These two lectures give an overview of organic synthesis using water as a solvent. This is aimed towards final year undergraduates and graduate students in chemistry

1. Organic Synthesis in Water
Kinetics, Mechanism and Synthesis
Graduate Lecture Series
Lecture 1
Dr Anthony Coyne
(anthony.g.coyne@gmail.com)

2. Outline
 Lecture 1 – Kinetics, Mechanism and Synthesis
• Background
• Mechanistic aspects
• Influence on rate of reaction
• Organic Synthesis
• Diels-Alder [4+2] cycloaddition reaction
• Huisgen [3+2] cycloaddition reaction
• Claisen rearrangement
• Epoxide ring opening
 Lecture 2 – Emerging areas of research
• Recap of Lecture 1
• Organic Synthesis
Emerging metal catalysed reactions
• Olefin metathesis
• Cyclopropanation reactions
• Chemical Biology Applications
• CuAAC reactions
• SPAAC reactions
• Sonogashira coupling
• Suzuki coupling

3. Why water as a reaction solvent?

4. Background
Acetonitrile (2009)
While acetonitrile is not a key solvent in organic synthesis it is just a matter of time that there will be a shortage
of a solvent that is used day to day

5. Background
Why water as a solvent for organic reactions?
Water is the universal solvent in Nature
Non-toxic
Non-flammable
Cheap and readily available
Can have interesting acceleration effects for certain reactions
Can be used in conjunction with some organic solvents
Applicable to a wide range of reaction types
Green Chemistry
Difficult to remove water from reactions
Solubility (not always a problem)
Not suitable for all organic reactions
Waste streams need to be treated
Pros
Cons

6. Background – Reactions using water as a solvent
Typically how have reactions been carried out using water as a solvent

7. Background – In the beginning
1828 – Friedrich Wöhler
1931 – Otto Diels and Kurt Alder
1948 – Robert Burns Woodward
Diels, O.; Alder, K. Justus Liebigs Ann. Chem. 1931, 490, 243
Woodward, R. B.; Baer, H. J. Am. Chem. Soc. 1948, 70, 1161
Wöhler, F. Annalen der Physik und Chemie. 1828, 88(2), 253

8. Background
1980 – Ronald Breslow – Columbia University
Rideout, D, Breslow, R, J. Am. Chem. Soc., 1980, 7816
Ph.D Harvard University
(R. B Woodward)
PostDoc – University of
Cambridge (Lord Todd)
Solvent k2 x 105 M-1s-1
Isooctane 5.94
Methanol 75.5
H2O 4400
Solvent k2 x 105 M-1s-1
Isooctane 1.90
Methanol 4.0
H2O 59.3
The reaction of cyclopentadiene and methyl vinyl ketone is over 700 times faster in water than in isooctane.
The corresponding reaction with acrylonitrile is only 30 times faster. Why is this?
k2 isooctane
k2 Water
= 740
k2 isooctane
k2 Water
= 31

9. Background
1996 – Jan Engberts – University of Groningen PhD. University of
Groningen
PostDoc – University of
Amsterdam
Solvent Additive k2 (M-1s-1)
Acetonitrile - 1.4 x 10-5
Water - 4.02 x 10-3
Acetonitrile Cu(NO3)2 0.472
Water Cu(NO3)2 1.11
k2 Acetonitrile
k2 Water = 287
k2 Acetonitrile
k2 Water
= 5.4
k2 Acetonitrile
k2 Water(Cu )
= 79285
Otto, S., Bertonicin F., Engberts, J.B.F.N., J. Am. Chem. Soc., 1996,118, 7702
2+
Uncatalysed Cu2+ catalysed
Reaction is accelerated 287 times faster in H2O compared to MeCN. When a Lewis acid (Cu(NO3)2) was added a large
rate acceleration observed however the reaction using water does not have a large rate acceleration as observed with the
uncatalysed reaction
Catalysis using water as a solvent possible however mechanistically can be difficult to study

10. Klign, J. E., Engberts, J.B.F.N. Nature. 2005, 435, 746
Background
2005 – K. Barry Sharpless – Scripps Research Institute
Narayan, S., Muldoon, J., Finn, M.G., et al. Angew. Chem. Int. Ed., 2005, 44, 3275
Solvent Conc (M) Time to completion
Toluene 2 > 120 h
Methanol 2 18 h
H2O 4.53 10 min
These reactions were classed as ‘on-water’ as they were seen to float on water surface

11. Background
2005 – K. Barry Sharpless
Narayan, S., Muldoon, J., Finn, M.G., et al. Angew. Chem. Int. Ed., 2005, 44, 3275
PhD. Stanford University
(E.E. Van Tamelen)
PostDoc – Stanford
University and Harvard
Solvent Reaction Time
Neat 36 h
H2O 3 h
Solvent Conc (M) Reaction
Time
Yield (%)
Toluene 1 144 79
Neat 3.69 10 82
H2O 3.69 8 81
Both reactants are liquids so there is not mixing issues
associated with neat reactions
The reaction ‘on-water’ is faster than the neat reaction.
No large rate acceleration observed with these
reactions. TSRI TSRI TSRI

