.6, 65.2, 72.4, 73.8, 74.5,
110.3, 121.1, 123.8, 126.7, 128.1, 128.6, 135.1, 136.2, 157.4, 169.0.
5,6-O-Isopropylidene-2-O-allyl-3-O-benzyl-L-ascorbic Acid (15C). This was prepared from 2A and allyl bromide (Scheme 11) in 80 % yield as a colorless semi-solid:
1 H-NMR (300 MHz, CDCl3) δ 1.36 (3H, s), 1.38 (3H, s), 4.03 (1H, dd, J = 8.6, 6.7 Hz),
4.11 (1H, dd, J = 8.6, 6.7 Hz), 4.30 (1H, dt, J = 6.7, 3.2 Hz), 4.54 (2H, m), 4.55 (1H, d, J
= 3.2 Hz), 5.26 (1H, dq, J = 10.3, 1.6 Hz), 5.34 (1H, dq, J = 17.2, 1.5 Hz), 5.48 (2H, br
13 s), 5.94 (1H, ddt, J = 17.2, 10.3, 6.1 Hz), 7.37 (5H, br s); C-NMR (75 MHz, CDCl3): δ
25.6, 25.8, 65.2, 72.5, 73.5, 73.9, 74.7, 110.3, 119.3, 121.4, 127.7, 128.5, 128.7, 129.1,
132.8, 155.9, 168.8.
5,6-O-Isopropylidene-3-O-acetyl-L-ascorbic Acid (1D). A mixture of 5,6-O- isopropylidene-L-ascorbic acid, 1 (1 g, 4.63 mmol) and 1.0 equivalents of pyridine (0.37 g, 4.63 mmol) in CH2Cl2 stirred at room temperature for 2 min. Then 1.0 equivalents of
129 acetyl chloride (0.36 g, 4.63 mmol) was added dropwise. The mixture was vigorously stirred until the solution became homogenous and was further stirred for 15 min at room temperature. The reaction was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was thoroughly washed with water and dried over anhydrous
Na2SO4 and the solvents were removed under reduced pressure. The product could not be obtained in its pure form with conventional silica gel column chromatography without a significant amount of 2D and 3D contaminations due to its isomerization.
5,6-O-Isopropylidene-2-O-acetyl-L-ascorbic Acid (2D). A mixture of 5,6-O- isopropylidene-L-ascorbic acid, 1 (1 g, 4.63 mmol) and 1.4 equivalents of pyridine (0.51 g, 6.48 mmol) in CH2Cl2 stirred at room temperature for 10 min. Then 1.2 equivalents of acetyl chloride (0.44 g, 5.55 mmol) was added dropwise. The mixture was vigorously stirred until the solution became homogenous and was further stirred for 2 h at room temperature. The reaction was diluted (4-fold) with water and extracted with ethyl acetate. The organic layer was thoroughly washed with water and dried over anhydrous
Na2SO4 and the solvents were removed under reduced pressure. The product was purified with conventional silica gel column chromatography using 5:2 n-hexane:ethyl
1 acetate to give 90% yield as a white solid: H-NMR (300 MHz, CDCl3) δ 1.38 (3H, s),
1.41 (3H, s), 2.36 (3H, s), 4.10 (1H, dd, J = 8.71, 6.74 Hz), 4.20 (1H, dd, J = 8.71, 6.74
Hz), 4.44 (1H, dt, J = 6.74, 2.81 Hz), 4.70 (1H, d, J = 2.81 Hz), 9.70-9.80 (1H, s): 13C-
NMR (75 MHz, CDCl3): δ 20.68, 25.54, 25.72, 65.28, 73.40, 74.57, 110.72, 115.39,
155.13, 166.23, 171.29.
5,6-O-Isopropylidene-2,3-O-diacetyl-L-ascorbic Acid (3D). This was isolated as a solid in the least polar fractions of 3D silica gel column separation: 1H-NMR (400 MHz,
130 CDCl3) δ 1.35 (3H, s), 1.39 (3H, s), 2.27 (3H, s), 2.30 (3H, s), 4.09 (1H, dd, J = 8.8, 6.6
Hz), 4.19 (1H, dd, J = 8.8, 6.6 Hz), 4.39 (1H, dt, J = 6.7, 2.6 Hz), 5.14 (1H, d, J = 2.6
13 Hz); C-NMR (100 MHz, CDCl3): δ 20.04, 20.42, 25.30, 25.62, 65.18, 72.86, 75.26,
110.81, 122.24, 150.82, 164.74, 165.41, 166.11.
