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Biocatalysis

Biased Borate Esterification during Nucleoside Phosphorylase-

Catalyzed Reactions: Apparent Equilibrium Shifts and Kinetic

Implications**

Felix Kaspar+,* Felix Brandt+, Sarah Westarp, Lea Eilert, Sebastian Kemper, Anke Kurreck,

Peter Neubauer, Christoph R. Jacob, and Anett Schallmey

Abstract: Biocatalytic nucleoside (trans-)glycosylations

catalyzed by nucleoside phosphorylases have evolved

into a practical and convenient approach to the prepara-

tion of modified nucleosides, which are important

pharmaceuticals for the treatment of various cancers

and viral infections. However, the obtained yields in

these reactions are generally determined exclusively by

the innate thermodynamic properties of the nucleosides

involved, hampering the biocatalytic access to many

sought-after target nucleosides. We herein report an

additional means for reaction engineering of these

systems. We show how apparent equilibrium shifts in

phosphorolysis and glycosylation reactions can be

effected through entropically driven, biased esterifica-

tion of nucleosides and ribosyl phosphates with inor-

ganic borate. Our multifaceted analysis further describes

the kinetic implications of this in situ reactant esterifica-

tion for a model phosphorylase.

Nucleosides and their analogs are central to the biological

and chemical sciences, as they serve a variety of biological

functions and represent a growing class of anti-cancer and

anti-viral pharmaceuticals.[1] Although the synthesis of

nucleosides typically proceeds via N-glycosylation of nucleo-

bases with heavily protected sugar synthons, there is an

increasing recognition for the inefficiency of the associated

synthetic routes.[2] Consequently, recent years have experi-

enced renewed interest in the biocatalytic synthesis of

natural and modified nucleosides. This includes, for in-

stance, the enzymatic preparation of halogenated purine

nucleoside synthons,[3] the diversification of alkylated pyr-

imidine nucleoside analogues,[4] the development of high-

yielding flow processes,[5] and the synthesis of the pharma-

ceuticals islatravir (anti-HIV)[6] and molnupiravir (anti-

Covid19)[7] in biocatalytic cascades. All these examples

employ nucleoside phosphorylases for key

(trans-)glycosylation reactions, which enable the installation

of ribosyl-based moieties on pyrimidine and purine nucleo-

bases in one step and without the need for any protecting

group chemistry.

Nucleoside phosphorylases catalyze the reversible phos-

phorolysis of nucleosides to the corresponding nucleobases

and pentose-1-phosphates (Scheme 1).[8] This reactivity can

be employed in reverse to transfer the glycosyl moiety from

one nucleoside (or a pentose-1-phosphate) to another

nucleobase, a reaction typically referred to as a (trans-

)glycosylation.[9] While such (trans-)glycosylation processes

are well established as synthetic tools,[10–21] they inherently

suffer from thermodynamic limitations, as the final yield of

these reactions is dictated solely by the substrate-dependent

thermodynamics of the respective (reverse) phosphorolytic

steps as well as the employed reaction conditions.[9,22,23]

Although some progress has been made to mitigate or

exploit the tight thermodynamic control in these systems

(e.g., via (by)product precipitation,[24] enzymatic product

removal[25] or application of recoverable excess reagent[26])

[*] Dr. F. Kaspar,+L. Eilert, Prof. Dr. A. Schallmey

Institute for Biochemistry, Biotechnology and Bioinformatics,

Technische Universität Braunschweig

Spielmannstraße 7, 38106 Braunschweig (Germany)

E-mail: [email protected]

[email protected]

Dr. F. Kaspar,+S. Westarp, Dr. A. Kurreck, Prof. Dr. P. Neubauer

Chair of Bioprocess Engineering, Institute of Biotechnology, Faculty

III Process Sciences, Technische Universität Berlin

Ackerstraße 76, 13355 Berlin (Germany)

F. Brandt,+Prof. Dr. C. R. Jacob

Institute of Physical and Theoretical Chemistry, Technische Uni-

versität Braunschweig

Gaußstraße 17, 38106 Braunschweig (Germany)

S. Westarp, Dr. A. Kurreck

BioNukleo GmbH

Ackerstraße 76, 13355 Berlin (Germany)

L. Eilert

Present address: Department Structure and Function of Proteins,

Helmholtz Centre for Infection Research

Inhoffenstraße 7, 38124 Braunschweig (Germany)

Dr. S. Kemper

Institute for Chemistry, Technische Universität Berlin

Straße des 17. Juni 135, 10623 Berlin (Germany)

[+] These authors contributed equally to this work.