12. Background – What is so significant about the rate acceleration using water?
A key aspect of the initial studies where water has been used as a solvent has focused on cycloaddition reactions.
Huisgen, R. Pure Appl Chem. 1980, 52, 2283
Huisgen, R., Seidel, H, Brüning, I, Chem. Ber. 1969, 102, 1102
Why are there large rate accelerations observed using water as a reaction solvent
and what is causing these?
The rate on going
from toluene to
ethanol only changes
by a factor of 5.6
This is because these reactions are solvent insensitive

13. Why are there large rate accelerations using water as a solvent?
Not fully understood however various theories have been put forward

14. Mechanistic Aspects – Hydrophobic Effect (Breslow)
k2 isooctane
k2 Water
= 740
k2 isooctane
k2 Water
= 31
Breslow and Rideout proposed that the rate acceleration observed for the reactions was due to the hydrophobic
effect.
Hydrophobic effect found widespread in nature and is important in
protein structure.
However this does not take into account the difference in rate between the reaction with methyl vinyl
ketone and acrylonitrile.
Rideout, D, Breslow, R, J. Am. Chem. Soc., 1980, 7816

15.  Jorgensen and co-workers examined
the reaction using computational
methods and suggested that the large
accelerations observed with vinyl
ketones was due to a significant
Hydrogen bonding effects
 Jorgensen calculated that the rate
acceleration was due to approximately
90% H-bonding with 10% from
hydrophobic effects
 Mechanism similar to H-bonding
organocatalysis
k2 isooctane
k2 Water
= 740
k2 isooctane
k2 Water
= 31
Mechanistic Aspects – H-Bonding Effects (Jorgensen)
Rideout, D, Breslow, R, J. Am. Chem. Soc., 1980, 7816
Jorgensen, W.L., J. Org. Chem., 1994, 59, 803
PhD Harvard
University
(E.J. Corey)
H-bonding occurs with the vinyl ketone however
not with the acrylonitrile

16. Mechanistic Aspects – ‘On-water’ Effect (Sharpless and Marcus)
Toluene: >120 h
H2O: 10 mins
 The ‘on-water’ is so named because the organic reactions are ‘floated’ on water
 Marcus and co-workers examined this reaction from a computational aspect . The large rate accelerations were
proposed to be due to the favorable H-bonding interactions to the transition state. The H-bonding was at the oil
water/interface where a proton protrudes into the ‘oil’ phase which acts as the catalyst.
 These reactions are very difficult to examine experimentally in order to explore the on-water effect.
Jung, Y, Marcus, R.A, J. Am Chem. Soc., 2007, 129, 5492
Jung, Y, Marcus, R.A, J. Chem. Phys. Cond Matt., 2010, 284117
PhD. McGill University
Nobel Prize: 1992
(Electron transfer
reactions)

17. Mechanistic Aspects – Influence on the rate of the reaction
k2 Toluene
k2 Water
= 40.9
1998 – M.R Gholami
k2 Acetonitrile
k2 Water
= 164
2004 – R.N. Butler
k2 Acetonitrile
k2 Water
= 15.3
1982 – R. Breslow
k2 Acetonitrile
k2 Water
= 211
Rideout, D, Breslow, R, J. Am. Chem. Soc., 1980, 7816
Gholami, M. R.; J. Chem. Res (S), 1999, 226
Butler, R.N. et al., J Chem. Soc Perkin Trans 2, 2002, 1807
In all of the above cases the reactants are soluble in water and the
kinetics can be measured
(‘in-water’ reactions)

18. ‘In-water’ versus ‘On-water’ – What is the difference?
‘In-water’ ‘On-water’
 Water is the only reaction medium - no
organic co-solvents are used.
 Reactions use insoluble substrates and
suspensions are observed.
 Typically reactants are present in
concentrations >0.1M or above.
 Mechanistically very difficult to study
 Large rate accelerations observed can be
substrate and reaction specific.
 Water is generally the reaction medium -
organic co-solvents are used to solubilise
reactants although significantly effects rate
acceleration.
 Reactions use soluble substrates
 Typically reactants are present in
concentrations < 0.1M or below.
 Mechanistically as reactants are in solution
these are easier to study
 Large rate accelerations observed can be
substrate and reaction specific.
Distinguishing between in-water and on water is very difficult however concentration of reactants and
solubility can be used as a guide

19. Mechanistic Aspects
Model Reactions using water as a solvent
Increase in rate of reaction
Increase in stereoselectivity
These can both be rationalized through various mechanisms
Exact mechanism is unknown and needs further research

20. What happens when you move to more complex reaction systems?

21. Pericyclic Reactions – Cycloaddition reactions
Concerted reactions and solvent insensitive
Key reactions in multi-step synthesis
Highly regio- and stereospecific
Can generate up to 4 new sterocentres in one synthetic step
Large number of diene/1,3-dipoles and diene/dipolarophiles available
Wide structural diversity
Diels-Alder [4+2] Cycloaddition Reaction
Huisgen [3+2] Cycloaddition Reaction