5,6-O-Isopropylidene-3-keto-2-(1-prop-2-enyl)-L-galactono-γ-lactone (1E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 4A in refluxing toluene for 6 h (Scheme 14): 1H-
NMR (300 MHz, CDCl3) δ 1.35 (3H, s), 1.40 (3H, s), 2.65 (2H, d, J = 7.4 Hz), 4.09 (1H, dd, J = 8.7, 6.8 Hz), 4.20 (1H, dd, J = 8.7, 6.8 Hz), 4.55 (1H, dt, J = 6.8, 2.0 Hz), 4.67
(1H, d, J = 2.0 Hz), 5.26 (1H, dq, J = 16.7, 1.4 Hz), 5.29 (1H, dq, J = 10.5, 1.0 Hz), 5.70
13 (1H, ddt, J = 16.7, 10.5, 7.5 Hz); C-NMR (75 MHz, CDCl3): δ 25.3, 25.5, 39.7, 64.8,
72.0, 74.5, 81.5, 111.4, 122.9, 127.6, 172.6, 205.5.
5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-prop-2-enyl)-L-galactono-γ-lactone
(3E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 9A in refluxing toluene for 6 h
1 (Scheme 14): H-NMR (300 MHz, CDCl3) δ 1.37 (3H, s), 1.42 (3H, s), 2.16 (3H, s), 2.80
(2H, m), 4.10 (1H, dd, J = 8.5, 7.1 Hz), 4.20 (1H, dd, J = 8.5, 7.1 Hz), 4.53 (1H, dt, J =
7.1, 1.6 Hz), 4.87 (1H, d, J = 1.7 Hz), 5.19 (1H, dq, J = 17.0, 1.5 Hz), 5.26 (1H, dq, J =
13 10.2, 1.5 Hz), 5.90 (1H, ddt, J = 17.2, 10.1, 7.0 Hz); C-NMR (75MHz CDCl3): δ 19.2,
25.5, 25.8, 34.5, 65.2, 73.7, 73.8, 82.1, 110.8, 120.8, 128.3, 169.7, 170.9, 201.0.
5,6-O-Isopropylidene-3-keto-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-galactono-γ- lactone (4E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 12A in refluxing toluene for 6 h
131 1 (Scheme 14): H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.40 (3H, s), 2.67 (2H, m),
4.00 (2H, m), 4.07 (1H, dd, J = 8.7, 6.9 Hz), 4.16 (1H, dd, J = 8.7, 6.9 Hz), 4.55 (1H, d, J
= 1.9 Hz), 4.65 (1H, dt, J = 6.9, 1.9 Hz), 5.20 (1H, dq, J = 10.5, 1.2 Hz), 5.22 (1H, dq, J
= 17.0, 1.2 Hz), 5.26 (1H, dq, J = 10.4, 1.2 Hz), 5.31 (1H, dq, J = 17.3, 1.6 Hz), 5.75 (1H, dddd, J = 17.0, 10.4, 7.9, 6.9 Hz), 5.90 (1H, ddt, J = 17.3, 10.4, 5.7 Hz); 13C-NMR (75
MHz, CDCl3): δ 25.4, 25.7, 41.2, 64.9, 69.2, 74.2, 79.9, 81.5, 110.9, 118.0, 122.2, 127.8,
133.2, 171.4, 206.1. Anal. Calcd for C15H20O6: C, 60.80; H, 6.80. Found: C, 60.66; H,
6.70.
5,6-O-Isopropylidene-3-keto-2-(1-prop-2-enyl)-2-O-methyl-L-galactono-γ-lactone
(2E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 11A in refluxing toluene for 6 h
1 (Scheme 14): H-NMR (300 MHz, CDCl3) δ 1.32 (3H, s), 1.38 (3H, s), 2.63 (2H, m),
3.34 (3H, s), 4.06 (1H, dd, J = 8.5, 7.0 Hz), 4.17 (1H, dd, J = 8.5, 7.0 Hz), 4.54 (1H, d, J
= 1.7 Hz), 4.65 (1H, dt, J = 7.0, 1.7 Hz), 5.20 (1H, dt, J = 18.2, 1.0 Hz), 5.25 (1H, dt, J =
13 10.1, 1.0 Hz), 5.73 (1H, dddd, J = 18.1, 10.2, 8.0 Hz); C-NMR (75 MHz, CDCl3): δ
25.5, 25.6, 41.0, 55.8, 64.9, 74.3, 80.4, 81.5, 110.9, 122.1, 127.8, 171.2, 206.1.
5,6-O-Isopropylidene-3-keto-2-(1-methyl-1-prop-2-enyl)-L-galactono-γ-lactone
(5E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 3A in refluxing toluene for 6 h
1 (Scheme 14): H-NMR (300 MHz, CDCl3) δ 1.19 (3H, d, J = 7.0 Hz), 1.35 (3H, s), 1.41
(3H, s), 2.69-2.81 (1H, m), 4.08 (1H, dd, J = 8.7, 6.8 Hz), 4.17 (1H, dd, J = 8.7, 6.8 Hz),
4.52 (1H, dt, J = 6.8, 2.1 Hz), 4.61 (1H, d, J = 2.2 Hz), 5.25 (1H, dt, J = 17.9, 1.0 Hz),
132 5.29 (1H, dt, J = 9.8, 1.0 Hz), 5.76 (1H, ddd, J = 17.9, 9.8, 8.2 Hz); 13C-NMR (75 MHz,
CDCl3): δ 12.5, 25.4, 25.5, 44.3, 64.8, 74.3, 74.4, 81.8, 111.1, 120.0, 134.3, 172.8, 205.7.
