Continuous multiple liquid-liquid separation: Diazotization of amino acids in flow

Article abstract of DOI:10.1021/ol301930h

A second-generation laboratory-scale, modular liquid-liquid separation device based on computer-controlled high-pressure pumps and a high-resolution digital camera has been invented. The diazotization of amino acids to produce valuable chiral hydroxyacids is demonstrated in flow for the first time. The use of a triple-separator system in conjuction with the developed diazotization process allows the safe and efficient production and automated isolation of multigram quantities of valuable chiral hydroxyacids.

Full text of DOI:10.1021/ol301930h

ORGANIC
LETTERS
2012
Vol. 14, No. 16
4246–4249
Continuous Multiple LiquidꢀLiquid
Separation: Diazotization of Amino
Acids in Flow
Dennis X. Hu, Matthew O’Brien, and Steven V. Ley*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
CB21EW, United Kingdon
svl1000@cam.ac.uk
Received July 16, 2012
ABSTRACT
A second-generation laboratory-scale, modular liquidꢀliquid separation device based on computer-controlled high-pressure pumps and a high-
resolution digital camera has been invented. The diazotization of amino acids to produce valuable chiral hydroxyacids is demonstrated in flow for
the first time. The use of a triple-separator system in conjuction with the developed diazotization process allows the safe and efficient production
and automated isolation of multigram quantities of valuable chiral hydroxyacids.
In the past decade, continuous-flow chemistry has
emerged as a powerful complement to batch chemistry
on the laboratory scale due to relative advantages some of
which include: increased safety, more accurate tempera-
ture control, ease of reaction scale-up, and amenability
to automation.1 The rapid emergence of new enabling
technologies such as tube-in-tube gas reactors,2 photoche-
mical reactors,3 copper tube reactors,4 flow IR cells,5
agitating cell and ultrasound reactors,6 and cryo-flow
reactors,7 have rapidly and dramatically expanded the
potential scope of flow chemistry’s applications.
(1) For recent reviews, see: (a) Hartman, R. L.; McMullen, J. P.;
Jensen, K. F. Angew. Chem., Int. Ed. 2011, 50, 7502. (b) Valera, F. E.;
Quaranta, M.; Moran, A.; Blacker, J.; Armstrong, A.; Cabral, J. T.;
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Ceylan, S.; Kirschning, A. Adv. Synth. Catal. 2012, 354, 17.
(d) Baumann, M.; Baxendale, I. R.; Ley, S. V. Mol. Diversity 2011, 15,
613. (e) Han, X.; Poliakoff, M. Chem. Soc. Rev. 2012, 41, 1428. (f) Mak,
X. Y.; Laurino, P.; Seeberger, P. H. Beilstein J. Org. Chem. 2009, 5, No.
19. (g) Yoshida, J.; Kim, H.; Nagaki, A. ChemSusChem 2011, 4, 331.
(h) Webb, D.; Jamison, T. F. Chem. Sci. 2010, 1, 675. (i) Hartman, R. L.;
Jensen, K. F. Lab Chip 2009, 9, 2495. (j) Fukuyama, T.; Rahman, T.;
Sato, M.; Ryu, I. Synlett 2008, 151.
(2) CO2: (a) Polyzos, A.; O’Brien, M.; Petersen, T. P.; Baxendale,
I. R.; Ley, S. V. Angew. Chem., Int. Ed. 2011, 50, 1190. H2: (b) O’Brien,
M.; Taylor, N.; Polyzos, A.; Baxendale, I. R.; Ley, S. V. Chem. Sci.
2011, 2, 1250. CO: (c) Koos, P.; Gross, U.; Polyzos, A.; O’Brien, M.;
Baxendale, I. R.; Ley, S. V. Org. Biomol. Chem. 2011, 9, 6903.