22. Mechanistic Aspects – Influence on the stereoselectivity
Solvent Dienophile endo/exo
Cyclopentadiene (excess) Methyl vinyl ketone 3.85:1
Methyl acrylate 2.9:1
Ethanol Methyl vinyl ketone 8.5:1
Methyl acrylate 5.2:1
Water (0.15M) Methyl vinyl ketone 21.4:1
Methyl acrylate 9.3:1
Water (0.30M) Methyl vinyl ketone 18.6:1
Methyl acrylate 5.9:1
How can water be useful as a reaction solvent in organic synthesis?
1982 – Ronald Breslow
Enhancement of the endo/exo ratio and this is more pronounced in the cases of the vinyl ketones. Useful in organic
synthesis step where the endo isomer is required. Concentration of the reactants has some effect on endo:exo ratio
Breslow, R. Maitra, U, Rideout, D, Tet. Lett, 1984, 25(12), 1239

23. Pericyclic Reactions – Diels-Alder Cycloaddition reactions
Solvent Time (hr) Yield (%)
Toluene 168 Trace
Water 1 77
The cis isomer is formed however this isomerises to the trans isomer
Addition of THF, MeOH or 1,4-Dioxane causes a fall off in the rate of reaction
Reaction has excess diene present and the concentration of this is maintained at 1.0M
Increase in concentration of diene gives a fall off in rate of reaction
Reaction needs to be stirred vigorously
The use of the sodium salt adds an extra step to convert to the methyl ester
Grieco, P.A. et al, J. Org. Chem, 1983, 48, 3139

24. What about in more complex cases?
Pericyclic Reactions – Diels-Alder [4+2] cycloaddition reaction
Quassinoid natural product
R Solvent Diene conc t (h) Yield (%) endo:exo
Et Benzene 1M 288 52 0.85:1
Et H2O 1M 168 82 1.3:1
Na H2O 1M 8 83 2.0:1
Na H2O 2M 5 100 3.0:1
Grieco, P.A. et al, Tet Lett, 1983, 24, 1897
Concentration critical for these type of reactions
Significant increase in rate stereoselectivity in comparison to the reaction in organic solvents

25. Pericyclic Reactions – Diels-Alder [4+2] Intramolecular cycloaddition reaction
Solvent Temp (oC) Ratio
Toluene 90 75:25
Water 90 40:60
Reaction in water shows different selectivity
compared to toluene.
Solvent Reaction Time
Chloroform 10 days
Water 2 days
Williams, D.R. et al, Tetrahedron Lett, 1985, 26, 1362
Lovastatin
Witter, D.J., Vederas, J.C., J. Org. Chem, 1996, 61, 2613
Reaction accelerated on going from chloroform to water.
No change in stereoselectivity
Possible assembly for lovastatin core by Aspergillus terrus MF 4845

26. Pericyclic Reactions – Huisgen [3+2] cycloaddition reaction
Mechanistically identical to the Diels-Alder [4+2] cycloaddition reaction
Typically 1,3-Dipoles are highly unstable and need to be generated in-situ
There are some 1,3-dipoles that are stable at room temperature
This makes carrying out reactions using water as a reaction solvent more difficult and exploring the
mechanistic aspects

27. Pericyclic Reactions – Huisgen [3+2] cycloaddition reaction
Dipolarophile Solvent Yield endo/exo
Acetonitrile 96 3:1
Water 95 7:1
Acetonitrile 80 3:1
Water 95 16:1
Acetonitrile 65 8:1
Water 91 10:1
Unusually these 1,3-dipoles are highly stable and can be isolated and stored.
Stability is due to the electron withdrawing nature of the two cyano groups
Reaction with vinyl ketones is highly endo selective using water a a reaction solvent
(Same trend as observed with cyclopentadiene)
Azomethine ylides
Butler, R.N. et al, J. Chem. Soc. Perkin Trans 2, 2002, 1807

28. Pericyclic Reactions – Huisgen [3+2] cycloaddition reaction - Azides
Thermal cycloaddition
Cu and Ru Catalysed (CuAAC and RuAAC)
Strain Promoted cycloaddition (SPAAC) – Lecture 2
1,4 and 1,5 isomer formed in a 1:1
ratio. Need to be heated over 80oC
Cu - 1,4-isomer
Ru - 1,5-isomer
Strain of the double or triple bond of the
dipolarophiles gives rise to a rate increase for
the reaction. No metal or heat required for the
reaction.
Azide chemistry has undergone a renaissance with the advent of the CuAAC, RuAAC and SPAAC
(Sharpess, Fokin, Meldal and Bertozzi)

29. Azides Kinetics – Engberts
Pericyclic Reactions – Huisgen [3+2] cycloaddition reaction
Novartis – Rufinamide synthesis
Solvent k rel
Hexane 1
EtOH 1.6
H2O/NCP (99:1) 53.2
Solvent Temp (oC) Yield (%)
Neat 80 72
N-Heptane 80 46
EtOH 77 40
H2O 80 98
The cycloaddition reaction was found to be over 50 times faster in water than in hexane. 1% NCP was added to help
solubilise the azide
HCl is a side product of this reaction. In
organic solvents this polymerises the
chloroacrylonitrile. In water a two phase
system is observed where the HCl is dissolved
in the water layer
Engberts, J.B.F.N et al., Tet. Lett., 1995, 36, 5389
Portmann, R., WO98022423, 1998

30. Pericyclic Reactions – Huisgen [3+2] cycloaddition reaction
Synthesis of Biotin – De Clercq
Monomeric streptavidin and bound
biotin
(KD = 10-14 M)
The cycloaddition precursor was synthesized from L-cysteine.
When heated in water this undergoes cycloaddition reaction followed by elimination of N2
The seven membered ring opens up using water to form the N-benzylated biotin.
DeClercq, P. J., et al, Tetrahedron Lett., 1994, 35, 2615

31. Pericyclic Reactions – Claisen [3,3] sigmatropic rearrangement
Concerted reactions and solvent insensitive
Key reactions in multi-step synthesis
A number of different variants – N (azaClaisen), S (thiaClaisen)
Claisen
Aza-Claisen
Thia-Claisen
Typically require high temperatures which can lead to decomposition products
Substitution on the substrate can lower the activation energy for reaction.