5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-methyl-1-prop-2-enyl)-L-galactono-γ- lactone (6E). This was obtained as a major diastereomer with a small amount of a minor diastereomer161 from the Claisen rearrangement of pure 8A in refluxing toluene for 6 h
1 (Scheme 14): H-NMR (300 MHz, CDCl3) δ 1.23 (3H, d, J = 6.8 Hz), 1.39 (3H, s), 1.48
(3H, s), 2.16 (3H, s), 2.95 (1H, dq, J = 7.8, 6.8 Hz), 4.08-4.18 (2H, m), 4.46-4.55 (2H, m), 5.28 (1H, dt, J = 17.0, 1.1 Hz), 5.31 (1H, dt, J = 10.4, 1.1 Hz), 5.71 (1H, ddd, J =
13 17.0, 10.4, 7.8 Hz); C-NMR (75 MHz, CDCl3): δ 12.4, 19.3, 25.3, 26.7, 41.4, 65.2,
75.1, 77.3, 84.8, 110.0, 120.6, 132.9, 169.3, 170.3, 200.9.
5,6-O-Isopropylidene-3-keto-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ-lactone
(7E). This was synthesized as the major product of 1 and trans-cinnamyl chloride reaction using the standard procedure for 1C (Scheme 15), and isolated as a yellowish
1 viscous oil: H NMR (400 MHz, CDCl3) δ 1.29 (3H, s), 1.30 (3H, s), 3.53 (1H, d, J = 2.0
Hz), 3.86 (1H, dd, J = 8.6, 7.0 Hz), 3.89 (1H, dd, J = 8.6, 7.0 Hz), 3.92 ( 1H, d, J = 2.0
Hz), 4.43 (1H, dt, J = 7.0, 2.0 Hz), 5.39-5.49 (2H, m), 6.35-6.50 (1H, m), 7.19-7.36 (5H,
13 m); C NMR (100 MHz, CDCl3): δ 25.2, 25.4, 56.7, 64.7, 74.0, 74.2, 81.9, 110.9, 122.3,
128.5, 128.7, 129.1, 131.1, 134.2, 172.0, 206.1.
5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ- lactone (8E). This was synthesized from 7E (Scheme 15) as a white semisolid in
1 quantitative yield using the standard procedure for 1F: H NMR (400 MHz, CDCl3) δ
1.32 (3H, s), 1.34 (3H,s), 2.14 (3H, s), 3.70 (1H, d, J = 8.4 Hz), 3.98 (1H, d, J = 9.3 Hz),
4.05 (2H, dd, J = 6.0, 1.2 Hz), 4.42 (1H, dt, J = 8.4, 6.0 Hz), 5.31 (1H, dq, J = 16.8, 1.6
133 Hz), 5.38 (1H, dq, J = 10.4, 1.2 Hz), 6.34 (1H, dddd, J = 16.8, 10.4, 9.2, 1.2 Hz), 7.20-
13 7.24 (3H, m), 7.33-7.38 (2H, m); C NMR (100 MHz, CDCl3): δ 19.2, 25.3, 26.6, 52.9,
65.1, 75.0, 76.7, 85.1, 109.8, 121.0, 128.8, 129.1, 129.3, 131.1, 133.8, 169.1, 170.3,
201.8.
5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ- lactone (9E). This was obtained as a major product from refluxing clean 10A in toluene for 6 h (Scheme 16), resulting in 100% conversion of the UV-active starting material to a major less polar non-UV-active product with minute traces of minor diastereomers:161,218
1 H NMR (300 MHz, CDCl3) δ 1.27 (3H, s), 1.45 (3H, s), 2.13 (3H, s), 2.97 (1H, dt, J =
6.8, 6.1 Hz), 3.82 (1H, dd, J = 8.8, 6.8 Hz), 3.97 (1H, dd, J = 8.8, 6.8 Hz), 4.05 (1H, d, J
= 8.6 Hz), 4.75 (1H, d, J = 6.8 Hz), 5.17 (1H, dt, J = 17.0, 1.1 Hz), 5.30 (1H, dt, J = 10.3,
1.1 Hz), 6.43 (1H, ddd, J = 17.0, 10.2, 8.6 Hz), 7.21-7.27 (2H, m), 7.32-7.40 (3H, m); 13C
NMR (75 MHz, CDCl3): δ 19.3, 25.4, 26.4, 51.3, 64.9, 72.7, 76.0, 83.9, 110.2, 119.9,
128.5, 129.3, 129.8, 132.5, 134.9, 169.3, 170.6, 201.5.
5,6-O-Isopropylidene-3-keto-2-O-acetyl-2-(1-phenyl-1-prop-2-enyl)-L-galactono-γ- lactone (10E). This was obtained as a minor product from silica gel column separation of
9E (10E & 8E are the same compound with identical sets of 1H- & 13C-NMR spectra).
