Design of an in vitro biocatalytic cascade for the manufacture of islatravir

Article abstract of DOI:10.1126/science.aay8484

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Full text of DOI:10.1126/science.aay8484

RESEARCH
the phosphate to the 5 position. The resulting
sugar 5-phosphate becomes a substrate for de-
oxyribose 5-phosphate aldolase (DERA), which
performs a retro-aldol cleavage into glycer-
aldehyde 3-phosphate and acetaldehyde. This
sequence can rapidly assemble nucleosides
from simple starting materials when run in
reverse (17). Using the salvage pathway to
synthesize islatravir requires all three enzymes
to act on non-natural substrates bearing a fully
substituted carbon at C-4 of the 2-deoxyribose
ring. The full sequence of enzymes has not
been used to both construct a non-natural
sugar and attach a nucleobase. Nonetheless,
we were encouraged by previous applications
of PNP and PPM to produce non-natural nu-
cleosides (17, 19) as well as the ability of DERA
to make non-natural sugars (20).
The reversibility of the reactions in the sal-
vage pathway allowed us to rapidly explore the
sequence in the retrosynthetic direction (19),
starting from islatravir. We began by evaluat-
ing a panel of purine phosphorylases for their
ability to cleave islatravir into the nucleobase
(2) and sugar 1-phosphate (3). The ethynyl sub-
stituent was accepted by several homologs,
with the native Escherichia coli PNP showing
the highest activity. Application of the E. coli
enzyme at high loading generated the other-
wise inaccessible 1-phosphate intermediate (3)
to use in testing phosphopentomutases. The
native E. coli PPM displayed the highest acti-
vity for converting this unnatural substrate to
the thermodynamically favored 5-phosphate
(4). With compelling proof of concept for the
two desired reactions in place, we then focused
on improving the activity of both enzymes by
means of directed evolution.
We used a homology model based on the
Bacillus cereus PPM crystal structure to gen-
erate a library of variants with single muta-
tions near the active site, which were screened
for improved activity in the reverse reaction.
In the best performing variant, the manganese-
binding D172 was replaced by a noncoordinating
alanine (fig. S1D). Recombination of beneficial
mutations from the first round—including a
change in the catalytic, phosphorous-transferring
residue T97S—resulted in a quintuple mutant
that exhibited ≥70-fold improvement over the
wild-type enzyme (Table 1 and figs. S22 to
S24). (Single-letter abbreviations for the amino
acid residues are as follows: A, Ala; C, Cys; D,
Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys;
L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg;
S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. In the
mutants, other amino acids were substituted
at certain locations; for example, T97S indicates
that threonine at position 97 was replaced by
serine.)
BIOCATALYSIS
Design of an in vitro biocatalytic cascade for the
manufacture of islatravir
Mark A. Huffman1*, Anna Fryszkowska1*, Oscar Alvizo2, Margie Borra-Garske2, Kevin R. Campos1,
Keith A. Canada1, Paul N. Devine1, Da Duan2, Jacob H. Forstater1, Shane T. Grosser1, Holst M. Halsey1,
Gregory J. Hughes1, Junyong Jo1, Leo A. Joyce1†, Joshua N. Kolev1, Jack Liang2, Kevin M. Maloney1,
Benjamin F. Mann1, Nicholas M. Marshall1‡, Mark McLaughlin1, Jeffrey C. Moore1, Grant S. Murphy1,
Christopher C. Nawrat1, Jovana Nazor2, Scott Novick2, Niki R. Patel1, Agustina Rodriguez-Granillo3§,
Sandra A. Robaire1, Edward C. Sherer3, Matthew D. Truppo1¶, Aaron M. Whittaker1,
Deeptak Verma3, Li Xiao3, Yingju Xu1, Hao Yang1
Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels
of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic
reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the
waste generated by purification of intermediates. They also allow reactions to be linked together to
overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates.
We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir.
