FLOW SYNTHESIS OF NATURAL PRODUCTS: ASCORBIC ACID
AND N5-OH-L-ORNITHINE SYNTHESIS
by
Maduabuchi Angus Modum
B.Sc., Federal University of Technology Owerri, 2021

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
CHEMISTRY

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
April 2025
© Maduabuchi Angus Modum, 2025

Abstract
Flow synthesis has emerged as a transformative approach in pharmaceutical manufacturing,
offering unique control over reaction parameters, enhanced safety, and improved efficiency.
This transformation from traditional batch processes to flow systems has the potential to
revolutionize the production of vital compounds, addressing the growing demand for
sustainable and cost-effective manufacturing methods in the pharmaceutical industry.
This study explored the application of flow chemistry to two distinct yet equally important
synthetic targets: L-ascorbic acid (vitamin C) and N5-OH-Ornithine, a key intermediate in
piperazic acid synthesis. The goal was to develop more efficient and scalable routes to these
compounds, which play crucial roles in nutrition and drug development.
For L-ascorbic acid synthesis, the study adapted a modified Reichstein process from batch to
flow conditions. Starting with D-glucose, four of the five steps, reduction, protection,
oxidation, and lactonization, were successfully implemented in flow systems. These steps
showed improved yields and shorter reaction times compared to batch methods. The second
step, enzymatic oxidation, remains under investigation. The secondary objective, protecting
L-Ornithine in the flow, faced challenges due to foaming and CO2 evolution, which caused
clogging in the flow system. Strategies to address these challenges include larger tubing,
continuous stirred tank reactors, and liquid-gas separators.
Success with L-ascorbic acid synthesis highlights the power of flow synthesis in improving
reaction efficiency and product yield. In contrast, the challenges encountered in L-Ornithine
protection highlights the complexities of gas-evolving reactions in flow systems. This work
contributes to the growing knowledge of applying flow chemistry in natural product
synthesis.
ii

Table of Contents

Abstract ..................................................................................................................................... ii
Table of Contents ..................................................................................................................... iii
List of Tables ......................................................................................................................... viii
List of Figures .......................................................................................................................... ix
List of Schemes ....................................................................................................................... xii
List of Abbreviations and Symbols........................................................................................ xiii
Acknowledgement ................................................................................................................. xvi
Dedication ............................................................................................................................. xvii
1.0 Introduction ..........................................................................................................................1
1.1 Flow Synthesis .....................................................................................................................1
1.1.1 Differences between Flow Synthesis and Batch Process ..................................................2
1.1.2 Benefits of Flow Synthesis ...............................................................................................3
1.1.3 Continuous Flow Synthesis...............................................................................................5
1.2 Flow Synthesis Components ................................................................................................6
1.2.1 Pumps................................................................................................................................7
1.2.2 Lines and Mixers...............................................................................................................8
1.2.3 Back Pressure Regulators .................................................................................................9
1.2.4 Inline Separators .............................................................................................................10
1.2.5 Drying in Flow ................................................................................................................10
1.2.6 Legends for Flow Components in Diagrams ..................................................................12
1.3 Challenges and Potential Solutions in Flow Synthesis ......................................................13
iii

1.4 Natural Products in Health and the Economy ....................................................................14
1.4.1 Value and Techniques for Localized Production of Natural Products ...........................19
1.5 Research Objective ............................................................................................................20
2.0 Flow Synthesis Method of Ascorbic Acid .........................................................................21
2.1 Chapter Overview ..............................................................................................................21
2.1.1 Value and Production of Ascorbic Acid .........................................................................21
2.1.2 Global Market and Applications .....................................................................................24
2.1.3 Industrial Production Methods ........................................................................................26
2.1.3.1 Reichstein Process .......................................................................................................26
2.1.3.2 Advantages and Limitations of Reichstein Process .....................................................30
2.1.4 Challenges in Traditional Synthesis................................................................................31
2.2 Developing Flow Synthesis of Ascorbic Acid ...................................................................32
2.2.1 D-glucose Reduction to Sorbitol in Batch and Flow ......................................................35
2.2.1.1 D-glucose Reduction to Sorbitol in Batch – Trial 1 ....................................................35
2.2.1.2 D-glucose Reduction to Sorbitol in Flow – Trial 2......................................................36
2.2.1.3 D-glucose Reduction to Sorbitol in Batch – Trial 3 ....................................................38
2.2.1.4 D-glucose Reduction to Sorbitol in Batch – Trial 4 ....................................................39
2.2.1.5 D-glucose Reduction to Sorbitol in Batch – Trial 5 ....................................................40
2.2.1.6 D-glucose Reduction to Sorbitol in Batch – Trial 6 ....................................................41
2.2.1.7 D-glucose Reduction to Sorbitol in Batch – Trial 7 ....................................................42
2.2.1.8 D-glucose Reduction to Sorbitol in Batch – Trial 8 ....................................................43
2.2.1.9 D-glucose Reduction to Sorbitol in Flow – Trial 9......................................................44
2.2.1.10 D-glucose Reduction to Sorbitol in Flow – Trial 10..................................................45
iv

2.2.1.11 Sorbitol Product Yields ..............................................................................................47
2.2.1.12 Optimization and Discussion of Reaction Conditions in Batch.................................49
2.2.1.12.1 Sorbitol Synthesis: Acetic acid ...............................................................................49
2.2.1.12.2 Sorbitol Synthesis: Reaction Time..........................................................................50
2.2.1.12.3 Sorbitol Synthesis: Amount of NaBH4 ...................................................................50
2.2.1.13 Comparison of Sorbitol Yield in Batch and Flow .....................................................51
2.2.2 L-Sorbose to Protected Sorbose in Batch and Flow........................................................52
2.2.2.1 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 1 ........................52
2.2.2.2 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 2 ........................54
2.2.2.3 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 3 ........................55
2.2.2.4 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 4 ........................56
2.2.2.5 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 5 .........................57
2.2.2.6 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 6 ........................58
2.2.2.7 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 7 .........................59
2.2.2.8 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 8 ........................60
2.2.2.9 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 9 .........................61
2.2.2.10 Diacetone-L-Sorbose Product Yields .........................................................................65
2.2.2.11. Optimization and Discussion of Reaction Conditions ..............................................69
2.2.2.11.1 Diacetone-L-Sorbose Synthesis: Temperature and Reaction Time ........................69
2.2.2.11.2 Diacetone-L-Sorbose Synthesis: Acetone to Sorbose Ratio ...................................71
2.2.2.12

Comparison of Diacetone-L-sorbose Yield in Batch and Flow ...........................72

2.2.3 Protected Sorbose (Diacetone-L-Sorbose - DAS) to 2-keto-L-gulonic Acid in Batch and
Flow .........................................................................................................................................73
v

2.2.3.1 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Batch – Trial 1 .....73
2.2.3.2 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Batch – Trial 2 .....74
2.2.3.3 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Flow – Trial 3 ......75
2.2.3.4 2-keto-L-gulonic Acid Yield and Comparison of Batch and Flow ..............................80
2.2.4 2-keto-L-gulonic Acid to Ascorbic Acid in Batch and Flow ..........................................82
2.2.4.1 2-keto-L-gulonic Acid to Ascorbic Acid Batch – Trial 1 ............................................82
2.2.4.2 2-keto-L-gulonic Acid to Ascorbic Acid Flow – Trial 2 .............................................83
2.2.4.3 Ascorbic Acid Product Yields and Comparison of Batch and Flow ...........................88
2.3 Conclusion .........................................................................................................................89
2.3.1 Comparison of Flow Methods with Batch Processes .....................................................89
2.3.2 Advantages and Limitations of Developed Protocols .....................................................90
2.3.3 Potential for Scale-up and Industrial Applications .........................................................91
3.0 Flow synthesis of N5-OH-L-Orn towards Chemoenzymatic synthesis of Piperazic acid .93
3.1 Chapter Overview ..............................................................................................................93
3.2. Piperazic Acid: A Versatile Building Block .....................................................................93
3.2.1 Biological Significance and Applications .......................................................................94
3.2.2 Current Synthetic Approaches and Limitations of Piperazic Acid .................................94
3.2.3.1 Biosynthesis of Piperazic Acid and the need for N5-OH-L-Orn .................................96
3.3 Developing the Flow Synthesis Strategy for N5-OH-L-Orn .............................................97
3.3.1 L-Ornithine Protection to N5-Cbz-Ornithine in Batch and Flow ...................................99
3.3.1.1 L-Ornithine Protection to N5-Cbz-Ornithine in Batch – Trial 1..................................99
3.3.1.2 L-Ornithine Protection to N5-Cbz-Ornithine in Flow – Trial 2 .................................100
3.3.1.2.1 L-Ornithine (I) to Ornithine-copper complex (II) solution .....................................100
vi

3.3.1.2.2 Ornithine-copper complex (II) solution protection to Cupric-ornithine complex ..101
3.4 Conclusion .......................................................................................................................102
3.4.1 Challenges and Limitations of Developed Flow Protocol ............................................102
3.4.2 Proposed Solutions to Overcoming Challenges of Developed Flow Protocol .............103
4.0 General Discussions and Conclusion ...............................................................................105
4.1 Achievement of Objectives and Summary of Key Findings ...........................................105
4.2 Further Studies and Future Recommendations ................................................................108
5.0 Experimental Section .......................................................................................................110
5.1 General Experimental Procedures....................................................................................110
5.2 Synthesis Procedures .......................................................................................................111
5.2.1 Preparation of D-sorbitol 2.2.1.10 ................................................................................111
5.2.2 Preparation of partially protected Sorbose 2.2.2.3 ........................................................111
5.2.3 Preparation of diacetone-L-sorbose 2.2.2.9 ..................................................................112
5.2.4 Preparation of 2-keto-L-gulonic acid 2.2.3.3 ................................................................113
5.2.5 Preparation of L-ascorbic acid 2.2.4.2 ..........................................................................114
References ..............................................................................................................................116
Appendix A: Spectroscopic Data ...........................................................................................128

vii

List of Tables
Table 1: Sorbitol reaction conditions and product yields for various trials in the batch and
flow. ........................................................................................................................................ 48
Table 2: Comparison of Sorbitol yield and the reaction parameters in Batch versus Flow.... 52
Table 3: 13C and 1H NMR Data for Partially Protected Sorbose (Batch 3) ............................ 67
Table 4: 13C and 1H NMR Data for Diacetone-L-sorbose (Flow 9) ....................................... 68
Table 5: Effect of Temperature and Residence Time on the L-Sorbose Acetonation Process in
the Batch ................................................................................................................................. 69
Table 6: Effect of Temperature and Residence Time on the L-Sorbose Acetonation Process in
the Flow .................................................................................................................................. 69
Table 7: Effect of Acetone to Sorbose ratio (A/S) on the Diacetone-L-Sorbose yield in the
Batch ....................................................................................................................................... 71
Table 8: Effect of acetone to Sorbose ratio (A/S) on the Diacetone-L-Sorbose yield in the
Flow ........................................................................................................................................ 71
Table 9: Comparison of Diacetone-L-sorbose yield and the Reaction parameters in Batch
versus Flow ............................................................................................................................. 72
Table 10: 13C and 1H NMR Data for 2-keto-L-gulonic acid (Flow 3) .................................... 80
Table 11: Comparison of 2-keto-L-gulonic acid yields and the Reaction parameters in Batch
versus Flow ............................................................................................................................. 82
Table 12: 13C and 1H NMR Data for Ascorbic acid (Flow 2) ................................................. 87
Table 13: Comparison of Ascorbic acid yields and the Reaction parameters in Batch versus
Flow ........................................................................................................................................ 89

viii

List of Figures
Figure 1: Flow Process or Flow Synthesis simple diagram. ..................................................... 2
Figure 2: Difference between Batch process and Flow Process ............................................... 3
Figure 3: Comparison /Differences between traditional batch process, flow process and
Continuous flow process ........................................................................................................... 6
Figure 4: The syringe pump used in the laboratory. ................................................................. 7
Figure 5: T-mixer and Y-mixer (left to right) used in the laboratory. ...................................... 8
Figure 6: Back Pressure Regulator Used in the laboratory setup (75 psi). ............................... 9
Figure 7: Molecular sieve columns (4A and 3A - top to bottom) used in the laboratory setup.
................................................................................................................................................. 11
Figure 8: Standard Flow Reactor Component/ Equipment ..................................................... 12
Figure 9: Chemical structure of L-ascorbic acid (vitamin C) ................................................. 22
Figure 10: The oxidation process of L-ascorbic acid to DHA losing protons and electrons .. 23
Figure 11: Regioselective oxidation of D-Sorbitol at C5 using Gluconobacter Oxydans to LSorbose.................................................................................................................................... 28
Figure 12: Sorbitol 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz)........................ 36
Figure 13: Sorbitol 1H-NMR for Flow 2 recorded in DMSO-d6 (300 MHz). ........................ 38
Figure 14: Sorbitol 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz)........................ 39
Figure 15: Sorbitol 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz)........................ 40
Figure 16: Sorbitol 1H-NMR for Batch 5 recorded in DMSO-d6 (300 MHz)........................ 41
Figure 17: Sorbitol 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz)........................ 42
Figure 18: Sorbitol 1H-NMR for Batch 7 recorded in DMSO-d6 (300 MHz)........................ 43
Figure 19: Sorbitol 1H-NMR for Flow 9 recorded in DMSO-d6 (300 MHz). ........................ 45
ix

Figure 20: Sorbitol 1H-NMR for Flow 10 recorded in DMSO-d6 (300 MHz). ...................... 47
Figure 21: Structure of L-sorbose before the protection of its hydroxyl groups .................... 53
Figure 22: DAS Product 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz). .............. 54
Figure 23: DAS Product 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz). .............. 55
Figure 24: DAS Product 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz). .............. 56
Figure 25: DAS Product 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz). .............. 57
Figure 26: DAS Product 1H-NMR for Flow 5 recorded in DMSO-d6 (300 MHz). .............. 58
Figure 27: DAS Product 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz). .............. 59
Figure 28: DAS Product 1H-NMR for Flow 7 recorded in DMSO-d6 (400 MHz). ............... 60
Figure 29: Diacetone-L-sorbose 1H-NMR for Flow 9 recorded in CDCl3 (400 MHz). .......... 62
Figure 30: Diacetone-L-sorbose 13C-NMR for Flow 9 recorded in CDCl3 (400 MHz). ......... 63
Figure 31: Diacetone-L-sorbose COSY for Flow 9 recorded in CDCl3 (400 MHz). .............. 63
Figure 32: Diacetone-L-sorbose HSQC for Flow 9 recorded in CDCl3 (400 MHz). .............. 64
Figure 33: Diacetone-L-sorbose HMBC for Flow 9 recorded in CDCl3 (400 MHz). ............. 64
Figure 34: The structure of the partially protected sorbose and the NMR (2-D) correlation. 67
Figure 35: The structure of obtained diacetone-L-sorbose and the NMR (2-D) correlation. . 67
Figure 36: The structures of the potential products: partially protected L-sorbose, diacetoneL-sorbose, and 1,2:4,6-di-O-isopropylidene derivative .......................................................... 70
Figure 37: 2-keto-L-gulonic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz). . 74
Figure 38: 2-keto-L-gulonic acid 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz). . 75
Figure 39: 2-keto-L-gulonic acid 1H-NMR for Flow 3 recorded in DMSO-d6 (400 MHz). .. 77
Figure 40: 2-keto-L-gulonic acid 13C-NMR for Flow 3 recorded in DMSO-d6 (400 MHz). . 78
Figure 41: 2-keto-L-gulonic acid HSQC for Flow 3 recorded in DMSO-d6 (400 MHz). ...... 78
x

Figure 42: 2-keto-L-gulonic acid COSY for Flow 3 recorded in DMSO-d6 (400 MHz). ...... 79
Figure 43: 2-keto-L-gulonic acid HMBC for Flow 3 recorded in DMSO-d6 (400 MHz). ..... 79
Figure 44: The structure of the 2-keto-L-gulonic acid and the NMR (2-D) correlation. ........ 80
Figure 45: Ascorbic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz). ............. 83
Figure 46: Ascorbic acid 1H-NMR for Flow 2 recorded in DMSO-d6 (400 MHz). .............. 85
Figure 47: Ascorbic acid 13C-NMR for Flow 2 recorded in DMSO-d6 (400 MHz). ............. 85
Figure 48: Ascorbic acid COSY for Flow 2 recorded in DMSO-d6 (400 MHz). .................. 86
Figure 49: Ascorbic acid HSQC for Flow 2 recorded in DMSO-d6 (400 MHz). .................. 86
Figure 50: Ascorbic acid HMBC for Flow 2 recorded in DMSO-d6 (400 MHz). ................. 87
Figure 51: The structure of the Ascorbic acid and the NMR (2-D) correlation. .................... 87

xi

List of Schemes
Scheme 1: The Reichstein process for vitamin C synthesis. The L-configuration of ascorbic
acid is derived from D-glucose by inversion of the carbon skeleton. ..................................... 30
Scheme 2: The proposed methodology for the synthesis of L-ascorbic acid from D-glucose 33
Scheme 3: The proposed methodology (batch and flow process) for the synthesis of Dsorbitol from D-glucose .......................................................................................................... 34
Scheme 4: The proposed methodology/three-batch reaction for vitamin C synthesis from Lsorbose .................................................................................................................................... 34
Scheme 5: The proposed methodology/ flow reaction for vitamin C synthesis from L-sorbose
................................................................................................................................................. 34
Scheme 6: Reduction of D-glucose to D-sorbitol in the flow – trial 2 ................................... 37
Scheme 7: Reduction of D-glucose to D-sorbitol in the flow – trial 10 ................................. 46
Scheme 8: The protection of L-sorbose to diacetone-L-sorbose in the flow .......................... 62
Scheme 9: The Oxidation of Diacetone-L-Sorbose to 2-KGA in the flow, using KMnO4 .... 77
Scheme 10: The lactonization and enolization of 2-KGA to L-ascorbic acid in the flow ...... 84
Scheme 11: Reaction Scheme for the Synthesis of N5-OH-Ornithine (Adapted from
Neumann et al.). ...................................................................................................................... 98
Scheme 12: Proposed Reaction scheme for the synthesis of N5-Cbz-Ornithine (IV) from LOrnithine (I) ............................................................................................................................ 99
Scheme 13: The protection of L-Ornithine to Ornithine-copper complex (II) solution in the
flow ....................................................................................................................................... 101
Scheme 14: The protection of Ornithine-copper complex (II) solution to Cupric-ornithine
complex in the flow .............................................................................................................. 102
xii

List of Abbreviations and Symbols
13

C

Carbon 13 NMR

1

H

Proton NMR

°C

degrees Celsius

δ

chemical shift in parts per million

ID

One dimensional

2D

Two dimensional

2-KGA

2-keto-L-gulonic acid

(Boc)2O

Di-tert-butyl dicarbonate

Boc

tert-butyloxycarbonyl

BPR

Back Pressure Regulator

CAGR

Compound annual growth rate

Cbz-cl

Benzyl Chloroformate

Cbz-Osu

Benzyl N-succinimidyl carbonate

CDCl3

Deuterated chloroform

COSY

Correlated Spectroscopy

CO2

Carbon dioxide

CSTR

Continuous Stirred Tank Reactor

Cu (II)

Copper (II)

CuSO4

Copper (II) sulfate

DAB

Deacetylbaccatin

DAS

Diacetone-L-Sorbose

DHA

Dehydro-L-ascorbic acid

DMSO-d6

Deuterated dimethyl sulfoxide

EDTA

Ethylenediaminetetraacetic acid
xiii

Eq.