5,6-O-Isopropylidene-2-keto-3-(1-prop-2-enyl)-L-galactono-γ-lactone (1F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 1C in refluxing toluene for 12 h
218 1 (Scheme 17): H-NMR (400 MHz, CDCl3) δ 1.30 (3H, s), 1.32 (3H, s), 2.54 (2H, dt, J
= 7.2, 1.2 Hz), 3.13 (1H, br), 4.10 (1H, dd, J = 8.8, 6.8 Hz), 4.17 (1H, dd, J = 15.2, 8.8
Hz), 4.52 (1H, dt, J = 13.2, 6.8 Hz), 4.70 (1H, d, J = 1.2 Hz), 5.25 (1H, dq, J = 17.2, 3.2
134 Hz), 5.35 (1H, dq, J = 10.4, 1.6 Hz), 5.81 (1H, ddd, J = 10.4, 7.2, 3.2 Hz); 13C-NMR (100
MHz, CDCl3): δ 24.6, 24.8, 39.6, 64.4, 73.1, 77.2, 80.8, 111.3, 121.9, 129.0, 159.4,
193.9.
5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone
(2F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 7C in refluxing toluene for 24 h
218 1 (Scheme 17): H-NMR (400 MHz, CDCl3) δ 1.32 (3H, s), 1.34 (3H, s), 2.16 (3H, s),
2.76 (1H, m), 2.97 (1H, m), 4.04 (1H, dd, J = 8.6, 7.2 Hz), 4.14 (1H, dd, J = 8.6, 7.2 Hz),
4.28 (1H, dt, J = 7.2, 1.6 Hz), 4.94 (1H, d, J = 1.6 Hz), 5.23 (2H, m), 5.62-5.73 (1H, m);
13 C-NMR (100 MHz, CDCl3): δ 20.0, 25.0, 25.2, 36.9, 65.1, 72.6, 78.4, 81.1, 111.1,
121.9, 128.1, 158.2, 170.6, 186.3.
5,6-O-Isopropylidene-2-keto-3-O-methyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone
(3F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 12C in refluxing toluene for 24 h
218 1 (Scheme 17): H-NMR (300 MHz, CDCl3) δ 1.33 (6H, s), 2.51 (1H, dd, J = 15.0, 8.3
Hz), 2.66 (1H, ddt, J = 14.9, 5.6, 1.3 Hz), 3.70 (3H, s), 3.99 (1H, dd, J = 8.4, 7.0 Hz),
4.14 (1H, dd, J = 8.4, 7.0 Hz), 4.49 (1H, dt, J = 7.0, 1.3 Hz), 4.58 (1H, d, J = 1.3 Hz),
5.26 (1H, dq, J = 17.1, 1.3 Hz), 5.31 (1H, dq, J = 10.5, 1.3 Hz), 5.75 (1H, dddd, J = 17.0,
13 10.3, 8.3, 5.6 Hz); C-NMR (75 MHz, CDCl3): δ 24.7, 25.2, 35.0, 52.8, 65.0, 72.5, 79.3,
79.5, 111.3, 121.7, 129.1, 159.9, 192.2.
5,6-O-Isopropylidene-2-keto-3-O-benzyl-3-(1-prop-2-enyl)-L-galactono-γ-lactone
(4F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 15C in refluxing toluene for 24 h
135 218 1 (Scheme 17): H-NMR (300 MHz, CDCl3) δ 1.34 (3H, s), 1.35 (3H, s), 2.57 (1H, dd,
J = 14.8, 8.5 Hz), 2.75 (1H, ddt, J = 14.8, 5.5, 1.4 Hz), 4.00 (1H, dd, J = 8.3, 7.3 Hz),
4.11 (1H, dd, J = 8.3, 7.3 Hz), 4.55 (1H, dt, J = 7.3, 1.2 Hz), 4.63 (1H, d, J = 1.2 Hz),
4.90 (1H, d, J = 10.9 Hz), 5.25 (1H, m), 5.30 (1H, d, J = 10.8 Hz), 5.31 (1H, d, J = 18.6
Hz), 5.82 (1H, dddd, J = 18.8, 10.3, 8.5, 5.5 Hz), 7.30 (5H, br s); 13C-NMR (75 MHz,
CDCl3): δ 24.9, 25.4, 36.3, 64.9, 67.1, 72.7, 79.3, 79.4, 111.2, 121.9, 127.5, 127.9, 128.4,
129.2, 137.5, 159.8, 192.2.
5,6-O-Isopropylidene-2-keto-3-(1-methyl-1-prop-2-enyl)-L-galactono-γ-lactone
(5F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 3C in refluxing toluene for 24 h
218 1 (Scheme 17): H-NMR (400 MHz, CDCl3) δ 1.15 (3H, d, J = 6.9 Hz), 1.29 (3H, s),
1.32 (3H, s), 2.71 (1H, m), 3.23 (1H, br), 4.08 (1H, dd, J = 8.7, 7.1 Hz), 4.18 (1H, dd, J =
8.4, 7.1 Hz), 4.52 (1H, dt, J = 7.1, 1.5 Hz), 4.75 (1H, d, J = 1.5 Hz), 5.18 (2H, m), 5.72-
13 5.85 (1H, m); C-NMR (100 MHz, CDCl3): δ 13.2, 24.8, 24.9, 43.8, 64.4, 73.4, 78.9,
80.0, 111.2, 118.6, 135.3, 159.5, 195.3.