[**]A previous version of this manuscript has been deposited on a

preprint server (https://doi.org/10.26434/chemrxiv-2022-xjvpw).

published by Wiley-VCH GmbH. This is an open access article under

the terms of the Creative Commons Attribution License, which

permits use, distribution and reproduction in any medium, provided

the original work is properly cited.

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How to cite: Angew. Chem. Int. Ed. 2023, 62, e202218492

International Edition: doi.org/10.1002/anie.202218492

German Edition: doi.org/10.1002/ange.202218492

and irreversible phosphorolysis of 6-oxopurines is well

established,[10,27,28] there currently exists no general method

for the direct manipulation of glycosylation equilibria.

During our development of the PUB module for

continuous high-throughput phosphate detection in bio-

chemical assays,[29] we serendipitously found that the pres-

ence of borate effects a series of inhibitory phenomena

during pyrimidine nucleoside phosphorolysis as well as an

apparent equilibrium shift caused by biased borate esterifi-

cation of nucleosides over ribose 1-phosphate (Rib1P).

While saccharides are well known to undergo complex

equilibrium reactions with borate in aqueous solution,[30,31]

comparably little is known about in situ competition of such

processes. Similarly, although the literature offers some

examples for biocatalytic applications of equilibrium shift

phenomena through preferential esterification of one reac-

tant with borate (namely, the enzymatic epimerization of

fructose,[32] lactose,[33] galactose,[34,35] and arabinose[36]) the

exact species involved in these processes remain largely

elusive, as do the kinetic implications of this esterification

on the enzyme-level. Despite the precedents for borate

inhibiting NAD+-dependent enzymes in a non-competitive

fashion,[37,38] the molecular determinants and mechanisms of

these phenomena have remained equally elusive. To shed

light on the thermodynamic and kinetic implications of

borate ester formation on nucleoside phosphorylase-cata-

lyzed reactions and provide a characterized precedent for

biased borate esterification, we herein report a multifaceted

analysis of this reaction system with spectroscopic and

computational approaches. Furthermore, we examined the

synthetic utility of this biased borate esterification, as

concentration-dependent apparent equilibrium shifts pro-

vide an additional means for reaction engineering in

biocatalytic glycosylation reactions.

Our investigation was sparked by the serendipitous

observation that phosphorolysis reactions with 5-bromour-

idine (1a) consistently exhibited noticeably lower equili-

brium conversions in the presence of moderate concentra-

tions of borate (Figures 1A and B). For instance, a reaction

containing 400 μM 1a, 4 mM (10 equivalents) phosphate and

40 μgmL1(0.45 mol%) of the well characterized pyrimidine

nucleoside phosphorylase from Geobacillus thermoglucosi-

dasius (GtPyNP),[4,39–42] serving as a model enzyme (see the

Supporting Information for details), reached its equilibrium

at 70% conversion after 10 min in glycine-buffered solution

(Figure 1B). In contrast, the same reaction additionally

containing 20 mM borate took almost 20 min to reach an

equilibrium at 47% conversion, as monitored by multi-

wavelength UV spectroscopy employing principles of spec-

tral unmixing.[43,44] This effect was not rooted in enzyme

inactivation, as GtPyNP showed an identical melting point

and fully retained its catalytic activity after prolonged

incubation in borate-containing buffers (Figures S1 and S2).