32. In Nature
Chorismate to Prephenate- enzyme catalysed by
Chorismate mutase
kcat
= 106
kuncat
Chorismate mutase
Pericyclic Reactions – Claisen [3,3] sigmatropic rearrangement
Reaction is water at 75oC at pH 5 has a t1/2 of 10
mins
k(H2O)/k(MeOH) = 100
This Claisen rearrangement occurs at a much faster rate in
water in comparison to methanol.
This reaction occurs even without the presence of the
enzyme Chorismate mutase
Reaction with no enzyme
Andrews, P.R., et al, Biochemistry, 1973, 12, 3492

33. Pericyclic Reactions – Claisen [3,3] sigmatropic rearrangement
Brandes, E. et al., J. Org. Chem., 1989, 54, 515
Rearrangement of allyl vinyl ethers – effect of water on the rate of reaction.
R Solvent Rate
(k x 10-5s-1)
Yield
(%)
Na H2O 18 85
Na MeOH 0.79 -
Me C6H12 0.084 -
The Claisen rearrangement of the sodium carboxylate allyl vinyl ether is over 200 times faster that the corresponding
reaction in cyclohexane (methyl ester)
Rearrangement of naphthyl ethers – effect of water on the rate of reaction.
Solvent Yield (%)
Toluene 16
DMF 21
MeOH 56 (+14%)
neat 73
H2O 100
Narayan, S., Muldoon, J., Finn, M.G., et al. Angew. Chem. Int. Ed., 2005, 44, 3275
‘On-water’ reaction is faster than other polar solvents. Product
can be filtered off when water is used
No large rate accelerations observed as in the case of
quadricyclane and DEAD

34. Claisen Rearrangement
This Claisen rearrangement occurs at a much faster rate in water in comparison to toluene.
The corresponding a-anomeric reaction occurs in a similar time.
When the reaction was carried out in toluene only decomposition products were observed. The NaBH4 was added
to reduce the aldehyde.
Lubineau, A., et al, J. Chem. Soc. Perkin Trans 1, 1992, 1631
Lubineau, A. et al., Tetrahedron Lett., 1990, 31, 4147
Rearrangement of allyl vinyl ethers – effect of water solubilising groups

35. Claisen Rearrangement
Gambogin
Solvent T (oC) t (h) Conversion (%)
Ethanol 65 4 0
Methanol 65 4 0
MeOH/H2O (1:1) 65 4.0 100
MeOH/H2O (1:2) 100 0.5 100
H2O - - ppt of SM
Synthesis of Gambogin (Nicolaou)
First isolated in 1996 from
Gamboge resin from
Garcinia hamburgi
MIC (Hela) = 6.25 mg/mL
MIC (HLA) = 3.13 mg/mL
Organic co-solvent
present as reaction in
pure water causes
precipitation of the
starting material

36. Nucleophilic Ring Opening
Epoxides and aziridines are excellent synthetic intermediates
Readily converted to other functional groups such as diols, aminoalcohols and diamines
Can be done enantioselectively
Could also be described as a ‘Click’ reaction as they are highly efficient and give high yields
In Nature nucleophilic ring opening has been proposed as a key biosynthetic step in the formation of
some natural products
Epoxides
Aziridines

37. Nucleophilic Ring Opening
Cane, Celmer, Westley Proposed Mechanism
Monensin Brevetoxin
Jamison T.J. et al, Mar. Drugs, 2010, 8, 763

38. Nucleophilic Ring Opening – Epoxides (water as a nucleophile)
What happened when you heat an epoxide in water?
Monoepoxide
Bisepoxide
Qu, J et al, J Org Chem, 2008, 73, 2270
Could this be a competing pathway in epoxide ring opening with other nucleophiles in water?

39. Nucleophilic Ring Opening – Epoxides (Other nucleophiles)
Monoepoxide
No reaction occurred in either toluene or diethylether
Bonollo et al, Green Chem, 2006, 8, 960

40. Nucleophilic Ring Opening – Cascade Sequence
Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Angew Chem. Int. Ed.,
2001, 40, 2004
Cited 5609 times (Dec 2014)
Rostovtsev, V.V.; Green, L.G.; Fokin, V.V; Sharpless, K.B.
Angew Chem. Int. Ed., 2002, 41, 2596
Cited 4971 times (Dec 2014)

41. Epoxide Ring Opening – Biomimetic Approach
Conditions Ratio (endo: exo)
Cs2CO3, MeOH 1:2.7
AcOH, Toluene 1.6:1
Ethylene glycol 9:1
Methanol 8:1
Water > 10:1
Brevetoxin B Ph.D Harvard University
(Stuart Schreiber)
PostDoc Harvard University
(Eric Jacobsen)
Jamison T.J. et al, Science, 2007, 317, 1189