5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1-methyl-1-prop-2-enyl)-L-galactono-γ- lactone (6F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 9C in refluxing toluene for
218 1 24 h (Scheme 17): H-NMR (400 MHz, CDCl3) δ 1.10 (3H, d, J = 7.0 Hz), 1.33 (3H, s), 1.35 (3H, s), 2.16 (3H, s), 3.17 (1H, m), 4.04 (1H, dd, J = 8.8, 7.1 Hz), 4.13 (1H, dd, J
= 8.8, 7.1 Hz), 4.30 (1H, dt, J = 7.2, 2.4 Hz), 4.83 (1H, d, J = 2.4 Hz), 5.15-5.28 (2H, m),
13 5.64-5.81 (1H, m); C-NMR (100 MHz, CDCl3): δ 12.8, 19.9, 25.2, 25.5, 42.1, 65.5,
73.4, 77.3, 79.7, 111.0, 119.7, 134.7, 158.0, 170.9, 187.5.
136 5,6-O-Isopropylidene-2-keto-3-O-methyl-3-(1-methyl-1-prop-2-enyl)-L-galactono-
γ-lactone (7F). This was obtained as a major diastereomer with an insignificant amount of a minor diastereomer from the Claisen rearrangement of pure 13C in refluxing toluene
218 1 for 24 h (Scheme 17): H-NMR (300 MHz, CDCl3) δ 1.10 (3H, d, J = 6.8 Hz), 1.33
(6H, s), 2.70 (1H, dq, J = 9.0, 6.8 Hz), 3.73 (3H, s), 3.98 (1H, dd, J = 8.3, 7.3 Hz), 4.14
(1H, dd, J = 8.3, 7.1 Hz), 4.45 (1H, dt, J = 7.2, 1.3 Hz), 4.63 (1H, d, J = 1.3 Hz), 5.14
(1H, dt, J = 17.0, 1.3 Hz), 5.17 (1H, dt, J = 10.3, 1.3 Hz), 5.69 (1H, ddd, J = 17.0, 10.2,
13 9.1 Hz); C-NMR (75 MHz, CDCl3): δ 13.6, 24.9, 25.4, 42.3, 54.6, 65.2, 72.9, 78.5,
81.1, 111.2, 119.3, 135.9, 159.8, 192.6.
5,6-O-Isopropylidene-2-(1-prop-2-enyl)-L-gulono-γ-lactone (1G). To a stirred solution of diastereomerically pure 1E (1.50 g, 5.85mmol) in absolute EtOH (80 mL) at -
o o 79 C was added NaBH4 (244 mg, 6.44 mmol). The reaction mixture was kept at -79 C for 5 min and then allowed to rise to room temperature for a period of no more than 30 min. The reaction mixture was concentrated under reduced pressure to about 25 mL and diluted with a mixture of 1:1 cold water and ethyl acetate (250 mL) and stirred for 30 min. The ethyl acetate layer was separated and the aqueous layer was extracted two times with ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous
Na2SO4, and the solvents were removed under reduced pressure. The residue was chromatographed on silica gel using 3:1 n-hexane/ethyl acetate to give 1.39 g (92%) of pure 1G as a white semisolid and ~200 mg of its diastereomer (Scheme 20): 218 1H-NMR
(400 MHz, CDCl3) δ 1.40 (3H, s), 1.43 (3H, s), 2.56 (1H, dd, J = 14.5, 8.8 Hz), 2.62 (1H, dd, J = 14.5, 6.2 Hz), 2.77 (1H, d, J = 4.5 Hz), 3.65 (1H, br), 4.03 (1H, dd, J = 8.4, 7.0
Hz), 4.15 (1H, t, J = 6.4 Hz), 4.17 (1H, m), 4.37 (1H, dt, J = 7.0, 4.5 Hz), 4.44 (1H, dd, J
137 = 6.4, 4.5 Hz), 5.30 (1H, d, J = 12.0 Hz), 5.31 (1H, d, J = 16.4 Hz), 5.90-6.00 (1H, m);
13 C-NMR (100 MHz, CDCl3) δ 25.5, 25.9, 36.4, 65.2, 74.3, 75.9, 77.2, 79.8, 110.7,
121.1, 130.9, 175.4. Anal. Calcd for C12H18O6: C, 55.81; H, 7.02. Found: C, 55.73; H,
7.04.