Five enzymes were engineered through directed evolution to act on non-natural substrates. These
were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a
three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of
the previously reported routes.
he structural complexity of new drug
molecules and the growing desire to
develop green and efficient synthetic
processes demand innovation and excel-
lence in organic chemistry (1). Enzyme
engineering of multiple enzymes to act on
unnatural substrates at industrially relevant
concentrations. Ongoing advances in directed
evolution (6, 7), combined with an increas-
ing abundance of genomic information, have
now brought this goal within reach, enabling
us to construct a nine-enzyme cascade to manu-
facture the investigational HIV treatment
islatravir.
The nucleoside analog islatravir (MK-8591,
EFdA, 1) is an HIV reverse transcriptase trans-
location inhibitor with a previously undeveloped
mechanism of action (8, 9). Its extraordinary
potency and long duration of action promise
utility in reduced-frequency dosing regimens
for HIV treatment and preexposure prophy-
laxis (10). Several synthetic routes to islatravir
have been published, each requiring between
12 and 18 steps (11–15). Inefficiencies in the
previous syntheses arose from the need for
multiple protecting-group manipulations as
well as the difficulty of controlling anomeric
stereochemistry in 2′-deoxyribonucleosides.
Enzymatic reactions often eliminate the need
for protecting groups while at the same time
providing precise control over stereoselectivity.
We therefore envisaged that biocatalysis might
effectively address the synthetic challenges of
islatravir.
T
catalysis incorporated into pharmaceutical
manufacturing represents one such innova-
tion, providing benefits that include unparal-
leled selectivity, increased atom economy, and
improved safety (2, 3). But a truly transform-
ative potential lies in combining two or more
enzymatic steps into biocatalytic cascade se-
quences (4, 5). Biocatalytic cascades save re-
sources by avoiding isolation of intermediates.
They also allow thermodynamically unfavora-
ble steps to be coupled to more favorable re-
actions and can avoid enzyme inhibition by
consuming inhibitory intermediates as they
are formed. Cascades are enabled by the ex-
ceptional chemoselectivity of enzymes and
their compatibility with a common set of mild
aqueous reaction conditions. Designing chem-
ical syntheses around the use of multienzyme
cascades could revolutionize the manufacture
of drugs. Putting this vision into practice re-
quires a capacity for rapid identification and
1Process Research and Development, Merck & Co., Inc.,
Rahway, NJ 07065, USA. 2Codexis, Inc., 200 Penobscot
Drive, Redwood City, CA 94063, USA. 3Computational and
Structural Chemistry, Discovery Chemistry, Merck & Co.,
Inc., Kenilworth, NJ 07033, USA.
*Corresponding author. Email: mark_huffman@merck.com (M.A.H.);
anna.fryszkowska@merck.com (A.F.) †Present address: Arrowhead
Pharmaceuticals, 502 South Rosa Road, Madison, WI 53719, USA.
‡Present address: Invenra, 505 South Rosa Road, Madison, WI 53719,
USA. §Present address: Schrodinger, 222 Third St, Cambridge, MA
02142, USA. ¶Present address: Janssen Research & Development,
Spring House, PA 19477, USA.
The bacterial nucleoside salvage pathway
(Fig. 1A) provides an attractive biocatalytic
retrosynthetic scheme for deoxyribonucleo-
sides (16, 17). The biological sequence degrades
purine 2′-deoxyribonucleosides using three en-
zymes (18). First, purine nucleoside phospho-
rylase (PNP) displaces the nucleobase with
phosphate to give deoxyribose 1-phosphate.
Phosphopentomutase (PPM) then transfers
The improved PPM variant enabled us to
evolve PNP for improved activity. We screened
a library of variants with single mutations near
the active site for formation of islatravir in
Huffman et al., Science 366, 1255–1259 (2019)
6 December 2019
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RESEARCH
| REPORT
the tandem forward reaction from sugar 5-
phosphate (4), with the 1-phosphate (3) being
generated in situ by the newly evolved PPM.
A single amino acid change, M64A, near the
alkyne-bearing C-4 of the docked substrate
(fig. S1E) provided a PNP variant with about
350-fold improved activity (Table 1 and figs.
S26 and S27). With the two active enzymes,
we focused our attention on evaluating the
practicality of the glycosylation cascade.