(d) Mercadante, M. A.; Leadbeater, N. E. Org. Biomol. Chem. 2011, 9,
6575. Syngas: (e) Kasinathan, S.; Bourne, S. L.; Tolstoy, P.; Koos, P.;
O’Brien, M.; Bates, R. W.; Baxendale, I. R.; Ley, S. V. Synlett 2011, 2648–
2651. C2H4: (f) Bourne, S. L.; Koos, P.; O’Brien, M.; Martin, B.;
Schenkel, B.; Baxendale, I. R.; Ley, S. V. Synlett 2011, 2643. O2:
(g) Petersen, T. P.; Polyzos, A.; O’Brien, M.; Ulven, T.; Baxendale,
I. R.; Ley, S. V. ChemSusChem 2012, 5, 274.
(3) (a) Hook, B. D. A.; Dohle, W.; Hirst, P. R.; Pickworth, M.; Berry,
M. B.; Booker-Milburn, K. I. J. Org. Chem. 2005, 70, 7558.
(b) Vasudevan, A.; Villamil, C.; Trumbull, J.; Olson, J.; Sutherland,
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Seeberger, P. H. Org. Lett. 2011, 13, 5008. (d) Levesque, F.; Seeberger,
P. H. Angew. Chem., Int. Ed. 2012, 51, 1706. (e) Bou-Hamdan, F. R.;
Seeberger, P. H. Chem. Sci. 2012, 3, 1612. (f) Tucker, J. W.; Zhang, Y.;
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J. G.; Baxendale, I. R.; Ley, S. V. Chem. Sci. 2011, 2, 765. (b) Carter,
C. F.; Lange, H.; Ley, S. V.; Baxendale, I. R.; Wittkamp, B.; Goode,
J. G.; Gaunt, N. L. Org. Process Res. Dev. 2010, 14, 393. (c) Carter, C. F.;
Baxendale, I. R.; O’Brien, M.; Pavey, J. B. J.; Ley, S. V. Org. Biomol.
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r
10.1021/ol301930h
Published on Web 08/06/2012
2012 American Chemical Society

One of the chief focuses of our research group has been
the development of enabling technologies for flow chem-
istry which reduce manual downstream processing tasks
such as isolation and purification. For example, we and
others have advocated the use of polymer-supported re-
agents and scavengers to capture impurities in-line,8 allow-
ing either the direct collection of pure material from flow
reactors or direct use of the reaction stream in multistep
sequences.9 In certain “simple” workup processes such as
extraction and acid or base washes, however, liquid-phase
workup procedures would be preferable to solid-phase
procedures if they could be readily conducted in flow.
We and other groups have therefore sought to develop
tools for automated in-line aqueous workups and phase
separations for flow synthesis as a complementary down-
stream processing technique to solid-phase techniques.10
Recently, we reported the invention of a syringe pump
and camera-based liquidꢀliquid phase separation tool
and its application to reactions requiring a single phase
separation.11 Here, we disclose the development of a
modular second-generation high-pressure pump system
which allows solvent-independent, multiple consecutive phase
separations and its application to the diazotization of amino
acids in flow.
Scheme 1. Detailed Schematic of a Single Extractor
(Configuration A: Heavy Phase Further Processed)
The basic schematicfor a singleextractor isshownabove
(Scheme 1). The reaction stream from a flow reactor enters
the first extractor which mixes the reaction stream with
an extraction solvent. The biphasic stream then enters the
top of a small (3 mm diameter) glass separating column
Scheme 2. Detailed Schematic of a Single Extractor
(Configuration B: Lighter Phase Further Processed)
(6) (a) Browne, D. L.; Deadman, B. J.; Ashe, R.; Baxendale, I. R.;
Ley, S. V. Org. Process Res. Dev. 2011, 15, 693. (b) Sedelmeier, J.; Ley,
S. V.; Baxendale, I. R.; Baumann, M. Org. Lett. 2010, 12, 3618. (c) Noel,
T.; Naber, J. R.; Hartman, R. L.; McMullen, J. P.; Jensen, K. F.;
Buchwald, S. l. Chem. Sci. 2011, 2, 287. (d) Cintas, P.; Mantegna, S.;
Gaudino, E. C.; Cravotto, G. Ultrason. Sonochem. 2010, 17, 985.