5,6-O-Isopropylidene-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-gulono-γ-lactone
(4G). This was synthesized from diastereomerically pure 4E in 90% yield as a semisolid
1 using the same procedure as for 1G: H-NMR (400 MHz, CDCl3) δ 1.38 (3H, s), 1.46
(3H, s), 2.47 (1H, m), 2.77 (1H, m), 3.01 (1H, d, J = 4.6 Hz), 3.83 (1H, dd, J = 8.4, 6.8
Hz), 4.21 (1H, dd, J = 8.8, 6.8 Hz), 4.25 (1H, t, J = 7.0 Hz), 4.28-4.31 (1H, m), 4.39 (1H, dt, J = 8.0, 4.6 Hz), 4.48 (1H, dd, J = 7.0, 4.6 Hz), 4.55-4.60 (1H, m), 5.19-5.24 (2H, m),
13 5.25-5.26 (2H, m), 5.72-5.82 (1H, m), 5.86-5.96 (1H, m); C-NMR (100 MHz, CDCl3) δ
25.2, 26.6, 35.7, 65.5, 65.7, 71.9, 74.9, 78.2, 81.5, 110.1, 117.5, 120.8, 129.9, 133.7,
173.2. Anal. Calcd for C15H22O6: C, 60.39; H, 7.43. Found: C, 60.52; H, 7.63.
5,6-O-Isopropylidene-2-(1-methyl-1-prop-2-enyl)-L-gulono-γ-lactone (5G). This was synthesized from diastereomerically pure 5E in 91% yield as a semisolid using the
1 same procedure as for 1G: H-NMR (400 MHz, CDCl3): δ 1.27 (3H, d, J = 7.2 Hz), 1.40
(3H, s), 1.43 (3H, s), 2.64 (1H, d, J = 4.5), 2.75 (1H, m), 4.03 (1H, dd, J = 8.8, 7.0 Hz),
4.15 (1H, dd, J = 8.8, 7.0 Hz), 4.23 (1H, t, J = 6.2 Hz), 4.35 (1H, dt, J = 7.0, 4.5 Hz), 4.45
(1H, dd, J = 6.2, 4.5 Hz), 5.26 (1H, d, J = 10.4 Hz), 5.29 (1H, d, J = 9.8 Hz), 5.99 (1H,
13 ddd, J = 17.6, 10.4, 8.0 Hz); C-NMR (100 MHz, CDCl3) δ 14.5, 25.6, 25.9, 41.9, 65.3,
74.7, 76.9, 77.7, 80.5, 110.7, 118.5, 138.4, 175.2. Anal. Calcd for C13H20O6: C, 57.34; H,
7.40. Found: C, 57.40; H, 7.22.
138 5,6-O-Isopropylidene-2-(1-phenyl-1-prop-2-enyl)-L-gulono-γ-lactone (7G). This was synthesized from diastereomerically pure 7E in 90% yield (crude) as a semisolid using the same procedure as for 1G: Since analytically pure 7G could not be obtained by traditional chromatographic techniques, it was characterized as acetate (7H) which was easily obtained in its pure form (see below).
The Product of NaBH4 Reduction of 5,6-O-Isopropylidene-2-keto-3-O-acetyl-3-(1- prop-2-enyl)-L-galactono-γ-lactone (I). This was synthesized from diastereomerically pure 2F in 60% yield as a white semisolid using the same procedure as for 1G (Scheme
218 1 21): H-NMR (400 MHz, CDCl3) δ 1.42 (3H, s), 1.46 (3H, s), 2.25 (3H, s), 2.48 (1H, dd, J = 14.2, 8.0 Hz), 2.54 (1H, dd, J = 14.0, 7.5 Hz), 4.05 (1H, dd, J = 8.4, 6.8 Hz), 4.09
(1H, s), 4.20 (1H, dd, J = 8.4, 6.8 Hz), 4.30 (1H, d, J = 3.0 Hz), 4.53 (1H, dt, J = 6.8, 3.0
Hz), 5.24 (2H, dd, J = 15.2, 7.2 Hz), 5.52 (1H, br), 5.82-5.93 (1H, m); 13C-NMR (100
MHz, CDCl3) δ 20.5, 25.7, 40.9, 65.7, 71.8, 73.5, 77.7, 79.9, 111.1, 120.8, 130.8, 169.4,
170.1.
5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-prop-2-enyl)-L-gulono-γ-lactone (1H).
To a stirred solution of a mixture of diastereomerically pure 1G (1.20 g, 4.65 mmol), 4-
DMAP (284 mg, 2.32 mmol) and triethylamine (3.3 mL, 23 mmol) in dichloromethane
(30 mL) at -79 oC was added acetic anhydride (1.76 mL, 18.6 mmol). The reaction mixture was stirred for 3 h at -79 oC and then quenched with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic layer was washed with water and brine, and then dried over anhydrous Na2SO4. The solvents were removed under reduced pressure and the residue was chromatographed on silica gel using
10:1 n-hexane/ethyl acetate to give pure 1H (1.59 g, quant.) as a white semisolid
139 218 1 (Scheme 20): H-NMR (400 MHz, CDCl3) δ 1.37 (3H, s), 1.43 (3H, s), 2.15 (3H, s),
2.16 (3H, s), 2.58 (1H, m), 2.60 (1H, m), 3.98 (1H, dd, J = dd, J = 9.0, 6.0 Hz), 4.06 (1H, dd, J = 9.0, 7.0 Hz), 4.30 (1H, dd, J = 8.0, 6.0 Hz), 4.43 (1H, dt, J = 11.6, 6.0 Hz), 5.27
13 (1H, m), 5.83 (1H, m), 5.94 (1H, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ 20.4, 20.5,
25.0, 26.1, 36.2, 64.6, 72.5, 74.6, 78.1, 81.3, 110.5, 121.4, 128.8, 169.3, 169.8, 169.9.