The observed apparent equilibrium shift persisted in the

reverse direction of the reaction (starting from the products

Rib1P and 2a, Figure S6) and additional experiments

established that the reaction system consistently behaved

according to lower apparent equilibrium constants of

phosphorolysis (K’), whose magnitude depended on the

concentration of borate (Figure 1C). Since such drastic

equilibrium shifts are unprecedented for phosphorolysis

systems, we suspected that the formation of a dominant

secondary species would actively remove the nucleoside 1 a

from this equilibrium in a thermodynamically controlled

fashion. Indeed, the conversion data phenotypically follow-

ing a Boltzmann-type relationship (Figure 1D) could be

described well with a thermodynamic model accounting for

the presence of an additional equilibrium system in which

1a is partially transformed to a borate ester (Figure 1E, see

the Supporting Information for details and equations).

Although borate esters of ribose are known to persist in

dilute aqueous solution,[45–47] these stable esters generally

involve the anomeric hydroxyl group, which is absent in 1a.

Nevertheless, Kim et al.[48,49] observed borate esters of the

nucleoside-based cofactor NAD+and the natural trinucleo-

tides by mass spectrometry, indicating that analogous

species might be formed by recruitment of the 2’- and/or 3’-

hydroxyl groups. In addition, structurally similar nucleoside

boronate esters have been reported by Smietana[50,51] and

others.[52] Based on these precedents, we hypothesized that

the cyclic borate ester 1a* would be the predominant

species causing the observed equilibrium shift (Figure 1F).

To probe if borate esters like 1 a* would be feasible

species to effect apparent equilibrium shifts under dilute

aqueous reaction conditions, we turned to density functional

theory (DFT) calculations and NMR spectroscopy. DFT

calculations (using B3LYP/TZ2P[53–56] in combination with a

COSMO[57] solvent model for water, see the Supporting

Information for details) suggested that the formation of the

2’- or 3’-borate monoesters of 1a would be highly disfavored

Scheme 1. Nucleoside phosphorolysis and strategies for apparent

equilibrium shifts. NB=nucleobase.

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processes, while the formation of the proposed five-mem-

bered cyclic diester 1a* offers around 40 kJmol1net gain in

Gibbs free energy, indicating that this esterification could

feasibly proceed against the concentration gradient of water

(Figure 1F). Indeed, direct evidence for the presence of 1a*

could be obtained by NMR spectroscopy. When 1 a (10 mM)

was incubated with 20 mM borate in glycine buffer at pH 9,

a dominant borate-32

containing species in equilibrium with free borate could

be observed by 11B NMR (Figure S10), while 1H NMR

showed additional signals indicative of a modification of the

ribosyl moiety of 1a (Figure 1G). Furthermore, slight

changes in the coupling constants across the sugar ring

implied the introduction of ring torsion, consistent with the

configurational changes required in 1a* (Table S4). In

addition to this ester, we observed two distinct minor

components in the mixture, which exhibited identical

coupling constants to 1a* and existed in similar concen-

trations (ca. 1:1.2 ratio). Based on the well-characterized

diastereomeric borate ester dimers of methyl apiose

described by Ishii and Ono[59] and the report of purine

nucleoside solubilization as 2:1 complexes with borate by

Tsuji and colleagues,[60] we tentatively ascribe these species

as the cis- and trans-isomers 1a** (Figures 1G and F).

Overall, the Gibbs free energies obtained experimentally by

NMR spectroscopy or equilibrium state calculations based

on UV data (ΔRGca. 32 kJmol1, see the Supporting

Information for details) are comparable to those obtained

by DFT calculation. Although an analogous six-membered

ester (and potentially its dimers) between the 3’- and 5’-

hydroxyl groups would introduce similar configurational

changes to those ascribed to 1a*, DFT calculations revealed

that its formation is much less exothermic and consequently

disfavored. Similarly, the analogous five-membered borate

ester of Rib1P is less favored than 1a* (which is requisite

for the observed equilibrium shift), as supported by DFT

and NMR data (Figures 1F and S10). In addition to the

lower entropic gain, we expect that steric conflicts between

the phosphate group and the borate ester as well as charge

repulsion account for the less favored formation of Rib1P*

compared to 1a*. Additionally, we observed no trace of

dimers of Rib1P* by NMR, which is likely a result of the

highly disfavored formal 5 charge of these species.