Because the PPM-catalyzed equilibrium favors
the starting 5-phosphate, the phosphate transfer
and glycosylation reactions must be performed
simultaneously. Despite the improved activity
of both enzymes, the tandem reaction plateaued
at <50% yield. A reverse reaction starting from
islatravir reached 40% conversion, demon-
strating that the reaction was equilibrium
limited. In addition, the inorganic phosphate
byproduct of the glycosylation reaction is
known to inhibit PPM (21). Removing the
inorganic phosphate as it is formed provided
an effective solution to the equilibrium and
inhibition problems (22). Addition of sucrose
phosphorylase (SP) and sucrose to the reaction
converts free phosphate to glucose 1-phosphate
and shifts the entire equilibrium forward. The
resulting three-enzyme cascade runs to full
conversion at substrate concentrations as high
as 200 mM (scheme S4B).
zymes in the retro-aldol reaction. Several DERA
homologs displayed high activity in the retro-
aldol of the alkynylated sugar. After further
evaluation in the forward aldol reaction, we
selected the DERA from Shewanella halifaxensis
for its high activity, complete stereoselectivity
in the formation of the new C–C bond (> 99%
de), and kinetic selectivity favoring reaction
with the (R)-enantiomer of the aldehyde. The
(S)-aldehyde was very slowly converted to the
(3S,4S) diastereomer. This wild-type DERA was
also active in reaction with the nonphosphory-
lated aldehyde (7). Despite extensive research
on applications of DERA enzymes, we did not
find any reports of reactions of aldehydes with
a fully substituted a-carbon.
The limitation of the S. halifaxensis DERA
was that it did not tolerate the high concen-
trations of acetaldehyde required for a practical
synthesis. Two rounds of directed evolution
addressed this constraint, yielding an improved
variant that retained high activity at an acet-
aldehyde concentration >400 mM (Table 1).
The engineered enzyme contained several new
mutations, including C47V (fig. S1C). This cys-
teine residue is found near the active site in
other DERA homologs and acts as a regulator
of the enzyme activity, binding the product of
acetaldehyde self-aldol condensation (23).
With the repurposed nucleoside salvage en-
zymes in hand, we focused on an efficient syn-
thesis of 2-ethynylglyceraldehyde 3-phosphate
(5). Known biochemical pathways to its natural
analog (R)-glyceraldehyde phosphate do not al-
low for facile incorporation of a C-2-substitutent.
Retrosynthetic analysis suggested that 5 could
be accessed from the simple achiral building
block 2-ethynylglycerol (6) through oxidation
and phosphorylation reactions (Fig. 1B). In
theory, these transformations could occur in
either order. We explored enzymes for all
four of these reactions and did not find any
with activity for oxidation of 8. We therefore
pursued the path of oxidation followed by
phosphorylation.
Discovery of an enzyme with phosphorylation
activity toward 7 required extensive screening
of a broad spectrum of kinases that naturally
act on sugars and primary alcohols. Ultimately,
we identified a pantothenate kinase (PanK)
from E. coli with very low-level activity toward
the (R)-enantiomer of aldehyde 7. Applying
directed evolution, we were able to rapidly
increase the activity by introducing two muta-
tions (L277I and I281M) in the helix of the
pantothenate binding site (fig. S1B). Further
engineering improved the enzyme’s activity
and stability. The evolved variant displayed
greater than 100-fold higher activity (fig. S19)
and pro-(R) kinetic selectivity (E = 8) (scheme
S19), achieving full conversion at 200 mM
concentration (Table 1). Practical use of the
kinase in vitro required regeneration of its
cofactor adenosine triphosphate. We chose
We continued exploring the retrosynthetic
degradation pathway by screening DERA en-
A
Bacterial nucleoside salvage pathway applied to islatravir (1). Unnatural structural elements are highlighted in green.
NH2
N
N
N
H
N
F
NH2
N
HO
2
5
N
N
–HO3PO
–HO3PO
O
O
OPO3H–
HO
O
4
1
2
OH
PNP
PPM
DERA
3
N
+
F
O
HO
5
HO
HO
3
4
O
–
H2PO4
+
HO
islatravir (1)
B
Biocatalytic retrosynthetic analysis of aldehyde intermediate (5).