(7) Browne, D. L.; Baumann, M.; Harji, B. H.; Baxendale, I. R.; Ley,
S. V. Org. Lett. 2011, 13, 3312.
(8) (a) Baxendale, I. R.; Ley, S. V.; Mansfield, A. C.; Smith, C. D.
Angew. Chem., Int. Ed. 2009, 48, 4017. (b) Lange, H.; Capener, M. J.;
Jones, A. X.; Smith, C. J.; Nikbin, N.; Baxendale, I. R.; Ley, S. V. Synlett
2011, 869. (c) Baumann, M.; Baxendale, I. R.; Kirschning, A.; Ley, S. V.
Heterocycles 2010, 82, 1297. (d) Baumann, M.; Baxendale, I. R.; Martin,
L. J.;Ley, S. V. Tetrahedron 2009, 65, 6611. (e) Mennecke, K.; Kirschning,
A. Beilstein J. Org. Chem. 2009, 5, No. 21. (f) Smith, C. D.; Baxendale,
I. R.; Tranmer, G. K.; Baumann, M.; Smith, S. C.; Lewthwaite, R.; Ley,
S. V. Org. Biomol. Chem. 2007, 5, 1562. (g) Kirschning, A.; Solodenko,
W.; Mennecke, K. Chem.;Eur. J. 2006, 12, 5972. (h) Hodge, P. Ind. Eng.
Chem. Res. 2005, 44, 8542. (i) Ley, S. V.; Baxendale, I. R.; Bream, R. N.;
Jackson, P. S.; Leach, A. G.; Longbottom, D. A.; Nesi, M.; Scott, J. S.;
Storer, R. I.; Taylor, S. J. J. Chem. Soc., Perkin Trans. 1 2000,
3815–4195.
(9) For natural product syntheses employing solid-supported re-
agents in flow, see: (a) Baxendale, I. R.; Deeley, J.; Griffiths-Jones,
C. M.; Ley, S. V.; Saaby, S.; Tranmer, G. K. Chem. Commun. 2006, 2566.
(b) Baxendale, I. R.; Griffiths-Jones, C. M.; Ley, S. V.; Tranmer, G. K.
Synlett 2006, 3, 427. (c) Baumann, M.; Baxendale, I. R.; Brasholz, M.;
Hayward, J. J.; Ley, S. V.; Nikbin, N. Synlett 2011, 1375.
through a stainless steel tube within a T-piece connector.
Inside the phase-separating column is a small, colored
polymer float of an intermediate density between the
organic and aqueous phases such that it remains at the
interface. The bottom of the phase-separating column is
connected to a computer-controlled high-pressure pump
with a back pressure regulator (75ꢀ100 psi). When the
flow rate of the high-pressure pump is lower than that of
the mixed incoming streams, the upper layer overflows out
of the top of the separating column. The computer uses a
digital high-resolution camera to monitor the colored bead
in the column and provides damped feedback to the high-
pressure pump according to the level of the interface
(10) (a) Hall, J. F. B.; Han, X.; Poliakoff, M.; Bourne, R. A.; George,
ꢀ
M. W. Chem. Commun. 2012, 48, 3073. (b) Sprecher, H.; Payan,
M. N. P.; Weber, M.; Yilmaz, G.; Wille, G. J. Flow Chem. 2012, 2, 20.
(c) Castell, O. K.; Allender, C. J.; Barrow, D. A. Lab Chip 2009, 9, 388.
(d) Hornung, C. H.; Mackley, M. R.; Baxendale, I. R.; Ley, S. V. Org.