Anal. Calcd for C16H22O8: C, 56.13; H, 6.48. Found: C, 56.35; H, 6.38.
5,6-O-Isopropylidene-3-O-acetyl-2-O-(1-prop-2-enyl)-2-(1-prop-2-enyl)-L-gulono-
γ-lactone (4H). This was synthesized from diastereomerically pure 4G in quantitative yield as a white semisolid using the same procedure as for 1H: 1H-NMR (400 MHz,
CDCl3) δ 1.37 (3H, s), 1.46 (3H, s), 2.11 (3H, s), 2.58 (1H, dt, J = 14.8, 7.6 Hz), 2.71
(1H, dt, J = 14.8, 7.6 Hz), 3.69 (1H, dd, J = 8.8, 6.8 Hz), 4.01 (1H, dd, J = 8.8, 6.8 Hz),
4.19 (1H, m), 4.22 (1H, m), 4.33 (1H, m), 4.43 (1H, dd, J = 8.0, 4.2 Hz), 5.12-5.16 (2H, m), 5.26-5.30 (2H, m), 5.51 (1H, d, J = 4.2 Hz), 5.78-5.90 (2H, m); 13C-NMR (75 MHz,
CDCl3) δ 20.6, 25.1, 26.5, 37.4, 65.2, 66.7, 72.4, 74.5, 79.2, 79.4, 110.3, 116.2, 120.8,
129.8, 134.0, 169.2, 172.4. Anal. Calcd for C17H24O7: C, 59.99; H, 7.11. Found: C, 60.23;
H, 7.39.
5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-methyl-1-prop-2-enyl)-L-gulono-γ- lactone (5H). This was synthesized from diastereomerically pure 5G in quantitative yield
1 as a white semisolid using the same procedure as for 1H: H-NMR (400 MHz, CDCl3) δ
1.21 (3H, d, J = 7.2 Hz), 1.37 (3H, s), 1.43 (3H, s), 2.14 (3H, s), 2.15 (3H, s), 2.75-2.84
(1H, m), 3.90 (1H, dd, J = 8.8, 6.4 Hz), 4.04 (1H, dd, J = 8.8, 6.4 Hz), 4.31 (1H, dd, J =
8.0, 6.0 Hz), 4.42 (1H, dt, J = 12.8, 6.4 Hz), 5.16 (1H, d, J = 14.8 Hz), 5.17 (1H, d, J =
13 12.8 Hz), 5.89-5.94 (1H, m), 5.96 (1H, d, J = 8.0 Hz); C-NMR (100 MHz, CDCl3) δ
140 14.9, 20.5, 20.6, 25.0, 26.1, 41.6, 64.5, 73.0, 74.9, 78.7, 82.9, 110.4, 118.2, 135.8, 169.4,
169.8, 170. Anal. Calcd for C17H24O8: C, 57.30; H, 6.79. Found: C, 57.48; H, 6.50.
5,6-O-Isopropylidene-2,3-O-diacetyl-2-(1-phenyl-1-prop-2-enyl)-L-gulono-γ- lactone (7H). This was synthesized from diastereomerically pure 7G in quantitative yield
1 as a white semisolid using the same procedure as for 1H: H-NMR (400 MHz, CDCl3) δ
1.34 (3H, s), 1.39 (3H, s), 1.94 (3H, s), 2.11 (3H, s), 3.91 (1H, dd, J = 9.0, 6.4 Hz), 4.00
(1H, dd, J = 9.0, 6.4 Hz), 4.08 (1H, dd, J = 8.0, 6.2 Hz), 4.31 (1H, dt, J = 11.9, 6.4 Hz),
5.20 (1H, d, J = 16.9 Hz), 5.26 (1H, d, J = 10.4 Hz), 6.11 (1H, d, J = 8.0 Hz), 6.22 (1H, ddd, J = 16.9, 10.4, 9.0 Hz), 7.26-7.35 (2H, m), 7.39-7.41 (3H, m); 13C-NMR (75 MHz,
CDCl3) δ 20.3, 20.6, 24.9, 26.0, 52.4, 64.5, 72.2, 74.4, 77.6, 83.1, 110.4, 119.3, 127.7,
128.5, 130.2, 134.2, 136.4, 169.1, 169.4, 169.7. Anal. Calcd for C22H26O8: C, 63.15; H,