= Purine Nucleoside Phosphorylase
HO
PNP
[P]
[O]
O
HO
PPM = Phosphopentomutase
7
DERA
[P]
= Deoxyribose 5-Phosphate Aldolase
= Phosphate transferase
HO
HO
–HO3PO
HO
OH
O
[O]
= Oxidoreductase
5
6
HO
–HO3PO
OH
[O]
[P]
8
Fig. 1. Biocatalytic retrosynthetic planning. (A) Purine nucleoside degradation pathway applied retrosynthetically to islatravir (1). (B) Retrosynthesis of the
glyceraldehyde phosphate analog leading to a simple achiral building block.
Huffman et al., Science 366, 1255–1259 (2019)
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RESEARCH
| REPORT
acetyl phosphate as an economical source of
activated phosphate paired with a thermo-
stable acetate kinase (AcK) from Thermotoga
maritima (Fig. 2).
and F464L (fig. S1A). Multiple additional muta-
tions throughout the enzyme improved protein
expression, stability, and activity and alleviated
product inhibition. With higher activity came
an increase in overoxidation of the product.
The overoxidation led to an increase in the
enantiopurity of 7 throughout the course of
the reaction (up to 99% enantiomeric excess),
albeit at the cost of yield (fig. S10).
that in situ product consumption would mini-
mize the inhibition suffered by the oxidase.
Unfortunately, the pantothenate kinase lacked
the required chemoselectivity, rapidly phos-
phorylating triol 6.
The final piece to complete the biocatalytic
pathway was the desymmetrizing oxidation of
2-ethynylglycerol (6). Among a broad range of
oxidoreductases, we identified evolved var-
iants of galactose oxidase (GOase M1 and F2)
that chemoselectively formed the monoalde-
hyde 7 with limited overoxidation (Table 1).
These copper-dependent enzymes were pre-
viously engineered for improved expression
in E. coli (M1-variant) (24) and broadened
substrate scope (F2-variant) (25). Unfortunately,
both M1 and F2 GOase variants favored for-
mation of the undesired (S)-enantiomer with
8:92 and 40:60 R:S ratios, respectively. The
(S)-selectivity is consistent with the stereo-
chemistry of the natural substrate D-galactose.
Nevertheless, the relaxed stereoselectivity of
the F2-variant suggested that its enantiopre-
ference could be reversed, and the GOase F2
became the starting point for evolution.
Synthetic applications of GOases require two
additional redox enzymes: a catalase to dis-
proportionate the hydrogen peroxide bypro-
duct and a peroxidase to maintain the correct
oxidation state of copper (26). We chose com-
mercially available bovine catalase and horse-
radish peroxidase and used these enzymes
during evolution of the GOase. Over 12 rounds
of oxidase engineering, we targeted an im-
provement in activity, a reduction in prod-
uct inhibition, and a reversal of the innate
enantiopreference. The engineered oxidase
displayed 11-fold improved activity and a
reversed 90:10 R:S selectivity (Table 1). The
change in enantioselectivity resulted mainly
from two mutations in the active site: W290Y
Isolating the intermediates 5 and 7 proved
to be challenging because of their high solu-
bility in water. Therefore, we focused on deve-
loping a process in which a single aqueous
solution was carried through the entire se-
quence. This approach necessitated evaluating
the impact of each component on the remain-
ing steps. The use of acetyl phosphate as the
phosphorous donor requires neutralization of
the liberated acetic acid, generating a solution
with elevated ionic strength. The high salt con-
tent inhibits downstream enzymes—in partic-
ular, the hexameric PNP (figs. S15 and S16). To
enable these reactions to proceed effectively,
we carried out further PNP evolution under
high-salt conditions and diluted the kinase
product solution to reduce its ionic strength
before the next step.