Equivalents

FDA

Food and Drug Administration

FT-IR

Fourier Transform Infrared Spectroscopy

H2SO4

Sulfuric acid

HCl

Hydrochloric acid

HSQC

Heteronuclear Single Quantum Coherence

HMBC

Heteronuclear Multiple Bond Correlation

KMnO4

Potassium permanganate

KOH

Potassium hydroxide

mCPBA

meta-Chloroperoxybenzoic acid

MCM-41

Mobil Composition of Matter-41

MeOH

Methanol

NaBH4

Sodium borohydride

NaOH

Sodium hydroxide

NaHCO3

Sodium bicarbonate

Na2SO4

Sodium sulfate

NMR

Nuclear Magnetic Resonance

N-N

Nitrogen to nitrogen

NRPS

Nonribosomal peptite synthetase

PCF

Plant cell fermentation

Piz

Piperazic acid

Ppm

Parts per million

Psi

Pounds per square inch

PTFE

Polytetrafluoroethylene

RDA

Recommended dietary allowance
xiv

RT

Room temperature

Ru

Ruthenium

ZSM-5

Zeolite Socony Mobil-5

xv

Acknowledgement
I am deeply grateful to Almighty God for his unwavering guidance, strength and
perseverance throughout my journey. My heartfelt appreciation goes to my supervisor, Dr.
Kalindi Morgan, for her constant support since the beginning of this journey. Her mentorship
and guidance have been instrumental in shaping my research and academic growth, and I feel
lucky to have such an energetic and amazing supervisor.
I would also like to sincerely thank my supervisory committee members, Dr. Todd
Whitcombe, Dr. Andrea Gorrell and Dr. Oliver Iorhemen, for their kind assistance,
encouragement, and insightful feedback whenever needed. Their contributions and feedback
have greatly enriched my work. I gratefully acknowledge MITACS for its financial support,
which enabled the beginning of this research.
To my colleagues in the Morgan lab, schoolmates, and friends, thank you for making this
journey enjoyable and memorable. Your camaraderie and support have been invaluable.
I am grateful to my mother, Mrs. Uche Modum, for her endless prayers and support. Her love
and encouragement have been a guiding light in my life.

xvi

Dedication
In loving memory of my beloved late father, Engr. Patrick Modum, whose unwavering
guidance and profound belief in my potential, charted the course of my life's journey. This
thesis stands as a testament to his enduring influence.

xvii

1. Introduction
1.1 Flow Synthesis
The past 100 years have witnessed a remarkable leap forward in our comprehension and
creation of organic compounds. However, there is a striking contrast in research laboratory
practices compared to commercial or industrial production. Despite the advancements in
organic chemistry knowledge, laboratory methods and equipment have remained largely
unchanged. Researchers continue to rely on traditional tools such as round bottom flasks,
magnetic stir bars, and reflux condensers, with many processes still requiring significant
hands-on effort. This highlights a gap between theoretical progress and practical
implementation in organic chemistry. While batch chemistry, the traditional approach, can
achieve the desired chemical changes (transformations) in most cases without requiring
alternative methods like flow chemistry, a growing body of research highlights the numerous
advantages of flow synthesis.1–10
Flow synthesis, also known as flow chemistry, as shown in Figure 1, is a chemical
production method where reactants are pumped through a reactor,11,12 generating a stream of
products. In contrast to traditional batch processes, where reactions occur in a fixed volume,
flow chemistry enables a dynamic and continuous reaction environment. Reactants are
precisely mixed in specialized reactors, often microreactors or tubular systems, which
provide excellent control over reaction parameters5,6 such as temperature, pressure, and
residence time. As the reaction mixture flows through the system, it undergoes controlled
transformations, resulting in a constant and steady product output. Furthermore, flow
synthesis facilitates real-time monitoring and adjustment of reaction conditions10–12,
improving the final product's consistency and quality. These features make flow chemistry an
1

increasingly attractive option for both research and industrial applications, offering potential
improvements in efficiency, sustainability, and process control compared to traditional batch
methods.

Figure 1: Flow Process or Flow Synthesis simple diagram.

1.1.1 Differences between Flow Synthesis and Batch Process
Flow synthesis and batch processing exhibit fundamental differences that significantly
impact their application in various industries. In flow synthesis, each portion of the
reaction mixture is processed individually in a controlled manner, allowing for easier
detection and correction of defects. In batch processing, all reactants are mixed in a single
vessel, and the reaction is completed before the final product is analyzed.13–16 This
characteristic of flow synthesis enhances quality control and enables immediate
adjustments to the process, ensuring consistent quality throughout production.
Furthermore, flow synthesis can lead to cost savings by reducing waste, inventory, and
transportation costs due to its scalability and compact setup, as well as increasing
productivity13,16 and stability, offering better adaptability to customer needs compared to
batch processing. Additionally, flow systems require less space, make better use of raw
materials, and produce a higher volume of consistent products,14,16 making them more
2

cost-effective and increasingly popular in manufacturing.16,17 Flow processes are
inherently safer in many cases due to the smaller quantities of materials at any given
time.17 This is particularly advantageous when dealing with hazardous or highly reactive
compounds. Figure 2 below is a simple diagram that shows the difference between the
batch and flow processes.

Figure 2: Difference between Batch process and Flow Process

1.1.2 Benefits of Flow Synthesis
Flow synthesis offers many benefits over traditional batch processing.18,19 One primary
advantage of flow synthesis is enhanced safety,10,19 which allows for better control of
process parameters and improved product quality control.6,19 This heightened safety is
attributed to the precise management of key reaction parameters such as mixing, heating,
and residence time, which are more attainable in flow systems than conventional batch
reactions. As a result, the potential for unwanted side reactions and hazardous conditions is
3

minimized, making flow synthesis an attractive option for industries where safety is
paramount.18–20 An additional benefit is that flow synthesis contributes to greater
sustainability in chemical and pharmaceutical manufacturing. Reducing waste and energy
consumption associated with flow processes aligns with the increasing emphasis on
sustainable practices within the industry. It is an appealing choice for companies seeking to
enhance their sustainability profile.18,20 Another key advantage of flow synthesis is its
ability to deliver faster reactions, increased flexibility, and automation, rendering it suitable
for a wide range of industries, particularly pharmaceuticals.18
In conclusion, the benefits of flow synthesis have led to its growing popularity in various
industries, including pharmaceuticals and manufacturing. As such, this thesis utilized this
strategy for vitamin C production in collaboration with an industrial partner. The nature of
the flow process can lead to improved productivity and quality control, addressing some of
the limitations of the Reichstein process in batch production.14 The flow production
method may offer potential advantages in addressing some of these limitations, although
further research and development are needed to fully assess the viability of Vitamin C
production.2, 14

4

1.1.3 Continuous Flow Synthesis
Continuous flow synthesis is a method of chemical production where reactants and their
resulting products are continuously fed through a series of reactors,3,4,21 generating a
collection of products at the outlet without interruption rather than mixing them in a single
batch. The reaction occurs in an unbroken stream, with no clear separation between
individual reaction volumes, and they mix and react under tightly controlled conditions
such as temperature, pressure, and flow rate. Continuous flow synthesis represents a pivotal
advancement in the field of chemical synthesis,21–23 revolutionizing the production of
pharmaceuticals and fine chemicals. It is often preferred for large-scale production and
processes that benefit from steady-state conditions,22,23 while segmented flow, a flow
chemistry technique in which a continuous stream of liquid or gas is divided into discrete
segments, can be advantageous for high-throughput experimentation24 and reactions that
require precise control over individual reaction volumes. The choice between continuous
and segmented flow often depends on the reaction's specific requirements, the production
scale, and the desired level of control over individual reaction volumes.25,26 Traditional
batch processes are time-consuming and energy-intensive, and the transition from
traditional batch mode to modern continuous flow chemistry signifies a significant shift in
the approach to chemical synthesis.17,21,23,27,28
Historically, continuous flow reactors have been used industrially for over 100 years8,11,15
to synthesize bulk chemicals and are now increasingly applied to synthesizing
pharmaceuticals from multiple sources such as marine, plant and bacterial natural
products.17 The application or research on continuous flow chemistry continues to thrive in
5

academic and industrial laboratories, driven by developing efficient and innovative
syntheses. This discipline has diversified and expanded to provide novel and practical
solutions to various fields, including synthetic organic chemistry, biochemistry, renewable
fuels, and materials synthesis.18,19,21,22,28

Traditional Batch Process

A

+

B

Reaction 1

C

C

+

D

Reaction 2

E

E

+

F

Reaction 3

G

Continuous Flow Synthesis

D
A

C

F
E

G

B
Reaction 1

Reaction 2

Reaction 3

Figure 3: Comparison /Differences between traditional batch process, flow process and Continuous flow
process

1.2 Flow Synthesis Components
Flow synthesis offers a modern and efficient approach to chemical reactions. The
6

components of flow synthesis systems typically include pumps, flow meters,
cooling/heating units, valves, separators, and regulators. These components are essential
for maintaining the continuous flow of reagents, controlling the reaction temperature, and
ensuring the safe and efficient operation of the system.27
1.2.1 Pumps
Continuous flow systems rely on pumps to maintain a consistent flow of reagents. These
pumps are essential components that ensure the precise and continuous delivery of
reactants, solvents, and other fluids, thereby enabling the efficient operation of the flow
synthesis process.26,27 Various types of pumps are used in continuous flow systems,
including syringe, peristaltic, and diaphragm pumps. The selection and use of pumps in
continuous flow systems is critical for achieving precise fluid control and ensuring the
reliability and long-term operation of the continuous flow process.26,27,29,30 Syringe pumps
(see Figure 4) offer high-precision fluid delivery and are often used in laboratory-scale
continuous flow setups.27 In this project, syringe pumps were utilized as proof of concept.

Figure 4: The syringe pump used in the laboratory (New Era Pump Systems Inc.).

7

1.2.2 Lines and Mixers
Lines and mixers are required in continuous flow systems to ensure the efficient and
homogeneous mixing of solid and liquid feed components.31 In this context, lines refer to
the piping and tubing that transport the various components to the mixer. In contrast, mixers
are the devices responsible for combining these components into a uniform mixture. In-line
mixers are widely used in various industries and designed to be integrated into the
processing system, allowing ingredients to be mixed as they flow through the mixer.27,31
This continuous flow of ingredients ensures a seamless and uninterrupted mixing process,
making in-line mixers ideal for applications that demand high precision, such as
pharmaceuticals, cosmetics, and personal care products. The Y-mixer and T-mixer (Figure
5) are inline mixers26,27 used for producing homogenous mixtures. The Y-mixer is widely
used to produce homogeneous mixtures from bulk components, offering an efficient
solution for mixing solid and liquid feed components.27,32 Conversely, T-mixers, micro-T
mixers, and oriented Y-mixers are primarily discussed in the context of microfluidics and
their mixing performances with single fluids, focusing on pressure drop and energy
considerations.33,34

Figure 5: T-mixer and Y-mixer (from IDEX Health and Science LLC.).

8

1.2.3 Back Pressure Regulators
A back pressure regulator (BPR), as shown in Figure 6, is a device used to control and
maintain pressure in a system27 by regulating the flow of a gas, liquid, or multiphase fluid.
BPRs are commonly employed in various industrial processes, including continuous flow
systems, to ensure stable and controlled pressure conditions. The BPR works by limiting
the upstream pressure, thereby maintaining the desired pressure at the outlet, and it does so
by adjusting the orifice area to control the flow.35,36 Back pressure regulators offer several
advantages over traditional pressure control methods, making them an asset in various
industrial applications. One key benefit is their ability to enable remote pressure control37,38
at a safe distance, enhancing operational safety and flexibility. This feature allows
operators to adjust and maintain pressure levels from a distance, reducing the risk of
exposure to potentially hazardous environments. Additionally, BPRs have a low-pressure
drop under dynamic conditions,38 ensuring efficient pressure control without significantly
affecting the flow rate. This characteristic is particularly advantageous in processes where
maintaining a consistent flow rate is critical. Furthermore, BPRs can be used with lowpressure inert gases, making them suitable for various applications, including those
requiring inert gases for specific process requirements.35,39

Figure 6: Back Pressure Regulator (IDEX Health and Science) Used in the Laboratory Setup (75 psi).

9

1.2.4 Inline Separators
Inline separators are crucial components in various industrial processes,40 playing a
significant role in the continuous flow separation of solvents and other substances. These
devices are designed to be integrated directly into pipelines, allowing for seamless and
efficient separation without interrupting the flow of materials. The primary mechanism of
the inline separators involves creating a centrifugal spinning action within the flow stream.
The spinning motion effectively separates liquid entrainment droplets in gases,40 making
these separators essential for steam, air, and gas processing applications. The centrifugal
force generated within the separator causes heavier liquid droplets to be thrown outwards
toward the walls of the separator. At the same time, the lighter gas continues to flow
through the center. Using inline separators in continuous flow processes offers several
advantages, including efficient separation, which reduces waste by effectively separating
substances during the flow process and improves process performance by maintaining the
purity of products.40,41 Inline separators are instrumental in supporting the transition
towards more sustainable and efficient manufacturing practices, particularly in industries
such as pharmaceuticals and specialty chemicals, where the responsible use of solvents and
other substances is a key consideration.19,40,42,43 By enabling continuous separation and
recycling of solvents, inline separation contributes to reduced solvent consumption,
improved product quality, enhanced safety and lower environmental impact, aligning with
green chemistry principles.
1.2.5 Drying in Flow
Flow drying involves uninterrupted movement of the material through the drying system,
10

allowing for a homogeneous and efficient drying process. In the context of drying, molecular
sieves, silica gel, and other drying agents are used to remove moisture from solvents and
other materials.44 Molecular sieves are crystalline metal aluminosilicates with uniform pores
that selectively absorb water and other polar molecules.44 They are commonly used in
drying processes to achieve very low moisture levels in solvents and gases.44,45 Molecular
sieves are available in different types, such as type 3A, 4A, and 5A, each with a specific
pore size and adsorption capacity.44,45 Type 3A molecular sieves are commonly used for
drying polar liquids and gases, while type 4A molecular sieves are suitable for drying nonpolar liquids and gases. Type 5A molecular sieves are used for drying and purifying gases
and fluids in the presence of water. The choice of molecular sieve depends on the specific
application and the level of moisture removal required.44

Figure 7: Molecular sieve columns (4A and 3A - top to bottom) used in the laboratory setup.

Silica gel is another popular drying agent with a high affinity44 for water molecules. It is
often used in desiccators and as a drying agent in various industrial processes. The
difference between molecular sieves and silica in continuous-flow drying is their specific
properties and performance characteristics. Silica gel, which has an affinity for water
molecules, removes moisture from solvents and gases and is available in different pore
sizes, and it can be regenerated for reuse. However, it is generally less effective than
molecular sieves in removing water and may not maintain water uptake at elevated
11

temperatures as effectively as molecular sieves, as its ability to continue absorbing water
diminishes. Therefore, while both molecular sieves and silica are used as drying agents,
molecular sieves are often preferred for applications requiring high water absorption
capacity, selective adsorption, and the ability to maintain water uptake at elevated
temperatures, such as in compressed air drying and gas dehydration processes.45–47

1.2.6 Legends for Flow Components in Diagrams
Throughout this thesis, diagrams are used to indicate flow components. As shown in Figure
8, the individual components are represented using the following standardized symbols or
legends.

Figure 8: Standard Flow Reactor Component/ Equipment

12

1.3 Challenges and Potential Solutions in Flow Synthesis
While having numerous advantages, flow synthesis also presents several challenges. One of
the key challenges is the precise control of reaction parameters,18,48 such as temperature,
pressure, and flow rates. The precise control of reaction parameters is a fundamental
challenge in continuous flow synthesis,48 as it directly impacts the quality and efficiency of
the production process. Achieving and maintaining the necessary control over these
reaction parameters requires advanced instrumentation and monitoring systems to meet the
desired conditions consistently. Another challenge is the scale-up and integration of
processes from the laboratory to industrial scale,18,23 which can be difficult due to the need
for specialized equipment and engineering considerations.
Safety and hazard mitigation are also significant concerns in continuous flow systems,
especially when handling reactive or hazardous chemicals and in the event of a system
failure or blockage. Ensuring the compatibility of catalysts and reactors in continuous flow
systems,21,23 particularly when using heterogeneous catalysts, is also a challenge that can
affect the efficiency and productivity of the process. Addressing these challenges is crucial
for the widespread adoption and successful implementation of flow synthesis in various
industries, including pharmaceuticals and specialty chemicals.18,21,23 It requires a
multidisciplinary approach, incorporating advanced process control, engineering solutions,
safety protocols, a comprehensive understanding of the specific requirements and operating
conditions of flow systems, research and development efforts to advance the capabilities of
flow synthesis in industrial, as well as the development and implementation of advanced
solutions to overcome these limitations.18,21,23,48,49
13

1.4 Natural Products in Health and the Economy
To organic and medicinal chemists, natural products are the small organic molecules (less
than 2000 g/mol) known as secondary metabolites produced by living organisms for
ecological adaptations. Their role in helping their producing organism adapt to their
environment has driven their chemical complexity. Their chemical complexity, rich with
heteroatoms and stereocenters, is involved in their biological activities, which is important
in their historical and current role as medicines. Natural products have been pivotal in
human history, serving as medicinal, nutritional, and culturally significant sources for
thousands of years. Natural products (secondary metabolites) are the most successful
source of potential drug candidates.50–54 Despite a recent decline in their utilization within
drug discovery and development processes,50 natural products remain an unparalleled
source of structural diversity. This unique characteristic offers significant advantages over
conventional combinatorial chemistry approaches, particularly in identifying novel, low
molecular weight lead compounds. It is worth noting that less than 10% of the world's
biodiversity has been assessed for potential biological activity, suggesting that a vast
reservoir of useful natural lead compounds remains unexplored.53
Using natural products for medicinal purposes has a rich history spanning thousands of
years. Ancient civilizations, including Mesopotamia, Egypt, and China, documented the
medicinal uses of plants and other natural sources in texts like cuneiform tablets (2600
BC), the Ebers Papyrus (2900 BC), and Chinese Materia Medica.53 These early records
detailed hundreds of plant-based drugs and their applications, covering various medical
conditions and treatments.53 The establishment of privately owned pharmacies by Arabs in
14

the 8th century marked a significant development in pharmacy history.53 Persian scholars,
notably Avicenna, made substantial contributions to the field, with his Canon Medicinae
influencing pharmacy and medicine for centuries in Europe and the Islamic world.53 This
rich history underscores the enduring importance of natural products in human medicine. It
highlights the cross-cultural exchange of knowledge that has shaped our understanding of
medicinal plants over the millennia.
The systematic study of natural products began in the 19th century with Friedrich
Sertürner's isolation of morphine in 1805, marking the start of modern natural product
chemistry and pharmacology. Throughout the 19th and early 20th centuries, numerous
important drugs were isolated from natural sources, including quinine, salicin (aspirin's
precursor), and penicillin, revolutionizing healthcare55–66 The modern development of
aspirin began in 1853 when Charles Frédéric Gerhardt first synthesized acetylsalicylic acid
by treating sodium salicylate with acetyl chloride.56,67 However, it wasn't until 1897 that
Felix Hoffmann at Bayer Company refined the synthesis process, motivated by his father's
intolerance to salicylic acid.67 Hoffmann's method involved replacing the hydrogen from
the hydroxy group on salicylic acid's benzene ring with an acetyl group, creating a more
stomach-friendly compound.67 The synthesis of aspirin is an esterification reaction where
salicylic acid reacts with acetic anhydride, typically catalyzed by small amounts of sulfuric
or phosphoric acid. This process yields aspirin and acetic acid as a byproduct.56,67 Bayer
began marketing aspirin globally by 1899.56,67 Penicillin, another revolutionary drug, was
discovered by Alexander Fleming in 1928 when he noticed a mold contaminating his
Staphylococcus culture plates, which had antibacterial properties.57

15

Vitamins are typically part of a broader, cultural definition of natural products
encompassing anything derived from a living organism and having health benefits. Most
vitamins are still small organic molecules but are essential to the growth and repair of the
producing organisms. Vitamins, particularly vitamin C, play vital roles in numerous
physiological processes. Vitamin C, for instance, functions as a cofactor for enzymes
involved in collagen synthesis, neurotransmitter production, and carnitine biosynthesis.68,69
It is also a powerful antioxidant, protecting cells from oxidative stress.68 The biosynthesis
of vitamin C is essential for most organisms. Still, humans and some other primates have
lost this ability due to mutations in the gene encoding the enzyme L-gulono-γ-lactone
oxidase.68 This evolutionary loss has made humans dependent on dietary sources of vitamin
C, highlighting the importance of understanding its biosynthesis and regulation in plants
and other organisms. The ability to synthesize or obtain adequate amounts of vitamins,
including vitamin C, is crucial for maintaining health, supporting growth, and enabling
proper cellular function across various species.
The pharmaceutical industry has been one of the primary beneficiaries of natural product
research.65,66 Between 1981 and 2014, 33% of all Food and Drug Administration (FDA)approved small-molecule drugs were either natural products or their derivatives.66 In
certain therapeutic areas, the impact of natural products is even more pronounced. For
instance, in anti-cancer drugs, over 60% of approved drugs between 1981 and 2014 were
derived from natural products.66 Some of the most economically significant drugs derived
from natural products include Paclitaxel (Taxol), a potent anti-cancer drug, with annual
sales exceeding $1 billion at its peak. Paclitaxel (Taxol) was originally isolated from the
bark of the Pacific yew tree (Taxus brevifolia). However, the low yield and ecological
16

concerns associated with harvesting yew bark led to the development of alternative
production methods. Bristol-Myers Squibb (BMS) pioneered a semisynthetic approach
using 10-deacetylbaccatin III (10-DAB), extracted from the renewable leaves and twigs of
the European yew (Taxus baccata).70 This process involves 11 chemical transformations
and 7 isolations to convert 10-DAB into paclitaxel.71 BMS developed a plant cell
fermentation (PCF) technology to improve further sustainability, which cultivates specific
Taxus cell lines in aqueous media under controlled conditions.71 The PCF process
eliminates the need for chemical transformations as it does not rely on traditional chemical
synthesis methods, instead leverages biological processes within plant cell cultures to
directly produce the target compound (paclitaxel), reducing the use of hazardous chemicals
and solvents while allowing year-round production.71 This innovative approach has
significantly enhanced paclitaxel production's efficiency and environmental sustainability,
ensuring a stable supply for cancer treatment.
Artemisinin is an anti-malarial drug derived from sweet wormwood (Artemisia annua) that
has saved millions of lives worldwide.72 It is now produced through semisynthesis to meet
global demand. The process involves extracting artemisinic acid from genetically
engineered yeast, which is then converted to artemisinin through photochemical
oxidation.72,73 This method, developed by Sanofi, has significantly increased artemisinin
production capacity.72,73
Statins, a cholesterol-lowering drug inspired by compounds found in red yeast rice, has
global sales of billions of dollars annually.66 Statins are primarily produced through
chemical synthesis. The most common statin, atorvastatin (Lipitor), is synthesized in a
17

multi-step process starting from (R)-4-cyano-3-hydroxybutyric acid, with a key step
involving an asymmetric aldol reaction to form the statin core structure.74 Other statins, like
simvastatin, are produced by semisynthesis from lovastatin, obtained through fermentation
of Aspergillus terreus.75,76
Red yeast rice, the natural source of statins, contains monacolins produced by fermenting
rice with Monascus purpureus. Monacolin K, structurally identical to lovastatin, is the
primary active compound.77 However, due to variability in monacolin content and potential
contamination, the FDA has issued warnings against using red yeast rice products as statin
substitutes.76,78 The pharmaceutical industry has developed more controlled and
standardized methods for statin production to ensure consistent quality and efficacy. The
economic impact of these and other natural product-derived drugs produced through a
combination of methods, with a trend towards increased use of semisynthesis and total
synthesis, is substantial, not only in direct sales but also in their contribution to public
health and reduced healthcare costs.
The food and beverage industry relies heavily on natural products for flavours, colours, and
preservatives. Essential oils, extracts, and other natural compounds enhance the taste and
aroma of foods and drinks, for example, vanillin, which is the primary component of
vanilla flavour. Although synthetically produced, it is still a natural product that has
become a major global commodity.79 Natural products are also increasingly important in
cosmetics and personal care. The production of natural products involves complex
biosynthetic pathways in living organisms, primarily utilizing building blocks derived from
acetyl coenzyme A, shikimic acid, mevalonic acid, and 1-deoxyxylulose-5-phosphate.60
18