42. Epoxide Ring Opening
Triepoxide and Bisepoxide
Where next for this methodology?
Could this be used in the synthesis of brevetoxin or larger polyether natural products such as Maitotoxin?
Jamison T.J. et al, Science, 2007, 317, 1189

43. Lecture 1 - Overview
Large rate accelerations and increase in stereoselectivites observed using water as
a reaction solvent which are not found using organic solvents.
The reasons for these accelerations are not fully understood and need further
mechanistic study
Can be applied to more complex systems

44. Organic Synthesis in Water
Emerging Areas of Research
Graduate Lecture Series
Lecture 2
Dr Anthony Coyne
(anthony.g.coyne@gmail.com)

45. Outline
 Lecture 1 – Kinetics, Mechanism and Synthesis
• Background
• Mechanistic aspects
• Influence on rate of reaction
• Organic Synthesis
• Diels-Alder [4+2] cycloaddition reaction
• Huisgen [3+2] cycloaddition reaction
• Claisen rearrangement
• Epoxide ring opening
 Lecture 2 – Emerging areas of research
• Recap of Lecture 1
• Organic Synthesis:
Emerging metal catalysed reactions
• Olefin metathesis
• Cyclopropanation reactions
• Chemical Biology Applications
• CuAAC reactions
• SPAAC reactions
• Sonogashira coupling
• Suzuki coupling

46. Complexity of reactions using water as a solvent
Level of
complexity
Claisen Rearrangement
(1 reactant)
Lecture 1
Diels-Alder Reaction
(2 reactants)
Lecture 1
Olefin Metathesis (Cross Metathesis)
(2 reactants and catalyst)
Lecture 2
Suzuki Reaction
(2 reactants and catalyst, ligand, base)
Lecture 2

47. Diels-Alder Reaction – Cu catalysed
Solvent Additive k2 (M-1s-1)
Acetonitrile - 1.4 x 10-5
Water - 4.02 x 10-3
Acetonitrile Cu(NO3)2 0.472
Water Cu(NO3)2 1.11
Otto, S., Bertonicin F., Engberts, J.B.F.N., J. Am. Chem. Soc., 1996,118, 7702
Reactions using catalysis are more complex to understand when water is used as a reaction
solvent
Lecture 1
Uncatalysed Reaction
Increase in rate of reaction when water is used as
the solvent
Cu catalysed Reaction
No large rate increase is observed on
changing to water as a solvent.
Lewis acid catalysis predominates?

48. Olefin Metathesis
Ruthenium Catalysts (Grubbs and Hoyveda) Robert Grubbs
Ph.D Columbia University
(Ron Breslow)
PostDoc Stanford
University
(James Collman)
Amir Hoyveda
Ph.D Yale University
(Stuart Schreiber)
PostDoc Harvard
University
(David Evans)

49. Olefin Metathesis
Ruthenium Catalysts - Modified with water solubilizing groups
In many cases catalysts have been modified where one of the ligands was appended with a water solubilizing
group.
Where the catalyst is modified using a PEG chain the molecular weight can be up to 5000 g/mol
Catalysts are only used in simple reactions and major focus of research has been focused on the catalysts and
not on reactions
Still an emerging area of research
Insoluble in water

50. Olefin Metathesis
Ring Closing Metathesis – Water soluble catalysts
Grubbs R.H et al, J. Org. Chem. 1998, 63, 9904
Solvent Catalyst
(mol%)
Conversion
(%)
MeOH 5 90
H2O 5 60
H2O 10 90
No reaction was observed with the diallylmalonate due to methylidene instability
When substituted with a phenyl ring the reaction goes in high conversion
The catalysts were shown to be stable in water over a number of days

51. Olefin Metathesis
CM and ROMP – Water soluble catalysts
Ring Opening Metathesis Polymerisation (ROMP) Cross Metathesis (CM)
Hoyveda-Grubbs type catalyst
This catalyst is highly versatile and can be used sucessfully in CM, ROMP and RCM reactions
Attempts to synthesis the phosphine derived catalyst was not possible (Grubbs I)
Grubbs R.H et al, J. Am. Chem. Soc. 2006, 128, 3509
Catalyst synthesis is straightforward

52. Olefin Metathesis
Dimerisation of Vancomycin– Grubbs I Catalyst
Nicolaou, K.C., et al, Chem.. Eur. J., 2001, 7, 3824
Using a modified Vancomycin it was possible to
homodimerise this using olefin metathesis
Typically the yields for this reaction were 30-60% with
the only product formed is the homodimer
No other side reactions observed even though there is
a large number of different functional groups on the
vancomycin scaffold
Grubbs I catalyst was used without any
modification

53. Olefin Metathesis
CM in Biological Systems
A surface cysteine residue undergoes a dethiolation to give a dehydroalanine. This can undergo
conjugate addition reactions with thiols. This can also undergo reaction with allylmercaptan to give a
double bond which is an effective substrate for olefin metathesis
No modified catalyst used,
t-Butanol needed for solubility,
Cysteine needs to be on the surface of the protein
Uses natural amino acids
Davis, B.G.; Bernardes, G.J.L, et al, J. Am Chem Soc,. 2008, 130, 9642
Davis, B.G.; Bernardes, G.J.L, et al, J. Am Chem Soc., 2008, 130, 5052