With a total of nine enzymes used in the
synthesis and no intermediate isolations, ma-
nagement of cumulative protein content was
critical. The final filtration of crystallized
islatravir became difficult as the amount of pro-
tein in the mixture increased. The homogeneous
nature of the oxidation and phosphorylation
reactions opened the possibility of immobiliz-
ing their enzymes to allow easy removal after
these steps. For this purpose, we applied an
affinity immobilization technique that relies
on the capture of polyhistidine-tagged pro-
teins on a metal-containing solid support (27).
The recombinant oxidase and kinase enzymes
were immobilized in this way, which had the
added benefit of eliminating nontagged pro-
teins during immobilization and minimizing
At this point, we had evolved enzymes for
each step of a fully biocatalytic sequence from
ethynyl glycerol (6) to islatravir. We then turned
to strategic considerations around how best
to integrate these individual reactions into the
overall in vitro synthesis. As discussed above,
the three reversible reactions catalyzed by PPM,
PNP, and SP must take place concurrently to
achieve a favorable equilibrium. Extending the
simultaneous cascade to include the reversible
aldol addition could provide additional syn-
ergy. Compatible pH and temperature ranges
would allow all four enzymes to function in
the same solution. Sucrose phosphorolysis as a
thermodynamic driving force could pull forward
all the equilibria, including the aldol reaction.
These factors enabled us to lower the excess
of acetaldehyde to levels tolerated by all the
enzymes. In this way, we could operate a sim-
ultaneous four-enzyme cascade in which the
nucleoside degradation pathway runs in reverse,
driven to high conversion by phosphate removal
(Fig. 2). Islatravir crystallized from the reac-
tion mixture and could be isolated directly
through filtration in greater than 95% purity
and 76% yield from 5.
The oxidation and phosphorylation reactions
are essentially irreversible, so directly coupling
them with the downstream cascade provides
no thermodynamic benefit. We considered a
tandem GOase-kinase combined reaction, so
Table 1. Properties and performance of evolved enzymes used in the biocatalytic pathway.
Starting enzyme
Conversion
Evolved variant
Source
Rounds of
evolution acids changed (no.)
Global amino
Loading
(%)*
Conversion
Loading
(%)*
Enzyme
Oxidase
Evolution focus
....................................o...r..g..a...n..i.s...m........................................................................................................................................................................................................................................................................................
(selectivity)
Variant M1:33%†‡
(8:92 R:S)
(selectivity)
80%†§
(90:10 R:S)
Fusarium
graminearum
Stereoselectivity
..................................
(GOase)
12
34
100
20
Variant F2: 8%†‡
(40:60 R:S)
Activity
............................................................................................................................................................................................................................................................................................................................................
.P..a...n..K....................................................................................................................................................................................................................................................................................................................................
E. coli
Activity
Acetaldehyde
3
10
<1% (5:1 R:S)†¶
97%**[>98:1:1 (3S4R):
(3R4R):(3S4S)]
0.5%††
0.18%‡‡
10
>95% (10:1 R:S)†#
97%**[>98:1:1 (3S4R):
(3R4R):(3S4S)]
34%††
62%‡‡
(>99.5:0.5 dr)
10
DERA
S. halifaxensis
2
11
5
0.2
............................................................................t..o...l.e..r..a..n...c..e...............................................................................................................................................................................................................................................
.P..P...M......................................................................................................................................................................................................................................................................................................................................
E. coli
Activity
2
5
0.5
0.5
PNP
E. coli
Activity
4
7
0.125
0.125
..................................................................................................................................................................................................(..>...9...9....5...:.0.....5....d...r..)..............................................................................................................
*Enzyme loading refers to the mass of lyophilized clarified cell lysate relative to the mass of the reaction substrate. Results may reflect improvements in enzyme expression as well as
activity.
CuSO4, 25°C, 4 hours.
mM MnCl2, pH 7.5, 40°C, 18 hours.
†Reaction with nonimmobilized enzymes.
‡GOase-M1-Strep and F2-Strep: 172 mM 6, pH 7.5, 0.2 mM CuSO4, 25°C, 4 hours.
¶44 mM 7, pH 7.5, 20°C, 18 hours. #235 mM 7, pH 6.4, 20°C, 18 hours.
‡‡13 mM 1b, pH 7.5, 40°C, 16 hours.