These pathways create various secondary metabolites with diverse biological activities.
However, pharmaceutical companies often employ alternative methods for large-scale
production due to the challenges of extracting and isolating compounds from natural
sources.80
Pharmaceuticals and vitamins are primarily produced through three major approaches:
Chemical synthesis involves creating compounds through controlled chemical reactions,
often used for less complex molecules or those needed in large quantities.60,80
Semisynthesis, which combines natural product extraction with chemical modification. For
example, paclitaxel is produced by extracting 10-deacetylbaccatin from yew needles and
performing a four-stage synthesis.80 The third approach to the production is fermentation
and biotechnology. Increasingly, genetically engineered microorganisms are used to
produce pharmaceuticals and vitamins. For instance, artemisinic acid, a precursor to the
antimalarial drug, is now produced using engineered yeast.81 This method is particularly
favoured for its sustainability and scalability.81 These production methods aim to overcome
the limitations of traditional natural product extraction, such as low yields, environmental
variability, and sustainability concerns, while still harnessing the unique structural
complexity and bioactivity of natural products.80
1.4.1 Value and Techniques for Localized Production of Natural Products
In recent years, there has been a growing interest in the localized production of natural
products, driven by sustainability concerns, supply chain security, and the desire for higherquality and more diverse compounds. Localized production of natural products offers
significant value82 utilizing local biomass resources can enhance sustainability and reduce
19

transportation costs. Key techniques include biorefinery processes, which integrate various
biomass conversion methods to efficiently produce biofuels, chemicals, and
pharmaceuticals. Microbial fermentation is another versatile technique, allowing for
producing high-value products like antibiotics and vitamins from local resources, adaptable
to meet local demand.83 Localized production can enable the creation of a wider variety of
natural products, including rare or endangered species that may not be viable for largescale cultivation. This diversity is particularly valuable in drug discovery, where novel
compounds can lead to breakthrough treatments.84 Additionally, phytochemical extraction
enables the harvesting of bioactive compounds from native plants,85 contributing to the
production of cosmetics and pharmaceuticals. Finally, incorporating green chemistry
principles ensures these production techniques minimize environmental impact, promoting
a more sustainable approach to natural product synthesis. Together, these techniques or
methodologies cultivate a resilient framework for localized production, driving economic
and environmental benefits.

1.5 Research Objective
The objectives of this thesis were:
•

To develop a flow synthesis method to synthesize L-ascorbic acid from D-glucose.

•

To achieve the protection of L-ornithine for synthesizing N5-Hydroxy-L-Ornithine
towards the chemoenzymatic synthesis of Piperazic acid.

Moreover, this thesis will compare the flow synthesis and batch process methods for
synthesizing L-ascorbic acid.
20

2. Flow Synthesis Method of Ascorbic Acid
2.1 Chapter Overview
The flow synthesis of L-ascorbic acid (vitamin C) explored in this study presents an
alternative approach to traditional batch processes. It provides insights into alternative
reaction conditions compared to traditional batch processes, demonstrating potential
reaction time and yield improvements. This chapter examines the initial efforts in applying
flow chemistry techniques to specific steps in synthesizing L-ascorbic acid from D-glucose,
comparing these flow processes with their batch counterparts. The conversion of selected
batch reaction steps to continuous flow processes is investigated, focusing on synthesizing
key intermediates in the L-ascorbic acid production pathway. Reaction parameters and
yields will be compared between batch and flow methods for specific synthetic steps. The
results of these flow chemistry experiments on selected steps in the ascorbic acid synthesis
will be presented, as well as reaction times and yields for these specific transformations
between batch and flow methods.
2.1.1 Value and Production of Ascorbic Acid
Vitamins are organic substances crucial for normal physiological functions in living
organisms, particularly in higher forms of animal life.86 These micronutrients are required
in small quantities for various critical processes within the body, including growth,
development, and overall health maintenance.86 Unlike macronutrients such as proteins,
carbohydrates, and lipids, vitamins generally cannot be synthesized by the body in
sufficient amounts to meet its needs. They must be obtained through diet or
supplementation.86 There are 13 essential vitamins, each serving distinct yet often
21

overlapping bodily functions.87 These vitamins are categorized according to their solubility:
fat-soluble vitamins (A, D, E, and K) and water-soluble vitamins (C and the B-complex
vitamins).87
L-ascorbic acid (Figure 9), also known as vitamin C, is one of these essential vitamins. As a
water-soluble vitamin, it is not stored in the body and must be regularly consumed through
diet or supplementation.87,88,89,90 Ascorbic acid is primarily used to treat and prevent
scurvy, an illness resulting from a severe lack of this essential nutrient.90,91 Scurvy
manifests symptoms such as fatigue, joint pain, gum disease, and poor wound healing.90–92
Beyond its crucial role in combating scurvy, ascorbic acid offers a range of health benefits.
It supports the immune system,92,93 helps the body fight infections and illnesses, forms and
maintains bones, cartilage, skin, and blood vessels, and acts as an antioxidant.92 It is also
essential for the formation of collagen,94 a protein that is vital for the health of the skin,
bones, and blood vessels. Vitamin C is an antioxidant, protecting the body from damage
caused by free radicals, which may contribute to aging and developing certain diseases.95
Among all vitamins, it stands out as the simplest in structure and was one of the pioneering
vitamins to be isolated, characterized, purified, and have its molecular structure
elucidated.96 L-ascorbic acid is the biologically active form of Vitamin C. The
recommended dietary allowances (RDAs) for vitamin C vary by age and gender, with fruits
and vegetables89 being the best sources of this vitamin.

Figure 9: Chemical structure of L-ascorbic acid (vitamin C)

22

Vitamin C exists primarily as L-ascorbic acid in nature, with its primary interconversion
occurring between L-ascorbic acid and its oxidized form, dehydro-L-ascorbic acid (DHA),
through a reversible redox reaction (Figure 10).97,98 This oxidation process involves losing
electrons and protons sequentially, transforming L-ascorbic acid to a semidehydroascorbate radical and then to DHA. The oxidation of L-ascorbic acid to DHA
occurs in two steps: L-ascorbic acid loses one electron and one proton to form an
intermediate called semidehydroascorbate (also known as ascorbyl radical), then
semidehydroascorbate loses another electron and proton to form DHA.97,98 This oxidation
process can occur spontaneously in the presence of oxygen. Still, it is accelerated by
various factors such as heat, light, and metal ions like iron or copper.99 The reaction is pHdependent, with oxidation occurring more rapidly in alkaline conditions.100 L-ascorbic acid
demonstrates biological activity, making it the sole form valuable for medical
applications.97

Figure 10: The oxidation process of L-ascorbic acid to DHA losing protons and electrons

Historically, vitamin C deficiency, known as scurvy,91,96 was a significant health concern,
particularly among sailors and explorers who lacked access to fresh fruits and vegetables
during long voyages, which led to the need for vitamin C production.96 Its symptoms
include hemorrhaging from mucous membranes, a significant decrease in red blood cell
23

count leading to anemia, and, if left untreated, is ultimately fatal.89,91,96 In 1937, Dr. Albert
Szent-Györgyi was awarded the Nobel Prize in Physiology or Medicine for his
groundbreaking work isolating the vitamin C molecule from red peppers and elucidating its
crucial role in preventing scurvy.94,97 Ascorbate, the less acidic or salt form of vitamin C,101
is synthesized by many vertebrates. In fish, amphibians, and reptiles, ascorbate is
synthesized in the kidneys, whereas in mammals, the liver is responsible for its
production.101
2.1.2 Global Market and Applications
The global market for ascorbic acid has been experiencing steady growth, driven by
increasing awareness of its health benefits and expanding applications across multiple
sectors. The global ascorbic acid market was valued at USD 1.7 billion in 2023 and is
projected to reach USD 2.9 billion by 2033, growing at a compound annual growth rate
(CAGR) of 5.60%.102 This growth is attributed to several factors, including rising consumer
health consciousness, increasing demand for fortified food and beverages, and expanding
applications in pharmaceuticals and personal care products.102
Roughly 110 kilotons of vitamin C are produced annually89,95 which is used primarily by
the pharmaceutical industry (50 %), followed by the use of antioxidants in food (25 %) and
beverages sector (15 %). Only about 10 % of the vitamins are used for animal feed
applications.95 This contrasts with all other vitamins, where the feed sector is the major
application area of vitamins. More than 80 % of today’s world demand for vitamin C is
satisfied by Chinese producers, with the remaining production distributed across the
European Union (particularly Germany and the Netherlands), India, the United States,
24

Mexico, and a few other countries.103 However, these collectively represent a small fraction
of global vitamin C manufacturing. In the late 1950s, Chinese production capacity was only
30 tons annually.95 Supply was controlled by big European and Japanese manufacturers
such as Roche, BASF, Merck, and Takeda Pharmaceutical.89,95 By the early 1990s, 26
Chinese manufacturers had already gained one-third of the world’s vitamin C market.89,95
Recognizing the threat, the established producers tried to prevent Chinese companies from
entering the market through several severe price cuts. By 2002, only four Chinese
manufacturers had survived, and the price had hit record lows.89,95 Since 2002, the
production and export activities of Chinese vitamin C manufacturers have been under the
supervision of the trade association CCCMHPIE (China Chamber of Commerce for Import
and Export of Medicines and Health Products),89,95 which is close to the Ministry of
Commerce of the central Chinese Government. Today’s leading Chinese producers are
Northeast Pharmaceutical Group Co., Ltd (NEPG) of Shenyang Liaoning Province,
Weisheng Pharmaceutical Company (CSPC) of Shijiazhuang, Hebei Province, Welcome
Pharmaceutical Company (NCPC), Shijiazhuang, Hubei Province, and Jiangsu Jiangshan
Pharmaceutical (new name is Aland Nutraceutical Co., Ltd.) of Jingjiang, Jiangsu
Province.89,95 The global market for ascorbic acid is robust and diverse, with applications
spanning multiple industries. Its role in promoting health and wellness and its functional
properties in food preservation and personal care ensure a steady demand. As consumer
awareness grows and new applications emerge, the ascorbic acid market is set to experience
further expansion. However, industry players must navigate challenges such as
sustainability concerns and regulatory requirements to capitalize on these opportunities
fully.
25

2.1.3 Industrial Production Methods
Traditionally, the industrial production of ascorbic acid has relied on the ReichsteinGrussner process.89,96 Since its first chemical synthesis in 1933,89,96 the industrial
production of ascorbic acid has evolved considerably, driven by increasing demand across
various sectors, including pharmaceuticals, food and beverages, and cosmetics.
2.1.3.1 Reichstein Process
The Reichstein process is a chemical and microbial method for the industrial production of
L-ascorbic acid (vitamin C) from D-glucose. It was designed in 193395 with optimizations
over time95 and has been a mainstay in vitamin C production for over 60 years.95,104 The
process as shown in Scheme 1 involves several steps, including the hydrogenation of Dglucose to D-sorbitol, followed by microbial oxidation or fermentation of sorbitol to Lsorbose,89,95 the conversion of L-sorbose into 2-keto-L-gulonic acid, which is a key
intermediate89 in the production of vitamin C and, finally, the lactonization of 2-keto-Lgulonic acid to give ascorbic acid.
Various reduction strategies are used to reduce D-glucose to D-sorbitol.105–107,107,108 One of
these strategies is catalytic transfer hydrogenation105,106 which involves using a catalyst to
facilitate hydrogen transfer, leading to the efficient reduction of D-glucose to D-sorbitol. Dglucose is hydrogenated to D-sorbitol using a catalyst such as nickel.105,106 Under high
temperature and pressure, this reduction has been achieved most recently in industrial
settings. This process has been optimized to achieve a high yield of D-sorbitol.89,106 Another
strategy is utilizing supported metal catalysts such as ruthenium (Ru) supported on MCM41107,108 or ZSM-5,108 is an effective strategy for the conversion of D-glucose into D26

sorbitol. Another strategy is the use of carbonized cassava dregs- supported ruthenium
nanoparticles as a catalyst107 which has been studied for the efficient conversion of Dglucose into D-sorbitol, demonstrating the influence of carbonization temperature on the Dglucose conversion and D-sorbitol yield. These strategies demonstrate the diverse
approaches employed in reducing D-glucose to D-sorbitol, each with its own advantages
and considerations.
The next step is the microbial oxidation or fermentation of sorbitol to L-sorbose. The most
prominent industrial method of producing L-sorbose is the biotransformation of D-sorbitol
to L-sorbose in Gluconobacter or Acetobacter species.109,110 During the process, two
hydrogen atoms were removed from one D-sorbitol molecule to form one L-sorbose
molecule. This two-step fermentation method used in the industrial production of vitamin C
includes both chemical reaction and biological fermentation steps, making it difficult to
replicate synthetically.111 The microbial oxidation or fermentation of D-sorbitol to L-sorbose
is important to oxidize the correct secondary alcohol due to the regiospecificity of the
enzymatic reaction. This process involves converting the alcohol group at the C-5 position
of D-sorbitol to a ketone group at the C-5 position of L-sorbose, as shown in Figure 11. This
regiospecificity is essential for producing L-sorbose,109 which is a crucial intermediate in the
industrial synthesis of vitamin C (ascorbic acid). Achieving this regiospecific oxidation
synthetically would be challenging due to the need for highly selective and controlled
chemical reactions109 to specifically target the C-5 position of D-sorbitol without affecting
other functional groups or carbon positions. Microbial fermentation offers a more efficient
and selective approach to achieve this regiospecific oxidation, as the enzymes involved in
the process exhibit high selectivity for the targeted alcohol group,109 leading to near27

quantitative yields of L-sorbose.
Doing this process synthetically is challenging due to the complexity of the biochemical
reactions involved and the need for specific enzymes and microorganisms to catalyze the
conversion.111 Furthermore, the use of protective groups and the specific conditions
required for microbial oxidation or fermentation add to the complexity of synthetic
production.

Figure 11: Regioselective oxidation of D-Sorbitol at C5 using Gluconobacter Oxydans to L-Sorbose

D-sorbitol, which is a sugar alcohol, is oxidized to L-sorbose by G. Oxydans.89 This
microbial oxidation process is preferred over chemical oxidation due to the microbial
enzymes' specificity. Microorganisms possess highly specific enzymes that can selectively
oxidize the C-5 alcohol group of D-sorbitol to form L-sorbose,109 while chemical oxidation
may lead to overoxidation/ the formation of undesired by-products.89,112 Therefore, the use of
microbial fermentation in the Reichstein process ensures the production of L-sorbose,112
which is a key intermediate in the synthesis of vitamin C.113 Subsequently, the 4 hydroxyl
groups in L-sorbose are protected by forming an acetal95 with acetone and an acid, leading to
the production of diacetone-L-sorbose.95 The next step involves organic oxidation with
28

potassium permanganate to produce diprogulic acid, which is then heated with water to yield
2-keto-L-gulonic acid. The process is then completed with a ring-closing step or gamma
lactonization with water removal.89,95

29

Scheme 1: The Reichstein process for vitamin C synthesis. The L-configuration of ascorbic acid is derived
from D-glucose by inversion of the carbon skeleton.

2.1.3.2 Advantages and Limitations of Reichstein Process
The Reichstein process is known for its high conversion efficiency and chemically stable
intermediate production. As discussed earlier, it is a well-established and widely used
method that has been used for over six decades, providing a reliable approach to vitamin C
production.89,114 Secondly, the process has demonstrated commercial viability and has been
used to meet the global demand for L-ascorbic acid.95
However, despite its advantages, the process presents several challenges and limitations.
First, it is characterized by a long production time, which can impact overall productivity
and efficiency.114 The process involves complex chemical and microbial steps, leading to
30

operational challenges and specialized expertise requirements.89,114 For example, the
hydrogenation step in the process necessitates high temperature and pressure, resulting in
increased energy consumption and operational costs.89,114 Furthermore, the process uses
toxic chemicals, raising environmental and safety concerns.14 The continuous operation of
the process can also be challenging, further impacting its efficiency and costeffectiveness.114
Despite these drawbacks, the Reichstein process remains a significant industrial route for
Vitamin C production due to its high overall yield and other economic factors. Efforts are
being made to find alternatives to this process, such as two-step fermentation, which has
been shown to have lower capital and operating costs. Therefore, while the Reichstein
process has been instrumental in vitamin C production, it is important to address these
challenges and limitations to improve the overall efficiency and sustainability of the
production process. Nevertheless, the Reichstein process is only efficient if produced in
massive industrial processes.89,111,115,116 Canada has no local production process and is
vulnerable to worldwide shortages.117 This calls for a more efficient method of vitamin C
production that is not dependent on fermentation. Given the benefits of flow synthesis, this
thesis explores this method for vitamin C production.
2.1.4 Challenges in Traditional Synthesis
While long-established and versatile, traditional batch synthesis faces several significant
challenges in modern chemical manufacturing. One primary issue is the inconsistency in
product quality between batches, as replicating exact conditions for each batch is
difficult.16,118 This variability can lead to increased quality control costs and potential
product rejections. Batch processes are also time-consuming and resource-intensive, with
31

considerable downtime between batches for cleaning and preparation.16,118 The fixed batch
sizes often result in underutilized equipment, reducing overall efficiency.118 From a safety
perspective, batch reactions can pose risks due to accumulating reactive intermediates or
heat, especially in exothermic processes.118 Scalability is another major challenge; what
works well in a laboratory setting may not translate directly to industrial-scale production,
often requiring extensive and costly process development.16,118 Environmental concerns are
also significant, as batch processes typically generate more waste and consume more
energy than continuous methods.118 Additionally, batch synthesis often struggles with
reactions requiring precise temperature control or involving highly reactive
intermediates.118 The pharmaceutical industry faces regulatory hurdles when transitioning
from batch to continuous processes, as defining a "batch" for regulatory filings becomes
complex in continuous manufacturing.118 These challenges collectively push the industry
towards exploring more efficient and sustainable alternatives, such as flow chemistry,
which offers potential solutions to many of these issues.
2.2 Developing Flow Synthesis of Ascorbic Acid
The proposed flow synthesis of ascorbic acid would involve a series of interconnected
steps, each requiring careful optimization. This research investigates the feasibility of
translating key steps in the ascorbic acid synthesis pathway from batch flow processes.
This study focuses on specific transformations within the overall synthesis to evaluate
potential advantages in reaction efficiency, yield, and process control that flow chemistry
might offer compared to traditional batch methods. The proposed flow synthesis relies
heavily on the Reichstein process since it is an established methodology, with the key goal
of translating the batch synthesis (Scheme 2) into a flow methodology. To do so, each
32

synthetic reaction must first be developed using batch methodology and then translated to
flow synthesis techniques.

Scheme 2: The proposed methodology for the synthesis of L-ascorbic acid from D-glucose

Scheme 3 shows the proposed method for synthesizing L-sorbitol from D-glucose in batch
and flow. D-glucose will be reduced to D-sorbitol using the reducing agent sodium
borohydride (NaBH4). In Scheme 4, the synthesis is done in the batch, and the reaction is
carried out in a round bottom flask, first for the acetonation of L-sorbose to diacetone-Lsorbose, then the oxidation of diacetone-L-sorbose to 2-keto-L-gulonic acid using
potassium permanganate, and then the lactonization reaction of 2-keto-L-gulonic acid to
obtain ascorbic acid.