Consequently, the formation of the borate ester 1a* should

be expected to dominate in direct competition during a

phosphorolysis reaction, which we could confirm by subject-

ing phosphorolysis reaction mixtures with increasing borate

concentrations to NMR analysis (Figure S13). Further

thermodynamic experiments supported entropic effects as a

driving force for this biased esterification. Arrhenius plots of

the 1a–1a* equilibrium obtained by 1H NMR revealed a

drastic temperature-dependence, favoring the presence of

the free nucleoside at higher temperatures (Figure S11). In

agreement with these observations, Arrhenius plots for the

borate esterification derived from equilibrium shifts in the

phosphorolysis reaction indicated an approach to an ener-

getic balance between 1a* and Rib1P* at higher temper-

atures (Figure S5). Collectively, these results describe 1a* as

the dominant borate ester in this reaction system, respon-

sible for depleting the pool of free nucleoside in dilute

aqueous solution at room temperature. Consistent with this

Figure 1. APhosphorolysis of 5-bromouridine (1a), and B–Eapparent

equilibrium shifts observed in the presence of borate. FDFT data.

G1H NMR of 1a (10 mM) with or without 20 mM borate in 200 mM

glycine buffer at pH 9 and 25°C. See the Supporting Information for

experimental details, raw data and equations.[58] [a] Not confidently

accessible by DFT due to their high charge.

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conclusion, the 2’-deoxy nucleoside deoxy-1a, incapable of

forming 1a* or 1a**, did not show an equilibrium shift

during its phosphorolysis in the presence of borate or any

reaction with borate discernible by NMR (Figure S15).

Next, we sought to identify the cause of the apparently

reduced reaction rates in the presence of borate by enlisting

kinetic studies and molecular dynamics (MD) simulations.

This effect was especially prominent with borate concen-

trations greater than 20 mM and led to decreases in the

reaction rate of GtPyNP by more than a factor of four, as

illustrated in Figure 2D. Since high borate concentrations

primarily yield 1a* in solution (and not the enzymatic

substrate 1a), we initially entertained the hypothesis that

this phenomenon was a function of the decreased concen-

tration of the free nucleoside substrate and the associated

apparent decrease of affinity. However, the Michaelis–

Menten kinetics obtained for 1a (with phosphate in excess)

proved inconsistent with this hypothesis. While a reduction

in available substrate concentration should primarily result

in an increase in the apparent Michaelis–Menten constant

KM, we observed no change in KMbut instead a sharp

decline of the rate constant under saturating substrate

concentrations (kcat, Figure 2B). With saturating concentra-

tions of both substrates, the observed rate constant kobs

exhibited a similar Boltzmann-type decrease as observed for

the apparent equilibrium constant, which could be described

well by an equilibrium model expressing the observed rate

constant as a function of kcat and the esterified fraction of 1 a

(Figure 2D, see the Supporting Information for equations).

As this decrease of kobs was further completely absent for

deoxy-1a in the presence of borate (Figures 2C and E), we

concluded that borate alone does not inhibit GtPyNP, but

rather the borate ester 1a*. If GtPyNP could bind but not

convert 1a*, we reasoned that the position of the equili-

brium between 1a and 1a* would determine the ratio of

potentially active enzyme (1a bound to GtPyNP) versus

inactive enzyme (1a* bound to GtPyNP), assuming that

catalysis is a rate-limiting step. Given the highly solvent-

exposed active site of pyrimidine nucleoside phosphorylases

in the open state, we expected that GtPyNP should be able

to accommodate the slightly twisted and sterically more

demanding borate ester 1a* and potentially allow its

equilibration with the free nucleoside substrate 1a while

bound to the enzyme. Phenomenologically, such a process

would resemble a classical non-competitive inhibition,

consistent with our kinetic data. Indeed, MD simulations

(using GROMACS[61,62] with the CHARMM36[63] force field)