§GOAse-13BB-His: 258 mM 6, pH 7.5, 0.2 mM
**142 mM 5, 420 mM acetaldehyde, pH 7.2, 30°C, 24 hours. ††15 mM 4, 5
Huffman et al., Science 366, 1255–1259 (2019)
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RESEARCH
| REPORT
NH2
N
O
N
NH2
N
+
O
Me
1.5 eq
H
N
H
N
N
N
F
Me
PanKRd4BB
co-immobilized
OPO3H–
AcK
O2 (air)
0.85 eq
HO
HO
HO
HO
N
F
HO
–HO3PO
O
O
O
HO
OH
GOaseRd13BB
catalase HRP
DERARd3BB
PPMRd3BB
+
–
PNPRd5BB
H2PO4
HO
6
7
5
1
sucrose
76% yield
97% ee
67% assay
yield from 6
SP
fructose
glucose 1-phosphate
51% overall yield from 6 to 1
Auxiliary enzymes
Horseradish Peroxidase from Amoracia rusticana
Evolved enzymes
evolved Galactose Oxidase from Fusarium graminearum
GOaseRd13BB
PanKRd4BB
HRP
evolved Pantothenate Kinase from E. coli
catalase Catalase from Bos taurus
evolved Deoxyribose 5-Phosphate Aldolase
from Shewanella halifaxensis
Acetate Kinase from Thermotoga maritima
DERARd3BB
AcK
SP
Sucrose Phosphorylase from Alloscardovia omnicolens
evolved Phosphopentomutase from E. coli
PPMRd3BB
PNPRd5BB
Denotes an immobilized version of an enzyme
evolved Purine Nucleoside Phosphorylase from E. coli
BB denotes backbone of the respective evolution round
Fig. 2. Fully assembled biocatalytic pathway. Evolved enzymes are in colored boxes, and wild-type auxiliary enzymes are in white boxes.
undesired side reactions catalyzed by host-cell
enzymes. Immobilization was not pursued for
the aldol and glycosylation enzymes because
the product crystallizes during this step.
tecting groups. We envision a growing adop-
tion of cascade biocatalysis as a strategy for the
sustainable synthesis of complex non-natural
molecules such as pharmaceuticals. The appli-
cation of enzyme cascades to a diverse range
of molecular structures will rely on further ad-
vances in the pace of protein engineering (28)
and the continuing discovery of new enzymatic
transformations (7, 29).
20. C. L. Windle, M. Müller, A. Nelson, A. Berry, Curr. Opin. Chem.
Biol. 19, 25–33 (2014).
21. K. Hammer-Jespersen, A. Munch-Petersen, Eur. J. Biochem. 17,
397–407 (1970).
22. W. Tischer, H.-G. Ihlenfeldt, O. Barzu, H. Sakamoto,
E. Pistotnik, P. Marlière, S. Pochet, Enzymatic synthesis of
deoxyribonucleosides, U.S. patent no. US07229797 B1 (2007).
23. M. Dick et al., Chem. Sci. 7, 4492–4502 (2016).
24. L. Sun, I. P. Petrounia, M. Yagasaki, G. Bandara, F. H. Arnold,
Protein Eng. Des. Sel. 14, 699–704 (2001).
25. J. B. Rannes et al., J. Am. Chem. Soc. 133, 8436–8439 (2011).
26. A. Toftgaard Pedersen et al., Org. Process Res. Dev. 19,
1580–1589 (2015).
The biocatalytic synthesis produces only
a single stereoisomer of islatravir in a self-
correcting manner. Oxidative desymmetriza-
tion establishes the fully substituted carbon
center with 90:10 selectivity, and the ratio is
increased by further oxidation of the minor
enantiomer. Every subsequent step provides
an opportunity to amplify the stereochemical
purity. The kinase and aldolase enzymes react
with kinetic selectivity toward the (R)-aldehydes
7 and 5, respectively, allowing upgrade of the
enantiomer ratio. The aldolase creates the
second stereogenic center with great preci-
sion. Last, only the (3S,4R)-diastereomer of the
sugar phosphate 4 can react further, and the
glycosylation sets the anomeric center with
perfect selectivity.