33

Scheme 3: The proposed methodology (batch and flow process) for the synthesis of D-sorbitol from Dglucose

Scheme 4: The proposed methodology/three-batch reaction for vitamin C synthesis from L-sorbose

Scheme 5: The proposed methodology/ flow reaction for vitamin C synthesis from L-sorbose

34

Scheme 5 above shows the method for synthesizing ascorbic acid from L-sorbose in flow
systems. In Scheme 5, the reactions are done individually, as described in Scheme 4, but in
the flow. Ascorbic acid is obtained after the lactonization of 2-keto-L-gulonic acid.
The following subsections will discuss the development of the discrete flow synthesis steps
utilizing first batch techniques and then transitioning to flow techniques to conduct the
reactions of D-glucose to D-sorbitol and from L-sorbose to ascorbic acid, examining the
challenges encountered and the strategies employed to overcome them. The goal of the
research was to prepare for continuous flow synthesis of Vitamin C. Continuous flow
synthesis methodology consists of directly feeding the products of one reaction as the
starting material of the next. Although the work described is for discrete flow synthesis
steps, minimal purification was undertaken after each step to use the product of the
preceding reaction in the subsequent reaction to prepare and plan for transitioning this work
to continuous flow synthesis.

2.2.1 D-glucose Reduction to Sorbitol in Batch and Flow
2.2.1.1 D-glucose Reduction to Sorbitol in Batch – Trial 1
For the batch synthesis, which was done first to obtain parameters for the flow, D-glucose
(1.01 g, 5.61 mmol) was dissolved in 20 mL of dH2O. NaBH4 (0.1142 g, 3.02 mmol)
dissolved in 10 mL of dH2O was added to this solution. The solution was allowed to react
at room temperature with constant stirring for 20 minutes, as this reaction time was
sufficient for conversion based on preliminary optimization studies. After this, to react any
excess NaBH4 in the reaction mixture, glacial acetic acid (6 mL) was added dropwise until
35

universal pH indicator paper indicated the reaction mixture was acidified below 7. Once
moderately acidic, the solvent evaporated to dryness under reduced pressure. Excess H2O
was removed, leaving D-sorbitol as a white powder (0.821 g, 4.51 mmol, 80%) and
analyzed by NMR (300 MHz, DMSO-d6) and IR. See Figure 12 and Appendices A 1, A 2
and A 3.

Figure 12: Sorbitol 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

2.2.1.2 D-glucose Reduction to Sorbitol in Flow – Trial 2
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps,
as shown in Scheme 6. D-glucose (1.02 g, 5.66 mmol) was dissolved in 20 mL of dH2O and
added to the flow at a 2 ml/min flow rate from a first Pump A. NaBH4 (0.1407 g, 3.72 mmol)
dissolved in 10 mL of dH2O was added to the flow simultaneously at a 1 ml/min flow rate
36

from a second Pump B. The reaction mixture introduced into the flow system via a T-mixer
(T-mixer 1) was passed through a reactor coil maintained at ambient temperature (20 ± 2°C).
This temperature was consistently maintained throughout the reaction zone to ensure uniform
conditions. Glacial acetic acid (5 mL) was added to the flow at a 0.5 ml/min flow rate from a
third Pump C. The product was obtained following a 10-minute residence time in the reactor,
and the solvent was evaporated to dryness under reduced pressure. Excess H2O was removed,
leaving D-sorbitol as a white powder (1.02 g, 5.59 mmol, 100%) analyzed by NMR (300
MHz, DMSO-d6). See Figure 13 and Appendices A 4 and A 5.

Scheme 6: Reduction of D-glucose to D-sorbitol in the flow – trial 2

37

Figure 13: Sorbitol 1H-NMR for Flow 2 recorded in DMSO-d6 (300 MHz).

2.2.1.3 D-glucose Reduction to Sorbitol in Batch – Trial 3
D-glucose (1.01 g, 5.61 mmol) was dissolved in 20 mL of dH2O. NaBH4 (0.1097 g, 2.90
mmol) dissolved in 10 mL of dH2O was added to this solution. The solution was allowed to
react at room temperature with constant stirring for 30 minutes. After this, Glacial acetic
acid (1.5 mL) was added dropwise until the universal pH indicator paper indicated that the
reaction mixture was moderately acidic. The product was obtained, and the solvent was
evaporated to dryness under reduced pressure. Excess H2O was removed, leaving Dsorbitol as a white powder (1.02 g, 5.59 mmol, 100%) analyzed by NMR (300 MHz,
DMSO-d6) and IR. See Figure 14 and Appendices A 6, A 7, A 8 and A 9.

38

Figure 14: Sorbitol 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz).

2.2.1.4 D-glucose Reduction to Sorbitol in Batch – Trial 4
Glacial acetic acid was not used for this trial, and the reaction time was increased to 40
minutes. D-glucose (1.02 g, 5.66 mmol) was dissolved in 20 mL of dH2O. NaBH4 (0.1093
g, 2.89 mmol) dissolved in 10 mL dH2O was added to this solution. The solution was
allowed to react at room temperature with constant stirring for 40 minutes. The product was
obtained, and the solvent was evaporated to dryness under reduced pressure. Excess H2O
was removed, leaving D-sorbitol as a white powder (1.01 g, 5.54 mmol, 100%) analyzed by
NMR (300 MHz, DMSO-d6) and IR. See Figure 15 and Appendices A 10 to A 13.

39

Figure 15: Sorbitol 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz).

2.2.1.5 D-glucose Reduction to Sorbitol in Batch – Trial 5
Glacial acetic acid was not used for this trial, and the reaction time was increased to 60
minutes. D-glucose (1.10 g, 6.10 mmol) was dissolved in 20 mL of dH2O. NaBH4 (0.1480
g, 3.91 mmol) dissolved in 10 mL of dH2O was added to this solution. The solution was
allowed to react at room temperature with constant stirring for 60 minutes. The product was
obtained, and the solvent was evaporated to dryness under reduced pressure. Excess H2O
was removed, leaving D-sorbitol as a white powder (1.04 g, 5.70 mmol, 93%) analyzed by
NMR (300 MHz, DMSO-d6). See Figure 16 and Appendices A 14 and A 15.

40

Figure 16: Sorbitol 1H-NMR for Batch 5 recorded in DMSO-d6 (300 MHz).

2.2.1.6 D-glucose Reduction to Sorbitol in Batch – Trial 6
In this trial, the amount of NaBH4 was increased. D-glucose (1.05 g, 5.83 mmol) was
dissolved in 20 mL of dH2O. NaBH4 (0.1755 g, 4.64 mmol) dissolved in 10 mL of dH2O
was added to this solution. The solution was allowed to react at room temperature with
constant stirring for 60 minutes. The product was obtained, and the solvent was evaporated
to dryness under reduced pressure. Excess H2O was removed, leaving D-sorbitol as a white
powder (1.03 g, 5.65 mmol, 97%) analyzed by NMR (300 MHz, DMSO-d6). See Figure 17
and Appendices A 16 and A 17.

41

Figure 17: Sorbitol 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz).

2.2.1.7 D-glucose Reduction to Sorbitol in Batch – Trial 7
In this trial, the amount of NaBH4 used for the reaction and reaction time was increased. Dglucose (1.02 g, 5.66 mmol) was dissolved in 20 mL of dH2O. NaBH4 (0.2005 g, 5.30
mmol) dissolved in 10 mL of dH2O was added to this solution. The solution was allowed to
react at room temperature with constant stirring for 105 minutes. The product was obtained,
and the solvent was evaporated to dryness under reduced pressure. Excess H2O was
removed, leaving D-sorbitol as a white powder (1.02 g, 5.59 mmol, 99%) analyzed by
NMR (300 MHz, DMSO-d6). See Figure 18 and Appendix A 18.

42

Figure 18: Sorbitol 1H-NMR for Batch 7 recorded in DMSO-d6 (300 MHz).

2.2.1.8 D-glucose Reduction to Sorbitol in Batch – Trial 8
In this trial, the reaction time was increased, NaBH4 was increased at intervals, and aliquots
were collected to monitor the sorbitol synthesis. D-glucose (1.07 g, 5.94 mmol) was
dissolved in 20 mL of dH2O. NaBH4 (0.1137 g, 3.01 mmol) dissolved in 10 mL of dH2O was
added to this solution. The solution was allowed to react at room temperature with constant
stirring for 60 minutes. After 60 minutes, one-third of the reaction (~10 mL) was removed.
The product obtained was evaporated to dryness under reduced pressure. Excess H2O was
removed, leaving D-sorbitol as a white powder called 8-A (0.35 g, 1.97 mmol, 100%)
analyzed by NMR (300 MHz, DMSO-d6). See Appendices A 19 and A 20. To the remaining
mixture, an additional amount of NaBH4 (0.0303 g, 0.801 mmol) was added and stirred for
43

45 minutes (105 minutes total). Half of the reaction (~10 mL) was removed. The product
obtained was evaporated to dryness using a rotary evaporator. Excess H2O was removed,
leaving D-sorbitol as a white powder called 8-B (0.35 g, 1.98 mmol, 100%) analyzed by
NMR (300 MHz, DMSO-d6). See Appendices A 21 and A 22. Additional NaBH4 (0.0300 g,
0.793 mmol) was added to the remaining aliquot and allowed to react for an additional 45
minutes (150 minutes total). The product was obtained, and the solvent was evaporated to
dryness under reduced pressure. Excess H2O was removed, leaving D-sorbitol as a white
powder called 8-C (0.35 g, 1.98 mmol, 100%) analyzed by NMR (300 MHz, DMSO-d6). See
Appendix A 23.
2.2.1.9 D-glucose Reduction to Sorbitol in Flow – Trial 9
Using the batch parameters in trial six, which was optimal for the glucose reduction
compared to previous trials, a second trial for the flow was done. A flow system was set up
using PTFE tubing (0.75 mm inner diameter) and syringe pumps. D-glucose (1.04 g, 5.77
mmol) was dissolved in 20 mL of dH2O and added to the flow at a 2 ml/min flow rate from a
first Pump A. NaBH4 (0.1758 g, 4.65 mmol) dissolved in 10 mL of dH2O was added to the
flow simultaneously at a 1 ml/min flow rate from a second Pump B. The reaction mixture
introduced into the flow system via a T-mixer (T-mixer 1) was passed through a reactor coil
maintained at ambient temperature (20 ± 2°C). This temperature was consistently maintained
throughout the reaction zone to ensure uniform conditions. The product was obtained
following a 10-minute residence time in the reactor, and the solvent was evaporated to
dryness under reduced pressure. Excess H2O was removed, leaving D-sorbitol as a white
powder (1.05 g, 5.76 mmol, 100%) analyzed by NMR (300 MHz, DMSO-d6). See Figure 19
and Appendix A 24.

44

Figure 19: Sorbitol 1H-NMR for Flow 9 recorded in DMSO-d6 (300 MHz).

2.2.1.10 D-glucose Reduction to Sorbitol in Flow – Trial 10
In this trial, the residence time in the flow was increased to let the reaction come to
completion. A flow system was set up using PTFE tubing (0.75 mm inner diameter) and
syringe pumps, as shown in Scheme 7. D-glucose (1.03 g, 5.71 mmol) was dissolved in 10
mL of dH2O and added to the flow at a 0.5 ml/min flow rate from a first Pump A. NaBH4
(0.1753 g, 4.63 mmol) dissolved in 10 mL of dH2O was added to the flow simultaneously at
a 0.5 ml/min flow rate from a second Pump B. The reaction mixture introduced into the flow
system via a T-mixer (T-mixer 1) was passed through a reactor coil maintained at ambient
temperature (20 ± 2°C). This temperature was consistently maintained throughout the
reaction zone to ensure uniform conditions. The product was obtained following a 20-minute
45

residence time in the reactor, and the solvent was evaporated to dryness under reduced
pressure. Excess H2O was removed, leaving D-sorbitol, a white powder (1.04 g, 5.70 mmol,
99%) analyzed by NMR (300 MHz, DMSO-d6). See Figure 20 and Appendices A 62 – A 65.

Scheme 7: Reduction of D-glucose to D-sorbitol in the flow – trial 10

46

Figure 20: Sorbitol 1H-NMR for Flow 10 recorded in DMSO-d6 (300 MHz).

2.2.1.11 Sorbitol Product Yields
The reduction of D-glucose using NaBH4 successfully yielded D-sorbitol, as confirmed by IR
and NMR spectroscopic analysis. The reaction trials consistently produced product yields
exceeding 100%, except for trial 1 (Table 1). The low yield is attributed to the degradation of
the product into a brown mass due to excessive heating.

47

Table 1: Sorbitol reaction conditions and product yields for various trials in the batch and flow.

Trial

Reaction
Process

Acetic
acid

Reaction
Time
(min)

Amount
(mmol)
D-glucose

Amount
(mmol)
NaBH4

Crude
Yield (%)

3.02

Crude
Yield
(mmol)
D-sorbitol
4.51

1

Batch

Yes

40

5.61

2

Flow

Yes

10

5.66

3.72

5.59

100

3

Batch

Yes

50

5.61

2.90

5.59

100

4

Batch

No

40

5.66

2.89

5.54

100

5

Batch

No

60

6.11

3.91

5.70

93

6

Batch

No

60

5.83

4.64

5.65

97

7

Batch

No

105

5.66

5.30

5.59

99

8-A

Batch

No

60

1.98 (5.94)

3.01

1.97

100

8-B

Batch

No

105

1.98 (5.94)

3.81

1.98

100

8-C

Batch

No

150

1.98 (5.94)

4.60

1.98

100

9

Flow

No

10

5.77

4.65

5.76

100

10

Flow

No

20

5.71

4.63

5.70

99

80

Yields at or surpassing 100% can be primarily attributed to residual water content.119 This
phenomenon is linked to D-sorbitol's nature as a humectant, characterized by its high
affinity for water molecules.119 D-sorbitol structure features multiple hydroxyls (-OH)
groups, enabling extensive hydrogen bonding with water molecules,119,120 a process known
as sugar hydration. The strong interaction between sorbitol and water likely resulted in
incomplete water removal during drying, leading to artificially inflated yields. 119,120 This

48

persistence of water, despite drying efforts, is a direct consequence of the robust sugar
hydration effect. The product may also contain unreacted starting materials, side products,
or inorganic salts from the reaction mixture that were not completely removed, as
purification has not been done.
2.2.1.12 Optimization and Discussion of Reaction Conditions in Batch
2.2.1.12.1 Sorbitol Synthesis: Acetic acid
Trials 1 and 3, done in the batch, and trial 2 in flow, employed acetic acid to neutralize
excess NaBH4 and halt further reactions. The presence of acetic acid is evidenced by
distinctive 1H-NMR chemical shifts at 1.89 ppm for trials 1 and 2 and 1.87 ppm for trial 3
(See Figures 12, 13 and 14, respectively). Corroborating 13C-NMR data reveals strongly
deshielded signals at 172.25 ppm (1 and 2) and 172.48 ppm (3), further confirming acetic
acid's presence (See Appendices A 2, A 5 and A 7). Notably, these acetic acid-containing
trials also exhibited signals indicative of unreacted glucose, the anomeric carbons for both
α and β isomers, appearing at 4.90 ppm (d, J = 3.3 Hz) and 4.26 ppm (d, J = 7.8 Hz). These
chemical shifts are significantly lower than those typical of glycosyl donors (6.0-7.0 ppm),
suggesting the formation of glucosides rather than free glucose. The introduction of acetic
acid promoted Fischer glycosylation,119,121 leading to glucose self-condensation. This
reaction is known to be catalyzed under acidic conditions, explaining the observed
glucoside formation in these trials.
The trials without acetic acid demonstrated a marked decrease in glucoside formation, as
evidenced by the reduced intensity of downfield anomeric doublets corresponding to both α
and β isomers (See Figures 15 to 20 and Appendices A 10 – A 23). While these acid-free

49

trials successfully minimized glucoside production, some glucose remained unreacted,
varying depending on reaction duration and NaBH4 concentration. Although unreacted
glucose was still present, these trials exhibited lower residual reagent levels than trials 1, 2
and 3, as indicated by the diminished intensity of the anomeric signals. The higher
proportion of unreacted glucose in trials 1, 2 and 3 can be attributed to competition
between Fischer glycosylation and glucose reduction reactions. This competitive effect
arose from the acidic conditions introduced by acetic acid, which simultaneously promoted
glycosylation while hindering the reduction process.
2.2.1.12.2 Sorbitol Synthesis: Reaction Time
Generally, extending the reaction duration promotes more complete glucose reduction. A
comparison between trials 8-A and 8-B revealed that a 45-minute increase in reaction time
in the batch resulted in a significant decrease in unreacted glucose, as evidenced by the
reduced intensity of 1H-NMR anomeric doublets (See Appendices A 19 and A 21,
respectively). However, trial 8-C illustrates that excessive reaction times can increase
undesired side reactions (See Appendix A 23). This suggests an optimal reaction window
exists, balancing maximal glucose reduction against the onset of secondary product
formation.
2.2.1.12.3 Sorbitol Synthesis: Amount of NaBH4
Similar to reaction time, increasing the amount of NaBH4 generally enhances glucose
reduction. However, trials with a NaBH4-to-glucose ratio exceeding 0.8 showed an
increased presence of unknown impurities, likely resulting from additional side reactions.
For instance, trials 7 and 8-C, which used ratios of 0.9 and 0.8, exhibited significant
impurity formation (See Appendices A 18 and A 23, respectively). Conversely, insufficient
50

NaBH4 led to incomplete glucose reduction, as evidenced by the prominent anomeric
proton signals observed in the 1H-NMR spectra of trials 1, 2, and 3 (See Appendices A 1, A
4, and A 6, respectively). This highlights the need for an optimal NaBH4 ratio to balance
effective glucose reduction with minimal side reactions.
2.2.1.13 Comparison of Sorbitol Yield in Batch and Flow
Based on a comprehensive analysis of various reaction parameters in the batch during this
optimization, including the presence or absence of acetic acid, the NaBH4 to D-glucose
ratio, and reaction duration, trials 5 and 6 yielded the most favourable outcome of sorbitol
yields in the batch (See Table 1). These trials employed NaBH4 to D-glucose ratios of 0.64
and 0.79, respectively, with a consistent reaction time of 60 minutes for both. Drawing
from these results, the optimal conditions for this reaction in the batch were proposed and
are;
•

NaBH4 to D-glucose ratio: 0.7

•

Acetic acid: Omitted

•

Reaction duration in batch: 60 minutes

These parameters balance efficient glucose reduction and minimal side product formation,
offering a refined protocol for synthesizing D-sorbitol from D-glucose. Implementing a
flow setup using the optimized parameters yielded remarkable results, achieving
comparable product quality with a significantly reduced reaction or residence time of just
20 minutes, in contrast to the 60-minute duration required for the batch process. This
substantial reduction in residence time highlights the superior efficiency of the flow
system. The enhanced performance of the flow reaction can be attributed primarily to the
more effective mixing dynamics inherent to flow processes. The reagents are continuously
51

and intimately mixed in flow reactors as they travel through the reactor channels, ensuring
uniform concentration throughout the reaction medium. This efficient mixing promoted
rapid mass transfer and heat exchange and allowed for faster completion of the glucose
reduction. Additionally, the continuous nature of the flow process enabled better control
over reaction parameters. It reduced the likelihood of concentration or temperature
fluctuations, which could lead to side reactions or incomplete conversion. Consequently,
the flow process reduced the reaction time and improved product consistency and yield, as
seen in Table 2, demonstrating the advantages of flow chemistry in optimizing this Dglucose to D-sorbitol reduction process.
Table 2: Comparison of Sorbitol yield and the reaction parameters in Batch versus Flow

D-Glucose to Sorbitol Reduction - Best Results in Batch versus Flow
Batch

Flow

Reaction/ Residence time
(mins)

60

20

D-sorbitol Yield (mmol)

5.65

5.70

Total Yield (%)

97

99

2.2.2 L-Sorbose to Protected Sorbose in Batch and Flow
2.2.2.1 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 1
The L-sorbose protection (the hydroxyl groups at C-2, C-3, C-4, and C-6, as shown in Figure
21) was done by reacting with acetone.

52

Figure 21: Structure of L-sorbose before the protection of its hydroxyl groups

L-sorbose (0.625 g, 3.46 mmol) was dissolved in 25 mL of acetone (acetone to sorbose ratio
of 97: 1). H2SO4 (1 mL) was added dropwise to this solution until universal pH indicator
paper indicated the reaction mixture was acidified. The solution was allowed to react at room
temperature with constant stirring for 180 minutes. After this, sodium bicarbonate (20 mL)
was added dropwise until the universal pH indicator paper indicated that the reaction mixture
was neutralized. The solvent was evaporated to dryness under reduced pressure. Excess
acetone was removed, leaving a white powder (0.41 g, 1.58 mmol, 45 %) analyzed by NMR
(300 MHz, DMSO-d6). See Figure 22/ Appendix A 31.

53

Figure 22: DAS Product 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

2.2.2.2 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 2
In this trial, the effect of acetone volume on the reaction completion and yield was
investigated by increasing the volume of acetone from 25 mL to 50 mL and the reaction time.
L-sorbose (0.624 g, 3.46 mmol) was dissolved in 50 mL of acetone (acetone to sorbose ratio
of 194: 1). H2SO4 (1.2 mL) was added dropwise to this solution until universal pH indicator
paper indicated the reaction mixture was acidified. The solution was allowed to react at room
temperature with constant stirring for 240 minutes. After this, sodium bicarbonate (20 mL)
was added dropwise until the universal pH indicator paper indicated that the reaction mixture
was neutralized. The solvent was evaporated to dryness under reduced pressure. Excess
acetone was removed, leaving a white powder (0.51 g, 1.95 mmol, 56 %) analyzed by NMR
(300 MHz, DMSO-d6). See Figure 23/ Appendix A 32.