54. Metal catalysed cyclopropanation
Key Reaction
Carbenes generally unstable in water but when stabailzed by complexation to a transition metal
this increases their lifetime
Diazo compounds can be either generated in situ, synthesised or in some cases be purchased
The formation of both trans and cis cyclopropanes are passible and if a chiral ligand is used then this
reaction can be carries out enantioselectively
Cyclopropane containing natural products

55. Metal catalysed cyclopropanation
Rhodium(II) catalysed cyclopropanation – Water soluble catalysts
[Rh(OAc)2]2
Catalyst Yield (%) dr (trans/cis)
[Rh(OAc)2]2 26 1.6:1
[Rh(O2C tBu)2]2 61 1.5:1
[Rh(O2C(CH2)6Me)2]2 72 1.5:1
Water soluble catalysts gave low yields and poor
diastereoselectivity
This is due to [Rh(OAc)2]2 having a preference for
the aqueous layer and not dissolving in the
‘organic’ layer
Water insoluble catalysts gave higher yields
than water soluble catalysts
There is a biphasic system (due to 2 eq of
styrene) where the water insoluble catalysts go
into the organic layer
No change in diastereoselectivity
Wurz, R. P., Charette, A. B., Org Lett., 2002, 4, 4531

56. Metal catalysed cyclopropanation
Rhodium(II) catalysed cyclopropanation – Water soluble catalysts
Run Conversion (%) Yield (%) dr (trans/cis)
1 100 90 10:1
4 90 88 9:1
When the catalyst is recycled for a 4th time there is a negligible drop
in yield and diastereoselectivity
Water soluble catalysts gave excellent yields and trans diastereoselectivity
The porphyrin catalyst was modified using four sugar functional group which aided water solubility.
The catalyst could be recycled
Loading of 1 mol% is high as the catalyst has a molecular weight of 1450 (C73H80N4O21Ru)
Che, C-M et al., J. Am. Chem. Soc, 2010, 132, 1886

57. Metal catalysed cyclopropanation
Rhodium(II) catalysed cyclopropanation – Water soluble catalysts
55%
Uses natural amino acid
Incorporation of an alkene possible onto
Ubiquitin as lysine can be tethered using a N-
hydroxysuccinimide ester
Subsequent cyclopropanation using a diazo
modified dansyl tag carried out in 55% yield
using the water soluble Ru-porphyrin catalyst
Che, C-M et al., J. Am. Chem. Soc, 2010, 132, 1886

58. Metal catalysed cyclopropanation
From synthetic porphyrin to natural porphyrin systems
Porphyrins are found in cytochrome P450s (CYPs) which catalyse a wide range of oxidations such as
hydroxylations, epoxidations and heteroatom oxidation.
Could Iron be used as a metal for the generation of the carbene intermediate?
Carriera and co-workers showed that simple substrates can undergo cyclopropanation using iron catalyst
Typically enzymes are highly specific for substrates and how can this be overcome?
1450 Da 106169 Da (P450BM3)
Carriera, E. M et al., Org Lett, 2012, 14, 2162

59. Metal catalysed cyclopropanation
Cytochrome P450’s as cyclopropanation catalysts (Frances Arnold) B.S: Mechanical and Aerospace
Engineering (Princeton)
PhD: Chemical Engineering (Berkeley)
Post Doc: Biophysical Chemistry
(Caltech)
cis
trans
92 P450BM3 variants screened with this reaction
Catalyst Yield (%) dr (trans/cis) ee cis ee trans
P450BM3 1 63:37 27 2
H2-5-F10 59 84:16 41 63
H2A10 33 40:60 95 78
By making mutations this can greatly enhance the reactivity and selectivity of these enzymes
Amount of enzyme used needs to be reduced from 0.2 mol%
Arnold, F.H. et al., Science., 2013, 339, 307

60. Metal catalysed cyclopropanation
Cytochrome P450’ BM3 as cyclopropanation catalysts (Frances Arnold)
BM3-Hstar (T268A-C400H-L437W-V78M-L181V)
Arnold, F.H. et al., CatSciTech, 2014, 339, 307
By making five mutations this can greatly enhance
the reactivity and selectivity of these enzymes.
A noticeable mutation was on the cysteine where
this was mutated to a a histidine.This gave improved
rates for in vivo reactions
Levomilnacipran

61. Metal catalysed reactions
It is possible to carry metal-catalysed reactions using water as a reaction solvent.
These can be applied to complex reaction systems
The solubility of the catalyst/ligand combination can play a major part in the outcome of the reaction.
Typically the rate acceleration and enhanced stereoselectivity observed with pericyclic
reaction is not observed in metal catalysed reactions.
Still an emerging area of research

62. Can organic reactions be carried out in biological systems?
Bioorthogonal chemistry

63. Bioorthogonal Reactions
What are bioorthogonal reactions?
The term bioorthogonal chemistry refers to a chemical reaction that can occur inside of living systems without
interfering with native biochemical processes
Carolyn Bertozzi
University of Califonia, Berkeley
Joseph Fox
University of Delaware
Scott Hilderbrandt
Harvard Medical School
Azide-Alkyne cycloadditon (CuAAC/SPAAC)
1,2,4,5-Tetrazine-Alkyne cycloaddition
Ralph Weissleder
Harvard Medical School