Process Res. Dev. 2007, 11, 399. (e) Sahoo, H. R.; Kralji, J. G.; Jensen,
K. F. Angew. Chem., Int. Ed. 2007, 46, 5704. (f) Kralji, J. G.; Sahoo,
H. R.; Jensen, K. F. Lab Chip 2007, 7, 256. (g) Kolehmainen, E.;
Turunen, I. Chem. Eng. Process. 2007, 46, 834. (h) Atallah, R. H.;
Ruzicka, J.; Christian, G. D. Anal. Chem. 1987, 59, 2909.
(11) O’Brien, M.; Koos, P.; Browne, D. L.; Ley, S. V. Org. Biomol.
Chem. 2012, in press. DOI: 10.1039/C2OB25912E.
Org. Lett., Vol. 14, No. 16, 2012
4247

Scheme 3. Flow Diazotization with Multiple Extraction Setupa
a A solution of amino acid in aq sulfuric acid is mixed with an aqueous solution of sodium nitrite in a Uniqsis flow reactor and reacted in a heated coil.
The exit stream is treated by three ethyl acetate extractors in series, and the aqueous exit stream is allowed to collect into a flask containing urea to quench
excess sodium nitrite.
relative to the height of the column selected by the user.
The computer increases the exit flow rate when the inter-
face is too high and reduces the flow rate whenthe interface
is too low (see Supporting Information for source code,
photos, and video). Due to the damped feedback and a
rapid computer communication rate, the level of the inter-
face is essentially indefinitely stable.
WhiletheconfigurationdescribedandshowninScheme1
specifically takes the more dense phase on for further
processing (e.g., DCM in a DCM/aqueous mixture or
H2O in a H2O/Et2O mixture), it is also possible to take
the lighter phase on for further processing using the
configuration shown in Scheme 2.
Since each separator has an input and two controlled
outputs, several can be placed in series for multiple separa-
tions by connecting the output of one to the input of another.
The control program has been written to allow a single
computer and camera to control three or more separators in
series simultaneously based on the user’s one-time definition
of the columns of interest and column configuration. The
final output can be connected to another flow reactor for
further reactions, as the final flow rate remains controlled
and accurate due to the final high-pressure pump.
We decided to test the multiple separator system in
conjuction with the flow synthesis of chiral R-hydroxyacids
(Scheme 3), which are highly useful chiral synthons and
valuable precursors to numerous natural products and
pharmaceutical agents. Importantly, the hydrophobic hy-
droxyacid, hydroxyvaline (1, Table 1), has an experimentally
determined partition coefficient of ∼0.74 between ethyl
acetate and water, necessitating multiple extractions. In
batch, the classic diazotization of amino acids is typically
conducted at 0 °C due to the release of gas and allowed to stir
for several hours to allow the reaction to go to completion.12
The use of flow apparatus allowed us to safely conduct
the diazotization reactions at 60 °C, allowing the reactions
to complete between 10 and 60 min in the reactor coil before
direct extraction. Impressively, the separators’ control pro-
gram was unaffected by the interfacial turbulence caused
by the vigorous evolution of nitrogen gas through the
first extractor column, surviving several 24þ h runs of
the diazotization reaction without manual assistance.
The results of flow diazotization, displacement, and
multiple extraction of select amino acids on scales >1 g
are summarized in Table 1. The flow preparation and
extraction was highly efficient and remained stereoselec-
tiveatthe elevatedtemperature used inmostinstances. The
diazotizations were typically conducted at a concentration
of 0.25 M postreagent-mixing (Method A, see Supporting
Information); higher dilution and higher flow rates were
necessary to keep more hydrophobic substrates and their
products solubilized (Method B). In some cases, acetone
was introduced to prevent crystallization of the less
polar hydroxyacid products within the coil (Method C).