54

Figure 23: DAS Product 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz).

2.2.2.3 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 3
In this trial, the volume of acetone and reaction time was increased. L-sorbose (0.625 g, 3.46
mmol) was dissolved in 60 mL of acetone (acetone to sorbose ratio of 233: 1). H2SO4 (1.2
mL) was added dropwise to this solution until universal pH indicator paper indicated the
reaction mixture was acidified. The solution was allowed to react in an ice-cold water bath
with constant stirring for 300 minutes. After this, sodium bicarbonate (20 mL) was added
dropwise until the universal pH indicator paper indicated that the reaction mixture was
neutralized. The solvent was evaporated to dryness under reduced pressure. Excess acetone
was removed, leaving a white powder (0.54 g, 2.07 mmol, 60 %) analyzed by NMR (400
MHz, DMSO-d6). See Figure 24, Appendices A 58 to A 61.

55

Figure 24: DAS Product 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz).

2.2.2.4 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 4
In this trial, the volume of acetone and reaction time was increased. L-sorbose (0.625 g, 3.46
mmol) was dissolved in 80 mL of acetone (acetone to sorbose ratio of 311: 1). H2SO4 (1.5
mL) was added dropwise to this solution until universal pH indicator paper indicated the
reaction mixture was acidified. The solution was allowed to react in an ice-cold water bath
with constant stirring for 300 minutes. After this, sodium bicarbonate (30 mL) was added
dropwise until the universal pH indicator paper indicated that the reaction mixture was
neutralized. The solvent was evaporated to dryness under reduced pressure. Excess acetone
was removed, leaving a white powder (0.70 g, 2.68 mmol, 87 %) analyzed by NMR (300
MHz, DMSO-d6). See Figure 25/ Appendix A 34.

56

Figure 25: DAS Product 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz).

2.2.2.5 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 5
Flow synthesis was done using the batch parameters in trial 4. A flow system was set up
using PTFE tubing (0.75 mm inner diameter) and syringe pumps. L-sorbose (0.625 g, 3.46
mmol) was dissolved in 80 mL of acetone and added to the flow at a 4 ml/min flow rate from
a first Pump A. H2SO4 (1.5 mL) diluted in acetone (20 mL) was added to the flow
simultaneously at a 1 ml/min flow rate from a second Pump B. The reaction mixture
introduced into the flow system via a T-mixer (T-mixer 1) was passed through a reactor coil
in an ice bath (0 - 5 C). This temperature was consistently maintained throughout the reaction
zone to ensure uniform conditions. Sodium bicarbonate (20 mL) was added to the flow
simultaneously at a 1 ml/min flow rate from a third Pump C. The product was obtained
following a 10-minute residence time in the reactor, and the solvent was evaporated to

57

dryness using a rotary evaporator. Excess acetone was removed under reduced pressure,
leaving a white powder (0.69 g, 2.65 mmol, 76 %) analyzed by NMR (300 MHz, DMSO-d6)
and Mass Spectrometry. See Figure 26, Appendices A 35 and A 36, respectively.

Figure 26: DAS Product 1H-NMR for Flow 5 recorded in DMSO-d6 (300 MHz).

2.2.2.6 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 6
In this trial, reaction time was increased. L-sorbose (0.626 g, 3.47 mmol) was dissolved in 80
mL of acetone (acetone to sorbose ratio of 311: 1). H2SO4 (1.5 mL) was added dropwise to
this solution until universal pH indicator paper indicated the reaction mixture was acidified.
The solution was allowed to react in an ice-cold water bath with constant stirring for 360
minutes. After this, sodium bicarbonate (30 mL) was added dropwise until the universal pH
indicator paper indicated that the reaction mixture was neutralized. The solvent was
evaporated to dryness under reduced pressure using a rotary evaporator. Excess acetone was
58

removed, leaving a white powder (0.81 g, 3.11 mmol, 90 %) analyzed by NMR (300 MHz,
DMSO-d6). See Figure 27 / Appendix A 37.

Figure 27: DAS Product 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz).

2.2.2.7 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 7
Flow synthesis was done using the batch parameters in trial 6, which was optimal for the Lsorbose protection compared to previous trials, and the residence time was increased. A flow
system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps. Lsorbose (0.625 g, 3.46 mmol) was dissolved in 80 mL of acetone and added to the flow at a 4
ml/min flow rate from a first Pump A. H2SO4 (1.5 mL) diluted in acetone (20 mL) was added
to the flow simultaneously at a 1 ml/min flow rate from a second Pump B. The reaction
mixture introduced into the flow system via a T-mixer (T-mixer 1) was passed through a
reactor coil in an ice bath (0 - 5 °C). This temperature was consistently maintained
throughout the reaction zone to ensure uniform conditions. Sodium bicarbonate (20 mL) was
59

added to the flow simultaneously at a 1 ml/min flow rate from a third Pump C. The product
was obtained following a 20-minute residence time in the reactor, and the solvent was
evaporated to dryness under reduced pressure. Excess acetone was removed, leaving a white
powder (0.74 g, 2.84 mmol, 82 %) analyzed by NMR (400 MHz, DMSO-d6) and Mass
Spectrometry. See Figure 28, Appendices A 38 and A 39, respectively.

Figure 28: DAS Product 1H-NMR for Flow 7 recorded in DMSO-d6 (400 MHz).

2.2.2.8 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Batch – Trial 8
In this trial, the reaction was done in an ice bath; aliquots were collected at intervals from 30
minutes to monitor the L-sorbose protection, using the acetone to sorbose (A/S) ratio of
311:1 to see if reaction time does affect the protection aside from the A/S ratio and reaction
temperature. L-sorbose (0.64 g, 3.55 mmol) was dissolved in 80 mL of acetone (acetone to
60

sorbose ratio of 311: 1). H2SO4 (1.5 mL) was added dropwise to this solution until universal
pH indicator paper indicated the reaction mixture was acidified. The solution reacted in an
ice-cold water bath with constant stirring for 30 minutes. At times 30 minutes, 1 hour, 2
hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours,
and 8.5 hours, aliquots were removed, and acetone was removed under reduced pressure to
yield fractions 8 A (0.023 g), 8 B (0.023 g), 8 C (0.023 g), 8 D (0.026 g), 8 E (0.026 g), 8 F
(0.026 g), 8 G (0.028 g), 8 H (0.031 g), 8 I (0.031 g), 8 J (0.031 g), 8 K (0.031 g), 8 L (0.031
g) and 8 M (0.032 g), respectively. See Appendices A 40 to A 52.
2.2.2.9 L-Sorbose Protection to Diacetone-L-Sorbose (DAS) Flow – Trial 9
In this trial, the residence time was increased from 20 to 30 minutes. A flow system was set
up using PTFE tubing (0.75 mm inner diameter) and syringe pumps. L-sorbose (0.625 g, 3.46
mmol) was dissolved in 80 mL of acetone and added to the flow at a 4 ml/min flow rate from
a first Pump A. H2SO4 (1.5 mL) diluted in acetone (20 mL) was added to the flow
simultaneously at a 1 ml/min flow rate from a second Pump B. The reaction mixture
introduced into the flow system via a T-mixer (T-mixer 1) was passed through a reactor coil
in an ice bath (0 - 5 °C). This temperature was consistently maintained throughout the
reaction zone to ensure uniform conditions. Sodium bicarbonate (20 mL) was added to the
flow simultaneously at a 1 ml/min flow rate from a third Pump C. The product was obtained
following a 30-minute residence time in the reactor, and the solvent was evaporated to
dryness under reduced pressure. Excess acetone was removed, leaving a white powder (0.86
g, 3.30 mmol, 95 %) analyzed by NMR (400 MHz, CDCl3). See Figure 29-33, Appendices A
53, A 54, A 55, A 56 and A 57.

61

Scheme 8: The protection of L-sorbose to Diacetone-L-Sorbose in the flow

Figure 29: Diacetone-L-sorbose 1H-NMR for Flow 9 recorded in CDCl3 (400 MHz).

62

Figure 30: Diacetone-L-sorbose 13C-NMR for Flow 9 recorded in CDCl3 (400 MHz).

Figure 31: Diacetone-L-sorbose COSY for Flow 9 recorded in CDCl3 (400 MHz).

63

Figure 32: Diacetone-L-sorbose HSQC for Flow 9 recorded in CDCl3 (400 MHz).

Figure 33: Diacetone-L-sorbose HMBC for Flow 9 recorded in CDCl3 (400 MHz).

64

2.2.2.10 Diacetone-L-Sorbose Product Yields
L-sorbose protection to yield diacetone-L-sorbose highly depends on reaction conditions,
particularly the acetone-to-sorbose ratio and temperature control. Optimal conversion was
achieved with an acetone-to-sorbose molar ratio of 311:1, conducted in a cold-water bath.
Experiments with lower acetone-to-sorbose ratios resulted in incomplete protection (See
Tables 6 and 7), as evidenced by distinctive 1H-NMR spectral features. Specifically, trials 1,
2, and 5 exhibited characteristic methyl peaks at 1.30 ppm and 1.40 ppm, integrating to 6 H,
indicative of partial protection (See Figures 22, 23 and 26, respectively). Complementary
13

C-NMR data corroborated these findings, showing additional peaks beyond the 12 carbon

signals characteristic of DAS, confirming incompletely protected species and unreacted
sorbose (Appendix A 59). In contrast, fully protected diacetone-L-sorbose displays an
integration of 12 H for its methyl protons, corresponding to the expected 12 methyl protons
in the molecule, as shown in Figures 27 and 29.
The synthesis of diacetone-L-sorbose (DAS) was optimized through a systematic
investigation of reaction time in batch conditions, employing an acetone-to-sorbose ratio of
311:1. Optimal conversion was achieved within a narrow window of 6 to 6.5 hours, yielding
fully protected DAS with minimal partially protected intermediates and no detectable side
products, as evidenced by spectroscopic analyses. These trials (8 H, 8 I and 9) exhibited
characteristic methyl peaks at 1.40 ppm, 1.47 ppm and 1.54 ppm, with integrations of 6 H, 3
H and 3 H, respectively, integrating to 12 H altogether, indicative of DAS methyl protons.
(Appendices A 47, A 48 and A 53). The 13C-NMR spectrum displayed 12 carbon peaks
(Figure 30 /Appendix 54). At the same time, HMBC experiments revealed the expected
correlations between the methyl carbons and the quaternary carbons at 98 ppm and 112 ppm

65

on both sides of the diacetone-L-sorbose molecule (Figure 33 / Appendix A 57), which was
notably absent in the HMBC spectra of the partially protected product (Trials 1, 2, and 3), as
only one quaternary carbon at 112 ppm correlated with the methyl carbon, showing that only
a part was protected. The other quaternary carbon at 98 ppm, which was to correlate with
methyl carbon, was not evident in the HMBC spectra (Appendix A 61). Using the optimal
acetone to sorbose ratio of 311:1 at a reaction time of less than 6 hours gave a mixture of
partially protected product, and DAS as indicated by the extra peak at 1.60 ppm which is the
partially protected product peak, in the 1H-NMR spectra (Appendices A 43 – A 46).
Conversely, extending the reaction time beyond 6.5 hours led to the formation of DAS
accompanied by side products, as indicated by extra peaks in the 1H-NMR spectra at specific
chemical shifts (1.30 ppm, 1.60 ppm, 1.70 ppm and 2.10 ppm). See Appendices A 49 to A
52.
For the flow, the 30-min residence time gave DAS with fewer side products as shown by
characteristic methyl peaks at 1.40 ppm, 1.47 ppm and 1.54 ppm, with integrations of 6 H, 3
H and 3 H, respectively, integrating to 12 H altogether, indicative of DAS methyl protons.
(Appendix A 53). Residence times below 30 mins gave a mixture of DAS and partially
protected product, which was also supported by mass spectra, showing the DAS of molecular
weight 260 g/mol and partially protected product (as shown in Figure 34) of 220 g/mol. See
Figure 26 / Appendix A 35 and Appendix A 36 for the 1H-NMR and mass spectrum,
respectively, for trial 5. For trial 7, See Figure 28 / Appendix A 38 and Appendix A 39 for
the 1H-NMR and mass spectrum, respectively.

66

Figure 34: The structure of the partially protected sorbose and the NMR (2-D) correlation.

The NMR Data for the partially protected sorbose is shown in Table 3 below, and Figure 34
above shows the 2 D correlation as seen in Table 3 and Appendices A 58 – A 61. The data
showed that only one part of the sorbose was protected.
Table 3: 13C and 1H NMR Data for Partially Protected Sorbose (Batch 3)

Carbon #

13

C δ, ppm

1

H δ, ppm

COSY

HMBC

1

27.1

1.296

H2

H2

2

28.0

1.399

H1

H1

3

59.4

3.588, 3.4876

H6

-

4

62.5

3.5017, 3.4533

-

-

5

73.9

3.9769

H6, H7

-

6

82.3

4.0831

H3, H5

H7

7

85.1

4.2829

H5

H4’

8

111.2

-

-

H1, H2

9

114.7

-

-

H4, H4’, H5

Figure 35: The structure of obtained diacetone-L-sorbose and the NMR (2-D) correlation.

67

Figure 35 above shows the COSY, HSQC and HMBC Spectra correlation of obtained DAS
for Flow 9, and the NMR data is shown in Table 4, while NMR spectra for the product are
shown in Figures 29, 30, 31, 32 and 33.
Table 4: 13C and 1H NMR Data for Diacetone-L-sorbose (Flow 9)

Carbon #

13

C δ, ppm

1

H δ, ppm

COSY

HMBC

1

18.6

1.4272

H4

H4

2

26.5

1.3609

H3

H3

3

27.3

1.4976

H2

H2

4

29.0

1.3609

H1

H1

5

60.3

4.0546

H9

H1

6

63.7

3.8185

OH

H9

7

72.3

4.0903

H8

H8, H9

8

73.3

4.3171

H7, H9

H5, H7, H9

9

85

4.4671

H5, H8

H6

10

97.5

-

-

H2, H4, H5

11

112.0

-

-

H2, H3, H7

12

114.5

-

-

H6, H7, H8, H9

A set of flow chemistry conditions, shown in Table 5 and Table 6, were screened for this
protection step throughout the process, and the best conditions obtained were T = -15 to 0° C,
Acetone to Sorbose ratio of 311:1, and a residence time of 30 mins. Before optimizing the
best conditions, the effect of temperature and the process duration on the sorbose acetonation
reaction was studied.

68

2.2.2.11. Optimization and Discussion of Reaction Conditions
2.2.2.11.1 Diacetone-L-Sorbose Synthesis: Temperature and Reaction Time
Incomplete protection occurred when the reaction was done at room temperature and less
than 5 hours reaction time for batch, but DAS was synthesized fully when the reaction was
carried out in an ice bath.
Table 5: Effect of Temperature and Residence Time on the L-Sorbose Acetonation Process in the Batch

Temperature regime, °C

Process duration, min

DAS yield, %

20 (rt)

60

Incomplete protection

20 (rt)

180

Incomplete protection

20 (rt)

240

Incomplete protection

20 (rt)

300

Incomplete protection

-15, 0 (ice bath)

300

87

-15, 0 (ice bath)

360

90

Table 6: Effect of Temperature and Residence Time on the L-Sorbose Acetonation Process in the Flow

Temperature regime, °C

Residence time, min

DAS yield, %

20 (rt)

10

Incomplete protection

20 (rt)

15

Incomplete protection

-15, 0 (ice bath)

10

76

-15, 0 (ice bath)

20

82

-15, 0 (ice bath)

30

95

Protecting L-sorbose with acetone to form diacetone-L-sorbose presented a significant
challenge due to the potential formation of undesired or isomeric products. A critical issue in
this step is the possible formation of 1,2:4,6-di-O-isopropylidene derivative instead of the
desired diacetone-L-sorbose under certain reaction conditions, as structures are shown in
Figure 36. This phenomenon has been observed and reported in the literature,122 highlighting
69

researchers' common struggles when dealing with complex protection strategies.122 Extensive
optimization efforts addressed this challenge, carefully controlling reaction parameters such
as temperature, catalyst concentration, and reaction time. These optimizations were crucial to
ensure the selective formation of diacetone-L-sorbose while minimizing the production of its
isomeric counterpart. Throughout the process, rigorous NMR analysis was employed to
verify the structure of the obtained product, allowing for the differentiation between the
desired diacetone-L-sorbose and the potentially formed 1,2:4,6-di-O-isopropylidene
derivative. This meticulous approach was necessary given these compounds' structural
similarity and potential impact on subsequent synthetic steps. After numerous iterations and
refinements of the reaction conditions, the successful synthesis of the correct diacetone-Lsorbose isomer was achieved and confirmed through comprehensive NMR characterization,
as shown in Table 4 and Figure 35.
The structures of potential products and by-products from the reaction, diacetone-L-sorbose,
partially protected L-sorbose and 1,2:4,6-di-O-isopropylidene derivative, which we were
trying to avoid, are given below.

Figure 36: The structures of the potential products: partially protected L-sorbose, diacetone-L-sorbose, and
1,2:4,6-di-O-isopropylidene derivative

70

2.2.2.11.2 Diacetone-L-Sorbose Synthesis: Acetone to Sorbose Ratio
The yield of DAS increased with the relative content of acetone, and the protection of the Lsorbose was incomplete when less acetone was used. The effect of the acetone to sorbose
ratio on the DAS yield was studied in batch and flow (Tables 7 and 8) under optimum
temperature conditions. Raising the acetone to sorbose (A/S, v/w) ratio from 97:1 to 311:1
resulted in complete protection of the L-sorbose and increased DAS yield for the flow and
the batch synthesis.
Table 7: Effect of Acetone to Sorbose ratio (A/S) on the Diacetone-L-Sorbose yield in the Batch

A/S Ratio
97: 1

Process Duration,
min
180

Acetonation
Temperature, °C
-15, +20

DAS yield, %
Incomplete protection

194: 1

240

-15, +20

Incomplete protection

233: 1

300

-15, +20

Incomplete protection

311: 1

300

-15, +20

87

311: 1

360

-15, +20

90

Table 8: Effect of acetone to Sorbose ratio (A/S) on the Diacetone-L-Sorbose yield in the Flow

A/S Ratio

Residence time,

Acetonation

min

temperature, °C

97: 1

10

-15, +20

Incomplete protection

194: 1

15

-15, +20

Incomplete protection

233: 1

20

-15, +20

Incomplete protection

233: 1

30

-15, +20

Incomplete protection

311: 1

10

-15, +20

76

311: 1

20

-15, +20

82

311: 1

30

-15, +20

95

71

DAS yield, %

2.2.2.12 Comparison of Diacetone-L-sorbose Yield in Batch and Flow
Based on a comprehensive analysis of various reaction parameters in the batch during this
optimization, including the acetone to sorbose ratio, the acetonation temperature, and
reaction duration, trials 6, 8 H, and 8 I yielded the most favourable outcome of DAS yields.
These trials employed acetone to a sorbose ratio of 311: 1, a 6-hour reaction time for trials
6 and 8 H, and a 6.5-hour reaction time for 8 I. Drawing from these results, the optimal
conditions for this reaction in the batch were proposed and are; Acetone to Sorbose ratio of
311: 1, acetonation temperature of 0 °C to 5 °C and a reaction duration of 6 hours.
These parameters balance the efficient, full L-sorbose protection and minimal side product
formation, offering a refined protocol for protecting L-sorbose using acetone to obtain
diacetone-L-sorbose. Implementing a flow setup using the optimized parameters yielded
remarkable results, achieving improved yield and a significantly reduced reaction or
residence time of just 30 minutes in trial 9, in contrast to the 6-hour duration required for
the batch process. See Appendices A 53 – A 57.
Table 9: Comparison of Diacetone-L-sorbose yield and the Reaction parameters in Batch versus Flow

L-sorbose protection to Diacetone-L-sorbose: Results in Batch versus Flow
Batch

Flow

Reaction/ Residence time
(mins)

360

30

DAS Yield (mmol)

3.11

3.30

Total Yield (%)

90

95

72

2.2.3 Protected Sorbose (Diacetone-L-Sorbose - DAS) to 2-keto-L-gulonic Acid in Batch
and Flow
2.2.3.1 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Batch – Trial 1
This step focused on the oxidation of protected sorbose to 2-keto-L-gulonic acid. DiacetoneL-sorbose (0.91 g, 3.49 mmol) was dissolved in 50 mL of dH2O. The reaction mixture was
placed in an ice bath. NaOH (1.50 g, 37.5 mmol) was added dropwise until universal pH
indicator paper indicated the reaction mixture was alkaline. KMnO4 (0.80 g, 5.06 mmol) was
dissolved in 50 mL of dH2O to achieve a 0.1 mol/L concentration. The KMnO4 solution was
added dropwise to the diacetone-L-sorbose solution for 15 minutes with constant stirring.
The reaction mixture was then heated to 50° C and allowed to react at this temperature. After
2 hours, the reaction was cooled to room temperature and excess KMnO4 was quenched by
the slow addition of Sodium bisulphite (2.73 g, 26.23 mmol) until the purple colour of
KMnO4 disappeared. The mixture was acidified to pH 2 using concentrated H2SO4 (2 mL)
and monitored with universal pH indicator paper. The solution was filtered, and the filtrate
was concentrated under reduced pressure using a rotary evaporator. The concentrate was
extracted with ethyl acetate (10 mL). The organic layer was dried over anhydrous Na2SO4
and filtered, and the solvent was evaporated to dryness under reduced pressure using a rotary
evaporator. Excess ethyl acetate was removed, leaving 2-keto-L-gulonic acid, a white powder
(0.58 g, 2.98 mmol, 85%) analyzed by NMR (300 MHz, DMSO-d6). See Figure 37 and
Appendices A 66 to A 68.