based on our recently disclosed crystal structure of GtPyNP

in complex with uridine (PDB ID 7m7k,[4] see the Support-

ing Information for details) yielded several insights in

support of the proposed model. First, an analysis of the

clustered states over 50 ns simulation time indicated that the

borate ester 1a* can be bound in analogy to 1a via hydrogen

bonds with the amide motif of the nucleobase. Secondly, this

analysis also showed that the average state in which the

enzyme-1a and the enzyme-1a* complexes resided during

the simulation time displayed a quite solvent-exposed active

site, feasibly permitting esterification and hydrolysis proc-

esses to happen in situ. Thirdly, an examination of the

distances between the domains responsible for active site

closure indicated markedly reduced molecular motion of the

enzyme-1a* complex compared to the enzyme-1a complex

Figure 2. A–CMichaelis–Menten plots of the phosphorolysis of 1a and

deoxy-1a,Dand Ephosphorolysis rate under saturating conditions. F–

HMD simulations of GtPyNP with bound 1a or 1a*. See the

Supporting Information for experimental details, raw data and

equations.[58]

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(Figures 2F–H and S19). While GtPyNP with bound 1a

exhibited oscillatory opening and closing motions on a

timescale of around 11 ns (similar dynamics are known for

the closely related thymidine phosphorylases),[64–70] binding

of 1a* largely arrested this process. Specifically, binding of

1a* locks the enzyme in an open conformation by displacing

a catalytically essential arginine residue, which reversibly

adopts an inward position. This appears to halt domain

movement and yield an inactive enzyme, while the ester-

ification of the 2’- and 3’-OH groups of 1a further obstructs

access to the anomeric carbon, collectively preventing

productive phosphorolysis. As indicated by the clustered

structures, 1a* also slightly obstructs access to the

phosphate binding site of GtPyNP. Accordingly, we exper-

imentally observed a decreased affinity for phosphate in the

presence of 1a*, in addition to the lower kcat values

stemming from pseudo-non-competitive inhibition by 1a*

(Figure 2B). Experimentally accessed and computed rate

constants for the various processes involved in the catalytic

cycle also proved consistent with the hypothesized mecha-

nism. Enzyme opening/closing (ca. 0.1 ns1) occurs on a

much shorter time scale than the esterification of 1a (ca.

3 s1, Figure S12), catalysis (ca. 6 s1) and substrate binding

by GtPyNP (ca. 16 s1, see the Supporting Information).

Thus, the comparably slow substrate release (ca. 0.2 s1)

suggests that in situ hydrolysis of 1a* does happen

(hydrolysis pathway, Scheme 2), while we expect that the

pathway comprising dissociation of 1a* and productive

binding of 1a (dissociation pathway) is also populated

significantly (see the Supporting Information for further

discussions of the inhibition hypotheses). In contrast to the

phosphorolysis, the kinetics in the glycosylation direction

remained essentially unchanged in the presence of borate

(Figures S7 and S8), providing further support for the minor

role of Rib1P* in this reaction system. Consistent with the

entropically driven formation of 1 a* from 1a, we observed

decreased inhibition of the phosphorolysis reaction at higher

temperatures, as evident from Eyring plots obtained with

different borate concentrations (Figure S9). Taken together,

these results support an inhibitory mechanism phenotypi-

cally resembling a non-competitive inhibition, where rapid

equilibration of 1a to its borate ester 1a* (both in solution

and potentially while bound to the enzyme) reversibly

decreases the fraction of catalytically active GtPyNP so that

throughput in its catalytic cycle is primarily regulated by the

position of the 1a–1a* equilibrium (Scheme 2). Preliminary

data for other pyrimidine nucleoside phosphorylases as well

as other nucleosides suggests that this inhibitory mechanism

is likely not limited to GtPyNP and 1a (Figures S1 and S18).