The full in vitro biocatalytic cascade uses
five engineered enzymes and four auxiliary
enzymes to stereoselectively assemble islatravir
from simple achiral building blocks in 51%
overall yield. The atom economy far exceeds
that of previous syntheses of this target, and
the number of steps is less than half. The en-
tire sequence takes place under mild condi-
tions in a single aqueous solution without the
isolation of intermediates. This extraordinary
efficiency was made possible by the ability to
identify and engineer enzymes that can build
complex structures with excellent stereo- and
chemoselectivity and without the need for pro-
REFERENCES AND NOTES
27. M. P. Thompson et al., Tetrahedron 75, 327–334 (2019).
1. K. R. Campos et al., Science 363, eaat0805 (2019).
2. R. A. Sheldon, J. M. Woodley, Chem. Rev. 118, 801–838 (2018).
3. N. J. Turner, L. Humphreys, Biocatalysis in Organic Synthesis
(The Royal Society of Chemistry, 2018).
4. J. H. Schrittwieser, S. Velikogne, M. Hall, W. Kroutil, Chem. Rev.
118, 270–348 (2018).
5. S. P. France, L. J. Hepworth, N. J. Turner, S. L. Flitsch, ACS
Catal. 7, 710–724 (2017).
6. F. H. Arnold, Q. Rev. Biophys. 48, 404–410 (2015).
7. F. H. Arnold, Angew. Chem. Int. Ed. 57, 4143–4148 (2018).
8. H. Ohrui et al., Nucleosides Nucleotides Nucleic Acids 26,
1543–1546 (2007).
9. E. Michailidis et al., J. Biol. Chem. 289, 24533–24548 (2014).
10. S. E. Barrett et al., Antimicrob. Agents Chemother. 62,
e01058-18 (2018).
11. M. McLaughlin et al., Org. Lett. 19, 926–929 (2017).
12. M. Kageyama, T. Nagasawa, M. Yoshida, H. Ohrui, S. Kuwahara,
Org. Lett. 13, 5264–5266 (2011).
13. K. Fukuyama, H. Ohrui, S. Kuwahara, Org. Lett. 17, 828–831
(2015).
14. M. Kageyama et al., Biosci. Biotechnol. Biochem. 76, 1219–1225
(2012).
28. M. D. Truppo, ACS Med. Chem. Lett. 8, 476–480 (2017).
29. P. N. Devine et al., Nat. Rev. Chem. 2, 409–421 (2018).
ACKNOWLEDGMENTS
We acknowledge the help and support of the following people.
Merck team: J. McIntosh for providing wild-type acid
phosphatase and acetate kinase constructs; R. Cohen, X. Wang,
M. Reibarkh, and P. Dormer for nuclear magnetic resonance
analysis support; R. Patel, C. Mastykarz, W. Pan, J. Gouker,
I. Farasat, J. Russell, and L. Do for supporting protein
engineering and directed evolution workflows; A. Kassim,
T. Andreani, and R. Matthew for help with the preparation of
synthetic intermediates; and E. Margelefsky, K. Mattern,
M. Miller, and H. Rose for reaction optimization support in scale-
up experiments. Codexis team: A. Ortega, A. Sowell-Kantz,
H. Maniar, J. Slaton, C. Micklitsch, L. Miller, and M. Krawczyk
for library screening support; V. Mitchell, C. Selim, and
A. Petkova for library construction support; N. Subramanian and
N. Dellas for library design support; J. Riggins for analytical
development support; J. Vroom and S. Sivaramakrishnan
for enzyme characterization support; and D. Entwistle for
chemistry support. Funding: This work was funded by Merck
& Co., Inc. Author contributions: M.A.H. and A.F. carried out
and supervised chemistry development and prepared the
manuscript. K.R.C., P.N.D., K.M.M, and M.D.T. supervised
chemistry development. O.A. and K.A.C. supervised enzyme
evolution. J.H.F., S.T.G., G.J.H., J.N.K., J.C.M., M.M, C.C.N.,
N.R.P., S.A.R., A.M.W., Y.X., and H.Y. carried out chemistry
development. M.B.-G., D.D., J.L., N.M.M., G.S.M., J.N., and S.N.
carried out enzyme evolution. H.M.H., J.J., L.A.J., and B.F.M.
developed analytical methods to allow chemistry development
15. S. Kohgo, H. Ohrui, M. Matsuoka, H. Mitsuya, 4′-C-substituted-
2-haloadenosine derivative, U.S. patent no. US7625877 B2
(2009).