73

Figure 37: 2-keto-L-gulonic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

2.2.3.2 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Batch – Trial 2
For this trial, acetone was used as the solvent to minimize water, especially in the flow.
Diacetone-L-sorbose (0.34 g, 1.30 mmol) was dissolved in 20 mL of acetone. The reaction
mixture was placed in an ice bath. NaOH (0.50 g, 12.5 mmol) was added dropwise until
universal pH indicator paper indicated the reaction mixture was alkaline. KMnO4 (0.35 g,
2.21 mmol) dissolved in 20 mL of acetone was added dropwise to the diacetone-L-sorbose
solution for 10 minutes with constant stirring. The reaction mixture was then heated to 50° C
and allowed to react at this temperature. After 2 hours, the reaction was cooled to room
temperature, and excess KMnO4 was quenched by the slow addition of sodium bisulphite
(1.20 g, 11.53 mmol) till the disappearance of KMnO4 purple colour. The mixture was
acidified to pH 2 using concentrated H2SO4 (1 mL) and monitored with universal pH
74

indicator paper. The solution was filtered, and the filtrate was concentrated under reduced
pressure using a rotary evaporator. The concentrate was extracted with ethyl acetate (6 mL).
The organic layer was dried over anhydrous Na2SO4 and filtered, and the solvent was
evaporated to dryness under reduced pressure using a rotary evaporator. Excess ethyl acetate
was removed, leaving 2-keto-L-gulonic acid, a white powder (0.22 g, 1.13 mmol, 88%)
analyzed by NMR (300 MHz, DMSO-d6). See Figure 38 and Appendices A 69 to A 71.

Figure 38 2-keto-L-gulonic acid 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz).

2.2.3.3 Diacetone-L-Sorbose (DAS) Oxidation to 2-keto-L-gulonic Acid Flow – Trial 3
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps
(Scheme 9). Diacetone-L-sorbose (0.33 g, 1.26 mmol) was dissolved in 20 mL of acetone.
NaOH (0.50 g, 12.5 mmol) was added to this mixture and the solution was added to the flow
at a 1 ml/min flow rate from a first pump A. KMnO4 (0.35 g, 2.21 mmol) dissolved in 10 mL
75

of acetone was added to the flow simultaneously at a 0.5 ml/min flow rate from a second
pump B. The reaction mixture introduced into the flow system via a T-mixer (T-mixer 1) was
passed through a reactor coil in a hot water bath at an elevated temperature of 50° C. This
temperature was consistently maintained throughout the reaction zone to ensure uniform
conditions. Sodium bisulphite (1.20 g, 11.53 mmol) dissolved in 5 mL of dH20 was added to
the flow simultaneously at a 0.25 ml/min flow rate from a third pump C. H2SO4 (1 mL)
diluted in 10 mL of acetone was added to the flow simultaneously at a 0.5 ml/min flow rate
from a fourth pump D, and was then allowed to react with the reaction mixture at a second
reactor coil for 20 minutes. In the drying unit, 3 A° and 4 A° molecular sieves trapped the
aqueous layer. The sieves were pre-dried in the oven at 160° C for 16 hours before use. The
product was obtained following a 40-minute residence time in the reactor, and the solvent
was evaporated to dryness under reduced pressure using a rotary evaporator. Excess acetone
was removed, leaving 2-keto-L-gulonic acid, a white powder (0.22 g, 1.13 mmol, 91%)
analyzed by NMR (400 MHz, DMSO-d6) and mass spectrometry. See Figures 39 to 43 and
Appendices A 72 to A 77.

76

Scheme 9: The Oxidation of Diacetone-L-Sorbose to 2-KGA in the flow, using KMnO4

Figure 39: 2-keto-L-gulonic acid 1H-NMR for Flow 3 recorded in DMSO-d6 (400 MHz).

77

Figure 40: 2-keto-L-gulonic acid 13C-NMR for Flow 3 recorded in DMSO-d6 (400 MHz).

Figure 41: 2-keto-L-gulonic acid HSQC for Flow 3 recorded in DMSO-d6 (400 MHz).

78

Figure 42: 2-keto-L-gulonic acid COSY for Flow 3 recorded in DMSO-d6 (400 MHz).

Figure 43: 2-keto-L-gulonic acid HMBC for Flow 3 recorded in DMSO-d6 (400 MHz).

79

Table 10: 13C and 1H NMR Data for 2-keto-L-gulonic acid (Flow 3)

Carbon #

13

C δ, ppm

1

H δ, ppm

COSY

HMBC

1

175.1

-

-

-

2

213.5

-

-

H3

3

76.89

4.168

H4

H4

4

74.4

3.671, 4.751

H3, H5

H3, H5, H5’, H6

5

74.1

3.869, 4.077

H4, H6

H3, H4, H6

6

64.4

3.205

H5

H5

Figure 44: The structure of the 2-keto-L-gulonic acid and the NMR (2-D) correlation.

2.2.3.4 2-keto-L-gulonic Acid Yield and Comparison of Batch and Flow
2-keto-L-gulonic acid was successfully synthesized from diacetone-L-sorbose, and the NMR
analysis of the product obtained revealed an intriguing mixture of 2-keto-L-gulonic acid
(Figures 39 to 43 / Appendices A 72 to A 76). The 13C NMR spectrum (See Figure 40 /
Appendix A 73) exhibited a striking array of signals, including a prominent peak at δ 213
ppm, characteristic of the ketone carbon in the open-chain form of 2-keto-L-gulonic acid.
Simultaneously, a signal at δ 102 ppm was observed, which can be assigned to the anomeric
carbon of the cyclic hemiacetal form.123 The spectrum also displayed additional peaks in the
80

60-80 ppm range, corresponding to the carbon atoms of both the open-chain and cyclic
structures. The proton NMR spectrum (see Figure 39 / Appendix A 72) revealed compelling
evidence for the cyclic structure. A sharp doublet is seen at 4.74 ppm, a characteristic of the
anomeric proton in the ring form. This signal stood out as a key indicator of cyclization.
Further supporting this interpretation, a cluster of overlapping multiplets spanning the 3.5 to
4.4 ppm region was observed. Together, these spectral features clearly show the existence of
the cyclic form in the reaction mixture. According to some literature, different solid forms of
2-keto-L-gulonic acid have been observed and sometimes, mixtures of two solid forms were
obtained, each containing one molecule of water of crystallization.124 This suggests that
different structural arrangements, including cyclic forms, can coexist in a single sample.124
Additionally, 2-keto-L-gulonic acid is known to form a monohydrate when crystallized from
water or water-containing organic solvents. The presence of water in the crystal structure
could facilitate the equilibrium between open-chain and cyclic forms.125 Many sugar
derivatives are also known to exist in equilibrium between open-chain and cyclic forms in
solution.125
The formation of cyclic 2-keto-L-gulonic acid can be rationalized through intramolecular
cyclization following the initial oxidation of diacetone-L-sorbose. It can be proposed that this
cyclization occurs spontaneously under the reaction conditions, driven by the thermodynamic
stability of the cyclic hemiacetal form. The potassium permanganate likely facilitates the
initial oxidation step, while the subsequent cyclization is an inherent property of the 2-ketoL-gulonic acid structure in solution. This result aligns with previous studies on similar sugar
derivatives, where cyclic forms are often observed in solutions due to intramolecular
reactions between carbonyl and hydroxyl groups.125 Aside from NMR, Mass spectrometry

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confirmed the product's identity, as seen in Appendix A 77. Implementing a flow setup using
the optimized parameters in the batch yielded remarkable results, achieving improved yield
and a significantly reduced reaction or residence time for the flow of just 40 minutes in trial
3, in contrast to the 2-hour duration required for the batch process, as shown in Table 11
below.

Table 11: Comparison of 2-keto-L-gulonic acid yields and the Reaction parameters in Batch versus Flow

Diacetone-L-sorbose to 2-keto-L-gulonic Acid: Batch versus Flow
Batch

Flow

Reaction/ Residence time
(mins)

120

40

DAS Yield (mmol)

1.13

1.13

Total Yield (%)

88

91

2.2.4 2-keto-L-gulonic Acid to Ascorbic Acid in Batch and Flow
2.2.4.1 2-keto-L-gulonic Acid to Ascorbic Acid Batch – Trial 1
This step was focused on the lactonization and enolization of 2-keto-L-gulonic acid to
ascorbic acid. 2-keto-L-gulonic acid (0.51 g, 2.62 mmol) was dissolved in 5 mL of methanol
and heated at an elevated temperature of 65° C to 100° C under reflux. H2SO4 (0.5 mL) was
added dropwise to this solution as a catalyst until universal pH indicator paper indicated the
reaction mixture was acidified. The solution was allowed to react with constant stirring for
180 minutes. After this, sodium bicarbonate (5 mL) was added dropwise until the universal
pH indicator paper indicated that the reaction mixture was moderately acidic. The product
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was obtained, and the solvent was evaporated to dryness under reduced pressure using a
rotary evaporator. Excess dH2O was removed, leaving a white powder (0.38 g, 2.15 mmol,
82%) analyzed by NMR (300 MHz, DMSO-d6). See Figure 45 and Appendices A 78 to A 80.

Figure 45: Ascorbic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

2.2.4.2 2-keto-L-gulonic Acid to Ascorbic Acid Flow – Trial 2
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps.
2-keto-L-gulonic acid (0.55 g, 2.83 mmol) was dissolved in 10 mL of methanol and added to
the flow at a 0.5 ml/min flow rate from a first Pump A. H2SO4 (0.5 mL) diluted in dH20 (5
mL) was added to the flow simultaneously at a 0.25 ml/min flow rate from a second Pump B.
The reaction mixture introduced into the flow system via a T-mixer (T-mixer 1) was passed
through a reactor coil in a hot water bath at an elevated temperature of 65° C to 100° C. This
temperature was consistently maintained throughout the reaction zone to ensure uniform
conditions. Sodium bicarbonate (5 mL) was added to the flow simultaneously at a 0.5 ml/min

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flow rate from a third Pump C. The product was obtained following a 20-minute residence
time in the reactor, and the solvent was evaporated to dryness under reduced pressure using a
rotary evaporator. Excess H20 was removed, leaving L-ascorbic acid, a white powder (0.42 g,
2.38 mmol, 85 %) analyzed by NMR (400 MHz, DMSO-d6) and Mass spectrometry. See
Appendix A 81 and A 82. The crude product (0.21 g, 1.19 mmol) was recrystallized with a
mixture of ethanol and water (3:1), and pure ascorbic acid (0.12 g, 0.68 mmol, 57%) was
obtained and analyzed by NMR (400 MHz, DMSO-d6). See Figures 46 to 50 / Appendix A
83 to A 87.

Scheme 10: The lactonization and enolization of 2-KGA to L-ascorbic acid in the flow

84

Figure 46: Ascorbic acid 1H-NMR for Flow 2 recorded in DMSO-d6 (400 MHz).

Figure 47: Ascorbic acid 13C-NMR for Flow 2 recorded in DMSO-d6 (400 MHz).

85

Figure 48: Ascorbic acid COSY for Flow 2 recorded in DMSO-d6 (400 MHz).

Figure 49: Ascorbic acid HSQC for Flow 2 recorded in DMSO-d6 (400 MHz).

86

Figure 50: Ascorbic acid HMBC for Flow 2 recorded in DMSO-d6 (400 MHz).

Table 12: 13C and 1H NMR Data for Ascorbic acid (Flow 2)

Carbon #

13

C δ, ppm

1

H δ, ppm

COSY

HMBC

1

62.4

3.728, 3.431

H2

H2, H3

2

68.8

4.867, 3.728, 3.431

H1, H3

H1, H3

3

75.0

4.717

H2

H2, H2’

4

118.4

-

-

H3

5

153.3

-

-

H3

6

171.1

-

-

H3

Figure 51: The structure of the Ascorbic acid and the NMR (2-D) correlation.

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2.2.4.3 Ascorbic Acid Product Yields and Comparison of Batch and Flow
Ascorbic acid was successfully synthesized from 2-keto-L-gulonic acid, as evidenced by the
NMR spectroscopic analysis (Figures 46 to 50 / Appendix A 83 to A 87). The NMR
spectrum of the product exhibited characteristic signals consistent with the structure of
ascorbic acid. Upon careful examination of the 1H-NMR spectrum (See Figure 46 / Appendix
A 83), a distinctive doublet emerged at around δ 4.71 ppm. This particular signal indicates
the proton attached to the C-4 carbon in the ascorbic acid structure. Two key doublets were
spotted hanging out between δ 3.42 and 3.44 ppm. These signals are expected from the
protons on C-5 and C-6 in ascorbic acid. The spectral data also revealed the presence of
hydroxyl protons, which appeared as broad singlets in the range of δ 8-11 ppm, further
corroborating the formation of ascorbic acid. Complementary 13C-NMR data after
recrystallization corroborated these findings, showing precisely the 6 carbon signals
characteristic of Ascorbic acid (Figure 47 / Appendix A 84). As much as the NMR strongly
supports ascorbic acid formation, mass spectrometry confirmed the product's identity, as seen
in Appendix A 82. The optimal condition for this step includes using a small amount of
water, as too much free water can negatively impact the reaction by increasing reaction time
and decreasing yield, partly due to the decomposition of L-ascorbic acid in the presence of
aqueous acid.69 A small amount of the base (5 ml) was used since ascorbic acid is more
stable in acidic conditions, so full neutralization was avoided to reduce the chances of side
reactions and reduction in the yield of ascorbic acid.
Implementing a flow setup using the optimized parameters in the batch yielded remarkable
results, achieving improved yield and a significantly reduced reaction or residence time for

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the flow of just 20 minutes in trial 2, in contrast to the 3-hour duration required for the batch
process.
Table 13: Comparison of Ascorbic acid yields and the Reaction parameters in Batch versus Flow

2-keto-L-gulonic Acid to Ascorbic Acid: Batch versus Flow
Batch

Flow

Reaction/ Residence time
(mins)

180

20

DAS Yield (mmol)

2.15

2.38

Total Yield (%)

82

85

2.3 Conclusion
2.3.1 Comparison of Flow Methods with Batch Processes
Results from our flow system demonstrated significant improvements over traditional batch
processes. The reduction of D-glucose to D-sorbitol was done in 20 minutes, compared to a
60-minute reaction time in the batch. The conversion of L-sorbose to diacetone-L-sorbose in
the flow reached a 95 % yield within a residence time of just 30 minutes, compared to a 90 %
yield in 360 minutes for the batch reaction. The subsequent oxidation step showed even more
dramatic improvements, with 2-keto-L-gulonic acid yields of 91 % achieved in a total
residence time of 40 minutes, a substantial reduction from the 2-hour reaction time typically
required in batch processes, which gave an 88 % yield. For the 2-keto-L-gulonic acid to
ascorbic acid conversion, the flow gave a slightly better yield (85 %) in 20 minutes of
residence time, while the batch gave an 82 % yield in around 180 minutes of reaction time.

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Higher yields and product quality were consistently maintained by adjusting flow rates,
reagent-to-solvent ratio, and temperatures. The overall yield of ascorbic acid from L-sorbose
reached 82-85%, surpassing the typical yields of 70-75% reported for industrial batch
processes. Additionally, though the flow approach did not reduce solvent usage significantly
compared to batch methods, it contributed to a more environmentally friendly production
process via efficient mixing and in-flow of reagents.
2.3.2 Advantages and Limitations of Developed Protocols
The developed flow synthesis protocols for ascorbic acid production demonstrate several
advantages over traditional batch processes. Primarily, the flow system achieved
remarkable improvements in reaction efficiency and speed. For instance, the conversion of
L-sorbose to diacetone-L-sorbose reached a 95% yield in just 30 minutes, compared to 90%
in 360 minutes for batch reactions. Similarly, the oxidation step yielded 91% of 2-keto-Lgulonic acid in 40 minutes, drastically reducing the typical 2-hour batch reaction time.
While showing a smaller improvement, the final conversion to ascorbic acid still achieved
a slightly higher yield (85%) in a fraction of the time required for batch processes. These
time reductions could significantly enhance production capacity and reduce energy
consumption. The 82-85% yield from L-sorbose to ascorbic acid surpasses typical
industrial batch yields, indicating the potential for increased production efficiency.
Furthermore, while the flow approach did not substantially reduce solvent usage, it
contributed to a more environmentally friendly process through efficient mixing and inflow reagent addition. However, the developed protocols also have limitations. The
marginal improvement in solvent usage suggests that further optimization in this area is
needed to enhance the environmental benefits. Additionally, the complexity of the flow
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system setup and the need for specialized equipment might present challenges in terms of
initial investment and operator training. The scalability of the process to industrial levels
and its long-term stability under continuous operation would require further investigation.
Despite these limitations, the significant improvements in reaction time, yield, and process
control make this flow synthesis approach a promising alternative to traditional batch
methods for ascorbic acid production.
2.3.3 Potential for Scale-up and Industrial Applications
The discrete flow synthesis steps from L-sorbose through to L-ascorbic acid have shown that
time for reaction completion is improved in flow vs batch synthesis. Furthermore, we
demonstrated that the crude products from each synthetic step could be used successfully in
the next reaction with minimal purification. Indeed, generally, only solvents were removed to
allow structural characterization. Thus, the improved reaction efficiency demonstrates that the
flow synthesis approach for ascorbic acid production has potential for scale-up and industrial
application. The improved reaction and reduced processing times observed in the laboratoryscale flow system suggest promising outcomes for larger-scale operations.
The next significant step in advancing the synthesis of ascorbic acid from D-glucose is
transitioning from discrete flow to continuous flow synthesis. This shift presents several
challenges that need to be addressed. Firstly, the lack of adequate pumps capable of handling
the diverse range of solvents and reagents across all steps must be resolved. Secondly, an inline
separator needs to be developed or optimized for efficient aqueous-organic phase separation,
particularly crucial in the diacetone-L-sorbose to 2-keto-L-gulonic acid step. Integrating such
a separator is vital for maintaining continuous operation without interrupting manual workup
procedures. Though using molecular sieves in the diacetone-L-sorbose to 2-keto-L-gulonic
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acid flow synthesis step is a step towards automation, it still retains elements of manual
workup. It may not be optimal for large-scale continuous flow synthesis as translating this into
a continuous process would require innovative inline purification techniques. The ongoing
research into the D-sorbitol to L-sorbose conversion presents a critical gap that must be bridged
for a truly continuous process. Nevertheless, the potential benefits of product consistency,
process safety, and operational efficiency make the flow synthesis of ascorbic acid a promising
candidate for industrial-scale implementation, particularly in the pharmaceutical and food
industries where high-purity ascorbic acid is in constant demand.