(12) For representative examples, see: (a) Deechongkit, S.; You, S.;
Kelly, J. W. Org. Lett. 2004, 6, 497–500. (b) Koppenhoefer, B.; Schurig,
V. Org. Synth. 1988, 66, 151. (c) Fu, S. J.; Birnbaum, S. A. I.; Greenstein,
J. P.; Fu, J.; Birnbaum, M. J. Am. Chem. Soc. 1954, 76, 6054. (d) Fischer,
E. Berichte 1906, 489.
4248
Org. Lett., Vol. 14, No. 16, 2012

The reaction could be conducted continuously on 20þ g
scales over 24 hþ periods without manual intervention or
detriment to yield. The enantiomer of hydroxyvaline de-
rived from D-valine generated by this process was used as the
starting material for our total synthesis of (ꢀ)-enniatin B.13
The poor yield for alanine (8, Table 1) is explained by the
extreme product water solubility The degradation in the
enantiomeric ratio of the hydroxyacid derived from serine
(7) appears to be due to the inductive effect of the β-oxygen
atom, which likely impedes SN2 substitution of the inter-
mediate R-lactone, increasing the relative rate of ring-
opening by acyl substitution. In all cases, the combination
of the flow reactor and the multiple extraction system
made each reaction sequence exceedingly simple to exe-
cute: a solution of amino acid in aqueous sulfuric acid and
a stock solution of aqueous sodium nitrite were connected
to a flow reactor, the pumps were started, and a final
solution of pure R-hydroxyacid in ethyl acetate was ob-
tained, dried, and concentrated. The aqueous phase, which
was automatically extracted and quenched in line, was
simply discarded. It should be noted that conducting the
same 20 g scale reaction in batch would require much more
careful thermal control and the manual manipulation of
over 3 L of solvent in a very large separating funnel.
In summary, a device capable of multiple liquidꢀliquid
extractions/phase separations has been developed and
applied to the first examples of amino acid diazotiza-
tion in flow on multigram scales. The device is easy to
assemble manually, and instructions along with the source
code for the machine are provided. Applications of the
device in other chemical reaction sequences are currently
being investigated in our laboratory and will be dis-
closed in due course. We believe the use of this device will
greatly accelerate synthetic preparations in researchlabora-
tories and enable more complex multistep sequences in the
future.
Table 1. Summary of the Results of Various Diazotization
Reactions in Flow
Acknowledgment. We thank the following for financial
support of this work: The BP Endowment (to S.V.L.),
the Winston Churchill Foundation of the United States
(to D.X.H.), and the EPSRC (M.O.B.). We also thank
Dr. Duncan L. Browne of the University of Cambridge
Department of Chemistry for helpful discussions on the
manuscript and Dr. Ulrike Gross (Boelringer-Ingelheim)
a Determined by chiral HPLC of the corresponding benzyl ester by
comparison with a racemic standard. b Determined by NMR. c Deter-
mined by NMR after derivitization of the corresponding benzyl ester
to both Mosher ester diastereomers. d Determined by NMR after
derivitization of the corresponding methyl ester to both Mosher
ester diastereomers. e From starting material of >95:5 er checked by
Mosher amide analysis after derivization to the corresponding methyl
ester (see Supporting Information).
€
and Max Bielitza (Fz. Juelich) for preliminary studies on
the diazotization reaction.
Supporting Information Available. Detailed machine
design with photos, an explanatory video showing the
full setup in operation, Python code with explanatory
comments, experimental details, compound characteriza-
tion, and supporting spectra are provided as Supporting
Information. This material is available free of charge via
the Internet at http://pubs.acs.org.
Notably, due to the short retention time of the reactions
and the rapid extraction of product from the acidic and
oxidizing aqueous layer, benzyl ether and ester protecting
groups largely survived the reaction conditions, with de-
protected byproducts washing out in the aqueous stream.
(13) Hu, D. X.; Bielitza, M.; Koos, P.; Ley, S. V. Tetrahedron Lett.
2012, 53, 4077.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 16, 2012
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