With a good understanding of the underlying processes

governing the kinetics and thermodynamics of the phosphor-

olysis of 1a in the presence of borate, we aimed to apply the

observed equilibrium shifts to other nucleosides, specifically

targeting glycosylation reactions. Assuming that other

nucleosides would behave in analogy to our model com-

pound 1a, we expected that conversion shifts in glycosyla-

tion reactions would provide a general strategy to improve

access to nucleosides from the precursor Rib1P (which could

either be supplied as an isolated compound or generated in

situ).[10,71–73] Indeed, DFT calculations for a representative

set of nucleosides suggested that a variety of pyrimidine and

purine nucleosides should undergo similar esterifications

with borate as 1a (Table S9), which could be confirmed by

1H NMR (ΔRGca. 32 to 34 kJmol1, Figure S14) and, for

two examples, with kinetic experiments (see the Supporting

Information and Figure S18 for details). Although we were

unable to translate the observed minor differences in Gibbs

free energies to conversion shifts in transglycosylations,

presumably due to a “kinetic lock” effect (see the Support-

ing Information and Figure S17 for details), glycosylation

reactions with various pyrimidine nucleobases 2a–fnicely

reflected the expected behavior and facilitated conversion

shifts of 6–17% in favor of the respective nucleoside

(Figure 3A). A similar effect could be observed for the

halogenated purine 2g when subjected to identical con-

ditions with the promiscuous purine nucleoside phosphor-

ylase from G. thermoglucosidasius. Lastly, an illustrative

two-factor optimization for the glycosylation of 5-iodouracil

(2d) and 5-ethynyluracil (2f, both are known for their

unfavorable glycosylation thermodynamics)[9,22] showed how

a balance of excess sugar donor and the “pull” effect of the

corresponding nucleoside borate ester can be employed to

improve the conversions in historically challenging nucleo-

base glycosylations (Figure 3B). For instance, when using

2 eq. of Rib1P, the conversion of 2f to its nucleoside 1f

could be improved from 74% to 89% through the

application of 50 mM borate, which, conventionally, would

have required the application of at least 6 eq. of Rib1P. As a

proof of synthetic utility, these conditions were transferred

to the semi-preparative scale. The glycosylation of 10 mM 2f

with 2 eq. Rib1P proceeds under heterogeneous conditions

with 42% conversion, which improved to 77% through the

application of 50 mM borate (Figure 3C). Purification by

reverse-phase HPLC cleanly yielded borate-free 1f from

this mixture in 40% isolated yield.

In conclusion, we characterized the equilibrium between

ribosyl nucleosides and their corresponding 2’,3’-borate

esters in aqueous solution, a phenomenon which facilitates

apparent equilibrium shifts during nucleoside phosphoroly-

Scheme 2. Proposed mechanism for rate decreases in the presence of

borate. For simplicity’s sake, the transformations of the catalytic cycle

are depicted as unidirectional, although they are naturally reversible.

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sis and glycosylation reactions due to a biased esterification

of nucleosides over the sugar phosphate Rib1P. This borate

esterification also causes decreases of the phosphorolysis

rate by pyrimidine nucleoside phosphorylases, most likely

via non-productive binding of the nucleoside borate ester to

the enzyme and its hydrolytic interconversion to the free

substrate. Collectively, the effects described herein shine

light on the activity of nucleoside-binding enzymes in the

presence of borate and provide an additional means for

reaction engineering in nucleobase glycosylation reactions.

We suspect that similar processes are at play during the

inhibitory processes observed for NAD+-dependent oxidor-

eductases and the equilibrium shifts previously reported for

various enzymatic sugar epimerizations. Similarly, we antici-

pate that biased borate esterification could provide an

avenue for engineering of a variety of different

(chemo-)enzymatic reaction systems.

Acknowledgements

The authors thank the NMR department of the TU

Braunschweig as well as Samantha Voges (TU Berlin) for

assistance with spectroscopic experiments and Dr. M. Rhia

L. Stone (Rutgers University) for comments on the manu-

script. F.K. gratefully acknowledges funding by the Deut-

sche Forschungsgemeinschaft (DFG, German Research

Foundation), project number 492196858. Open Access

funding enabled and organized by Projekt DEAL.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are openly

available in zenodo.org at https://doi.org/10.5281/zenodo.

7401178, reference number 7401178.

Keywords: Biocatalysis ·Borate Ester ·Equilibrium ·

Nucleoside Analogues ·Phosphorolyase

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Manuscript received: December 14, 2022

Accepted manuscript online: January 19, 2023

Version of record online: February 24, 2023

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