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3. Flow synthesis of N5-OH-L-Orn towards Chemoenzymatic synthesis of Piperazic acid
3.1 Chapter Overview
The flow synthesis of N5-OH-L-Ornithine represents a step towards the chemoenzymatic
synthesis of piperazic acid, a valuable building block in natural product synthesis and drug
discovery. This approach combines the efficiency of flow chemistry with the selectivity of
enzymatic catalysis. N5-OH-L-Ornithine acts as a key intermediate in the biosynthesis of
piperazic acid, where it is cyclized to form the characteristic nitrogen-nitrogen bond. Recent
studies have elucidated the enzymatic pathways involved in this process, with a piperazate
synthase playing a central role in converting N5-OH-L-Orn to piperazic acid.126 Thus, to
produce piperazic acid via this method, N5-OH-L-Orn needs to be made. One strategy is to
develop a flow synthesis method to produce N5-OH-L-Ornithine, which presents several
advantages, including improved reaction control, enhanced heat and mass transfer, and the
capacity to integrate without causing disruption. This approach follows the growing
movement towards more sustainable and efficient synthetic processes in the pharmaceutical
and fine chemical industries, potentially unlocking new opportunities in drug development
and natural product synthesis.
3.2. Piperazic Acid: A Versatile Building Block
Piperazic acid, a cyclic nonproteinogenic amino acid127 containing a rare nitrogen-nitrogen
bond,128,129 has emerged as a valuable component in bioactive natural products and drug
discovery. This unusual N-N bond makes piperazic acid the only known example of a
natural cyclic hydrazine amino acid.130 As N-N bonds are found in roughly 10% of
pharmaceutical structures,131 this unique structural feature has garnered significant
attention from researchers in recent years,132 leading to a deeper understanding of its
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biosynthesis, synthesis, incorporation into complex molecules, and potential applications.
Recent research has shed light on the enzymatic pathways involved in its formation.126,130
A key enzyme in this process is the piperazate synthase, responsible for cyclizing 5hydroxylated ornithine to form piperazic acid.130
3.2.1 Biological Significance and Applications
Natural products incorporating piperazic acid residues exhibit many potent biological
activities, including anti-malarial, anti-apoptotic, and antibacterial properties.127 These
compounds have been found in over 30 families of natural products, representing more
than 140 distinct molecules.127,133 In the pharmaceutical industry, piperazic acid is a
valuable building block for creating peptide-based natural product derivatives with
antibacterial and antifungal properties.133–135 Its potential ability to lock amino acids in
specific positions within peptides makes it a valuable tool for peptide engineering and
developing molecular probes.134 Finally, as an analogue of proline, it could be worth
exploring as an organocatalyst, but currently challenges of accessing this rare amino acid
have slowed research in this direction. The unique structural and functional properties of
piperazic acid have sparked interest in developing more efficient and cost-effective
methods for its production, including green biocatalysis and fermentation techniques, to
facilitate its broader application in medicinal chemistry and biotechnology.134

3.2.2 Current Synthetic Approaches and Limitations of Piperazic Acid
The current synthetic approaches to piperazic acid involve both traditional chemical
synthesis and biotechnological methods. Traditional chemical synthesis of piperazic acid
often faces challenges such as low yield, multiple complex steps, and challenges producing
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single enantiomers. These limitations have spurred interest in developing more efficient
and sustainable methods. Current synthetic approaches focus on innovative methodologies
that enhance efficiency and selectivity, yet they face notable limitations. Recent
advancements include a selective deprotection strategy that simplifies the synthesis of
piperazic acid-containing peptides,136 improving overall yields, new methods that
emphasize C–H functionalization and expand the structural diversity of piperazine
derivatives,137 which is crucial for drug discovery. Various synthetic routes, including
transition-metal-catalyzed and rearrangement reactions, have also been explored to enhance
the efficiency of piperazine synthesis.138,139 There are limitations, including the complexity
of protection strategies, as the need for multiple protecting groups complicates the
synthesis, often leading to increased steps and lower efficiency.136 Attempts have been
made to develop different piperazine synthases into biocatalysts. However, the patents
describing this work have been abandoned. 140,141 This attempt sought to couple the enzyme
for producing N5-OH-L-Ornithine with the piperazate synthase enzyme. Instead, this
project seeks to produce N5-OH-L-Ornithine via an efficient flow synthesis that could then
be coupled to a piperazate synthase immobilized in a packed bed reactor within a flow
system. Immobilization has been shown to increase the utility of biocatalysts.142–145 As
such, our approach may hold promise that could not be realized simply through coupling
enzymes. Despite these advancements, limitations remain, including the need for
specialized equipment and large-scale bioreactors to implement biotechnological processes
effectively. Furthermore, while these methods produce high-purity products, the scalability
to industrial levels requires further optimization.134,141 Overall, significant progress has
been made, but continued innovation is necessary to fully realize the potential of piperazic

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acid as a versatile building block in pharmaceuticals and natural product synthesis.
3.2.3.1 Biosynthesis of Piperazic Acid and the need for N5-OH-L-Orn
Piperazic acid biosynthesis has been a subject of significant interest due to its presence in
over 30 families of natural products, representing more than 140 compounds with potent
biological activities ranging from anti-malarial to anti-bacterial properties.128 The
biosynthesis of piperazic acid involves several key steps, with N5-hydroxy-ornithine (N5OH-L-Orn) playing a crucial role as an intermediate. A piperazate synthase forms the N-N
bond, a critical step in producing piperazic acid.127 The importance of N5-OH-L-Orn in this
process was elucidated in studies on Kutzneria spp. 744, where it was identified as a key
precursor in the biosynthetic pathway.146 The need for N5-OH-L-Orn in piperazic acid
biosynthesis stems from its role as a substrate for the piperazate synthase.128,130,146 The N5hydroxy ornithine group is necessary for the piperazate synthase enzyme to build the
nitrogen-nitrogen bond.128,130,146.136
Biocatalysis and chemoenzymatic approaches in flow systems offer significant advantages
for synthesizing complex molecules like piperazic acid. However, coupling multiple
enzymes in these systems presents several challenges.149 While two enzymes could
theoretically be used in a flow system to produce piperazic acid, this approach has inherent
difficulties. The co-immobilization of multiple enzymes on a single carrier can be
particularly challenging, as the carrier properties may affect each enzyme differently,
impacting their activity and stability.149 Furthermore, the need for precise control of
reaction conditions, such as pH and cofactor regeneration, becomes more complex in multienzyme systems.150 Flow biocatalysis can also face issues with enzyme stability over time
and incompatibility with certain reaction conditions, potentially limiting the efficiency of
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cascade reactions.151 Additionally, using water as the primary solvent may lead to substrate
solubility issues and dilute product streams, resulting in lower productivity.151 To overcome
these challenges, a synthetic approach to producing hydroxylated ornithine as a precursor
for piperazic acid synthesis may be more advantageous. This strategy could simplify the
reaction system, reduce the need for complex enzyme coupling, and allow for better control
over reaction conditions.152 By combining chemical synthesis with a single enzymatic step
in the flow, this approach could leverage the benefits of flow chemistry while avoiding the
complexities associated with multi-enzyme systems.151,153
3.3 Developing the Flow Synthesis Strategy for N5-OH-L-Orn
The flow synthesis of N5-OH-L-Ornithine encompasses carefully optimized steps, as
shown in reaction Scheme 11, designed to enhance efficiency and yield. By transitioning
from batch to flow processes, this approach aims to overcome limitations such as extended
reaction times, inconsistent product quality, and scalability issues often associated with
batch production.

97

Scheme 11: Reaction Scheme for the Synthesis of N5-OH-Ornithine (Adapted from Neumann et al.).

Scheme 11 outlines the proposed methodology for synthesizing N5-OH-L-Ornithine (VII)
from L-Ornithine (I), drawing inspiration from established batch protocols154 according to
an analogous procedure described for preparing Boc-Lys.155 This transition to flow
synthesis promises improved process control and product consistency and aligns with the
increasing need for more sustainable and efficient pharmaceutical manufacturing practices.
This thesis only focused on protecting L-Ornithine (I) until the derivative N5-CbzOrnithine (IV) is obtained, as shown in scheme 12 below. Benzyl N-succinimidyl
carbonate (Cbz-OSu) was preferred (scheme 12) over Benzyl chloroformate (Cbz-Cl)
(Scheme 11) for the synthesis of N5-Cbz-Ornithine due to its superior reactivity156 and
selectivity. This reagent allows for faster and more efficient protection of amino groups
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under milder reaction conditions,156 which is particularly beneficial when working with
sensitive substrates. Cbz-OSu typically yields higher amounts of the desired protected
amino acid156 while generating fewer side products, simplifying subsequent purification
steps. Its enhanced stability and ease of handling compared to the moisture-sensitive CbzCl make it a more practical choice in laboratory settings.156 Furthermore, Cbz-OSu
demonstrates better compatibility with other protecting groups and reaction conditions
often encountered in multi-step syntheses.156 These combined advantages, which are
improved reactivity, higher selectivity, better yields, reduced side products, increased
stability, and broader compatibility, make Cbz-OSu the reagent of choice for the protection
of amino groups in complex amino acid derivatives like N5-OH-Ornithine, especially in
scenarios where high yields and product purity are paramount.

Scheme 12: Proposed Reaction scheme for the synthesis of N5-Cbz-Ornithine (IV) from L-Ornithine (I)

3.3.1 L-Ornithine Protection to N5-Cbz-Ornithine in Batch and Flow
3.3.1.1 L-Ornithine Protection to N5-Cbz-Ornithine in Batch – Trial 1
Ornithine hydrochloride (350 mg, 2.08 mmol) was dissolved in 0.5M NaOH (4.5 mL). A
solution of CuSO4 (200 mg, 1.25 mmol, 0.60 equiv) in dH2O (18 mL) was added, and the
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mixture instantly changed to a deep blue colour. The solution was allowed to react at room
temperature with constant stirring for 5 hours. To the resulting ornithine-copper complex
(II) after the 5 hours reaction time, NaHCO3 (350 mg, 4.16 mmol, 2 equiv) was added,
followed by N-(Benzyloxycarbonyloxy) succinimide (720 mg, 2.89 mmol, 1.39 equiv) in
an ice bath. The pH was adjusted to 9 using 4 M NaOH, stirring the solution at room
temperature for 3 hours. The reaction mixture became foamy after adding Cbz-OSu, but the
colour remained unchanged. The blue precipitate (cupric-ornithine complex III) was
filtered after the 3-hour reaction time, washed with a small amount of water, and dried
using the freeze dryer and a blue solid product was obtained. The product was then
dissolved in 0.5 M EDTA solution (pH 8.0, 35 mL). After stirring the solution for 6 hours
at room temperature, the resulting white precipitate was collected by filtration, washed with
a small quantity of water, and dried using the freeze dryer to yield the N5-Cbz-Ornithine
product (IV) as white solids (180 mg, 0.68 mmol, 51% yield).
3.3.1.2 L-Ornithine Protection to N5-Cbz-Ornithine in Flow – Trial 2
3.3.1.2.1 L-Ornithine (I) to Ornithine-copper complex (II) solution
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps
(Scheme 13). Ornithine hydrochloride (350 mg, 2.08 mmol) was dissolved in 0.5 M NaOH
(4.5 mL) and added to the flow at a 0.15 ml/min flow rate from a first Pump A. CuSO4 (200
mg, 1.25 mmol, 0.60 equiv) dissolved in dH2O (18 mL), was added to the flow
simultaneously at a flow rate of 0.6 ml/min from a second Pump B. The reaction mixture
introduced into the flow system via a T-mixer (T-mixer 1) was passed through a reactor coil
at room temperature. This temperature was consistently maintained throughout the reaction
zone to ensure uniform conditions. The reaction mixture turned deep blue when the two
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solutions from both pumps met at T-mixer 1, and the product (Ornithine-copper complex (II)
solution) was obtained following a 30-minute residence time in the reactor (Scheme 13).

Scheme 13: The protection of L-Ornithine to Ornithine-copper complex (II) solution in the flow

3.3.1.2.2 Ornithine-copper complex (II) solution protection to Cupric-ornithine complex
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps
(Scheme 14). Ornithine-copper complex (II) solution (20 mL) was added to the flow at a 2
ml/min flow rate from a first Pump A. NaHCO3 (350 mg, 4.16 mmol) dissolved in dH2O (10
mL) was added to the flow simultaneously at a 1 ml/min flow rate from a second Pump B.
The reaction mixture introduced into the flow system via a T-mixer (T-mixer 1) was passed
through a reactor coil in an ice bath (0 - 5 °C). This temperature was consistently maintained
throughout the reaction zone to ensure uniform conditions. N-(Benzyloxycarbonyloxy)
succinimide (720 mg, 2.89 mmol) dissolved in acetone (10 mL) was added to the flow
simultaneously at a 1 ml/min flow rate from a third Pump C. The reaction mixture introduced
into the flow system via a T-mixer (T-mixer 2) was passed through a reactor coil in an ice
bath (0 - 5 °C). Foaming started immediately after the reaction mixture met at T-mixer 2, and
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the reaction did not proceed smoothly since precipitates started to form, which caused
clogging in the tubes. The reaction was not completed even when slower flow rates were
used, and no product was obtained in this step.

Scheme 14: The protection of Ornithine-copper complex (II) solution to Cupric-ornithine complex in the
flow

3.4 Conclusion
3.4.1 Challenges and Limitations of Developed Flow Protocol
The development of a flow synthesis protocol for N5-OH-ornithine, specifically from Lornithine to N5-Cbz-ornithine, encountered significant challenges, particularly during the
second step involving the protection of the ornithine-copper complex (II) solution with CbzOsu. The primary obstacles were foaming and precipitation, severely impacting the flow
process. Foaming, likely caused by the reaction between NaHCO3 and acidic by-products
such as N-hydroxysuccinimide (HOSu), which should be the by-product from the reaction,
resulted in CO2 generation and subsequent precipitation within the flow system. The
introduction of Cbz-OSu (Benzyl N-Succinimydyl Carbonate) in conjunction with sodium
bicarbonate into the system significantly amplified this effect, further compromising the
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stability and continuity of the flow process. Nevertheless, given the milder and more
sustainable reaction conditions promised by CBz-OSu, the next steps are to attempt the
reaction with no added base or a different base than sodium bicarbonate to maintain the
appropriate pH of the reaction. Should NaHCO3 prove to be essential for neutralization to
prevent acid buildup and maintain reaction equilibrium, a series of other alternatives are
contemplated. While potentially crucial for preventing the potential decomposition of
reactants or products, it simultaneously contributed to the foaming issue. This delicate
balance between maintaining pH and avoiding excessive gas generation highlights the
complexity of translating batch processes to flow systems.157 Precipitation from this reaction
led to severe clogging in the flow channels and tubes, persisting even when slower flow rates
were employed. The formation of solid precipitates in flow systems is a well-documented
challenge in continuous processing, particularly when dealing with inorganic salts or metal
complexes.158 These challenges underscore the need for innovative approaches in flow
chemistry when dealing with gas-evolving reactions and precipitation-prone processes.
3.4.2 Proposed Solutions to Overcoming Challenges of Developed Flow Protocol
Several innovative solutions can be implemented to address the challenges encountered in the
flow synthesis protocol for N5-Cbz-Ornithine to enhance process efficiency and reliability.
Increasing the tubing diameter to 1 mm or more would be a straightforward approach to
mitigate clogging risks, as it provides more space for precipitates and reduces the likelihood
of blockages.159 Integrating a gas-liquid separator is crucial for managing CO2 evolution,
allowing for gas-controlled venting while maintaining the liquid phase in the flow system.160
A particularly promising solution is the incorporation of a Continuous Stirred Tank Reactor
(CSTR) into the flow setup. CSTRs offer multiple advantages in addressing the observed
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challenges. The efficient mixing provided by an impeller or stirrer ensures homogeneous
conditions, reducing foaming and improving reaction uniformity.27 The stirring action also
keeps solids suspended, preventing precipitation-induced clogging that is problematic in
simple tubular reactors. Some CSTR designs feature wider outlets or specialized pumps to
handle slurries and precipitates effectively.161 The open or vented design of a CSTR
facilitates efficient gas release, which is crucial for managing CO2 evolution and reducing
foam buildup.162 This feature, combined with the gas-liquid separator, provides a
comprehensive solution for gas management in the flow system.
These proposed solutions, when implemented in combination, have the potential to
significantly improve the robustness and scalability of the flow synthesis protocol.
Integrating a CSTR with enhanced mixing capabilities, strategic gas management, and
careful selection of reagents addresses the core issues of foaming, precipitation, and
clogging. This approach solves immediate challenges and paves the way for more efficient
and scalable continuous flow processes in complex organic syntheses.23

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4. General Discussions and Conclusion
4.1 Achievement of Objectives and Summary of Key Findings
The primary objectives of this study were to develop a flow synthesis method for ascorbic
acid and to protect L-Ornithine towards the synthesis of N5-OH-Ornithine using flow
chemistry techniques. These objectives were pursued to advance the application of flow
chemistry in the synthesis of complex organic compounds, particularly those of
pharmaceutical relevance.
For the first objective, significant progress was made in translating the multi-step synthesis of
ascorbic acid from batch to flow conditions. The research successfully demonstrated the
feasibility of conducting several key steps of ascorbic acid synthesis in a flow process. This
study reported the flow synthesis steps of D-glucose to D-sorbitol, L-sorbose to L-ascorbic
acid and the attempted flow synthesis of the protection of L-Ornithine towards N5-OHOrnithine synthesis.
The flow synthesis method developed for ascorbic acid includes multiple reaction steps, each
requiring precise optimization of reaction parameters. These parameters included but were
not limited to residence time, reagent concentration, and flow rate. Through systematic
investigation and iterative refinement, optimal conditions were established for most synthetic
sequences. However, the second step of synthesizing L-ascorbic acid, the oxidation of Dsorbitol to L-sorbose, is still a work in progress by another researcher. Despite this limitation,
the successful implementation of the other steps in flow represents a substantial achievement.
The work has laid a solid foundation for future optimization efforts and has contributed
valuable insights into translating complex, multi-step syntheses from batch to flow processes.

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In the ascorbic acid synthesis project, as discussed in Chapter 2 of this thesis, the flow system
demonstrated marked improvements over conventional batch methods across multiple
reaction steps. The conversion of L-sorbose to diacetone-L-sorbose in flow conditions
achieved a 95% yield within a residence time of just 30 minutes. This gave a better result
than the batch reaction, which yielded 90% in 360 minutes. The reduction in reaction time,
coupled with an improved yield, shows the potential of flow chemistry in process efficiency.
The subsequent oxidation step in the flow to produce 2-keto-L-gulonic acid showed
improvements. The flow system achieved a yield of 91% within a total residence time of 40
minutes, which gave more yield than the traditional batch process, which required a 3-hour
reaction time to achieve an 88% yield. This shows the capacity of flow chemistry to
accelerate reaction rates and enhance product yields in complex oxidation processes.
For the final conversion of 2-keto-L-gulonic acid to L-ascorbic acid, while the yield
difference between batch and flow methods was less pronounced, the flow process still
offered advantages. The flow system produced a slightly higher yield of 85% in just 20
minutes of residence time compared to the batch process, which yielded 82% in
approximately 180 minutes. This result further reinforces the time-efficiency benefits of flow
chemistry, even in cases where yield improvements are marginal.
A key advantage of the flow system was the ability to perform real-time optimization of
reaction parameters. By adjusting flow rates, reagent-to-solvent ratios, and temperatures, as
seen at the L-sorbose to diacetone-L-sorbose step, consistently higher yields and product
quality were maintained throughout the process. The cumulative effect of these
improvements resulted in an overall yield of L-ascorbic acid from L-sorbose reaching 8285%. This yield surpasses the typical yields of 70-75% reported for industrial batch

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processes, demonstrating the potential of flow chemistry to enhance the efficiency of
ascorbic acid production at an industrial scale. While our flow approach did not significantly
reduce solvent usage compared to batch methods, it contributed to a more environmentally
friendly production process through efficient mixing and in-flow addition of reagents,
potentially reducing waste and improving overall process sustainability.
Turning to the second objective, the protection of L-Ornithine towards synthesizing N5-OHOrnithine in flow, the research yielded mixed results that nonetheless provide significant
contributions to the field. The initial phase of this work involved the successful development
of a flow synthesis protocol based on established batch methods.
As discussed in Chapter 3 of this thesis, the flow synthesis of N5-Cbz-Ornithine towards the
synthesis of N5-OH-Ornithine, encountered significant challenges that prevented the
completion of the full synthesis. However, important findings were still obtained. The first
step of the synthesis, the protection of L-ornithine, was successfully carried out in flow
conditions, achieving the desired transformation in under 30 minutes. This represents a
substantial time reduction compared to the batch process, which required 6 hours. This result
highlights the potential of flow chemistry to dramatically accelerate certain reaction steps,
even in complex amino acid derivatizations. However, the second step of this synthesis
presented considerable obstacles in the flow system. Using sodium bicarbonate (NaHCO3)
led to significant foaming and precipitation within the flow reactor, disrupting the continuous
process, as discussed previously. This challenge underscores critical consideration in flow
chemistry: the behaviour of reagents and products under continuous flow conditions can
differ markedly from batch processes, particularly when gas evolution or precipitation

107

occurs. While this issue was not fully resolved within the scope of this research, it provides
valuable insights into how this reaction can be modified within flow for the future.
The partial success in the flow synthesis of ascorbic acid demonstrates the potential for
applying continuous flow techniques to complex, multi-step syntheses of pharmaceutical
intermediates. The work has identified key areas for future optimization and has established a
framework upon which subsequent research can build. While the initial objectives of
continuous-flow synthesis were not met, the research has substantially contributed towards
the effort of producing the important vitamin, ascorbic acid, utilizing flow synthesis. The
next steps will build upon these efforts by optimizing reaction conditions, integrating an
inline separator, and implementing more advanced pumping systems/ reactor designs to
achieve a continuous-flow synthesis.
4.2 Further Studies and Future Recommendations
The study on the flow synthesis of ascorbic acid and the attempted flow synthesis of N5-OHOrnithine has provided valuable insights and opened up several avenues for further
investigation and improvement. This section outlines potential areas for future studies and
recommendations to address the challenges encountered and to further optimize the flow
chemistry processes developed in this work.
For the ascorbic acid synthesis project, a primary focus for future studies should be
implementing more advanced pumping systems. The limitations of the current pumping
equipment hindered the continuous-flow synthesis of ascorbic acid. Future work should
explore the use of high-performance pumps capable of handling higher pressures and
providing more precise flow control. This would help towards achieving a fully continuousflow synthesis of ascorbic acid. Another focus for future studies would be one of the main
108

challenges in continuous flow synthesis: the need for inline separation between steps.
Traditional batch processes often involve the isolation and purification of intermediates,
which is more complex in continuous-flow systems. An inline liquid-liquid extraction using
membrane-based separators can be utilized to address this.163 These can effectively separate
aqueous and organic phases continuously. Integrating continuous drying techniques such as
membrane pervaporation or molecular sieves27 should also be studied since drying in the
flow is one of the challenges of continuous flow.
In the N5-OH-Ornithine synthesis project, several recommendations can be made to address
the challenges of foaming and precipitation. A key area for investigation is the substitution of
NaHCO3 with NaOH as the base. This change might reduce or eliminate precipitation issues,
as NaOH is generally more soluble than NaHCO3164. However, careful consideration must be
given to the impact of this substitution on reaction kinetics and product stability. Studies
should be conducted to optimize NaOH's concentration and flow rate to maintain the desired
pH while minimizing side reactions. To further mitigate issues related to precipitation and
gas evolution, the use of larger diameter tubing (1 mm or more) should be explored. Wider
tubing could reduce the likelihood of clogging and improve the handling of heterogeneous
mixtures. However, this modification may affect residence times and heat transfer,
necessitating a comprehensive re-optimization of reaction conditions.165 Implementing a
liquid-gas separator in the flow system presents another promising avenue for improvement.
This addition could effectively manage the gas evolution observed in the reaction, preventing
disruptions to the flow and improving overall system stability. Various designs of liquid-gas
separators should be evaluated for their efficiency and compatibility with the specific
reaction conditions of the N5-OH-Ornithine synthesis.166
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5. Experimental Section
5.1 General Experimental Procedures
All reagents and solvents used were obtained or purchased from Thermo Fischer Scientific
and Sigma Aldrich. SGE Syringes, reactor coils (PTFE tubing), T-mixers, Y-mixers, and
back pressure regulators were purchased from IDEX Health & Science LLC. Scientific
syringe pumps were purchased from New Era Pump Systems Inc.
Synthesized products were isolated using a BÜCHI Rotavapor R-200 Rotary Evaporator and
dried in a desiccator or a BÜCHI Lyovapor L-200 Freeze Dryer. Synthesized products were
confirmed by analyzing the IR spectrum and 1H, 13C, DEPT-135, 1H-13C HSQC, and 1H-13C
HMBC NMR spectra. IR spectra were recorded using a Bruker ALPHA II FT-IR
spectrometer. One- and two-dimensional NMR spectra were recorded on a Bruker Fourier
300 MHz NMR spectrometer or 400 MHz Bruker Fourier spectrometer. All samples were
run in deuterated chloroform (99.96% isotopic purity) or deuterated DMSO (99.96% isotopic
purity), and NMR data was calibrated to the solvent signals of δH 7.24 ppm and δC 77.0 ppm
for deuterated chloroform, and δH 2.50 ppm and δC 39.52 ppm for deuterated DMSO. All
NMR spectra were processed by TopSpin 4.1.1 software for this thesis. Electrospray
ionization (ESI) mass spectrometry was run by staff in the Simon Fraser University (SFU)
Mass Spectrometry Facility, Department of Chemistry, on a Bruker Esquire ion trap mass
spectrometry.