64. Bioorthogonal Reactions
Bioorthogonal reactions requirements
Water as a solvent
Ambient temperature
Fast reaction kinetics
Synthetically accessible functionalities
Non-toxic reagents
No cross reactivity
Stable reactants and products
Stable at physiological pH
Cell permeable reagents

65. Bioorthogonal Reactions
Solvent k rel
Hexane 1
EtOH 1.6
H2O/NCP (99:1) 53.2
Azide-Norbornene cycloaddition (Engberts)
Engberts, J.B.F.N et al., Tet. Lett., 1995, 36, 5389
Solvent k rel
Toluene 1
EtOH 2.0
H2O/tBuOH (0.95 MF) 60.9
1,2,4,5-Tetrazine-styrene cycloaddition (Engberts)
Engberts, J.B.F.N et al., J Org. Chem, 1996, 61, 2001
Key observations from kinetic studies (Lecture 1)
Cu Catalysed azide-alkyne cycloaddition
(CuAAC) Sharpless, Fokin and Meldal (2002)
Strain Promoted cycloaddition (SPAAC)
Bertozzi (2004)
Solubility is an issue

66. Bioorthogonal Reactions – SPAAC
Influence of strain on reactivity?
Significant development carried out on a number of strained cyclooctynes.
Different substitution patterns on the ring can increase the rate of reaction
However in many cases the rates of the reactions have been determined in mixed organic and aqueous solvents.
There is the question as to whether this gives an adequate indication of the rate in the biological system
Solubility is a major issue in the development of strained ring systems
Using a strained dienophile/dipolarophile can have a significant effect on the rate of reaction

67. Bioorthogonal Reactions – Azides versus Tetrazines
Not present in biological systems
Possesses orthogonal reactivity to most biological
groups
Is a small functional group (only three atoms)
Does not have appreciable reactivity with water
Triazole products are stable
Why use azide and tetrazines in bioorthogonal reactions?
Not present in biological systems
Possesses orthogonal reactivity to most biological
groups
Very fast kinetics for inverse electron demand
cycloadditions (IED)
No metals needed
Bigger functional group and needs to be stabilized
with an aryl ring

68. Bioorthogonal Reactions –Azides versus Tetrazines
Selectivity of Bioorthogonal Reactions – Inverse electron demand cycloaddition reactions
Houk, K.N., et al., J. Am Chem. Soc., 2012, 134, 17904
k2 = 0.0064 M-1s-1 k2 = 210 M-1s-1
k2 = 2.10 M-1s-1
k2 Azide
k2 Tetrazine
= 32000
1,2,4,5-tetrazine does not
react with the cyclooctyne
trans-cyclooctene reacts over 32000 faster with the 1,2,4,5-tetrazine over the azide. However in the case of
cyclooctyne no reaction was observed with 1,2,4,5-tetrazine
The difference in reactivity between azides and tetrazines with various diene/dipolarophiles means that in
biological environments more than one tag/probe can introduced using a specific bioorthogonal reaction

69. Bioorthogonal Reactions –Azides versus Tetrazines
Reactivities of Strain Promoted Dienophile/Dipolarophiles
Dienophiles reported for their reactivity towards
1,2,4,5-Tetrazines
Dipolarophiles reported for their reactivity towards azides.
(Rates are measured in MeOH or MeCN)
There has been a wide range of different dipolarophile and dienophiles discovered for the strain-promoted
bioorthogoanal reactions.
Very much an active area of research however need further reliable rate data in water as this would reflect the
rates in biological systems
King, M, Wagner, A, Bioconjugate Chem., 2014, 25, 825

70. Bioorthogonal Reactions – SPAAC
Strained dipolarophiles - PEGylation of CalB
Van Delft, F. L., et al., Chem Comm., 2010, 46 97
CalB expressed with azidohomoalanine residues (5)
with one surface exposed residue
Cyclooctyne (DIBAC) synthesized in 10 steps
Corresponding tagging carried out under Cu(I)
conditions took 1-3 days and with no full conversion
observed (CuAAC).
Under SPAAC conditions the conversion was
complete after 3 hours
Solvent k (M-1 s-1)
CD3OD 0.31
D2O 0.36

71. Bioorthogonal Reactions – Azides
Strained dipolarophiles - In vivo imaging (Glycan Trafficking)
Bertozzi, C.R., et al., Proc. Nat Acad. Sci., 2007, 104, 16793
Visualization of dynamic processes in
living cells is possible
The visualization of the azidosugar
metabolism is possible using the
fluorescent tags
Both the azide sugar and strained alkyne
do not fluoresce but upon SPAAC reaction
the localization of these in a cell can be
visualized

72. Bioorthogonal Reactions – Azides
Strained dipolarophiles - In vivo imaging (Zebrafish)
Bertozzi, C.R., et al., Science., 2008, 320, 664
Scheme From – D. A Nagib, MacMillan Group, Princeton
Metabolic labeling was observed, similar to that of mammalian cells
Interestingly there was no observed toxicity resulting from use with Ac4GalNAz or DIFO reagents.
Organic reactions using water as a solvent in a living system