16. N. J. Turner, E. O’Reilly, Nat. Chem. Biol. 9, 285–288 (2013).
17. I. A. Mikhailopulo, A. I. Miroshnikov, Acta Naturae 2, 36–59
(2010).
18. M. G. Tozzi, M. Camici, L. Mascia, F. Sgarrella, P. L. Ipata, FEBS
J. 273, 1089–1101 (2006).
19. W. R. Birmingham et al., Nat. Chem. Biol. 10, 392–399 (2014).
Huffman et al., Science 366, 1255–1259 (2019)
6 December 2019
4 of 5

RESEARCH
| REPORT
and enzyme evolution. A.R.-G, E.C.S., D.V., and L.X. carried out
computational work to support enzyme discovery and evolution.
Competing interests: Merck & Co., Inc. has filed a patent
application covering the process: U.S. patent application no.
PCT/US2019/040316. Codexis, Inc. has filed patent applications
covering the evolved enzymes: U.S. patent application nos. PCT/
US2019/040353, PCT/US2019/040359, PCT/US2019/040369,
PCT/US2019/040376, and PCT/US2019/040379. Data and
materials availability: All data are available in the main text or the
supplementary materials.
Schemes S1 to S81
DNA and amino acid sequences of the enzymes
NMR Spectra
HR-MS Spectra
References (30–50)
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/366/6470/1255/suppl/DC1
Materials and Methods
Figs. S1 to S28
12 August 2019; accepted 24 October 2019
10.1126/science.aay8484
Tables S1 to S15
Huffman et al., Science 366, 1255–1259 (2019)
6 December 2019
5 of 5

Design of an in vitro biocatalytic cascade for the manufacture of islatravir
Mark A. Huffman, Anna Fryszkowska, Oscar Alvizo, Margie Borra-Garske, Kevin R. Campos, Keith A. Canada, Paul N.
Devine, Da Duan, Jacob H. Forstater, Shane T. Grosser, Holst M. Halsey, Gregory J. Hughes, Junyong Jo, Leo A. Joyce,
Joshua N. Kolev, Jack Liang, Kevin M. Maloney, Benjamin F. Mann, Nicholas M. Marshall, Mark McLaughlin, Jeffrey C. Moore,
Grant S. Murphy, Christopher C. Nawrat, Jovana Nazor, Scott Novick, Niki R. Patel, Agustina Rodriguez-Granillo, Sandra A.
Robaire, Edward C. Sherer, Matthew D. Truppo, Aaron M. Whittaker, Deeptak Verma, Li Xiao, Yingju Xu and Hao Yang
Science 366 (6470), 1255-1259.
DOI: 10.1126/science.aay8484
Maximal efficiency from enzyme cascades
Enzymes are highly selective catalysts that can be useful for specific transformations in organic synthesis.
Huffman et al. combined designer enzymes in a multistep cascade reaction (see the Perspective by O'Reilly and Ryan).
The approach eliminates purification steps, recycles expensive cofactors, and couples favorable and unfavorable
reactions. With the target molecule islatravir, an experimental HIV drug, they optimized five enzymes by directed
evolution to be compatible with unnatural substrates and stable in the reaction conditions. Stereochemical purity was
amplified at every enzymatic step, and the final synthesis was both atom economical and efficient.
Science, this issue p. 1255; see also p. 1199
ARTICLE TOOLS
http://science.sciencemag.org/content/366/6470/1255
SUPPLEMENTARY
MATERIALS
http://science.sciencemag.org/content/suppl/2019/12/04/366.6470.1255.DC1
RELATED
CONTENT
http://science.sciencemag.org/content/sci/366/6470/1199.full
REFERENCES
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