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5.2 Synthesis Procedures
5.2.1 Preparation of D-sorbitol 2.2.1.10
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps,
as shown in Scheme 7. D-glucose (1.03 g, 5.71 mmol) was dissolved in 10 mL of dH2O and
added to the flow at a 0.5 ml/min flow rate from a first Pump A. NaBH4 (0.1753 g, 4.63
mmol) dissolved in 10 mL of dH2O was added to the flow simultaneously at a 0.5 ml/min
flow rate from a second Pump B. The reaction mixture introduced into the flow system via a
T-mixer (T-mixer 1) was passed through a reactor coil maintained at ambient temperature
(20 ± 2°C). This temperature was consistently maintained throughout the reaction zone to
ensure uniform conditions. The product was obtained following a 20-minute residence time
in the reactor, and the solvent was evaporated to dryness under reduced pressure. Excess H20
was removed, leaving D-sorbitol, a white powder (1.04 g, 5.70 mmol, 100%) analyzed by
NMR (300 MHz, DMSO-d6).
1

H NMR (300 MHz, DMSO-d6) δ = 7.0-9.0 (impurities), 5.84 (d, J = 6.9 Hz), 4.0-5.0

(multiplet), 2.5-4.0 (multiplet), 1.23 (s, impurity). 13C NMR (300 MHz, DMSO-d6) δ =
105.2, 76.2, 75.3, 74.6, 74.2, 73.7, 72.7, 71.9, 69.2, 64.8, 64.3, 63.8, 62.9.
5.2.2 Preparation of partially protected Sorbose 2.2.2.3
L-sorbose (0.625 g, 3.46 mmol) was dissolved in 60 mL of acetone (acetone to sorbose ratio
of 233: 1). H2SO4 (1.2 mL) was added dropwise to this solution until universal pH indicator
paper indicated the reaction mixture was acidified. The solution was allowed to react in an
ice-cold water bath with constant stirring for 300 minutes. After this, sodium bicarbonate (20
mL) was added dropwise until universal pH indicator paper indicated the reaction mixture

111

was neutralized. The solvent was evaporated to dryness under reduced pressure. Excess
acetone was removed, leaving a white powder (0.54 g, 2.07 mmol, 60 %) analyzed by NMR
(400 MHz, DMSO-d6).
1

H NMR (400 MHz, DMSO-d6) δ = 1.20 – 1.40 (6 H, 1.29 (s), 1.39 (s)), 3.40 – 3.64 (5 H,

multiplet), 3.75 (1 H, dd, J = 9.2, 6.7 Hz), 4.00 (d, J = 6.9 Hz), 4.1 (1 H, multiplet), 4.30 (s)).
13

C NMR (400 MHz, DMSO-d6) δ = 27.1, 28.0, 59.4, 62.5, 73.9, 82.3, 85.1, 111.2, 114.7.

ESIMS [M+Na] + calculated for C9H16O6Na 243.22, found 243.0837.
5.2.3 Preparation of diacetone-L-sorbose 2.2.2.9
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps.
L-sorbose (0.625 g, 3.46 mmol) was dissolved in 80 mL of acetone (acetone to sorbose ratio
of 311: 1) and added to the flow at a 4 ml/min flow rate from a first Pump A. H2SO4 (1.5
mL) diluted in acetone (20 mL) was added to the flow simultaneously at a 1 ml/min flow rate
from a second Pump B. The reaction mixture introduced into the flow system via a T-mixer
(T-mixer 1) was passed through a reactor coil in an ice bath (0 - 5 °C). This temperature was
consistently maintained throughout the reaction zone to ensure uniform conditions. Sodium
bicarbonate (20 mL) was added to the flow simultaneously at a 1 ml/min flow rate from a
third Pump C. The product was obtained following a 30-minute residence time in the reactor,
and the solvent was evaporated to dryness under reduced pressure. Excess acetone was
removed, leaving a white powder (0.86 g, 3.30 mmol, 95 %) analyzed by NMR (400 MHz,
CDCl3).
1

H NMR (400 MHz, CDCl3) δ = 1.30 – 1.55 (12 H, 1.36 (s, 6 H), 1.43 (s, 3 H), 1.49 (s, 3 H),

2.12 (1 H, dd, J = 7.0, 5.9 Hz), 3.82 (2 H, dd, J = 11.8, 7.0 Hz), 4.05 (2 H, dd, J = 3.3, 5.3
112

Hz), 4.09 (d, J = 1.7 Hz), 4.31 (d, J = 2.0 Hz), 4.46 (s)). 13C NMR (400 MHz, CDCl3) δ =
18.6, 26.5, 27.3, 29.0, 60.3, 63.7, 72.3, 73.3, 85.0, 97.6, 112.0, 114.5. ESIMS [M+Na] +
calculated for C12H20O6Na 283.290, found 283.1154.
5.2.4 Preparation of 2-keto-L-gulonic acid 2.2.3.3
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps
(Scheme 9). Diacetone-L-sorbose (0.33 g, 1.26 mmol) was dissolved in 20 mL of acetone.
NaOH (0.50 g, 12.5 mmol) was added to this mixture and the solution was added to the flow
at a 1 ml/min flow rate from a first Pump A. KMnO4 (0.35 g, 2.21 mmol) dissolved in 10 mL
of acetone was added to the flow simultaneously at a 0.5 ml/min flow rate from a second
Pump B. The reaction mixture introduced into the flow system via a T-mixer (T-mixer 1) was
passed through a reactor coil in a hot water bath at an elevated temperature of 50° C. This
temperature was consistently maintained throughout the reaction zone to ensure uniform
conditions. Sodium bisulphite (1.20 g, 11.53 mmol) dissolved in 5 mL of dH20 was added to
the flow simultaneously at a 0.25 ml/min flow rate from a third Pump C. H2SO4 (1 mL)
diluted in 10 mL of acetone was added to the flow simultaneously at a 0.5 ml/min flow rate
from a fourth Pump D, and was then allowed to react with the reaction mixture at a second
reactor coil for 20 minutes. In the drying unit, 3 A° and 4 A° molecular sieves trapped the
aqueous layer. The sieves were pre-dried in the oven at 160° C for 16 hours before use. The
product was obtained following a 40-minute residence time in the reactor, and the solvent
was evaporated to dryness under reduced pressure using a rotary evaporator. Excess acetone
was removed, leaving 2-keto-L-gulonic acid, a white powder (0.22 g, 1.13 mmol, 91%)
analyzed by NMR (400 MHz, DMSO-d6) and Mass spectrometry.

113

1

H NMR (400 MHz, DMSO-d6) δ = 3.10-3.22 (2 H, 3.20 (q, J = 10.6, 17.7, 10.8 Hz), 3.67 (1

H, dd, J = 7.2, 9.0 Hz), 3.85 (1 H, d, J = 9.0 Hz)), 4.05 (1 H, d, J = 9.6 Hz), 4.74 (1 H, d, J =
7.0 Hz).13C NMR (400 MHz, DMSO-d6) δ = 64.4, 74.1, 74.4, 76.8, 175.1, 213.5. ESIMS
[M+Na] + calculated for C6H10O7Na 217.140, found 217.0421 and ESIMS [M+K] +
calculated for C6H10O7K 233.140, found 233.0172.

5.2.5 Preparation of L-ascorbic acid 2.2.4.2
A flow system was set up using PTFE tubing (0.75 mm inner diameter) and syringe pumps.
2-keto-L-gulonic acid (0.55 g, 2.83 mmol) was dissolved in 10 mL of methanol and added to
the flow at a 0.5 ml/min flow rate from a first Pump A. H2SO4 (0.5 mL) diluted in dH20 (5
mL) was added to the flow simultaneously at a 0.25 ml/min flow rate from a second Pump B.
The reaction mixture introduced into the flow system via a T-mixer (T-mixer 1) was passed
through a reactor coil in a hot water bath at an elevated temperature of 65° C to 100° C. This
temperature was consistently maintained throughout the reaction zone to ensure uniform
conditions. Sodium bicarbonate (5 mL) was added to the flow simultaneously at a 0.5 ml/min
flow rate from a third Pump C. The product was obtained following a 20-minute residence
time in the reactor, and the solvent was evaporated to dryness under reduced pressure using a
rotary evaporator. Excess H20 was removed, leaving L-ascorbic acid, a white powder (0.42 g,
2.38 mmol, 85 %). The crude product (0.21 g, 1.19 mmol) was recrystallized with a mixture
of ethanol and water (3:1), and pure ascorbic acid (0.12 g, 0.68 mmol, 57%) was obtained
and analyzed by NMR (400 MHz, DMSO-d6).

114

1

H NMR (400 MHz, DMSO-d6) δ = 3.40-3.45 (2H, 3.42 (d, J = 6.0 Hz), 3.72 (s), 4.71 (1H,

d, J = 3.0 Hz).13C NMR (400 MHz, DMSO-d6) δ = 62.4, 68.8, 75.0, 118.4, 153.3, 171.1.
ESIMS [M+Na] + calculated for C6H8O6Na 199.12, found 199.0307 and [M+H] + calculated
as 177.12, found 177.0482.

115

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127

Appendix A: Spectroscopic Data

Fig. A 1 - Sorbitol 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

Fig. A 2 - Sorbitol 13C-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

128

Fig. A 3 - Sorbitol IR spectrum for Batch 1

Fig. A 4 - Sorbitol 1H-NMR for Flow 2 recorded in DMSO-d6 (300 MHz).

129

Fig. A 5 - Sorbitol 13C-NMR for Flow 2 recorded in DMSO-d6 (300 MHz).

Fig. A 6 - Sorbitol 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz).

130

Fig. A 7 - Sorbitol 13C-NMR for Batch 3 recorded in DMSO-d6 (300 MHz).

Fig. A 8 - Sorbitol HSQC for Batch 3 recorded in DMSO-d6 (300 MHz).

131

Fig. A 9 - Sorbitol IR spectrum for Batch 3

Fig. A 10 - Sorbitol 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz).

132

Fig. A 11- Sorbitol 13C-NMR for Batch 4 recorded in DMSO-d6 (300 MHz).

Fig. A 12 - Sorbitol HSQC for Batch 4 recorded in DMSO-d6 (300 MHz).

133

Fig. A 13 - Sorbitol IR spectrum for Batch 4

Fig. A 14- Sorbitol 1H-NMR for Batch 5 recorded in DMSO-d6 (300 MHz).

134

Fig. A 15 - Sorbitol 13C-NMR for Batch 5 recorded in DMSO-d6 (300 MHz).

Fig. A 16 - Sorbitol 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz).

135

Fig. A 17 - Sorbitol 13C-NMR for Batch 6 recorded in DMSO-d6 (300 MHz).

Fig. A 18 - Sorbitol 1H-NMR for Batch 7 recorded in DMSO-d6 (300 MHz).

136

Fig. A 19 - Sorbitol 1H-NMR for Batch 8-A recorded in DMSO-d6 (300 MHz).

Fig. A 20 - Sorbitol 13C-NMR for Batch 8-A recorded in DMSO-d6 (300 MHz).

137

Fig. A 21 - Sorbitol 1H-NMR for Batch 8-B recorded in DMSO-d6 (300 MHz).

Fig. A 22 - Sorbitol 13C-NMR for Batch 8-B recorded in DMSO-d6 (300 MHz).

138

Fig. A 23 - Sorbitol 1H-NMR for Batch 8-C recorded in DMSO-d6 (300 MHz).

Fig. A 24 - Sorbitol 1H-NMR for Flow 9 recorded in DMSO-d6 (300 MHz).

139

Fig. A 25 - Glucose Sample IR spectrum.

Fig. A 26 - Glucose Sample 1H-NMR recorded in DMSO-d6 (300 MHz).

140

Fig. A 27 - Glucose Sample 13C-NMR recorded in DMSO-d6 (300 MHz).

Fig. A 28 - Glucose Sample DEPT-135 recorded in DMSO-d6 (300 MHz).

141

Fig. A 29 - Sodium borohydride Sample IR spectrum

Fig. A 30 - Sodium borohydride Sample 1H-NMR recorded in DMSO-d6 (300 MHz).

142

Fig. A 31 - DAS Product 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

Fig. A 32 - DAS Product 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz).

143

Fig. A 33 - DAS Product 1H-NMR for Batch 3 recorded in DMSO-d6 (300 MHz).

Fig. A 34 - DAS Product 1H-NMR for Batch 4 recorded in DMSO-d6 (300 MHz).

144

Fig. A 35 - DAS Product 1H-NMR for Flow 5 recorded in DMSO-d6 (300 MHz).

Fig. A 36 - DAS Product Mass Spectrum for Flow 5

145

Fig. A 37 - DAS Product 1H-NMR for Batch 6 recorded in DMSO-d6 (300 MHz).

Fig. A 38 - DAS Product 1H-NMR for Flow 7 recorded in DMSO-d6 (400 MHz).

146

Fig. A 39 - DAS Product Mass Spectrum for Flow 7

Fig. A 40 - DAS product 1H-NMR for Batch 8-A recorded in CDCl3 (400 MHz).

147

Fig. A 41 - DAS product 1H-NMR for Batch 8-B recorded in CDCl3 (400 MHz).

Fig. A 42 -DAS product 1H-NMR for Batch 8-C recorded in CDCl3 (400 MHz).

148

Fig. A 43 - DAS product 1H-NMR for Batch 8-D recorded in CDCl3 (400 MHz).

Fig. A 44 - DAS product 1H-NMR for Batch 8-E recorded in CDCl3 (400 MHz).

149

Fig. A 45 - DAS product 1H-NMR for Batch 8-F recorded in CDCl3 (400 MHz).

Fig. A 46 - DAS product 1H-NMR for Batch 8-G recorded in CDCl3 (400 MHz).

150

Fig. A 47 - DAS product 1H-NMR for Batch 8-H recorded in CDCl3 (400 MHz).

Fig. A 48 - DAS product 1H-NMR for Batch 8-I recorded in CDCl3 (400 MHz).

151

Fig. A 49 - DAS product 1H-NMR for Batch 8-J recorded in CDCl3 (400 MHz).

Fig. A 50 - DAS product 1H-NMR for Batch 8-K recorded in CDCl3 (400 MHz).

152

Fig. A 51 - DAS product 1H-NMR for Batch 8-L recorded in CDCl3 (400 MHz).

Fig. A 52 - DAS product 1H-NMR for Batch 8-M recorded in CDCl3 (400 MHz).

153

Fig. A 53 – Diacetone-L-sorbose 1H-NMR for Flow 9 recorded in CDCl3 (400 MHz).

Fig. A 54 - Diacetone-L-sorbose 13C-NMR for Flow 9 recorded in CDCl3 (400 MHz).

154

Fig. A 55 - Diacetone-L-sorbose COSY for Flow 9 recorded in CDCl3 (400 MHz).

Fig. A 56 - Diacetone-L-sorbose HSQC for Flow 9 recorded in CDCl3 (400 MHz).

155

Fig. A 57 - Diacetone-L-sorbose HMBC for Flow 9 recorded in CDCl3 (400 MHz).

Fig. A 58 - DAS product 1H-NMR for Batch 3 recorded in DMSO-d6 (400 MHz).

156

Fig. A 59 - DAS product 13C-NMR for Batch 3 recorded in DMSO-d6 (400 MHz).

Fig. A 60 - DAS product HSQC for Batch 3 recorded in DMSO-d6 (400 MHz).

157

Fig. A 61 - DAS product HMBC for Batch 3 recorded in DMSO-d6 (400 MHz).

Fig. A 62 - Sorbitol 1H-NMR for Flow 10 recorded in DMSO-d6 (300 MHz).

158

Fig. A 63 - Sorbitol 13C-NMR for Flow 10 recorded in DMSO-d6 (300 MHz).

Fig. A 64 - Sorbitol 1H-NMR for Flow 10 recorded in DMSO-d6 (300 MHz).

159

Fig. A 65 - Sorbitol 13C-NMR for Flow 10 recorded in DMSO-d6 (300 MHz).

Fig. A 66 – 2-keto-L-gulonic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

160

Fig. A 67 - 2-keto-L-gulonic acid 13C-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

Fig. A 68 - 2-keto-L-gulonic acid HSQC for Batch 1 recorded in DMSO-d6 (300 MHz).

161

Fig. A 69 – 2-keto-L-gulonic acid 1H-NMR for Batch 2 recorded in DMSO-d6 (300 MHz).

Fig. A 70 - 2-keto-L-gulonic acid 13C-NMR for Batch 2 recorded in DMSO-d6 (300 MHz).

162

Fig. A 71 - 2-keto-L-gulonic acid HSQC for Batch 2 recorded in DMSO-d6 (300 MHz).

Fig. A 72 – 2-keto-L-gulonic acid 1H-NMR for Flow 3 recorded in DMSO-d6 (400 MHz).

163

Fig. A 73 - 2-keto-L-gulonic acid 13C-NMR for Flow 3 recorded in DMSO-d6 (400 MHz).

Fig. A 74 - 2-keto-L-gulonic acid HSQC for Flow 3 recorded in DMSO-d6 (400 MHz).

164

Fig. A 75 - 2-keto-L-gulonic acid COSY for Flow 3 recorded in DMSO-d6 (400 MHz).

Fig. A 76 - 2-keto-L-gulonic acid HMBC for Flow 3 recorded in DMSO-d6 (400 MHz).

165

Fig. A 77 - 2-keto-L-gulonic acid Mass spectrum for Flow 3

Fig. A 78 – Ascorbic acid 1H-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

166

Fig. A 79 – Ascorbic acid 13C-NMR for Batch 1 recorded in DMSO-d6 (300 MHz).

Fig. A 80 - Ascorbic acid DEPT-135 for Batch 1 recorded in DMSO-d6 (300 MHz).

167

Fig. A 81 – Ascorbic acid crude product 1H-NMR for Flow 2 recorded in DMSO-d6 (400 MHz).

Fig. A 82 – Ascorbic acid Mass Spectrum for Flow 2

168

Fig. A 83 – Ascorbic acid 1H-NMR for Flow 2 recorded in DMSO-d6 (400 MHz).

Fig. A 84 – Ascorbic acid 13C-NMR for Flow 2 recorded in DMSO-d6 (400 MHz).

169

Fig. A 85 - Ascorbic acid COSY for Flow 2 recorded in DMSO-d6 (400 MHz).

Fig. A 86 – Ascorbic acid HSQC for Flow 2 recorded in DMSO-d6 (400 MHz).

170

Fig. A 87 – Ascorbic acid HMBC for Flow 2 recorded in DMSO-d6 (400 MHz).

171