73. Bioorthogonal Reactions – 1,2,4,5-Tetrazines
Tetrazines in bioorthogonal reactions - Tetradoxin
12014 Da
12222 Da
100% Conversion
(5 mins)
Fox, J.F., et al., J. Am. Chem. Soc, 2008, 130, 13518
Tetradoxin is labelled with a maleimide derived trans-cyclooctene though coupling onto a cysteine residue on the surface
of the protein
Subsequent reaction with a 1,2,4,5-tetrazine led to complete conversion in 5 mins.
Reaction can be carried out in organic sovlents, water, cell lysate with identical results
Stability of the trans-cyclooctene over prolonged time is an issue so the reaction has to be quick

74. Other Metal catalysed reactions
Palladium Catalysed Reactions – Sonogashira (GFP)
No ligand
Sonogashira coupling carried out to label alkyne encoded GFP
with a rhodamine conjugated phenyl iodide.
Pd(NO3)2 did not have any effect on the cells over 1 hour
Using a ligands gave poorer yields
This is an interesting reaction as there are only a handful of
Sonogashira reactions carried out using water as a reaction
solvent
95%
Chen et al., J. Am. Chem. Soc, 2013, 135, 7330

75. Other Metal catalysed reactions
Palladium Catalysed Reactions – Suzuki Coupling (OmpC)
OmpC contained a genetically modified p-iodophenylalanine
OmpC is a membrane protein on the surface of E.coli
This modified OmpC was reacted with a boronic acid-fluorescein which was
carried out in 1hr using the palladium catalyst.
Suzuki reactions generally have water present as a co-solvent.
Davis, B.G. et al., J. Am. Chem. Soc, 2012, 134, 800

76. Bioorthogonal Reactions – Where next?
Within 8 years from the reporting of the first ‘click’ reaction there has been the application of this
methodology in living systems
The number of reactions in the bioorthogonal toolbox is increasing however there is the need for new
reaction types
Use of natural amino acids is key
A key aspect of this methodology is the discovery of new reactions that can be carried out using
water as a solvent
2002
Sharpless
Fokin
Meldal
2007 2008 2010

77. Conclusions
Organic reactions are possible using water as a solvent.
In some cases there are large rate accelerations observed however these can
be reaction and substrate specific.
Mechanistically the rationale for these accelerations is not fully understood
In the last decade a major application of organic reactions using water as a
solvent has been to bioorthogonal chemistry

78. Not discussed in these lectures
(NDI)3
(NDI)4
AO10 Dynamic Covalent Chemistry: A Tool For Synthesis, Molecular Recognition And Understanding Systems Behavior
(2L): Professor Jeremy Sanders (January 2015)
Dynamic Covalent Chemistry
Micellar Additives
Bruce Lipshutz
University of Califonia,
Santa Barbara

79. Reactions explored using water as a solvent

80. General References
D. Burtscher, K. Grela, Angew. Chem. Int. Ed., 2009, 48,
442 (Olefin Metathesis)
S. Otto, J.B.F.N. Engberts, Org Biomol. Chem., 2003, 1(16),
2809
C-J. Li, Chem. Rev., 2005, 105, 3095–3166 (General)
A. Chandra, V.V. Fokin, Chem. Rev., 2009, 109, 725
(General)
D. Dallinger, C. Oliver Kappe, Chem Rev, 2007, 107, 2563
(Microwave synthesis in water)
U. M. Lindström, Chem. Rev, 2002, 102, 2751
(Stereoselective reactions)
M. Raj, V. K. Singh, Chem. Comm., 2009, 6687
(Organocatalysis)
J. Paradowska, M. Stodulski, J. Mlynarski, Angew. Chem.
Int. Ed., 2009, 48, 4288 (Organocatalysis)
H. Hailes, Org. Proc. Res. Dev., 2007, 11, 114 (General)
‘Organic Reactions in water’ U. Marcus Lindströhm Ed.,
Blackwell Publishing, 2007
Science of Synthesis: Water in Organic Synthesis,
Shu Kobayshi Ed, Thieme, 2012
Organic Synthesis in Water, Paul A. Grieco Ed.,
Springer, 1999.
Aqueous Phase Organometallic Catalysis – Concepts
and Applications Boy Cornlis and Wolfgang Herrmann
Ed, Wiley, 1998 G. Molteni, Heterocycles, 2006, 68(10), 2177 (Huisgen
cycloaddition reactions)
R.N. Butler, A.G. Coyne, Chem. Rev., 2010, 110, 6302
(General – in-water/on-water)
Books
B. H. Lipshutz, S. Ghorai, Green Chem, 2014, 16, 3660
(General and Stereoselective reactions)

81. Water is the solvent used by nature for biological chemistry. Considering the enormous variety of biological
pathways and the complicated molecular structures and materials, including precise arrangements of multitudes of
asymmetric centers, which are found in biological systems, It is remarkable that up until recently organic synthesis has
mainly shunned water. In recent years there has been a resurgence in the use of water as a solvent in a wide variety of
reaction types.
The aim of these two lectures will be to explore these reactions where water has been applied as a solvent. The
initial focus will be the investigation of the kinetics of these reactions where interesting rate effects have been observed.
The focus will then move onto a range of reaction types such as the Diels-Alder [4+2] and Huisgen [3+2] cycloaddition
reactions, Claisen rearrangement, epoxide ring openings, olefin metathesis and cyclopropanation reactions. The final
part of this lecture series will focus on the development and application of biorthogonal reactions where the use of water
as the solvent is crucial.