6.26. Found: C, 63.46; H, 6.04.
The Product of NaBH3CN Reduction of 5,6-O-Isopropylidene-3-keto-2-(1-prop-2- enyl)-L-galactono-γ-lactone (X). To a stirred solution of diastereomerically pure 1E
(1.50 g, 5.85 mmol) and ammonium acetate (4.51 g, 58.5 mmol) in dry methanol (80 mL)
o at 25 C was added NaBH3CN (257 mg, 4.10 mmol) and 2.5 g of 4Å molecular sieve beads. The reaction mixture was vigorously stirred for 24 h at room temperature and remained cloudy throughout the reaction. The reaction mixture was concentrated under reduced pressure to about 20 mL and diluted with a mixture of cold brine-NaHCO3 solution and ethyl acetate, 1:5 respectively (250 mL) and stirred for 3 min. The ethyl acetate layer was separated and the aqueous layer was extracted two times with ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous Na2SO4, and the solvents were removed under reduced pressure. The residue was chromatographed on
141 silica gel using 3:1 n-hexane/ethyl acetate to give 70% of clean X as a white semisolid
1 and with spectroscopic traces of its diastereomer: H-NMR (400 MHz, CDCl3) δ 1.40
(3H, s), 1.43 (3H, s), 2.53-2.64 (2H, m), 4.04 (1H, dd, J = 8.8, 7.2 Hz), 4.16 (1H, dd, J =
8.8, 7.2 Hz), 4.19 (1H, t, J = 3.2 Hz), 4.37 (1H, dt, J = 6.6, 3.2 Hz), 4.44 (1H, dd, J = 6.6,
13 4.4 Hz), 5.23-5.33 (2H, m), 5.79-6.00 (1H, m); C-NMR (100 MHz, CDCl3): δ 25.54,
25.86, 36.34, 65.22, 74.30, 75.92, 76.76, 79.83, 110.64, 121.12, 130.88, 175.47.
The Product of NaBH3CN Reduction of 5,6-O-Isopropylidene-3-keto-2-(1-methy-l- prop-2-enyl)-L-galactono-γ-lactone (Y). This was synthesized from 5E in diastereomeric excess to give Y in 70% yield as a white semisolid using the same procedure as for X. Y was isolated as racemic mixtures of diastereomers: 13C-NMR (100
MHz, CDCl3) δ 13.87, 14.46, 25.50, 25.54, 25.84, 25.93, 41.19, 41.86, 65.16, 65.25,
74.44, 74.66, 76.83, 77.19, 77.69, 77.72, 78.89, 80.19, 80.46, 110.48, 110.68, 118.00,
118.52, 137.68, 138.38, 175.22, 175.35.
The Product of NaBH(OAc)3 Reduction of 5,6-O-Isopropylidene-2-keto-2-(1-prop-
2-enyl)-L-galactono-γ-lactone (Z). To a stirred solution of diastereomerically pure 1F
(1.50 g, 5.85 mmol) and 2.5 g of 4Å molecular sieve beads in methylene chloride (60 mL) at 0 oC was added phenethylamine (1.1 g, 8.78 mmol). The reaction mixture was vigorously stirred for 12-24 h at room temperature, until the imine formation was completed (determined by TLC analysis). The imine in methylene chloride was carefully treated with NaBH(OAc)3 (1.62 g, 7.61 mmol) for 12 h. The reaction mixtures remained cloudy throughout the reaction. The reaction mixture was concentrated under reduced pressure to about 20 mL and diluted with a mixture of cold brine-NaHCO3 and ethyl acetate, 1:5 respectively (250 mL) and stirred for 3 min. The ethyl acetate layer was
142 separated and the aqueous layer was extracted two times with ethyl acetate. The combined ethyl acetate extracts were dried with anhydrous Na2SO4, and the solvents were removed under reduced pressure. The residue was chromatographed on silica gel using 3:1 n-hexane/ethyl acetate to give 90% of clean Z as a white semisolid and with
1 spectroscopic traces of its diastereomer: H-NMR (400 MHz, CDCl3) δ 1.37 (3H, s), 1.39
(3H, s), 2.24 (1H, dd, J = 14.0, 7.2 Hz), 2.48 (1H, dd, J = 14.0, 7.2 Hz), 2.77-2.89 (2H, m), 2.99-3.06 (1H, m), 3.12-3.19 (1H, m), 3.82 (1H, s), 3.99 (1H, dd, J = 12.0, 8.0 Hz),
4.12 (1H, dd, J = 12.0, 8.0 Hz), 4.23 (1H, d, J = 2.0 Hz), 4.41 (1H, dt, J = 7.3, 2.0 Hz),
13 5.10-5.21 (2H, m), 5.76-5.87 (1H, m), 7.20-7.32 (5H, m); C-NMR (100 MHz, CDCl3):
δ 25.72, 25.73, 36.77, 39.14, 50.77, 65.34, 65.74, 74.11, 79.46, 79.65, 110.48, 120.59,
126.28, 128.48, 128.78, 131.81, 139.63, 174.60.
143
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163
APPENDIX
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212
213
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222
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225
226
227
228 The X-ray Crystal Structure of 9A
229 The X-ray Crystal Structure of 2E
230